The Effect And Mechanism Of Lipopolysaccharide (LPS) On Pyroptosis Of Human Skin Epithelial Cells Through Caspase-4 Pathway

Objective:To investigate the expression and significance of GSDMD and Caspase-4 protein in lipopolysaccharide(LPS)-induced pyroptosis in human skin epithelial cells(HSEC).To explore the role of Caspase-4 non-canonical inflammasome in LPS-induced secretion of inflammatory cytokines in HSEC.To explore the correlation and effect of Caspase-4 blocker Z-LEVD-FMK on lipopolysaccharide(LPS)-induced pyroptosis in HSEC cells,and to seek potential therapeutic targets.Methods:(1)The effect of LPS on the proliferation rate of HSEC at different concentrations and different time points was determined by Cell Counting Kit-8(CCK-8)in vitro to determine the optimal concentration and time of LPS on HSEC.(2)Western blot assay was used to detect the expression the expression levels of GSDMD、Caspase-4、Caspase-1、NLRP3 and other pyroptotic proteins were detected in the blank control group(HSEC without other treatment)、 Z-LEVD-FMK group(treated with 10 μg/m L Z-LEVD-FMK for 24h)、LPS group(treated with 50 μg/m L LPS for 24h)and LPS + Z-LEVD-FMK combined treatment group(treated with 50 μg/m L LPS + 10μg/m L Z-LEVD-FMK for 24h).(3)The expression levels of interleukin-1α、interleukin-1β and interleukin-18 in blank control group、Z-LEVD-FMK group、LPS group and LPS + Z-LEVD-FMK group were detected by enzyme-linked immuno sorbent assay(ELISA).Results:(1)CCK-8 results showed that compared with the blank control group,there was no significant difference in cell survival rate between the experimental group treated with less than 50μg/ml LPS at all time points(P > 0.05);After treated with 50,100,200 μg /ml LPS for 12,24,48 h,the survival rate of HSEC cells in the experimental group was significantly decreased,and the difference was statistically significan(P < 0.05)According to the IC50 value,the optimal LPS concentration and time were 50 μg/m L and 24 hours,respectively.(2)Western blot results showed that compared with the blank control group,the LPS group had significant increases in the relative expression of GSDMD、Caspase-4、Caspase-1and NLRP3 proteins(P < 0.05).Compared with the LPS group,the relative expressions of GSDMD 、 Caspase-4 、 Caspase-1 and NLRP3 proteins in the Z-levd-fmk+LPS group were significantly decreased(P < 0.05),and the expression of caspase-4 protein was significantly decreased.(3)The results of ELISA showed that the relative expressions of IL-18、 IL-1α and IL-1β in LPS group were significantly higher than those in blank control group(P < 0.05).Compared with the LPS group,the LPS+Z-LEVD-FMK group had significant reductions in the relative expression of IL-18、IL-1α and IL-1β(P < 0.05).Conclusion:LPS can induce pyroptosis in human skin epithelial cells,and release inflammatory factors to cause inflammatory response leading to cell damage and death.This process was inhibited by the Caspase-4 blocker Z-LEVD-FMK,indicating that the non-canonical pyroptosis pathway played an important role in LPS-induced pyroptosis.Therefore,controlling the pyroptosis of human skin epithelial cells,especially the non-canonical pyroptosis pathway may be a potential target for the treatment of skin and soft tissue infections.