G9a抑制剂BIX-01294对小鼠肝缺血再灌注损伤的影响及其机制

The influence and mechanism of G9a inhibitor BIX-01294 on hepatic ischemia refusion injury in mice

目的探讨G9a抑制剂BIX-01294在小鼠肝脏缺血再灌注中的影响及潜在机制。方法将雄性C57BL/6J小鼠随机抽样法等分成空白组、缺血再灌注(IR)组、不同剂量BIX-01294预处理组(10、30、50 mg/kg),每组8只,连续5 d腹腔注射给药,空白组及IR组给予等量无菌生理盐水,药物干预组给予等量药物,建立肝部分(70%)热缺血再灌注模型(缺血1 h,再灌注6 h),实验结束收集小鼠静脉血及缺血肝组织进行检测。结果苏木精-伊红(HE)染色显示,IR组肝小叶结构紊乱,肝细胞大面积变性坏死;IR+BIX组肝细胞坏死面积减小,肝组织结构损害较轻。IR+BIX(50 mg/kg)组血清[谷丙转氨酶(ALT):(348.82±95.4)U/L,谷草转氨酶(AST):(220.7±43.38)U/L]较IR组[ALT:(1384.96±183.61)U/L,AST:(1111.39±167.08)U/L]显著下降(P<0.01),以50 mg/kg效果最佳。与IR组[丙二醛(MDA):(1.76±0.54)nmol/mg prot;超氧化歧化酶(SOD):(348.05±54.07)U/mg prot;谷胱甘肽(GSH):(0.95±0.09)μmol/g prot]比较,IR+BIX(50 mg/kg)组MDA[(1.21±0.22)nmol/mg prot]显著降低(P<0.05),而SOD[(420.59±56.09)U/mg prot]及GSH[(1.08±0.07)μmol/g prot]明显升高(P均<0.05),差异有统计学意义。在对相关蛋白相对表达水平量的检测中,IR组c-Caspase-3、B淋巴细胞瘤/白血病-2相关X蛋白(bax)、Beclin-1及LC3-Ⅱ蛋白条带信号增强,B淋巴细胞瘤/白血病-2(bcl-2)、P62减弱,提示肝缺血再灌注损伤促进凋亡和自噬;而IR+BIX组与IR组比较,cleaved-Caspase-3、bax、Beclin-1、及LC3-Ⅱ降低,bcl-2、p62升高,提示BIX-01294预处理缓解了肝脏凋亡和自噬。结论BIX-01294预处理通过降低肝细胞的氧化应激水平、减轻细胞凋亡和自噬减轻小鼠肝脏缺血再灌注损伤。

Objective To study the influence of G9a also known as Euchromatic histone-lysine N-methyltransferase 2(EHMT2)inhibitor BIX-01294 on hepatic ischemia refusion(IR)injury in mice and the mechanism.Methods Totally,40 male C57BL/6J mice were randomly divided into sham group,IR group,pretreatment group with different doses of BIX-01294(10,30,50 mg/kg,intraperitoneal injection for 5 days).The sham group and IR group were given equal amounts of sterile saline,drug intervention group was given equal amounts of drugs,and partial(70%)hepatic IR model(ischemia for 1 h,reperfusion for 6 h)was established.At the end of the experiment,venous blood and ischemic liver tissue of mice were collected.Results Hematoxylin and eosin(HE)staining showed that in IR group,the structure of hepatic lobule was disordered,and large area of hepatocytes degenerated and necrotized.In IR+BIX group,the area of hepatocyte necrosis decreased and the damage of liver tissue structure was mild.The serum alanine aminotransferase(ALT)[(348.82±95.4)U/L]and aspartate aminotransferase(AST)[(220.7±43.38)U/L]in IR+BIX(50 mg/kg)group were significantly lower than those in IR group[ALT:(1384.96±183.61)U/L;AST:(1111.39±167.08)U/L,P<0.01],and 50 mg/kg was the best.As compared with the IR group[malondialdehyde(MDA):(1.76±0.54)nmol/mg prot;superoxide dismutase(SOD):(348.05±54.07)U/mg prot;glutathione(GSH):(0.95±0.09)μmol/gp prot],the MDA[(1.21±0.22)nmol/mg prot]in the IR+BIX(50 mg/kg)group was significantly decreased(P<0.05),while the SOD[(420.59±56.09)U/mg prot]and GSH[(1.08±0.07)μmol/gp prot]were significantly increased(P<0.05).The protein expression level of the signal of c-Caspase-3,B cell lymphoma/leukemia-2 associated X protein(bax),Beclin-1 and LC3-Ⅱprotein bands in IR group was enhanced,while that of B cell lymphoma/leukemia-2(bcl-2)and p62 was decreased,indicating that hepatic ischemia-reperfusion injury promoted apoptosis and autophagy.As compared with IR group,Caspase-3,bax,Beclin-1 and LC3-Ⅱin IR+BIX group were decreased,while bcl-2 and p62 were increased,indicating that pretreatment with BIX-01294 relieved liver apoptosis and autophagy.Conclusion BIX-01294 pretreatment can alleviate hepatic ischemia-reperfusion injury in mice by by reducing oxidative stress of hepatocytes,apoptosis and autophagy.