Abstract：ObjectiveTo explore the progression of diabetic macrovascular disease and the effects of Didangtang at different doses on it.MethodFour-week-old male apolipoprotein-E knockout （ApoE-/-） mice with diabetic macrovascular disease induced by exposure to high-fat diet combined with streptozotocin （STZ） were randomly divided into the model， simvastatin， as well as high-， medium-， and low-dose Didangtang groups. The age-matched ApoE-/- mice of the same batch only fed with a high-fat diet were classified into the ApoE-/- （model control） group， and C57BL/6 mice with the same genetic background receiving a regular diet into the normal group. The sampling was conducted at the 8th and 20th weeks of the experiment for observing the pathological characteristics of the aorta and the proportion of plaque area in mice of each group at different time points， followed by the comparison of blood glucose， blood lipid， and oxidized low-density lipoprotein （ox-LDL） levels. The aortic NOD-like receptor protein 3 （NLRP3） and cysteinyl aspartate-specific proteinase-1 （Caspase-1） protein expression was detected by Western blot assay， and the serum interleukin-1β （IL-1β）， interleukin-18 （IL-18）， interleukin-1α （IL-1α）， and tumor necrosis factor-α （TNF-α） levels by enzyme-linked immunosorbent assay （ELISA）.ResultThe comparison with the normal group revealed that the proportions of plaque area in the ApoE-/- group and the model group were increased （P<0.01）， while the proportion of plaque area in each administration group was significantly reduced in contrast to that of the model group （P<0.05）. The aortic NLRP3 and Caspase-1 protein expression levels as well as the serum IL-1β， IL-18， IL-1α， and TNF-α levels in the ApoE-/- group and the model group were significantly higher than those in the normal group （P<0.01）. Compared with the model group， each administration group exhibited a significant reduction in aortic NLRP3 and Caspase-1 protein expression and serum IL-1β， IL-18， IL-1α， and TNF-α levels （P<0.05）， with the strongest inhibitory effect detected in the medium-dose Didangtang group （P<0.05）.ConclusionDidangtang directly alleviates diabetic macrovascular disease possibly by down-regulating NLRP3 and Caspase-1 protein expression and easing the inflammatory cascade.
Keywords：diabetes;macrovascular disease;NOD-like receptor protein 3 （NLRP3） inflammasome;cysteinyl aspartate-specific proteinase-1 （Caspase-1）
Abstract：ObjectiveTo investigate the effect of Buyang Huanwutang （BHT） on proliferation and differentiation in neural stem cells （NSCs） after oxygen-glucose deprivation/reoxygenation （OGD/R） injury.MethodNSCs isolated from the hippocampus of SD rats were cultured and randomly divided into a normoxia group， a model group， a BHT group， a rapamycin （Rapa） group， and a combination group ［autophagy inhibitor 3-methyladenine （3-MA） combined with BHT］. The 20% blank serum was used in the normoxia group， and 20% BHT-medicated serum in the BHT group. The doses of Rapa and 3-MA were 1 μmol·L-1 and 5 mmol·L-1， respectively. The cells were subjected to OGD/R except those in the normoxia group. The cell morphology was observed under a light microscope. NSCs were confirmed by immunofluorescence detection of nestin expression. The viability and proliferation of NSCs were assessed by cell counting kit-8 （CCK-8） assay and 5-ethynyl-2-deoxyuridine （EdU） labeling， respectively. Furthermore， Ad-mCherry-GFP-LC3B fluorescence assay was performed to investigate autophagy. The effect of BHT on autophagy-related protein expression was detected by western blot assay. Brain derived neurotrophic factor （BDNF）， β-tubulin Ⅲ， and glial fibrillary acidic protein （GFAP） were evaluated by immunofluorescence assay.ResultOGD/R significantly reduced the cell viability of rat NSCs as compared with the normoxia group. Compared with the model group， the BHT group exhibited significantly improved viability of rat NSCs （P<0.01）. BHT induced the production of autophagosomes in NSCs after OGD. The BHT group showed increased expression of microtuble-associated protein 1 light chain 3Ⅱ (LC3Ⅱ） and Beclin-1 （P<0.05，P<0.01） and slightly changed p62 compared with the normoxia group， and significantly up-regulated LC3Ⅱ and Beclin-1 （P<0.05，P<0.01） and down-regulated expression of p62 （P<0.01） compared with the model group. The Rapa group had similar effect as the BHT group （P<0.05，P<0.01）， while the combination group inhibited the activity of autophagy （P<0.01）. As indicated by the results of ad-mCherry-GFP-LC3B， compared with the normoxia group， the model group showed increased fluorescence intensity （P<0.01）， and the BHT and Rapa groups could further increased the fluorescence intensity of autophagy （P<0.01）， while the combination group inhibited autophagy activity （P<0.01）. Immunofluorescence results revealed that compared with the normoxia group， the model group displayed significantly reduced positive cells of EdU， β-tubulin Ⅲ， GFAP， and BDNF （P<0.01）， and the BHT and Rapa groups exerted similar protective and promoting effects （P<0.05，P<0.01）， while the combination group partially blocked the neuroprotection and differentiation ability of BHT （P<0.05）.ConclusionBHT pretreatment can effectively protect rat NSCs against OGD-induced injury and promoted proliferation and differentiation by up-regulating autophagy.
Abstract：ObjectiveTo study the effect of Gegen Qinliantang （GQT） on the structure of intestinal flora in dysbacterial diarrhea rats by 16S rRNA sequencing.MethodSixty healthy SD rats were randomly and equally divided into a control group， a model group， high-， medium-， and low-dose GQT groups， and a Bifidobiogen group. The rat model was induced in the five groups except the control group by administration of mixed antibiotics （178.6 mg·kg-1 cefradine and 31.25 mg·kg-1 gentamicin sulfate） according to the dose. Drug intervention was carried out in each group （7.02， 3.51， and 1.755 g·kg-1 GQT for the high-， medium-， and low-dose GQT groups， 0.125 g·kg-1 bifidobacterium capsules for the Bifidobiogen group， and sterile distilled water for the control and model groups） with a volume of 10 mL·kg-1 for seven days. Colon contents of rats were obtained under anesthesia. The extracted fecal DNA underwent 16S rRNA high-throughput sequencing and the results were analyzed.ResultGQT was proved capable of adjusting the species number and Alpha and Beta diversity， improving the biological richness and diversity of the flora， and positively regulating three differential phyla （Firmicutes， Proteobacteria， and Bacteroidetes） and 14 differential genera （Bacteroides， Parabacteroides， Blautia， etc.） in rat model of dysbacterial diarrhea.ConclusionThe present study confirmed the regulatory effect of GQT on intestinal flora of dysbacterial diarrhea rats， and revealed the physiological and pathological mechanism between intestinal flora and dysbacterial diarrhea.
Abstract：ObjectiveTo explore the effects and mechanism of Chinese classical prescription Dahuang Zhechongwan on silicosis in mice.MethodThirty-six male Kunming mice of SPF grade were randomized into the normal control group， model control group， tetrandrine （Tet， 0.039 mg·kg-1） group， as well as high- （1.560 g·kg-1）， medium- （0.780 g·kg-1）， and low-dose （0.390 g·kg-1） Dahuang Zhechongwan groups， with six mice in each group. Mice in all groups except for the normal control group underwent static inhalation of silica （SiO2） dust for 40 consecutive days to induce fibrosis. After 28 days of intervention with corresponding drugs， the mice were sacrificed to collect the serum and lung tissues， with the former used for detecting tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， IL-6， and hydroxyproline （HYP） levels by enzyme-linked immunosorbent assay （ELISA） and the latter for observing the pathological changes. Meanwhile， the protein and mRNA expression levels of p38 mitogen-activated protein kinase （p38 MAPK）， nuclear transcription factor-κB （NF-κB）， transforming growth factor-β1 （TGF-β1）， α-smooth muscle actin （α-SMA）， Smad2， Smad3， and Smad7 in the lung tissues were determined by Western blot and real-time polymerase chain reaction （Real-time PCR）.ResultCompared with the normal group， the contents of TNF-α， IL-1β， IL-6 and HYP in the model group were significantly increased， the difference was statistically significant（P<0.05，P<0.01）； compared with the model group， the high-dose group of Dahuang Zhechongwan could significantly reduce the contents of TNF-α， IL-6 and HYP in the serum of mice（P<0.05， P<0.01）， indicating that Dahuang Zhechongwan could reduce the lung inflammation of silicosis mice. At the same time， compared with the normal group， the protein and mRNA expression levels of p38 MAPK， NF-κB p65， TGF-β1， α-SMA， Smad2 and Smad3 in the model group were significantly increased（P<0.05，P<0.01）， while the protein and mRNA expression levels of Smad7 were significantly decreased（P<0.01）； compared with the model group， the protein and mRNA expression levels of p38 MAPK， NF-κB p65， TGF-β1， α-SMA， Smad2 and Smad3 in the high-dose Dahuang Zhechongwan group were significantly increased the protein and mRNA expression levels were significantly decreased（P<0.05，P<0.01）， while Smad7 protein and mRNA expression levels were significantly increased（P<0.05，P<0.01）.ConclusionDahuang Zhechongwan ameliorates the alveolar inflammation， extracellular matrix （ECM） deposition， and fibrosis in mice with silicosis possibly by regulating the p38 MAPK/NF-κB/TGF-β1 pathway.
Keywords：Dahuang Zhechongwan;mouse model of silicosis;SiO2-induced fibrosis;p38 mitogen-activated protein kinase （p38 MAPK）/nuclear transcription factor-κB （NF-κB）/transforming growth factor-β1 （TGF-β1） pathway
Abstract：ObjectiveTo investigate the molecular mechanism of Qiyu Sanlong prescription （QYSL） in inhibiting the "addiction" of lung cancer A549 cells to miRNA21.MethodThe human lung cancer A549 cells were routinely passaged and divided into the blank group， blank serum group， QYSL-containing serum group， and siRNA group. The prepared QYSL-containing serum was used for intervention， with the optimal concentration and action time determined in previous studies. The protein and mRNA expression levels of miRNA21 and related molecules in its target phosphatase and tensin homolog deleted on chromosome 10 （PTEN）/phosphatidylinositol 3-kinase （PI3K） signaling pathway were detected by real-time polymerase chain reaction （Real-time PCR） and Western blot assay.ResultThe comparison with the blank serum group revealed that the mRNA expression levels of miRNA21 in the QYSL-containing serum group and the siRNA group were decreased， while the PTEN mRNA expression in the QYSL-containing serum group was increased， showing significant differences （P<0.01）. Compared with the blank serum， the QYSL-containing serum and siRNA significantly down-regulated PI3K and mammalian target of rapamycin （mTOR） mRNA expression （P<0.01）， whereas the QYSL-containing serum did not change the mRNA expression of protein kinase B （Akt）. The protein expression levels of PTEN in the QYSL-containing serum group and the siRNA group were obviously elevated in contrast to that in the blank serum group （P<0.05）. Meanwhile， the protein expression levels of phosphorylated Akt （p-Akt） and phosphorylated mTOR （p-mTOR） evidently declined in the QYSL-containing serum group （P<0.05）， but there was no significant reduction in total Akt and mTOR protein expression. The PI3K protein expression was slightly down-regulated， with no statistical significance.ConclusionQYSL inhibits the transcription of miRNA21， increases the expression of PTEN， and reduces the expression of key molecules in PI3K/Akt/mTOR signaling pathway， thus mildly inhibiting the "addiction" of lung cancer cells to oncogenes and blocking their proliferation.
Keywords：Qiyu Sanlong prescription （QYSL）;lung cancer A549 cells;miRNA21;phosphatase and tensin homolog deleted on chromosome 10 （PTEN）/phosphatidylinositol 3-kinase （PI3K） signaling pathway
Abstract：ObjectiveTo investigate the therapeutic effect and mechanism of modified Wenjingtang on endometriosis （EM） rats with kidney deficiency and blood stasis.MethodHealthy non-pregnant female Sprague-Dawley （SD） rats of SPF grade were randomly divided into the blank group and experimental group. After being modeled via soaking in ice water and subcutaneous injection of epinephrine hydrochloride， the ones in the experimental group were further divided into the sham operation group and EM model group， with the former only undergoing laparotomy and the latter further receiving autologous endometrial transplant for triggering EM. The successfully modeled rats with EM due to kidney deficiency and blood stasis were randomized into the positive drug （danazol， 63 mg·kg-1） group and low- （5 g·kg-1）， medium- （10 g·kg-1）， and high-dose （20 g·kg-1） modified Wenjingtang groups. The corresponding drugs were administered by gavage， once per day， for four weeks. Then the ectopic and eutopic endometrial tissues were stained with hematoxylin-eosin （HE） to observe the histopathological changes. The protein and mRNA expression levels of cysteinyl aspartate-specific proteinase-8 （Caspase-8）， matrix metalloproteinase-9 （MMP-9）， N-cadherin， and E-cadherin were detected by immunohistochemistry （IHC）， Western blotting， and real-time polymerase chain reaction （Real-time PCR）， respectively.ResultThe IHC and Western blot revealed that the protein expression levels of MMP-9 and N-cadherin in eutopic and ectopic endometrial tissues of the model group were significantly increased as compared with those in the sham operation group （P<0.01）， while the levels of Caspase-8 and E-cadherin was significantly decreased （P<0.01）. Compared with the model group， the danazol and low-， medium-， and high-dose modified Wenjingtang groups exhibited obviously down-regulated MMP-9 and N-cadherin protein expression in eutopic and ectopic endometrial tissues （P<0.05，P<0.01）， but up-regulated Caspase-8 and E-cadherin （P<0.05， P<0.01）. Real-time PCR uncovered that the mRNA expression levels of MMP-9 and N-cadherin in eutopic and ectopic endometrial tissues of the model group were significantly elevated as compared with those in the sham operation group （P<0.01）， whereas the levels of Caspase-8 and E-cadherin significantly declined （P<0.01）. The comparison with the eutopic endometrial tissue in the model group showed that the mRNA expression levels of MMP-9 and N-cadherin in the danazol group and high- and medium-dose modified Wenjingtang groups were significantly down-regulated， while those of Caspase-8 and E-cadherin were significantly up-regulated （P<0.05，P<0.01）.ConclusionModified Wenjingtang alleviates the immunosuppression and blocks the angiogenesis in EM rats with kidney deficiency and blood stasis syndrome by regulating the expression of such cytokines as Caspase-8， MMP-9， N-cadherin， and E-cadherin， thus exerting the therapeutic effect against EM. The above-mentioned micro-indicators are potential markers reflecting the disease （EM）， syndrome （kidney deficiency and blood stasis）， and pathological mechanisms （immunosuppression and angiogenesis）.
Abstract：ObjectiveTo explore the intervention effect of Yuxuebi tablet （YXB） on collagen-induced arthritis （CIA） in rats and its anti-inflammatory mechanism.MethodFollowing CIA modeling， the rats in the drug administration groups were separately treated with intragastric administration of YXB （0.1， 0.2， and 0.4 g·kg-1） and methotrexate （MTX， 0.4 mg·kg-1）， once a day. The incidence of CIA， mechanical pain threshold （MPT） and cold pain threshold （CPT） were evaluated once every three days. After continuous administration for 30 days， the peripheral blood of rats was collected for the determination of platelet （PLT） count and fibrinogen （FIB） content. The hematoxylin-eosin （HE） staining was conducted to analyze the pathological changes in joint tissues. The protein expression levels of interleukin （IL）-1β， IL-8， nuclear transcription factor-κB （NF-κB） p65， phosphorylated NF-κB （p-NF-κB） p65， Ras， and Raf-1 in joint tissues of CIA rats were detected by immunohistochemistry （IHC） and Western blot. The rheumatoid arthritis fibroblast-like synoviocytes （RA-FLS） were induced by tumor necrosis factor-α （TNF-α， 10 μg·L-1） in vitro and then subjected to transwell migration/invasion assay， followed by the detection of protein expression levels of Ras， Raf-1， and p-NF-κB p65 in RA-FLS by Western blot.ResultCompared with the control group， the model group exhibited an increased incidence of CIA， significantly decreased MPT （P<0.05，P<0.01）， elevated CPT （P<0.01） and PLT and FIB in the peripheral blood， worsened histopathological score of joints， enhanced RA-FLS migration and invasion， and up-regulated inflammatory factors （P<0.01）. The comparison with the model group revealed that YXB at different doses obviously reduced the incidence of CIA， increased MPT， down-regulated CPT and PLT and FIB in the peripheral blood （P<0.05，P<0.01）， ameliorated the pathological changes like synovial hyperplasia and bone and cartilage destruction （P<0.05，P<0.01）， and inhibited RA-FLS migration and invasion. Besides， the low-， medium-， and high-dose YXB reversed the IL-1β， IL-8， Ras， Raf-1， and p-NF-κB p65 expression in joint tissues of CIA rats to different extents， as well as the protein expression of Ras， Raf-1 and p-NF-κB p65 in RA-FLS （P<0.05，P<0.01）.ConclusionYXB reduces the incidence of CIA， ameliorates the clinical symptoms of RA and the pathological changes in joint tissues， and inhibits the formation of synovium， which may be attributed to its inhibition against Ras/Raf-1/NF-κB signaling pathway.
Abstract：ObjectiveTo investigate the hepatotoxicity of different doses of geniposide on the liver of rats and the effects on bile acid profile in serum， liver tissue and feces.MethodThe 60 Sprague Dawley rats， half male and half female， were randomly divided into 5 groups according to body weight： blank group and four different doses （50， 100， 200， 400 mg·kg-1） geniposide groups， 12 rats in each group. The rats were treated by gavage once a day for 7 consecutive days， and the serum， liver and cecal contents were collected on the 8th day of treatment. The activities of alanine aminotransferase （ALT）， aspartate aminotransferase （AST） and alkaline phosphatase （ALP）， the contents of albumin （ALB）， total bilirubin （TBIL）， total bile acid （TBA）， creatinine （Crea） and carbamide （Urea） were detected in each group. The sections of liver tissue were stained with hematoxylin-eosin（HE）， and the protein expressions of cytokeratin 7（CK7） and cytokeratin 19（CK19） were detected by immunohistochemistry. The protein expressions of CK7 and CK19 in the liver tissue were detected by Western blot. And the mRNA expressions of cholesterol 7α-hydroxylase （CYP7A1）， cholesterol 27α-hydroxylase （ CYP27A1） and cholesterol 12α-hydroxylase （CYP8B1） were detected by real-time PCR. The contents of 18 kinds of bile acids in serum， liver and cecal contents were determined by ultra-performance liquid chromatography-mass spectrometry（UPLC-MS）.ResultCompared with the control group， TBIL level in each dose of geniposide group was increasesd significantly （P<0.01）. ALT， AST activity and TBA content in 400 mg·kg-1 geniposide group were increased significantly （P<0.05， P<0.01）. HE staining showed that， compared with control group， there was bile duct reaction in the portal area and inflammatory cells infiltrate around bile duct in 200 mg·kg-1 and 400 mg·kg-1 geniposide groups， especially 400 mg·kg-1. The expressions of CK7 and CK19 in liver tissue of 400 mg·kg-1 geniposide group were significantly higher than those in the control group （P<0.05， P<0.01）. Compared with the control group， the contents of glycoursodeoxycholic acid （GUDCA） and glycohyodeoxycholic acid （GHDCA） in liver tissue of 400 mg·kg-1 geniposide group decreased significantly （P<0.05， P<0.01）， the contents of sodium taurochenodeoxycholate （TCDCA）， hyodeoxycholic acid （HDCA）， cholic acid （CA） and chenodeoxycholic acid （CDCA） in liver tissue increased significantly （P<0.01）， the contents of glycocholic acid hydrate （GCA）， glycochenodeoxycholic acid （GCDCA）， glycodeoxycholic acid hydrate （GDCA）， glycocholic acid （GLCA）， tauroursodeoxycholic acid （TUDCA）， GUDCA， GHDCA， ursodeoxycholic （UDCA） and taurolithocholic acid （TLCA） decreased， the proportions of TCDCA， HDCA， CA， CDCA and deoxycholic acid （DCA） in liver tissue increased， the contents of GHDCA and lithocholic acid （LCA） in serum decreased significantly （P<0.01）， while sodium taurohyodeoxycholate hydrate （THDCA）， taurocholic acid （TCA）， GCA， TCDCA， UDCA， CA， CDCA， DCA in serum decreased significantly （P<0.05， P<0.01）. The contents of CA， UDCA， CA， CDCA and DCA increased significantly （P<0.05）， the ratio of CA/DCA increased significantly （P<0.05）， and the ratio of CA and CDCA increased by 19.60% and 4.63%， respectively； Compared with the control group， the contents of all bile acids in cecal contents of 400 mg·kg-1 were decreased， and the contents of GCA， UDCA， HDCA， GCDCA， GDCA， TLCA， GLCA， CDCA， DCA and LCA were decreased significantly （P<0.05， P<0.01）. In addition， real-time PCR results showed that the mRNA expressions of CYP7A1， CYP27A1 in the 400 mg·kg-1 geniposide group were significantly higher than those in the control group （P<0.05， P<0.01）.ConclusionThe 400 mg·kg-1 geniposide can cause obvious hepatotoxicity in rats， and the bile acid profile in liver， serum and excrement changes significantly， and the changes of the each bile acid in liver， serum and feces are different. However， the causal relationship between the gardenoside-induced liver injury and the changes in bile acid profile are not clear. It needs to be further studied.
Abstract：ObjectiveTo detect the toxicity of water-eluted fraction from Siegesbeckiae Herba （SWEF） at different concentrations against MRC-5 human embryonic lung fibroblasts and its impacts on the expression of α7 nicotinic acetylcholine receptor （α7nAChR） and inflammatory factors， so as to figure out the active components responsible for toxicity and efficacy.MethodThe toxicities of SWEF at 1， 6， 10， 20， and 50 g·L-1 against MRC-5 cells were determined by cell counting kit-8 （CCK-8） assay combined with flow cytometry and Trypan blue staining. The changes in α7nAChR expression and inflammatory factor levels before and after α7nAChR gene silencing were detected to reveal the pharmacodynamic effect of SWEF on MRC-5 cells.ResultSWEF （≥6 g·L-1） obviously inhibited the viability of MRC-5 cells （P<0.01） and promoted their apoptosis and necrosis （P<0.01）， with the half-maximal inhibitory concentration （IC50） being 6.03 g·L-1. The determination of α7nAChR expression and inflammatory factor levels in MRC-5 cells showed that SWEF contained α7nAChR agonist-like substance， which enhanced α7nAChR mRNA and protein expression （P<0.05， P<0.01） and decreased the inflammatory factor levels （P<0.05， P<0.01）. SWEF down-regulated the inflammatory factors possibly by re-regulating α7nAChR mRNA expression， exhibiting a negative correlation between them （P<0.01）.ConclusionSWEF （≥6 g·L-1） is highly toxic to MRC-5 cells. Pharmacodynamic studies have confirmed that α7nAChR agonist-like substance contained in SWEF was responsible for the elevated α7nAChR expression and reduced inflammatory cytokines. It is inferred that excessive α7nAChR agonist-like substance may trigger the toxicity of Siegesbeckiae Herba.
Abstract：ObjectiveTo investigate the effect and mechanism of saikosaponin A （SSA） on the reversal of cisplatin （DDP） resistance in human lung cancer cell line A549/DDP.MethodsThe resistance of A549 and A549/DDP cells to DDP and the inhibitory effects of SSA against the proliferation of A549 and A549/DDP cells were detected using cell counting kit-8 （CCK-8）. The apoptosis rates of A549/DDP cells treated with SSA or DDP or SSA combined with DDP and the changes in reactive oxygen species （ROS） were determined by flow cytometry. The mRNA expression levels of C-myc， B-cell lymphoma 2 （Bcl-2） and cysteinyl aspartate-specific protease-3 （Caspase-3） were detected by real-time polymerase chain reaction （Real-time PCR）， followed by the determination of β-catenin transcriptional activity using the TopFish dual-luciferase reporter assay system and the measurement of β-catenin protein expression in A549/DDP cells by Western blot.ResultsThe results of CCK-8 assay showed that the DDP resistance of A549/DDP cells was 12.82 times that of A549 cells （P<0.05）. SSA inhibited the viability of A549 cells with the half maximal inhibitory concentration （IC50） being 34.9 μmol·L-1， and also suppressed the viability of A549/DDP cells in a concentration-dependent manner. Since the inhibition rate of 20 μmol/L SSA against A549/DDP cells was less than 10%， the reversal concentration was set at 20 μmol/L. Flow cytometry revealed that compared with the control， DDP alone increased the apoptosis rate of A549/DDP cells （P<0.05）， stimulated the accumulation of intracellular ROS （P<0.05）， down-regulated the mRNA expression levels of C-myc and Bcl-2 in A549/DDP cells， up-regulated Caspase-3 mRNA expression， and reduced the transcriptional activity of β-catenin （P<0.05）. Compared with the DDP group， the SSA+DDP group exhibited obviously increased apoptosis of A549/DDP cells， enhanced accumulation of intracellular ROS， down-regulated C-myc and Bcl-2 mRNA expression， up-regulated Caspase-3 mRNA expression （P<0.05）， and weakened β-catenin transcription （P<0.05）. DDP combined with SSA better decreased the β-catenin protein expression in contrast to that of control or DDP （P<0.05）.ConclusionsSSA enhances the sensitivity of A549/DDP cells to DDP possibly by inhibiting the activation of Wnt/β-catenin pathway.
Keywords：saikosaponin A （SSA）;A549/DDP cell;Wnt/β-catenin
Abstract：ObjectiveTo observe the effect of Yiqiyangyin Huoxuetongluo prescription on janus kinase 2/signal transducer and activator of transcription 3 （JAK2/STAT3） signaling pathway and cell apoptosis in rats with diabetic nephropathy （DN）， and to explore the mechanism of its intervention in DN.MethodA total of 100 SD rats were randomly divided into an experimental group （n=80） and a normal group （n=20）. The DN model was induced by high-sugar and high-fat diet combined with intraperitoneal injection of streptozotocin （STZ） in the experimental group， and confirmed by the pathological changes of kidney tissues in rats （three in each group） observed under light and electron microscopes. The model rats were randomly divided into a model group （normal saline， equal volume）， low-， medium-， and high-dose （5.775， 11.550， and 23.100 g·kg-1） Yiqiyangyin Huoxuetongluo prescription groups， and an irbesartan group （irbesartan tablets， 0.016 g·kg-1）. After drug intervention （i.g.， once a day for 16 consecutive weeks）， the 24-hour urine total protein （UTP）， serum total cholesterol （TC）， triglyceride （TG）， creatinine （SCr）， and blood urea nitrogen （BUN） levels of the rats were measured. Western blot was used to detect the protein expression of JAK2/STAT3 signaling pathway. Immunohistochemistry was used to determine the protein expression of B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， zonula occludens-1 （ZO-1）， and actinin-4 in rat kidney tissues.ResultCompared with the normal group， the model group exhibited elevated UTP， serum TC， TG， BUN， and SCr levels （P<0.05）， severe pathological damage of rat kidney tissues， up-regulated expression of phospho-JAK2 （p-JAK2）， phospho-STAT3 （p-STAT3）， and Bax， increased renal cell apoptosis， and diminished expression of Bcl-2， ZO-1， and actinin-4 （P<0.05）. Compared with the model group， the Yiqiyangyin Huoxuetongluo prescription group and the irbesartan group showed dwindled UTP， serum TC， TG， BUN， and SCr levels （P<0.05）， relieved pathological damage of rat kidney tissues， down-regulated p-JAK2， p-STAT3， and Bax expression， and up-regulated expression of Bcl-2， ZO-1， and actinin-4 （P<0.05）.ConclusionYiqiyangyin Huoxuetongluo prescription can reduce renal cell apoptosis and improve the prognosis of DN by inhibiting the activation of JAK2/STAT3 signaling pathway.
Keywords：Yiqiyangyin Huoxuetongluo prescription;diabetic nephropathy （DN）;Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway （JAK2/STAT3）;apoptosis
Abstract：ObjectiveTo observe the effects of modified Huangqi Biejiatang combined with auricular acupressure on diabetic peripheral neuropathy （DPN） due to Qi and Yin deficiency and serum myeloid differentiation factor 88/inhibitor of nuclear factor-κB （MyD88/IκB） signaling pathway.MethodOne hundred and forty cases were randomly divided into an observation group （n=70） and a control group （n=70）. In addition to routine treatments like dietary intervention and the regulation of fasting blood glucose （FBG） and blood pressure， the modified Huangqi Biejiatang combined with auricular acupressure was further provided in the observation group， while mecobalamine was administered in the control group. After four-week intervention， the toronto clinical scoring system （TCSS） score， traditional Chinese medicine （TCM） syndrome score， the conduction velocities of motor and sensory nerves （median nerve， common peroneal nerve， tibial nerve， and ulnar nerve）， glucose metabolism indexes ［fasting plasma glucose （FPG）， 2 h postprandial blood glucose （2 h PG）， and hemoglobin A1c （HbA1c）］， intestinal genera （Clostridium， Prauserella， Bacteroides， and Faecalibacterium）， as well as the serum MyD88， IκBα， and phosphorylated IκBα （p-IκBα） levels in the MyD88/IκB signaling pathway before and after treatment were observed in the two groups， for comparing their clinical efficacy and safety.ResultThe total effective rate of the observation group was 85.3% （58/68）， which was higher than 48.5% （32/66） of the control group （χ2=6.143， P<0.05）. The comparison with the control group revealed that the scores of TCSS and TCM syndrome， the levels of FPG， 2 h PG， HbA1c， MyD88， and p-IκBα， as well as the abundances of Clostridium and Prauserella in the observation group were decreased （P<0.05）， while the conduction velocities of motor and sensory nerves （median nerve， common peroneal nerve， tibial nerve， and ulnar nerve） were significantly accelerated （P<0.05）. Besides， the abundances of Bacteroides and Faecalibacterium and IκBα level were significantly elevated （P<0.05）. The incidence of adverse reactions in the observation group was 1.5% （1/68）， lower than 12.1% （8/66） in the control group （χ2=4.328， P<0.05）.ConclusionThe modified Huangqi Biejiatang combined with auricular acupressure alleviates DPN due to Qi and Yin deficiency， which may be attributed to the regulation of serum MyD88/IκB signaling pathway.
Abstract：ObjectiveTo study the effect mechanism of Guilu Erxian gum on Alzheimer's disease （AD） from the perspective of regulating perivascular space （PVS），and to explore the scientific connotation of "essence generating marrow".MethodThe 80 patients with AD diagnosed by western medicine and kidney deficiency and marrow empty syndrome diagnosed by traditional Chinese medicine were randomly divided into two groups，with 40 cases in each group. Both groups of patients were orally administered with cholinesterase inhibitor Alison，one tablet （5 mg） each time before sleep at night. On this basis，the control group additionally received placebo，while the treatment group was additionally treated with Guilu Erxian gum for 60 days. The Mini-Mental State Examination scale （MMSE）， Wechsler Memory Scale and Activity of Daily Living Scale （ADL） were used before treatment （0 d），as well as 31 d and 61 d after treatment. The number and diameter of PVS in midbrain，basal ganglia，deep insular white matter and semiovale center were counted and their diameters were measured with use of nuclear magnetic resonance imaging （MRI） for head. In addition，the curative effect was evaluated according to MMSE scores on 61 d.ResultThere was no significant difference between the two groups 31 d. On 61 d，MMSE and WMS scores increased，while ADL scores decreased as compared with the conditions on 0 d（P<0.01），and there were significant differences in the three indexes and clinical effective rate between two groups （P<0.05，P<0.01） . In addition，there was no significant difference in the number of PVS as compared with the number before treatment and in the comparison between the two groups after treatment，but there was a significant difference in the diameter of PVS（P<0.01）.ConclusionGuilu Erxian gum is effective in the treatment of AD，and it can improve the PVS diameter in patients，which may be related to the mechanism of "essence generating marrow ".
Abstract：ObjectTo observe the clinical efficacy of Yishen Tongluofang in treating oligosperm type male infertility with kidney deficiency and blood stasis syndrome and explore its effect on serum sex hormones and seminal plasma microenviro.MethodOne hundred and four patients were randomly divided into observation group and control group with 52 cases each. Patients in control group took compound Xuanju capsules orally， 3 capsules/time， 3 times/day. Patients in observation group took Yishen Tongluofang， 1 dose/day. Treatment courses continued three months and followed up for three months in both groups. The pregnancy situations of spouses within six months were recorded. Examination of semen parameters before and after treatment and score of kidney deficiency and blood stasis syndrome were conducted. The levels of seminal plasma zinc， fructose， elastase， acid phosphatase and α-glucosidase， serum follicle stimulating hormone （FSH）， luteinizing hormone （LH）， prolactin （PRT） and testosterone （T） were detected before and after treatment.ResultDuring the observation period of six months， the pregnancy rate of spouses in the observation group was 22.00%， higher than 10.00% in the control group， but the difference was not statistically significant （χ2=2.678，P>0.05）. The clinical efficacy in the observation group was better than that of the control group （Z=2.326，P<0.01）. Sperm concentration， sperm motility， sperm motility， normal morphological sperm and linear movement speed of the observation group were all superior to those of the control group （P<0.01）. The levels of zinc and fructose in seminal plasma of the observation group were higher than those of the control group （P<0.01）， and the score of kidney deficiency and blood stasis syndrome was lower than that of the control group （P<0.01）. Serum FSH， LH and PRT levels in the observation group were lower than those in the control group （P<0.01）， and the T level was higher than that in the control group （P<0.01）. The seminal plasma elastase of the observation group was lower than that of the control group， while the levels of acid phosphatase and α-glucosidase were higher than those of the control group （P<0.01）.ConclusionYishen Tongluofang can significantly improve sperm parameters， regulate the level of sex hormones and seminal plasma environment in patients with oligosperm type male infertility， and improve the tendency of spouse pregnancy. Its clinical efficacy is better，so it is worthy of further research and application.
Keywords：male infertility;oligospermia;asthenospermia;kidney deficiency and blood stasis syndrome;Yishen Tongluofang;sex hormones
Abstract：ObjectiveTo observe the effect of modified Bazhentang on the nutritional status and immune function of patients with Qi and blood deficiency syndrome in neoadjuvant chemotherapy （NAC） for gastric cancer.MethodOne hundred and ten patients were randomly divided into observation group and control group with 55 cases each. Both groups accepted FOLFOX6 protocol. Patients in control group took Jianpi Shengxue tablets orally， 3 tablets/time， 3 times/day. Patients in observation group received modified Bazhentang， 1 dose/day. The course of treatment was six weeks in both groups. Before and after treatment， scores were graded according to patient generated-subjective global assessment （PG-SGA）， Qi and blood deficiency syndrome， and the Revised Piper Fatigue Scale （PFS-R）. Levels of serum total protein （TB）， albumin （ALB）， prealbumin （PAB）， CD4+， CD8+， helper T lymphocyte 17 （Th17）， regulatory T cell （Treg）， immunoglobulin G （IgG）， IgM， and IgA were detected before and after therapy. Body mass index （BMI） and fat free mass index （FFMI） were measured before and after treatment. Weight loss was recorded， and the acute or subacute toxicity of anticancer drugs was evaluated.ResultThe degree of malnutrition in the observation group was lower than that in the control group （Z=2.401，P<0.01）. The levels of TB， ALB and PAB in the observation group were higher than those in the control group （P<0.01）. The CD4+， Treg and CD4+/CD8+ levels in the observation group were higher than those in the control group （P<0.01）. The CD8+， Th17 and Th17/Treg levels were lower than those in the control group （P<0.01）. Besides， the levels of IgM and IgA in the observation group were higher than those in the control group （P<0.01）. The PG-SGA score and weight loss in the observation group were lower than those in the control group （P<0.01）. The BMI and FFMI data of the observation group were higher than those of the control group （P<0.05）. The scores of PFS-R and Qi-blood deficiency syndrome were lower than those of the control group （P<0.01）. The incidence of nausea and vomiting in the observation group was 45.45% （25/55）， lower than 65.45% （36/55） in the control group （χ2=4.452，P<0.05）.ConclusionModified Bazhentang can be used to assist gastric cancer patients with NAC， which can improve nutritional status and immune function， promote immune balance， reduce clinical symptoms and fatigue， and reduce chemotherapy toxicity and side effects， so it is worthy of clinical use.
Keywords：gastric cancer;Qi and blood deficiency syndrome;neoadjuvant chemotherapy;Bazhentang;nutritional status;immune function
Abstract：ObjectiveTo explore the application value of modified Shiquan Dabutang in the treatment of elderly patients with osteoporotic intertrochanteric fractures （OIFs） due to Qi and blood deficiency by observing its impacts on inflammatory and bone metabolism indexes.MethodNinety-eight elderly patients admitted to our hospital for OIFs of Qi and blood deficiency syndrome from April 2018 to April 2020 were randomized into an observation group （n=49） and a control group （n=49）. Following the proximal femoral nail antirotation （PFNA） fixation， patients in the control group were treated with Guipiwan， while those in the observation group received the modified Shiquan Dabutang. The clinical efficacy， inflammatory and bone metabolism indexes， and complications were compared between the two groups after four weeks of treatment.ResultThe levels of such serum indexes as fibroblast growth factor-2 （FGF-2）， osteoprotegerin （OPG）， transforming growth factor-β1 （TGF-β1）， β-endorphin （β-EP）， bone-specific alkaline phosphatase （BALP）， and osteocalcin （BGP） in the observation group after treatment were significantly elevated as compared with those in the control group （P<0.05）， whereas the serum tumor necrosis factor-α （TNF-α） and D-dimer （D-D） declined （P<0.05）. The TCM symptom score in the observation group after treatment was obviously lower than that in the control group， while the Harris Hip Score （HHS） was higher （P<0.05）. The overall response rate of the observation group was 93.88% （46/49）， higher than 75.51% （37/49） of the control group （χ2=6.376， P<0.05）. The total incidence of incision infection， deep vein thrombosis of lower limbs， and pulmonary infection in the control group was 24.49% （12/49）， significantly higher than 6.12% （3/49） in the observation group （χ2=6.607， P<0.05）.ConclusionThe modified Shiquan Dabutang is able to alleviate inflammation， regulate bone metabolism， promote bone repair， and reduce the incidence of complications in elderly patients with OIFs due to Qi and blood deficiency.
Abstract：ObjectiveTo investigate the effect of Chloriti Lapis on metal elements in brain tissue and plasma of epileptic rats kindled by pentylenetetrazol （PTZ）， and to explore the possible material basis of Chloriti Lapis.MethodPTZ kindling method was used to establish epileptic rat model. Inductively coupled plasma mass spectrometry （ICP-MS） and inductively coupled plasma optical emission spectrometer （ICP-OES） were used to determine the contents of metal elements in brain tissue and plasma of the blank group， model group， carbamazepine group （0.1 g·kg-1） and Chloriti Lapis group （2 g·kg-1）. The data were statistically analyzed by SPSS 18.0 software.ResultCompared with the blank group， the contents of Sr， Sb and Ba in brain tissue of rats in the model group were significantly increased （P<0.05， P<0.01）， while the contents of Zn， Fe， Cu， K， Li， Co， Sn and Pb were significantly decreased （P<0.05， P<0.01）. Compared with the model group， the contents of Zn， Fe， K， Li， Co， As and Pb in brain tissue of rats in the Chloriti Lapis group were obviously increased （P<0.05， P<0.01）， while the contents of Sr and Sb were significantly decreased （P<0.01）. These results showed that Chloriti Lapis had positive effect on the regulation of the content of metal elements in rat brain tissue to normal level， the intervention effect was clear， and the overall effect was better than that of carbamazepine group. The determination of 21 metal elements in plasma showed that compared with the blank group， the levels of K， Sr and Cd in the model group were significantly increased （P<0.05）， and the contents of Li， Al， Ti and Cr were significantly decreased （P<0.05）. Compared with the model group， the contents of Ca， K， Li， Al and V in the Chloriti Lapis group were obviously increased （P<0.05， P<0.01）， and the contents of Fe， Ti， Sr and Cd were significantly decreased （P<0.05，P<0.01）. The correlation analysis of metal elements among the groups showed that there were 17 pairs of elements had positively correlation in the brain tissue of rats， 2 pairs of elements had significant negative correlation. In the plasma of rats， 8 pairs of elements had significant positive correlation and 6 pairs of elements had significant negative correlation.ConclusionThe metal element groups represented by Zn， Fe， K， Li， Co， As， Pb， Sr， Sb， Ca， Al， V， Ti and Cd may be the effective material basis for Chloriti Lapis to interfere PTZ-kindled epileptic model rats， which may be related to the influence of these metal element groups on the release of neurotransmitters and the electrical balance of neurons， the regulation of abnormal synchronous discharge induced by Na+， K+， Ca2+ channel disorders and intervention of metabolism pathways in brain tissue related to epilepsy. It can make the excitatory and inhibitory activities restrain each other， and finally reach the normal physiological state of neurons and cells. The intervention effect of Chloriti Lapis group was better than that of carbamazepine group.
Abstract：ObjectiveAn ultra-performance liquid chromatography coupled with Orbitrap Fusion Lumos Tribrid mass spectrometry （UPLC-Orbitrap Fusion Lumos Tribrid-MS） was applied to analyze the prototypes and their metabolites of Phellodendri Amurensis Cortex aqueous extract in the serum， urine and feces of normal rats， and to investigate the pharmacodynamic material basis of Phellodendri Amurensis Cortex in rats.MethodChromatographic separation was performed on the ACQUITY UPLC® CSHTM C18 column （2.1 mm×100 mm， 1.7 μm） with the mobile phase of 0.1% formic acid aqueous solution （A）-acetonitrile （B） for gradient elution （0-15 min， 2%-25%B； 15-25 min， 25%-50%B； 25-28 min， 50%-98%B）， flow rate was 0.3 mL·min-1， the injection volume was 10 μL and the column temperature was 40 ℃. Heated electrospray ionization （HESI） was used to collect data in the positive ion modes with the scanning range of m/z 100-1 000. By comparing chromatogram differences between the blank samples and the samples after administration， prototypes and their metabolites of biological samples after oral administration of Phellodendri Amurensis Cortex aqueous extract were identified.ResultAfter oral administration of Phellodendri Amurensis Cortex aqueous extract， a total of 70 compounds including 15 prototypes and 55 metabolites in rat serum， urine and feces were detected. Among them， 15 prototypes included 12 alkaloids and 3 limonoids， and 55 metabolites included 52 alkaloids and 3 limonoids. Desaturation， methylation， oxidation， sulfonation and glucuronide conjugation were observed as the primary metabolic pathways for the chemical constituents of Phellodendri Amurensis Cortex aqueous extract.ConclusionAlkaloids in Phellodendri Amurensis Cortex aqueous extract undergo phase Ⅰ and phase Ⅱ metabolism in rats， and limonoids mainly undergo phase Ⅰ metabolism in rats. This paper can provide experimental basis for further analyzing the process in vivo of Phellodendri Amurensis Cortex and elucidating its pharmacodynamic substance basis.
Keywords：Phellodendri Amurensis Cortex;alkaloids;ultra-performance liquid chromatography coupled with Orbitrap Fusion Lumos Tribrid mass spectrometry （UPLC-Orbitrap Fusion Lumos Tribrid-MS）;prototypes;metabolites;limonoids;rats
Abstract：ObjectiveTo compare the regulatory effects of Polygala tenuifolia and licorice-simmered P. tenuifolia on learning and memory， hypothalamus-pituitary-adrenal axis （HPA axis） function and neurotransmitters in rats with heart-kidney imbalance insomnia.MethodThe rat model of insomnia induced by multi-factor stimulation was established. After the model being made， the administration groups were given the extracts of P. tenuifolia and licorice-simmered P. tenuifolia by gavage （dose of 8 g·kg-1·d-1）， while the normal group and model group were given the same volume of normal saline for 7 days. Morris water maze was used to detect the changes of learning and memory ability of rats in each group. Enzyme-linked immunosorbent assay （ELISA） was used to determine the levels of corticotropin-releasing hormone （CRH）， adrenocorticotropic hormone （ACTH） and cortisol （CORT） in serum of rats from each group. The contents of 5-hydroxytryptamine （5-HT）， dopamine （DA）， γ-aminobutyric acid （GABA） and glutamic acid （Glu） in the hypothalamus of rats were determined simultaneously by ultra performance liquid chromatography tandem mass spectrometry （UPLC-MS/MS）.ResultCompared with the normal group， the spatial learning and memory ability of rats in the model group was decreased， the times and time of staying in target quadrant were significantly reduced （P<0.05， P<0.01）， the levels of CORT， CRH and ACTH in serum were significantly increased （P<0.01）， the contents of GABA， DA， 5-HT in hypothalamus tissue were significantly decreased （P<0.01）， and the content of Glu was significantly increased （P<0.01）. Compared with the model group， the spatial learning and memory ability of rats in the P. tenuifolia group and licorice-simmered P. tenuifolia group were increased， the times and time of staying in the target quadrant were significantly increased （P<0.05， P<0.01）， the levels of CORT， CRH and ACTH in serum were significantly decreased （P<0.05， P<0.01）， the contents of GABA， DA and 5-HT in hypothalamus tissue were significantly increased （P<0.05， P<0.01）， and the content of Glu was significantly decreased （P<0.05）. The recovery degree of each index in the licorice-simmered P. tenuifolia group was better than that in the P. tenuifolia group.ConclusionBoth P. tenuifolia and licorice-simmered P. tenuifolia can improve the learning and memory ability， improve the function of HPA axis， regulate the level of central neurotransmitters， and have the effect of calming the mind and improving the intelligence of rats with heart-kidney imbalance insomnia. The effect of licorice-simmered P. tenuifolia is better than that of P. tenuifolia.
Keywords：Polygala tenuifolia;licorice-simmered P. tenuifolia;insomnia;hypothalamus-pituitary-adrenal axis;neurotransmitters;ultra performance liquid chromatography tandem mass spectrometry （UPLC-MS/MS）
Abstract：ObjectiveDue to the limitation of traditional identification methods of Chinese medicinal materials， the study established a rapid method to identify Persicae Semen mixed with Armeniacae Semen Amarum by allele-specific polymerase chain reaction （PCR）.MethodBy comparing the ribosomal DNA internal transcribed spacer （ITS） gene sequences of Persicae Semen and Armeniacae Semen Amarum， single nucleotide polymorphism （SNP） sites were searched and specific primers were designed. Different Persicae Semen and Armeniacae Semen Amarum samples were amplified by PCR， the effects of annealing temperature， primer concentration and cycle number on the PCR reaction system were optimized， and the specificity and detection limit of this method were investigated. In addition， the established PCR method was used to detect the samples of Persicae Semen mixed with different proportion of Armeniacae Semen Amarum from different sources and producing areas.ResultA specific PCR method for identifying Persicae Semen mixed with Armeniacae Semen Amarum was established. When the annealing temperature was 63 ℃ and the number of primer cycles was 30， only Armeniacae Semen Amarum could be amplified with 432 bp specific band， while Persicae Semen samples did not have this band. The minimum detection limit of this method for Armeniacae Semen Amarum was 0.2 ng， and the detection limit for Armeniacae Semen Amarum adulterated in Persicae Semen was 1%.ConclusionThe established allele-specific PCR method can accurately detect whether there is Armeniacae Semen Amarum in Persicae Semen， which can provide experimental basis for the quality control of Persicae Semen and guarantee the safety of its clinical use.
Keywords：Persicae Semen;Armeniacae Semen Amarum;ribosomal DNA internal transcribed spacer （ITS）;gene sequence;polymerase chain reaction （PCR）;single nucleotide polymorphism （SNP） site;specific primer
Abstract：ObjectiveTo establish a qualitative and quantitative method for the determination of aristolochic acids in Aristolochia cinnabarina dried root tubers.MethodThe dried root tubers of A. cinnabarina was qualitative and quantitative analysis by ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry （UPLC-QTOF-MS/MS）. The analysis was performed on Waters ACQUITY UPLC-BEH C18 column （ 2.1 mm×100 mm， 1.7 μm） with the mobile phase of 0.1% formic acid aqueous solution （A）-acetonitrile （B） for gradient elution （0-1 min， 10%B； 1-9 min， 10%-30%B； 9-11 min， 30%-50%B； 11-15 min， 50%-90%B）. The flow rate was 0.45 mL·min-1， column temperature was 35 ℃， and the detection wavelength was 250 nm. Mass spectral data was acquired in positive mode of electrospray ionization （ESI）. At the same time， the UPLC fingerprints of aristolochic acids in 21 batches of A. cinnabarina dried root tubers were established， and the contents of 5 aristolochic acids in A. cinnabarina dried root tubers from different producing areas and different harvesting periods were determined.ResultA total of 17 compounds， including 8 aristolochic acids， 7 aristololactams and 2 4，5-dioxoaporphine alkaloids， were identified from A. cinnabarina dried root tubers by mass spectrometry data and bibliographic information. Ten common peaks were identified in the UPLC fingerprint， and they were tuberosinone-N-β-D-glucoside， aristolactam Ⅰa-N-β-D-glucoside， aristolochic acid Ⅳa-O-β-D-glucoside， aristolactam Ⅲa-N-β-D-glucoside， aristolactam Ⅰ-N-β-D-glucoside， aristolochic acid Ⅲa， aristolochic acid Ⅳa， aristolochic acid Ⅱ， aristolactam Ⅰ and aristolochic acid Ⅰ. According to the quantitative analysis， the results exhibited that aristolochic acid Ⅲa， aristolochic acid Ⅳa， aristolochic acid Ⅱ， aristolactam Ⅰ and aristolochic acid Ⅰ had good linear relationships in the linear range. The relative standard deviations （RSDs） of precision， stability and reproducibility tests were all less than 3.0%， the recovery was 97.06%-101.84% （RSD<3.0%）. The contents of aristolochic acid Ⅰ， aristolochic acid Ⅱ， aristolochic acid Ⅲa， aristolochic acid Ⅳa， and aristolactam Ⅰ in 21 batches of A. cinnabarina dried root tubers were 0.938 6-3.567 5， 1.377 6-3.688 1， 0.056 3-0.527 7， 0.108 8-0.305 5， 0.021 0-0.081 7 mg·g-1， respectively.ConclusionThe content of aristolochic acids in A. cinnabarina dried root tubers has a certain difference， the contents of aristolochic acid Ⅰ and Ⅱ are higher than other aristolochic acids. The established method is rapid， simple， accurate and reliable， which can provide reference for the quality control and evaluation of A. cinnabarina dried root tubers.
Keywords：Aristolochia cinnabarina dried root tubers;aristolochic acids;ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry （UPLC-QTOF-MS/MS）;fingerprint;aristololactams;4，5-dioxoaporphine alkaloids
Abstract：ObjectiveTo provide a scientific basis for the classification of Phyllanthi Fructus product grades.MethodA total of 30 batches of Phyllanthi Fructus currently available in the market were collected for quantification based on such appearance indexes as diameter， thickness， grain weight， and crust colour （L*， a*， and b* values）. The contents of gallic acid， corilagin， chebulagic acid， and ellagic acid were measured by high performance liquid chromatography （HPLC）， followed by descriptive statistical analysis （DSA）， analysis of variance （ANOVA）， and principal component analysis （PCA） to determine the importance of each main index and explore the correlations between the appearance indexes and internal components. The classification standard of Phyllanthi Fructus product grades was formulated， and its scientificity was verified in hepatocelular carcinoma HepG2 cells.ResultThe correlation analysis revealed that the crust colour L*， a*， and b* values were significantly negatively correlated with corilagin， chebulagic acid， and ellagic acid （|r|>0.5， P<0.01）， but irrelevant to gallic acid （|r|<0.1）. Considering the variable coefficient of each index， PCA results， and the requirement of gallic acid as quality indicator for Phyllanthi Fructus in Chinese Pharmacopoeia， the crust colour L*， a*， and b* values and gallic acid content were determined to be the classification indexes. The K-means cluster analysis confirmed that products with crust colour L*<44， a*<7， and b*<10 and gallic acid content >1.6% could be classified into the first class， and those failing to meet the above requirements into the second class. The cell experiment demonstrated that the half-maximal inhibitory concentration （IC50） of the first-class product against hepatocelular carcinoma HepG2 cells was lower than that of the second-class product. A colourimetric card was developed based on crust colour L*， a*， and b* values to provide a visual tool for on-site evaluation of Phyllanthi Fructus products.ConclusionThis study has initially established the classification standard of Phyllanthi Fructus product grades， which contributes to guiding price negotiation of Phyllanthi Fructus products based on quality grade and thus ensuring high quality and high price.
Abstract：ObjectiveTo explore the differences in rhizosphere microbial community structure between Fusarium wilt-infected and healthy Chrysanthemum morifolium plants.MethodThe rhizosphere soils of diseased and healthy C. morifolium plants were sampled and subjected to high-throughput 16S ribosomal DNA （rDNA） and internal transcribed spacer （ITS） sequencing， to identify the microbial community structure including bacteria and fungi.ResultFusarium wilt reduced the bacterial abundance and diversity but had no significant effect on fungal alpha-diversity.The proportions of Acidobacteria， Gemmatimonadetes， and Nitrospirae in rhizosphere soil of healthy C.morifolium plants were higher than those of diseased plants， while the proportions of Proteobacteria and Bacteroidetes were lower（P<0.05）. Fusarium fungi accounted for 27.49%， 14.53%， and 11.94% in diseased plants whereas 0.47%， 1.01%， and 0.67% in healthy plants.Pathogenic bacteria Pectobacterium and Dickeya were enriched in rhizosphere soil of diseased plants. The abundances of nitrifying， detoxifying， and photosynthetic bacteria in rhizosphere soil of healthy plants were higher than those of diseased plants.ConclusionFusarium wilt reduces the bacterial richness and diversity and triggers the enrichment of massive Fusarium fungi， Pectobacterium， and Dickeya. The proportion of beneficial bacteria in rhizosphere soil of healthy plants is significantly higher than that of diseased plants.
Abstract：ObjectiveTo investigate the taxonomic structure and diversity of endophytic fungi from Datura metel and screen the strains with anti-dermatophyte activities， so as to provide resources for the development of new lead compounds against dermatomycosis.MethodEndophytic fungi were isolated from the roots， stems and leaves of D. metel after tissue block incubation and then identified by morphological analysis and rDNA-internal transcribed spacer（ITS） sequencing. Their anti-dermatophyte activities were detected by agar diffusion assay.ResultA total of 292 strains of endophytic fungi were isolated from D. metel， belonging to 34 genera， with Fusarium （72.97%） in roots， Fusarium （37.25%） and Plectosphaerella （28.43%） in stems， and Colletotrichum （39.66%） in leaves as the dominant species. The isolation rate （89.23%）， colonization rate （84.62%）， and diversity index （1.82） of endophytic fungi in leaves were significantly higher than those in roots （70.48%， 70.48% and 1.23） and stems （69.39%，68.03% and 1.64）. The determination of anti-dermatophyte activities of 35 endophytic fungal fermented filtrates showed that the strains exhibiting inhibitory activities against Microsporum canis， Trichosporon mucoides， Trichophyton rubrum and Candida albicans accounted for 97.14%， 71.43%， 45.71%， and 25.71%， respectively. Among them， six strains （17.14%）， namely Fusarium sp. R1， Penicillium sp. R5， Aspergillus sp. R7， Metarhizium sp. S18， Diaporthe sp. S19， and Glomerella sp. L57， all inhibited the four types of cutaneous fungal pathogens.ConclusionThe endophytic fungi in D. metel are diverse， and the proportion of endophytic fungi possessing anti-dermatophyte activities is high， allowing them to serve as potential resources for the development of new anti-dermatophyte agents.
Abstract：ObjectiveTo elucidate the potential molecular markers and drug-compound-target mechanism of Epimedii Folium intervention on breast cancer stem cells（BCSCs） through chip analysis combined with network pharmacology and experimental validation.MethodRelevant drug information was retrieved in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） to obtain the active components and potential targets of Epimedii Folium. "Breast Cancer Stem Cells" were searched in Gene Expression Omnibus（GEO）database，and GSE98239 chip data were obtained through analysis and screening. Then GEO2R online analysis tool was used to obtain the differential genes to draw differential gene heat map and volcano map. The differential gene network map of Epimedii Folium intervention for breast cancer stem cells was constructed by Cytoscape 3.8.0，and Gene Ontology（GO） and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis of drug and disease genes were performed. Human breast cancer MDA-MB-231 cells were divided into 20%，40%，60% Epimedii Folium drug-containing serum group and control group. Cell counting kit-8（CCK-8），and Western blot were used to detect the effect of Epimedii Folium drug-containing serum intervention on cell activity and target protein expression in breast cancer cells.ResultTwenty-three active components including flavones，sterols，alkaloids and sesquiterpenoids were obtained from Epimedii Folium. It was found that Epimedii Folium interacted with B-cell lymphoma-2-like protein 1（BCL2L1），matrix metallopeptidase 2（MMP2），prostaglandin-endoperoxide synthase 2（PTGS2），vascular endothelial growth factor A（VEGFA），transforming growth factor beta receptor 1（TGFBR1） and other pivotal genes in breast cancer stem cells，participated in the induction of new angiogenesis and cell migration，enabled the continuous self-renewal of BCSCs，decreased apoptosis and cell migration，thus promoting the recurrence and metastasis of breast cancer. KEGG results showed that Epimedii Folium intervened in multiple differential expressed genes（DEGs）of transforming growth factor-β（TGF-β），vascular endothelial growth factor（VEGF），phosphoinositide 3kinase/protein kenase B（PI3K/Akt），mitogen-activated protein kinese（MAPK）and mammalian target of rapamycin（mTOR）subpathways in cancer signaling pathways to exert its efficacy in intervening breast cancer stem cells. Experiments showed that the survival rate of breast cancer cells was significantly reduced and the expression levels of TGFBR1 and Smad2 in breast cancer cells significantly decreased after the intervention of Epimedii Folium drug-containing serum（P<0.01）.ConclusionSeveral components in different concentrations of drug-containing serum of Epimedii Folium can synergistically act on target differentially expressed genes of breast cancer stem cells，and inhibit the proliferation of breast cancer cells by down-regulating the expression levels of TGFBR1，a key molecule in the TGF-β pathway，and Smad2，a downstream signal.
Keywords：network pharmacology;chip analysis;Epimedii Folium;breast cancer stem cells;mechanism
Abstract：ObjectiveTo evaluate the efficacy and safety of Chinese medicinal formulae in the treatment of antimicrobial-resistant pneumonia.MethodFollowing article retrieval from eight databases and data extraction by two reviewers， the methodological quality of the included trials was assessed and the outcome indicators were subjected to Meta-analysis using RevMan 5.3.ResultA total of 24 randomized controlled trials （RCTs） were included， involving 1 818 cases. Meta-analysis showed that Chinese medicinal formulae combined with western routine intervention was superior to the western routine intervention in improving the overall response rate （ORR） ［relative risk （RR）=1.27， 95% confidence interval （CI） （1.21， 1.34）， <0.000 01］， the bacterial clearance rate ［RR=1.49，95% CI （1.33， 1.66）， <0.000 01］， and the clinical pulmonary infection score （CPIS） ［mean difference （MD）=-1.64， 95% CI （-1.87， -1.41）， <0.000 01］. There was no significant difference in the incidence of adverse reactions ［RR=0.72， 95% CI （0.48， 1.07），=0.1］. The comparison with the western routine intervention also revealed that Chinese medicinal formulae better improved the ORR and CPIS.ConclusionAccording to the current research results， the Chinese medicinal formulae alone or combined with western routine intervention yielded more favorable clinical outcomes than western routine intervention in the treatment of antimicrobial-resistant pneumonia， without increasing the incidence of adverse events. Due to limited quality and quantity of the included RCTs， more high-quality trials are required to verify the above conclusions.
Abstract：ObjectiveTo explore the targets and relevant signaling pathways of Suoquanwan in the treatment of enuresis using network pharmacology，and animal expriments are applied to further define its mechanism of action.MethodTraditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP） database was used to screen out active chemical components of Suoquanwan，varieties of systematic biological databases were integrated to construct the "active component-disease-target" network relationship，and the common protein protein interaction network（PPI） network genes were functionally enriched. Quantitative real time polymerase chain reaction（Real-time PCR） and Western blot were used to verify the effect of Suoquanwan on AVPR2 and DRD2 gene.ResultA total of 32 active ingredients were screened from Suoquanwan. These active ingredients were interacted with 131 potential targets relating to Enuresis，which contained 14 core target genes，namely arginine vasopressin receptor 2 （AVPR2）， neurotrophic receptor tyrosine kinase 1（NTRK1）， dopamine receptor D2（DRD2）， opioid receptor mu 1（OPRM1）， 5-hydroxytryptamine receptor 1A（HTR1A）， 5-hydroxytryptamine receptor 1B（HTR1B），solute carrier family 6 member 4（SLC6A4），Adrenoceptor Alpha 2A（ADRA2A）， prostaglandin-endoperoxide synthase 2（PTGS2）， cholinergic receptor muscarinic 2（CHRM2）， solute carrier family 6 member 3 （SLC6A3）， 5-hydroxytryptamine receptor 6（HTR6）， solute carrier family 6 member 2（SLC6A2）， cytochrome P450 family 2 subfamily C member 19（CYP2C19）. Gene enrichments mainly involved to G protein-coupled receptor signaling pathway，regulation of trans-synaptic signaling，regulation of neurotransmitter transport and neuroactive ligand-receptor interaction. Real-time PCR and Western blot results showed that Suoquanwan could enhance the expression of AVPR2 in rat kidney，and weaken the expression of DRD2 in rat adrenal.ConclusionThe main chemical constituents in Suoquanwan may alleviate enuresis by regulating AVPR2 and DRD2 and then participating in the G protein-coupled receptor signaling pathway，regulation of trans-synaptic signaling，regulation of neurotransmitter transport and other biological processes.
Abstract：The historical evolution， fermentation technology and key links of Sojae Semen Praeparatum （SSP） were sorted out by consulting ancient books and modern literature， and the influencing factors and control methods of quality were analyzed and summarized in order to provide reference for the quality control of SSP. After analysis， it was found that in the fermentation process of SSP， fermentation strains， miscellaneous bacteria， temperature and humidity were all important factors affecting the quality of SSP. The condition control of "post fermentation" process has been paid more attention to in the past dynasties. In addition， the delicious SSP recognized in ancient times should be made from mold fermentation， and the breeding and application of fermented mold may be the key point to solve the quality problem of SSP. Therefore， based on the evaluation indexes of SSP in the past dynasties， it is of great significance to study and optimize the technological conditions such as strain， temperature and humidity in depth to improve the quality of SSP.
Abstract：Psoraleae Fructus is the dried and mature fruit of the legume Psoralea corylifolia. It is warm in nature， pungent and bitter in flavor， and attributive to the kidney and spleen meridians. Its main effect include warming the kidney and assisting Yang， absorbing Qi and relieving asthma， warming the spleen and relieving diarrhea， etc.， and it also can for external use of eliminating wind and freckle. Clinically， Psoraleae Fructus is mainly used for the treatment of impotence due to kidney deficiency， soreness of waist and knees， vitiligo， etc. The existing studies have shown that Psoraleae Fructus has a variety of pharmacological effect， such as anti-tumor， anti-oxidant， anti-bacterial， anti-inflammatory， promoting bone growth and protecting cardiovascular. But at the same time， many studies at home and abroad have found that taking Psoraleae Fructus and its compounds for a long time or in large doses can cause liver toxicity， phototoxicity， nephrotoxicity， etc. The most common is liver toxicity， most of the clinical reports on the toxicity of psoralen are caused by drug-induced liver injury events， which limits the clinical use of Psoraleae Fructus and can't exert its proper therapeutic effect. Therefore， it is particularly important to fully understand the toxicological mechanism of liver injury caused by Psoraleae Fructus and its attenuation methods. In this paper， by consulting the domestic and foreign related literatures in recent years that reported the hepatotoxicity of Psoraleae Fructus， the four aspects of clinical report on liver injury， hepatotoxic components， toxicological mechanisms and attenuation methods of Psoraleae Fructus were reviewed， including bile acid stasis and oxidative stress. The hepatotoxicity of Psoraleae Fructus was discussed in terms of reaction， mitochondrial damage， liver fat deformation， etc.， and the attenuation methods of Psoraleae Fructus were summarized from the aspects of compatibility attenuation and processing attenuation， aiming to comprehensively and objectively clarify Psoraleae Fructus. The potential toxicological mechanism of lipid-induced hepatotoxicity and research progress in attenuation were expected to provide a theoretical basis for further study of Psoraleae Fructus hepatotoxicity and clinical rational use of drugs.
Abstract：The theory of generation and restriction among five elements， as one of the basic theories in traditional Chinese medicine （TCM）， reveals that treating disease should focus on the root. Since its first record in Huangdi's Internal Classic （Huang Di Nei Jing）， this theory has been covered in many chapters of Synopsis of the Golden Chamber （Jin Gui Yao Lue） and further developed by physicians of later generations， allowing it to serve as a guide for clinical treatment of various diseases. Diabetic nephropathy （DN） is one of the most common complications of diabetes and also a main risk factor for death and disability by virtue of the long-term disease course and complex symptoms. At present， no specific drug is available in western medicine. Considering the close relationship of its complicated etiology and pathogenesis with the five zang organs， DN treatment should focus not only on the kidney， but also other zang organs. Guided by the theory of generation and restriction among five elements， this article believes that DN mainly results from kidney deficiency combined with spleen deficiency and its dysfunction in regulating the water passage. In addition， the exuberance of heart fire and the failure of liver to govern the free flow of Qi are also responsible for the occurrence of DN. Clinically， the therapeutic methods proposed based on theory of generation and restriction among five elements are recommended for DN treatment after the differentiation of actual manifestations into specific syndromes. Specifically， the method of replenishing Huo to nourish Tu is applicable to DN patients with spleen and kidney yang deficiency， the method of nourishing Shui to moisten Mu to those with liver and kidney yin deficiency， the method of mutual generation between Jin and Shui to those with lung and kidney yin deficiency， the method of banking up Tu to generate Jin to those with lung and spleen Qi deficiency， the method of purging the heart and tonifying the kidney to those with non-interaction between heart and kidney， and the method of banking up Tu to control Shui to those with spleen deficiency and fluid retention. Such timely and effective interventions are conducive to delaying the development of DN to end-stage renal disease （ESRD） and improving the clinical outcomes. This article discusses the application of the theory of generation and restriction among five elements in TCM to DN treatment， aiming to provide a theoretical basis for the future application of such new diagnosis and treatment ideas.
Keywords：generation and restriction among five elements;diabetic nephropathy （DN）;treatment based on syndrome differentiation
Abstract：Cystitis， one of the most common diseases in the urinary system， is manifested by urinary frequency， urinary urgency， and bladder pain， which are known as the classic symptom triad of bladder irritation， especially in women. In recent years， with the change of the lifestyle， the prevalence of bladder diseases in China is increasing year by year. According to the characteristics of etiology， pathogenesis， and clinical symptoms of cystitis， this paper listed the clinical diagnostic criteria in traditional Chinese medicine （TCM） and western medicine after consulting the relevant literature. Through the analysis of the existing animal model of cystitis， the fit between the model and clinical manifestations was evaluated， and the advantages and disadvantages were summarized. The models induced by "intraperitoneal injection of cyclophosphamide" and "Freund's complete adjuvant combined with bladder catheterization" were proved highly matched with manifestations despite some shortcomings such as long time and high cost. At present， the diagnostic criteria of cystitis are mainly based on western medicine， and the definitive diagnosis of the relevant types still depends on cystoscopy and tissue biopsy. The lack of TCM syndrome model limits the TCM research. Additionally， four diagnostic methods in TCM cannot be well applied to animal models because of the susceptibility to subjective factors. Behavioral tests can be used to determine the model index and develop the relevant behavior rating scale. Therefore， it is necessary to establish an animal model of cystitis in line with the clinical characteristics of western medicine and TCM syndrome differentiation， so as to better promote the study of cystitis.
Keywords：cystitis;clinical characteristics of traditional Chinese and western medicine;animal models