基于骨骼肌NLRP3/caspase-1/IL-1β、IL-18通路的黄连温胆汤改善IGT机制研究

Mechanism of Huanglian Wendan Decoction in improving impaired glucose tolerance based on skeletal muscle NLRP3/caspase-1/IL-1β,IL-18 pathway

基于骨骼肌Nod样受体蛋白3(Nod-like receptor protein 3,NLRP3)/炎性半胱氨酸天冬氨酸蛋白酶-1(cysteinyl aspartate specific proteinase-1,caspase-1)/白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-18(interleukin-18,IL-18)通路,探讨黄连温胆汤对糖耐量低减(impaired glucose tolerance,IGT)大鼠的影响机制。健康雄性SD大鼠予45%脂肪含量饲料喂养20周结合高温高湿环境及少动的不良生活方式复制IGT模型。分为空白组、模型组、盐酸二甲双胍组(阳性药组,0.05 g·kg^(-1)·d^(-1))及黄连温胆汤组(7.8 g·kg^(-1)·d^(-1)),连续灌胃给药4周。检测大鼠相关肥胖和血糖指数;ELISA法检测血清胰岛素水平,并计算胰岛素敏感指数(insulin sensitivity index,ISI)和胰岛素抵抗指数(insulin resistance index,IRI);real-time PCR、Western blot及免疫荧光技术检测骨骼肌组织核转录因子-κB(nuclear factor kappa B,NF-κB)、NLRP3、caspase-1、IL-1β及IL-18 mRNA和蛋白表达。与空白组比较,模型组骨骼肌组织中NF-κB、NLRP3、caspase-1、IL-1β、IL-18 mRNA与蛋白表达均有明显升高;与模型组比较,黄连温胆汤可有效调节IGT大鼠肥胖状态,减轻体质量,纠正餐后2 h血糖(2-hour plasma glucose,2hPG)、胰岛素抵抗指数(insulin resistance index,IRI)及胰岛素敏感指数(insulin sensitivity index,ISI)异常水平,降低骨骼肌组织中NF-κB、NLRP3、caspase-1、IL-1β、IL-18 mRNA与蛋白表达。以上结果结合前期研究表明,IGT的发生发展与炎症反应及骨骼肌细胞焦亡经典途径NLRP3/caspase-1/IL-1β、IL-18轴密切相关;推测黄连温胆汤改善胰岛素抵抗(insulin resistance,IR)的机制可能是通过NLRP3炎症小体通路以调节胰岛素受体信号通路异常,改善IR,从而逆转IGT病程。

This study investigated the mechanism of improving impaired glucose tolerance(IGT)of rats by Huanglian Wendan Decoction from the perspective of the skeletal muscle Nod-like receptor protein 3(NLRP3)/cysteinyl aspartate specific proteinase-1(caspase-1)/interleukin-1β(IL-1β),interleukin-18(IL-18)pathway.Healthy male SD rats were fed with the diet containing 45%fat for 20 weeks,accompanied by a high-temperature and high-humidity environment and an inactive lifestyle,for the establishment of the IGT rat model.The rats were divided into the blank control group,model control group,metformin hydrochloride group(positive drug group,0.05 g·kg^(-1)·d^(-1))and Huanglian Wendan Decoction group(7.8 g·kg^(-1)·d^(-1)).After continuous intragastric administration for 4 weeks,the obesity and glycemic indexes of all the rats were measured.The fasting serum insulin(FINS)level was determined by ELISA,with the insulin sensitivity index(ISI)and insulin resistance index(IRI)calculated.The mRNA and protein expression le-vels of nuclear factor kappaB(NF-κB),NLRP3,caspase-1,IL-1βand IL-18 in skeletal muscle tissue were detected by real-time polymerase chain reaction(PCR),Western blot and immunofluorescence.Compared with the blank control group,the model control group witnessed significantly increased mRNA and protein expression of NF-κB,NLRP3,caspase-1,IL-1βand IL-18.As revealed by the comparison with the model control group,Huanglian Wendan Decoction could effectively regulate the obesity status,reduce body weight,correct the abnormal levels of 2-hour plasma glucose(2 hPG),insulin resistance index(IRI),insulin sensitivity index(ISI),and lower the mRNA and protein expression of NF-κB,NLRP3,caspase-1,IL-1βand IL-18 in the skeletal muscle tissue of IGT rats.Combined with previous studies,the above results showed that the occurrence and development of IGT was closely related to inflammatory response and the classic pyroptosis pathway in skeletal muscle,such as NLRP3/caspase-1/IL-1β,IL-18.It is inferred that the mechanism of Huanglian Wendan Decoction was to alleviate insulin resistance(IR)and then reverse the course of IGT lies in the regulation of the abnormal insulin receptor signaling pathway based on the NLRP3 inflammasome pathway.