芪参活血颗粒含药血清通过抑制过度自噬改善脓毒症心肌细胞损伤

Qishen Huoxue granules containing serum can improve the damage of septic cardiomyocytes by inhibiting excessive autophagy

目的探讨芪参活血颗粒含药血清对脓毒症大鼠心肌细胞(H9C2)过度自噬的抑制作用及其对脓毒症心肌细胞的保护作用。方法12只SPF级Wistar大鼠,按芪参活血颗粒低、中、高剂量[分别相当于生药12.7、25.4、50.8 g/(kg·d)]灌胃制备含药血清。将传代培养的大鼠胚胎心肌细胞(H9C2)分为五组:对照组以含10%胎牛血清的DMEM培养;脂多糖(LPS)组以含10%胎牛血清的DMEM+1μg/ml的LPS培养;LPS+芪参活血颗粒低、中、高剂量组分别以含10%低、中、高剂量含药血清的DMEM预干预4 h后加入1μg/ml的LPS。共同培养4 h后,应用实时荧光定量PCR(qRT-PCR)和Western blot法检测自噬标志物Beclin-1、ATG5和LC3B的mRNA及蛋白表达;共同培育8、12、24、48 h后,采用CCK-8法检测各组细胞在不同时间点的细胞活性。结果与对照组比较,LPS组自噬标志物Beclin-1、ATG5 mRNA及Beclin-1、ATG5、LC3B蛋白表达在LPS干预细胞早期(4 h)即升高,差异有统计学意义(P<0.05)。与LPS组比较,LPS+芪参活血颗粒各剂量组均可降低LPS导致的Beclin-1、ATG5、LC3B的mRNA升高及蛋白的过表达,差异有统计学意义(P<0.05)。CCK-8法检测细胞活性实验显示:LPS组及LPS+芪参活血颗粒低、中、高剂量组在12、24 h及48 h细胞活性明显下降,与对照组差异有统计学意义(P<0.05);而LPS+芪参活血颗粒中剂量组24、48 h的细胞活性较LPS组明显上升,差异有统计学意义(P<0.05)。结论不同浓度芪参活血颗粒含药血清对LPS诱导的心肌细胞自噬均有抑制作用,且中剂量芪参活血颗粒灌胃大鼠获得的含药血清可通过抑制自噬改善脓毒症心肌损伤。

Objective To investigate the inhibitory effect of Qishen Huoxue granule containing serum on excessive autophagy of cardiomyocytes(H9c2)in septic rats and its protective effect on septic cardiomyocytes.Methods Twelve SPF grade Wistar rats were gavaged with low,medium and high doses of Qishen Huoxue granule[equivalent to crude drugs 12.7,25.4 and 50.8 g/(kg·d)].The cultured rat embryonic cardiomyocytes(H9c2)were divided into five groups:the control group was cultured with DMEM containing 10%fetal bovine serum(FBS);lipopolysaccharide(LPS)group was treated with DMEM containing 10%FBS+1 pg/ml LPS;LPS+Qishen Huoxue granule low,medium and high dose groups were pre intervened with DMEM containing 10%low,medium and high dose intragastric drug containing serum for 4 h,and then added 1 p.g/ml LPS.After co-cultured for 4 h,the mRNA and protein expression of autophagy markers Beclin-l,ATG5 and LC3B were detected by real time fluorescence quantitative polymerase chain reaction(qRT-PCR)and Western blot;After 8,12,24 and48 hours of co culture,the cell activity of cells in each group at different time points was detected by cell counting method(CCK-8)method.Results The expression of autophagy markers Beclin-1,ATG5 mRNA and Beclin-1,ATG5 and LC3B protein in LPS group increased in the early stage(4 h),which was statistically significant compared with the control group(P<0.05).However,LPS+Qishen Huoxue granule groups could reduce the overexpression of Beclin-1,ATG5 and LC3B mRNA and protein caused by LPS(P<0.05).CCK-8 method showed that the cell activity of LPS group LPS+Qishen Huoxue granule low,medium and high dose groups decreased significantly at 12 h,24 h and 48 h,which was significantly different from that of the control group(P<0.05);The cell activity of LPS+Qishen Huoxue granule medium dose group at 24 and 48 hours was significantly higher than that of LPS group(P<0.05).Conclusions The drug-containing serum of Qishen Huoxue granules at different concentrations had inhibitory effects on LPS-induced autophagy of car-diomyocytes,and the drug-containing serum obtained by intragastric administration of Qishen Huoxue granules in rats can improve the myocardial injury caused by sepsis by inhibiting autophagy.