益糖康对高糖诱导人肾小管上皮HK-2细胞上皮间质转化的影响

Effects of Yitangkang on epithelial mesenchymal transformation of human renal tubular epithelial cells HK-2 induced by high glucose

目的:探讨益糖康是否通过抑制肾小管上皮细胞发生上皮-间质转化(EMT)进而减轻糖尿病对肾脏的早期损伤。方法:40只SD雄性大鼠随机分成生理盐水组(1mL/100g)、益糖康组(2.10g/100g)、二甲双胍组(9.11g/100g)、二甲双胍+益糖康组(4.55g/100g二甲双胍+1.05g/100g益糖康煎剂),每组10只,分别给予相应药物灌胃,连续5d,腹主动脉取血,分离上清液。体外实验分组:NS组(10%生理盐水组血清)、YTK组(10%益糖康组血清)、Metformin组(10%二甲双胍组血清)和M+YTK组(10%二甲双胍+益糖康组血清)。高糖刺激人肾小管上皮HK-2细胞24h后,分别采用划痕实验、Transwell实验、Real-time PCR和Western Blot法检测细胞迁移力、体外侵袭力以及EMT相关基因表达水平的改变。结果:YTK组细胞划痕区的未汇合率显著高于NS组(P<0.05);YTK组穿膜细胞数显著低于NS组(P<0.05);M+YTK组穿膜细胞数显著低于Metformin组(P<0.05);与NS组比较,YTK、Metformin及M+YTK组细胞中E-cadherin mRNA表达水平显著升高(P<0.05,P<0.01);而Vimentin和α-SMA mRNA表达水平均显著降低(P<0.01),其中YTK组和Metformin组水平相当,而M+YTK组中Vimentin和α-SMA mRNA表达水平显著低于Metformin组(P<0.05)。结论:益糖康可能是通过抑制肾小管细胞发生EMT进而减轻糖尿病对肾脏的早期损伤,且中药复方益糖康联合西药二甲双胍对肾小管上皮细胞的EMT抑制效果较单独使用二甲双胍更为显著。

Objective: To investigate whether Yitangkang can reduce the early renal injury caused by diabetes by inhibiting the epithelial-mesenchymal transition(EMT) of renal tubular epithelial cells. Methods: A total of 40 male SD rats were randomly divided into normal saline group(1 mL/100 g), Yitangkang group(2.10 g/100 g), metformin group(9.11 g/100 g) and metformin+Yitangkang group(4.55 g/100 g metformin+1.05 g/100 g Yitangkang Decoction), with 10 rats in each group by gavage for 5 days. Blood was collected from abdominal aorta and supernatant was separated by centrifugation. The in vitro experiment groups: NS group(10%normal saline group serum), YTK group(10%Yitangkang group serum), Metformin group(10% metformin group serum) and M+YTK group(10%metformin+Yitangkang group serum). Human renal tubular epithelial HK-2 cells were stimulated by high glucose for 24 h, and the changes of cell migration capacity, invasion ability in vitro and expression level of EMT-related genes were detected by wounding healing assay, Transwell assay, Real-time PCR and Western Blot, respectively. Results: The non-confluence rate of wounding healing area in YTK group was significantly higher than that in NS group(P<0.05). Compared with NS group, the number of transmembrane cells in YTK group was significantly reduced(P<0.05). The number of transmembrane cells in M+YTK group was significantly lower than that in Metformin group(P<0.05). Compared with NS group, the mRNA expression levels of E-cadherin in YTK, Metformin and M+YTK groups were significantly increased(P<0.05, P<0.01). The mRNA expression levels of Vimentin and α-SMA in the YTK, Metformin and M+YTK groups were significantly lower than those in the NS group(P<0.01), and the expression levels of Vimentin and α-SMA in the YTK group were similar to those in the Metformin group. The mRNA expression levels of Vimentin and α-SMA in the M+YTK group were significantly lower than those in the Metformin group(P<0.05). Conclusion: Yitangkang may reduce the early renal injury caused by diabetes by inhibiting EMT of renal tubular cells. The effect of traditional Chinese medicine Yitangkang plus western medicine Metformin on inhibiting EMT is better than that of Metformin alone in renal tubular epithelial cells.