Effect and Mechanism of Yishen Tongluo Prescription on miR-514a-5p/TNFSF15 Signaling Pathway in MN Rats

Jing-yu MAO; Fei GAO; Xin-xin ZHANG; Hao LIU; Mei-jiao ZHAO; Su-zhi CHEN; Feng-wen YANG; Miao TAN; Jin-chuan TAN

doi:10.13422/j.cnki.syfjx.20212407

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Effect and Mechanism of Yishen Tongluo Prescription on miR-514a-5p/TNFSF15 Signaling Pathway in MN Rats

更新时间：2021-12-14

- Effect and Mechanism of Yishen Tongluo Prescription on miR-514a-5p/TNFSF15 Signaling Pathway in MN Rats
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 28, Issue 2, Pages: 121-130(2022)
- 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，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.20212407
  CLC： R2-0;R22;R285.5;R289;R33
- Published：20 January 2022，
  
  Published Online：29 October 2021，
  
  Received：25 July 2021，
- 稿件说明：
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毛竞宇,高飞,张欣欣等.益肾通络方对MN大鼠miR-514a-5p/TNFSF15信号通路的影响及机制[J].中国实验方剂学杂志,2022,28(02):121-130.

MAO Jing-yu,GAO Fei,ZHANG Xin-xin,et al.Effect and Mechanism of Yishen Tongluo Prescription on miR-514a-5p/TNFSF15 Signaling Pathway in MN Rats[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(02):121-130.
毛竞宇,高飞,张欣欣等.益肾通络方对MN大鼠miR-514a-5p/TNFSF15信号通路的影响及机制[J].中国实验方剂学杂志,2022,28(02):121-130. DOI： 10.13422/j.cnki.syfjx.20212407.

MAO Jing-yu,GAO Fei,ZHANG Xin-xin,et al.Effect and Mechanism of Yishen Tongluo Prescription on miR-514a-5p/TNFSF15 Signaling Pathway in MN Rats[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(02):121-130. DOI： 10.13422/j.cnki.syfjx.20212407.

摘要

目的

基于miR-514a-5p/肿瘤坏死因子超家族成员15（TNFSF15）信号通路探讨益肾通络方抑制膜性肾病（MN）大鼠肾小球足细胞凋亡的分子机制。

方法

80只SD大鼠采用预免疫和尾静脉注射阳离子化牛血清白蛋白（C-BSA）的方法建立MN大鼠模型，并使造模成功的MN大鼠随机分为模型组，益肾通络方高、中、低剂量组（26.44，13.22，6.61 g·kg

-1

）和贝那普利组（10 mg·kg

-1

），每组各10只，取健康大鼠20只作为正常组，各组分别予对应药物灌胃，每日1次，连续干预4周。给药结束后，实时荧光定量聚合酶链式反应（Real-time PCR）检测大鼠肾组织中miR-514a-5p及TNFSF15 mRNA表达水平；免疫组化（IHC）检测大鼠肾组织中足细胞标志蛋白足细胞裂孔膜蛋白（Nephrin），膜蛋白（Podocin），足萼蛋白（Podocalyxin），突触足蛋白（Synaptopodin），TNFSF15及足细胞凋亡相关蛋白B细胞淋巴瘤-2（Bcl-2）相关X蛋白（Bax），Bcl-2关联死亡启动子重组蛋白（BAD），大分子B细胞淋巴瘤（Bcl-XL）的表达水平；蛋白免疫印迹法（Western blot）检测大鼠肾组织中TNFSF15，Bax，BAD，Bcl-2，Bcl-XL的表达水平；原位末端标记法（TUNNEL）检测大鼠肾组织细胞凋亡率。

结果

与正常组比较，模型组大鼠肾组织中miR-514a-5p水平明显降低（

0.05），TNFSF15 mRNA水平明显升高（

0.05）；足细胞标志蛋白Nephrin，Podocin，Podocalyxin，Synaptopodin表达明显降低（

0.05）；TNFSF15，Bax，BAD蛋白表达水平明显升高（

0.05），Bcl-2，Bcl-XL蛋白表达水平明显降低（

0.05）；凋亡细胞明显增多（

0.05）。与模型组比较，益肾通络方各剂量组和贝那普利大鼠肾组织中miR-514a-5p水平明显升高（

0.05），TNFSF15 mRNA水平明显降低（

0.05）；足细胞标志蛋白Nephrin，Podocin，Podocalyxin，Synaptopodin表达明显升高（

0.05）；TNFSF15，Bax，BAD蛋白表达水平明显降低（

0.05），Bcl-2，Bcl-XL蛋白表达水平明显升高（

0.05）；凋亡细胞明显减少（

0.05）。

结论

益肾通络方可能通过miR-514a-5p/TNFSF15信号通路，减轻大鼠肾组织足细胞凋亡，缓解MN大鼠肾损伤。

Abstract

Objective

To explore the molecular mechanism of Yishen Tongluo prescription in inhibiting the apoptosis of glomerular podocytes in rats with membranous nephropathy （MN） based on the miR-514a-5p/tumor necrosis factor superfamily member 15 （TNFSF15） signaling pathway.

Method

Eighty SD rats were pre-immunized and injected with cationized bovine serum albumin （C-BSA） into the tail vein for inducing MN， and the successfully modeled MN rats were randomly divided into the model group， high-， middle-， and low-dose （26.44， 13.22， 6.61 g·kg

-1

） Yishen Tongluo prescription groups， and benazepril （10 mg·kg

-1

） group， with 10 rats in each group， and another 20 healthy rats were classified into the normal group. Rats in each group were gavaged with the corresponding drugs， once a day， for four successive weeks. After the administration， the 24-hour urine total protein （UTP） level， serum total cholesterol （TC）， triglyceride （TG）， albumin （ALB）， creatinine （SCr）， and urea nitrogen （BUN） levels were measured. The miR-514a-5p and TNFSF15 mRNA expression levels in the rat kidney tissue were detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and the expression levels of podocyte marker proteins Nephrin， Podocin， Podocalyxin， Synaptopodin， TNFSF15， and podocyte apoptosis-related proteins B lymphocytoma-2 （Bcl-2）-related X protein （Bax）， Bcl-2-associated death promoter （BAD） protein， and B-cell lymphoma-extra large （Bcl-XL） by immunohistochemistry （IHC）. Western blot was used to detect the expression levels of TNFSF15， Bax， BAD， Bcl-2， and BCL-XL in the rat kidney tissue. The apoptosis rate of rat kidney tissue was measured using the in situ end labeling method （Tunnel）.

Result

Compared with the normal group， the level of miR-514a-5p in the kidney tissue was significantly reduced （

0.05）， and the TNFSF15 mRNA expression was significantly increased （

0.05）. The expression levels of podocyte marker proteins Nephrin， Podocin， Podocalyxin， and Synaptopodin were down-regulated （

0.05）. The protein expression levels of TNFSF15， Bax， and BAD were increased （

0.05）， whereas the Bcl-2 and Bcl-XL protein expression levels were decreased （

0.05）. The number of apoptotic cells diminished significantly （

0.05）. Compared with the model group， the level of miR-514a-5p in the kidney tissue was significantly increased （

0.05）， while the level of TNFSF15 mRNA was significantly decreased （

0.05）. The expression levels of podocyte marker proteins Nephrin， Podocin， podocalyxin， and Synaptopodin were up-regulated （

0.05）， whereas the TNFSF15， Bax， and BAD protein expression levels were down-regulated （

0.05）. Bcl-2 and Bcl-XL protein expression levels rose （

0.05）. The number of apoptotic cells significantly decreased （

0.05）.

Conclusion

Yishen Tongluo prescription reduces the apoptosis of rat kidney podocytes and alleviates the kidney injury of MN rats through the miR-514a-5p/TNFSF15 signaling pathway.

关键词

膜性肾病益肾通络方足细胞miR-514a-5pTNFSF15

Keywords

membranous nephropathy （MN）Yishen Tongluo prescriptionpodocytesmiR-514a-5pTNFSF15

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