Protective Effect of Huazhuo Jiedu Huoxue Tongluo Prescription on Cerebral Ischemia-reperfusion Injury via Regulating Autophagy Based on JNK Signaling Pathway

ZHAO Minhan; CHU Xinqiao; CAO Xiaohui; HUO Ruiqing; SUN Kuo; LI Fangzhao; HAN Yufan; TIAN Junbiao

doi:10.13422/j.cnki.syfjx.20230793

您当前的位置：

首页 >

文章列表页 >

Protective Effect of Huazhuo Jiedu Huoxue Tongluo Prescription on Cerebral Ischemia-reperfusion Injury via Regulating Autophagy Based on JNK Signaling Pathway

更新时间：2023-05-17

- Protective Effect of Huazhuo Jiedu Huoxue Tongluo Prescription on Cerebral Ischemia-reperfusion Injury via Regulating Autophagy Based on JNK Signaling Pathway
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 7, Pages: 115-125(2023)
- 作者机构：
  
  1.河北中医学院，石家庄 050200
  2.中国中医科学院广安门医院，北京 100053
  3.石家庄市厚仁中医门诊部，石家庄 050011
  4.河北省中医院，石家庄 050000
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20230793
  CLC： R22;R242;R2-031;R285.5;R255.2;R743.3
- Received：06 January 2022，
  
  Published Online：11 May 2022，
  
  Published：05 April 2023
- 稿件说明：
移动端阅览
赵敏菡,储心乔,曹晓慧等.基于JNK信号通路调控细胞自噬探讨化浊解毒活血通络方对脑缺血再灌注损伤的保护作用[J].中国实验方剂学杂志,2023,29(07):115-125.

ZHAO Minhan,CHU Xinqiao,CAO Xiaohui,et al.Protective Effect of Huazhuo Jiedu Huoxue Tongluo Prescription on Cerebral Ischemia-reperfusion Injury via Regulating Autophagy Based on JNK Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(07):115-125.
赵敏菡,储心乔,曹晓慧等.基于JNK信号通路调控细胞自噬探讨化浊解毒活血通络方对脑缺血再灌注损伤的保护作用[J].中国实验方剂学杂志,2023,29(07):115-125. DOI： 10.13422/j.cnki.syfjx.20230793.

ZHAO Minhan,CHU Xinqiao,CAO Xiaohui,et al.Protective Effect of Huazhuo Jiedu Huoxue Tongluo Prescription on Cerebral Ischemia-reperfusion Injury via Regulating Autophagy Based on JNK Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(07):115-125. DOI： 10.13422/j.cnki.syfjx.20230793.

摘要

目的

研究化浊解毒活血通络方通过c-Jun氨基末端激酶（JNK）信号通路调控神经细胞自噬减轻脑缺再灌注损伤的作用机制。

方法

60只SD大鼠随机分为假手术组、大脑中动脉阻塞/再灌注（MCAO/R）组（模型组）、化浊解毒活血通络方（中药）组（25.0 g·kg

-1

）、JNK抑制剂SP600125（SP）组（5 mg·kg

-1

）、中药+SP组和JNK激动剂Anisomycin（Ani）组（15 mg·kg

-1

）6组。造模24 h后中药组和中药+SP组给予中药汤剂灌胃，连续给药3 d，其余各组灌胃等容积蒸馏水。神经功能评分评估神经功能缺损程度，2，3，5-氯化三苯基四氮唑（TTC）染色确定脑梗死体积；苏木素-伊红（HE）染色观察脑组织结构变化；尼氏染色检测神经元损伤；原位末端标记法（TUNEL）观察细胞凋亡；透射电镜观察自噬小体超微结构；蛋白免疫印迹法（Western blot）检测脑组织中微管相关蛋白1轻链3A/B（LC3A/B）、自噬相关基因5（Atg5）、自噬关键分子酵母Atg6同系物1（Beclin1）、p62、B细胞淋巴瘤-2（Bcl-2）、JNK、磷酸化（p）-JNK和c-Jun的蛋白表达；实时荧光定量聚合酶链式反应（Real-time PCR）检测各组大鼠脑组织中LC3A/B、Beclin1、p62、Atg5、Bcl-2、JNK、c-Jun mRNA表达量。

结果

与假手术组比较，模型组大鼠神经功能缺损评分明显升高（

0.05），脑组织梗死体积明显变大（

0.05），海马区及其周围脑组织形成典型梗死表现，皮层有大量神经元细胞变性、坏死、液化、固缩和深染，有炎性细胞浸润，电镜下可见炎症细胞浸润，线粒体、溶酶体、自噬体和自噬溶酶体严重肿胀。细胞凋亡情况较重，TUNEL阳性细胞明显增加（

0.05），脑组织内神经元核膜和核仁模糊，突起不连续，胞质尼氏体色浅且数量减少。Western blot结果显示大鼠脑组织LC3A/B、Beclin1、Atg5、JNK、p-JNK、c-Jun蛋白表达明显升高（

0.05），p62和Bcl-2蛋白表达明显降低（

0.05），Real-time PCR结果显示LC3A/B、Beclin1、Atg5、JNK、c-Jun mRNA表达明显增加（

0.05），p62、Bcl-2 mRNA表达明显降低（

0.05）。与模型组比较，中药组、SP组与中药+SP组大鼠神经功能缺损评分均明显降低（

0.05），梗死体积明显缩小（

0.05），脑组织病理状态均有所改善，尤以中药组改善明显；线粒体M嵴丰富，基本正常，内质网轻度扩张，高尔基体轻度肿胀，细胞核核膜轻度融合，溶酶体、自噬小体、自噬溶酶体可见，但数量较少，TUNEL阳性细胞明显减少（

0.05），凋亡情况有所减轻，尤以中药组最为显著，脑组织神经元核仁、核膜可辨，胞浆尼氏体有一定程度的脱失，但较模型组明显减轻。Western blot结果显示中药组、SP组与中药+SP组LC3A/B、Beclin1、Atg5、JNK、p-JNK、c-Jun蛋白表达明显降低（

0.05），中药组和SP组p62蛋白表达明显升高（

0.05），中药组和中药+SP组Bcl-2蛋白表达明显升高（

0.05）。Real-time PCR显示中药组、SP组和中药+SP组大鼠脑组织LC3A、LC3B、Beclin1、Atg5、JNK、c-Jun mRNA表达明显降低（

0.05），p62 mRNA表达明显增加（

0.05）；中药组和中药+SP组Bcl-2 mRNA表达明显增加（

0.05）。Ani组评分明显升高（

0.05），梗死体积显著增加，神经元细胞坏死情况加重，炎性浸润明显，神经元核膜融合、异型凸起，异染色质增加，边缘聚集，线粒体肿胀严重，内质网扩张，溶酶体增加，胞浆内囊泡增多，可见自噬体和自溶酶体，TUNEL阳性细胞增加（

0.05），凋亡情况较重，尼氏小体数量明显减少，脱失较重，着色浅，核仁核膜模糊；Ani组Atg5、c-Jun、JNK mRNA表达增加（

0.05），Beclin1、p62、Bcl-2 mRNA表达明显降低（

0.05）。与中药组和SP组比较，Ani组LC3A/B、Beclin1、Atg5、JNK、p-JNK、c-Jun蛋白表达明显升高（

0.05），p62、Bcl-2蛋白表达明显降低（

0.05）。与中药组比较，SP组和中药+SP组大鼠脑组织JNKmRNA表达降低（

0.05）；Ani组LC3A、LC3B、Atg5、c-Jun、JNK mRNA表达增加（

0.05），Bcl-2表达明显降低（

0.05）。与SP组比较，Ani组Atg5、c-Jun、JNK mRNA表达明显升高（

0.05），Beclin1、p62、Bcl-2 mRNA表达明显降低（

0.05）。

结论

化浊解毒活血通络方可上调LC3A/B、Beclin1、Atg5、JNK、p-JNK、c-Jun蛋白的表达，下调p62、Bcl-2蛋白表达；提高LC3A/B、Beclin1、Atg5、JNK、p-JNK、c-Jun mRNA表达，降低p62、Bcl-2 mRNA表达，表明化浊解毒活血通络方可通过JNK信号通路抑制自噬减少大鼠缺血/再灌注损伤。

Abstract

Objective

To investigate the mechanism of Huazhuo Jiedu Huoxue Tongluo prescription in alleviating cerebral ischemia-reperfusion injury via regulating nerve cell autophagy based on c-Jun N-terminal kinase（JNK）signaling pathway .

Method

Sixty SD rats were randomly divided into 6 groups： sham group， middle cerebral artery occlusion/reperfusion （MCAO/R） group （model group）， Huazhuo Jiedu Huoxue Tongluo prescription group ［traditional Chinese medicine （TCM） group（25.0 g·kg

-1

）］， JNK inhibitor SP600125 （SP） group（5 mg·kg

-1

）， TCM+SP group and JNK agonist Anisomycin （Ani） group（15 mg·kg

-1

）. After 24 h of modeling， TCM group and TCM+SP group were given TCM decoction （

） for 3 consecutive days， and the other groups were given equal volume of normal saline （

）. Neurological deficit was evaluated by neurological function score and cerebral infarct volume was determined by 2，3，5-triphenyltetrazole chloride （TTC） staining. Hematoxylin-eosin （HE） staining and Nissl staining were used to observe the structural changes of brain tissue and the damage of neurons， respectively. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay （TUNEL） was performed to detect cell apoptosis. The ultrastructure of autophagosomes was observed by transmission electron microscope. Western blot was employed to detect the protein expressions of microtubule-associated protein 1 light chain 3A/B （LC3A/B）， autophagy related 5 （Atg5）， the ortholog of yeast Atg6 （Beclin1）， p62， B-cell lymphoma 2 （Bcl-2）， JNK， phosphorylated （p）-JNK and c-Jun in brain tissue. The mRNA expressions of LC3A/B， Beclin1， p62， Atg5， Bcl-2， JNK and c-Jun were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）.

Result

Compared with the sham group， the model group had elevated neurological deficit score （

0.05）， enlarged cerebral infarct volume （

0.05）and typical infarction manifestations formed in hippocampal region and its surrounding brain tissue. In addition， there were a large number of neuronal cell degeneration， necrosis， liquefaction， nucleus pyknosis and deep staining， and inflammatory cell infiltration in the cortex in the model group， and severe swelling of mitochondria， lysosomes， autophagosomes and autophagolysosomes were clearly seen under electron microscope. TUNEL positive cells were increased （

0.05）， and cell apoptosis was severe. The nuclear membrane and nucleolus of neurons in brain tissue were blurred with discontinuous processes， and Nissl bodies in cytoplasm were stained light with reduced number. Western blot revealed that the model group had up-regulated protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun in brain tissue （

0.05）， while down-regulated protein expressions of p62 and Bcl-2 （

0.05）as compared with the sham group. Real-time PCR indicated that the mRNA expressions of LC3A/B， Beclin1， Atg5， JNK and c-Jun in the model group were higher （

0.05） while the mRNA expressions of p62 and Bcl-2 were lower （

0.05） than those in the sham group. Compared with the conditions in model group， the neurological deficit scores of TCM， SP and TCM+SP groups were lowered （

0.05）， and the cerebral infarct volume was reduced （

0.05）， with improved pathological status of brain tissue， especially in the TCM group. Furthermore， there were abundant and basically normal mitochondrial cristae， slightly dilated endoplasmic reticulum， slightly swollen golgi apparatus， slightly fused nuclear membrane， and few visible lysosomes， autophagosomes and autophagolysosomes. TUNEL positive cells were decreased （

0.05）， displaying reduced apoptosis， especially in the TCM group. The nucleolus and nuclear membrane of neurons in brain tissue were discernible， and Nissl bodies in cytoplasm was reduced to a certain degree as compared with those in the model group. Western blot showed a decrease in the protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun in the TCM group ，the SP group，and the TCM+SP group（

0.05），while an increase in the protein expressions of p62 in the TCM group and SP group（

0.05），and an increase in the protein expressions of Bcl-2 in the TCM group and TCM+SP group. By Real-time PCR， the mRNA expressions of LC3A， LC3B， Beclin1， Atg5， JNK and c-Jun had a down-regulation（

0.05） while the mRNA expression of p62 a up-regulation in the TCM group ，the SP group，and the TCM+SP group（

0.05），and the mRNA expression of Bcl-2 a up-regulation in the TCM group and the TCM+SP group（

0.05）.Scores of the Ani group were raised （

0.05）， and infarct volume was increased significantly， with aggravated neuronal cell necrosis and obvious inflammatory infiltration. Moreover， there were neuronal nuclear membrane fusion with abnormal protrusion， increased heterochromatin aggregation in edge， severe mitochondrial swelling， endoplasmic reticulum expansion， increased lysosomes， increased intracytoplasmic vesicles， and visible autophagosomes and autophagolysosomes. TUNEL positive cells were increased （

0.05）， representing severe apoptosis. The number of Nissl bodies dropped with light staining， and the nucleolus and nuclear membrane were blurred. Real-time PCR found that the mNRA expressions of Atg5， c-Jun， JNK were up-regulated （

0.05），while Beclin1， p62， Bcl-2 were were down-regulated in the Ani group （

0.05）. Compared with the TCM group and SP group，the protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK， c-Jun were decreased，and p62， Bcl-2 were increased in the Ani group（

0.05）. Compared with the TCM group，the mRNA expressions of JNK mRNA had a down-regulation in the SP group and TCM+SP group，while LC3A， LC3B， Atg5， c-Jun， JNK had an up-regulation（

0.05） and Bcl-2 had a down-regulation in the Ani group（

0.05）. Compared with the SP group，the mRNA expressions of Atg5， c-Jun， JNK had an up-regulation（

0.05）， and Beclin1， p62， Bcl-2 had a down-regulation in the Ani group（

0.05）.

Conclusion

Huazhuo Jiedu Huoxue Tongluo prescription significantly up-regulates the protein and mRNA expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun， and down-regulates the protein and mRNA expressions of p62 and Bcl-2， suggesting that the prescription can inhibit autophagy through JNK signaling pathway to reduce ischemia/reperfusion injury in rats.

关键词

Keywords

references

VIRANI S S ， ALONSO A ， APARICIO H J ， et al . Heart disease and stroke statistics-2021 update：A report from the American Heart Association ［J］. Circulation ， 2021 ， 143 （ 8 ）： e254 - e743 .

佚名 . 中国心血管健康与疾病报告（2019）节选二：脑血管病［J］. 心脑血管病防治， 2020 ， 20 （ 6 ）： 544 - 552 .

GOLDMAN S ， PRIOR S M ， BEMBENEK J P ， et al . Activation of blood coagulation and thrombin generation in acute ischemic stroke treated with rtPA ［J］. J Thromb Throm bolysis ， 2017 ， 44 （ 3 ）： 362 - 370 ．

WANCHAO S ， CHEN M ， ZHIGUO S ， et al . Protective effect and mechanism of Lactobacillus on cerebral ischemia reperfusion injury in rats ［J］. Braz J Med Biol Res ， 2018 ， 51 （ 7 ）： e7172 .

LYU M ， CUI Y ， ZHAO T ， et al . Tnfrsf12a-mediated atherosclerosis signaling and inflammatory response as a common protection mechanism of Shuxuening injection against both myocardial and cerebral ischemia-reperfusion injuries ［J］. Front Pharmacol ， 2018 ， 9 ： 312 .

DIAZ-CAñESTRO C ， MERLINI M ， BONETTI N R ， et al . Sirtuin 5 as a novel target to blunt blood-brain barrier damage induced by cerebral ischemia/reperfusion injury ［J］. Int J Cardiol ， 2018 ， 260 ： 148 - 155 .

霍瑞卿，赵敏菡，李芳钊，等 . 基于16S rRNA测序的肠道菌群探讨化浊解毒活血通络方对脑缺血再灌注损伤大鼠脑-肠轴的影响［J］. 中国实验方剂学杂志， 2022 ， 28 （ 1 ）： 121 - 130 .

何鹤，曾庆，黄国志 . 自噬——脑缺血中潜在的神经保护策略？［J］. 中国康复医学杂志， 2018 ， 33 （ 11 ）： 1360 - 1365 .

李希，万溪，高晶晶，等 . 化浊解毒活血通络方对脑缺血再灌注损伤大鼠ZO-1和occludin表达的影响［J］. 环球中医药， 2017 ， 10 （ 7 ）： 696 - 701 .

郭建文，张晓云，兰万成，等 . 陈绍宏教授"中风核心病机论" ［J］. 天津中医药， 2006 ， 23 （ 1 ）： 7 - 9 .

田军彪，赵层闪，高晶晶，等 . 化浊解毒活血通络方对脑缺血再灌注损伤大鼠血管内皮生长因子表达的影响［J］. 中华中医药杂志， 2015 ， 30 （ 9 ）： 3341 - 3343 .

赵见文，田军彪，李佃贵，等 . 化浊解毒活血通络方对脑缺血再灌注损伤小鼠血清IL-6和TNF- α 水平的影响［J］. 国际中医中药杂志， 2011 ， 33 （ 7 ）： 594 - 596 .

田军彪，万溪，高晶晶，等 . 化浊解毒活血通络方对大鼠脑缺血再灌注损伤后Bcl-2和Bax基因表达的影响［J］. 中华中医药杂志， 2017 ， 32 （ 6 ）： 2727 - 2730 .

田军彪，李希，万溪，等 . 化浊解毒活血通络方对脑缺血再灌注损伤大鼠PKC δ 、PKC θ 和PKC ε 表达的影响［J］. 中国老年学杂志， 2017 ， 37 （ 12 ）： 2867 - 2870 .

KRISHNA M ， NARANG H . The complexity of mitogen-activated protein kinases （MAPKs） made simple ［J］. Cell Mol Life Sci ， 2008 ， 65 （ 22 ）： 3525 - 3544 .

WANG M ， LI Y J ， DING Y ， et al . Silibinin prevents autophagic cell death upon oxidative stress in cortical neurons and cerebral ischemia-reperfusion injury ［J］. Mol Neurobiol ， 2016 ， 53 （ 2 ）： 932 - 943 .

CARLONI S ， BUONOCORE G ， BALDUINI W . Protective role of autophagy in neonatal hypoxia-ischemia induced brain injury ［J］. Neurobiol Dis ， 2008 ， 32 （ 3 ）： 329 - 339 .

SHENG R ， ZHANG L S ， HAN R ， et al . Autophagy activation is associated with neuroprotection in a rat model of focal cerebral ischemic preconditioning ［J］. Autophagy ， 2010 ， 6 （ 4 ）： 482 - 494 .

WEN Y D ， SHENG R ， ZHANG L S ， et al . Neuronal injury in rat model of permanent focal cerebral ischemia is associated with activation of autophagic and lysosomal pathways ［J］. Autophagy ， 2008 ， 4 （ 6 ）： 762 - 769 .

SHI R ， WENG J ， ZHAO L ， et al . Excessive autophagy contributes to neuron death in cerebral ischemia ［J］. CNS Neurosci Ther ， 2012 ， 18 （ 3 ）： 250 - 260 .

XU J ， KONG X ， XIU H ， et al . Combination of curcumin and vagus nerve stimulation attenuates cerebral ischemia/reperfusion injury-induced behavioral deficits ［J］. Biomed Pharmacother ， 2018 ， 103 ： 614 - 620 .

CHEN Z H ， CAO J F ， ZHOU J S ， et al . Interaction of caveolin-1 with Atg12-Atg5 system suppresses autophagy in lung epithelial cells ［J］. Am J Physiol Lung Cell Mol Physiol ， 2014 ， 306 （ 11 ）： L1016 - L1025 .

NISHIDA K ， KYOI S ， YAMAGUCHI O ， et al . The role of autophagy in the heart ［J］. Cell Death Differ ， 2009 ， 16 （ 1 ）： 31 - 38 .

CHEN Y ， LI Q ， LI Q ， et al . p62/SQSTM1，a central but unexploited target：Advances in its physiological/pathogenic functions and small molecular modulators ［J］. J Med Chem ， 2020 ， 63 （ 18 ）： 10135 - 10157 .

ZHOU Y Y ， LI Y ， JIANG W Q ， et al . MAPK/JNK signalling：A potential autophagy regulation pathway ［J］. Biosci Rep ， 2015 ， 35 （ 3 ）： e00199 .

SINHA S ， LEVINE B . The autophagy effector Beclin1：A novel BH3-only protein ［J］. Oncogene ， 2008 ，27 Suppl 1 （Suppl 1）： S137 - S148 .

SALMINEN A ， KAARNIRANTA K ， KAUPPINEN A . Beclin 1 interactome controls the crosstalk between apoptosis，autophagy and inflammasome activation：Impact on the aging process ［J］. Ageing Res Rev ， 2013 ， 12 （ 2 ）： 520 - 534 .

MILLER BRIAN C ， ZIJIANG Z ， STEPHENSON LINDA M ， et al . The autophagy gene ATG5 plays an essential role in B lymphocyte development ［J］. Autophagy ， 2008 ， 4 （ 3 ）： 309 - 314 .

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Research Progress on Protective Effect and Mechanism of Salidroside on Cerebral Ischemia Injury： A Review

Effect of Huazhuo Jiedu Huoxue Tongluo Prescription on Brain-gut Axis of Rats with Cerebral Ischemia-reperfusion Injury Based on 16S rRNA Sequencing of Intestinal Flora

Improved Learning and Memory in Sleep-deprived Mice by Anmeidan via Cellular Autophagy Regulation Through PI3K/Akt/mTOR Signaling Pathway

Traditional Chinese Medicine Prevents and Treats Cerebral Ischemia-reperfusion Injury by Regulating Nrf2 Signaling Pathway： A Review

Effect of Yishen Huayu Prescription on Autophagy of Transdifferentiated TCMK-1 Cells Based on SIRT1/FoxO1 Pathway

Related Author

XU Haoqun

ZHANG Wenjing

LOU Yuan

JIANG Yanan

YUAN Xin

WANG Yuan

Rui-qing HUO

Min-han ZHAO

Related Institution

Shandong University of Traditional Chinese Medicine（TCM）

Innovation Center of Classic TCM Formula

Hebei Provincial Hospital of Traditional Chinese Medicine

School of Traditional Chinese Medicine， Hubei University of Chinese Medicine

Engineering Research Center of Traditional Chinese Medicine Protection Technology and New Product Development for the Elderly Brain Health， Ministry of Education， Hubei University of Chinese Medicine

AI问答

Postal code：100700
Tel：010-84076882 Email：syfjx_2010@188.com
Technical support is provided by Beijing Founder electronics co., LTD 京ICP备11006657号-10 京公网安备11010802024621
It is recommended to read the content of this site in Chrome&IE9+. Please switch to extreme mode in browser 360.
Cookies We use cookies to help provide and enhance our service and tailor content. By continuing, you agree to the use of cookies.

⁰