摘要
目的探讨疏肝健脾活血方防治肝纤维化的可能作用机制。方法48只C57BL/6雄性小鼠随机分为正常组、模型组、疏肝健脾活血方组、CUGBP1敲减组,每组12只。CUGBP1敲减组一次性尾静脉注射CUGBP1敲减腺相关病毒,其余各组尾静脉注射对照腺相关病毒,每只小鼠注射量为100μl,病毒滴度不少于1×10^(12)vg/ml。4周后,除正常组外,其余各组小鼠腹腔注射20%四氯化碳(CCl_(4))2.5 ml/kg,每3天注射1次,连续4周,诱导肝纤维化模型;同时疏肝健脾活血方组小鼠予33.3 g/(kg·d)疏肝健脾活血方药液灌胃,正常组、模型组及CUGBP1敲减组予10 ml/kg蒸馏水灌胃,每天1次,连续4周。末次给药12 h后取材,采用HE染色、Masson染色、天狼星红染色进行肝脏病理学观察;检测血清中丙氨酸氨基转移酶(ALT)、门冬氨酸氨基转移酶(AST)水平及肝组织中羟脯氨酸(HYP)含量;检测肝组织中Ⅰ型胶原(CollagenⅠ)、α平滑肌激动蛋白(α-SMA)、磷酸化Smad2(p-Smad2)、磷酸化Smad3(p-Smad3)、Smad7、CUG结合蛋白1(CUGBP1)蛋白表达,肝组织中转化生长因子β1(TGF-β1)、CollagenⅠ、α-SMA、Smad7、CUGBP1 mRNA表达。结果与正常组比较,模型组小鼠肝组织病理显示肝细胞排列紊乱,有大量炎性细胞浸润与大量胶原纤维沉积,并且形成明显的纤维间隔;血清中ALT、AST水平及肝组织中HYP含量上升,肝组织中CollagenⅠ、CUGBP1、p-Smad2、p-Smad3、α-SMA蛋白表达升高,TGF-β1、CollagenⅠ、CUGBP1、α-SMA mRNA表达升高,Smad7蛋白及mRNA表达下降(P<0.05或P<0.01)。与模型组比较,疏肝健脾活血方组和CUGBP1敲减组小鼠肝组织病理显示有少量炎性细胞浸润,纤维组织增生不明显,纤维间隔稀疏;血清中ALT、AST水平及肝组织中HYP含量下降,上述蛋白及mRNA水平均有所改善(P<0.05或P<0.01)。与疏肝健脾活血方组比较,除Smad7 mRNA表达升高外,CUGBP1敲减组其余各指标差异均无统计学意义(P>0.05)。结论疏肝健脾活血方可能通过抑制肝组织CUGBP1表达,从而阻断TGF-β1/Smads信号后续转导,进而抑制肝纤维化进展。
Objective To explore the possible mechanism of Shugan Jianpi Huoxue Fprmula(疏肝健脾活血方,SJHF) in preventing and treating hepatic fibrosis.Methods Forty-eight C57 BL/6 male mice were randomly divided into normal group,model group,SJHF group and CUG binding protein 1(CUGBP1) knockout group,with 12 mice in each group.CUGBP1 knockout group was injected with CUGBP1 knockout adeno-associated virus(AAV9-shRNACUGBP1),while the other three groups were injected with control adeno-associated virus(AAV9-control) through tail vein in volume of 100μl with viral titers greater than 1×10^(12) vg/ml.Four weeks later,except for the normal group,mice were injected with 2.5 ml/kg of 20% carbon tetrachloride(CCl_(4)) intraperitoneally,once every three days for four weeks to induce hepatic fibrosis model;simultaneously,the SJHF group was given 33.3 g/(kg·d) of SJHF liquid by gavage,while the other three groups were given 100 ml/kg of distilled water by gavage,once a day for four weeks.Materials were collected 12 hours after the last medication,and the pathological morphology of the liver tissue was observed through HE staining,Masson staining and Sirius red staining.The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST) in serum,and the hydroxyproline(HYP) content in the liver tissue were detected.The protein expression of type I collagen(Collagen I),α-smooth muscle actin(α-SMA),phosphorylated Smad2(p-Smad2),phosphorylated Smad3(p-Smad3),Smad7,CUG binding protein 1(CUGBP1),as well as the mRNA expression of transforming growth factor β1(TGF-β1),Collagen I,α-SMA,Smad7,and CUGBP1 in the liver tissue were detected.Results Compared to those in the normal group,the pathology of the liver tissue in the model group was characterized by disordered arrangement of hepatocytes,a large amount of infiltrated inflammatory cells and deposited collagen fibers,and obvious fibrous septa;the levels of ALT and AST in serum and the content of HYP in liver tissue increased;the Collagen I,CUGBP1,p-Smad2,p-Smad3,and α-SMA protein expressions,as well as TGF-β1,Collagen I,CUGBP1,and α-SMA mRNA expressions increased,while Smad7 protein and mRNA expressions decreased(P<0.05 or P<0.01).Compared to those in the model group,he liver pathology in the SJHF group and the CUGBP1 knockout group showed a small amount of inflammatory cell infiltration,no obvious proliferation of fibrous tissue,and sparse fibrous septa;the levels of ALT and AST in serum,as well as the content of HYP in liver tissue decreased significantly,while all measured protein and mRNA expressions were improved(P<0.05 or P<0.01).Compared to the SJHF group,the CUGBP1 knockout group had higher Smad7 mRNA expression,but no significant differences in other outcomes were found between the groups(P>0.05).Conclusion SJHF may inhibit the progression of hepatic fibrosis by inhibiting the expression of CUGBP1 in the liver tissue and blocking the subsequent transduction of TGF-β1/Smads signal.
作者
揭阳
黄棪
王志丽
叶林茂
张俊杰
JIE Yang;HUANG Yan;WANG Zhili;YE Linmao;ZHANG Junjie(Zhejiang Chinese Medical University,Hangzhou,310053)
出处
《中医杂志》
CSCD
北大核心
2022年第13期1276-1282,共7页
Journal of Traditional Chinese Medicine
基金
国家自然科学基金(81973736)。
作者简介
通讯作者:张俊杰,zhangjunjie70@aliyun.com,(0571)86633105。
