Effect Of Berberine On TGF-β1 Induced Epithelial Mesenchymal Transition In HK-2 Cells By Regulating NLRP3 Inflammasome Pathway

Objective:To investigate the protective effect of berberine（BBR）on TGF-β1 induced human renal tubular epithelial cells（HK-2）to mesenchymal transition（EMT）,and to explore the effect of BBR on the expression of E-cadherin,α-smooth muscle actin（α-SMA）and NLRP3 inflammasome components NLRP3 and caspase-1 in HK-2 cells during EMT,providing a theoretical basis for the mechanism of BBR in attenuating the fibrosis of HK-2cells.Methods:（1）The docking sites of BBR and NLRP3 inflammasome components NLRP3,ASC and pro-caspase-1 were predicted by computer,and the possibility of BBR binding to the components was evaluated by the number of binding sites and function scores.（2）CCK8 assay was used to detect the effect of BBR on the proliferation of HK-2 cells and to screen the relative safe concentration of BBR on HK-2 cells.（3）At different concentrations of TGF-β1 with different length of stimulating time,changes in cell morphology of HK-2 cells were observed under the microscope;Western blot was used to detect the expression of E-cadherin andα-SMA,and the expression levels of NLRP3 and caspase-1 in the NLRP3 inflammasome pathway were also detected.（4）HK-2 cells were pre-incubated with BBR at low,medium and high concentrations for 18 hours,and then continued to be stimulated at appropriate concentrations of TGF-β1;The cell morphology was observed under the microscope,and the expression levels of E-cadherin,α-SMA,NLRP3 and caspase-1 were detected by Western blot.（5）The enzyme activity of caspase-1in HK-2 cells after BBR preprotecion was detected using Caspase-1 activity assay kit.（6）Enzyme-linked immunosorbent assay（ELISA）was used to detect the IL-1βcontent in the supernatant of HK-2 cells after BBR preprotection.Results:（1）According to the number of docking sites and function scores,BBR showed high affinity to NLRP3 and pro-caspase-1.（2）CCK8 assay showed that BBR inhibited the proliferation of HK-2 cells at a concentration of 50μmol·L^-1 or less with relatively safety for HK-2.（3）Western blot results showed that TGF-β1 could induce transdifferentiation of HK-2 cells,down-regulate of E-cadherin and up-regulate ofα-SMA,and could increase expression of NLRP3 and caspase-1.（4）Western blot showed that after BBR intervention,it could improve the induction of TGF-β1,up-regulate the expression of E-cadherin and down-regulate the expression ofα-SMA expression,and the expression of NLRP3 and caspase-1 could be reduced.（5）Compared with TGF-β1 group,BBR intervention decreased the enzyme activity of caspase-1 and the secretion of IL-1βin TGF-β1 induced HK-2 cells.Conclusion:BBR had high affinity with NRLP3 inflammasome.BBR could attenuate TGF-β1 induced transdifferention of HK-2 cells and down-regulate the expression of NLRP3 and caspase-1,which may be related to inhibiting the activation or assembly of NLRP3 inflammasome.