Bmscs Promote Apoptosis In Rat Hscs By Upregulation Of Urokinase-type Plasminogen Activator And Suppression Type-1Plasminogen Activator Inhibitor

Objective:To explore the mechanism that bone marrow mesenchymal stem cells regulated the apoptosis of hepatic stellate cells as viewed from uPA/PAI-1fibri no lytic syetem.Methods:BMSCs were isolated from bone marrow in rats and grown, propagated in culture flask in the passages4. HSCs and fiberoblast cells were recoveried and activated morphologically, a-SMA expression in HSCs was evaluated immune-histochemically. The co-culture of BMSCs with HSCs was performed by using a6-well Transwell membranes.The HSCs were seeded in the lower chamber with BMSCs and fiberoblast cells (2×105cells/ml) were seeded onto the Transwell membrane of the inner chamber.Cultures were maintained in HSCs in medium for24h,48h,72h. Four groups were divided randomly:①BMSCs and HSCs co-cultrue group②HSCs blank group③HSCs control group④UK122pretreated group:HSCs were pretreated with by uPA specific inhibitors -UK122six hours before co-culture with BMSCs;⑤BMSCs control group: BMSCs cultured alone. The concentration of uPA and PAI-1in the supernatants were analyzed via enzyme-linked immunosorbent assay (ELISA). The mRNA expression of uPA and PAI-1were evaluated with realtime fluorescence reverse transcription-polymerase chain reaction(RT-qPCR), MTT assay was performed to detect the effect of BMSCs on the proliferation of HSCs and to determine the perfect intervention concentration of UK122. The protein expressions of HGF-a in HSCs were evaluated by Western blot, the rate of apoptosis of HSCs were detected by Annexin-V-FITC/PI.Results:1. The level of PAI-1mRNA in BMSCs and HSCs co-cultrue group (24,48,72h:0.42±0.01,0.54±0.01,0.82±0.01) was obviously lower than HSCs blank group (24,48,72h:1.07±0.01,1.09±0.04,1.41±0.03)(P<0.01); The level of uPA mRNA in BMSCs and HSCs co-cultrue group (24,48,72h:1.55±0.06,1.96±0.05,2.95±0.03) was obviously higher than HSCs blank group (24,48,72h:1.07±0.06%,1.17±0.06,1.28±0.02)(P<0.01), and was related to the time of co-cult-ure o ELISA analysis of conditioned medium revealed that BMSCs stimulated HSCs to secrete uPA. With the co-culture time lasting, the inhibitory rate of HSCs proliferation with BMSCs and HSCs co-cultrue group(24,48,72h:10.95±0.44%,31.39±2.31%,46.1±2.77%) were significant higher than HSCs blank group(24,48,72h:1.07±0.06%,1.17±0.06,1.28±0.02) at different pointed time(P<0.01), a concentration of1μg/ml of UK122showed the strongest reduction of the inhibitory rate of HSCs proliferation in BMSCs group remarkablely.The result of HSCs by Annexin-V-FITC/PI and the protein expressions of active HGF in HSCs with BMSCs co-culture were significantly higher than fiberoblasts co-culture system and HSCs culture system at different pointed time that with the co-culture time lasting,(P<0.01). Blockage of uPA with UK122attenuated the effect of the BMSCs on HSCs.Conclusions:BMSCs increase HGF activity and promote apoptosis in the hepatic stellate cells by upregulation of uPA and downregulation of PAI-1.