Protective Effect And Mechanism Of N-3 Polyunsaturated Fatty Acids On Esophageal Epithelial Cell Against Acid Damage

Objective: Acid is one of the main factors of esophageal injury in reflux esophagitis.Our previous study has found that n-3 polyunsaturated fatty acids(PUFAs)inhibited inflammation in rats with RE.This study was aimed to observe the protective effect of n-3 PUFAs on esophageal epithelial cells against acid damage and to explore the molecular mechanisms.Methods: A model of esophageal epithelial cell acid damage was constructed by treating Het-1A cells with acidified medium(p H5.0)intermittently.After intervention,intracellular SOD activity and MDA content were detected,while the expression of Nrf2 was assessed by Western Blot and qPCR.Then the esophageal epithelial cell acid damage model was exposed to PUFAs with different ratios of n-6/n-3(9:1,3:1,1:1 and 1:3,respectively),and SOD activity and Nrf2 levels were measured.In the groups of 9:1 and1:1,the protein levels of Nrf2,NLRP3 and cleavedcaspase-1(p20)were detected by Western Blot while the mRNA levels of Nrf2,caspase-1,IL-1β and IL-18 were quantified by qPCR.IL-1β,IL-18 and LDH levels in the cell culture supernatant were measured by ELISA,and the pyroptosis rate was assessed by caspase-1 and TUNEL double staining.Finally,the cell of n-6/n-3PUFAs 1:1 group was intervened by agonists or inhibitors of Nrf2,respectively,which was used to detect Nrf2 expression and the above apoptosisrelated parameters.The Nrf2 expression and the apoptosis-related parameters were detected in n-6/n-3 PUFAs 1:1 group cells with Nrf2 agonists or inhibitors treatment,respectively.Results: After acidification intervention,the levels of MDA and Nrf2 were increased while that of SOD was decreased,and the differences were statistically significant(p<0.05).Among the groups of n-6/n-3 PUFAs 9:1,3:1and 1:1,Nrf2 mRNA levels and SOD activity increased with n-3 PUFAs ratio,reaching the highest at 1:1 group(p<0.05),but decreased in 1:3 group(p<0.05),while Nrf2 protein level was highest in the 1:1 group(p<0.05).Compared with the 9:1 group,the expression of NLRP3 and cleaved caspase-1(p20)and the pyroptosis rate of 1:1 group were decreased(p<0.05).IL-1β,IL-18,and LDH levels in the cell culture supernatant were also decreased in 1:1 group(p<0.05).In group of n-6/n-3 PUFAs 1:1,the expression of Nrf2 increased while protein levels of NLRP3 and cleaved caspase-1(p20)as well as pyroptosis rate decreased after Nrf2 agonist intervention(P<0.05);however,Nrf2 inhibitor intervention resulted in a decrease in the expression of Nrf2 and an increase in the levels of pyroptosis-related parameters and pyroptosis rate(p<0.05).There was no significant difference in all indexes between control and solvent group(p>0.05).Conclusions: N-3 PUFAs protects esophageal epithelial cells from acid damage and the protective effect is strongest when the ratio of n-6/n-3 PUFAs is 1:1 in this study.N-3PUFAs can inhibit NLRP3 inflammasome activation by up-regulating Nrf2 expression,followed by inhibition of pyroptosis to resist acid damage and exert a protective effect on esophageal epithelial cells.