Molecular Mechanism Of IFN-? Induced MiR-505 Targeting HMGB1 To Inhibit Endometrial Inflammatory Injury In Dairy Cows

The success or failure of pathogenic microorganisms invading uterine tissue is directly affected by the integrity of the endometrium.Inflammatory damage caused by pathogenic microorganisms led by pathogenic Escherichia coli invading the endometrial tissue of postpartum dairy cows is an important cause of infertility in dairy cows.Variety of factors affect the repair process of inflammatory injury in endometrial tissue.IFN-?,which is unique to ruminants,Preliminary research in this laboratory has confirmed that it also mediates the innate immune response in the uterus of mice to improve local inflammation.However,its protective effect on the inflammatory injury of endometrial tissue in dairy cows has not been thoroughly revealed,and whether it is related to injury-related pattern molecule;particularly there are few studies on the molecular mechanism of IFN-?interfering with endometrial inflammatory injury in dairy cows,to stimulate bovine endometrial tissue and endometrial epithelial cells(BEND or b EECs).First,different concentrations of IFN-? were used to intervene with the inflammatory injury model of endometrial tissue in dairy cows,and the optimal stimulating concentration of LPS and the intervention concentration of IFN-? were screened by molecular biological methods such as fluorescence quantification and tissue immunofluorescence,and its protective mechanism on inflammatory injury of dairy cow endometrial tissue was revealed.Secondly,the application of IFN-? to interfere with the inflammatory injury model of the cells on the endometrium of dairy cows,and immunoblotting and gene silencing were used to reveal the regulation of HMGB1/NF-?B signaling on inflammatory damage of b EECs.and secondly,the expression profile of characteristic miRNAs with IFN-?interfering in dairy cow endometrial epithelial cells is screened by high-throughput sequencing technology.Screen characteristic miRNAs based on sequencing results and previous research basis,utilizing bioinformatics software to anticipate miRNA candidate genes;HMGB1/NF-?B protection principle were disclosed by handling dual luciferase gene experiment and target gene knockdown and other experimental methods.It provides a scientific rationale for clinical application value of IFN-? and screening of molecular targets for the treatment of EII in dairy cows.The experiments are summarized as follows:1.Dairy cow endometrial tissue was promoted in vitro,so the endometrial tissue of dairy cows was successfully cultured,and 5,10,and 20 ?g/m L LPS were connected to provoke the endometrial tissue to establish an inflammatory injury model.At same time the results proved that the apoptotic level of endometrial tissue cells raised in the TUNEL experiment.With the addition of the concentration of IFN-?(25,50 and 100 ng/m L),In addition,IFN-? markedly suppressed the indication of TLR4 and HMGB1 can hinder the performance of HMGB1,thereby resisting the inflammatory damage to the endometrial tissue of dairy cows motivated via LPS.2.An inflammatory injury model of cow endometrial epithelial cells(b EECs or BEND)was established,and immunoblotting experiments proved that IFN-? significantly suppressesed the re-expression of HMGB1 and p-IKK and p-p65;Immunofluorescence experiments proved that the nucleus entry of NF-?B p65 was repressed.The transfection of b EECs with si-HMGB1 clarified the key role of HMGB1 in the inflammatory cascade of endometrial epithelial cells.These investigations revealed that IFN-? can of BALB/c mouse endometrial inflammatory injury model also achieved the same research conclusion.3.The high-throughput sequencing results proved that when IFN-? interfered with b EECs,the conservative specific miRNA expression profile changed significantly.Among them,miRNA expression indicated two trends with up-regulation and down-regulation under LPS stimulation;bta-miR-505,bta-miR-488,and bta-miR-31.However,under the intervention of IFN-?,the expression of bta-miR-505 was evidently up-regulated,while those of bta-miR-182,bta-miR-488,and bta-miR-31 have no significant change.In R software revealed that it participates in intracellular signal transduction and has a two-way regulation function.The KEGG annotation revealed that different expressing miRNA target genes are mainly involved in the process of cell life,and mediate innate immune regulation and participate in cell replication and repair processes.4.The clinical manifestations indicated that the formulation of miR-505 notably declined in endometrial inflammatory injury tissue and mouse endometrial inflammatory injury tissue,as well as LPS-induced inflammatory injury of b EECs;It is worth noting that IFN-? intervention reversed the decrease in miR-505 expression.When miR-505 was overexpressed,si RNA interference of target genes also revealed the same conclusion.Conclusion: IFN-? by upregulating the expression of miR-505,and targeting the non-coding region of HMGB1 negatively modulated the expression of HMGB1,and restrain the sensitization of HMGB1/NF-?B signaling.