Regulation Of Lactiplantibacillus Plantarum LP-315 On The Immune Response To DSS-induced Inflammatory Bowel Disease In Mice

Objective To investigate the intervention effect of Lactiplantibacillus plantarum LP-315 on intestinal barrier and immune regulation of IBD model in mice.The probiotic characteristics and functional mechanism of this lactic acid bacteria were analyzed to provide new ideas and theoretical basis for the application of probiotics in daily health care and clinical treatment.Methods Forty SPF 6-week-old male C57BL/6J mice were randomly divided into 5 groups:blank group(NC group),model group(DSS group),probiotic group(LP group),drug group(MS group)and probiotic combined drug group(PM group),with 8 mice in each group.NC group and DSS group were given 200μL saline daily,LP group and MS group were given the same volume of LP-315 and 5-ASA solution,and PM group was given respectively 100μL LP-315 and 5-ASA solution.Except for the NC group drinking sterile water,the other groups were free to drink 2.3%DSS solution for 7 days.After 7 days,they were replaced with 0.8%DSS solution to drink until the 21st day to construct the enteritis model.During the intervention period,the body weight,fecal traits and hematochezia of mice were observed and recorded,and DAI score was performed.After the mice were sacrificed,the colon pathological sections,goblet cells and tight junction proteins(Occludin,ZO-1)were stained by HE staining,PAS staining and immunofluorescence staining,respectively.The proportion of spleen CD4~+T cell subsets(Th1/Th2/Th17)was analyzed by flow cytometry.The levels of cytokines(IL-1β,TNF-α,IFN-γ,IL-4,IL-6,IL-10,IL-17A)and oxidative stress-related enzymes(MPO,GSH-Px)in colon tissue were measured by ELISA.Results(1)DSS group mice had increased disease activity,obvious symptoms of bloody diarrhea,severe colonic mucosal defects,and diffuse infiltration of inflammatory cells in the lamina propria.In contrast,LP-315 intervention is beneficial to symptom improvement and histological remission.The DAI score of LP group decreased significantly on the 21st day(P<0.01),and there was no significant difference in the score of PM group.Both LP group and PM group could significantly alleviate the decrease of colon length/weight ratio caused by DSS(P<0.05;P<0.05),and reduce the histopathological score(P<0.01;p<0.01).However,the improvement of the above parameters in the MS group was not significant.(2)The intestinal barrier of mice in the DSS group was significantly damaged.The positive fluorescence of TJs protein was dim and the distribution was extremely scattered.The goblet cells were almost completely destroyed and positive staining was observed sporadically.In contrast,LP-315intervention is beneficial to the recovery of intestinal barrier structure and function.The fluorescence distribution and average intensity of each treatment group were improved to some extent,only the expression of Occludin in LP group was significantly different(P<0.01).The number and distribution density of goblet cells were significantly improved in LP group and PM group,and the positive staining almost filled the whole crypt.The number of goblet cells in MS group increased,but the distribution was uneven.(3)In the DSS group,the spleen was enlarged and the color was deepened.The CD4~+T cell subsets in the spleen showed excessive polarization of Th1 and Th17 and inhibition of Th2 polarization.The colonic cytokine network was disordered and the expression of stress-related enzymes was abnormal.In contrast,LP-315intervention is beneficial to immune regulation and immune homeostasis.CD4~+T cell subsets:Th1/Th2 ratio in LP group,PM group and MS group was significantly decreased(P<0.01;P<0.01;P<0.05),but the ratio of the former two was closer to that of NC group.The proportion of Th17 in different treatment groups was decreased,and the decrease in LP group and PM group was significant(P<0.01;P<0.01).Cytokine profile and oxidative stress:The pro-inflammatory cytokine IFN-γwas significantly decreased and the anti-inflammatory cytokine IL-4 was significantly increased in LP group(P<0.05;P<0.05).There was no significant difference in the decrease of IL-17A and the increase of IL-10.The levels of IL-1β,TNF-αand MPO in PM group were significantly decreased(P<0.01;P<0.05;P<0.05),the level of IL-4was significantly increased(P<0.05),and the levels of IL-1βand IL-6 in MS group were significantly decreased(P<0.05;P<0.05).(4)The correlation analysis immune markers and pathological phenotypes showed that pro-inflammatory immune markers were positively correlated with pathological parameters,while anti-inflammatory markers were the opposite.Conclusions Lactiplantibacillus plantarum LP-315 showed a positive improvement effect on DSS-induced IBD model in mice,and its combination with 5-ASA had a specific advantage in regulating the levels of multiple cytokines.It may be that LP-315 combined with drugs on the basis of mediating immune regulation and balancing immune microenvironment further strengthened the anti-inflammatory effect.