全反式维甲酸对糖尿病肾病模型大鼠转化生长因子β1基因甲基化水平影响的实验研究

Effects of all-trans retinoic acid on methylation level of TGF-β1 gene in diabetic nephropathy rat models

目的:探讨全反式维甲酸(ATRA)对糖尿病肾病(DN)模型转化生长因子β1(TGF-β1)基因甲基化水平的影响。方法:将30只成年雄性SD大鼠随机分为对照组、DN组、DN+ATRA组,每组10只。采用高糖高脂饮食加腹腔注射小剂量链脲佐菌素建立DN大鼠模型,对照组和DN组给予等体积0.9%氯化钠溶液灌胃,DN+ATRA组给予5 mg/kg ATRA灌胃,连续干预4周。观察各组大鼠24 h尿蛋白和血液生化指标,肾脏组织行HE染色和PAS染色,选择MS-PCR检测各组大鼠肾脏中TGF-β1基因的DNA甲基化水平,选择逆转录聚合酶链式反应(RT-PCR)、Western blot法及免疫组织化学检测肾脏组织TGF-β1 mRNA及蛋白表达。结果:与对照组比较,DN组大鼠空腹血糖(FBG)、血肌酐(Scr)、尿素氮(BUN)、24 h尿蛋白升高(均P<0.05)。与DN组比较,DN+ATRA组大鼠FBG、BUN、Scr、24 h尿蛋白降低,但高于对照组(均P<0.05)。HE染色和PAS染色DN组肾小球形状不规则,系膜基质增多着玫瑰红色,部分肾小管有萎缩且存在管腔扩张,肾小管周围有较多炎症细胞浸润和组织增生,DN+ATRA组大鼠肾脏组织病变情况明显减轻。DN组大鼠肾脏组织TGF-β1基因的DNA甲基化水平相较于对照组显著降低,DN+ATRA组大鼠肾脏组织TGF-β1基因的DNA甲基化水平相较于DN组显著升高(P<0.05)。对照组大鼠肾脏组织TGF-β1mRNA及蛋白水平显著低于DN组,DN+ATRA组大鼠肾脏组织TGF-β1mRNA和蛋白水平显著高于对照组,低于DN组(均P<0.05)。结论:ATRA能通过升高TGF-β1基因的DNA甲基化水平,抑制DN大鼠肾脏组织TGF-β1mRNA和蛋白表达,改善DN大鼠肾脏组织的纤维化程度。

Objective:To investigate the effects of all-trans retinoic acid(ATRA)on the methylation level of transforming growth factorβ1(TGF-β1)gene in diabetic nephropathy(DN)model.Methods:Thirty adult male SD rats were randomly divided into control group,DN group,DN+ATRA group,with 10 rats in each group.A rat model of DN was established by high-sugar and high-fat diet plus intraperitoneal injection of low-dose streptozotocin.The normal group and DN group were given gavage with the same volume of 0.9%sodium chloride solution,and 5 mg/kg ATRA was given for gastric gavage in the DN+ATRA group for 4 weeks.The 24-hour urine protein and blood biochemical indexes of rats in each group were observed.HE staining and PAS staining were performed for kidney tissue.The methylation level of TGF-β1 gene in the kidneys of each group was detected by MS-PCR.RT-PCR,Western blot and immunohistochemistry were used to detect the expressions of TGF-β1 mRNA and protein in renal tissue.Results:Compared with control group,the levels of FBG,Scr,BUN and 24-hour urine protein were increased in DN group(all P<0.05).Compared with DN group,the levels of FBG,BUN,Scr and 24-hour urine protein were decreased in DN+ATRA group,but higher than those in control group(all P<0.05).HE staining and PAS staining showed that the glomerular shape was irregular,the mesangial matrix was increased and the rose-red color appeared,some renal tubules were atrophic and luminal dilatation existed,and there were more inflammatory cell infiltration and tissue proliferation around the renal tubules in DN group;the pathological changes of renal tissue in DN+ATRA group were significantly alleviated.The DNA methylation level of TGF-β1 gene in renal tissue of rats in DN group was significantly lower than that in the control group,and the DNA methylation level of TGF-β1 gene in renal tissue of rats in DN+ATRA group was significantly higher than that in DN group(all P<0.05).The l evels of TGF-β1 mRNA and protein in control group were significantly lower than those in DN group,and those in the DN+ATRA group were significantly higher than those in control group and lower than those in DN group(all P<0.05).Conclusion:ATRA can inhibit the expressions of TGF-β1 mRNA and protein in the kidney of DN rats by increasing the level of DNA methylation of TGF-β1 gene,and improve the degree of fibrosis in the kidney of DN rats.