酒精性肝硬化患者脂肪因子及外泌体蛋白的表达

Adipokine and expression of exosome protein in patients with alcoholic liver cirrhosis

目的探讨酒精性肝硬化(alcoholic liver cirrhosis,ALC)患者脂肪因子及外泌体蛋白表达的特点。方法选取2014年1月1日至2016年12月31日于在西安交通大学第一附属医院门诊复查的ALC患者为病例组(66例),选择同期在本院进行健康体检的性别、年龄、体重指数(body mass index,BMI)均匹配的个体为对照组(60例)。应用全自动生化分析仪检测肝功能,主要包括总胆红素、白蛋白、胆固醇、甘油三酯、PT、丙氨酸氨基转移酶(alanine aminotransferase,ALT)和天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)。采用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测病例组和对照组血清脂联素和视黄醇结合蛋白4(retinol binding protein 4,RBP4)水平。采用流式细胞术检测外泌体Twist1蛋白和结缔组织生长因子(connective tissue growth factor,CTGF)的表达。脂肪细胞因子及外泌体蛋白与肝功能指标的相关性采用Pearson相关性分析。结果病例组脂联素水平显著高于对照组[(12.35±5.38)mg/L vs(9.79±4.33)mg/L],RBP4水平显著低于对照组[(6.20±1.97)mg/L vs(7.41±2.35)mg/L],Twist1蛋白显著低于对照组(15.4±0.39 vs 23.9±0.27),CTGF显著高于对照组(78.3±5.47 vs 43.2±1.59),差异均有统计学意义(P均<0.05)。Pearson相关性分析表明,所有研究对象脂联素水平与ALT、AST、总胆红素、PT呈正相关(r> 0.3,P <0.05);RBP4和外泌体Twist1蛋白与总胆红素和PT均呈负相关(r <-0.3,P <0.05);外泌体CTGF与ALT、AST、PT呈正相关(r> 0.3,P <0.05),而与总胆红素呈负相关(r=-0.492,P=0.019)。与对照组、Child-Pugh A级和Child-Pugh B级的患者相比,Chile-Pugh C级的患者脂联素水平显著升高[(19.22±7.55)mg/L vs(9.79±4.33)mg/L vs(8.32±4.47)mg/L vs(9.26±4.31)mg/L],而RBP4水平显著降低[(2.97±1.12)mg/L vs(6.67±1.93)mg/L vs(6.59±1.77)mg/L vs(7.41±2.35)mg/L],差异有统计学意义(P均<0.05)。对照组、Child-Pugh A级、Child-Pugh B级和Child-Pugh C级患者Twist1蛋白呈逐渐降低趋势(Slope=-3.995,P <0.0001),CTGF呈逐渐升高趋势(Slope=19.39,P <0.0001)。结论脂联素、RBP4、Twist1蛋白和CTGF与ALD的发生密切相关,可能是潜在的预防或延缓肝硬化发生的重要靶点。

Objective To investigate the characteristics of adipokine and expression of exosome protein in patients with alcoholic liver cirrhosis(ALC).Methods A total of 66 patients with ALC reviewed in outpatient clinic of the First Affiliated Hospital of Xi’an Jiaotong University from January 1st,2014 to December 31st,2016 were selected as case group.Those who underwent medical examination in our hospital was selected as control group(60 cases),whose gender,age and body mass index(BMI)were matched in the same period.Automatic biochemical analyzer was used to detect liver function,including total bilirubin,albumin,cholesterol,triglyceride,prothrombin time(PT),alanine aminotransferase(ALT)and aspartate aminotransferase(AST).Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of adipokine and retinol binding protein 4(RBP4)in peripheral blood.Flow cytometry was used to detect the expression of Twist1 protein and connective tissue growth factor 2(CTGF).Correlation of adipocyte factor,exocrine protein and liver function indexes were analyzed by Pearson correlation analysis.Results The adipokine level of patients in case group was significantly higher than that in control group[(12.35±5.38)mg/L vs(9.79±4.33)mg/L]and the RBP4 level was significantly lower than that in control group[(6.20±1.97)mg/L vs(7.41±2.35)mg/L],the differences were statistically significant(all P<0.05).Twist1 protein level of patients in case group was significantly lower than that in control group(15.4±0.39 vs 23.9±0.27)and the CTGF level was significantly higher than that in control group(78.3±5.47 vs 43.2±1.59),the differences were statistically significant(all P<0.05).Pearson correlation analysis showed that the level of adiponectin was positively correlated with ALT,AST,total bilirubin and PT(r>0.3,P<0.05),RBP4 and Twist1 protein were negatively correlated with total bilirubin and PT(r<-0.3,P<0.05),CTGF was positively correlated with ALT,AST and PT(r>0.3,P<0.05),and was negatively correlated with total bilirubin(r=-0.492,P=0.019).The level of adipokine in patients with Child-Pugh C grade was significantly higher[(19.22±7.55)mg/L vs(9.79±4.33)mg/L vs(8.32±4.47)mg/L vs(9.26±4.31)mg/L]and the level of RBP4 was significantly lower[(2.97±1.12)mg/L vs(6.67±1.93)mg/L vs(6.59±1.77)mg/L vs(7.41±2.35)mg/L]than those of control group,Child-Pugh A group and Child-Pugh B group,the differences were statistically significant(all P<0.05).The fluorescence intensity of Twist1 protein in control group,Child-Pugh A grade,Child-Pugh B grade and Child-Pugh C grade decreased gradually(Slope=-3.995,P<0.0001),and the fluorescence intensity of CTGF increased gradually(Slope=19.39,P<0.0001).Conclusions Adipokine,RBP4,Twist1 protein and CTGF are closely related to the occurrence of ALC,which may be the potential treatment target for the prevention or delay in the occurrence of liver fibrosis.