Research On The Intervention Mechanisms Of Lactobacillus Fermentum On LPS-induced Inflammatory Injury Of Endometrial Epithelial Cells In Bos Taurus

[Objective] One of the reasons for the huge losses in dairy farming in recent years is endometriosis,which is more common in postpartum and mostly hidden.Researchers have been researching safer and more effective treatments.The application of probiotics has been a hot topic in recent years.Its benefits are non-toxic and safe.It does not produce drug residues in animals.When animals do not have diseases,they can also strengthen the body's immune system and prevent diseases.The role.This experiment aims to study the regulation of LPS-induced bovine endometrial cells through the determination of inflammatory factors and inflammatory pathways to provide the basis for the clinical application of Lactobacillus fermentum.[Materials and Methods] The cow endometrial cells were stably cultured in the laboratory,and the optimal concentration and time of LPS acting on the cells were determined by CCK-8 method;the accuracy of the purchased bacteria was determined by 16 S rDNA sequence analysis and homology comparison,and CCK was used.The optimal concentrations of-8 cells of L.fermentum and LPS were used together.The levels of IL-1?,IL-6,IL-8 and TNF-? were measured by ELISA to determine the change of inflammatory cytokine secretion.Real time fluorescence was used.Quantitative PCR was used to detect the expression of IL-1?,IL-6,IL-8,inflammatory cytokines of TNF-?,and TLR4,NF-?B(P65),and MyD88 inflammatory pathway genes [Results] Using the CCK-8 assay,it was found that LPS did not show toxic effects in a short duration of action.On the contrary,low-level stimulation stimulated cell growth slightly,but it prolonged inflammation in bovine endometrial epithelial cells.When the effect was 24 h and the concentration was 80?g/mL,the relative survival rate of bovine endometrial epithelial cells was significantly lower than that of the control group(p<0.05).When the concentration was greater than 80?g/mL,Compared with the control group,the relative survival rate of bovine endometrial epithelial cells decreased more significantly(p<0.01).When the action time was 48 h,LPS exerted a potent toxicity effect,compared with the control group,bovine utero The relative survival rate of membrane cells was significantly reduced(p<0.01);LPS and Lactobacillus fermentans were used in combination with bovine endometrial cells.Lactobacillus fermentum could alleviate the cytotoxicity caused by LPS for a short time.The effect time was 2h and the concentration was 108 CFU/mL.The relative survival rate of membrane epithelial cells was significantly increased(p<0.05),but the long-term stimulation of Lactobacillus fermentum also played a toxic role;by ELISA enzyme-linked immunosorbent assay found: compared with the control group,LPS group TNF The secretion of ?-,IL-1?,IL-6 and IL-8 in inflammatory cytokines was significantly increased(p<0.01).Compared with LTA group,IL-1? and IL-6 were detected in LPS+LF group.The secretion of inflammatory cytokines,IL-8 and TNF-? were significantly decreased(p<0.05).Compared with the control group,the LF group could increase the secretion of four inflammatory cytokines to varying degrees(p> 0.05,p<0.05),but compared with the increase in LPS group,the LF group was normal stress response.This indicated that Lactobacillus fermentum could alleviate the excessive secretion of inflammatory cytokines from bovine endometrial cells induced by LPS.The real-time fluorescence quantitative PCR assay showed that compared with the control group,the relative expression levels of IL-1?,IL-6,IL-8,TNF-?,TLR4,MyD88,and NF-kB(p65)mRNA in the LPS group Significant increase(p<0.01);relative expression of TNF-?,IL-1?,IL-6,IL-8,TLR2,MyD88,NF-?B(p65)mRNA in LPS+LF group compared with LPS group The amount was significantly reduced(p<0.01);compared with the control group,mRNA expression levels of the seven genes in the LF group increased to varying degrees(p>0.05,p<0.05,p<0.01),but were lower than LPS Group,within normal stress response.Thus,it was shown that Lactobacillus fermentum can significantly reduce the high expression of inflammatory related gene mRNA induced by LPS.[Conclusion] 1,80?g/mL LPS incubated cells for 24 h,can establish a good endometrial inflammatory damage model;2,fermentative Lactobacillus can reduce the inflammatory factor IL-1?,IL-6,IL-8 and TNF-? content,At the same time,it can reduce the gene expression of TLR4,NF-?B,MyD88 inflammatory pathway genes and IL-1?,IL-6,IL-8,and TNF-? inflammatory factors,indicating that this Lactobacillus fermentum LPS induced bovine uterine cells The inflammation model has a regulatory role and provides a theoretical basis for clinical application.