目的: 观察可溶性Klotho蛋白对高糖诱导的信号转导及转录激活蛋白3（signal transducer and activator of transcription,STAT3）信号通路磷酸化和肾小管上皮细胞纤维化相关基因表达的影响。方法: 采用体外培养的HK2肾小管上皮细胞,分别设立高糖组（D-葡萄糖30 mmol/L）、正常糖对照组（D-葡萄糖 5 mmol/L）、高渗透压对照组（D-葡萄糖5 mmol/L+甘露醇25 mmol/L）、Klotho干预高糖组和HO3867（STAT3磷酸化抑制剂）干预高糖组,应用蛋白印迹法、实时荧光定量PCR（real time fluorescence quantitative PCR, rt-fqPCR）等检测方法分别观察STAT3磷酸化水平变化和肾小管上皮细胞纤维化相关因子的表达水平变化。结果: ①rt-fqPCR检测结果表明,与正常糖对照组相比,高糖组 HK2细胞内源性Klotho mRNA表达显著下降（P<0.01）,而高渗透压对照组无改变（P>0.05）;②蛋白印迹检测结果表明,与正常糖组相比,高糖可上调HK2细胞内的STAT3磷酸化水平（P<0.01）,而可溶性Klotho蛋白干预可显著抑制高糖引起的STAT3磷酸化激活（P<0.01）;STAT3磷酸化抑制剂（HO3867）干预可显著抑制高糖诱导的STAT3磷酸化水平升高（P<0.01）;③rt-fqPCR检测结果表明,高糖组HK2细胞表达的纤维化相关因子（α-平滑肌肌动蛋白、Ⅳ型胶原蛋白α1、纤维连接蛋白）较正常糖对照组升高（P<0.01）。Klotho蛋白可抑制高糖诱导的HK2细胞表达纤维化相关因子（α平滑肌肌动蛋白、Ⅳ型胶原蛋白α1、纤维连接蛋白）（P<0.01）。应用STAT3磷酸化抑制剂（HO3867）可部分下调高糖诱导的纤维化相关因子表达（P<0.01）。结论: 可溶性Klotho蛋白可抑制高糖诱导的肾小管上皮细胞纤维化,其机制可能是部分通过抑制STAT3磷酸化通路实现的。
Objective: To observe the effect of Klotho on high glucose-activated phosphorylation of signal transducer and activator of transcription 3 (STAT3) and expression of fibrosis related gene in renal tubular epithelial cells under high glucose. Methods: Human proximal tubular epithelial cells (HK2) were cultured and incubated with high glucose (HG, D-glucose 30 mmol/L), normal glucose (NG, D-glucose 5 mmol/L), high mannitol (HM, D-glucose 5 mmol/L and mannitol 25 mmol/L), and Klotho or HO3867 in the presence of high glucose. STAT3 activation and expression of target genes were assayed by Western blot or real time fluorescence quantitative PCR(rt-fqPCR). Results: Compared with NG or HM, a significant decrease of endogenous Klotho gene level was detected after HG treatment(P<0.01). Western blot analysis revealed that the level of STAT3 phosphorylation was significantly increased after HG treatment compared with NG or HM(P<0.01). The activation of STAT3 induced by high glucose was markedly reduced by Klotho or HO3867 treatment(P<0.01). The level of mRNA expression of fibrosis related gene(α-SMA, Col4A1, FN) was significantly increased after high glucose treatment in HK-2 cells (P<0.01), while was markedly inhibited by Klotho treatment(P<0.01). HO3867, the inhibitor of STAT3, partially decreased the mRNA levels of above mentioned genes (P<0.01). Conclusions: Soluble Klotho could attenuate high glucose-induced renal fibrosis, which might be achieved partially through inhibition of STAT3 pathway.
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