Protective Effect And Mechanism Of Konjac Mannan Oligosaccharide And Bacillus Subtilis On Lps Induced Intestinal Oxidative Damage

Oxidative stress can cause animal systemic and metabolic diseases.The system of intestinal antioxidant enzyme plays an important role in oxidative stress.A large number of studies show that konjac mannan oligosaccharides can promote intestinal bacteria and inhibit the growth of harmful bacteria,and also has the ability of scavenging oxygen free radicals.Bacillus subtilis has many kinds of probiotic functions on animal body,and plays an important role in enhancing immune function,reducing oxidative stress and preventing the growth and colonization of pathogens.Our laboratory preliminary study found that konjac mannan oligosaccharide played a significant role in promoting the growth of Bacillus subtilis.The combination of them had significant repair effect on intestinal epithelial cell activity and inflammatory injury.On the basis of previous research,this paper used LPS to stimulate colon epithelial Caco-2 cells,to establish the model of oxidative damage to investigate the repair effect of konjac mannan oligosaccharides and Bacillus subtilis on oxidative damage of intestinal epithelial cells,and establish the injury model in mice induced by LPS,to further explore the combined effect of the animal’s body repair and protection coordination effect.The main contents are as follows:1.Constructed an oxidative injury model of intestinal epithelial cells.By using different concentrations of LPS to stimulate Caco-2 cells for different time,the oxidation index and antioxidant index of cells were detected.It was found that LPS increased the expression of MDA and decreased the activity of antioxidant enzyme SOD,and finally determined that the best concentration of LPS induced oxidative damage in Caco-2 cells was 2 g/m L,and the action time was 8h.Compared with the control group,LPS could increase the expression of NOX2 and produce more reactive oxygen species.The expression of Nrf2 was up-regulated and the expression of Keap1 was down regulated.The expression of downstream antioxidant genes SOD1,GPx1 was also upregulated,indicating that the Nrf2/Keap1 antioxidant enzyme system was activated.2.The effect of konjac mannan oligosaccharides and Bacillus subtilis on the injury of intestinal epithelial cells.The results showed that compared with the LPS-injured group,the SOD enzyme activity and GSH content of the cell culture supernatant of the konjac manno-oligosaccharide treatment group were significantly increased,but there was no significant change in the two antioxidant enzymes in the cell lysate.Q-PCR showed that konjac mannooligosaccharides had no significant effect on the expression of oxidant genes,suggesting that konjac mannooligosaccharides may play an antioxidant role in cell culture,probably through their own physicochemical properties and in cells.Reactions such as free radicals are not involved in the cellular antioxidant system.Compared with the LPS-injured group,SOD enzyme activity and GSH content were significantly increased in supernatants and lysates of Bacillus subtilis treated cells.Compared with the control group,the expression of Nrf2 and downstream antioxidant genes SOD1 and GPx1 was significantly up-regulated in the positive control group of Bacillus subtilis,and the Nrf2 and downstream antioxidant genes SOD1 and GPx1 were also treated by Bacillus subtilis compared to the LPS injured group.Expression is significantly raised.This suggests that Bacillus subtilis may up-regulate the expression of antioxidant genes by affecting the Nrf2 signaling pathway of cells and increase the anti-oxidation function of cells.Compared with the LPS-injured group,the expression of MDA was down-regulated,SOD enzyme activity and GSH content were increased in the combined treatment group of oligosaccharides and probiotics.Compared with the probiotic-treated group,the combination of oligosaccharides and probiotics could down-regulate the expression of MDA.Up-regulation of SOD enzyme activity had no significant effect on GSH content.3.The protective effect of konjac mannan oligosaccharide and Bacillus subtilis on mice damage model.(1)Compared with the LPS control group,the activities of three antioxidant enzymes of T-AOC,SOD and GSH in the liver of mice were significantly increased compared with those in the control group.The addition of probiotics significantly increased the activity of superoxide dismutase and total antioxidant enzymes,but had no significant effect on the content of GSH in the liver.The combined treatment of oligosaccharides and probiotics can significantly increase the activity of three antioxidant enzymes in the liver,and is more effective than the addition of probiotics alone.(2).Compared with the LPS control group,oligosaccharide and Bacillus subtilis irrigation could significantly reduce the content of MDA in the serum of mice,and the combination of the two groups was more effective.HE staining of mouse ileum revealed that LPS induced obvious injury to ileum tissue,intestinal villus rupture and tissue shrinkage,and addition of oligosaccharides and Bacillus subtilis could repair the injury of ileal tissue in mice to varying degrees.(3)The addition of Bacillus subtilis and konjac mannan oligosaccharide significantly inhibited the expression of inflammatory factors TNF-and IL-6 in mice ileum,and the combined treatment of oligosaccharide and probiotics was more effective.(4)Compared with the LPS control group,the content of GSH and the activity of SOD in the three groups were significantly improved.Gavage treatment group could significantly inhibit the upregulation of NOX2 gene in intestinal tract of mice induced by LPS,and the combination of gavage group and gavage group reduced more significantly than that of single gavage oligosaccharide or probiotics.The preconditioning group of gavage has a good protective effect on the inhibition of the expression of antioxidant enzyme system by LPS.The expression of Clauding-1 protein was detected by western,blot than that of LPS control group,the expression of intragastric administration of oligosaccharide and probiotics can significantly increase the Clauding-1 protein,Bacillus subtilis and konjac mannan oligosaccharides on LPS induced intestinal TJ damage has a good protective effect.In conclusion,LPS can cause oxidative damage of colon epithelial Caco-2 cells;Bacillus subtilis,konjak mannan and its combination had significant effects on repairing oxidative damage of cells;Bacillus subtilis and konjac mannan oligosaccharides with adding more obvious than the use of probiotics,konjac mannan oligosaccharides alone.Gavage of Bacillus subtilis and konjac mannan oligosaccharide have good protective effect on LPS acute injury model in mice.