Mechanism of Salvianolate in Inducing Autophagy in Podocytes of Rats with Membranous Nephropathy via AMPK/SIRT1/PGC-1<italic style="font-style: italic">α </italic>Signaling Pathway

ZHANG Yao; GAO Fei; TAN Miao; YANG Fengwen; CHEN Suzhi; REN Meifang; YUAN Guodong; TAN Jinchuan

doi:10.13422/j.cnki.syfjx.20230142

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Mechanism of Salvianolate in Inducing Autophagy in Podocytes of Rats with Membranous Nephropathy via AMPK/SIRT1/PGC-1α Signaling Pathway

更新时间：2023-05-17

- Mechanism of Salvianolate in Inducing Autophagy in Podocytes of Rats with Membranous Nephropathy via AMPK/SIRT1/PGC-1α Signaling Pathway
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 8, Pages: 203-213(2023)
- 作者机构：
  
  1.河北中医学院，石家庄 050200
  2.河北中医学院第一附属医院，河北省中医院，石家庄 050011
  3.河北医科大学第四医院，石家庄 050000
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20230142
  CLC： R2-0;R33;R289;R692
- Published：20 April 2023，
  
  Published Online：18 January 2023，
  
  Received：12 September 2022，
- 稿件说明：
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张尧,高飞,檀淼等.丹参多酚酸盐通过AMPK/Sirt1/PGC-1α信号通路诱导膜性肾病大鼠足细胞自噬的机制[J].中国实验方剂学杂志,2023,29(08):203-213.

ZHANG Yao,GAO Fei,TAN Miao,et al.Mechanism of Salvianolate in Inducing Autophagy in Podocytes of Rats with Membranous Nephropathy via AMPK/SIRT1/PGC-1α Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(08):203-213.
张尧,高飞,檀淼等.丹参多酚酸盐通过AMPK/Sirt1/PGC-1α信号通路诱导膜性肾病大鼠足细胞自噬的机制[J].中国实验方剂学杂志,2023,29(08):203-213. DOI： 10.13422/j.cnki.syfjx.20230142.

ZHANG Yao,GAO Fei,TAN Miao,et al.Mechanism of Salvianolate in Inducing Autophagy in Podocytes of Rats with Membranous Nephropathy via AMPK/SIRT1/PGC-1α Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(08):203-213. DOI： 10.13422/j.cnki.syfjx.20230142.

摘要

目的

通过观察丹参多酚酸盐对膜性肾病（MN）大鼠肾组织中腺苷酸活化蛋白激酶（AMPK）、沉默信息调节因子（Sirt1）、过氧化物酶体增殖物激活受体

辅激活因子-1

（PGC-1

）蛋白的表达及细胞自噬和凋亡的情况，探讨其治疗MN的可能的分子机制。

方法

80只雄性SD大鼠随机分为正常组，模型组，盐酸贝那普利组（10 mg·kg

-1

），丹参多酚酸盐低、中、高剂量组（16.7、33.3、66.7 mg·kg

-1

），通过尾静脉注射阳离子化牛血清白蛋白（C-BSA）的方法造模。造模成功后，各组按照相应比例剂量连续给药4周后留取24 h尿、血清和肾组织，尿液用于检测24 h尿蛋白定量（24 h UTP）、血清用于检测血尿素氮（BUN）、血肌酐（SCr）、白细胞介素-6（IL-6）、肿瘤坏死因子-

（TNF-

）、C反应蛋白（CRP）、谷胱甘肽过氧化物酶（GSH-Px）、超氧化物歧化酶（SOD）、丙二醛（MDA）的含量。采用光镜、电镜、免疫荧光法观察肾脏病理学变化，蛋白免疫印迹法（Western blot）检测大鼠肾组织磷酸化（p）-AMPK、AMPK、p-Sirt1、Sirt1、PGC-1

蛋白表达水平；免疫组化（IHC）检测大鼠肾组织自噬特异性基因-1（Beclin-1）、微管相关蛋白1轻链3（LC3）Ⅱ、泛素结合蛋白（p62）、B细胞淋巴瘤（Bcl）-2、Bcl-2相关X蛋白（Bax）、胱天蛋白酶（Caspase）-7蛋白表达水平。

结果

与正常组比较，模型组大鼠24 h UTP、IL-6、TNF-

、CRP、MDA水平显著升高（

0.01），SOD和GSH-Px水平显著降低（

0.01），BUN、SCr变化差异无统计学意义；与模型组比较，丹参多酚酸盐低中高剂量组和贝那普利组大鼠24 h UTP、IL-6、TNF-

、CRP、MDA水平显著降低（

0.01），SOD和GSH-Px水平显著升高（

0.01）。在苏木素-伊红（HE）、马松（Masson）染色、免疫荧光及电镜下观察可见模型组大鼠肾组织病理损伤明显，贝那普利和丹参多酚酸盐治疗后，肾组织细胞的病理损伤逐渐改善。与正常组比较，模型组大鼠肾脏p-AMPK/AMPK、p-Sirt1/Sirt1、PGC-1

、Bcl-2、Beclin-1、LC3Ⅱ表达显著降低（

0.01），Bax、Caspase-7、p62的表达显著增加（

0.01）；与模型组比较，贝那普利和丹参多酚酸盐治疗后大鼠肾脏p-AMPK/AMPK、p-Sirt1/Sirt1、PGC-1

、Bcl-2、Beclin-1、LC3Ⅱ表达显著升高（

0.01），Bax、Caspase-7、p62的表达显著降低（

0.01）。

结论

丹参多酚酸盐对MN大鼠肾保护作用，这可能与激活AMPK/Sirt1/PGC-1

通路，上调自噬，减少凋亡有关。

Abstract

Objective

To observe the effect of salvianolate on the protein expressions of adenosine monophosphate （AMP）-activated protein kinase （AMPK）， silent information regulator 1 （SIRT1） and peroxisome proliferator-activated receptor-

coactivator-1

（PGC-1

）， autophagy and apoptosis

in kidney tissue of rats with membranous nephropathy （MN）， and to explore its possible molecular mechanism against MN.

Method

Eighty male SD rats were randomly divided into normal group， model group， benazepril hydrochloride group （10 mg·kg

-1

）， and salvianolate low-， medium-， and high-dose groups （16.7， 33.3 and 66.7 mg·kg

-1

）. The rats were modeled by injection of cationized bovine serum albumin （C-BSA） into the tail vein. After successful modeling， rats in the administration groups were given corresponding doses of drugs for 4 consecutive weeks， and then 24-hour urine， serum and kidney tissue were collected for the detection of 24-hour urinary protein （UTP）， blood urea nitrogen （BUN）， serum creatinine （SCr）， interleukin-6 （IL-6）， tumor necrosis factor-

（TNF-

）， C reactive protein （CRP）， glutathione peroxidase （GSH-Px）， superoxide dismutase （SOD）， and malondialdehyde （MDA）. The pathological changes of kidneys were observed by light microscope， electron microscope and immunofluorescence. Western blot was used to detect the protein expressions of phospho-AMPK （p-AMPK）， AMPK， phospho-SIRT1 （p-SIRT1）， SIRT1 and PGC-1

in rat kidney tissue. The protein expressions of autophagy-specific gene （Beclin-1）， microtubule-associated protein 1 light chain 3 （LC3） Ⅱ， ubiquitin-binding protein （p62）， B cell lymphoma （Bcl-2）， Bcl-2-associated X （Bax）， and cysteine aspartic protease-7 （Caspase-7） in rat kidney tissue were determined by immunohistochemistry （IHC）.

Result

Compared with the conditions in the normal group， the levels of UTP， IL-6， TNF-

， CRP and MDA in the model group were increased （

0.05） while the levels of SOD and GSH-Px were decreased （

0.05）， and there was no difference in BUN and SCr. Compared with the model group， the administration groups had lowered UTP， IL-6， TNF-

， CRP and MDA （

0.05） while elevated SOD and GSH-Px （

0.05）. It could be seen from hematoxylin and eosin （HE） staining， Masson staining， immunofluorescence and electron microscopy that the pathological damage of rat kidney tissue in the model group was significant， but after treatment with benazepril hydrochloride and salvianolate， the pathological damage of kidney cells was gradually improved. The expressions of p-AMPK/AMPK， p-SIRT1/SIRT1， PGC-1

， Bcl-2， Beclin-1 and LC3Ⅱ in rat kidney in the model group were lower than those in the normal group （

0.05） while the expressions of Bax， Caspase-7 and p62 were higher （

0.05）. Compared with the model group， benazepril hydrochloride group and salvianolate groups had an up-regulation in the expressions of p-AMPK/AMPK， p-SIRT1/SIRT1， PGC-1

， Bcl-2， Beclin-1 and LC3Ⅱ in the kidney （

0.05） while a down-regulation in the expressions of Bax， Caspase-7 and p62 （

0.05）.

Conclusion

The protective effect of salvianolate on the kidneys of MN rats may be related to the activation of AMPK/SIRT1/PGC-1

signaling pathway， the up-regulation of autophagy and the reduction of apoptosis.

关键词

丹参多酚酸盐膜性肾病腺苷酸活化蛋白激酶（AMPK）/沉默信息调节因子（Sirt1）/过氧化物酶体增殖物激活受体γ辅激活因子-1α（PGC-1α）通路自噬凋亡

Keywords

salvianolatemembranous nephropathyadenosine monophosphate （AMP）-activated protein kinase （AMPK）/silent information regulator 1 （SIRT1）/peroxisome proliferator-activated receptor-γ coactivator-1α （PGC-1α） signaling pathwayautophagyapoptosis

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⁰

Mechanism of Salvianolate in Inducing Autophagy in Podocytes of Rats with Membranous Nephropathy via AMPK/SIRT1/PGC-1α Signaling Pathway

DOI：10.13422/j.cnki.syfjx.20230142

摘要

Abstract

关键词

Keywords

references