| Objectives: Tubulointerstitial fibrosis is the principal hallmark and basic pathological change in most types of Clinical chronic progressive renal diseases. It is characterized by disruption of tubular basement membrane and multiplication of myofibroblast cell as well as the accumulation of extracellular matrix. Injury of basement membran is a beginning and multiplication of myofibroblast cells is the premise, in the end, collagen fibers is increased. Therefore multiplication of myofibroblast cells is an important mechanism of renal fibrosis.It has been largely demonstrated that EMT play a key role in multiplication of myofibroblast cells and the progression of chronic renal fibrosis. Epithelial-myofibroblast transition (EMT) of tubular epithelial cells, characterized by loss of epithelial cells characteristics and gain of myofibroblast characteristics. As we knew the intracellular signaling pathways involved in EMT were complex and incompletely understood.Janus kinase/signal transducer and activators of transcription (JAK/ STAT) is an important signaling pathway, which has been known to mediate the signaling of numerous cytokinase and to be implicated in the regulation of a wide range of cellular processes such as proliferation, differentiation and apoptosis. It was reported that angiotensin II and high glucose could induce activation of JAK/STAT in the glomerular mesangial cells of rat cultrued in vitro. There were few reports about JAK/STAT signaling pathway in tubulointerstitial fibrosis. Phosphoinositide3-kinase/Protein kinaseB (PI3K/ AKT) signaling is an important pathway. It mediates epithelial-myofibroblast transition in numerous carcinoma cells. It also mediates cell proliferation through EGFR in renal epithelial cells. Tubular EMT can be induced by advanced glycation end products, TGFβ1, angiotensin II. And TGFβ1 is probably the key inducer of EMT because TGFβ1 signaling is sufficient to induce EMT in cultured epithelial cells. It has not been known whether JAK/STAT or PI3K/AKT involved in EMT. In order to got a better understanding of it, We therefore cultured the normal rat tubular epithelial cell line (NRK-52E), then examined whether TGFβ1-induced EMT by JAK/STAT and PI3K/AKT pathway.Methods:Detection of EMT, JAK/STAT and PI3K/AKT signaling pathway of NRK-52E cells cultured in vitro.1 NRK-52E cultured by TGFβ1(10ng/ml). Extracted total protein and mRNA from cells which after cultured for 0h, 6h, 12h, 24h and 48h. Then detected the total protein and mRNA of CK18 andα-SMA by Westernblot and RT-PCR analysis.2 NRK-52E cultured by TGFβ1(10ng/ml). Extracted total protein from cells which after cultured for 0min, 10min, 30min, 1h, 2h, 4h. Then detected the total protein of AKT, P-AKT, JAK, P-JAK2, STAT1 and P-STAT1 by Westernblot analysis.3 Use inhibitors of JAK/STAT and PI3K/AKT to intervene cell growth progress. AG490 is a specific inhibitor of JAK/STAT and LY294002 is a specific inhibitor of PI3K/AKT. Then divided cells into four groups. They were Normal group, TGFβ1(10ng/ml) group, TGFβ1(10ng/ml)+LY294002 (20μM) group and TGFβ1(10ng/ml)+AG490(10μM) group. Then extracted total protein from cells which after cultured 1h, and detected AKT and P-AKT by Westernblot and ICC analysis. Extracted total protein from cells which after cultured 30min, and detect JAK, P-JAK2, STAT1 and P-STAT1 by Westernblot and ICC analysis.4 Use AG490 and LY294002 to intervene cell growth progress. And divided cells into six groups. They were Normal groups, TGFβ1(10ng/ml) group,TGFβ1(10ng/ml)+LY294002(20μM) group, TGFβ1(10ng/ml)+ AG490 (10μM) group, LY294002(20μM) group and AG490 (10μM) group. Then extracted total protein and mRNA from cells which after cultured 48h, and detected CK18 andа-SMA by Westernblot and RT-PCR analysis.Results:1 Expression ofα-SMA and CK18 in NRK-52E after TGFβ1(10ng/ml) stimulated .Western Bloting indicated that the expression ofα-SMA was increased from cultured for 6h, and the analysis for the relative level ofα-SMA showed a gradual and stable increased with the prolonging of time. The expression of CK18 decreased from 6h, and inversely with prolonging of time.RT-PCR and Western presented the same trend.2 Expression of AKT, p-AKT, JAK, p-JAK, STAT1 and p-STAT1 in NRK-52E after TGFβ1 stimulated.Western Bloting indicated that the expression of AKT, JAK2, STAT1 didn't change. But p-AKT, p-JAK2, p-STAT1 were increased quickly after cultured for 10min, and p-JAK2, p-STAT1 achieved the peak at 30min; p-AKT achieved the peak at 1 hour. Then decreased gradually.3 Effect of TGFβ1, TGFβ1+LY294002, TGF-β1+AG490 on the expres- sion of AKT, p-AKT in NRK-52E at stimulated time of 1h. and JAK, p-JAK, STAT1, p-STAT1 in NRK-52E at stimulated time of 30min .Western Bloting and ICC indicated that p-AKT, p-JAK2, p-STAT1 lowly expressed in Normal group, but highly expressed in TGFβ1 group. In T+AG goup had a lower expression than T goup but higher than Normal group. In T+LY group p-AKT, p-STAT1 had a lower expression than TGFβ1 goup, but p-JAK2, had a high expression like in TGFβ1 goup.4 Effect of TGFβ1, TGFβ1+LY294002, TGFβ1+AG490 on the expres- sion ofα-SMA and CK18 in NRK-52E at stimulated time of 48h by westernblot and RT-PCR analysis.Western Bloting and RT-PCR indicated thatα-SMA rarely expressed in N group, but highly expressed in TGFβ1 group. In T+LY and T+AG goup had a lower expression. On the contrary, CK18 had a high expression in Normal gorup, but a low expression in TGFβ1 group. The expression in T+LY and T+AG was lower than Normal group but higher than TGF-β1 group. Conclusions: The results documented that TGFβ1 could induce EMT of NRK-52E cells in vitro, and this effect is related to phosphorylation of AKT, JAK2 and STAT1. AG490 and LY294002 which wre inhibitors of JAK/ STAT and PI3K/AKT signaling pathway, could partially inhibited TGFβ1- induced EMT. And this effect is related to the inhibition of phosphorylation of AKT, JAK2 and STAT1. p-JAK2 might be an upstream factor of phosphorylation of AKT. But it needed further studies to understand the relations between signaling pathway.
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