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  

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

作　　者：马伯艳[1] 辛相如 陆阁玲 李寒 姜北[1] MA Boyan;XIN Xiangru;LU Geling;LI Han;JIANG Bei(Heilongjiang University of Chinese Medicine,Harbin 150040,China)

机构地区：[1]黑龙江中医药大学,哈尔滨150040

出　　处：《中国实验方剂学杂志》2022年第18期1-11,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(81873231)。

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-1β(IL-1β)、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-1β、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(P<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(P<0.01)。与空白组比较,IR-HepG2细胞IL-1β�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-1β(IL-1β)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 wa

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 

分 类 号：R2-0[医药卫生—中医学] R22R285.5R289R33