| BackgroundInflammatory bowel diseases(IBD),including Crohn’s disease(CD)and ulcerative colitis(UC),are characterized by chronic and uncontrolled intestinal inflammation.Due to progressive activation of the immune system,IBD can lead to additional potential complications such as fibrosis,stenosis,or colon malignancy.Therefore,anti-inflammatory drugs composed of 5-aminosalicylic acid and glucocorticoids are conventional therapies for IBD.Because long-term use of these drugs has clear side effects,many alternative therapeutic approaches such as probiotics and fecal bacteria transplantation have become research focuses.Certain influential studies have highlighted that Lactobacillus rhamnosus GG(LGG)and its supernatant benefit IBD patients and IBD-like mouse models.Given that the outcome of probiotic therapy depends on the stage of the disease and the overall health status of IBD patients,strategies employing LGG-derived secretory protein,named HM0539,are more and more investigated.Research scopeWe previously found that the HM0539,a secretory protein from LGG culture supernatant,protects the intestinal barrier by the upregulation of intestinal mucin and zonula occludens-1(ZO-1)expression in E.coli K1-induced,DSS-induced and LPS/Dgalactosamine induced colitis,respectively.However,the detailed mechanism remains elusive.In the study,we aim to investigate the anti-inflammatory effects of HM0539 on the expression of TLR4/MyD88/NF-κB in LPS-induced RAW264.7 cells and its role in DSS-induced or OXA-induced colitis.MethodWe first evaluated the effects of HM0539 on LPS-induced macrophage viability via the MTT assay.The inhibitory effects of HM0539 on LPS-induced activation of TLR4/MyD88/NF-κB signaling cascades and COX2/iNOS protein levels were analyzed by Western blot.Next,we quantitated the extracellular accumulation of PGE2,NO,IL-1β,IL-6,IL-18,and TNF-α levels in RAW264.7 cells at the indicated time.Intracellular ROS levels were measured by fluorescence intensity and flow cytometry.After TLR4 and MyD88 overexpression,the TLR4/MyD88/NF-κB signaling responses and downstream cytokines will be determined by Western blot and ELISA,respectively.The effects of HM0539 on the clinical symptoms of DSS-induced and OXAinduced colitis in mice were evaluated by daily body weight change,hematochezia score,disease activity index and colon length loss.Conditions of colon tissue damage were determined by H&E staining.Oxidative stress markers,such as NO,MPO,MDA,and SOD,were determined by respective assays.The protein levels of the TLR4/MyD88/NF-κB pathway and Bax/Bcl-2/caspase-3 were determined by WB.ResultHM0539 improved the cell viability and ameliorated morphological changes in LPS-induced RAW264.7 macrophages by downregulating the protein levels of TLR4/MyD88/NF-κB signaling pathways and COX2,iNOS levels.The accumulation of PGE2,NO,ROS,IL-1β,IL-6,IL-18,and TNF-α accumulation also reduced by HM0539.HM0539 pretreatment reduced the effect of TLR4 overexpression and MyD88 overexpression on TLR4/MyD88/NF-KB signaling responses and the production of downstream cytokine in LPS-induced RAW264.7 cells.HM0539 could significantly improve the clinical symptoms of DSS-induced and OXA-induced colitis in mice.Treatment with HM0539 mitigated DSS-induced colon tissue damage.HM0539 alleviated DSS-induced oxidative stress in mice.Treatment with HM0539 downregulated protein levels of TLR4/MyD88/NF-κB signaling pathway in the colon tissue of DSS-induced mice.Last but not least,HM0539 downregulated Bax and caspase-3 in colon tissue of DSS-induced mice,meanwhile upregulated Bcl-2 levels in mice.ConclusionHM0539,a novel derived protein from Lactobacillus rhamnosus GG,showed remarkable anti-inflammatory effects through inhibition of TLR4/MyD88/NF-κB signaling in LPS-induced RAW264.7 macrophages and DSS-induced colitis in mice,which provide potential for IBD treatment. |
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