Ropivacaine alleviates LPS-induced apoptosis of ulcerative colitis cell line NCM-460

Abstract

Objective To investigate the impact of ropivacaine on apoptosis of lippolysaccharide(LPS) -induced ulcerative colitis cell line NCM-460 and on activity of nucleotide oligomerization domain (NOD)-like receptor protein-3 (NLRP3) inflammatome.Methods Human colon epithelial cell line NCM-460 was cultured in vitro and divided into control group (no intervention), model group (10 μg/mL LPS treatment), low/medium/high concentration ropivacaine group (10 μg/mL LPS and 0.5, 1, 1.5 mmol/L ropivacaine co-treatment, respectivoly). Cell viability was determined by cell counting kit 8 (CCK-8) and the appropriate concentration was selected. The cells were then divided into control group, model group, ropivacaine group (10 μg/mL LPS and 1.5 mmol/L ropivacaine treatment) and ropivacaine + inhibitor group (10 μg/mL LPS, 1.5 mmol/L ropivacaine and 1 μmol/L NF-κB pathway inhibitor BAY 11-7082 treatment), inhibitor group (10 μg/mL LPS+1 μmol/L NF-κB pathway inhibitor BAY 11-7082 treatment) and ropivacaine + activator group (10 μg/mL LPS, 1.5 mmol/L ropivacaine and 1 μmol/L NF-κB pathway activator Prostratin), all groups were treated for 24 h. The level of IL-6, IL-8 and TNF-α were detected by enzyme-linked immunosorbent assay (ELISA). The proliferation rate was detected by EdU incorporation. Hoechst 33258 staining microscopy was used to detect the apoptosis rate. Level of cyclinD1, caspase-3, NLRP3 and NF-κB pathway-related proteins were detected by Western blot. Results Compared with the control group, the cell viability of the model group was significantly decreased and the cell viability of high-concentration experimental group was increased after adding ropivacaine (P＜0.05). So, 1.5 mmol/L ropivacaine was selected for the follow-up experiment. Compared with the control group, the concentration of inflammatory cytokines IL-6, IL-8, TNF-α, apoptosis rate and the protein expression of caspase-3, NLRP3 and phosphorylated p-NF-κB in model group were all significantly increased (P＜0.05), while the proliferation rate and cycilnD1 protein expression were decreased (P＜0.05). Compared with model group, the concentrations of IL-6, IL-8, TNF-α, apoptosis rate and the expression of caspase-3, NLRP3 and p-NF-κB protein in ropivacaine group and inhibitor group were significantly decreased (P＜0.05), while the proliferation and cycilnD1 protein expression were increased(P＜0.05). Compared with ropivacaine group, the trend of the above indexes in ropivacaine + inhibitor group was more significant(P＜0.05), and the trend of these indexes in ropivacaine + agonist group was significantly reversed(P＜0.05). Conclusions Ropivacaine can inhibit the activation of NLRP3 inflammasome and block the signal transduction of NF-κB pathway, further inhibit LPS-induced apoptosis of NCM-460 cells and promote proliferation.

Keywords

• human colon epithelial cell line|lipopolysaccharide|ropivacaine|proliferation|apoptosis