高糖条件下氧化应激-AMPK-Cx43-NLRP3途径调节大鼠胃平滑肌细胞外基质重构的机制研究  

Mechanism of oxidative stress-AMPK-Cx43-NLRP3 pathway regulating extracellular matrix remodeling of rat gastric smooth muscle cells under high glucose condition

作　　者：张高源 宋佰慧[2] 包伊特格乐 张默函[1] ZHANG Gaoyuan;SONG Baihui;BAO Yitegele;ZHANG Mohan(Department of Histology&Embryology,College of Medicine Yanbian University,Yanji 133000,China;Department of Changchun Medical College of Basic Medicine,Changchun 130031,China)

机构地区：[1]延边大学医学院组织胚胎学教研室,吉林延吉133000 [2]长春医学高等专科学校,吉林长春130031

出　　处：《中国病理生理杂志》2024年第1期68-73,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.82060154);吉林省自然科学基金项目(No.YDZJ202201ZYTS147);吉林省教育厅科学技术研究项目(No.JJKH20221351KJ)。

摘　　要：目的:探讨高糖条件下通过氧化应激-AMP活化蛋白激酶(AMPK)-缝隙连接蛋白43(Cx43)-核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)通路对胃平滑肌细胞外基质重构的调节作用。方法:将体外培养的大鼠原代平滑肌细胞分为正常糖组、高糖组、高糖+NLRP3抑制剂MCC950(15 nmol/L)组、高糖+Cx43半通道阻滞剂GAP19(100μmol/L)组、高糖+AMPK抑制剂Compound C(CC;10μmol/L)组和高糖+抗氧化剂α-硫辛酸(α-LA;100μmol/L)组,均培养48 h后检测。Western blot检测细胞中caspase-1、基质金属蛋白酶2(MMP-2)、NLRP3、Cx43、转化生长因子β1(TGF-β1)、TGF-β3、金属蛋白酶组织抑制物1(TIMP-1)、嘌呤能P2X7受体(P2X7R)和磷酸化AMPK(pAMPK)蛋白水平;ELISA检测细胞培养液中腺苷三磷酸、白细胞介素1β、I型胶原(Col I)和ColⅢ含量。结果:与高糖组相比,高糖+MCC950组MMP-2和TGF-β3表达水平显著降低(P<0.01),TGF-β1和TIMP-1表达水平显著升高(P<0.01);高糖+GAP19组NLRP3和caspase-1表达水平显著降低(P<0.01),而P2X7R表达无显著差异;高糖+CC组NLRP3、caspase-1、P2X7R、总Cx43和胞膜Cx43蛋白水平,以及胞膜Cx43/胞浆Cx43比值均显著降低(P<0.01);高糖+α-LA组p-AMPK、caspase-1、NLRP3、P2X7R、总Cx43和胞膜Cx43蛋白水平,以及胞膜Cx43/胞浆Cx43比值均显著下降(P<0.05)。结论:高糖通过氧化应激-AMPK-Cx43-NLRP3通路调节Col I和ColⅢ含量,进而参与了胃平滑肌细胞外基质的重构。AIM:To investigate the regulatory effect of oxidative stress-AMP-activated protein kinase(AMPK)-connexin 43(Cx43)-nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)pathway on extracellular matrix(ECM)remodeling of gastric smooth muscle cells under high glucose(HG)condition.METHODS:Primary rat smooth muscle cells cultured in vitro were divided into normal glucose group,HG group,HG+NLRP3 inhibitor MCC950(15 nmol/L)group,HG+Cx43 hemichannel blocker GAP19(100μmol/L)group,HG+AMPK inhibitor Compound C(CC;10μmol/L)group,and high glucose+antioxidantα-lipoic acid(α-LA;100μmol/L)group,which were all cultured for 48 h for detection.The protein levels of caspase-1,matrix metalloproteinase-2(MMP-2),NLRP3,Cx43,transforming growth factor-β1(TGF-β1),TGF-β3,tissue inhibitor of metalloproteinase-1(TIMP-1),purinergic P2X7 receptor(P2X7R)and phosphorylated AMPK(p-AMPK)in the cells were detected by Western blot.The levels of adenosine triphosphate,interleukin-1β,collagen type I(Col I)and collagen typeⅢ(ColⅢ)in the cell culture medium were determined by ELISA.RESULTS:Compared with HG group,the levels of MMP-2 and TGF-β3 in HG+MCC950 group were decreased(P<0.01),while the levels of TGF-β1 and TIMP-1 were increased(P<0.01).The levels of NLRP3 and caspase-1 in HG+GAP19 group were decreased(P<0.01),while the expression of P2X7R was not changed.The levels of NLRP3,caspase-1,P2X7R,total Cx43 and membrane Cx43,and the ratio of membrane Cx43 to cytoplasmic Cx43 were decreased in HG+CC group(P<0.01).The levels of p-AMPK,caspase-1,NLRP3,P2X7R,total Cx43 and membrane Cx43,and the ratio of membrane Cx43 to cytoplasmic Cx43 were decreased in HG+α-LA group(P<0.05).CONCLUSION:High glucose regulates the content of Col I and ColⅢthrough the oxidative stress-AMPK-Cx43-NLRP3 pathway,thereby participating in the extracellular matrix remodeling of gastric smooth muscle cells.

关 键 词：糖尿病胃轻瘫 胃平滑肌 细胞外基质 氧化应激-AMPK-Cx43-NLRP3信号通路 

分 类 号：R573[医药卫生—消化系统] R587.2[医药卫生—内科学] R363.2[医药卫生—临床医学]