大鼠肝纤维化病理过程中Smads锚着蛋白表达变化

Dynamic characteristics of SARA during liver fibrogenesis in rats

目的观察肝纤维化病理过程中肝脏Smads锚着蛋白(SARA)的表达变化及其与肝纤维化的关系。方法大鼠每kg体重10μl二甲基亚硝胺腹腔注射,1次/d,每周连续3 d,共4周,复制大鼠肝纤维化模型。模型大鼠分别设首次造模后1、3 d、1,2、3、4周末,与造模停止后1、2、4周,共9个时间段为观察组。每组5～8只,另设正常大鼠10只为对照组。天狼猩红染色观察肝组织胶原沉积,盐酸水解法测定肝组织羟脯氨酸(HYP)含量,免疫组织化学染色观察肝组织SARA蛋白表达,Western blot法分析肝组织转化生长因子(TGF)β1、α-平滑肌肌动蛋白(α-SMA)和SARA蛋白的表达,并进行以上指标的相关性分析。结果随二甲基亚硝胺染毒持续,模型大鼠肝脏胶原增生(Hyp含量)与沉积增加,4周末时达到高峰,可见宽大纤维间隔与假小叶；而后随染毒停止,肝脏胶原沉积与纤维间隔有所减轻。模型组4、5、6、8周肝组织Hyp平均含量(μg/g)分别为193．0±39．2、188．5±39．9．174．4±21．2、163．6±31．5,对照组分别为125．6±19．5,t值在3．43～4．9,P值均＜0．01。免疫组织化学染色发现,SARA主要表达于正常与纤维化肝脏的肝窦周围间质细胞,随肝纤维化发展SARA阳性染色细胞数量减少,随染毒停止与肝纤维化恢复,SARA渐恢复至正常组水平。Western blot发现,随肝纤维化形成,模型大鼠肝组织TGFβ1、α-SMA蛋白表达逐渐增加,而SARA蛋白表达逐渐减少；肝纤维化恢复过程中,SARA渐恢复接近正常水平,TGFβ1与α-SMA蛋白表达有所下降。在肝纤维化形成与恢复过程中,SARA蛋白与Hyp含量、TGFβ1与α-SMA表达均呈明显负相关。结论SARA蛋白主要表达于肝脏间质细胞；随大鼠肝纤维化发展,SARA表达减少,SARA蛋白与肝纤维化形成呈负相关关系。

Objective To investigate the dynamic characteristics of Smad anchor for receptor activation （SARA） expression during liver fibrogenesis in rats and the relationship between SARA and liver fibrosis. Methods Liver fibrosis was induced in 74 rats by intraperitoneal injection of dimethylnitrosamine （DMN） with a dosage of 10 μl/kg body weight, once a day, 3 days per week for 4 weeks. The model rats were randomly divided into 9 groups for studying the changes： 1 d, 3 ds, 1 week, 2 weeks, 3 weeks and 4 weeks after starting the ip injections （intoxicating phase）, and 1 week, 2 weeks and 4 weeks after stopping the injections （5^th w, 7^th w and 8^th w, recovery phase）. Each group included 5-8 rats. In addition, 10 non-treated rats served as normal controls. The rat liver tissues were examined. Collagen deposition was stained with Sirius red, and hydroxyproline （Hyp） was measured with Jamall＇s method. SARA spatial expression in the livers was detected by immunohistochemistry, and the expressions of TGF β1, α -SMA and SARA protein were detected by Western blot. The relationships of SARA with Hyp, TGF β1 and α-SMA were analyzed. Results During the intoxicating phase, the rat hepatic collagen production （Hyp content） and deposition increased as DMN intoxication continued, and there was marked fibrous septum and pseudo-lobule formation at the end of the 4^th w. During the recovery phase, the rat hepatic collagen deposition and fibrous septum formation were lessened, but the Hyp content in the livers of the model rats at the end of 4^th w, 5^th w, 6^th W and 8^th w was still higher than that of the controls （193.04± 39.15, 188.49 ±39.92, 174.39±21.22, 163.59± 31.47 vs 125.64± 19.51; t from 3.43 to 4.9, P 〈 0.01）. SARA was mainly stained positively in interstitial cells surrounding the hepatic sinusoids in both normal and fibrotic livers, and the number of the positive stained cells decreased as liver fibrosis developed, and gradually returned to normal after stopping the intoxication. The expressions of TGF 151 and α-SMA were gradually increased, as shown with Western blot, but SARA decreased as liver fibrosis developed. The expressions of TGF 151 and α-SMA were slightly decreased, SARA expression recovered to the normal level after stopping the DMN intoxication. During liver fibrosis developing and recovery phases, SARA was significantly negatively correlated with TGF 151 and α-SMA expressions and Hyp contents. Conclusions SARA was mainly expressed in the liver interstitial cells, and it was negatively correlated with liver fibrosis formation.