Intervention effect of coix seed polysaccharides on an in vitro inflammation model of ulcerative colitis and its mechanism: a study based on the NLRP3/caspase-1 pathway

Abstract:

Objective To investigate the intervention effect of coix seed polysaccharides（Coixan） on an in vitro inflammation model of ulcerative colitis（UC） and its mechanism from the perspective of inflammation theory.Methods NCM460 cells were cultured in vitro and were induced by different doses（0，2.5，5，10，20，and 40 μg/mL） of lipopolysaccharide（LPS） to obtain the optimal induction dose of 20 μg/mL，and NCM460 cells were induced by different concentrations（0，5，10，20，40，and 80 μmol/L） of Coixan to obtain the optimal concentrations for promoting proliferation（10，20，and 40 μmol/L）. The cells were randomly divided into blank group，model group（LPS induction），LPS+low-，middle-，and high-dose Coixan（10，20，and 40 μmol/L） groups，and LPS+salicylazosulfapyridine（SASP）（200 μg/mL） group，and after all cells except those in the blank group were stimulated with LPS（20 μg/mL） for 48 hours to establish an in vitro cell inflammation model of UC，Coixan and SASP were used for 48 hours of intervention. ELISA was used to measure the levels of the inflammatory factors interleukin-1β（IL-1β） and tumor necrosis factor-α（TNF-α） in supernatant；immunofluorescence assay was used to measure the expression level of the pyroptosis-related protein caspase-1；qRT-PCR was used to measure the gene expression levels of NLRP3，caspase-1，GSDMD-N，and IL-1β associated with pyroptosis in the NLRP3/caspase-1 pathway；Western blot was used to measure the protein expression levels of NLRP3，caspase-1，and IL-1β associated with pyroptosis in each group.Results Compared with 0 μg/mL LPS，20 μg/mL LPS for 48 hours of induction moderately inhibited the growth of NCM460 cells（P<0.01），and meanwhile，there was a significant increase in the content of the inflammatory factors IL-1β and TNF-α at 48 hours of intervention（P<0.01）；therefore，20 ug/mL LPS induction for 48 hours was selected for modeling. Compared with 0 μmol/L Coixan，there was an increase in cell viability after intervention with Coixan at concentrations of 10，20，and 40 μmol/L，especially Coixan at concentrations of 10 and 20 μmol/L（P<0.05）. Compared with the normal control group，the model group had significant increases in the levels of the inflammatory factors IL-1β and TNF-α，the protein expression levels of NLRP3，caspase-1，and IL-1β，and the gene expression levels of NLRP3，caspase-1，GSDMD-N，and IL-1β（P<0.01）；compared with the model group，the middle- and high-dose groups and the SASP group had significant reductions in the secretion of IL-1β and TNF-α（P<0.01）. Compared with the model group，the middle- and high-dose groups and the SASP group had a significant reduction in the expression level of the pyroptosis-related protein caspase-1（P<0.01）；compared with the model group，the low-，middle-，and high-dose groups and the SASP group had significant reductions in the protein and gene expression levels of NLRP3 and caspase-1（P<0.05 or P<0.01），and the middle and high-dose groups and the SASP group had significant reductions in the gene expression levels of GSDMD-N and IL-1β（P<0.01）；compared with the model group，the middle- and high-dose groups and the SASP group had significant reductions in the expression levels of the pyroptosis-related proteins NLRP3，caspase-1，and IL-1β（P<0.01）.Conclusions Coixan may exert a protective effect on human intestinal epithelial cells by downregulating the expression levels of the pyroptosis-related factors NLRP3，caspase-1，GSDMD-N，and IL-1β and inhibiting the pyroptosis of intestinal epithelial cells.