Study on the mechanism by which <italic style="font-style: italic">Yishen Tongluo</italic> Formula inhibits podocyte pyroptosis via the NLRP3/CASP1/GSDMD pathway in mice with diabetic kidney disease

ZHAO Liang; XIANG Shixie; XIE Zhishen; WANG Pan; SHI Xiancong; WANG Jiajun; DUAN Yafei; ZHANG Zhenqiang; CHEN Jie; XU Jiangyan; ZHANG Xiaowei

doi:10.3969/j.issn.1006-2157.2023.11.008

您当前的位置：

首页 >

文章列表页 >

Study on the mechanism by which Yishen Tongluo Formula inhibits podocyte pyroptosis via the NLRP3/CASP1/GSDMD pathway in mice with diabetic kidney disease

Experimental Studies | 更新时间：2023-12-08

- Study on the mechanism by which Yishen Tongluo Formula inhibits podocyte pyroptosis via the NLRP3/CASP1/GSDMD pathway in mice with diabetic kidney disease
- Journal of Beijing University of Traditional Chinese Medicine Vol. 46, Issue 11, Pages: 1529-1540(2023)
- 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：
  
  ZHANG Xiaowei, Ph.D., Associate Researcher. Academy of Chinese Medical Sciences, Henan University of Chinese Medicine, No.156, Jinshui Donglu Road, Jinshui District, Zhengzhou 450046. E-mail: zhangxw2020@163.com
- 基金信息：
  
  National Key R&D Program of China(2020YFE0201800);Natural Science Foundation of China(82104471);Henan Provincial Key Research and Development Program(221111520300);Key R&D and Promotion Projects of Henan Province(222102310647;232102310454);Henan Provincial Program for Innovative Research Team (in Science and Technology) at University(21IRTSTHN026)
- DOI：10.3969/j.issn.1006-2157.2023.11.008
  CLC： R285.5
- Received：01 June 2023，
  
  Published：30 November 2023
- 稿件说明：
移动端阅览
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：

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

references

DAEHN IS , DUFFIELD JS . The glomerular filtration barrier: a structural target for novel kidney therapies [J]. Nat Rev Drug Discov , 2021 , 20 ( 10 )： 770 - 788 .

SU PP , LIU DW , ZHOU SJ , et al . Down-regulation of Risa improves podocyte injury by enhancing autophagy in diabetic nephropathy [J/OL]. Military Med Res , 2022 , 9 ( 1 )： 23 [ 2023-01-23 ]. https://mmrjournal.biomedcentral.com/articles/10.1186/s40779-022-00385-0 .

QI W , KEENAN HA , LI Q , et al . Pyruvate kinase M2 activation may protect against the progression of diabetic glomerular pathology and mitochondrial dysfunction [J]. Nat Med , 2017 ， 23 ( 6 )： 753 - 762 .

ISERMANN B , VINNIKOV IA , MADHUSUDHAN T , et al . Activated protein C protects against diabetic nephropathy by inhibiting endothelial and podocyte apoptosis [J]. Nat Med , 2007 , 13 ( 11 )： 1349 - 1358 .

连心逸，柳红芳，苗桂珍，等．芪地糖肾颗粒对糖尿病肾脏疾病患者蛋白尿的治疗及其对尿裂隙素、足细胞素的作用 [J]. 北京中医药大学学报， 2022 ， 45 ( 12 )： 1223 - 1229 .

SHAO F . Gasdermins: making pores for pyroptosis [J]. Nat Rev Immunol ， 2021 , 21 ( 10 )： 620 - 621 .

CHENG Q , PAN J , ZHOU ZL , et al . Caspase-11/4 and gasdermin D-mediated pyroptosis contributes to podocyte injury in mouse diabetic nephropathy [J]. Acta Pharmacol Sin , 2020 , 42 ( 6 )： 954 - 963 .

BROZ P , PELEGRÍN P , SHAO F . The gasdermins, a protein family executing cell death and inflammation [J]. Nat Rev Immunol , 2019 ， 20 ( 3 )： 143 - 157 .

宋瑞婧，张欣欣，高飞，等．加味升降散对糖尿病肾病大鼠TXNIP/NLRP3通路及足细胞焦亡的影响 [J]. 中药药理与临床， 2022 ， 38 ( 4 )： 2 - 9 .

靳贺超，梁胜然，张冠文，等．基于TXNIP/NLRP3/GSDMD信号通路探讨当归补血汤对糖尿病肾病大鼠足细胞焦亡的影响 [J]. 中国实验方剂学杂志， 2022 ， 28 ( 3 )： 49 - 57 .

赵靓，张效威，谢治深，等．基于网络药理学探讨益肾通络方治疗糖尿病肾脏疾病的药效作用机制及关键调控通路的验证 [J]. 北京中医药大学学报， 2022 ， 45 ( 8 )： 824 - 834 .

ZHANG XW , ZHAO L , XIANG SX , et al . Yishen Tongluo formula alleviates diabetic kidney disease through regulating Sirt6/TGF-β 1 /Smad2/3 pathway and promoting degradation of TGF-β 1 [J/OL]. J Ethnopharmacol , 2023 ， 307 ： 116243 [ 2022-01-23 ]. https://linkinghub.elsevier.com/retrieve/pii/S0378874123001113 .

WENDT TM , TANJI N , GUO J , et al . RAGE drives the development of glomerulosclerosis and implicates podocyte activation in the pathogenesis of diabetic nephropathy [J]. Am J Pathol , 2003 ， 162 ( 4 )： 1123 - 1137 .

BENZING T , SALANT D . Insights into Glomerular Filtration and Albuminuria [J]. New Engl J Med , 2021 ， 384 ( 15 )： 1437 - 1446 .

HASEGAWA K , WAKINO S , SIMIC P , et al . Renal tubular Sirt1 attenuates diabetic albuminuria by epigenetically suppressing Claudin-1 overexpression in podocytes [J]. Nat Med , 2013 ， 19 ( 11 )： 1496 - 1504 .

TOMITA I , KUME S , SUGAHARA S , et al . SGLT2 Inhibition Mediates Protection from Diabetic Kidney Disease by Promoting Ketone Body-Induced mTORC1 Inhibition [J]. Cell Metab , 2020 ， 32 ( 3 )： 404 - 419 .

ISERMANN B , VINNIKOV IA , MADHUSUDHAN T , et al . Activated protein C protects against diabetic nephropathy by inhibiting endothelial and podocyte apoptosis [J]. Nat Med , 2007 ， 13 ( 11 )： 1349 - 1358 .

YOSHII H , UCHINO H , OHMURA C . Clinical usefulness of measuring urinary polyol excretion by gas-chromatography/mass-spectrometry in type 2 diabetes to assess polyol pathway activity [J]. Diabetes Res Clin Pract , 2001 , 51 ： 115 - 123 .

KOUZUMA T , TAKAHASHI M , ENDOH T ． An enzymatic cycling method for themeasurement of myo-inositol in biological samples [J]. Clin Chim Acta , 2001 , 312 ： 143 - 151 .

CHEN JG , GUI DK , CHEN YF , et al . Astragaloside Ⅳ improves high glucose-induced podocyte adhesion dysfunction via alpha3beta1 integrin upregulation and integrin-linked kinase inhibition [J]. Biochem Pharmocal , 2008 ， 76 ( 6 )： 796 - 804 .

CHEN JQ , YUAN SL , ZHOU J , et al . Danshen injection induces autophagy in podocytes to alleviate nephrotic syndrome via the PI3K/AKT/mTOR pathway [J/OL]. Phytomedicine , 2022 ， 107 ： 154477 [ 2023-01-23 ]. https://linkinghub.elsevier.com/retrieve/pii/S0944711322005669 .

LIU X , LIU Y , YANG Y , et al . Antioxidative Stress Effects of Salvia przewalskii Extract in Experimentally Injured Podocytes [J]. Nephron ， 2016 ， 134 ( 4 )： 253 - 271 .

WU L , LIU CJ , CHANG DY , et al . Annexin A1 alleviates kidney injury by promoting the resolution of inflammation in diabetic nephropathy [J]. Kidney Int , 2021 ， 100 ： 107 - 121 .

WU CQ , LU W , ZHANG Y , et al . Inflammasome Activation Triggers Blood Clotting and Host Death through Pyroptosis [J]. Immunity ， 2019 ， 50 ( 6 )： 1401 - 1411 .

WAN JY , LIU DW , PAN SK , et al . NLRP3-mediated pyroptosis in diabetic nephropathy [J/OL]. Front Pharmacol , 2022 ， 13 ： 998574 [ 2023-01-23 ]. https://www.frontiersin.org/articles/10.3389/fphar.2022.998574/full .

XIA SY , ZHANG ZB , MAGUPALLI VG , et al . Gasdermin D pore structure reveals preferential release of mature interleukin-1 [J]. Nature , 2021 ， 593 ( 7860 )： 607 - 611 .

Views

252

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Mechanism of Yishen Tongluo Formula regulating the TLR4/MyD88/NF-κB signaling pathway to ameliorate pyroptosis in diabetic nephropathy mice

Study on the mechanism of astragaloside Ⅰ inhibiting podocyte pyroptosis in diabetic kidney disease

Exploring the pathogenesis of "internal heat leading to zheng" in diabetic kidney disease from the perspective of "glucose toxicity" and its differential diagnosis and treatment

Diagnosis and treatment guideline of integrated traditional Chinese and western medicine for diabetic kidney disease

Discussion on the role of latent heat in diabetic kidney disease based on symptomatic treatment based on pathogenesis differentiation

Related Author

ZHANG Yifei

CAO Zijing

ZHANG Zeyu

BAI Xuehui

TANG Jingyi

XI Junyu

LIU Weijing

LIU Zhongjie

Related Institution

Beijing Puren Hospital

Dongzhimen Hospital, Beijing University of Chinese Medicine

School of Traditional Chinese Medicine, Henan University of Chinese Medicine

Collaborative Innovation Center of Research and Development on the Whole Industry Chain of Yu-Yao of Henan Province, Henan University of Chinese Medicine

Henan University of Chinese Medicine

AI问答

Postal code：100029
Tel：(010)53911857 Email：jbutcm@bucm.edu.cn
Technical support is provided by Beijing Founder electronics co., LTD 京ICP备09064830号-19 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

Study on the mechanism by which Yishen Tongluo Formula inhibits podocyte pyroptosis via the NLRP3/CASP1/GSDMD pathway in mice with diabetic kidney disease

DOI：10.3969/j.issn.1006-2157.2023.11.008

摘要

Abstract

关键词

Keywords

references