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目的观察转录共激活子p300和蛋白激酶C(PKC)β2在高糖及甲基乙二醛糖化终产物(AGEs)诱导人系膜细胞(HMCs)活性氧(ROS)产生和纤维连接蛋白(FN)蛋白表达中的作用及相互关系。方法将培养的HMCs分为以下几组:①正糖组(NG)、高糖组(HG)、渗透压组(LG)、正糖+血清白蛋白组(BSA)、正糖+糖化终产物组(AGEs);②高糖+空载体组(HN)、高糖+PKCβ2转染组(PO)、高糖+PKCβ2抑制剂CGP53353组(PI)、糖化终产物+空载体组(AN)、糖化终产物+PKCβ2转染组(APO)、糖化终产物+PKCβ2抑制剂CGP53353组(API);③正糖+p300抑制剂组(NG+Gar)、高糖+p300抑制剂组(HG+Gar)、血清白蛋白+p300抑制剂组(BSA+Gar)、糖化终产物+p300抑制剂组(AGEs+Gar),以上各组细胞均培养2 d。荧光显微镜及荧光酶标仪检测细胞内ROS水平;Western blot法检测各组细胞p300、PKCβ2及FN蛋白表达。结果 HG组及AGEs组p300、PKCβ2蛋白表达及ROS水平增加,分别较NG组及BSA组增加了1.04倍、1.26倍、0.78倍和1.45倍、1.07倍、0.71倍(P<0.05)。同HG组及AGEs组相比,HG+Gar组及AGEs+Gar组ROS水平显著降低,分别为HG组及AGEs组的43%和39%(P<0.05)。PO组及APO组分别较HG组和AGEs组p300、PKCβ2蛋白表达进一步上调,分别为HG组及AGEs组的1.19倍、1.73倍和1.23倍、1.69倍(P<0.05);PI组和API组p300、PKCβ2蛋白表达显著下调(P<0.05);HN组和AN组p300、PKCβ2蛋白表达分别较HG组及AGEs组差异无统计学意义;HG+Gar组及AGEs+Gar组p300、FN蛋白表达显著下降,分别为HG组及AGEs组的31%、43%和37%、29%(P<0.05)。结论高糖及MGO-糖化终产物通过诱导HMCs转录共激活子p300激活参与系膜细胞氧化应激水平上调及FN蛋白表达。
Objective To investigate the effects of transcription co-activator p300 and protein kinase C (PKC) β2 on the production of reactive oxygen species (ROS) and fibronectin (FN) in human mesangial cells (HMCs) induced by high glucose and methylglyoxal glycosylation end products ) Protein expression and their relationship. Methods The cultured HMCs were divided into the following groups: ①Normal Glucose, HG, LG, BSA, (AGEs); ② high glucose + empty vector group (HN), high glucose + PKCβ2 transfected group (PO), high glucose + PKCβ2 inhibitor CGP53353 group Glycosylation end products + PKCβ2 transfection group (APO), glycation end products + PKCβ2 inhibitor CGP53353 group (API); ③Normal glucose + p300 inhibitor group (NG + Gar), high glucose + p300 inhibitor group ), Serum albumin + p300 inhibitor group (BSA + Gar), advanced glycation end products + p300 inhibitor group (AGEs + Gar). All the above groups were cultured for 2 days. Fluorescence microscopy and fluorescent microplate reader to detect intracellular ROS levels; Western blot method to detect p300, PKCβ2 and FN protein expression in each group. Results The levels of p300 and PKCβ2 protein and ROS in HG and AGEs groups increased by 1.04 times, 1.26 times, 0.78 times and 1.45 times, 1.07 times and 0.71 times respectively (P <0.05) compared with those in NG and BSA groups. Compared with HG group and AGEs group, ROS levels in HG + Gar group and AGEs + Gar group were significantly lower than that in HG group and AGEs group (43% vs 39%, respectively, P <0.05). Compared with HG group and AGEs group, the expression of p300 and PKCβ2 protein in PO group and APO group were increased by 1.19 times, 1.73 times, 1.23 times and 1.69 times (P <0.05), respectively, compared with HG group and AGEs group (P <0.05) p300 and PKCβ2 protein expression in HG group and AN group were significantly lower than those in HG group and AGEs group (P <0.05). There was no significant difference in p300 and PKCβ2 protein expression between HN group and AN group Decreased significantly to 31%, 43% and 37%, 29% (P <0.05) respectively in HG group and AGEs group. Conclusion High glucose and MGO-glycosylated end products are involved in the up-regulation of oxidative stress and FN protein expression in mesangial cells by inducing p300 activation of transcriptional co-activator of HMCs.