Palosuran改善四氯化碳肝硬化模型大鼠肝纤维化的机制研究

Mechanism of Palosuran in the Improvement of CCl_4-induced Liver Fibrosis in Rats

目的探究尾加压素Ⅱ(UⅡ)受体拮抗剂Palosuran对四氯化碳(CCl4)肝硬化模型大鼠肝纤维化的改善作用。方法 2015年1—12月,将32只SPF/VAF级雄性Wistar大鼠随机分为对照组、4周模型组、8周模型组、治疗组,各8只。4周模型组、8周模型组分别腹腔注射0.2 ml/100 g CCl4溶液(分别连续4周、8周)建造肝硬化模型,造模后分别处死;对照组腹腔注射等量0.9%氯化钠溶液(连续8周),结束注射后处死;治疗组腹腔注射等量CCl4溶液(连续4周)建造肝硬化模型,之后改为Palosuran灌胃(4周),第8周末处死。取各组肝组织进行天狼猩红染色,观察各组肝组织病理组织学变化、统计肝纤维化半定量计分;检测各组肝组织羟脯氨酸(Hyp)水平;采用RT-qPCR检测各组肝组织Ⅰ型胶原(ColⅠ)mRNA表达水平;采用ELISA检测各组血清ColⅠ水平;采用免疫组化法检测各组肝组织α平滑肌肌动蛋白(α-SMA)阳性细胞计数。结果对照组仅在肝内汇管区及静脉壁周围可见胶原沉积;4周模型组可见肝脏结构紊乱、纤维组织增生、假小叶形成;8周模型组可见肝小叶结构破坏,纤维组织增生明显,将肝小叶分隔成大小不等的肝细胞团,肝细胞索排列弥漫,中央静脉周围带大片肝细胞气球样肿胀,胞质疏松淡染,肝窦受压变窄;治疗组纤维间隔细小,增生的纤维组织明显减少。治疗组肝纤维化半定量计分[(8.6±2.5)分]低于8周模型组[(16.1±2.2)分](P<0.05)。治疗组肝组织Hyp水平[(135.6±20.9)ng/g]低于8周模型组[(332.4±2.6)ng/g](P<0.05);治疗组肝组织ColⅠmRNA表达水平(103.63±7.96)及血清ColⅠ水平(8.75±1.67)均低于8周模型组(160.00±17.85,16.13±2.03)(P<0.05)。治疗组肝组织α-SMA阳性细胞计数[(110.35±11.09)个]少于8周模型组[(210.57±38.09)个](P<0.05)。结论 Paolsuran可以改善CCl_4肝硬化模型大鼠的肝纤维化程度,其机制可能是减少肝脏α-SMA阳性细胞。

Objective To investigate the effects of urotensin Ⅱ receptor antagonist palosuran on the improvement of CCl4-induced liver fibrosis in rats.Methods This study was conducted between January and December 2015.Thirty-two SPF/VAF grade male Wistar rats were randomly divided into four groups,normal control group〔receiving intragastric administration of sodium chloride solution （0.9%） 0.2 ml/100 g for 8 weeks〕;4-week CCl4 cirrhotic model group（receiving intragastric administration of CCl4 solution 0.2 ml/100 g for 4 weeks）,8-week CCl4 cirrhotic model group （receiving intragastric administration of CCl4 solution 0.2 ml/100 g for 8 weeks） and palosuran-treated group （receiving intraperitoneal injection of CCl4 0.2 ml/100 g for 4 weeks,then intragastric administration of palosuran 300 mg·kg.-1·d.-1 for another 4 weeks） with 8 rats in each.After the intervention,the rats in all groups were sacrificed and the liver tissues were taken out.The pathological manifestations in the liver tissue specimens with sirius red staining were detected,and semiquantitative scores of hepatic fibrosis was recorded.Hydroxyproline（Hyp） content in the liver tissue specimens was determined.Collagen type Ⅰ （ColⅠ） mRNA levels in the liver tissue specimens were assessed by RT-qPCR.Serum levels of ColⅠ were assessed by ELISA.The expressions of α-SMA-positive cells in liver tissue specimens were determined by means of immunohistochemistry.Results The pathological manifestations found：the normal control group exhibited only a small amount of collagen expression in the surrounding area of hepatic portal vein and hepatic portal area;the 4-week cirrhotic model group presented disordered structure of liver,proliferation of fibrous tissue and formation of pseudolobuli;the 8-week cirrhotic model group displayed destroyed hepatic lobular structure,marked proliferation of fibrous tissues,liver cell clusters separating the hepatic lobules into different sizes,diffusely arranged hepatic cords,massive balloon-like liver cells appearing in the central vein area,loosened and slightly colored cell cytoplasm,and narrowed liver sinusoid due to compression;the palosuran-treated group showed tiny fiber interval and significantly reduced proliferation of fibrous tissue.Compared with the 8-week cirrhotic model group,the palosuran-treated group had much lower degree of liver fibrosis showed by the semiquantitative scores 〔（8.6±2.5）vs.（16.1±2.2）,P〈0.05〕,significantly lower Hyp content〔（135.6±20.9）ng/g vs.（332.4±2.6）ng/g,P〈0.05〕,obviously lower ColⅠ mRNA levels 〔（103.63±7.96） vs.（160.00±17.85）,P〈0.05 〕,substantially lower serum ColⅠ levels〔（8.75±1.67） vs.（16.13±2.03）,P〈0.05 〕,much less α-SMA-positive cells in liver〔（110.35±11.09）vs.（210.57±38.09）,P〈0.05〕.Conclusion Paolsuran could relieve the CCl4-induced liver fibrosis in rats.The mechanism may be associated with the reduction of expressions of α-SMA-positive cells in liver.