LPS-Induced Activation Of The CGAS-STING-TBK1-IRF3 Pathway Is Regulated By Mitochondrial Dysfunction In Chronic Endometritis

OutlineChronic Endometritis(CE)is an infectious and inflammatory lesion of the endometrium caused by pathogenic bacteria.Apart from subtle symptomatology such as abdominal pain,fever,abnormal menstruation,patients of bearing age with CE suffers from infertility or recurrent abortion.The signal communication of the embryos and the endometrium is disturbed by immune cells and inflammatory factors when the endometrium becomes infected,which affected embryo implantation.The treatment of CE still relies on antibiotic management.Although this way is effective for many patients,however,symptoms of chronic endometritis persist in a certain proportion of patients after standard antibiotic treatment.Thus,seeking for other effective methods to manage CE to avoid the overuse of antibiotics is an urgent problem to be solved.To search the pathogenesis of CE,Western Blot and Immunohistochemical Staining were firstly conducted,and we found the levels of cGAS-STING-TBK1-IRF3 pathway proteins were apparently increased.Inflammatory factors including IL-6,IL-8,IL-1β,IFN-β1 were significantly higher by real time PCR in the endometrium of patients with Chronic Endometritis compared with those in the control group.In addition,we also disclosed the translocation of STING and IRF3 in human endometrial stromal cells when the cGASSTING-TBK1-IRF3 pathway kept active by Western Blot and Immunofluorescence Staining.For further investigating that how LPS regulated the cGAS-STING-TBK1-IRF3 pathway,mitochondrial function was detected.We discovered that LPS up-regulated the reactive oxygen species,down-regulated the mitochondrial membrane potential,and induced mitochondrial DNA leaking into cytoplasm.At the same time,transfecting mitochondrial DNA into human endometrial stromal cells activated the cGAS-STING-TBK1-IRF3 pathway.It is the first study to disclose that mitochondrial dysfunction participated in chronic endometritis and mitochondrial DNA activated the cGAS-STING-TBK1-IRF3 pathway to induce the production of inflammatory factors.LPS-Induced Activation of the cGAS-STING-TBK1-IRF3 Pathway is Regulated by Mitochondrial Dysfunction in Chronic EndometritisObjective:The aim of this study was to investigate the role of mitochondrial dysfunction and the cGAS-STING-TBK1-IRF3 pathway in Chronic Endometritis.Methods:(1)In this chapter,we collected 9 cases of endometrium of CE as the experimental group and 9 matched normal endometrium as the control group.Six pairs of endometrium were used to analyze the activation of the cGAS-STING-TBK1-IRF3 pathway by Western Blot and the related inflammatory factors by real time PCR.The other 3 pairs of samples were made into paraffin sections to conduct IHC to detect the level of STING protein in endometrium.(2)In vitro experiments,we used LPS to stimulate HESCs to analyze the cGAS-STINGTBK1-IRF3 pathway proteins by Western Blot and the related inflammatory factors by real time PCR.In addition,we also detected the translocation of STING and IRF3 by Western Blot after separation of cytoplasmic and nuclear proteins and Immunofluorescence Staining.(3)DCFH-DA was used to detect reactive oxygen species and JC-10 to analyze mitochondrial membrane potential after LPS stimulated HESCs.In addition,Quant-iTTM PicoGreenTM dsDNA Assay Kits was used to analyze mitochondrial DNA after LPS stimulated HESCs.(4)Extracted the mitochondrial DNA and transfected it into HESCs.Western Blot was conducted to analyze the change of the cGAS-STING-TBK1-IRF3 pathway.(5)LPS was used to infuse the uterine cavity of C57BL/6JGpt mice to build the mice model of CE.The appearance of the uterus was observed after dissection,Western Blot was conducted to analyze the cGAS-STING-TBK1-IRF3 pathway proteins and real time PCR to analyze the related inflammatory factors.Results:(1)The level of the cGAS-STING-TBK1-IRF3 proteins were apparently increased by Western Blot and STING also expressed higher in endometrium of CE by IHC.In real time PCR,the experimental group had higher IL-6,IL-8,IL-1β,IFN-βl compares with the control group.(2)In vitro experiments,LPS activated the cGAS-STING-TBK1-IRF3 pathway proteins of HESCs by Western Blot and induced the production of IL-6,IL-8,IL-1β,IFN-β1 by real time PCR.In addition,we also found that STING encompassed the nucleus and IRF transferred from cytoplasm into nucleus after LPS-stimulation by Immunofluorescence Staining and Western Blot.(3)We found that reactive oxygen species increased,mitochondrial membrane potential decreased and mtiochondrial DNA leaked into cytoplasm by Immunofluorescence Staining after LPS-stimulation.(4)Transfecting mitochondrial DNA into HESCs induced the activation of the cGASSTING-TBK1-IRF3 pathway proteins by Western Blot.(5)Uterine hyperemia of mice after LPS-stimulation,which was related to STING gene.TBK1 was activated by Western Blot and inflammatory factors including IL-6,IL-8,IL1β,IFN-β1 were increasing by real time PCR after LPS intrauterine infusion.Conclusion:LPS induced mitochondrial dysfunction,and mitochondrial DNA leakage activated the cGAS-STING-TBK1-IRF3 pathway proteins which induced the production of inflammatory factors included IL-6,IL-8,IL-1β,IFN-β1 in Chronic Endometritis.