The Effect Of Procyanidins On Proliferation And Differentiation Of NIH3T3 Cells Induced By TGF-β

Objective: To explore the effect of procyanidins on the proliferation and differentiation of NIH3T3 cells induced by TGF-β1 and on the changes of PI3K/AKT pathway.To provide a new idea for the early intervention of pulmonary fibrosis.Methods: Mouse embryo fibroblasts(NIH3T3 cells)were selected as the subjects cultured under suitable conditions in vitro.The cells were seeded into 96-well plates and then randomly divided into five groups.Group A(blank control group)was cultured without TGF-β1,procyanidins or LY294002.Group B(TGF-β1 group)was cultured only with TGF-β1(5 μg/L).Group C(procyanidins+TGF-β1 group)was pre-treated with procyanidins(50 μg/m L)for 1 hour and then was treated with TGF-β1(5 μg/L).Group D(LY294002+TGF-β1 group)was pre-treated firstly with LY294002(10 μmol/L)for 1 hour and then was cultured with TGF-β1(5 μg/L).Group E(LY294002+procyanidins+TGF-β1 group)was pre-treated firstly with LY294002(10 μmol/L)and then was treated with procyanidins(50 μg/m L)for 1 hour.Finally,the TGF-β1(5 μg/L)was added in group E.The above five groups were cultured under suitable conditions in vitro for 24 hours.Cell survival was detected by Cell Counting Kit-8 after 12 hours,24 hours,and 48 hours.The morphological changes of five groups were observed.The relative m RNA expression levels of PI3 K and AKT was detected.And the protein expression of α-SMA,Col-I,phospho-PI3 K,and phospho-AKT was detected.Results:1.Cell morphology: NIH3T3 cells of group A were spindle or star fibroblasts under inverted biological microscope.The cells of other groups were myofibroblasts differentiated into flat structure,especially of group B.2.Cell viability(CCK8 assay): The cell viability of group B was higher than group A after 12 hours,24 hours,and 48 hours.The cell viability of group C and group D was higher than group A but lower than group B after 24 hours and 48 hours.The cell survival of group E was lower than group B at three time points(P<0.05).3.Western-Blot results showed that the protein expression of theα-SMA and Col-I in the four experimental groups was higher than that in the group A.In comparison with group B,the expression of two proteins above was lower in group C,group D,and group E.The protein expression of α-SMA in the group E was lower than that in the group C and group D(P<0.05).There was no significant difference between group C and group D.The protein expression of phospho-PI3 K and phospho-AKT in the group B,group C,group D,and group E was higher than that in the blank control group.The protein expression of phosphopotein from high to low was group B,group C,group D,and group E(P<0.05).4.qRT-PCR results showed that the relative m RNA expression levels of PI3 K and AKT in the group B,C,D and E was higher than that in group A.The relative m RNA expression levels of PI3 K and AKT from high to low were group B,group C,group D,and group E(P<0.05).Conclusion: Procyanidins may inhibit the proliferation and differentiation of NIH3T3 cells by deregulating TGF-β1-PI3K/AKT pathway.