| Endometritis is a kind of uterine inflammatory injury that is commonly induced by mechanical trauma or the invasion of pathogenic microorganisms in dairy cows.It can lead to failed embryo implantation,repeated abortions,infertility,and other diseases,which significantly lead to the reduction of reproductive effectiveness in dairy cows,is detrimental to the dairy industry’s healthy and sustainable growth,and the global dairy industry suffers significant financial losses as a consequence.In spite of the fact that research on cow endometritis has been steadily expanding over the last couple of years,there are numerous pathogenic bacteria and the pathophysiology is not well understood,as a result,present therapeutic options have significant limits.Antimicrobial drugs have high efficiency in killing pathogenic bacteria,making them the preferred option for treating endometritis in dairy cows in the past.However,non-resistant breeding is being promoted at present,it is forbidden to use a large number of antimicrobial drugs,so new treatment approaches are in urgent demand to supplant them.Micro RNA(miRNA)can indirectly engage in a range of biological regulatory mechanisms via target genes,including cell differentiation,proliferation,apoptosis,and tumorigenesis,which has extremely strong effect on maintaining the health of organisms and avoiding illness.miR-26 a is a multifunctional micro RNA from the miR-26 family that represents a major regulatory role in acute nephritis and lung damage.Nevertheless,the function and regulatory mechanism of miR-26 a in dairy cow endometritis remain unknown.In this paper,the inflammatory endometrial tissue of dairy cows was taken as the research object,focusing on the relationship between miR-26 a and its potential target gene MAP3K8.Searching for the miR-26 a and MAP3K8 regulation mechanism in the inflammatory endometrium of dairy heifers,exploring novel ways for treating endometritis,and providing new suggestions for other researchers.The main research contents and experimental results are as follows:(1)Uterus horn tissue of healthy cows and cows with obvious clinical symptoms of endometritis were collected,through histopathological observation,MPO fluorescence activity measurement,and RT-q PCR experiment,it was determined that the uterine tissue structure of the disease group was severely abnormal,a large number of inflammatory cells infiltrated,MPO activity increased markedly,and the inflammatory factors such as IL-1β,IL-6,and TNF-α also increased in expression,implying that there was indeed an inflammatory reaction in the uterine tissue of the diseased dairy cows.The expression of miR-26 a decreased in the disease group,while the levels of m RNA,protein and fluorescence level of MAP3K8 decreased in the diseased group.The above findings imply that the expression levels of miR-26 a and MAP3K8 are associated with inflammatory development,which may affect the occurrence of endometritis in dairy cows.(2)Utilizing target gene prediction databases such as Target Scan and miRDB,it was found that there may be a targeting connection between MAP3K8 and miR-26 a.The luciferase activity of wild and mutant MAP3K8 3’-UTR double luciferase reporter gene vectors was measured.The results showed that the luciferase activity of wild-type vector decreased when miR-26 a mimics was transfected,indicating that miR-26 a has a silencing impact on MAP3K8 and validating their targeting connection.(3)LPS was used to produce the inflammatory model of bovine endometrial epithelial cell lines(b EECs).Experiments with CCK-8 demonstrated that LPS would not adversely affect the activity of b EECs.RT-q PCR experiment showed that the expression level of miR-26 a was highly correlated with the stimulation time and dose of LPS.miR-26 a mimics were transfected into b EECs,it was discovered that MAP3K8 was decreased in m RNA and protein levels,as well as the phosphorylation levels of proteins located in the downstream of the MAPK pathway,such as ERK and MEK,and the release of major inflammatory factors.However,the results were reversed when miR-26 a inhibitor was transfected.Transfection of si-MAP3K8 silenced MAP3K8 and significantly inhibited the expression of its downstream protein and the release of inflammatory factors,which was highly consistent with the transfection of miR-26 a mimics.And it is affirmed that MAP3K8 has an important biological function in the process of regulating endometritis by miR-26 a.(4)In order to further prove the influence of MAP3K8 on endometritis,this study successfully established a mouse model of acute endometritis by injecting LPS.It was found that the m RNA,protein and fluorescence levels of MAP3K8 in the uterus of mice stimulated by LPS increased significantly,and the phosphorylation levels of ERK and MEK increased as well as the inflammatory factors,which was consistent with the results obtained in the uterus samples of dairy cows.Conclusion: The above results showed that miR-26 a inhibited the activation of MAPK signaling pathway by targeting MAP3K8,and alleviated the damage of inflammatory reaction to the endometrium of dairy cows. |
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