| Objective:To explore the effect of berberine on the transdifferentiation of fibroblasts into myofibroblasts and its mechanism in the pathogenesis of pulmonary fibrosis.Methods:The effects of BBR on the proliferation activity of normal HLF cells were detected to determine the experimental concentration.The cells was divided into four groups:control group,Transforming growth factor-β1(TGF-β1)group,BBR group and TGF-β61+BBR group.The morphological changes of HLF cells were observed.The expression of collagen-1 in HLF cells was detected by Sirius red staining.The expression of alpha smooth muscle actin(α-SMA)in HLF cells was detected by immunofluorescence.α-SMA,Collagen-1,Matrix Metalloproteinase 7(MMP7)and Fibronectin(FN)expression levels were detected by Western Blot and qRT-PCR after stimulation of HLF cells.The P-Smad2/3 protein levels in each experimental group were stimulated by different time(0,30,60,120min),and Western Blot and qRT-PCR were used to detect the protein and gene expression levels of Transforming growth factor-β receptor Ⅱ(TGF-βR2).Results:Berberine could inhibit the proliferation of HLF cells(IC50=451.3 μm),especially the fibroblasts stimulated by TGF-β1(IC50=106.6μm).When the concentration of berberine reached 50 μm,the cell proliferation rate decreased most obviously.After stimulation with TGF-β1,the morphology of HLF cells changed from long spindle shape to slender spindle shape,and the boundary between cells changed from clear to fuzzy;after the addition of berberine,the number of HLF cells decreased slightly;after the combined stimulation,the number of cells decreased significantly,and the cell morphology was not as smooth and neat as the negative control.The protein expression levels of α-SMA,Collagen-I,MMP7 and FN were significantly increased(P=0.002,<0.0001,0.0181,0.0001);There was no significant difference in the expression of BBR histone between the two groups;TGF-β1 and BBR at the same time were higher than TGF-β1 group;The protein expression levels of SMA,Collagen-I,MMP7 and FN were significantly decreased(P=0.0013.<0.0001,0.0494,<0.0001).The results of qRT-PCR showed that the expression levels of α-SMA,Collagen-I,MMP7,and FN genes in HLF cells stimulated by TGF-β1 increased significantly,and the difference was statistically significant(p values were 0.0003,0.0093,0.0024,<0.0001),Berberine can reverse this change(the p value is<0.0001,0.0274,0.0247,<0.0001).Western Blot was used to detect the phosphorylation level of SMAD2/3 protein at different medication times(0,30,60,120min),and the results showed that the simultaneous addition of TGF-β1 and BBR group can significantly reduce the SMAD2/3 protein phosphorylation protein in the TGF-β1 group The expression level of(the p-values are 0.0094,0.0038,and 0.0027 at 30,60,and 120 minutes,respectively).Western Blot showed that TGF-βR2 protein expression in HLF cells stimulated by TGF-β1 increased(p<0.0001);BBR histone expression levels were not significantly different from normal controls;TGF-β1 and BBR were added at the same time compared with TGF-β1 The histone expression level was significantly reduced(p<0.0001).The qRT-PCR results showed that the TGF-βR2 gene expression level of HLF cells stimulated by TGF-β1 increased(p=0.0016),and the gene expression level of the BBR group was not significantly different from that of the normal control;The expression level of β1 group decreased significantly(p=0.0202).Conclusion:BBR regulates the pathogenesis of pulmonary fibrosis by inhibiting the transdifferentiation of HLF cells into myofibroblasts.The mechanism may be related to the inhibition of the activation of the TGF-βR/SMAD2/3 signaling pathway. |
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