瘀血痹含药血清调控NLRP3介导心肌细胞焦亡的作用机制

Study on Mechanism of Yuxuebi Capsule(瘀血痹胶囊)-Containing Serum Regulating NLRP3-Mediated Pyroptosis on Myocardial Cell

目的探讨瘀血痹含药血清对脂多糖诱导的HL-1心肌细胞焦亡的保护作用机制及对NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)通路的影响。方法将40只SD大鼠随机分为4组:瘀血痹胶囊低、中、高剂量组分别给予0.1、0.2、0.4 g/kg灌胃,空白组给予等量蒸馏水灌胃。连续灌胃10 d后制备含药血清。将心肌细胞HL-1分为空白组,脂多糖(Lipopolysaccharide,LPS)处理组,LPS+瘀血痹含药血清低、中、高剂量组,LPS+MCC950组。收集上述各组细胞,CCK8检测细胞活力,检测各组细胞Caspase-1 activity表达情况,RT-PCR检测各组细胞NLRP3、消皮素D(Gasdermin-D,GSDMD)、白介素1β(interleukin 1β,IL-1β)、半胱氨酸蛋白酶-1(Caspase-1)基因表达及Western Blotting检测NLRP3、IL-1β、Caspase-1、cleaved-GSDMD蛋白表达。结果与空白组比较,LPS处理组心肌细胞活力下降,Caspase-1 activity表达显著升高(P<0.01),NLRP3、GSDMD、IL-1β、Caspase-1 mRNA表达升高(P<0.01),NLRP3、IL-1β、Caspase-1、cleaved-GSDMD蛋白表达升高(P<0.01);与LPS处理组相比,给予NLRP3抑制剂MCC950组和瘀血痹含药血清组均可改善上述指标变化。结论瘀血痹胶囊能有效抑制NLRP3炎症小体表达,减少心肌细胞焦亡改善心肌损伤,其机制可能与调控NLRP3通路相关。

Objective To explore the protective mechanism of Yuxuebi Capsule(瘀血痹胶囊)-containing serum on lipopolysaccharide-induced HL-1 cell and its impact on NOD-like receptor thermal protein domain associated protein 3(NLRP3)pathway.Methods Forty SD rats were randomly divided into 4 groups:the low,medium and high dose groups of Yuxuebi Capsule which were give the drug with the corresponding doses of 0.1 g/kg,0.2 g/kg and 0.4 g/kg by gavage respectively.The blank group was given the same amount of distilled water by gavage.The drug-containing serum was prepared after continuous intragastric administration for 10 days.Cardiomyocyte HL-1 was divided into blank group,lipopolysaccharide(LPS)treatment group,LPS+low,medium and high dose of Yuxuebi Capsule-containing serum groups and LPS+MCC950 group.The cells of the above groups were collected,the cell viability was detected by CCK8 and the expression of Caspase-1 activity was detected.The gene expressions of NLRP3,gasdermin D(GSDMD),interleukin-1β(IL-1β)and Caspase-1 were detected by Reverse transcription-polymerase chain reaction(RT-PCR)and Western Blotting was used to detect the protein expressions of NLRP3,IL-1β,Caspase-1 and cleaved GSDMD.Results Compared with those of the blank group,the viability of HL-1 cells was significantly reduced,and the levels of NLRP3,GSDMD,IL-1βand Caspase-1 were increased significantly in the LPS group(P<0.01).The expression of caspase1 activity was increased significantly.Compared with LPS group,the MCC950 group and Yuxuebi Capsule-containing serum groups can improve the above indicators.Conclusion Yuxuebi Capsule can effectively inhibit the NLRP3 expression,reduce the myocardial cell pyroptosis and improve myocardial injury.The mechanism may be related to NLRP3 pathway.