Therapeutic Effect And Mechanism Of Clostridium Butyricum On Escherichia Coli-induced Endometritis In Mice

Endometritis is a common disease in dairy cows and one of the common causes of infertility in dairy cows,accounting for more than 60% of infertility in adult heifers.According to statistics,the incidence of endometritis in dairy cows in China is about15% to 60%,which seriously affects the fertility and performance of dairy cows,increases the dead paw rate of dairy cows,and causes large economic losses to the livestock breeding industry.The treatment of endometritis is still based on antibiotics,but the heavy use of antibiotics often leads to problems such as milk waste,antibiotic resistance and drug residues.Therefore,it is very important to develop antibiotic alternatives.The role of uterine flora in endometritis is receiving increasingly widespread attention.It has been found that,under normal conditions,there are specific microbial communities in the reproductive tract,including the uterus,which play a crucial role in the development of the uterus and resistance to pathogenic infections,and good results have been achieved in the treatment of endometritis by uterine infusion of probiotics.Therefore,this study aimed to analyze the uterine secretion flora of healthy and endometriotic cows,screen potential probiotics,verify their therapeutic effects and mechanisms on endometritis,and lay the foundation for the development of microecological preparations for the prevention and treatment of endometritis in dairy cows.Firstly,we collected uterine secretions from healthy and endometritis cows from a cattle farm in Shandong Province,and then performed microbiome analysis.The results revealed significant differences between the microbial composition of uterine secretions from healthy and endometritis cows.At the phylum level,Firmicutes,Bacteroidetes,Proteobacteria,Fusobacteriota and Actinobacteria were more abundant in the two groups of samples.At the genus level,Escherichia-Shigella abundance was higher in the endometritis group than in the healthy group,while Clostridium abundance was lower in the endometritis group than in the healthy group.LEf Se analysis showed that Pseudomonas,Rikenellaceae and Burkholderiaceae were enriched in the uterus of dairy cows with endometritis,while Clostridiaceae,in particular,Clostridium butyricum is abundant in the uterus of healthy dairy cows.Subsequently,to investigate the effect of Clostridium butyricum and its culture supernatant on endometritis.We established a mouse endometritis model by infusing E.coli into the uterus of mice based on the results of uterine microbiome analysis and previous research methods.The mice were then injected with Clostridium butyricum and its culture supernatant separately in the uterus and tested for uterine inflammatory response,while ampicillin treatment was used as a positive control group.The results showed that Clostridium butyricum and its culture supernatant significantly inhibited E.coli-induced histopathological damage,uterine index,and uterine tissue concentrations of TNF-α and IL-1β in mice the anti-inflammatory effect of culture supernatant was superior to that of viable Clostridium butyricum and ampicillin.In addition,Clostridium butyricum and its culture supernatant significantly reduced the E.coli load in the uterus,but they were not as effective as antibiotics.Culture supernatant had a better antimicrobial effect than viable Clostridium butyricum.Finally,to investigate the anti-endometritis effective components of Clostridium butyricum culture supernatant,short-chain fatty acids in the supernatant were analyzed by targeted metabolomics.The results showed that butyric acid was the main shortchain fatty acid.Subsequently,we compared the therapeutic effects of sodium butyrate and Clostridium butyricum culture supernatant on E.coli induced endometritis in mice.The results showed that sodium butyrate could significantly inhibit E.coli induced endometritis in mice,and there was no statistical difference between its therapeutic effect and that of Clostridium butyricum culture supernatant,indicating that butyrate was the main anti-endometritis molecule in Clostridium butyricum culture supernatant.To explore the anti-endometritis mechanism of sodium butyrate,we examined the signalling pathways such as NF-κB and Nrf2/HO-1,and the results showed that sodium butyrate significantly inhibited E.coli-induced activation of NF-κB signalling pathway,promoted protein expression of Nrf2/HO-1,and reduced the level of oxidative stress.These results suggest that Clostridium butyricum may inhibit E.coli-induced endometritis in mice by regulating NF-κB and Nrf2/HO-1 signalling pathways through the metabolite butyric acid.In conclusion,this study showed that the uterine microbiota of postpartum cows with endometritis differed from that of healthy cows,and the uterus of diseased cows contained reduced levels of Clostridium butyricum.Clostridium butyricum could inhibit E.coli-induced endometritis in mice by modulating NF-κB and Nrf2/HO-1antioxidant signalling pathways through its metabolite butyric acid.This suggests that Clostridium butyricum or butyric acid may be a new treatment agent for cow endometritis in the future.