| Aim The aim of this present study was to explore the effects and the possible machanism of mesenchymal stem cells(MSCs) on epithelial-to-mesenchymal transition(EMT) induced by transforming growth factor-?1(TGF-?1), especially the role of hepatocyte growth factor(HGF) secreted by MSCs.Methods In vitro experiment,(1) MSC surface antigens were identified by flow cytometry, and did adipogenic and osteogenic differentiation.(2)The cultured HK-2 cells were divided into five groups: ?control, ?TGF-?1(8ng/ml), ?TGF-?1+ MSCs, ? TGF-?1+MSCs+anti-HGF antibody(0.5ug/ml), ? TGF-?1+MSCs+ PY294002(10umol/L). After 24 h, the morphological changes were observed by inverted microscopy. The expression of ?-smooth muscle actin(?-SMA), E-cadherin, HGF and p-Akt were analyzed by real time polymerase chain reaction(real time PCR) and western blot(WB) analysis, respectively. In vivo experiment, the mice were divided into four groups: ?control(n=10) ?UUO(n=10) ?UUO+MSC(n=10) ?UUO+ MSC+anti-HGF antibody(n=10). The mice in MSC treated group were received an injection of MSCs(1×106 cells) via tail vein with or without treatment of anti-HGF antibody(0.5ug) at 1st and 3rd day after ureteral obstruction. At the 7th and 14 th d, the mice were killed and collected blood, urine and renal tissue. Urea nitrogen(BUN), serum creatinine(Scr), uric acid(UA) and proteinuria were measured by automatic biochemistry analyzer. Histopathology of kidney was also analyzed and immunohistochemical method was used to detect the expression of ?-SMA, E-cadherin and HGF.Results In vitro experiment :(1) MSC surface antigen of CD34 and CD45 were negative, CD105 was positive. Cultured with adipogenic induction complete medium after 12 days, cells containing much of lipid droplet emerged, which showed red stained by Oil Red. When cultured with osteogenic induction complete medium after 14 days, they formed many calcium nodus, which showed orange stain by alizarin bordeaux.(2) MSCs and HK-2 cells were able to express HGF. After TGF-?1 induction, the level of HGF was significantly higher on MSC and lower on HK-2 cells(P<0.01).(3) The HK-2 cells presented fibroblast-like appearance and had a characteristic spindle shape after stimulated with TGF-?1, and the level of ?-SMA was significantly increased, E-cadherin decreased remarkably(P<0.01). However, the cells stimulated with TGF-?1 were cocultured with MSC demonstrated relative well-preserved architecture, the increase in E-cadherin, p-Akt and the decrease in ?-SMA were attenuated remarkably(P<0.05). Furthermore, adding anti-HGF antibody and pretreated with PI3 K inhibitor PY294002, the HK-2 cells morology also presented fibroblast-like appearance, and abolished the inhibition of the increase of ?-SMA and the decrease of E-cadherin(P<0.05) and p-Akt(P<0.01).In vivo experiment:(1). The levels of BUN, Scr, UA and proteinuria were significantly increased in UUO group(P<0.01), however, these indexes were remarkably decreased in MSC group(P<0.01), while neutralization with anti-HGF antibody, BUN, Scr, UA and proteinuria were rebounded(P<0.01).(2).The renal sections showed histological features of serious injury, consisting of severe tubular swelling, necrosis and a significant increase in tubulointerstitial collagen deposition in UUO group. After administration with MSC, the kidney demonstrated less tubular swelling and necrosis, tubulointerstitial fibrosis and less collagen deposition. In addition, the protective effects were abrogated by a neutalizing antibody against HGF. The renal sections showed the similar features of serious injury as UUO group.(3) Inmmmunohistochemical detection of the expression of ?-SMA, E-cadherin and HGF. In UUO group, the level of ?-SMA was significantly increased(P<0.05), while E-cadherin was decreased significantly(P<0.01), and the expression of HGF was decreased(P<0.05); when treatment with MSCs, the expression of ?-SMA was decreased while E-cadherin and HGF had a significantly increased(P<0.05); However, neutralizating HGF with anti-HGF antibody abolished the inhibition of the increase of ?-SMA and the decrease of E-cadherin and HGF(P<0.01).Conclusion This study showed that MSCs could restore TGF-?1-induced EMT by secreting HGF via the PI3K/Akt pathway. |
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