加味温胆汤抑制NLRP3介导的内皮细胞焦亡防治冠心病痰瘀互结证机制研究

Mechanism of Jiawei Wendan decoction inhibiting NLRP3-mediated endothelium pyroptosis to prevent and treat for coronary heart disease of syndrome of phlegm-blood stasis

目的基于NLRP3/Caspase-1通路,观察加味温胆汤对内皮细胞焦亡的调控作用,探讨加味温胆汤防治冠心病痰瘀互结证的作用机制。方法随机将20只雄性SD大鼠分为空白血清组和加味温胆汤血清组,空白血清组给予蒸馏水灌胃,加味温胆汤血清组给予加味温胆汤8.64 g/kg灌胃,1次/d,连续灌胃7 d。末次灌胃3 h后,腹主动脉穿刺取血,分离血清。取人冠状动脉内皮细胞,将其分为4组:空白组加入空白血清培养,模型组加入脂多糖(LPS)、空白血清、三磷酸腺苷(ATP)培养,MCC950组加入LPS、空白血清、MCC950、ATP培养,加味温胆汤组加入LPS、加味温胆汤血清、ATP培养。MTT法检测各组细胞活力,免疫组织化学法检测细胞中NLRP3、Caspase-1、白细胞介素-1β(IL-1β)阳性表达情况,RT-qPCR法检测细胞中NNLRP3、Caspase-1、IL-1βmRNA表达量。结果与空白组比较,模型组细胞活力显著降低(P<0.05),NLRP3、Caspase-1、IL-1β阳性表达积分和mRNA表达量均明显升高(P均<0.05);与模型组比较,MCC950组和加味温胆汤组细胞活力均显著增高(P均<0.05),NLRP3、Caspase-1、IL-1β阳性表达积分和mRNA表达量均明显降低(P均<0.05)。结论加味温胆汤可能通过调控NLRP3/Caspase-1通路,减轻炎症反应,提高细胞活力而抑制由LPS/ATP诱导的人冠状动脉内皮细胞焦亡。

Objective It is to observe the regulating effect of Jiawei Wendan decoction on endothelial pyroptosis based on NLRP3/Caspase-1 pathway,to explore the mechanism of this decoction in preventing and treating coronary heart disease of syndrome of phlegm-blood stasis syndrome.Methods Twenty male SD rats were randomly divided into blank serum group and Jiawei Wendan decoction serum group,the blank serum group was given distilled water by gavage,Jiawei Wendan decoction serum group was given Jiawei Wendan decoction 8.64 g/kg by gavage,both once per day,and continuously treated for 7 days.3 hours after the last administration,abdominal aorta puncture was used to take blood and separate serum.Human coronary artery endothelial cells were cultured and divided into 4 groups:the blank group cultured with blank serum,model group cultured with lipopolysaccharide(LPS)+blank serum+adenosine triphosphate(ATP),MCC950 group culctured with LPS+blank serum+MCC950+ATP,and Jiawei Wendan decoction group culctured with LPS+Jiawei Wendan decoction serum+ATP.The cell viability in each group was detected by MTT assay.The positive expression of NLRP3,Caspase-1 and interleukin-1β(IL-1β)was detected by immunocytochemistry,and the expression of NNLRP3,Caspase-1 and IL-1βmRNA were detected by RT-qPCR.Results Compared with the blank group,the cell viability of the model group was decreased significantly(P<0.05),the positive expression scores of NLRP3,Caspase-1 and IL-1βprotein were increased significantly(all P<0.05),and the transcription levels of of NLRP3,Caspase-1 and IL-1βmRNA were increased significantly(all P<0.05).Compared with the model group,the cell viability of MCC950 group and Jiawei Wendan decoction group was significantly increased(P<0.05),the expression of NLRP3,Caspase-1 and IL-1βprotein was significantly decreased(P<0.05),and the transcription levels of NLRP3,Caspase-1 and IL-1βmRNA were significantly decreased(all P<0.05).Conclusion Jiawei Wendan decoction can effectively inhibit the LPS-ATP-induced apoptosis of endothelial cells,which may be due to the regulation of NLRP3/Caspase-1 pathway,the reduction of cellular inflammatory response and the enhancement of cell viability.