Mechanism of Huanglian Wendantang in Improving IR-HepG2 Based on NLRP3/Caspase-1 Signaling Pathway

Boyan MA; Xiangru XIN; Geling LU; Han LI; Bei JIANG

doi:10.13422/j.cnki.syfjx.20221301

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Mechanism of Huanglian Wendantang in Improving IR-HepG2 Based on NLRP3/Caspase-1 Signaling Pathway

更新时间：2022-08-11

- Mechanism of Huanglian Wendantang in Improving IR-HepG2 Based on NLRP3/Caspase-1 Signaling Pathway
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 28, Issue 18, Pages: 1-11(2022)
- 作者机构：
  
  黑龙江中医药大学，哈尔滨 150040
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20221301
  CLC： R2-0;R22;R285.5;R289;R33
- Published：20 September 2022，
  
  Published Online：16 June 2022，
  
  Received：23 February 2022，
- 稿件说明：
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马伯艳,辛相如,陆阁玲等.基于NLRP3/Caspase-1信号通路探讨黄连温胆汤改善HepG2细胞胰岛素抵抗的作用机制[J].中国实验方剂学杂志,2022,28(18):1-11.

MA Boyan,XIN Xiangru,LU Geling,et al.Mechanism of Huanglian Wendantang in Improving IR-HepG2 Based on NLRP3/Caspase-1 Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(18):1-11.
马伯艳,辛相如,陆阁玲等.基于NLRP3/Caspase-1信号通路探讨黄连温胆汤改善HepG2细胞胰岛素抵抗的作用机制[J].中国实验方剂学杂志,2022,28(18):1-11. DOI： 10.13422/j.cnki.syfjx.20221301.

MA Boyan,XIN Xiangru,LU Geling,et al.Mechanism of Huanglian Wendantang in Improving IR-HepG2 Based on NLRP3/Caspase-1 Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(18):1-11. DOI： 10.13422/j.cnki.syfjx.20221301.

摘要

目的

探究黄连温胆汤含药血清对胰岛素抵抗体外模型细胞焦亡的作用及机制。

方法

设置黄连温胆汤含药血清组和空白血清组，将SD大鼠随机分为2组，按照体表面积换算分别予7.8 g·kg

-1

·d

-1

的黄连温胆汤药液和同体积的生理盐水灌胃，提取并配制空白血清及不同浓度的含药血清；采用棕榈酸钠处理HepG2细胞构建胰岛素抵抗模型，随机分为空白组、模型组、盐酸二甲双胍组、空白血清组、黄连温胆汤含药血清高剂量组、黄连温胆汤含药血清中剂量组、黄连温胆汤含药血清低剂量组，培养24 h后应用1×10

-7

mol·L

-1

胰岛素处理各组细胞15 min，收集细胞上清，采用葡萄糖氧化酶（GOD-POD）法测定各组细胞葡萄糖的含量，并计算葡萄糖消耗量及抑制率，噻唑蓝（MTT）比色法检测细胞增殖情况，筛选黄连温胆汤含药血清最佳剂量。将HepG2细胞随机分为空白组、模型组、黄连温胆汤含药血清组，酶联免疫吸附测定法（ELISA）检测各组细胞白细胞介素-1

（IL-1

）、白细胞介素-18（IL-18）的含量，实时荧光定量聚合酶链式反应（Real-time PCR）、蛋白免疫印迹法（Western blot）检测各组NOD样受体蛋白3（NLRP3） mRNA和蛋白的表达情况。机制部分将HepG2细胞随机分为空白组、空载体组、NLRP3过表达组、空载体+IR组、空载体+IR+黄连温胆汤含药血清组、NLRP3过表达+IR组、NLRP3过表达+IR+黄连温胆汤含药血清组，GOD-POD法测定各组细胞葡萄糖的含量，并计算葡萄糖消耗量。ELISA检测各组细胞IL-1

、IL-18释放水平；Real-time PCR、Western blot技术检测各组细胞胱天蛋白酶-1（Caspase-1）、消皮素D（GSDMD）及NLRP3 mRNA及蛋白的表达；免疫荧光技术检测NLRP3、GSDMD和Caspase-1的表达。

结果

与空白组比较，模型组葡萄糖消耗量显著下降（

0.01）；与模型组比较，黄连温胆汤含药血清高剂量组葡萄糖消耗量升高最为显著（

0.01）。与空白组比较，IR-HepG2细胞IL-1

、IL-18释放水平和NLRP3 mRNA与蛋白表达明显升高（

0.05，

0.01）；与模型组比较，黄连温胆汤含药血清可明显降低IR-HepG2细胞上清IL-1

、IL-18、NLRP3 mRNA与蛋白的表达（

0.05，

0.01）。与空白组、空载体组比较，NLRP3过表达可显著降低细胞葡萄糖消耗量（

0.01）；与空载体+IR组比较，NLRP3过表达可明显上调IL-1

、IL-18水平和NLRP3、Caspase-1、GSDMD的mRNA及蛋白水平（

0.05，

0.01）；与NLRP3+IR组比较，黄连温胆汤含药血清可明显逆转上述指标（

0.05，

0.01）。

结论

高脂诱导IR-HepG2细胞的胰岛素敏感性与炎症及NLRP3表达密切相关。黄连温胆汤含药血清通过靶向抑制NLRP3/Caspase-1信号通路改善IR-HepG2细胞焦亡，为胰岛素抵抗及2型糖尿病的预防与治疗提供新靶点。

Abstract

Objective

To explore the effect of the serum containing Huanglian Wendantang on pyroptosis

in vitro

model of insulin resistance and its mechanism.

Method

SD rats were randomly divided into two groups， namely Huanglian Wendantang-containing serum group and blank serum group， and given 7.8 g·kg

-1

·d

-1

Huanglian Wendantang and equal volume of normal saline by intragastric administration according to body surface area. Blank serum and medicated serum with different concentration were extracted and prepared. HepG2 cells were treated with sodium palmitate to construct the model of insulin resistance （IR）， and they were randomly divided into control group， model group， metformin hydrochloride group， blank serum group， and Huanglian Wendantang-containing serum high-， medium-， and low-dose groups. After 24 h of cultivation， the cells of each group were treated with insulin for 15 min at concentration of 1×10

-7

mol·L

-1

， and the cell supernatant was collected. The glucose oxidase （GOD-POD） kit was used to determine the glucose content of each group， and calculate the glucose consumption and inhibition rate. The methyl thiazolyl tetrazolium （MTT） assay was used to detect the cell proliferation， thus screening out the optimal dose of serum containing Huanglian Wendantang. HepG2 cells were randomly divided into control group， model group， and Huanglian Wendantang-containing serum group. The levels of interleukin-1

（IL-1

） and interleukin-18 （IL-18） in each group were determined by enzyme-linked immunosorbent assay （ELISA）， and the mRNA and protein expression levels of NOD like receptor protein 3 （NLRP3） in each group were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. In terms of the mechanism， HepG2 cells were randomly divided into control group， empty vector group， NLRP3 overexpression group， empty vector + IR group， empty vector + IR + Huanglian Wendantang-containing serum group， NLRP3 overexpression + IR group， and NLRP3 overexpression + IR + Huanglian Wendantang-contain serum group. GOD-POD method was used to measure the glucose content of each group cells， and calculate the glucose consumption. ELISA was used to determine the release of IL-1

and IL-18 in each group. Real-time PCR and Western blot assay were used to determine the mRNA and protein expressions of cysteinyl aspartate specific proteinase-1 （Caspase-1）， gasdermin D （GSDMD）， and NLRP3. Immunofluorescence assay was used to detect NLRP3， GSDMD， and Caspase -1 expressions.

Result

As compared with the control group， the glucose consumption in the model group was significantly decreased （

0.01）. As compared with the model group， the increase of the glucose consumption of IR-HepG2 cells was the most significant in the Huanglian Wendantang-containing serum high-dose group （

0.01）. As compared with the control group， the IL-1

and IL-18 release levels and the mRNA and protein expressions of NLRP3 in IR-HepG2 cells were significantly increased （

0.05，

0.01）. Huanglian Wendantang effectively reduced IR-HepG2 cell supernatant IL-1

， IL-18， and NLRP3 mRNA and protein expressions as compared with the model group （

0.05，

0.01）. Overexpression of NLRP3 significantly reduced the cell glucose consumption as compared with the control group and the empty vector group （

0.01）， and significantly up-regulated the IL-1

and IL-18 levels and the mRNA and protein levels of NLRP3， Caspase-1， and GSDMD as compared with the empty vector + IR group （

0.05，

0.01）. Huanglian Wendantang-containing serum effectively reversed the above indicators as compared with the NLRP3 + IR group （

0.05，

0.01）.

Conclusion

High fat-induced insulin sensitivity of IR-HepG2 cells is closely related to inflammation and NLRP3 expression. Huanglian Wendantang-containing serum improves IR-HepG2 cell pyroptosis through the targeted inhibition of NLRP3/Caspase-1 signaling pathway， which provides new therapeutic targets for the prevention and treatment of IR and type 2 diabetes mellitus （T2DM）.

关键词

细胞焦亡NOD样受体蛋白3（NLRP3）黄连温胆汤胰岛素抵抗HepG2细胞系

Keywords

pyroptosisNOD like receptor protein 3 （NLRP3）Huanglian Wendantanginsulin resistanceHepG2 cell line

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