基于NLRP3/CASP1/GSDMD通路探讨益肾通络方抑制糖尿病肾脏疾病小鼠足细胞焦亡的作用机制

赵靓; 向世勰; 谢治深; 王潘; 石贤聪; 王佳俊; 段亚飞; 张振强; 陈洁; 徐江雁; 张效威

doi:10.3969/j.issn.1006-2157.2023.11.008

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基于NLRP3/CASP1/GSDMD通路探讨益肾通络方抑制糖尿病肾脏疾病小鼠足细胞焦亡的作用机制

实验研究 | 更新时间：2023-12-08

- 基于NLRP3/CASP1/GSDMD通路探讨益肾通络方抑制糖尿病肾脏疾病小鼠足细胞焦亡的作用机制
- Study on the mechanism by which Yishen Tongluo Formula inhibits podocyte pyroptosis via the NLRP3/CASP1/GSDMD pathway in mice with diabetic kidney disease
- 北京中医药大学学报 2023年46卷第11期页码：1529-1540
- 作者机构：
  
  1.河南中医药大学中医药科学院　郑州　450046
  2.马来西亚管理与科技大学国际学术部
- 作者简介：
  
  赵靓，女，在读硕士生
  # 张效威，男，博士，助理研究员，主要研究方向：代谢性疾病及靶器官损伤的中药活性成分发现及作用机制，E-mail: zhangxw2020@163.com
- 基金信息：
  
  国家重点研发计划项目(2020YFE0201800);国家自然科学基金项目(82104471);河南省重点研发专项(221111520300);河南省重点研发与推广专项（科技攻关）(222102310647;232102310454);河南省高校科技创新团队支持计划项目(21IRTSTHN026)
- DOI：10.3969/j.issn.1006-2157.2023.11.008
  中图分类号： R285.5
- 收稿日期：2023-06-01，
  
  纸质出版日期：2023-11-30
- 稿件说明：
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赵靓, 向世勰, 谢治深, 等. 基于NLRP3/CASP1/GSDMD通路探讨益肾通络方抑制糖尿病肾脏疾病小鼠足细胞焦亡的作用机制[J]. 北京中医药大学学报, 2023,46(11):1529-1540.

ZHAO Liang, XIANG Shixie, XIE Zhishen, et al. Study on the mechanism by which Yishen Tongluo Formula inhibits podocyte pyroptosis via the NLRP3/CASP1/GSDMD pathway in mice with diabetic kidney disease[J]. Journal of beijing university of traditional chinese medicine, 2023, 46(11): 1529-1540.
赵靓, 向世勰, 谢治深, 等. 基于NLRP3/CASP1/GSDMD通路探讨益肾通络方抑制糖尿病肾脏疾病小鼠足细胞焦亡的作用机制[J]. 北京中医药大学学报, 2023,46(11):1529-1540. DOI： 10.3969/j.issn.1006-2157.2023.11.008.

ZHAO Liang, XIANG Shixie, XIE Zhishen, et al. Study on the mechanism by which Yishen Tongluo Formula inhibits podocyte pyroptosis via the NLRP3/CASP1/GSDMD pathway in mice with diabetic kidney disease[J]. Journal of beijing university of traditional chinese medicine, 2023, 46(11): 1529-1540. DOI： 10.3969/j.issn.1006-2157.2023.11.008.

摘要

目的

观察益肾通络方(熟地黄、黄芪、山萸肉、丹参、大黄、鬼箭羽、血竭)对

小鼠足细胞及小鼠肾足细胞MPC-5焦亡的影响，探讨其抑制糖尿病肾脏疾病足细胞焦亡改善足细胞损伤的可能作用机制。

方法

①体内实验。以

小鼠(模型组及各给药组)及同窝对照

小鼠(对照组)为研究对象。

小鼠按体质量随机分为5组，除模型组外，其余4组分别按照每日1.0、2.5、5.0 g/kg剂量给予益肾通络方及10 mg/kg缬沙坦灌胃，模型组及对照组给予等量蒸馏水。治疗8周后，透射电子显微镜观察小鼠肾脏组织中足细胞数量与形态变化情况；对肾脏组织进行TUNEL染色，同时利用免疫荧光技术标记足细胞标志蛋白Wilms瘤基因1(WT-1)；酶联免疫吸附测定法(ELISA)检测小鼠血清白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)水平；利用实时荧光定量PCR、Western blotting检测小鼠肾脏组织中NOD样受体热蛋白结构域相关蛋白3(NLRP3)、胱天蛋白酶-1(CASP1)、焦孔素D(GSDMD)mRNA及蛋白表达和激活情况。②体外实验。以糖基化终末期产物(AGEs)诱导的小鼠肾足细胞MPC-5为研究对象，利用CCK-8法及乳酸脱氢酶(LDH)释放检测技术，评价细胞成活率及细胞膜完整性；利用Q-PCR、Western blotting检测MPC-5细胞中NLRP3、CASP1、GSDMD的mRNA及蛋白表达和激活情况。

结果

①体内实验。与对照组相比，模型组小鼠肾脏足细胞足突出现融合，基底膜明显增厚，肾脏组织TUNEL染色阳性细胞比例增加，WT-1阳性表达率下降，血清中IL-1β、IL-18含量增加，NLRP3、CASP1、GSDMD、IL-1β及IL-18 mRNA表达上调，NLRP3蛋白表达上调，CASP1、GSDMD被激活(均

0.05)；与模型组相比，益肾通络方不同剂量干预后，肾脏组织的足细胞损伤情况缓解，肾脏组织TUNEL阳性细胞明显减少，血清中IL-1β、IL-18含量下降，NLRP3、CASP1、GSDMD mRNA表达下调，NLRP3蛋白表达下调，CASP1、GSDMD通路蛋白激活被抑制(均

0.05)。②体外实验。与对照组相比，模型组MPC-5细胞活性下降，LDH释放增加，NLRP3、CASP1、GSDMD、IL-1β及IL-18 mRNA表达上调，NLRP3蛋白表达上调，CASP1、GSDMD被激活(均

0.05)；给予益肾通络方作用后，足细胞损伤情况改善，LDH释放率下降，NLRP3、CASP1、GSDMD mRNA表达下调，NLRP3蛋白表达下调，CASP1、GSDMD通路蛋白激活被抑制(均

0.05)。

结论

益肾通络方可能通过抑制NLRP3/CASP1/GSDMD信号通路的激活，减少足细胞焦亡的发生，进而发挥改善糖尿病肾脏疾病小鼠足细胞损伤的作用。

Abstract

Objective

This study aimed to observe the effect of

Yishen Tongluo

Formula

which consists of prepared rehmannia root

milkvetch root

asiatic cornelian cherry fruit

danshen root

rhubarb root and rhizome

ramuli euonymi

and draconis resin

on podocytes in

mice and the murine renal podocytes cell line(MPC-5)

and to explore the potential mechanism of

Yishen Tongluo

Formula in improving podocyte injury in diabetic kidney disease.

Methods

(ⅰ)

In vivo

. The

mice and littermate control

mice were used as the study objects. The

mice were divided into five groups according to their bodyweights

in which four groups were treated with

Yishen Tongluo

Formula (1.0

2.5

5.0 g/kg) or valsartan (10 mg/kg) once a day. The model group and the control group were given the same amount of distilled water. After 8 weeks of treatment

the number and morphology of podocytes in the kidney were assessed by transmission electron microscopy

and the podocyte marker protein (WT-1) was visulized by immunofluorescence and TUNEL staining. The serum levels of interleukin-1β (IL-1β) and interleukin-18(IL-18) were measured by enzyme-linked immunosorbent assay. The mRNA and protein expression and activation of NLRP3

CASP1 and GSDMD were measured by Q-PCR and Western blotting. (ⅱ)

In vitro

. MPC-5 cells induced with advanced glycation end products (AGEs). CCK-8 and lactate dehydrogenase (LDH) release detection techniques were used to detect LDH and evaluate the cell survival rate and membrane integrity. The mRNA levels of NLRP3

CASP1 and GSDMD in MPC-5 cells were measured by Q-PCR and Western blotting.

Results

(ⅰ)

In vivo

. Compared with the renal podocytes of

mice

those of

mice were fused

the basement membrane was thickened and the percentage of TUNEL-positive cells was increased

the positive expression rate of WT-1 was decreased

IL-1β and IL-18 levels in the serum were increased

the mRNA levels of NLRP3

CASP1 and GSDMD were upregulated

the protein expression of NLRP3 was upregulated

and CASP1 and GSDMD were activated(

0.05). Compared with the model group

the podocyte injury in renal tissue was alleviated

the number of TUNEL positive cells in renal tissue was decreased

and the serum IL-1β and IL-18 levels decreased. NLRP3

CASP1

GSDMD

IL-1β and IL-18 mRNA levels reduced

the protein expression of NLRP3 and inhibited CASP1 and GSDMD pathway activation(all

0.05). (ⅱ)

In vitro

. Compared with the control group

the survival rate of MPC-5 cells in the AGEs model group decreased

LDH release increased

the mRNA levels of NLRP3

CASP1 and GSDMD were upregulated

the protein expression of NLRP3 was upregulated

and CASP1 and GSDMD were activated(all

0.05). After administration of

Yishen Tongluo

Formula

podocyte injury was improved

LDH release decreased

the mRNA levels of NLRP3

CASP1

GSDMD

IL-1β and IL-18 and the expression of NLRP3 protein decreased and CASP1 and GSDMD were inhibited(all

0.05).

Conclusion

Yishen Tongluo

Formula can improve podocyte injury in mice with diabetic kidney disease by inhibiting the activation of the NLRP3/CASP1/GSDMD signaling pathway and reducing podocyte scorch death.

关键词

Keywords

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