| Endometritis is a superficial diffuse inflammation of the uterine tissue,with an incidence ranging from 37% to 54% in dairy cows,causing huge economic losses to the cattle industry.Endometritis can lead to reduced milk production,hinder follicle growth,disrupt the ovulation process,and delay ovulation.It can also lead to irregular ovarian cycles that can reduce the reproductive capacity of dairy cows and,in more severe cases,can lead to infertility.Endometritis occurs when the uterine epithelial cells are damaged,resulting in a massive influx of pathogenic microorganisms and local colonization of the uterus.At present,the clinical treatment of cow endometritis mainly relies on the combination of several antibiotics.However,due to the emergence of antibiotic resistance and residues in dairy products,the development of safe,effective and green drugs has become very important.Alternative immunomodulatory therapy is expected to be an effective alternative to antibiotics.MicroRNAs(mi RNAs),belonging to a class of evolutionarily conserved small non-coding RNA of ～22 nucleotides,are widely involved in endometritis pathogenesis and development by regulating gene expression at the post-transcriptional level.The mi RNAs are expressed in various organs and cells,including endometrium,regulating pro and anti-inflammatory actions.Seven hundred and six mi RNAs have been reported in bovine endometrium,including miR-24,which constituted the mi RNA network regulating more than two-thirds of all protein-coding genes.MicroRNAs depictsamajor component of the innate immune system’s regulatory networks also serve as the first line of defence against both innate and adaptive immunological responses.MicroRNAs,being a gene regulator,may be adopted in regulating some cogent genes fingered in the pathogenesis of endometritis.There are several genes,and protein-coding genes reportedly aid the occurrence and prolongation of endometritis,and these include the cell receptors(TLR),cell signaling genes(TRAF6 and TBK1)and endometrial protein-coding genes(LGALS9 and SMAD9).The above mention genes have been reported in the pathogenesis and diagnosis of endometritis.However,the role of the stable transcript of miR-24;miR-24-3p in the regulation of endometritis and investigate its anti-inflammatory effects on lipopolysaccharide(LPS)-induced inflammation,and the possible molecular mechanisms,series of researches were carried out in this thesis to achieve this set goal.1.Exploration of Toll-like Receptor 4(TLR4)downregulation by miR-24-3p to modulate LPS-induced Endometritis through the NF-κB signaling pathway.The effect of miR-24-3p in down-regulating TLR4 was investigated.LPS was infused into mice uteri to induce endometritis and LPS-treated bovine endometrial epithelial cells(BEECs),and H&E staining of the uterine tissue were performed.Histopathological analysis inflamed uteri revealed LPS induced severe pathological changes,suggested that mouse endometritis model was well established.MiR-24-3p mimics,negative control,inhibitor,negative control,Si-TLR4,and Si-Negative control with GP Transfect-Mate were added to BEEC following the manufacturer’s description 6hours later,3ug/ml LPS was added to the required plates for 24 hours.The expressions of miR-24-3p and TLR4 were measured via quantitative real-time polymerase chain reaction(RT-qPCR)and western blot(WB).At the same time,inflammatory cytokines secretion was assessed via ELISA(enzyme-linked immunosorbent assay)and RT-qPCR techniques.MiR-24-3p overexpression or inhibition suppressed or elevated LPS-induced pro-inflammatory cytokines,and overexpression deactivated the NF-κB pathway.Knockdown of TLR4 inhibited LPS-induced inflasmmatory responses.The interaction between miR-24-3p and TLR4 was validated by luciferase reporter assay,immunofluorescence assay,and RT-qPCR.Bta-miR-24-3p was decreased with increased TLR4 level.Subsequently,we further verified that miR-24-3p repressed TLR4 expression by binding to the 3′-UTR of TLR4 m RNA.Cloning of TLR4(pcDNA3.1(+)TLR4)into BEECs with transfection of miR-24-3p corroborates the effect of miR-24-3p on inflammation.2.Evaluate the negative regulation of lipopolysaccharide-induced endometrial inflammatory response by miR-24-3p targeting TNF receptor-associated factor 6(TRAF6).Whole mice uteri and bovine endometrial epithelial cells(BEECs)were separately stimulated with LPS.The BEECs were also transfected with miR-24-3p mimic,negative control,SiTRAF6,SiNC;pcDNA3.1empty and pcDNA3.1(+)TRAF6 separately with LPS stimulation.The expression levels of miR-24-3p and TRAF6 were measured via RT-qPCR and WB techniques.Bothinflammatory cytokines secretion and expression in LPS-induced inflammatory response was assessed by ELISA and qRT-PCR respectively.Bioinformatics analysis and dual-luciferase reporter assay validated the interaction between miR-24-3p and TRAF6.The activation of the NF-κB/MAPK pathway and p65 phosphorylation was investigated by western blot and immunofluorescence assay.The expression of miR-24-3p was decreased,and TRAF6 was elevated with an increased level of pro-inflammatory cytokines in LPS-treated BEECs and mice uterus.The overexpression of miR-24-3p suppressed LPS-induced secretion of inflammatory cytokines(IL-1β,IL-6,IL-8 and TNF-α)and deactivation of NF-κB/MAPK pathways.The downregulation of TRAF6(SiTRAF6)inhibited LPS-induced inflammatory response in BEECs.TRAF6 is validated as a target of miR-24-3p,and miR-24-3p reversed the overexpression of cloned TRAF6 on inflammation response in BEECs.3.Study of the therapeutic potential of miR-24-3p in attenuating LPS-induced endometrial inflammatory responses by targeting TBK1.The mice uteri and bovine endometrial epithelial cells(BEECs)were separately stimulated with LPS.The BEECs were also transfected with miR-24-3p mimic,negative control,SiTBK1 and SiNC;pcDNA3.1empty and pcDNA3.1(+)TBK1 separately with LPS stimulation.The study revealed that miR-24-3p and TBK1 expression in lipopolysaccharide(LPS)-triggered mice endometritis model and LPS-stimulated endometrial epithelial cells were significantly decreased and increased respectively with the elevated secretion of pro-inflammatory cytokines.The result showed that overexpression of miR-24-3p markedly decreased the expression of TBK1 and pro-inflammatory cytokines.In addition,overexpression of miR-24-3p also suppressed NF-κB p65 activation by targeting the TBK1,adaptor molecule mediated pathway.Bioinformatics analysis validated the molecular interaction between miR-24-3p and TBK1,and a dual-luciferase reporter assay confirmed that miR-24-3p might directly target the 3’-untranslated region of TBK1.Silencing TBK1(SiTBK1)yields a similar result as obtained following the overexpression of miR-24-3p.Recombinant overexpression of TBK1 could not alter the effect of miR-24-3p on the inflammatory response in BEECs.4.Investigating the involvement of MiR-24-3p/ SMAD family member 9(SMAD9) molecular interactions in regulating endometrial inflammation.The RT-qPCR assay indicated that SMAD9 expression in the uterine tissues of mice with endometritis and BEECs with LPS stimulation was significantly elevated.The overexpression of miR-24-3p significantly decreased the expression of SMAD9 and inflammatory cytokines(TNF-α,IL-6,IL-8,and IL-1β)and the phosphorylation of NF-κB p65 and IκBα.Similar results were also obtained following the knockdown of SMAD9(Si-SMAD9).Furthermore,a dual-luciferase reporter assay further validated that miR-24-3p inhibited SMAD9 expression by binding directly to the 3′-UTR of SMAD9.Likewise,overexpression cloning of SMAD9,(pcDNA3.1(+)SMAD9)could not halt the anti-inflammatory potential of miR-24-3p.5.Effect of bta-miR-24-3p on Galectin-9 Expression in LPS-Stimulated Bovine Endometrial Epithelial Cells through TLR4/NF-κBsignaling Pathway.LPS-treated bovine endometrial epithelial cells(BEECs)were cultured to investigate the role of bta-miR-24-3p.The expression levels of bta-miR-24-3p and galectin-9(LGALS9)were measured by RT-qPCR was downregulated and upregulated,respectively.The LPS-induced inflammatory response was assessed by the elevated secretion of inflammatory cytokines measured using ELISA and quantitative RT-qPCR.WB assessed activation of nuclear factor-κB(NF-κB)and TLR4 pathway.The interaction between bta-miR-24-3p and LGALS9 was validated by bioinformatics analysis and a luciferase reporter assay.LPS-induction in BEECs with bta-miR-24-3p was overexpressed,leads inhibition of pro-inflammatory cytokines,LGALS9 expression,and TLR4/NF-B pathway deactivation.Knockdown of LGALS9(Si-LGALS9)inhibited the LPS-induced inflammatory response in BEECs.LGALS9 was validated as a target of bta-miR-24-3p.Cloned overexpression of LGALS9(pcDNA3.1(+)LGALS9)failed to alter the effect of bta-miR-24-3p on the inflammatory response in BEECs.Overall,bta-miR-24-3p attenuated the LPS-induced inflammatory response via targeting LGALS9.In conclusion,miR-24-3p was incriminated and suppressed in the pathogenesis of endometritis,regulating the expression of inflammatory promoter endometrial specific genes and signaling pathways with their adaptor molecules.The overexpression of miR-24-3p and knockdown of the specific genes attenuated the LPS-induced endometrial inflammation with the deactivation of the cellular inflammatory signaling pathways.The possible anti-inflammatory mechanism of miR-24-3p was related to the inhibition of TBK1 and TRAF6-dependent,elevated endometrial specific genes(LGALS9 and SMAD9),and TLR4/NF-κB/MAPK signaling pathways.Therefore,the mechanism involves upregulating miR-24-3p that can target the different genes and signaling pathways,thereby counteracting the effect of inflammatory reaction is an important therapeutic baseline. |
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