小鼠肝组织中胰岛素样生长因子结合蛋白相关蛋白1在肝纤维化发生及发展中的作用

Effect of insulin-like growth factor binding protein-related protein 1 in the formation and development of hepatic fibrosis

目的 探讨小鼠肝组织中胰岛素样生长因子结合蛋白相关蛋白1（IGFBPrP1）在肝纤维化发生及发展过程中的作用.方法 采用腹腔注射硫代乙酰胺制备小鼠肝纤维化模型,按时间将小鼠分为模型4、5、6周组（各10只）,并分别设立正常对照组（各6只）,采用Masson染色检测肝组织胶原沉积,免疫组织化学检测肝组织中IGFBPrP1、α-平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原蛋白（Collagen Ⅰ）、纤维连接蛋白（FN）、TGF-β1和Smad3蛋白表达和分布,同时以Western blot检测IGFBPrP1、α-SMA和Smad3蛋白表达.采用单因素方差分析、Pearson等级相关检验进行分析,以P〈0.05为差异有统计学意义.结果 免疫组织化学检测结果发现,模型组小鼠肝组织中IGFBPrP1由0.21±0.03上升到5.03±0.09,α-SMA由0.11±0.04上升到10.09±0.18,Collagen Ⅰ由0.22±0.01上升到11.01±±0.16,FN由0.31±0.09上升到19.81±1.62,TGF-β1由0.49±0.02上升到5.97±0.19,Smad3由0.22±0.03上升到2.03±0.07,这些检测因子在模型组的表达与正常对照组比较,随时间的增加而明显增强（F=783.141,998.200,886.715,935.242,931.241,697.118,P〈0.05）.在肝纤维化形成过程中,IGFBPrP1的表达与α-SMA、Collagen Ⅰ、FN、TGF-β1和Smad3的表达均呈正相关（r=0.906,0.927,0.988,0.947,0.977,P〈0.05）.Western blot检测结果发现,模型组小鼠肝组织的IGFBPrP1蛋白表达量由0.23±0.01上升到0.92±0.07,α-SMA蛋白表达量由0.36±0.02上升到1.39±0.03,FN蛋白表达量由0.03±0.00上升到0.12±0.02,Smad3蛋白表达量由0.09±0.01上升到0.56±0.04,模型组小鼠的蛋白表达量均较正常对照组明显升高（F=57.316,201.214,103.871,72.966,P〈0.05）.在肝纤维化形成过程中,IGFBPrP1的表达与α-SMA、FN和Smad3的表达均呈正相关（r=0.982,0.924,0.965,P〈0.05）.结论 IGFBPrP1随着肝纤维化程度的加重,其表达水平逐渐上调,IGFBPrP1的促肝纤维化作用可能与促进肝星状细胞激活、使细胞外基质的重要组成成分Collagen Ⅰ和FN的合成与分泌增加及影响TGF-β1/Smad3信号通路有关.

Objective To investigate the effect of insulin-like growth factor binding protein-related protein 1 （ IGFBPrP1 ） in the formation and development of hepatic fibrosis. Methods Hepatic fibrosis model of mice was made by intraperitoneal injecting with thioacetamide, then the mice were sacrificed four, five and six weeks later （10 mice were sacrificed at each time point, model groups）. Mice in the control groups were treated by normal saline （6 mice were sacrificed at each time point）. Collagen accumulation in liver tissues was detected by Masson stain. Distribution and dynamic expressions of IGFBPrP1, alpha-smooth muscle actin （ α-SMA ）,Collagen Ⅰ , fibronectin （ FN）, TGF-β1 and Smad3 in different groups were detected by immunohistochemistry.The expressions of IGFBPrP1, α-SMA and Smad3 were detected by Western blot. All data were analyzed using the analysis of variance （ANOVA）, Pearson rank correlation coefficient. Results The expressions of IGFBPrP1 in the liver tissues were increased from 0.21 ±0.03 to 5.03 ±0.09, α-SMA from 0. 11 ±0.04 to 10.09 ±0. 18,Collagen Ⅰ from 0.22 ±0.01 to 11.01 ±0. 16, FN from 0.31 ±0.09 to 19.81 ±1.62, TGF-β1 from 0.49 ±0.02 to 5.97 ± 0. 19, and Smad3 from 0.22 ± 0.03 to 2.03 ± 0.07. Compared with the control groups, the expressions of IGFBPrP1, α-SMA, Collagen Ⅰ , FN, TGF-β1 and Smad3 in the model groups were significantly increased as time passed by （ F = 783. 141,998. 200,886. 715,935. 242, 931. 241,697. 118, P 〈 0. 05 ）. During the formation of hepatic fibrosis, the expression of IGFBPrP1 was positively correlated with the expressions of α-SMA,Collagen Ⅰ , FN, TGF-β1 and Smad3 （ r = 0. 906, 0. 927, 0. 988, 0. 947, 0. 977, P 〈 0.05 ）. The results of Western blot showed that the protein expression of IGFBPrP1 was increased from 0. 23 ± 0.01 to 0.92 ± 0.07,α-SMA from 0.36 ± 0. 02 to 1.39 ± 0.03, FN from 0.03 ± 0.00 to 0.12 ± 0.02, and Smad3 from 0.09 ± 0. 01 to 0.56 ±0.04. The protein expressions of IGFBPrP1, α-SMA, FN and Smad3 were significantly increased in the model groups when compared with the control groups （F =57. 316, 201. 214, 103. 871, 72. 966, P 〈0.05）.During the formation of hepatic fibrosis. IGFBPrP1 was positively correlated with the expressions of α-SMA, FN and Smad3 （r = 0. 982, 0. 924, 0. 965, P 〈 0.05 ）. Conclusions The expression of IGFBPrP1 increases as the aggravation of the fibrosis. IGFBPrP1 promotes the formation and development of hepatic fibrosis by activating hepatic stellate cells, accelerating the synthesis and secretion of Collagen Ⅰ and FN which are the principal components of extracellular matrix, and affecting the TGF-β1/Smad3 pathway.