解毒通络方通过TGF-β1/Smad和ROCK通路对大鼠心肌纤维化的改善作用

doi:10.13481/j.1671-587X.20210402

Abstract

Abstract: Objective

To explore the effect of Jiedu Tongluo Decoction on the myocardial fibrosis in the rats， and to clarify its mechanism.

Methods

A Total of 30 SPF Wistar rats were randomly divided into normal control group， model group， captopril group，low and high doses of Jiedu Tongluo Decoction groups （n=6）. The myocardial fibrosis model was established by subcutaneous injection of isoproterenol hydrochloride （5 mg·kg^－1）. The rats in captopril group and low and high doses of Jiedu Tongluo Decoction groups were treated with 0.005 g·kg^－1·d^－1 captopril and 1， 5 g·kg^－1·d^－1 Jiedu Tongluo Recipe for 28 d，and the rats in control group and model group were given the equal volume of distilled water by gavage. Hematoxylin-eosin （HE） staining and Masson tri-chrome staining were applied to observe the pathomorphology of myocardium tissue of the rats in various groups，and the percentages of collagen fiber areas were calculated.The　hydroxyproline levels in myocardium tissue of the rats were determined by alkaline hydrolysis method， and the angiotensin Ⅱ（Ang Ⅱ） levels in myocardium tissue of the rats were detected by enzyme-linked immunosorbent assay （ELISA）；the expression levels of transforming growth factor-β1 （TGF-β1） and the LIM kinase （LIMK）proteins were detected by immunohistochemistry； the expression levels of Smad2， Smad3， and Rho-associated protein kinase （ROCK） mRNA in myocardium tissue of the rats were detected by Real-time fluorescence quantitative PCR （RT-qPCR），and the expression levels of Smad2/3， phosphorylated Smad2/3 （p-Smad2/3） and ROCK proteins in myocardium tissue of the rats were analyzed by Western blotting method.

Results

The myocardium tissue showed the typical fibrosis changes 28 d after isoproterenol injection； compared with normal control group， the percentage of collagen fiber area and the levels of hydroxyproline and Ang Ⅱ in myocardium tissue of the rats in model group were significantly increased （P<0.05），the expression levels of TGF-β1，and LIMK were increased（P<0.05）， the expression levels of Smad2， Smad3， and ROCK mRNA were increased（P<0.05）， and the expression levels of p-Smad2/3 and ROCK proteins were significantly increased （P<0.05）. Compared with model control group， the percentages of collagen fiber areas and the levels of hydroxyproline and Ang Ⅱ in myocardium tissue of the rats in captopril group and low and high doses of Jiedu Tongluo Decoction groups were significantly decreased （P<0.05），and the expression levels of TGF-β1 and LIMK proteins were decreased（P<0.05），the expression levels of Smad2， Smad3， and ROCK mRNA were decreased（P<0.05），and the expression levels of p-Smad2/3 and RCOK proteins of the rats were significantly decreased（P<0.05）.There were no significant differences in the above indexes of the rats between different doses of Jiedu Tongluo Decoction groups and captopril group（P>0.05）.

Conclusion

Jiedu Tongluo Decoction can inhibit the process of myocardial fibrosis by inhibiting the TGF-β1/Smad2/3 and ROCK pathway.

Key words: Jiedu Tongluo, myocardial fibrosis, transforning growth factor-β1, Smad2/3 pathway, Rho-associated protein kinase

CLC Number:

R285.5

Dan ZHANG,Ju HUI,Jiajuan GUO. Improvement effect of Jiedu Tongluo Decoction on myocardial fibrosis in rats through TGF-β1/Smad2/3 and ROCK pathways[J].Journal of Jilin University(Medicine Edition), 2021, 47(4): 826-833.

Figures/Tables 8

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References 25

1	任玺勃，南征. 任继学医学全书［M］. 北京：中国医药科技出版社， 2014：142-143.
2	刘静秋，黄永生. 夏膝口服液对自发性高血压大鼠血压、ET、NO及全血黏度的影响［J］. 中国老年学杂志， 2009， 29（14）： 1816-1817.
3	刘静秋，袁丁，黄永生. 夏膝颗粒对自发性高血压大鼠血清PⅠCP的影响［J］. 中国老年学杂志， 2009， 29（7）： 887-888.
4	FRANGOGIANNIS N G. Cardiac fibrosis： Cell biological mechanisms， molecular pathways and therapeutic opportunities［J］. Mol Aspects Med， 2019， 65：70-99.
5	PARICHATIKANOND W， LUANGMONKONG T， MANGMOOL S， et al. Therapeutic targets for the treatment of cardiac fibrosis and cancer： focusing on TGF-β signaling［J］.Front Cardiovasc Med，2020， 7： 34.
6	YU B， SLADOJEVIC N， BLAIR J E， et al. Targeting Rho-associated coiled-coil forming protein kinase （ROCK） in cardiovascular fibrosis and stiffening［J］. Expert Opin Ther Targets， 2020， 24（1）： 47-62.
7	LIVAK K J， SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^－ΔΔCT method［J］. Methods， 2001， 25（4）： 402-408.
8	惠菊，徐培斌，潘阳，等. 解毒通络方对异丙肾上腺素诱导大鼠心肌纤维化作用及机制研究［J］. 中国中西医结合杂志， 2020， 40（9）：1107-1111.
9	赵丽晶，康晶，安英，等. 鸢尾黄素对大鼠心肌纤维化的作用及其机制［J］. 吉林大学学报（医学版）， 2017， 43（2）： 288-292， 465.
10	李少春，马丽娜，陈坚，等. 大蒜辣素对大鼠心肌梗死后纤维化的影响及与TGFβ/Smads信号通路的关系［J］. 中国中药杂志， 2016， 41（13）： 2517-2521.
11	崔英子，郭家娟，李相军，等. 潜阳解毒通络方对自发性高血压大鼠心肌纤维化的影响［J］. 中国继续医学教育， 2015， 7（28）：178-180.
12	MA Z G， YUAN Y P， WU H M， et al. Cardiac fibrosis： new insights into the pathogenesis［J］. Int J Biol Sci， 2018， 14（12）： 1645-1657.
13	CHEN C， LI R X， ROSS R S， et al. Integrins and integrin-related proteins in cardiac fibrosis［J］. J Mol Cell Cardiol， 2016， 93： 162-174.
14	JOHANSEN A K Z， MOLKENTIN J D. Hippo signaling does it again： arbitrating cardiac fibroblast identity and activation［J］. Genes Dev，2019，33（21/22）：1457-1459.
15	YOUSEFI F， SHABANINEJAD Z， VAKILI S， et al. TGF-β and WNT signaling pathways in cardiac fibrosis： non-coding RNAs come into focus［J］. Cell Commun Signal， 2020， 18（1）： 87.
16	MENG J X， QIN Y Y， CHEN J Z， et al. Treatment of hypertensive heart disease by targeting Smad3 signaling in mice［J］. Mol Ther Methods Clin Dev， 2020， 18： 791-802.
17	XUE K， ZHANG J， LI C， et al. The role and mechanism of transforming growth factor beta 3 in human myocardial infarction-induced myocardial fibrosis［J］. J Cell Mol Med， 2019， 23（6）： 4229-4243.
18	CHEN R R， FAN X H， CHEN G， et al. Irisin attenuates angiotensin II-induced cardiac fibrosis via Nrf2 mediated inhibition of ROS/ TGFβ1/Smad2/3 signaling axis［J］. Chem Biol Interact， 2019， 302： 11-21.
19	HANNA A， HUMERES C， FRANGOGIANNIS N G. The role of Smad signaling cascades in cardiac fibrosis［J］. Cell Signal， 2021， 77：109826.
20	SHAHBAZI R， BARADARAN B， KHORDADMEHR M，et al. Targeting ROCK signaling in health，malignant and non-malignant diseases［J］.Immunol Lett，2020，219：15-26.
21	SANTOS G L， HARTMANN S， ZIMMERMANN W H，et al.Inhibition of Rho-associated kinases suppresses cardiac myofibroblast function in engineered connective and heart muscle tissues［J］.J Mol Cell Cardiol，2019， 134：13-28.
22	MORIN P， WICKMAN G， MUNRO J， et al. Differing contributions of LIMK and ROCK to TGFβ-induced transcription， motility and invasion［J］. Eur J Cell Biol， 2011， 90（1）： 13-25.
23	CHEN Q H， GIMPLE R C， LI G N， et al. LIM kinase 1 Acts as a profibrotic mediator in permanent atrial fibrillation patients with valvular heart disease［J］.J Biosci， 2019， 44（1）： 16.
24	HUANG W J， TAN M L， WANG Y， et al. Increased intracellular Cl^－ concentration improves airway epithelial migration by activating the RhoA/ROCK pathway［J］. Theranostics， 2020， 10（19）： 8528-8540.
25	LIU Y B， FAN Y， LI J L， et al. Combination of LCZ696 and ACEI further improves heart failure and myocardial fibrosis after acute myocardial infarction in mice［J］.Biomedecine Pharmacother，2021，133： 110824.

Group	Hydroxyproline	Ang Ⅱ
Normal control	0.16±0.03	263.9±45.9
Model	0.42±0.04^*	638.2±99.7^*
Captopril	0.30±0.04^△	389.9±64.2^△
Jiedu Tongluo Decoction
Low dose	0.34±0.03^△	366.6±99.8^△
High dose	0.28±0.02^△#	332.8±77.8^△

Group	TGF-β1	LIMK
Normal control	0.65±0.21	0.35±0.11
Model	11.10±1.41^*	12.84±1.29^*
Captopril	5.00±2.83^*Δ	8.22±1.92^*Δ
Jiedu Tongluo Decoction
Low dose	3.90±1.49^*△	6.41±1.51^*△
High dose	1.40±1.51^*△	8.59±1.99^*△

Group	p-Smad2/3	ROCK
Normal control	0.51±0.14	0.86±0.18
Model	1.13±0.12^*	1.26±0.20^*
Captopril	0.88±0.13^*△	0.85±0.15^△
Jiedu Tongluo
Low dose	0.85±0.16^*△	1.01±0.18
High dose	0.74±0.11^*△	0.95±0.20^△

[1]	WAN Qi, YU Baogang. Effects of miR-125b on proliferation and migration of cardiac fibroblasts by TLR4/NF-κB signaling pathway [J]. Journal of Jilin University(Medicine Edition), 2020, 46(02): 286-291.
[2]	WANG Liping, WANG Jianhui, LI Shumin, YANG Xiuhong. Effect of N-acetyl cysteine pathway on expression of intermediate conductance Ca²⁺-activated K⁺ channels in cardiac fibroblasts of AGT-REN double transgenic hypertension mice [J]. Journal of Jilin University Medicine Edition, 2017, 43(05): 881-886.
[3]	ZHAO Lijing, KANG Jing, AN Ying, XU Bo, WANG Yanchun. Eeffect of tectorigenin on myocardial fibrosis in rats and its mechanism [J]. Journal of Jilin University Medicine Edition, 2017, 43(02): 288-292.
[4]	GOU Xiangbo, GUO Jing, BAI Jing. Protective effect of total flavones of buckwheat flowers and leaves on myocardial fibrosis of type 2 diabetic ratsand its mechanism [J]. Journal of Jilin University Medicine Edition, 2016, 42(04): 694-698.
[5]	AN Ying, LU Qian, XU Congfei, WANG Yanchun, HAN Liqin. Inhibitory effect of Rhodiola total flavonoids on proliferation of cardiac fibroblasts in rats [J]. Journal of Jilin University Medicine Edition, 2015, 41(06): 1190-1194.
[6]	AN Ying, YANG Qian, HUANG Jianlu, JIANG Kai, ZENG Qingrong, SONG Wenxin, WANG Yanchun. Improvement effect of Vitacamphorae Injection on myocardial fibrosis in rats [J]. Journal of Jilin University Medicine Edition, 2015, 41(04): 737-741.
[7]	CHU Chun, ZENG Ou, LUO Jian, LI Fang, XIAO Ting, ZHOU Songbai, YANG Jun. Effects of hydrogen sulfide on expressions of MT1-MMP and TIMP-2 proteins and myocardial fibrosis in myocardium tissue of diabetic rats and their mechanisms [J]. Journal of Jilin University Medicine Edition, 2015, 41(02): 235-239.
[8]	ZHAO Jin-xiang1,ZHAO Li-yan2,WANG Xiu-ge1,GOU Lin-hua1,NAN Zheng1. Effects of JieDu TongLuo BaoShen capsule on serum adiponectin and expression levels of adiponectin and MCP-1 proteins in kidney tissue of type 2 diabetic rats [J]. Journal of Jilin University Medicine Edition, 2014, 40(01): 65-69.
[9]	WU Lan,ZHANG Chun-yan,SUN Jing-hui,SONG Li-jun. Effect of irbesartan on periostin expression in myocardium tissue of VMC mice in chronic phase and its mechanism of antimyocardial fibrosis [J]. Journal of Jilin University Medicine Edition, 2013, 39(6): 1153-1158.
[10]	LI Feng, GAO Hai-cheng, SHENG Xiao-tong, SUN Wen-yi, CHEN Yuan-yuan, YANG Xiu-na. Effect of sodium ferulate on \|Rho/Rock signal pathway and its protective effect on liver tissue [J]. J4, 2012, 38(5): 889-893.
[11]	MIAO Chun-sheng\|JING Hai-yan\|YIN Li1\|LI Xi-ning\|REN Li-qun. Effect of apocynum venetum extract on ERK pathway in rat myocardial fibrosis induced by AngⅡ [J]. J4, 2012, 38(4): 618-622.
[12]	JIA Chun-shu,CHEN Ai-jun,LI Min,LI Xiang-Jun,MIAO Chun-sheng,LI Xi-ning,RELi-qun. Effects of selenium and vitamin E on myocardial fibrosis in rats fed on grains from Keshan disease endemic area [J]. J4, 2011, 37(2): 211-214.
[13]	GAO Hai-Cheng, KONG Ning, GAO Hai-Mei, REN Li-Qun. Effects of sodium ferulate on tTG expression in rat \|ischemic myocardium [J]. J4, 2010, 36(6): 1071-1074.
[14]	MIAO Chun-Sheng, GAO Hai-Cheng, LI Xiang-Jun, YIN Li, LI Yun-Qian, LI Cai. Expression of p53 in myocardium tissue of myocardial fibrosis rats induced with isoproterenol \|and its significance [J]. J4, 2010, 36(6): 1064-1066.
[15]	JIANG Tie-Chao, ZOU Ying-Gang, YU Xiao-Yan, WANG Hong-Zhi, YANG Ping. Expression \|of NF-κB in rats with \|myocardial 　ischemic fibrosis and its significance  [J]. J4, 2010, 36(5): 879-881.

Improvement effect of Jiedu Tongluo Decoction on myocardial fibrosis in rats through TGF-β1/Smad2/3 and ROCK pathways

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