Abstract：ObjectiveBased on fingerprint， index component content and dry extract yield， a quality evaluation method for substance benchmark of Xiebaisan was established to study the key quality attributes， to explore the quantitative transfer relationship between decoction pieces and substance benchmark， and to preliminarily formulate the quality standard of substance benchmark of Xiebaisan.MethodThe substance benchmark of Xiebaisan was prepared according to the records of ancient formulas， fingerprints of 15 batches of decoction pieces and substance benchmarks were collected by high performance liquid chromatography （HPLC） and the index components were determined with the mobile phase of acetonitrile-0.05% phosphoric acid solution for gradient elution. The dry extract yield， fingerprint similarity and transfer rate of index components were combined to study the quantity value transmitting.ResultTen characteristic peaks were identified in fingerprint of the substance benchmark and two characteristic peaks from stir-fried Mori Cortex， four characteristic peaks from baked Lycii Cortex， four characteristic peaks from Glycyrrhizae Radix et Rhizoma Praeparata cum Melle. Mulberroside A， liquiritin and glycyrrhizic acid were used as index components for the determination， the contents of mulberroside A， liquiritin and glycyrrhizic acid in substance benchmark of Xiebaisan were 2.69%-4.26%， 0.09%-0.17% and 0.09%-0.16%， and their transfer rates were （31.37±4.14）%， （36.12±4.03）% and （12.25±0.88）%， respectively. The similarity of fingerprint of substance benchmarks was good， the fingerprint similarities of 14 batches of substance benchmarks and control fingerprint were >0.9. The dry extract yield of substance benchmark of Xiebaisan ranged from 8.09% to 11.29%.ConclusionThe established quality evaluation method of substance benchmark of Xiebaisan is scientific and reasonable， and the transfer process of decoction pieces to substance benchmarks is stable and controllable. The preliminary quality standard of the substance benchmark can provide basis and reference for the development of modern preparations of Xiebaisan in the future.
Abstract：ObjectiveTo observe the effect of Yinhuotang （YHT） on the depression-like behavior of mice with bilateral ovariectomy （OVX） induced by chronic stress and explore its action mechanism based on estradiol （E2）-estrogen receptor β （ERβ） pathway.MethodThe experiment was divided into two parts. In the first part， mice were randomly divided into the sham operation （Sham） group， model （OVX） group， positive drug （E2， 0.13 mg·kg-1） group， and YHT （23.4 g·kg-1） group. The OVX model was reproduced by OVX combined with chronic unpredictable mild stress （CUMS）. On the 8th day after OVX， the mice in each group were exposed to CUMS and treated with drugs. The changes in immobility time， horizontal movement score， and vertical movement score of mice in each group were observed in forced swimming test （FST）， tail suspension test （TST） and open-field test （OFT）， respectively. Serum and brain E2 levels were detected by enzyme-linked immunosorbent assay （ELISA）， the aromatizing enzyme （Cyp19） mRNA expression by real-time fluorescence polymerase chain reaction （Real-time PCR）， the expression of ERα and ERβ in dentate gyrus of hippocampus by immunohistochemistry （IHC）， and the total ERα and ERβ levels in the brain by Western blotting. In the second part， the mice were divided into the Sham group， OVX group， YHT group， and blocker （Y+B， 23.4 g·kg-1+100 μg·kg-1） group. Mice in the Y+B group were first treated with intragastric administration of YHT and then with intraperitoneal injection of ERβ blocker （PHTPP） on the next day. The changes in immobility time， horizontal motor score， and vertical motor score were observed in the three behavioral tests.ResultCompared with the Sham group， the OVX group displayed significantly increased immobility time， decreased horizontal and vertical movement scores （P<0.01）， down-regulated serum and brain E2 levels （P<0.01） and Cyp19 mRNA expression in the brain （P<0.01）， and up-regulated total ERβ in dentate gyrus and brain （P<0.01）. However， there was no significant change in total ERα expression in the dentate gyrus or brain. As revealed by comparison with the OVX group， the immobility time of the E2 group was decreased significantly， while the horizontal and vertical movement scores were increased significantly （P<0.01）. The E2 levels in the serum was significantly elevated （P<0.01）. The Cyp19 mRNA expression in the brain and the total ERα expression in the dentate gyrus and brain were not significantly changed， while the expression levels of total ERβ in dentate gyrus and brain were significantly increased （P<0.01）. In the YHT group， the immobility time declined significantly， and the horizontal and vertical movement scores rose significantly （P<0.01）. The serum E2 did not increase， whereas the brain E2 increased significantly （P<0.01）. The expression of Cyp19 gene in the brain was significantly increased （P<0.05，P<0.01）. There was no significant change in the total ERα of dentate gyrus and brain， but the expression levels of total ERβ in dentate gyrus and brain were significantly increased （P<0.05，P<0.01）. PHTPP reversed the effects of YHT on OVX mice in FST， TST and OFT.ConclusionYHT promotes the synthesis and release of endogenous estrogen in brain and improves the depression-like behavior of OVX mice induced by chronic stress， which is possibly related to the activation of E2/ERβ pathway.
Abstract：ObjectiveTo investigate the effect of Banxia Xiexintang on the epithelial-mesenchymal transition （EMT） of human peritoneal mesothelial cell line （HMrSV5） induced by gastric cancer-derived exosomes （Exo）.MethodBanxia Xiexintang-containing serum was prepared and the human gastric cancer NCI-N87-derived exosomes （NCI-N87-Exo） were extracted， followed by their identification by transmission electron microscopy and Western blotting and labeling with 1，1-dioctadecyl-3，3，3，3- tetramethylindocarbocyanine perchlorate （Dil）. The cells were divided into the blank group， model group， and low-， medium-， and high-dose （13.5，27，54 g·kg-1） Banxia Xiexintang groups. HMrSV5 cells in the blank group were cultured alone， the ones in the model group with 100 mg·L-1 NCI-N87-Exo， and those in the low-， medium-， and high-dose Banxia Xiexintang groups with 100 mg·L-1 NCI-N87-Exo plus low-， medium-， and high-dose 10% Banxia Xiexintang-containing serum， respectively. Confocal laser microscope was used to observe the uptake of NCI-N87-Exo by HMrSV5 cells at 24 h， 48 h and 72 h. Seventy-two hours later， the morphological changes in HMrSV5 cells were observed. The protein expression levels of E-cadherin， cytokeratin 19 （CK19）， α-smooth muscle actin （α-SMA）， elastin， and transforming growth factor-β1 （TGF-β1）， Smad2/3， and p-Smad2/3 were assayed by Western blot.ResultIt was observed under the transmission electron microscope that NCI-N87-Exo showed an oval or dish-shaped vesicle structure with a particle size ranging from 40 to 80 nm. Exo marker proteins CD9 and CD63 were highly expressed while calreticulin was not expressed， implying that the NCI-N87-Exo was confirmed. After 24 h， 48 h， 72 h of co-culture， it was observed under the fluorescence microscope that NCI-N87-Exo were taken up by HMrSV5 cells， which was positively correlated with time. Compared with the blank group， Banxia Xiexintang significantly inhibited the uptake of NCI-N87-Exo by HMrSV5 cells， with better effect noticed in the middle- and high-dose Banxia Xiexintang groups（P<0.05，P<0.01）. After intervention with Banxia Xiexintang-containing serum， the HMrSV5 cells were arranged densely， and the intercellular space was significantly reduced， with the most obvious changes present in the high-dose Banxia Xiexintang group. Western blot revealed that the protein expression levels of E-cadherin and CK19 in HMrSV5 cells after being intervened with the medium- and high-dose Banxia Xiexintang-containing serum were increased significantly as compared with those in the blank group， whereas the levels of α-SMA and Elastin were decreased significantly （P<0.01）. Banxia Xiexintang-containing serum at the low， medium， and high doses remarkably down-regulated TGF-β1 and p-Smad2/3 protein expression（P<0.05，P<0.01）. However， there was no significant change in Smad2/3.ConclusionNCI-N87-Exo can be taken up by HMrSV5 cells to induce EMT. Banxia Xiexintang can inhibit the uptake of NCI-N87-Exo by HMrSV5 cells and the resulting EMT induced by NCI-N87-Exo， which is related to the regulation of TGF-β1/Smads signaling pathway.
Abstract：ObjectiveTo study the efficacy and mechanism of Zishenwan （ZSW） against pyroptosis and epithelial-mesenchymal transition （EMT） of renal tubular epithelial cells in diabetic nephropathy （DN） mice， so as to provide evidence for the treatment of DN with ZSW.MethodThe db/db mice with spontaneous diabetes were randomly divided into the model group， dapagliflozin （1.0 mg·kg-1） group， and high-， medium-， and low-dose （6.0， 3.0， 1.5 g·kg-1） ZSW groups. The non-diabetic db/m mice were classified into the normal group. The ones in the model and normal groups were given an equal volume of deionized water by gavage， while those in the other groups were intervened with the corresponding drugs for 12 weeks. The fasting blood glucose （FBG） level was tested at tail vein once every two weeks. The levels of urine albumin-creatinine ratio （ACR）， β-N-acetyl-D-glucosaminidase （NAG）， and cystatin C （CysC） were detected once every four weeks. After 12 weeks of administration， the blood sampled from eyeballs was used for measuring the blood urea nitrogen （BUN） and serum creatinine （SCr）. The pathological changes in renal tissues were observed by light microscopy and transmission electron microscopy. The expression of EMT markers in the renal tubular epithelium was analyzed by immunohistochemistry （IHC）. The in situ terminal end-labeling （TUNEL） staining was conducted to analyze the nuclear damage of renal tubular epithelial cells. The protein and mRNA expression levels of EMT markers， nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3） inflammasome and pyroptosis-related inflammatory cytokines in renal tissues were separately assayed by Western blot and Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）.ResultCompared with the normal group， the model group displayed significantly increased FBG， BUN， serum SCr， ACR， NAG， and CysC （P<0.01）， impaired renal tissues， altered EMT marker expression intensities and levels （P<0.01）， and elevated TUNEL-positive rate and protein and mRNA expression levels of pyroptosis-related inflammatory cytokines and NLRP3 inflammasome （P<0.01）. Compared with the model group， ZSW and dapagliflozin significantly decreased the levels of FBG， BUN， serum SCr， ACR， NAG， and CysC （P<0.01）， relieved the pathological injuries in renal tissues， changed the EMT marker expression intensities （P<0.01） and protein and mRNA expression levels （P<0.05， P<0.01）， and down-regulated the TUNEL-positive rate （P<0.01） of renal tubular epithelial cells as well as the protein and mRNA expression levels of pyroptosis-related inflammatory cytokines （P<0.01） and NLRP3 inflammasome （P<0.05， P<0.01）.ConclusionZSW alleviates DN possibly by inhibiting pyroptosis and EMT in renal tubular epithelial cells.
Abstract：ObjectiveTo explore the effect of Chaihu Jia Longgu Muli Tang on the hippocampus of rats with chronic stress depression based on the brain-derived neurotrophic factor (BDNF)/tyrosine kinase B (TrkB)/cyclic adenosine phosphate response element-binding protein (CREB) pathway.MethodSixty SD rats were divided into a blank group (n=10) and an experimental group (n=50) for the induction of the chronic stress depression model. The rats in the experimental group were further divided into the following five groups: a model group， a fluoxetine hydrochloride group (0.003 g·kg-1), and low-(1.625 g·kg-1), medium-(3.25 g·kg-1), and high-dose (6.5 g·kg-1) Chaihu Jia Longgu Muli Tang groups. The rats were administered correspondingly by gavage once a day for eight weeks. Behavioral tests were performed to evaluate the depression state of the rats before modeling， after modeling， and after drug administration. Hematoxylin-eosin (HE) staining was used to observe the morphological changes in the hippocampus of rats. The immunohistochemical (IHC) staining was used to quantitatively detect BDNF protein expression in the rat hippocampus. The mRNA and protein expression of BDNF， TrkB， and CREB in the rat hippocampus was detected by the real-time fluorescence-based quantitative PCR (Real-time PCR) and Western blot， respectively.ResultCompared with the blank group， the model group showed decreased sucrose preference rate (P<0.05)， declining horizontal and vertical scores (P<0.05)， and prolonged immobility time and floating time (P<0.05). Additionally， HE staining results revealed that hippocampal neuron structure was damaged. IHC staining showed that the mRNA and protein expression of BDNF， TrkB， and CREB was significantly decreased (P<0.05). Compared with the model group， the fluoxetine hydrochloride group and the Chaihu Jia Longgu Muli Tang groups displayed elevated sucrose preference rate (P<0.05)， increased horizontal and vertical scores (P<0.05)， and shortened immobility time and floating time (P<0.05). Furthermore, the hippocampal neuron structure was significantly repaired. IHC staining showed that the mRNA and protein expression of BDNF， TrkB， and CREB was significantly increased (P<0.05).ConclusionChaihu Jia Longgu Muli Tang can significantly improve the depression-like behaviors of rats after chronic stress stimulation and enhance the regeneration and repair of neurons in the hippocampus. The underlying mechanism may be related to the up-regulation of the BDNF/TrkB/CREB signaling pathway in the hippocampus of rats.
Keywords：Chaihu Jia Longgu Muli Tang;depression;hippocampus;brain-derived neurotrophic factor （BDNF）/tyrosine kinase B （TrkB）/cyclic adenosine phosphate response element binding protein （CREB）;mechanism study
Abstract：ObjectiveTo observe the improving effect of Danggui Shaoyaosan on diminished ovarian reserve （DOR） in rats triggered by Tripterygia wilfordii polyglycoside tablet combined with stress， and to explore the role of transforming growth factor-β1 （TGF-β1）/Smads signaling pathway in such improvement.MethodForty-eight female SD rats with normal sexual cycle were selected and randomly divided into a normal group （n=8） and a modeling group （n=40）， and the ones in the modeling group were given Tripterygium wilfordii polyglycoside tablets （50 mg·kg-1） combined with random stress for 15 d. After successful modeling， the rats were randomized into the model group， low-， medium-， and high-dose （3.96， 7.92， 15.84 g·kg-1） Danggui Shaoyaosan groups， and estradiol valerate group （0.09 mg·kg-1）， with eight in each group. Under the premise of stress exposure， they were separately gavaged with the normal saline， low-， medium- and high-dose Danggui Shaoyaosan， and estradiol valerate for 15 successive days. The estrous cycle of rats in each group was observed daily. After intervention， the rats were sacrificed and the ovarian visceral index was calculated. The pathological changes in ovarian tissues were observed by hematoxylin eosin （HE） staining. The protein expression levels of TGF-β1 and TGF-β1 receptor （TGF-β1R） in the ovarian tissues of rats were measured by immunohistochemistry （IHC）， and the mRNA expression levels of Smad2， Smad3， and Smad7 in the ovarian tissues by real-time polymerase chain reaction （Real-time PCR）.ResultCompared with the normal group， the model group exhibited disordered estrus cycle （P<0.05）， reduced visceral index （P<0.01）， and down-regulated TGF-β1 and TGF-β1R protein and Smad2 and Smad3 mRNA expression in the ovarian tissues （P<0.01）， and up-regulated Smad7 mRNA expression （P<0.01）. Compared with the model group， Danggui Shaoyaosan at the low， medium， and high doses and estradiol valerate improved the estrus cycle of rats to varying degrees （P<0.05） and increased the visceral index， with better effects observed in the medium-group and high-dose Danggui Shaoyaosan groups （P<0.05，P<0.01）. Besides， the protein expression levels of TGF-β1 and TGF-β1R and the mRNA expression levels of Smad2 and Smad3 in the ovarian tissues were elevated to varying degrees （P<0.01）， and the Smad7 mRNA expression declined （P<0.01）. The improvements in TGF-β1 and TGF-β1R protein expression of the medium-dose Danggui Shaoyaosan group and estradiol valerate group were more obvious.ConclusionDanggui Shaoyaosan significantly improves ovarian reserve in DOR rats， which is closely related to the regulation of TGF-β1/Smads signaling pathway.
Abstract：objectiveTo observe the activation of microglia and the expression of inflammatory factors in hippocampus of mice with depression-like behavior after mother-infant separation （MS） combined with lipopolysaccharide （LPS） stress， and to explore the possible anti-depression mechanisms of Wenyang （WY）， Jieyu （JY）， and Wenyang Jieyu （WYJY） prescriptions from the perspective of warming Yang and relieving depression.MethodSeventy offspring mice were randomly divided into a normal group （n=10）， a LPS stress group （n=10）， and a modeling group （n=50）. After undergoing 8 h·d-1 mother-infant separation during postnatal day 5 （PD5）–PD14， mice in the modeling group were further divided into the MS + LPS group， WY group， JY group， WYJY group， and fluoxetine （FLU） group， with 10 in each group. The birth date of the offspring mice was recorded as PD0. The mice in the normal， LPS， and MS + LPS groups were fed a normal diet during PD21–PD90， while those in the other groups were treated with the mixtures of corresponding drugs and feed， followed by seven-day intraperitoneal injection of LPS since PD91 for inducing depression. The depression-like behavior of mice in each group was detected in the open-field， O-maze， and social interaction tests. The protein expression of microglia-specific ionized calcium-binding adaptor molecule 1 （Iba-1） in the hippocampus was assayed by immunohistochemistry， and the mRNA expression of interleukin-1β （IL-1β）， interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， Iba-1， and glucocorticoid receptor （GR） by real-time fluorescence quantitative PCR （Real-time PCR）.ResultCompared with the normal group， the LPS group exhibited significantly reduced residence time at the central area within 5 min （P<0.01） and shortened total exercise distance （P<0.01）. In the MS + LPS group， the open-arm activity time and the total activity distance decreased significantly （P<0.01， P<0.05）， whereas the training， discrimination and exploration time increased significantly （P<0.01）. The expression of Iba-1 in hippocampal CA1 region of mice in the LPS and MS + LPS groups was remarkably elevated （P<0.01）. Compared with the LPS group， the MS + LPS group displayed significantly prolonged distance of 5-min exercise （P<0.05）， increased training， discrimination and exploration time （P<0.05， P<0.01）， and up-regulated Iba-1 expression in hippocampal CA1 area （P<0.01）. As revealed by comparison with the MS + LPS group， both the total 5-min exercise distance （P<0.01） and the training and discrimination time （P<0.01， P<0.05） of mice in each administration group was significantly shortened. The discrimination and exploration time of mice in the JY， WYJY， and FLU groups was significantly reduced （P<0.01）， and the expression of Iba-1 in hippocampal CA1 region of mice in each administration group was significantly down-regulated （P<0.01）.ConclusionThe warming Yang and relieving depression method helps to inhibit the occurrence and development of depression due to its efficacy in activating microglia in hippocampus of depression mice and lowering the expression of IL-1β， IL-6， and TNF-α.
Keywords：mother-infant separation （MS）;lipopolysaccharide （LPS）;warming Yang and relieving depression;microglia;hippocampus
Abstract：ObjectiveTo explore the possible mechanism of Astragali Radix-Curcumae Rhizoma （AC） in inhibiting tumor growth in the orthotopic transplantation model of colon cancer in mice.MethodThe molecular docking technology was used to predict the intermolecular interaction between the main active components of AC and the pathway target proteins， such as stromal cell-derived factor-1 （SDF-1）， C-X-C motif chemokine receptor 4 （CXCR4）， and nuclear factor kappa-B p65 （NF-κB p65）. The orthotopic transplantation model of CT26.WT colon cancer was established in mice for in vivo experimental verification. Sixty BALB/c male mice were randomly divided into a sham operation group， a model group， a 5-fluorouracil （5-Fu， 30 mg·kg-1） group，and low- （0.32 g·kg-1）， medium- （0.64 g·kg-1）， and high-dose （1.28 g·kg-1） AC groups， with 10 mice in each group. The sham operation group and the model group received normal saline by gavage. The corresponding drugs were administered by gavage in the 5-Fu group and by intraperitoneal injection in the AC groups. After intervention for 15 days， the tumor in situ was completely stripped， and the colon tissues 5-6 cm in length adjacent to the tumor were taken. The tumor volume was measured and calculated. The pathological changes of tumor tissues and colon tissues were observed by Hematoxylin-Eosin （HE） staining. Western blot was used to detect the protein expression of SDF-1， CXCR4， p-NF-κB p65 in colon tissues. Western blot and Real-time quantitative polymerase chain reaction （Real-time PCR） were used to detect SDF-1， CXCR4， NF-κB p65， Cyclin D1， oncogene c-Myc protein and mRNA expression in tumor tissues.ResultCompared with the model group， 5-Fu and AC groups showed reduced tumor volumes in situ （P<0.05， P<0.01）， with the tumor inhibition rate in the 5-Fu group as high as （61.38±2.34）%. The tumor-inhibiting effect was optimal in the medium-dose AC group， with the tumor inhibition rate of （43.43±3.71）%. Compared with the model group， 5-Fu and AC groups showed relieved pathological changes of tumor and colon tissues. Specifically， AC down-regulated the protein expression levels of SDF-1， CXCR4， and p-NF-κB p65 in colon tissues （P<0.01）， and down-regulated the protein and mRNA expression levels of SDF-1， CXCR4， NF-κB p65， Cyclin D1， and c-Myc in tumor tissues （P<0.05， P<0.01）.ConclusionAC can inhibit the growth of orthotopic transplantation tumor of colon cancer， and its intervention mechanism may be related to the regulation of related protein and mRNA expression in the SDF-1/CXCR4/NF-κB signaling pathway.
Abstract：ObjectiveTo investigate the effect of astragaloside Ⅳ（AST Ⅳ）and Notoginseng total saponins （NTS） combined with bone marrow mesenchymal stem cell （BMSC） transplantation on neural repair and angiogenesis in rats with cerebral ischemia.MethodThe rats were randomly divided into a sham operation group， a model group， low- and high-dose AST Ⅳ + NTS groups， a BMSC infusion group， and low- and high-dose BMSC infusion+AST Ⅳ （10 and 20 mg·kg-1） + NTS group （25， 50 mg·kg-1）. BMSCs were isolated and purified by whole bone marrow adherent culture. The positive expression of surface markers of BMSCs （CD29， CD90， CD34， and CD45） was detected by flow cytometry. The focal cerebral ischemia model was established by middle cerebral artery occlusion （MCAO）. The PKH26-labeled BMSCs were injected into the tail vein of rats in the BMSC infusion group， once a day. The rats in the combination groups received BMSC injection once a day and intragastric administration of drugs twice a day. Other groups were administered twice a day by gavage. The sham operation group and the model group received the same amount of normal saline. Symptoms and signs of neurological deficits were assessed by the Longa method and the cerebral infarction rate was determined by TTC staining. The survival and vascularization ［double positive expression of PKH26/vascular endothelial growth factor （VEGF）］ after transplantation of BMSCs were observed by the immunofluorescence method. The protein expression of Ang1 and TGF-β1 was measured by Western blot.ResultBMSCs were properly isolated and cultured. The identification of surface markers CD29， CD90， CD34， and CD45 was consistent with the characteristics of BMSCs. The neurological deficit score and cerebral infarction rate of the model group were significantly increased （P<0.01）. All drugs and cell transplantation could alleviate the above pathological changes in varying degrees. The strongest effect was observed in high-dose BMSC infusion+AST Ⅳ+NTS group （P<0.01）， which was superior to those in the AST Ⅳ+NTS groups or the BMSC infusion group. BMSC injection helped cells survive in the ischemic brain tissues and promoted angiogenesis， and this effect could be enhanced by the combination with drugs. After cerebral ischemia， the expression of Ang1 and TGF-β1 was increased， and the effect in the BMSC infusion+AST Ⅳ+NTS groups was the strongest （P<0.01）.ConclusionAST Ⅳ combined with NTS can promote the survival of transplanted BMSCs and facilitate angiogenesis after target repair of damaged blood vessels after cerebral ischemia. The mechanism may be related to the improvement of the local microenvironment in the brain after cerebral ischemia and the promotion of the survival and differentiation of transplanted stem cells.
Keywords：astragaloside Ⅳ;Notoginseng total saponins;bone marrow mesenchymal stem cells;cerebral ischemia;angiogenesis
Abstract：ObjectiveTo explore the mechanism of energy changes in the three stages of the formation of coronary heart disease due to blood stasis in rat model from the perspective of mitochondrial fusion-fission dynamic changes.MethodThirty healthy male rats were divided into the blank control group （n=6） and model group （n=24） using SPSS 21.0 simple random sampling method. The rats in the blank control group were fed an ordinary diet， while those in the model group a high-fat diet. After seven days of adaptive feeding， the rats were treated with intragastric administration of vitamin D3 （VitD3） at 300 000 U·kg-1 and then at 200 000 U·kg-1 14 d later. The high-fat diet continued for 21 d， and six rats were randomly selected as samples for the pre-stage blood stasis syndrome group， followed by model verification and sampling. The remaining rats continued to receive the high-fat diet for 30 d， and six were randomly selected and categorized into the sub-stage blood stasis syndrome group， followed by model verification and sampling. The rest of rats were classified into the heart blood stasis syndrome group. While continuing the high-fat diet， they were also treated with multipoint subcutaneous injection of isoproterenol （ISO，5 mg·kg-1） for three consecutive days. One week later， the electrocardiogram （ECG） was recorded for determining whether the modeling was successful and the samples were taken at the same time. The changes in mitochondrial morphology and quantity were observed under a transmission electron microscope. The expression of mitochondrial dynamics-related proteins was measured by Western blot and the cellular localization of related proteins by immunofluorescence assay.ResultThe levels of total cholesterol and low-density lipoprotein cholesterol in the pre-stage and sub-stage blood stasis syndrome groups were significantly increased as compared with those in the blank control group （P<0.05）. The blood rheology index in the pre-stage blood stasis syndrome group was significantly elevated in contrast to that in the blank control group （P<0.05）. The three-layered membrane of the aorta in the blank group was intact. However， the tunica media of the pre-stage blood stasis syndrome group began to show obvious calcification， with a small number of inflammatory cells adhering to the intima. The subintima and media smooth muscles in the sub-stage blood stasis syndrome group exhibited cavity structures. The three-layered structure of the arterial wall in the heart blood stasis syndrome group was severely damaged. The ECG of the blank control group revealed the regular appearance of P wave，regular QRS waveform （no broadening or deformity）， and no obvious ST-segment depression or elevation. The ECG of the pre-stage blood stasis syndrome group showed no obvious abnormalities as compared with that of the blank control group. In the sub-stage blood stasis syndrome group， the ECG showed an upward trend of the J point and slight ST-segment elevation， with the elevation≤0.1 mV. The ECG in the heart blood stasis syndrome group displayed significant ST-segment depression （>0.1 mV） and J point depression >0.1 mV. The mitochondria in the blank control group were normal in size and morphology， with clear and dense cristae， whereas those in the pre-stage blood stasis syndrome group were fusiform with sparse cristae. Some mitochondria in the sub-stage blood stasis syndrome group were significantly elongated， and even vacuole-like changes were present. In the heart blood stasis syndrome group， the mitochondria were ruptured. As demonstrated by comparison with the blank control group， the expression levels of mitofusin 2 （Mfn2）， dynamin-related protein 1 （Drp1）， and fission protein 1 （Fis1） in the model group were significantly up-regulated （P<0.05，P<0.01）. Compared with the pre-stage blood stasis syndrome group， the heart blood stasis syndrome group exhibited down-regulated Mfn2 （P<0.05）. Compared with the blank control group and the pre-stage blood stasis syndrome group， the sub-stage blood stasis syndrome group and the heart blood stasis syndrome group displayed down-regulated optic atrophy 1（OPA1） （P<0.05，P<0.01）. The Drp1 and Fis1 protein expression declined significantly in the sub-stage blood stasis syndrome group in comparison with that in the pre-stage blood stasis syndrome group （P<0.05，P<0.01）. The expression levels of Mfn2 and Drp1 in the heart blood stasis syndrome group were lower than those in the sub-stage blood stasis syndrome group （P<0.01）. The comparison with the blank control group showed that Mfn2 and OPA1 were extensively accumulated in mitochondria of both the pre-stage and sub-stage blood stasis syndrome groups， while the red-stained Mfn2 was significantly reduced in the heart blood stasis syndrome group. The Drp1/Fis1 fluorescence was weak in the blank group and the pre-stage blood stasis syndrome group but strong in the sub-stage blood stasis syndrome group and heart blood stasis syndrome group.ConclusionThe cardiomyocyte mitochondria dynamics changes with the change in energy demand of cardiomyocytes. Mfn2 is dominated by fusion effect in the early stage of the formation of coronary heart disease due to blood stasis. With the gradual development of this disease， Mfn2 begins to mediate mitochondrial autophagy. OPA1 plays a role in intimal fusion and cristae integrity. The decreased OPA1 expression is closely related to the accelerated progression of coronary heart disease differentiated into blood stasis syndrome. The process by which Drp1 and Fis1 separate damaged mitochondria to prepare for mitochondrial autophagy contributes to alleviating the imbalance between the energy demand and supply of human body.
Keywords：mitochondrial dynamics;coronary heart disease due to blood stasis;mitofusin 2 （Mfn2）;optic atrophy 1（OPA1）;dynamin-related protein 1 （Drp1）;fission protein 1 （Fis1）;energy metabolism
Abstract：ObjectiveTo explore the acute toxicities and hepatotoxicities of aqueous extracts of Taxilli Herba from Morus alba， Toxicodendron trichocarpum， Camellia oleifera， Salix babylonica， Melia azedarach， and Nerium indicum against zebrafish model and the effect of different hosts on the toxicity of Taxilli Herba， hoping to provide a theoretical basis for the safe use of Taxilli Herba.MethodThe normally developed AB zebrafish at 3-day post fertilization was selected for acute toxicity study. According to the results of preliminary toxicity experiments， the zebrafishes were treated with aqueous extracts of Taxilli Herba from different hosts at six doses， and their mortality was calculated 72 h later. GraphPad Prism 6.0 was used for plotting the dose-toxicity curve， followed by the calculation of their median lethal concentration （LC50） and 10% lethal concentration （LC10）. The gz15Tg/+（AB） liver fluorescent protein transgenic zebrafish with normal development at 4-day post fertilization was applied for the hepatotoxicity study. The zebrafishes were divided into the low-， medium-， and high-dose groups of aqueous extracts of Taxilli Herba from six hosts， the positive control （acetaminophen） group， and the blank （embryo amniotic fluid） group， and then treated with the corresponding drugs. Seventy-two hours later， the liver morphology and fluorescent area changes in zebrafish were observed. And the activities of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were detected.ResultThe results of acute toxicity test demonstrated that the LC50 values of water extracts of Taxilli Herba from M. alba， T. trichocarpum， C. oleifera， S. babylonica， M. azedarach， and N. indicum were 1.24， 0.94， 0.51， 0.38， 0.11， 0.09 g·L-1， respectively， and the LC10 values were 0.70， 0.60， 0.35， 0.28， 0.08， 0.07 g·L-1， respectively. As revealed by hepatotoxicity test， compared with the blank group， the positive control group exhibited liver morphological changes， decreased fluorescent area （P<0.01）， and elevated ALT and AST activities （P< 0.01）， suggesting that acetaminophen was hepatotoxic to zebrafish. However， there was no change in the liver morphology or fluorescent area of zebrafish in the low-， medium-， and high-dose groups of water extracts of Taxilli Herba from M. alba， and the ALT and AST activities were decreased. By contrast， the liver morphology and fluorescent areas in the medium- and high-dose groups of water extracts of Taxilli Herba from T. trichocarpum， C. oleifera， S. babylonica， M. azedarach， and N. indicum changed to varying degrees （P<0.05， P<0.01）. Besides， the activities of both ALT and AST were also enhanced. These indicated that Taxilli Herba from M. alba had no hepatotoxicity to zebrafish， while that from T. trichocarpum， C. oleifera， S. babylonica， M. azedarach， and N. indicum showed varying degrees of hepatotoxicity to zebrafish.ConclusionThe toxicity of Taxilli Herba is host-dependent. Taxilli Herba from M. alba has no hepatotoxicity， but that from the other five hosts shows varying degrees of hepatotoxicity. Standardizing the host source may be an important measure to realize the medication safety of Taxilli Herba.
Abstract：ObjectiveTo explore whether palmatine interferes with the proliferation and apoptosis of colon cancer HCT116 cells by binding to G-quadruplex in the promoter region of MYC proto-oncogene and its possible molecular mechanism.MethodFluorescence spectrum was used to analyze the binding ability of palmatine to MYC G-quadruplex. Circular dichroism analysis was conducted to confirm the effect of palmatine on the configuration of MYC G-quadruplex， followed by the prediction of their binding mode based on molecular docking and the localization analysis of palmatine in HCT116 cells under a fluorescence microscope. The effects of palmatine on MYC gene transcription and MYC protein expression were determined by real-time fluorescence quantitative polymerase chain reaction and Western blot， respectively. The effects of palmatine on the viability and apoptosis of HCT116 cells were further assayed by thiazolyl blue tetrazolium bromide （MTT） assay and flow cytometry.ResultAs revealed by fluorescence spectrum and molecular docking analysis， palmatine might bind to G-quadruplex of MYC gene through stacking. Circular dichroism analysis showed that palmatine could maintain the parallel configuration of MYC G-quadruplex. It was discovered in fluorescence imaging that palmatine was distributed in the nucleus and bond to G-quadruplex of MYC gene. In addition， palmatine inhibited MYC gene transcription， MYC protein expression， as well as the viability of HCT116 cells， and promoted the apoptosis of HCT116 cells.ConclusionPalmatine is able to bind to MYC G-quadruplex to further inhibit the expression of MYC gene and protein expression， which may be one of the molecular mechanisms of palmatine in suppressing the proliferation of colon cancer HCT116 cells and facilitating their apoptosis.
Abstract：ObjectiveTo investigate the action mechanism of Yinchenhao Tang against type 2 diabetes mellitus （T2DM） complicated with non-alcoholic fatty liver disease （NAFLD） in MKR mice.MethodForty eight-week-old MKR mice were fed a high-fat diet for eight weeks and then divided into the model group，original Yinchenhao Tang （17.16 g·kg-1） group，Yinchenhao Tang group at a specified dose （4.68 g·kg-1） in teaching materials，and positive drug ［metformin + simvastatin， （65+2.6）×10-3 g·kg-1］ group. Another 10 MKR mice of the same age were classified into the blank group and 10 FVB mice into the normal group. After eight weeks of intragastric administration in each group，the liver wet weight，oral glucose tolerance test （OGTT），serum inflammatory factors interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α），and changes in blood lipid and liver function were determined. Hematoxylin-eosin （HE） staining was conducted for observing the morphological changes in liver tissue under a transmission electron microscope，followed by the detection of Toll-like receptor 4 （TLR4），myeloid differentiation factor 88 （MyD88），and nuclear transcription factor-κB （NF-κB） protein expression by Western blot.ResultCompared with the model group，the medication groups exhibited significantly reduced liver wet weight index （P<0.01），improved OGTT result （P<0.05），and down-regulated serum IL-6 and TNF-α levels （P<0.01）. In terms of morphological changes，Yinchenhao Tang protected the hepatocyte structure and alleviated hepatocyte steatosis. Moreover， Yinchenhao Tang obviously down-regulated the protein expression levels of TLR4，MyD88，and NF-κB in liver tissue of MKR mice with T2DM combined with NAFLD （P<0.05），and the down-regulation of TLR4 and NF-κB in the original Yinchenhao Tang group was better than that in the Yinchenhao Tang group at a specified dose in teaching materials （P<0.05）.ConclusionYinchenhao Tang is able to reduce inflammatory factor levels and down-regulate TLR4，MyD88，and NF-κB expression in liver tissue to relieve the pathological liver injury and interfere with T2DM combined with NAFLD of MKR mice. It exerts a certain liver-protective effect by lowering the blood lipids and delaying the hepatic inflammation.
Abstract：ObjectiveTo observe the changes in oxidative stress and transforming growth factor-β1 （TGF-β1）/Smad signaling pathway in hippocampal tissue of senile depressed mice after chronic unpredictable mild stress and to explore the possible anti-depression mechanism of Bushen Shugan prescription.MethodNinety five-month-old mice were randomly divided into six groups， namely the normal group， senile depression model group， high-， medium-， and low-dose Bushen Shugan prescription groups， and fluoxetine group， with 15 in each group. Mice in all groups， except for the normal group， were exposed to chronic unpredictable mild stress （CUMS） for inducing the senile depression. Since the first day of modeling， the mice in the high-， medium- and low-dose Bushen Shugan prescription groups were gavaged with 19.5， 9.75， 4.87 g·kg-1 Bushen Shugan prescription， the ones in the fluoxetine group with 0.033 g·kg-1 fluoxetine， and those in the normal and senile depression model groups with an equal volume of normal saline for 21 successive days. The behavioral responses of mice in each group were evaluated in the open field test （OFT）， and the hippocampal tissues of mice were collected for testing the relevant indexes. The superoxide dismutase （SOD） content was determined by WST-1 method， malondialdehyde （MDA） content by TBA method， glutathione （GSH） content by micro enzyme-linked immunosorbent assay （ELISA）， and mRNA expression of TGF-β1， Smad2， Smad3， and Smad7 by Real-time polymerase chain reaction （Real-time PCR）.ResultCompared with the normal group， the senile depression model group exhibited significantly lowered horizontal and vertical scores in OFT， decreased SOD and GSH contents in hippocampal tissues， elevated MDA （P<0.05）， up-regulated TGF-β1， Smad2， and Smad3 mRNA expression， and down-regulated Smad7 （P<0.05）. Compared with the senile depression model group， Bushen Shugan prescription at the high， medium， and low doses and fluoxetine all increased SOD and GSH contents in mouse hippocampal tissues， decreased the MDA content （P<0.05）， down-regulated the mRNA expression of TGF-β1， Smad2， and Smad3 in hippocampal tissues， and up-regulated the Smad7 mRNA expression （P<0.05）. The comparison with the high-dose Bushen Shugan prescription group showed that the SOD and GSH contents in hippocampal tissues of mice in the medium- and low-dose Bushen Shugan prescription groups declined significantly， while the MDA contents rose significantly （P<0.05）. Besides， the mRNA expression levels of TGF-β1， Smad2 and Smad3 in the hippocampal tissues of mice in the medium- and low-dose Bushen Shugan prescription groups were significantly up-regulated， and those of Smad7 were significantly down-regulated （P<0.05）.ConclusionBushen Shugan prescription alleviates the depression symptoms in aged SAPM8 mice possibly by regulating the hippocampal oxidative stress and TGF-β1/Smad signaling pathway.
Abstract：ObjectiveTo investigate the effect of Ranae Oviductus (RO) on ovarian follicular development， phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway， and pregnancy function in rats, and the estrogen-like mechanism of OR.MethodSeventy female Wistar rats were randomly divided into a normal group， a progynova+ luteohormone group (1 mg·kg-1+40 mg·kg-1), a clomiphene group (10 mg·kg-1), and high-dose(400 mg·kg-1) and low-dose(200 mg·kg-1) RO groups. Rats were administered correspondingly by gavage for eight weeks. After seven weeks of intragastric administration, the estrus cycle of all rats was measured. After eight weeks of intragastric administration， four rats from each group were selected to give birth. For other rats, blood was collected on the day of estrus， and the serum levels of estradiol (E2)，progesterone (P)， testosterone (T)， follicle-stimulating hormone (FSH)， and luteotropic hormone (LH) were detected. Uterus and ovaries were extracted and weighed to calculate organ index. One ovary was made into pathological sections, and the follicles at different developmental stages and corpus luteum were counted. Real-time fluorescence-based quantitative polymerase chain reaction（Real-time PCR）and Western blot were performed on the other ovary to detect mRNA and protein changes in the PI3K/Akt signaling pathway. Forty female Kunming mice were randomly divided into a normal group and RO groups (400 mg·kg-1) with 14 days， 28 days， and 56 days of intervention. Mice in the RO groups were raised with male mice in cages after intragastric administration of OR for 14， 28， and 56 days， respectively. After 18 days, the number of intrauterine fetuses on both sides and the number of stunted fetuses were counted.ResultAfter eight weeks of intragastric administration of OR， the rats showed decreased uterine index (P<0.05)， declining serum LH (P<0.05)， reduced luteum (P<0.01)， dwindled primary follicles (P<0.05)， and increased rate of follicle atresia (P<0.01). Additionally， more luteal or interstitial glands degenerated into interstitial structures in the ovarian cortex in a short time. The mRNA expression levels of PI3K and Akt in the ovary were elevated (P<0.01)， while the mRNA expression levels of mTOR and PTEN were reduced (P<0.01). The phosphorylation level of Akt protein showed a downward trend without significant difference. For the rats， the number of fetuses was decreased (P<0.05). The pregnancy rate of mice was decreased to varying degrees after administration of RO for different durations， with the lowest in the 14 day RO group， as low as 30%. After 28 days of intragastric administration of RO， the difference in left and right uterine pregnancy increased (P<0.05).ConclusionLong-term administration of RO can lead to premature ovarian failure by over-stimulating the ovary， which is similar to clomiphene. Short-term administration can result in decreased pregnancy rate， excessive ovulation on one side， and inhibition of ovulation on the other side. The influence on follicles needs further exploration.
Abstract：ObjectiveTo investigate the effect of shikonin on uterine leiomyoma in rats and its molecular mechanism.MethodSixty female SD rats， of SPF grade and weighing 200-250 g， were randomly divided into the control group， model group， low-， medium-， and high-dose （5， 10， 20 mg·kg-1） shikonin groups， and mifepristone group. A rat model of uterine leiomyoma was established， and the changes in uterine wet weight， uterine coefficient， and smooth muscle thickness were detected after drug administration for four successive weeks. The pathological changes in uterine tissue were observed by hematoxylin-eosin （HE） staining. The contents of estrogen receptor （ER） and progesterone receptor （PR） in serum and uterus were measured by enzyme-linked immunosorbent assay （ELISA）， and the protein expression levels of ER， PR， phosphorylated extracellular signal-regulated kinase （p-ERK）， and ERK in the uterine tissue were assayed by Western blot.ResultCompared with the control group， the model group exhibited significantly increased uterine wet weight， uterine coefficient， and smooth muscle thickness （P<0.01）， uterus deformity， focal smooth muscle cell necrosis and hyperplasia， neutrophil infiltration. elevated serum and uterine ER and PR （P<0.01）， and up-regulated p-ERK protein expression in the uterine tissue （P<0.01）. Compared with the model group， shikonin at the middle and high doses and mifepristone significantly reduced the uterine wet weight， uterine coefficient， and smooth muscle thickness （P<0.01）， relieved the pathological changes in uterus，and lowered serum and uterine ER and PR， and down-regulated the p-ERK protein expression in the uterine tissue （P<0.05）. In addition， the uterine wet weight， smooth muscle thickness， serum ER， and uterine PR and p-ERK protein expression in the low-dose shikonin group were significantly lower than those in the model group （P<0.05）.ConclusionShikonin produces the anti-uterine leiomyoma activity possibly by inhibiting the activation of ERK pathway.
Abstract：ObjectiveTo study the clinical effect of Qinghao Fuzheng Jiedu decoction on systemic lupus erythematous （SLE）.MethodA total of 109 SLE patients admitted to the Rheumatology and Immunology Department of Wuhan No. 1 Hospital from December 2019 to October 2020 were selected and divided into an observation group （55 cases） and a control group （54 cases） using the random number table. Two cases in the observation group dropped out， leaving a total sample of 53， and one case in the control group dropped out， with 53 cases finally included. Patients in the control group were treated with prednisone tablet and azathioprine. On this basis， those in the observation group further received Qinghao Fuzheng Jiedu decoction. The clinical efficacy， traditional Chinese medicine （TCM） syndrome score， TCM syndrome efficacy， immunoglobulin （Ig） G， IgA， IgM， and complements C3 and C4 of the two groups were compared. The conversion of positive antinuclear antibody （ANA） and anti-double-stranded deoxyribonucleic acid antibody （DS-DNA） titers to negative in two groups after treatment was analyzed.ResultThe total clinical efficacy rate of the observation group was significantly higher than that of control group （92.45% vs 73.58%，χ2=6.692，P<0.05）. Before treatment， there were no significant differences in IgG， IgA， IgM， complements C3 and C4， and serum ANA and ds-DNA titers between two groups. After treatment， the levels of IgG， IgA， and IgM and serum ANA and ds-DNA titers in both groups obviously declined， whereas the levels of complements C3 and C4 rose （P<0.05）. Besides， the levels of IgG， IgA， and IgM and serum ANA and ds-DNA titers in the observation group were lower than those in the control group， while the levels of complements C3 and C4 were higher （P<0.05）. The negative rates of ANA and ds-DNA in observation group were significantly higher than those in control group （χ2=8.040，P<0.05）. TCM syndrome scores were decreased in both groups after treatment （P<0.05）， and the score in observation group was lower than that in control group （P<0.05）. In terms of TCM syndrome efficacy， the total effective rate of observation group was significantly increased as compared with that of the control group （94.34% vs 50.94%，χ2=25.112，P<0.05）.ConclusionQinghao Fuzheng Jiedu decoction is effective in treating SLE and has a certain clinical application value.
Keywords：systemic lupus erythematous（SLE）;Qinghao Fuzheng Jiedu decoction;clinical efficacy;traditional Chinese medicine （TCM） syndrome score;immunoglobulin
Abstract：ObjectiveTo observe the clinical efficacy of modified Xiaoyao Erxian decoction in the treatment of mood disorders among perimenopausal syndrome due to kidney deficiency and liver depression and its effects on monoamine neurotransmitters and brain-derived neurotrophic factor （BDNF）.MethodAccording to the random number table， 108 patients were divided into a control group （54 cases） and an observation group （54 cases）. Control group were treated with Shugan Jieyu capsule orally， two capsules per time， two times per day， while those in the observation group received the modified Xiaoyao Erxian decoction， one bag per day， for 12 consecutive weeks. Before and after treatment， the Hamilton Anxiety Scale （HAMA）， 17-item Hamilton Depression Rating Scale （HAMD-17）， modified Kupperman Index （KI）， Pittsburgh Sleep Quality Index （PSQI）， Menopause-specific Quality of Life （MENQOL） and kidney deficiency and liver depression syndrome scores were calculated. The levels of estradiol （E2）， follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， BDNF， 5-hydroxytryptamine （5-HT）， norepinephrine （NE）， and dopamine （DA） were detected， followed by safety evaluation.ResultThe HAMA， HAMD-17， KI， kidney deficiency and liver depression syndrome， PSQI， and MENQOL scores of the observation group were lower than those of the control group （P<0.01）， whereas the 5-HT， E2， DA， NE， and BDNF levels in the observation group were higher （P<0.01）. The clinical efficacy of the observation group was superior to that in the control group （Z=2.184， P<0.05）. No adverse reactions occurred after the oral administration of Chinese medicinal preparations.ConclusionThe modified Xiaoyao Erxian decoction effectively alleviates the mood disorders and other related symptoms of perimenopausal syndrome due to kidney deficiency and liver depression by regulating the monoamine neurotransmitters， BDNF， and E2， without inducing obvious side effects.
Abstract：ObjectiveTo observe the rehabilitation effect of modified Guipitang administration combined with traditional Chinese medicine （TCM） hot pressing in patients with deficiency of Qi and blood syndrome breast cancer postoperative， and investigate its effect on immune function and tumor markers.MethodOne hundred and fifty-four patients were divided into observation group （77 cases） and control group （77 cases） by random number table. Two groups were given comprehensive treatment measures after operation. Patients in control group additionally took Bazhen granules orally， 8 g/time， 2 times/day， for eight weeks. Patients in observation group additionally took Guipitang orally for syndrome differentiation， 1 dose/day for eight weeks. The chest， shoulders and upper limbs of the affected side were hot-pressed with TCM， 20 min/time， 2 times/ day， 5 days a week， the first 4 weeks. The occurrence of lymphedema， subcutaneous fluid， poor skin flap growth， sleep disturbance， shoulder joint dysfunction， etc. Were recorded in both groups. Before and after treatment， the scores of European organization for research and treatment of cancer quality of life questionnaire core-30（EORTC QLQ-30）， and scores of cancer-induced fatigue and Qi and blood deficiency were graded. T lymphocyte subsets （CD3+， CD4+， CD8+ levels and CD4+/CD8+）， regulatory T cells （Treg）， inhibitory T cells （Ts）， cytotoxic T cells （Tc）， human growth differentiation factor 3 （GDF3）， serum carbohydrate antigen 153 （CA153）， carcinoembryonic antigen （CEA） and human epidermal growth factor -2 （HER-2） levels were detected before and after treatment.ResultAfter treatment， the observation group incidence of lymphedema， subcutaneous effusion， poor skin flap growth， sleep disturbance and shoulder joint dysfunction was 8（10.39%）， 9（11.69%）， 11（14.29%）， 25（32.47%） and 8 （10.39%） respectively in the observation group， all lower than 18（23.38%）， 20（25.97%）， 23（29.87%）， 46（59.74%） and 19（24.68%） in the control group（P<0.05， P<0.01）. The scores of overall quality of life and function scores in the observation group were higher than those in the control group （P<0.01）， hile symptom score was lower than that in the control group （P<0.01）. The scores of cancer-induced fatigue deficiency of Qi and blood syndrome in the observation group were lower than those in the control group （P<0.01）. The Tc， CD3+， CD4+， CD4+/CD8+ levels in the observation group were higher than those of the control group （P<0.01）， while the Treg， Ts， CD8+ levels were lower than those in the control group （P<0.01）. The GDF3， CA153， CEA， HER-2 levels in the observation group were lower than those in the control group （P<0.01）.ConclusionOn the basis of conventional comprehensive interventions of western medicine， Guipitang combined with TCM hot pressing for breast cancer patients after surgery can reduce postoperative complications， reduce fatigue， postoperative symptoms and TCM syndromes. Besides， it can enhance the immune function of the body， improve the quality of life， promote postoperative recovery， and inhibit the expression of tumor markers， thus improving the prognosis of patients.
Keywords：breast cancer;postoperative recovery;deficiency of Qi and blood syndrome;Guipitang;traditional Chinese medicine hot pressing;immune function
Abstract：ObjectiveUltra-high performance liquid chromatography-quadrupole/orbitrap high resolution mass spectrometry （UHPLC-Q-Orbitrap-MS） was used to rapidly analyze and assign the chemical constituents of Naizilai granules.MethodAn ACQUITY UPLC BEH Shield RP C18 column （2.1 mm×100 mm， 1.7 µm） was selected for chromatographic analysis， the mobile phase was 0.1% formic acid aqueous solution （A） and acetonitrile （B） for gradient elution （0-3 min， 1%B； 3-16 min， 1%-11%B； 16-30 min， 11%-34%B； 30-37 min， 34%-52%B； 37-42 min， 52%-100%B； 42-44 min， 100%B）， flow rate was 0.3 mL·min-1 and the column temperature was 35 ℃. Mass spectrometry data of Naizilai granules were collected in positive and negative ion modes， the chemical constituents of this preparation were speculated and identified according to the precise molecular weight， secondary fragmentation and other information， combined with reference substance and literature data.ResultA total of 175 compounds were identified and speculated， including 72 flavonoids， 77 organic acids， 15 sesquiterpenes， 6 coumarins and 5 other compounds. Among these identified chemical constituents， there were 154 from Artemisia rupestris， 64 from Hyssopus cuspidatus， 33 from Cordia dichotoma， 42 from Viola tianshanica， 56 from Lactuca sativa， 65 from Mentha haplocalyx， 78 from Matricaria chamomilla， 28 from Ziziphus jujuba， 7 of which were common components of these eight herbs.ConclusionThe established analytical method can realize the rapid and accurate identification of the chemical constituents in Naizilai granules， and basically covers the main constituents of each medicinal material in the formula， so as to provide a basis for improving the quality evaluation system of the preparation and lay a foundation for elucidating the pharmacodynamic mechanism.
Keywords：Naizilai granules;ultra-high performance liquid chromatography-quadrupole/orbitrap high resolution mass spectrometry （UHPLC-Q-Orbitrap-MS）;flavonoids;organic acids;sesquiterpenoids;chemical constituents;coumarins
Abstract：ObjectiveTo carry out germplasm resource evaluation and new variety breeding of Murraya paniculata and improve the germplasm quality， so as to ensure the demand， yield and quality of medicinal materials.MethodFollowing resource investigation and collection， 17 traits of 107 M. paniculata germplasm samples， like plant type， basal diameter， leaf shape， leaf length， and leaf width were determined and then subjected to principal component analysis and factor analysis for screening the principal component factors. Nine primary traits were selected as variables for further cluster analysis using Ward's method and Euclidean distance. According to the characteristics of medicinal parts， the core germplasms were screened out. Then the contents of auxin， zeatin， zeatin nucleoside， isopentenyl adenine， isopentenyl adenine riboside， dihydrozeatin， and dihydrozeatinriboside in the leaves were measured by ultra-performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）， followed by their correlation analysis with agronomic trait.ResultThe variation coefficients of petiole length， branching number， and basal diameter were large. The nine main factors could be classified into four categories， with a contribution rate of 72.822%. The cluster analysis with Ward's method and Euclidean distance showed that 107 germplasm samples were clustered into six clusters and 61 core germplasms were identified. Such traits as leaf length， leaf width， petiole length， leaf surface， and petiole color were found to play an important role in the classification of M. paniculata germplasms. The content of zeatin nucleoside exhibited significant positive correlations with leaf length （P<0.01）， petiole length （P<0.01）， and leaf width （P<0.05）.ConclusionThese results have laid the foundation for further selection and breeding of M. paniculata new varieties.
Abstract：ObjectiveTo establish the high performance liquid chromatography （HPLC） fingerprint of Citri Sarcodactylis Fructus， and to search for makers to characterize the quality difference of Citri Sarcodactylis Fructus from different origins coupled with chemometrics.MethodThe analysis was performed on a Thermo Hypersil GOLD C18 column （4.6 mm×250 mm， 5 μm） with mobile phase consisted of acetonitrile-0.05% phosphoric acid solution for gradient elution， and the detection wavelength was set at 254 nm. A total of 31 batches of samples were analyzed to establish the HPLC fingerprint of Citri Sarcodactylis Fructus. Similarity evaluation was performed by Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System （2012 edition） to confirm the common peaks， which were identified by comparison of reference substances. On the basis， chemometrics methods were used to analyze and evaluate the quality of Citri Sarcodactylis Fructus from different origins. At the same time， 3 batches of 5 species of decoction pieces from the genus Citrus in the family Rutaceae， including Citri Sarcodactylis Fructus， Aurantii Fructus Immaturus， Aurantii Fructus， Citri Reticulatae Pericarpium Viride and Citri Reticulatae Pericarpium， were randomly collected for evaluating the effectiveness and reliability of the established HPLC fingerprint of Citri Sarcodactylis Fructus.ResultHPLC fingerprint of Citri Sarcodactylis Fructus was established and 22 common peaks were identified. And seven common peaks among them were identified as 6，7-dimethoxycoumarin， diosmin， hesperidin， byakangelicin， 5，7-dimethoxycoumarin， bergapten and oxypeucedanin. Except for 2 batches of samples， the similarities of fingerprints between other 29 batches of samples were >0.9. The 31 batches of Citri Sarcodactylis Fructus were basically divided into 3 groups by cluster analysis and principal component analysis， which were consistent with the classification of three different producing areas. Eight differential markers were screened by orthogonal partial least squares discriminant analysis and four of them （5，7-dimethoxycoumarin， bergapten， 6，7-dimethoxycoumarin and diosmin） were identified by reference substances. Similarity evaluation of 5 species of decoction pieces from genus Citrus in the family Rutaceae was carried out by taking the reference fingerprint of Citri Sarcodactylis Fructus as treference chromatogram， similarity of Citri Sarcodactylis Fructus decoction pieces was 0.892-0.977， and the similarities of the other 4 kinds of decoction pieces were 0.215-0.517.ConclusionThe established fingerprint method is reasonable， effective and accurate for quality control of Citri Sarcodactylis Fructus， the characterization information is more comprehensive combined with chemometrics.
Abstract：ObjectiveTo explore the mechanism of Xiaojinwan in treating breast cancer bone metastases through cell experiments and bioinformatic analysis.MethodThe inhibitory effect of Xiaojinwan on MCF-7 cell viability was detected by cell counting kit-8 （CCK-8） assay. The key components and targets responsible for Xiaojinwan in inhibiting breast cancer bone metastases were predicted by network pharmacology and molecular docking. The active components and targets of Xiaojinwan were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCSMP） and SwissTarget Prediction， and the breast cancer bone metastases-related targets from GeneCards and DisGeNET. The results were imported into STRING for constructing a protein-protein interaction （PPI） network， followed by Gene ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis using DAVID. A network of the active components of Xiaojinwan-breast cancer bone metastases-related targets-pathways was constructed using Cytoscape 3.7.2. AutoDock 4 was employed for molecular docking. The protein expression levels of matrix metallopmteinase-9 （MMP-9）， hypoxia-inducible factor 1α （HIF1A）， and androgen receptor （AR） were assayed by Western blot.ResultXiaojinwan inhibited the viability of MCF-7 cells and acted on breast cancer bone metastases through such processes as redox and protein autophosphorylation. KEGG enrichment analysis showed that HIF-1， vascular endothelial growth factor （VEGF） and phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） pathways were involved. As verified by molecular docking， the active components such as eucalyptin stably bound to AR and MMP-9. Western blot indicated that Xiaojinwan dose-dependently inhibited the expression of MMP-9 and HIF1A proteins in MCF-7 cells.ConclusionXiaojinwan acts on AR and MMP-9 through HIF， VEGF and other related signaling pathways， thereby improving hypoxia in tumor microenvironment， inhibiting angiogenesis， and reducing cell invasion and viability.
Abstract：The present study systematically evaluated the efficacy and safety of Pushen capsule （PC） in the treatment of hyperlipidemia （HPL） to provide the basis for its clinical application in the future. The randomized controlled trials （RCTs） of PC in treating HPL were comprehensively retrieved from four Chinese databases and three English databases. The included RCTs were evaluated using the Cochrane risk-of-bias tool， followed by the Meta-analysis by RevMan 5.3. Twenty-four RCTs were included， with 2 634 patients involved. The patients in the experimental group were treated with PC， PC combined with conventional therapy，or PC combined with other Chinese patent medicine. The cases in the control group were treated conventionally or by other Chinese patent medicine. Meta-analysis results showed that PC alone or in combination was superior to the treatment in the control group in improving total cholesterol （TC），triglyceride （TG），high-density lipoprotein cholesterol （HDL-C），low-density lipoprotein cholesterol （LDL-C），and total response rate. Fourteen trials reported adverse reactions， including seven reporting specific results. The adverse reactions were dominated by epigastric discomfort， but the difference was not statistically significant. However， affected by the quality of the included trials，the evidence strength of the conclusion of this study is graded low. More high-quality RCTs reported in detail are needed for further confirmation.
Abstract：The application of modern scientific theories and technologies to explore the mechanism of Chinese medicine and its compounds is one of the key issues in realizing the modernization of traditional Chinese medicine （TCM） research. Chinese medicine and its compounds produce comprehensive pharmacodynamics through multiple components acting on multiple targets， the core of clarifying the mechanism is to solve the key scientific problems of static correlation and dynamic integration verification between the components and the target network topology. At present， the effective method to clarify the mechanism of Chinese medicine and its compounds is to statically correlate the topological network of in vitro components and targets through network pharmacology. Although there are also component-target verification studies， they often learn from research idea of single component-single target， it is urgent to establish a quantitative integration and overall verification method that conforms to the characteristics of TCM. According to supramolecular Qi chromatography theory of TCM， the microscopic mechanism of interaction between Chinese medicine and human body is actually the two supramolecular host and object groups （the active ingredient group of Chinese medicine and the target group of human body） based on imprinted template， which shows the macroscopic properties and pharmacodynamics. Based on this， the author proposes to use supramolecular Qi chromatography theory as the guidance， combined with supramolecular chemistry， network dynamics， quantitative pharmacology and other methods to quantitatively integrate and verify the compositions and the target groups with imprinted template as the core predicted by network pharmacology， looking for the optimal quality markers， greatly reducing the difficulty of multi-component-multi-target experimental verification of Chinese medicine and its compounds.
Abstract：The recycling of traditional Chinese medicine （TCM） wastes is an important research topic to be solved urgently in the industrialization of TCM resources. Rhei Radix et Rhizoma is a bulk Chinese herb mainly derived from Rheum palmatum，R. tanguticum，and R. officinale. At present，these three medicinal plants have been cultivated on a large scale and widely used in the fields of medicine，health care，food，cosmetics，and veterinary medicine，with an annual demand of more than 5 500 tons（1 ton=1 000 kg）. However，a large number of wastes such as non-medicinal parts and residues produced in the production and deep processing are discarded because there is no effective way of utilization，resulting in serious waste of resources and environmental pollution. The non-medicinal parts contain not only the chemical components and pharmacological effects similar to those of roots and rhizomes but also a variety of amino acids，mineral elements，and conventional nutrients. They have a long history of use，and the content of some resource components is higher than that in roots and rhizomes. In particular，their stems and leaves exhibit great potential to be consumed as food and medicine due to high safety. Besides，the content of anthraquinones in Rhei Radix et Rhizoma residue is high and it possesses good antibacterial activity. It can be seen that the waste from the industrialization of Rhei Radix et Rhizoma has high utilization value. Hence，based on the relevant literature and investigation on the application of producing areas in China and abroad，the paper summarized the utilization status of their medicinal and non-medicinal parts，the waste production in the industrialization，as well as the active substances and utilization ways and put forward the multi-level and multi-path utilization strategy of Rhei Radix et Rhizoma wastes，in order to provide reference for the rational development and application of Rhei Radix et Rhizoma resources and promote the effective utilization and green development of the corresponding wastes.
Keywords：Rhei Radix et Rhizoma;non-medicinal parts;medicinal residue;waste;resource value;resource utilization
Abstract：Betulinic acid （BA） is a lupane pentacyclic triterpene extracted from a variety of Chinese herbs such as Betulae Platyphyllae Cortex， Astragali Radix， Paeoniae Radix Alba， Jujubae Fructus， Sanguisorbae Radix， Eucommiae Cortex， Glycrrhizae Radix et Rhizoma， Aucklandiae Radix， and Ziziphi Spinosae Semen. It has attracted wide attention from doctors because of its low toxicity， high efficacy， and multiple functions. BA has been found to possess a significant anti-tumor biological activity， and it is expected to become a potential drug for the treatment of malignant tumors. So far， a number of studies have shown that BA is able to promote apoptosis， inhibit proliferation， metastasis and invasion， and induce cell cycle arrest via multiple mechanisms， thus resisting various malignant tumors such as ovarian cancer， breast cancer， gastric cancer， lung cancer， colorectal cancer， and prostate cancer. It exerts the anti-tomor effect by regulating the expression of cancer suppressor genes p53 and p21， triggering the generatoipn of reactive oxygen species （ROS）， down-regulating the expression of nuclear transcription factor-κB （NF-κB）， adjusting the B lymphocytoma-2 （Bcl-2） family to cause tumor cell apoptosis， and regulating transcription factor Sp1/3/4 to induce apoptosis. Its anti-proliferative activity is mainly achieved via the regulation of cyclin B， cyclin D and cyclin dependent kinases CDK and CDC. Its efficacy in inhibiting metastasis and invasion is mainly realized by regulating matrix metalloproteinase （MMP） and matrix metalloproteinase inhibitor （TIMP）， up-regulating E-cadherin， down-regulating N-cadherin and blocking the epithelial-mesenchymal transformation （EMT）. In addition， BA also induces cell cycle arrest， affects tumor metabolic reprogramming， and activates autophagy to inhibit tumor. Although there are a large number of studies on BA against tumors and its efficacy has been proved strong， the systematic review on its anti-tumor effect is still lacking. Therefore， this study reviewed the anti-tumor effect and mechanism of BA， in order to provide reference for its subsenquent research.
Abstract：Pyroptosis， also known as cell inflammatory necrosis， is different from apoptosis， necrosis， and other forms of cell death in morphological characteristics， occurrence， and regulatory mechanism. It is a new type of programmed cell death dependent on Caspase， which has been discovered and confirmed in recent years. Studies have shown that pyroptosis is closely related to the occurrence of liver diseases， and is critical in alcoholic liver disease （ALD）， non-alcoholic fatty liver disease （NAFLD）， liver fibrosis， and liver cancer. Pyroptosis causes inflammatory injury of hepatocytes to promote the occurrence and development of liver diseases by activating Caspase-1， cleaving the effector gasdermin-D （GSDMD）， and releasing pro-inflammatory cytokines， such as interleukin-18 （IL-18） and interleukin-1β （IL-1β） mainly through the classical NOD-like receptor protein 3 （NLRP3） inflammasome pathway. Clinically， Chinese medicine in the treatment of liver diseases has unique efficacy and low side effects. In the intervention on liver diseases， Chinese medicine blocks the pyroptosis signaling pathway to relieve the liver inflammation by inhibiting the assembly and activation of NLRP3 inflammasome multiprotein complex， blunting the activity of Caspase-1 or Caspase-4/Caspase-5/Caspase-11， and inhibiting the cleavage of GSDMD to reduce the release of pro-inflammatory cytokines such as IL-18 and IL-1β. Therefore， in-depth investigation of pyroptosis facilitates unveiling its role in the occurrence， development， and prognosis of liver diseases， and the enhancement or inhibition of pyroptosis may provide a new strategy for the prevention and treatment of liver diseases by Chinese medicine. At present， NLRP3 inflammasome， a key protein in the classic pyroptosis signaling pathway， has become an anti-liver disease target of Chinese medicine. This study briefly summarized the relationship between pyroptosis and liver diseases and reviewed the intervention of monomers， compound prescriptions， effective fractions and extracts of Chinese medicine in recent years to provide important guidance for further exploring the pathogenic mechanism of pyroptosis and the treatment of liver diseases with Chinese medicine.
Keywords：Chinese medicine;liver diseases;pyroptosis;NOD-like receptor protein 3 （NLRP3） inflammasome
Abstract：Diabetic cardiomyopathy occurs in diabetic patients and is different from hypertensive heart disease， coronary atherosclerotic heart disease， and other cardiac abnormalities. The main clinical symptoms are systolic and diastolic cardiac dysfunction， myocardial fibrosis， congestive heart failure， and angina pectoris. As one of the main complications of diabetes， its incidence and fatality rates have been on the rise year by year. However， modern medicine still fails to figure out its pathogenesis and no specific drug is available， which has seriously affected the survival and quality of life of patients. Cardiomyocytes contain a large number of mitochondria， which participate in cardiac energy metabolism and other biological activities and occupy an important position in the development of diabetic cardiomyopathy. Mitochondrial quality control mainly involves mitochondrial oxidative stress， mitochondrial dynamics， mitochondrial autophagy， and intracellular calcium regulation， which is an important condition for stabilizing the normal mitochondrial structure and exerting normal mitochondrial functions. In recent years， the efficacy of traditional Chinese medicine in intervening in mitochondrial quality control through multiple angles， pathways， and targets to affect the structure and function of myocardial mitochondria and significantly improve the clinical symptoms of patients with diabetic cardiomyopathy has attracted wide attention from scholars. Therefore， this paper reviewed the experimental studies and/or clinical observations concerning the treatment of diabetic cardiomyopathy with effective compounds of Chinese herbs and/or Chinese herbal compounds in the past ten years to further explain the pathogenesis of diabetic cardiomyopathy， clarify the regulatory mechanism of traditional Chinese medicine in mitochondrial quality control， and summarize the scientific connotations and shortcomings of traditional Chinese medicine in the treatment of diabetic cardiomyopathy， hoping to provide certain ideas and methods for further clinical application of traditional Chinese medicine in the treatment of diabetic cardiomyopathy.