Mechanism Of Caspase-1 Inhibitor Ac-YVAD-cmk Reducing Chondrocyte Pyroptosis By Down Regulating GSDMD Protein Expression

Objective:1.Observe the effect of Caspase-1 on chondrocyte pyroptosis in chondrocytes;2.To explore the potential mechanism of Caspase-1 promoting chondrocyte pyroptosis;3.To explore the possible mechanism of Caspase-1 inhibitor Ac-YVAD-cmk inhibiting chondrocyte pyroptosis.Methods:In this experiment,C28 chondrocytes were subcultured with culture medium(1%liquid containing penicillin and streptomycin,5-10% fetal bovine serum,90%-95%DMEM without fetal bovine serum).The cell lines were divided into four groups according to the experimental treatment conditions.The normal control group:(adding the same volume of saline as the inducer);Ac-YVAD-cmk induction group(10 μ M Ac-YVAD-cmk);Ac-YVAD-cmk + LPS +ATP induction group(10 μM Ac-YVAD-cmk、10 μg / ml LPS and 5mm ATP);LPS + ATP induction group(10 μg / ml LPS and 5 m M ATP).After drug intervention,the cells in each group were tested for CCK-8 cell proliferation toxicity,LDH,Western blot and immunohistochemistry of cell climbing tablets to detect the contents of caspase-1,GSDMD and GSDMD-N.Results:1.CCK-8 cell proliferation-toxicity test The results showed that compared with the normal control group,the cell survival rate of Ac-YVAD-cmk + LPS + ATP induction group and LPS + ATP induction group decreased significantly(P < 0.01).Compared with LPS + ATP induction group,the cell survival rate of Ac-YVAD-cmk + LPS + ATP induction group was significantly increased(P < 0.01).2.LDH activity test:The results showed that compared with the normal control group,the LDH released by Ac-YVAD-cmk + LPS + ATP induction group and LPS + ATP induction group increased significantly(P < 0.01).Compared with LPS + ATP induced group,LDH release in Ac-YVAD-cmk + LPS + ATP induced group decreased significantly(P <0.01).3.Western Blot analysis of the contents of caspase-1,GSDMD and GSDMD-N.(1)Caspase-1: compared with the blank control group,the relative gray value of Ac-YVAD-cmk + LPS + ATP induction group and LPS + ATP induction group was significantly higher(P < 0.01).Compared with LPS + ATP induction group,the relative gray value of Ac-YVAD-cmk + LPS + ATP induction group decreased significantly(P < 0.01).(2)GSDMD: compared with the blank control group,the relative gray value of Ac-YVAD-cmk + LPS + ATP induction group and LPS + ATP induction group was significantly higher(P < 0.01).Compared with LPS + ATP induction group,the relative gray value of Ac-YVAD-cmk + LPS + ATP induction group decreased significantly(P < 0.01).(3)GSDMD-N: compared with the blank control group,the relative gray value of Ac-YVAD-cmk + LPS + ATP induction group and LPS + ATP induction group was significantly higher(P < 0.01).Compared with LPS + ATP induction group,the relative gray value of Ac-YVAD-cmk + LPS + ATP induction group decreased significantly(P < 0.01).4.The fluorescence intensity of caspase-1,GSDMD and GSDMD-N was detected by immunohistochemistry.(1)Caspase-1: compared with the normal control group,the fluorescence intensity of Ac-YVAD-cmk + LPS + ATP induction group and LPS + ATP induction group was significantly higher(P < 0.01).Compared with LPS + ATP induced group,the fluorescence intensity of Ac-YVAD-cmk + LPS + ATP induced group decreased significantly(P < 0.01).(2)GSDMD: compared with the normal control group,the fluorescence intensity of Ac-YVAD-cmk + LPS + ATP induction group and LPS + ATP induction group was significantly higher(P < 0.01).Compared with LPS + ATP induced group,the fluorescence intensity of Ac-YVAD-cmk + LPS + ATP induced group decreased significantly(P < 0.01).(3)GSDMD-N: compared with the normal control group,the fluorescence intensity of Ac-YVAD-cmk + LPS + ATP induction group and LPS + ATP induction group was significantly higher(P < 0.01).Compared with LPS + ATP induced group,the fluorescence intensity of Ac-YVAD-cmk + LPS + ATP induced group decreased significantly(P < 0.01).Conclusion:1.The activation of Caspase-1 induced by LPS combined with ATP is an important factor in promoting chondrocyte pyroptosis;2.Caspase-1 inhibitor Ac-YVAD-cmk reduces the production of chondrocyte pyroptosis by inhibiting the expression of GSDMD protein,which will provide a new strategy for the clinical treatment of OA.