Study On Lactobacillus Paracasei And Its Exopolysaccharide In Alleviating Colitis In Mice

Ulcerative colitis(UC)is a chronic inflammatory disease of the intestinal tract caused by various factors such as environment,mental health,and poor work habits.Its pathogenesis is still unknown,but its onset causes severe damage to the intestinal mucosa,leading to changes in the intestinal environment,abnormal immune response,and symptoms such as diarrhea,weight loss,and bloody stools.At present,drugs can be selected according to the patient’s own condition to alleviate the condition,but after taking the drug,patients will have a series of side reactions that will lead to a decrease in the quality of life,and at the same time,the frequency and measurement of its use will cause problems such as increased medical burden.A growing number of trials have demonstrated the efficacy of probiotic complex lactic acid bacteria exopolysaccharides in alleviating intestinal inflammation,with potential research value in modulating the immune system and improving host health.Previous studies by our group found that Lactobacillus paracasei JY062and its extracellular polysaccharide can maintain intestinal flora balance,anti-inflammatory and other health functions.However,the research foundation on the mechanism of JEC alleviating UC is relatively weak,especially the experience on intestinal mucosal barrier and body inflammation after combination is insufficient.L.paracasei JY062 isolated from traditional Tibetan fermented dairy products and its extracellular polysaccharide were studied.A human colon cancer cell model stimulated by lipysaccharide was established to study whether L.paracasei JY062 combined with extracellular polysaccharide can relieve intestinal inflammation,and to explore its possible mechanism of action.DSS was used to induce the establishment of UC mouse model,an in-depth study on the anti-inflammatory and immunomodulatory effects of L.paracasei JY062 complex extracellular polysaccharide on UC mice was conducted,providing a theoretical basis for the development of mixed microbial preparations to alleviate UC Through the above research,the following results are obtained:(1)Protective effect of JEC on LPS-induced Caco-2 cells.After JEC intervention,transcription levels of anti-inflammatory factors TGF-β,IL-10,ZO-1,tight junction protein Claudin-1,Occludin and MUC2 upregulated,and m RNA expression levels of pro-inflammatory factors TNF-α,IFN-γ,IL-6,IL-17,IL-1βand tight junction protein Claudin-2 decreased.Meanwhile,JEC reduced the apoptosis rate from 30.7%to 14.2%.It indicates that JEC can alleviate the intestinal barrier damage caused by LPS.(2)The alleviating effect of JEC on inflammatory response in UC mice.Compared with the DSS group,the JEC group showed a significant increase in thymic index(p<0.05),a highly significant decrease in splenic index(p<0.01),and a highly significant decrease in DAI score(p<0.0001);shortened swelling of the colon,disappearance of cupped cells,inflammatory cell infiltration,and mucosal damage were alleviated.JEC had a significant inhibitory effect on MPO activity and LPS levels(p<0.0001,p<0.01).It indicates that JEC can effectively reduce the inflammatory response in UC mice.(3)The enhancing effect of JEC on intestinal mucosal barrier function in UC mice.After DSS induction,the levels of pro-inflammatory factors TNF-α,IFN-γ,IL-1β,IL-6 and IL-17 in the colon were significantly increased(p<0.05),and the levels of anti-inflammatory factors IL-10 and TGF-βwere significantly decreased(p<0.05),JEC can effectively regulate the content of immune factors and improve the inflammatory response.Immune dysfunction caused abnormal expression of Ig G,Ig M and Ig A levels,and immunoglobulin levels decreased in the JEC group(p<0.001,p<0.0001).MUC2,ZO-1,Occludin and Claudin-1 protein expression were significantly upregulated after JEC intervention(p<0.05),effectively reversing the overproduction of Claudin-2,where the m RNA levels of ZO-1 and Occludin were close to those of the normal group.JEC was more effective in regulating macrophage imbalance caused by DSS,which polarized pro-inflammatory M1macrophages into anti-inflammatory M2 macrophages,as evidenced by a decrease in the recruitment of M1 colonic macrophages represented by CD68~+CD86~+and an increase in the number of M2colonic macrophages represented by CD206~+CD68~+,accompanied by JEC enhanced the differentiation rate of Treg cells(p<0.001,p<0.0001)and decreased the proportion of Th17 cells(p<0.01)in different tissues,demonstrating that JEC can effectively improve the Treg/Th17 immune imbalance.The above results demonstrate that JEC can enhance intestinal barrier function and effectively alleviate intestinal inflammation.(4)The regulatory effect of JEC on the intestinal microenvironment of UC mice.Com pared with the DSS group,JEC could increase the Alpha diversity index,and Beta diversit y analysis also showed significant differences in intestinal flora,and among the intestinal f lora composition,Firmicutes,Bacteroidota,Odoribacter,Lachnospiraceae＿NK4A136＿group,u nclassified＿Muribaculaceae,Akkermansia increased in abundance and Defluviicoccus,Strepto coccus and other harmful bacteria decreased in abundance,enhancing the intestinal microbi al barrier,with Akkermansia and Proteobacteria levels close to the NC group.By measurin g SCFAs,it was found that JEC could increase the contents of acetic acid,propionic acid,butyric acid,isobutyric acid,valeric acid and isovaleric acid to different degrees,among which the effect of enhancing the contents of propionic acid and isovaleric acid was more significant(p<0.05),and the concentrations of isobutyric acid and valeric acid were close t o those of the NC group,indicating that JEC could regulate intestinal metabolic functions and maintain the homeostasis of intestinal internal environment by upregulating the content s of SCFAs.(5)Inhibitory effects of JEC on inflammatory signaling pathways in UC mice.Analysis by RT-PCR combined with Western blotting revealed that JEC reduced the gene expression levels of TLR4,My D88,NF-κB p65,protein expression of TLR4,My D88,p-IκBα,NF-κB p65 and p-NF-κB p65,and inhibited the degradation of IκBαcaused by modeling drugs(p<0.05).Therefore,it is hypothesized that JEC attenuates intestinal inflammation and improves intestinal immune function in UC mice by inhibiting TLR4/My D88/NF-κB pathway.