Janus激酶抑制剂对高糖诱导的肾小管上皮细胞转分化的作用

Effect of JAK/STAT Pathway Activation on High Glucose-induced Transdifferentiation in Renal Proximal Tubular Epithelial Cells

目的评估Janus激酶抑制剂AG490对高糖诱导肾小管上皮细胞转分化及转化生长因子β1(TGF-β1)表达的影响。方法体外培养人肾近曲小管上皮细胞株(HKC),分别给予高糖和高糖+AG490干预,Western blot检测平滑肌肌动蛋白(α-SMA)、E-钙粘素(E-Cadherin)及信号蛋白STAT1、STAT3、磷酸化STAT1(p-STAT1)和p-STAT3的表达,酶联免疫吸附法测定细胞上清液中TGF-β1和Ⅰ型胶原的分泌,逆转录聚合酶链反应检测TGF-β1 mRNA表达。结果与低糖对照组比较,高糖培养的HKC中α-SMA、p-STAT1和p-STAT3表达明显上调,E-Cadherin表达明显下调,TGF-β1 mRNA表达增加,细胞培养上清液中TGF-β1和Ⅰ型胶原分泌增加。AG490明显下调p-STAT1和p-STAT3表达的同时,明显抑制高糖刺激HKC中α-SMA表达的升高,减轻E-Cadherin表达下降程度,降低TGF-β1 mRNA表达及TGF-β1和Ⅰ型胶原的分泌。结论JAK参与了高糖诱导的HKC转分化,并刺激TGF-β1和细胞外基质分泌,JAK抑制剂AG490能有效拮抗其作用。

Objective To evaluate the effect of JAK/STAT signaling pathway activation on the transdifferentiation and secretion of transforming growth factor-β1 （ TGF-β1 ） induced by high glucose in renal proximal tubular epithelial cells. Methods Human kidney cells （HKC） were cultured and then divided into four groups： low glucose （LG） group, high glucose （HG） group, high mannitol （ LG ＋ M） group, and HG ＋ AG490 group. Immunoprecipitation and Western blot analysis were used to determine the expression of tryosine phosphorylated Janus kinase 2 （ p-JAK2 ） . The protein expressions of STAT1, STAT3, p-STAT1, and p-STAT3 and the expressions of α-SMA and E-Cadherin were observed by Western blot. The contents of TGF-β1, fibronectin and type Ⅰ collagen in the supematants of the cultured HKC were detected by enzyme-linked immunosorbent assay （ELISA）. The expression of TGF-β1 mRNA was measured by reverse transcription and poly-merase chain reaction （RT-PCR）. Results Compared with LG group, the expressions of JAK2, p-STAT1, p- STAT3, and TGF-β1 mRNA were significantly increased in HG group from 6 to 72 hours. Meanwhile, the contents of TGF-β1 and collagen I in the supernatants and the expression of α-SMA increased and the expression of E-Cadherin decreased. The expressions of JAK2, p-STAT1, p-STAT3, and TGF-β1 mRNA as well as the levels of TGF-β1 and collagen Ⅰ in the supernatant s in HG ＋ AG490 group were significantly lower than in the HG group. The expressions of α-SMA and E-Cadherin were also decreased in HG ＋ AG490 group. Conclusion Activation of JAK/STAT signaling pathway may be involved in the high glucoseinduced transdifferentiation and overproduction of TGF-β1 and ECM proteins in HKCs.