Research On The Relieving Effect Of Lactobacillus Rhamnosus On Lipopolysaccharide-induced Intestinal Inflammation

Intestinal inflammation is one of the main gastrointestinal diseases which plagues humans.In recent years,the incidence in the world has increased year by year.Conventional therapeutic drugs have many side effects and are not suitable for long-term administration.Many studies have found that lactic acid bacteria(LAB)as an adjuvant drug have a positive effect in the treatment of intestinal inflammation.As one of the most widely studied LAB,Lactobacillus rhamnosus has a variety of probiotic functions.At present,some in vivo and in vitro tests have proven that L.rhamnosus can improve gastrointestinal function and relieve intes tinal inflammation.However,its specific mechanism is still unclear.Therefore,it is of great significance to analyze the mechanism of L.rhamnosus in relieving intestinal inflammation.In this study,L.rhamnosus JL-1 isolated from healthy infant feces was used as the research object.The adhesion capacity of the strain was evaluated by establishing an in vitro adhesion model of Caco-2 cells.In addition,the viability of the strain in acidic environment,bile salt environment and simulated gastrointestinal juice was evaluated by simulating the gastrointestinal environment.Then,the potential probiotic properties of the strain were further explored by whole genome sequencing and comparative genomics.We investigated the effects of L.rhamnosus JL-1 on intestinal inflammation by using lipopolysaccharide(LPS)induced Caco-2 cells inflammation model and mouse intestinal inflammation model.Finally,related regulatory signaling pathways were studied to clarify the mechanism.We obtained the following results through the study:(1)L.rhamnosus JL-1 has strong adhesion ability and ability to tolerate gastrointestinal environment.Its adhesion rate to Caco-2 cells was as high as 92.89%,which was significantly higher than that of other tested L.rhamnosus strains.The survival rates of the strain were 95.76% and 82.90% after 3 h incubation at p H 2.0 and p H 3.0,respectively.After culturing in 0.3%,0.5%,and 1.0% bile salt environments for 4 h,the survival rates were 97.88%,90.82%,and 90.01%,respectively.The survival rates of the strain were 95.65% and 88.07% when cultured in simulated gastric juice and intestinal juice,respectively.Therefore,L.rhamnosus JL-1 has the potential for colonization in the gastrointestinal tract.(2)The basic characteristics of the L.rhamnosus JL-1 genome were clarified,and the functional characteristics of the strain were excavated from the genetic level.The genome of L.rhamnosus JL-1 contains a circular chromosome and no plasmid.The length of the genome is 3007502 bp and GC content is 46.69%.A total of 2935 coding genes,59 t RNA genes and 15 r RNA operons were predicted in the genome.Comparative genomic analysis revealed that there were genes related to cell adhesion,acid tolerance and bile salt tolerance in the genome.There were 3700 pan genes,1740 core genes and 1960 dispensable genes in the 11 L.rhamnosus genomes.The core gene evolution tree was constructed by using maximum likelihood method and the result indicated that the genetic relationship between L.rhamnosus JL-1 and L.rhamnosus 4B15 were closer.By colinearity analysis,it was found that compared with other genomes,L.rhamnosus JL-1 and L.rhamnosus GG have good colinearity.There were no regions of translocation and inversion in the genome,which further proved the potential functional characteristics of the strain at the gene level.(3)L.rhamnosus JL-1 could reduce the inflammatory response of Caco-2 cells induced by LPS.LPS was used to construct Caco-2 cells inflammation model,and the effect of L.rhamnosus JL-1 on the in vitro inflammation model was investigated.The results show that L.rhamnosus JL-1 could reduce inflammation response by inhibiting the m RNA expression of proinflammatory factors TNF-?,IL-1? and IL-6 in LPS-induced Caco-2 cells(p<0.05),and the effects were dose-dependent to some extent.NF-?B signaling inhibitor PDTC reduced m RNA expression of TNF-?,IL-1?,and IL-6(p<0.01),which demonstrated that L.rhamnosus JL-1 reduced inflammatory response in LPS-induced Caco-2 by inhibiting NF-?B signaling pathway.(4)L.rhamnosus JL-1 could reduce intestinal inflammation and intestinal mucosal damage in mice induced by LPS.Mouse intestinal inflammation model was constructed by LPS to explore the effect of L.rhamnosus JL-1 on the in vivo inflammation model.The result showed that L.rhamnosus JL-1 relieved the pathological changes such as mental atrophy,weight loss,and intestinal wall thinning in mice,which effectively reduced intestinal inflammation and intestinal mucosal injury.m RNA expression of pro-inflammatory factors TNF-?,IL-1?,and IL-6 in ileum tissue were significantly reduced(p<0.05),and the effects were dose-dependent to some extent.(5)The mechanism of L.rhamnosus JL-1 in relieving intestinal inflammation induced by LPS was preliminarily analyzed.Real-time RT-PCR was used to analyze the expression of TLR4 and NF-?B genes in the TLR4-NF-?B signaling pathway.It was found that L.rhamnosus JL-1 can regulate the expression of downstream factors by inhibiting TLR4-NF-?B signaling pathway,thereby reducing intestinal inflammation and intestinal mucosal damage induced by LPS.