p67^(phox)在肝纤维化中的作用及其机制  

The role of p67^(phox) in hepatic fibrosis and the underlying mechanism

作　　者：马立业 程欣欣 徐哲龙[1] MA Liye;CHENG Xinxin;XU Zhelong(Department of Pathophysiology,School of Basic Medical Sciences,Tianjin Medical University,Tianjin300070,China)

机构地区：[1]天津医科大学基础医学院病理生理学系,天津300070

出　　处：《天津医科大学学报》2024年第6期514-521,共8页Journal of Tianjin Medical University

基　　金：国家自然科学基金项目(81970255)。

摘　　要：目的:探讨NADPH氧化酶的胞浆亚基p67^(phox)在肝纤维化中的作用及其机制。方法:用四氯化碳(CCl_(4))腹腔注射的方法建立小鼠肝脏纤维化模型,随机将小鼠分为5组:野生型组(WT)、对照组(NCF2^(fl/fl)-Control)、模型组(NCF2^(fl/fl)-CCl_(4))、NCF_(2)基因敲除加模型组(NCF2^(LKO)-CCl_(4))和NCF2过表达加模型组(NCF2^(OE)-CCl_(4)),每组6只小鼠。分别用HE染色、Masson染色、天狼猩红染色观察肝脏的病理学改变;免疫组化法检测肝脏平滑肌肌动蛋白(α-SMA)表达水平;Western印迹检测肝脏组织中p67^(phox)、纤维化相关蛋白α-SMA和信号传导及转录激活因子(STAT)3、蛋白激酶B(AKT)、细胞外调节蛋白激酶(ERK)通路蛋白的表达。结果:与NCF2^(fl/fl)-Control组相比,注射CCl_(4)引起小鼠肝纤维化及损伤,p67^(phox)表达下降(t=2.96,P<0.05)。与NCF2^(fl/fl)-CCl_(4)组相比,NCF2^(LKO)-CCl_(4)组肝脏α-SMA表达增强(t=3.6,P<0.05)。此外,与NCF2^(fl/fl)-CCl_(4)组相比,NCF2^(LKO)-CCl_(4)组AKT通路相关蛋白(pPDK1、AKT、p-AKT^(473)、p-AKT^(308))表达上升(t=3.17、1.68、3.34、2.47,均P<0.05),STAT3通路相关蛋白(STAT3、p-STAT3^(705))表达升高(t=3.95、2.67,均P<0.05),ERK通路相关蛋白(p-c-Raf、p-ERK)表达增加(t=3.68、1.96,均P<0.05)。与NCF2^(fl/fl)-CCl_(4)组相比,NCF2^(OE)-CCl_(4)组小鼠肝脏病理损伤及纤维化程度减轻,p-PDK1、AKT、p-AKT^(473)及p-AKT^(308)表达下降(t=2.31、3.53、3.54、2.71,均P<0.05),STAT3及p-STAT3^(705)表达减少(t=3.47、2.5,均P<0.05),p-c-Raf和p-ERK蛋白表达下降(t=2.5、6.27,均P<0.05)。结论:p67^(phox)表达下调,通过激活STAT3、AKT及ERK通路来诱导肝纤维化的发生。Objective:To investigate the role of cytoplasmic subunit p67^(phox)of NOX in hepatic fibrosis and its underlying mechanism.Methods:The mouse hepatic fibrosis model was established by intraperitoneal injection of CCl_(4).Mice were grouped into five groups:WT group,NCF2^(fl/fl)-Control group,NCF2^(fl/fl)-CCl_(4)group,NCF2^(LKO)-CCl_(4)group and NCF2_(OE)-CCl_(4)group,with 6 mice in each group.HE,Masson and Sirius red staining were adopted to observe the pathological changes of liver tissue.Expression ofα-smooth muscle actin(α-SMA)was detected by immunohistochemistry.Expressions of p67^(phox)and fibrosis-related proteins includingα-SMA,signal transducer and activator of transcription(STAT)3,AKT and extracellular regulated protein kinases(ERK)were detected by Western blotting.Results:CCl_(4)injection induced liver fibrosis and injury,compared with NCF2^(fl/fl)-Control group,the expression of p67^(phox)was decreased(t=2.96,P<0.05).Compared with NCF2^(fl/fl)-CCl_(4)group,NCF2^(LKO)-CCl_(4)group had increased expression ofα-SMA(t=3.6,P<0.05).In addition,compared with NCF2^(fl/fl)-CCl_(4)group,NCF2^(LKO)-CCl_(4)group showed increased expression of the AKT signaling proteins(P-PDK1,AKT,p-AKT^(473),and p-AKT^(308))(t=3.17,1.68,3.34,2.47,all P<0.05),enhanced expression of the STAT3 signaling proteins(STAT3 and p-STAT3^(705))(t=3.95,2.67,both P<0.05),and upregulated expression of the ERK signaling proteins(p-c-Raf and p-ERK)(t=3.68,1.96,both P<0.05).Compared with NCF2^(fl/fl)-CCl_(4)group,NCF2_(OE)-CCl_(4)group showed less pathological damages and fibrosis.Moreover,NCF2_(OE)-CCl_(4)group had reduced expression of P-PDK1,AKT,p-AKT^(473) and p-AKT^(308)(t=2.31,3.53,3.54,2.7,all P<0.05),decreased expression of STAT3 and p-STAT3^(705)(t=3.47,2.5,both P<0.05),and reduced expression of p-c-Raf and p-ERK(t=2.5,6.27,both P<0.05).Conclusion:Down-regulation of p67^(phox)induces hepatic fibrosis by activating STAT3,AKT and ERK signaling pathways.

关 键 词：p67^(phox) 信号转导及转录激活因子3 蛋白激酶B 细胞外调节蛋白激酶 肝纤维化 

分 类 号：R363[医药卫生—病理学]