摘要
目的 基于miR-29b/转化生长因子β(TGF-β)/Smad信号通路探讨复方血栓通对糖尿病视网病变(DR)大鼠的的作用机制.方法 100只雄性SD大鼠,其中80只建立糖尿病模型,20只作为正常组.通过腹腔注射1％链脲佐菌素(STZ)成功建立糖尿病DR大鼠模型72只,按照随机数字表法将其分为模型组、二甲双胍组、复方血栓通1组、复方血栓通2组,每组各18只.二甲双胍组通过灌胃二甲双胍184 mg/kg,复方血栓通1组通过灌胃复方血栓通片100 mg/kg,复方血栓通2组通过灌胃复方血栓通片200 mg/kg,给药10周.正常组大鼠随意给予正常饮食和饮用水,而模型组、二甲双胍组、复方血栓通1组、复方血栓通2组大鼠喂以高脂肪饮食,直至第16周末.测定并比较各组大鼠血清胰岛素与血糖水平,比较各组大鼠血清细胞因子[肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β和IL-6]、超氧化物歧化酶(SOD)、丙二醛水平.实时荧光定量PCR与蛋白印迹法分别检测各组大鼠视网膜组织中miR-29b表达与TGF-β/Smad信号通路蛋白表达.结果 与正常组相比,模型组大鼠血清胰岛素及30、60、90 min内血糖均明显升高,差异均有统计学意义(P＜0.05);与模型组比较,二甲双胍组、复方血栓通1组、复方血栓通2组大鼠血清胰岛素及30、60、90 min内血糖水平均显著降低,差异均有统计学意义(P＜0.05).与正常组相比,模型组大鼠血清TNF-α、IL-1β和IL-6水平均显著升高,差异均有统计学意义(P＜0.05);与模型组比较,二甲双胍组、复方血栓通1组、复方血栓通2组大鼠血清TNF-α、IL-1β和IL-6水平均显著降低,差异均有统计学意义(P＜0.05).与正常组相比,模型组血清中的血清SOD活性显著降低,丙二醛含量显著升高,差异均有统计学意义(P＜0.05);与模型组比较,二甲双胍组、复方血栓通1组、复方血栓通2组大鼠血清SOD活性显著增加,丙二醛含量降低,差异均有统计学意义(P＜0.05).正常组大鼠眼球视网膜视盘结构清晰,各层细胞排列整齐;模型组大鼠部分视网膜细胞水肿,层次模糊,排列稀疏紊乱,盘间隙呈空泡状,核染色质致密;二甲双胍组、复方血栓通1组、复方血栓通2组大鼠上述视网膜病变显著逆转.与正常组相比,模型组大鼠视网膜组织中miR-29b、TGF-β、p-Smad-3蛋白水平均显著升高,差异均有统计学意义(P＜0.05);与模型组比较,二甲双胍组、复方血栓通1组、复方血栓通2组大鼠视网膜组织中miR-29b、TGF-β、p-Smad-3蛋白水平均显著降低,差异均有统计学意义(P＜0.05).二甲双胍组、复方血栓通1组、复方血栓通2组组间大鼠以上各指标比较,差异均无统计学意义(P＞0.05).结论 复方血栓通可显著改善大鼠视网膜病变通过抑制机体氧化应激与炎症因子水平,其机制可能与抑制miR-29b/TGF-β/Smad信号通路相关.
Abstract
Objective To explore the mechanism of Fufang Xueshuantong on diabetes retinopathy(DR)in rats based on miR-29b/transforming growth factor-β(TGF-β)/Smad signaling pathway.Methods Among 100 male SD rats,80 rats were used to establish diabetes model,and 20 rats were used as normal group.72 diabetes DR rats were successfully established by intraperitoneal injection of 1％streptozotocin(STZ).They were divided into the model group,the metformin group,the compound Xueshuantong 1 group and the compound Xueshuantong 2 groups according to the random number table method,with 18 rats in each group.The metformin group was treated with 184 mg/kg of metformin by gavage,the Fufang Xueshuantong 1 group was treated with 100 mg/kg of Fufang Xueshuantong Tablets,and the Fufang Xueshuantong 2 group was treated with 200 mg/kg of Fufang Xueshuantong Tablets for 10 weeks.The normal group rats were given normal diet and drinking water at will,while the model group,metformin group,compound Xueshuantong 1 group,and compound Xueshuantong 2 group rats were fed a high-fat diet until the 16th weekend.The levels of serum insulin and blood glucose in each group of rats were measured and compared,and the levels of se-rum cytokines[tumor necrosis factor-α(TNF)-α,interleukin(IL)-1β and IL-6],superoxide dismutase(SOD),and malondialdehyde in each group of rats were compared.The expression of miR-29b and TGF-β/Smad signaling pathway protein expression in retinal tissues of rats in each group were detect using real time fluorescence quantitative PCR and Western blotting,respectively.Results Compared with the normal group,the serum insulin and blood glucose levels within 30,60,and 90 minutes in the model group rats were significantly increased,and the differences were statistically significant(P＜0.05);compared with the model group,the serum insulin and blood glucose levels within 30,60,and 90 minutes of rats in the metformin group,compound Xueshuantong 1 group,and compound Xueshuantong 2 group were significantly reduced,and the differences were statistically significant(P＜0.05).Compared with the normal group,the levels of TNF-α,IL-1 β and IL-6 in the serum of the model group rats significantly increased,and the differences were statistically significant(P＜0.05);compared with the model group,the serum levels of the levels of TNF-α,IL-1 β and IL-6 in the metformin group,compound Xueshuantong 1 group,and compound Xueshuantong 2 group were decreased significantly,and the differences were statistically significant(P＜0.05).Compared with the normal group,the serum SOD activity in the model group was significantly reduced,and the content of malondialdehyde was significantly increased,and the differences were statistically significant(P＜0.05);compared with the model group,the serum SOD activity of rats in the metformin group,compound Xueshuantong 1 group,and compound Xueshuantong 2 group significantly increased,while the content of malondialdehyde decreased,and the differences were statistically significant(P＜0.05).The structure of the retina and optic disc in the normal group of rats was clear,and the cells in each layer were arranged neatly.Part of the retinal cells in the model group rats were edematous,with blurred layers and sparse and disordered arrangement.The intervertebral spaces were vacuolated,and the nuclear chromatin was dense.The retinopathy of rats in the metformin group,compound Xueshuantong group 1,and compound Xueshuantong group 2 was significantly reversed.Compared with the normal group,miR-29b,TGF-β and p-Smad-3 in the retinal tissue of the model group rat were significantly increased,and the differences were statistically sig-nificant(P＜0.05);compared with the model group,miR-29b,TGF-β and p-Smad-3 in the retinal tissue of rats in the metformin group,compound Xueshuantong 1 group,and compound Xueshuantong 2 group were significantly reduced,and the differences were statistically significant(P＜0.05).There were no statistically significant difference in the above indicators among the metformin group,compound Xueshuantong 1 group,and compound Xueshuantong 2 groups in rats(P＞0.05).Conclusion Fufang Xueshuantong significantly improves the levels of oxida-tive stress and inflammation in rat retina,and its mechanism is related to the inhibition of miR-29b/TGF-β/Smad signaling pathway related.
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