Lactobacillus Rhamnosus GG Inhibits The Intestinal Inflammatory Response Induced By Highly Pathogenic Klebsiella Pneumoniae

Objective:To clarify the inhibitory effect of Lactobacillus rhamnosus GG(LGG)and its supernatant on the inflammatory response caused by highly pathogenic Klebsiella pneumoniae,and further verify that LGG can activate Toll-like receptors(Toll-like receptors,TLRs)and the downstream nuclear factor kappa B(NF-κB)molecules further inhibit the related molecular mechanisms of inflammatory response caused by highly pathogenic K.pneumoniae,providing new clues for the prevention and treatment of highly pathogenic K.pneumoniae infection in clinic.Methods:1.The MTT method was used to assess the effect of LGG supernatant with varying pH values on Caco-2 cell activity.2.To clarify the process of highly pathogenic K.pneumoniae infecting intestinal cells(Caco-2),three modes of action were used: pretreatment of LGG and LGG supernatant(adding LGG and LGG supernatant to incubate cells before K.pneumoniae),co-incubation(adding LGG and LGG supernatant to incubate cells simultaneously with highly pathogenic K.pneumoniae),and treatment(adding LGG and LGG supernatant to incubate cells after K.pneumoniae.3.LDH release was used to confirm the protective effect of LGG and LGG supernatant on Caco-2 cell damage caused by highly pathogenic K.pneumoniae.4.The effects of LGG and its supernatant on the expression of TNF-α,IL-8,IL-1β,IL-10 and TGF-β mRNA in Caco-2 induced by highly pathogenic K.pneumoniae were detected by real-time quantitative PCR.5.Enzyme-linked immunosorbent assay(ELISA)was used to detect the changes of the concentrations of cytokines IL-10 and TNF-α in the supernatant of Caco-2 cells induced by highly pathogenic K.pneumoniae.6.The effects of LGG and LGG supernatant on the mRNA expression of myeloid differentiation primary response gene 88(My D88)mRNA Caco-2 cell receptor TLR2,TLR4,and TLRs signal pathway induced by highly pathogenic K.pneumoniae were investigated using real-time quantitative PCR.7.Real-time quantitative PCR was used to detect the effects of LGG and LGG supernatant on the mRNA expression of NF-κB and SIGIRR in Caco-2 cells induced by highly pathogenic K.pneumoniae.8.Western blot was used to detect the effects of LGG and LGG supernatant on the expression of NF-κB protein and nuclear translocation of NF-κB p-65 subunit in Caco-2 cells induced by highly pathogenic K.pneumoniae.9.After infecting C57BL/6N male mice with highly pathogenic K.pneumoniae for 24 hours,the mice were gavage-treated for 7 days with LGG and LGG supernatant,and their daily weight changes were recorded.The mice were killed and dissected after 7 days,and the colon tissues were collected for future experiments.10.Real-time quantitative PCR was used to detect the effects of LGG and its supernatant on the expression of cytokines TNF-α,IL-6,IL-18,IL-1β and IL-10 mRNA in colon tissue caused by highly pathogenic K.pneumoniae.11.Real-time quantitative PCR was used to detect the effects of LGG and LGG supernatant on the expression of TLR2 and TLR4 mRNA in colon tissue induced by highly pathogenic K.pneumoniae.12.The effects of LGG and its supernatant on the expression of NF-κB protein and nuclear translocation of NF-κB p-65 subunit in colon tissue induced by highly pathogenic K.pneumoniae were detected by real-time quantitative PCR and Western blot.Results:1.LGG and LGG supernatant decreased the expression of TNF-α,IL-8 and IL-1β of Caco-2,and increased the expression of IL-10 and TGF-β of anti-inflammatory cytokines.In these three treatment modes,LGG and LGG supernatant increased the expression of TLR2 mRNA,inhibited the increase of TLR4 expression caused by K.pneumoniae,further inhibited the transcription of NF-κB,promoted the transcription expression of the negative regulator of NF-κB SIGIRR,and inhibited the nuclear translocation of NF-κB p-65 subunit in cytoplasm.2.After oral administration of K.pneumoniae,the weight of mice decreased;The expression of pro-inflammatory cytokines TNF-α,IL-6,IL-18 and IL-1β in intestinal tissue increased,while the expression of anti-inflammatory cytokine IL-10 decreased.Oral administration of LGG and LGG supernatant for 7 days significantly reduced the transcription of pro-inflammatory cytokines in mouse colon tissue and promoted the expression of IL-10.In addition,oral administration of LGG and LGG supernatant increased transcription of receptor TLR2,decreased transcription expression of TLR4 and NF-κB,and inhibited nuclear translocation of NF-κB p-65 in the cytoplasm of colon tissue.Conclusion:Through the NF-κB signaling pathway,LGG and its supernatant affect the expression of pro-inflammatory and anti-inflammatory cytokines,effectively reducing the inflammatory response caused by highly pathogenic K.pneumoniae.