黄连温胆汤对IGT大鼠骨骼肌细胞焦亡经典通路的影响  

Effect of Huanglian Wendantang on Classical Pathway of Skeletal Muscle Pyroptosis in IGT Rats

作　　者：田旭升[1] 李云凤 李寒 王宁[1] 陆阁玲 陈冰冰 辛相如 马伯艳[1] TIAN Xu-sheng;LI Yun-feng;LI Han;WANG Ning;LU Ge-ling;CHEN Bing-bing;XIN Xiang-ru;MA Bo-yan(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》2021年第18期1-8,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(81873231);黑龙江省自然科学基金面上项目(H2018057)。

摘　　要：目的:基于NOD样受体蛋白3(NLRP3)/半胱氨酸天冬氨酸蛋白水解酶-1(Caspase-1)/消皮素D(GSDMD)/白细胞介素-1β(IL-1β)/白细胞介素-18(IL-18)经典通路,探讨黄连温胆汤对糖耐量低减(IGT)大鼠骨骼肌细胞焦亡的影响,并阐释其可能作用机制。方法:健康雄性SD大鼠予45%脂肪热能的高脂饲料长期喂养20周诱导IGT大鼠模型。造模成功后将实验分为4组,分别为正常组、模型组、阳性药组(盐酸二甲双胍,0.05 g·kg^(-1)·d^(-1))和黄连温胆汤组(7.8 g·kg^(-1)d^(-1)),正常组和模型组则给予相应体积蒸馏水,各组给药体积均为10 mL·kg^(-1)d^(-1),连续灌胃给药4周。实验结束后取血分离血清并取大鼠骨骼肌进行液氮保存。采用酶联免疫吸附测定法(ELISA)检测血清IL-1β和IL-18含量;实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)分别检测骨骼肌组织NLRP3,Caspase-1,GSDMD mRNA及蛋白表达水平;免疫荧光法观测骨骼肌组织GSDMD,IL-1β和IL-18蛋白表达;苏木素-伊红(HE)染色观察大鼠骨骼肌组织病理学变化。结果:与正常组比较,模型组大鼠血清中IL-1β,IL-18含量和骨骼肌组织中NLRP3,Caspase-1,GSDMD mRNA及蛋白表达水平均显著升高(P<0.01);免疫荧光检测显示IGT大鼠骨骼肌组织GSDMD,IL-18和IL-1β蛋白含量显著上升(P<0.01);HE染色可见骨骼肌组织具有明显病理形态变化。与模型组比较,黄连温胆汤和盐酸二甲双胍组均可有效降低IGT大鼠血清IL-18和IL-1β含量和骨骼肌组织NLRP3,Caspase-1,GSDMD mRNA和蛋白表达(P<0.01);免疫荧光技术结果表明黄连温胆汤能有效降低IGT大鼠骨骼肌组织GSDMD,IL-1β和IL-18蛋白含量(P<0.01);HE染色结果表明黄连温胆汤能改善大鼠骨骼肌组织形态变化。结论:黄连温胆汤对IGT大鼠骨骼肌细胞焦亡具有抑制作用,其作用机制与调节细胞焦亡经典途径中NLRP3/Caspase-1/GSDMD/IL-1β/IL-18通路具有重要关系。Objective: To explore the effects of Huanglian Wendantang(HLWDT)on pyroptosis of skeletal muscle in rats with impaired glucose tolerance(IGT)and to explain the mechanism based on NOD-like receptor protein 3(NLRP3)/cysteine aspartate-specific protease-1(Caspase-1)/gasdermin D(GSDMD)/interleukin-1β(IL-1β)/IL-18 signaling pathway. Method:The SD male rats were fed with 45% high-fat diet for20 weeks to induce the IGT model. After modeling,the rats were randomly divided into a blank group,a model group,a positive control group(metformin hydrochloride,0.05 g·kg^(-1) d^(-1)),and an HLWDT(7.8 g·kg^(-1) d^(-1))group based on the body weight of rats. The blank group and the model group were fed with the same volume of distilled water. The dose for each group was set as 10 mL·kg^(-1) d^(-1). After four weeks of continuous gavage,blood was collected and serum was separated. The skeletal muscles of rats were stored in liquid nitrogen. Subsequently,serum IL-1β and IL-18 were detected by the enzyme-linked immunosorbent assay(ELISA). The mRNA and protein expression levels of NLRP3,Caspase-1,and GSDMD were detected by real-time quantitative PCR(Real-time PCR)and Western blot,respectively. The expression of GSDMD,IL-1β,and IL-18 proteins in skeletal muscle tissues was detected by immunofluorescence. Hematoxylin-eosin(HE)staining was used to observe the pathological changes of skeletal muscles. Result: Compared with the blank group,the model group showed increased IL-1β and IL-18 in serum,and NLRP3,Caspase-1,and GSDMD gene and protein expression in skeletal muscle tissues(P<0.01). Immunofluorescence assay showed that GSDMD,IL-18,and IL-1β protein expression in skeletal muscle tissues of the model group was significantly elevated(P<0.01). HE staining showed obvious pathological changes in skeletal muscles. Compared with the model group,the HLWDT group and the positive control group could decrease IL-1β and IL-18 in serum and NLRP3,Caspase-1,and GSDMD gene and protein expression in skeletal muscle tissues(P<0.01). In additio

关 键 词：黄连温胆汤 骨骼肌细胞焦亡 糖耐量低减(IGT) 炎症反应 

分 类 号：R2-0[医药卫生—中医学] R22