GlcNAc经O-糖基化依赖性途径抑制大鼠阴茎海绵体平滑肌细胞中MAPKs通路  

Glc NAc inhibits MAPKs pathway in rat corpus cavernosum smooth muscle cells via O-glycosylation-dependent pathway

作　　者：刘博深 王轶[1] 郭林民 孙中义[1] LIU Boshen;WANG Yi;GUO Linmin;SUN Zhongyi(Department of Urology,Institute of Surgery Research,Third Affiliated Hospital,Army Medical University (Third Military Medical University),Chongqing,400042,China)

机构地区：[1]陆军军医大学(第三军医大学)第三附属医院(野战外科研究所)泌尿外科,重庆400042

出　　处：《第三军医大学学报》2019年第3期206-212,共7页Journal of Third Military Medical University

基　　金：国家国际科技合作专项项目(2014DFR30860);陆军军医大学科技成果转化基金项目(2016XZH16);陆军军医大学第三附属医院(野战外科研究所)1135人才项目基金(2016)~~

摘　　要：目的探讨β-N-乙酰氨基葡萄糖(β-N-acetylglucosamine,GlcNAc)对大鼠阴茎海绵体平滑肌细胞促分裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)通路的影响。方法分离健康成年大鼠阴茎组织,高糖培养基诱导阴茎海绵体平滑肌细胞(cavernous smooth muscle cells,CSMC)分离生长,免疫荧光、免疫组化鉴定CSMC,GlcNAc处理CSMC 12 h,分别于0、10 min、1、2、4、8、12 h收集细胞裂解液,O-(连接) N-乙酰葡糖胺转移酶(O-N-acetylglucosamine transferase,OGT)抑制剂Alloxan、O-(连接) N-乙酰葡糖胺水解酶(O-GlcNAcase,OGA)抑制剂Thiamet G及GlcNAc处理CSMC 72 h,Western blot检测蛋白总糖基化水平及MAPKs通路相关蛋白在12、72 h的磷酸化水平。结果成功分离培养大鼠CSMC,GlcNAc显著提升了CSMC细胞O-糖基化水平(P <0. 05)。处理细胞后12 h,GlcNAc显著抑制大鼠CSMC的ERK1/2、JNK1/2/3和p38的蛋白磷酸化水平(P <0. 05),处理细胞后72 h,GlcNAc显著抑制了大鼠CSMC的ERK1/2和p38蛋白磷酸化水平(P <0. 05),但不影响其总蛋白的表达水平(P>0. 05),且与Thiamet G的作用相同。另外,GlcNAc与Thiamet G能协同抑制ERK1/2、JNK1/2/3和p38的蛋白磷酸化水平(P <0. 05)。结论 GlcNAc经O-糖基化依赖性途径抑制阴茎海绵体平滑肌细胞中MAPKs通路。Objective To investigate the effect of β-N-acetylglucosamine( Glc NAc) on the mitogenactivated protein kinases( MAPKs) pathway in rat penile cavernosum smooth muscle cells. Methods After penile tissue was collected from healthy adult rats,cavernous smooth muscle cells( CSMC) were isolated and cultured under high glucose medium. Immunofluorescence( IF) assay and immunocytochemical( ICC) assay were used to identify CSMC. Then the cells were treated with Glc NAc for 12 h,the cell lysate was collected at0,10 min,and 1,2,4,8 and 12 h. Western blotting was employed to detect the total glycosylation level of protein and phosphorylation of MAPKs pathway-associated proteins after the cells were treated with O-Nacetylglucosamine transferase( OGT) inhibitor,Alloxan,O-GlcNAcase( OGA) inhibitor,Thiamet G,and Glc NAc. Results The rat CMSC were successfully isolated. Compared with the control cells,Glc NAc treatment significantly increased the O-glycosylation level of CSMC( P < 0. 05). At 12 h,Glc NAc significantly inhibited ERK1/2,JNK1/2/3 and p38( P < 0. 05),while at 72 h,Glc NAc significantly inhibited the phosphorylation of ERK1/2 and p38 protein in rat CSMC( P < 0. 05),but did not affect the levels of total proteins( P > 0. 05),which was the same effect of Thiamet G. In addition,Glc NAc and Thiamet G synergistically inhibited the phosphorylation levels of ERK1/2,JNK1/2/3 and p38( P < 0. 05). Conclusion Glc NAc inhibits MAPKs pathway in CSMC via O-glycosylation-dependent pathway.

关 键 词：海绵体平滑肌细胞 GLCNAC O-糖基化 MAPKS 

分 类 号：R33[医药卫生—人体生理学] R322.64[医药卫生—基础医学]