摘要
目的:探讨TGF-β1/Smad3信号轴的氧连糖基化修饰(O-连接的N-乙酰葡萄糖胺修饰)对体外培养的小鼠心脏成纤维细胞(MCFs)在缺氧/复氧(H/R)后活力和分化的影响及机制。方法:原代培养的MCFs缺氧6 h再恢复常氧24 h建立细胞H/R模型,通过感染O-连接的N-乙酰葡萄糖胺转移酶(OGT)重组腺病毒提高MCFs氧连糖基化水平,将MCFs随机分组为空载腺病毒预处理+常氧(Ad. Null+Ctrl)组、空载腺病毒预处理+H/R(Ad. Null+H/R)组、OGT腺病毒预处理+常氧(Ad. OGT+Ctrl)组和OGT腺病毒预处理+H/R(Ad. OGT+H/R)组。RT-qPCR检测结缔组织生长因子(CTGF)、α-平滑肌肌动蛋白(α-SMA)、白细胞介素1β(IL-1β)和IL-18 mRNA水平;Western blot检测p-Smad3、Smad3、OGT和炎症介质蛋白水平;免疫共沉淀实验检测Smad3修饰状态及蛋白间相互结合;CCK-8法检测细胞活力;免疫荧光共聚焦显示Smad3亚细胞定位。结果:H/R致使MCFs氧连糖基化水平下降,过表达OGT显著缓解了H/R诱导的细胞氧连糖基化水平降低(P<0.05)。提高氧连糖基化水平抑制H/R诱导的MCFs活力和向肌成纤维细胞分化,减少I型胶原合成和炎症介质IL-1β/IL-18表达(P<0.05);TGF-β1信号轴胞内关键分子Smad3氧连糖基化水平升高,并且Smad3磷酸化及核转位受阻(P<0.05)。结论:氧连糖基化修饰竞争性抑制Smad3 Ser423/425磷酸化并使Smad3滞留于胞质,负向调控TGF-β1/Smad3信号轴,抑制H/R对体外培养的小鼠心脏成纤维细胞功能变化的诱导。
AIM:To investigate the effect of O-linked N-acetylglucosamine(O-GlcNAc)modification(OGlcNAcylation)of TGF-β1/Smad3 signaling axis on viability and differentiation of cultured mouse cardiac fibroblasts(MCFs)after hypoxia/reoxygenation(H/R).METHODS:Primarily cultured MCFs were incubated under hypoxic condition for 6 h and then exposed to normoxia for another 24 h to establish H/R cell model. Increased global O-GlcNAcylation was achieved by infection with O-GlcNAc transferase(OGT)adenovirus. The cells were randomly divided into null adenovirus preconditioning with normoxia(Ad. Null+Ctrl)and H/R(Ad. Null+H/R)groups,and OGT adenovirus preconditioning with normoxia(Ad. OGT+Ctrl)and H/R(Ad. OGT+H/R)groups. The mRNA levels of connective tissue growth factor(CTGF),α-smooth muscle actin(α-SMA),interleukin-1β(IL-1β)and IL-18 were detected by RT-qPCR. The protein levels of p-Smad3,Smad3,OGT,IL-1β and IL-18 were detected by Western blot. The O-GlcNAcylation of Smad3 and interaction with OGT/Smad4 were detected by co-immunoprecipitation. The cell viability was measured by CCK-8 staining. Subcellular localization of Smad3 was revealed by confocal immunofluorescence.RESULTS:H/R caused a significant decrease in O-GlcNAcylation of MCFs,while overexpression of OGT notably alleviated the reduction of O-GlcNAcylation induced by H/R(P<0. 05). Increased O-GlcNAcylation inhibited the viability and differentiation of MCFs into myofibroblasts,and decreased the synthesis of collagen type I and the expression of IL-1β and IL-18 induced by H/R(P<0. 05). O-GlcNAcylation blocked the phosphorylation and nuclear accumulation of Smad3 which is the key component of TGF-β1 signaling axis.CONCLUSION:O-GlcNAcylation inhibits the phosphorylation and nuclear translocation of Smad3,thus inhibiting TGF-β1/Smad3 signaling axis,and attenuates the functional changes of MCFs induced by H/R in vitro.
作者
金慧
谢中杰
张丽娜
龚开政
张振刚
李如君
Jin Hui;Xie Zhong-jie;Zhang Li-na;Gong Kai-zheng;Zhang Zhen-gang;Li Ru-jun(Department of Cardiology,The Affiliated Hospital of Yangzhou University,Yangzhou 225001,China;Department of Cardiology,The First People's Hospital of Taizhou,Taizhou 318020,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2022年第2期222-229,共8页
Chinese Journal of Pathophysiology
基金
江苏省自然科学基金资助项目(No.BK20200937)
扬州市科技计划资助项目(No.YZ2019058)
扬州市绿扬金凤计划资助项目(No.YZLYJFJH2017YB118)。
