Effects Of Three Strains Of Lactobacillus On LPS-induced Inflammation Of Cow Endometrial Epithelial Cells

Endometritis is one of the important diseases affecting the production of dairy cows,and effective prevention and treatment of the disease is essential.Antibiotics are currently widely used to treat the disease,but long-term use can lead to problems such as strain resistance and drug residues.Therefore,new preventive and therapeutic drugs need to be developed to prevent and control endometritis.In this paper,three strains Lactobacillus johnsonii,Lactobacillus salivarius and Lactobacillus weissella were used to prevent LPS-induced inflammatory response in cow endometrial epithelial cells.Firstly,the growth curve of three Lactobacillus strains was plotted by the plate counting method,and the change of Lactobacillus OD₆₀₀ was also examined.Then the Oxford Cup method was used to detect the inhibitory effect of the three Lactobacillus supernatants on the main pathogens of cow endometritis.The results showed that the supernatants of the three strains of Lactobacillus had significant inhibitory effects on the main pathogenic bacteria（P<0.05）.After that,the cow endometrial epithelial cells were treated with 1μg/mL LPS for 0,3,6,9,12,and 24 h,and the cell death rate was calculated by measuring the release amount of LDH.None of the treatments in the time period caused significant damage to the cells（P>0.05）.The expressions of cytokines IL-8 and TNF-αwere detected by ELISA and fluorescence.The results showed that the expression of cytokine IL-8 and TNF-αprotein and mRNA increased significantly after treatment of cells at 1μg/mL for 3 h.（P<0.05）,in order to successfully establish an in vitro inflammation model.Three suspensions of Lactobacillus were diluted to 10⁴,10⁵,10⁶ CFU/mL.The results of LDH showed no significant damage to cells（P>0.05）.After co-culture with cow endometrial epithelial cells for 3 h,1μg/mL LPS was added for 3 h to detect the secretion of chemokine IL-8 and pro-inflammatory factor TNF-αprotein and mRNA.The results showed that the expression of IL-8 and TNF-αincreased significantly after LPS treatment with the addition of10⁴ CFU/mL of Lactobacillus johnsonii for 3 h（P<0.01）.The results of pretreatment with Lactobacillus salivarius and Lactobacillus faecalis treated with 10⁵ and 10⁶ CFU/mL were similar,and there was no significant change in IL-8 and TNF-αprotein and mRNA expression（P>0.05）.In order to explore the anti-inflammatory mechanism of Lactobacillus johnsonii on the endometrial epithelial cells of dairy cows,the expression levels of p-p65,p65,p-IκB-αand IκB-αwere detected by Western blot.The results showed that the ratio of p-p65/p65 and p-IκB-α/IκB-αwas significantly decreased after the addition of 10⁴ CFU/mL of Lactobacillus johnsonii for 3 h（P<0.05）.The anti-inflammatory effect of Lactobacillus is accomplished by the NF-κB signaling pathway.In summary,10⁴ CFU/mL Lactobacillus johnsonii has the effect of preventing endometritis in dairy cows,which exhibits a good anti-inflammatory effect and can be used as an alternative strain for the development of microecological preparations.