| Objective:To study the effects of BLA on the proliferation and apoptosis of LX-2cells;to analyze the secretion of LX-2 extracellular matrix and its underlying mechanism after acetaldehyde induction by BLA.Method: 1.LX-2 cells were cultured in vitro with different BLA concentrations(01.17,2.34,4.69,9.38,18.75,37.50,75,150,300,600 g/mL)for 24 and 48 hours.The absorbance values of 450 nm in each well were determined by CCK-8 method and the IC50 values were calculated for 24 h and 48 h.The concentration of BLA was screened according to IC50 value,and the experiment was divided into BLA group(18.75 g/mL)and Control group(DMEM complete medium of 10% FBS).2.Morphological changes of each group were observed by inverted microscope,and nuclear changes were observed by DAPI staining by inverted fluorescence microscope.Flow cytometry was used to detect the apoptosis and cell cycle of each group.3.Bax,Bcl-2 and Caspase-3 protein expressions were detected by Western Blot.4.Acetaldehyde further activates LX-2 cells:LX-2 cells were stimulated with culture solutions containing different concentrations(0,10,20,50,100,200,400,600,800 ?mol/L)acetaldehyde for 24 h.CCK-8 test was used to detect the effect of different concentrations of acetaldehyde on the proliferation of LX-2 cells.5.ELISA to detect the effects of BLA on the synthesis and secretion of Col-? and Col-? in LX-2 cells activated by acetaldehyde:The experiment was divided into three groups:Normal Controls(NC)group,400 ?mol/L acetaldehyde group,and BLA group(BLA concentration: 18.75 ?g/mL,acetaldehyde concentration: 400 ?mol/L).The amount of Col-? and Col-? secretion in the cell supernatant was measured.6.Western Blot was used to detect the effect of BLA on the expression of ?-SMA,TGF-?1,MMP-1,TIMP-1 protein in acetaldehyde-activated LX-2 cells:Western blot was performed on?-SMA,TGF-?1,MMP-1,TIMP-1 in NC group,acetaldehyde(400 ?mol/L)groupand BLA group.The expression levels of four proteins were analyzed.Result: According to CCK-8,the inhibiting concentration(IC50)of BLA on LX-2cells for 24 h was 20.89 g/mL,and the IC50 at 48 hours was 19.20 ?g/mL.Flow cytometry was used to detect the cell cycle,and the results showed that compared with the control group,the BLA group had fewer cells in the S phase and G2/M(P<0.05)and more cells in the G1 phase(P <0.05).Western blot results showed that the expression of Bax and Caspase-3 was significantly increased and the expression of Bcl-2 was significantly decreased in the BLA group compared with the Ctrl group(P<0.05).We further induced the activation of LX-2 cells with different concentrations of acetaldehyde,and found that the effect of promoting cell proliferation was most obvious when the concentration of acetaldehyde was 400 ?mol/L.When BLA was used for intervention,the content of Col-? and Col-? in the cell supernatant was detected by ELISA.)the content of Col-? and Col-? in the NC group were:(10.18± 0.76)ng/m L,and(21.83 ± 1.51)ng/mL,the Col-??Col-? content of acetaldehyde group was significantly higher than the NC group(P <0.05).The contents of Col-I and Col-? in the BLA group were:(17.39 ± 1.30)and(26.52 ± 1.86)ng/mL,which were significantly lower than the acetaldehyde group(P<0.05)but significantly higher than the NC(P <0.05).Western Blot was used to control ?-SMA,TGF-?1,MMP-1,TIMP-1 in NC group,acetaldehyde(400 ?mol/L)group and BLA group.The expression levels of several proteins showed that the expression levels of ?-SMA and TGF-?1 proteins in the blank control group were very low,and the expression levels of ?-SMA and TGF-?1 proteins in LX-2 cells stimulated by acetaldehyde were significantly higher than those in the NC group.The expression of ?-SMA and TGF-?1 protein in BLA group was significantly lower than that in acetaldehyde group,but higher than that in NC group;The expression of MMP-1 in the acetaldehyde group was significantly lower than that in the NC group,while the expression of TIMP-1 was significantly higher than that in the NC group;the expression of MMP-1in the BLA group was significantly higher than that in the acetaldehyde group,but lower than that in the NC group.The expression level of TIMP-1 protein wassignificantly lower in the acetaldehyde group than in the NC group.Conclusion:1.BLA can inhibit the proliferation of LX-2 and promote its apoptosis.2.BLA can effectively reduce the synthesis and secretion of Col-I and Col-? in LX-2cells induced by acetaldehyde,possibly by increasing the expression of MMP-1 and decreasing the expression of TIMP-1,so as to increase the ratio of MMP-1/ TIMP-1.3.BLA may inhibit LX-2 cell activation by inhibiting TGF-?1 signaling pathway. |
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