高迁移率族蛋白B1在小鼠肝再生中对细胞增殖的作用及机制

Involvement of high-mobility group box 1 in cellular proliferation during liver regeneration in mice

目的:研究高迁移率族蛋白B1(HMGB1)在小鼠部分肝切除术(PH)后的再生过程中对细胞增殖的作用及机制。方法:将21只雄性BALB/c小鼠,随机分为假手术组和6个PH组,每组3只。假手术组小鼠只打开腹腔暴露肝脏并不切除肝脏;PH组小鼠进行70%肝切除术,并分别于术后第6小时以及第1、2、3、5、7天处死。另外采用丙酮酸乙酯(EP)作为HMGB1的特异性抑制剂进行干预实验,9只雄性BALB/c小鼠,随机分为假手术组、PH组和EP干预组,每组3只,EP干预组小鼠在70%肝切除术后按40mg/(kg·d)给予EP腹腔注射,术后第2天处死。取各组小鼠肝组织提取总蛋白及核浆蛋白,采用Western blot法分别检测提取的不同蛋白样品中HMGB1、TLR4、RAGE、NF-κB p65、Cyclin D1、PCNA的蛋白表达水平。结果:小鼠于进行70%PH后的1周内,肝组织内胞核中HMGB1的蛋白表达水平,在术后第2天上升至顶峰(P<0.05),之后逐渐回落,细胞质中该蛋白表达水平的变化与其一致。干预实验中,相比较假手术组,术后第2天PH组中TLR4、RAGE、NF-κB p65、Cyclin D1、PCNA的蛋白表达水平也明显提高(P<0.05)。EP干预后,虽然肝组织内胞核蛋白中的HMGB1以及总蛋白中TLR4与RAGE的表达水平与PH组比较没有明显改变,但胞浆蛋白中的HMGB1和下游NF-κB p65、Cyclin D1、PCNA的蛋白表达水平出现了显著下降(P<0.05)。结论:HMGB1在小鼠肝再生过程中对细胞的增殖发挥了调控作用,该蛋白可能作为调控肝再生的潜在干预靶点。

OBJECTIVE: To study the effect and regulatory mechanism of high-mobility group box 1(HMGB1) on cellular proliferation during liver regeneration after partial hepatectomy(PH) in mice.METHODS:Using the classic mouse model of 70% PH,21 male BALB/c mice were randomly divided into the sham operation and the six PH groups(n=3). Mice in the sham operation group, the abdominal cavities were open surgically to expose but without removal of the liver,while mice in the PH group received 70%PH and were killed at 6 h,and at 1,2,3,5 and 7 days after the operation,respectively. In addition,ethyl pyruvate(EP) was used as the specific inhibitor of HMGB1 for intervention. In this experiment,9 male BALB/c mice were randomly divided into sham operation, PH and ethyl pyruvate(EP) intervention(n=3)groups. Mice in EP intervention group were intraperitoneally injected with EP at 40 mg/(kg·d) after 70%hepatectomy, and were killed 2 days after operation. Total protein and nuclear or cytoplasmic proteins were extracted from liver tissues of mice from each group, and the protein expression levels of HMGB1, TLR4,RAGE,NF-κB p65,Cyclin D1 and PCNA were detected by Western blot. RESULTS:Within one week after mice had PH,the protein expression level of HMGB1 in cellular nuclei reached the peak on the 2 nd day after operation(P<0.05),then decreased gradually. Changes of protein expression levels in the cytoplasm was consistent with that trend. In addition,the protein expression levels of TLR4,RAGE,NF-κB p65,Cyclin D1 and PCNA in the PH group were also significantly higher than those in the sham operation group(P<0.05).After the intervention of EP,the expression levels of HMGB1 in the nuclear protein of liver tissues and theexpression levels of TLR4 and RAGE in the total protein did not change significantly. However,the expressionlevels of HMGB1 in the cytoplasmic protein and the protein expression levels of NF-κB p65,Cyclin D1 andPCNA decreased significantly(P<0.05). CONCLUSION: HMGB1 played a regulatory role in cellularproliferation during mice liver regeneration,and this protein may be used as a potential important interventiontarget for the regulation of liver regeneration.