解毒凉血方含药血清调节TGFβ1/Smad信号通路对急性肝衰竭肝细胞凋亡的抑制作用

Inhibitory effect of Jiedu Liangxue prescription-medicated serum on apoptosis of hepatocytes in acute liver failure by regulating TGFβ1/Smad signaling pathway

目的:研究解毒凉血方含药血清对TNFα联合ActD诱导肝衰竭肝细胞凋亡的抑制作用及机制。方法:采用人正常肝细胞Chang liver及肿瘤细胞HepG2筛选解毒凉血方含药血清的有效比例。干预组采用有效比例的解毒凉血方含药血清预刺激24 h,然后采用TNFα联合ActD刺激Chang liver模拟急性肝衰竭肝细胞凋亡,流式细胞术检测各组细胞的凋亡率,免疫荧光实验检测凋亡肝细胞TBFβ1的表达及分布,免疫印迹法检测TGFβ1/Smad信号通路中TGFβ1、p-TβRⅡ、Smad7及p-Smad2/3的表达水平。结果:15%(体积比)解毒凉血方含药血清对TNFα联合ActD诱导的肝细胞凋亡具有抑制作用,15%解毒凉血方含药血清组细胞凋亡率显著低于模型组及空白血清组(P<0.01);与模型组及空白血清组比较,15%解毒凉血方含药血清预处理组胞质中TGFβ1、p-TβRⅡ以及Smad2/3磷酸化(p-Smad2/3)均低水平表达,而Smad7表达水平较高(均P<0.05)。结论:解毒凉血健脾方含药血清对TNFα联合ActD刺激诱导的急性肝衰竭肝细胞凋亡具有抑制作用,其机制可能与调节TGFβ1/Smad信号通路有关。

Objective:To study the inhibitory effect and mechanism of Jiedu Liangxue prescription-medicated serum on apoptosis of hepatocytes induced by TNFα&ActD in liver failure.Methods:Chang liver cells and tumor cells(HepG2)were used to screen the effective proportion of Jiedu Liangxue prescription-medicated serum.The effective ratio of Jiedu Liangxue prescription-medicated serum was used to stimulus Chang liver cells 24 hours beforehand,and then using the TNFα&ActD induce Chang liver cell apoptosis.The flow cytometry was used to detect apoptosis rate of liver cell each group.Immunofluorescence test was performed to detect the expression and distribution of TGFβ1 in apoptotic hepatocytes,and TGFβ1,p-TβR-Ⅱ、Smad7 and P-Smad2/3 in TGFβ1/Smad signaling pathways were tested by Western Blot.Results:The serum containing 15%volume of Jiedu Liangxue prescription could inhibit the apoptosis of hepatocytes induced by TNFα&ActD.The apoptosis rate of liver cell in 15%Jiedu Liangxue prescription-medicated serum group were significantly lower than those of model group and blank serum group(P<0.01);Comparing with the TNFα/ActD group and the blank serum group,the expression levels of TGFβ1,P-TβR-Ⅱand p-Smad2/3 in 15%Jiedu Liangxue prescription-medicated serum group were lower,and Smad7 expression level was higher(P<0.05).Conclusion:The Jiedu Liangxue prescription-medicated serum can inhibit the apoptosis of hepatocytes induced by TNFα&ActD-induced acute liver failure,and the mechanism may be related to the regulation of TGFβ1/Smad signaling pathway.