The Mechanism Of Hmgb1 Promoting Decidualization In Mouse

Backgroud: Pregnancy is an extremely accurate and complicated process in mammals,which involves embryo implantation,placentation,embryonic development and delivery.Embryo implatation activate endometrial stromal cells sorrounding embryo to proliferate and differentiate into decidual cells.Decidualization is a key process in embryo implantation.There are two distinct areas in decidualization,an avascular primary decidualization zone and a widely vessel-distributed secondary decidualization zone after embryo implantation.Hmgb1 is a necessary factor in life survival.Many of studies about effects of Hmgb1 on pregnancy focus on Hmgb1 influencing blastocyst development,whereas few describe the effects of Hmgb1 on uterine microenvironment in pregnancy.Our previous sequencing results indicated that Hmgb1 had increasing expression in decidualization.Therefore,our recent study is to explore the relationship between Hmgb1 and endometrial decidualization and the outcomes of decidualization and pregnancy if knocking down Hmgb1 expression by si-Hmgb1.In addition,endometrial stromal cells polyploidization is necessary with decidualization.Reduced polyploid cells inhibits decidualization.Hyper-activation of mitochondria is positively linked with polyploidy development in decidualization.Mitochondrial dysfunction caused by injured polyploidization decidual cell may lead to infertility.Hmgb1 can also regulate mitochondrial quality control.Therefore,our recent study is to explore if Hmgb1 influences decidualization by regulating mitochondrial quality.Also,mitochondrial quality control include mitochondrial energy metabolism system,mitophagy and mitochondria fusion and fission,which are important to sustain basic activity of cell.Studies have indicated that autophagy plays a significant part in embryo implantation,embryo development,plantation and delivery.Hmgb1 is a key regulator in autophagy.Therefore,our study is to explore whether autophagy affected Hmgb1 involved in decidualization.Also,Hmgb1 in nucleus can promote ER/PR transcription factor to bind DNA.ER/PR plays a signifcant role in decidualization.Thus,our study is to explore if Hmgb1 regulates ER/PR transcription factor in decidualization.And,Hmgb1 released from nucleus to extracellular can regulates immune response through bingding to viarious receptor such as RAGE,TLR4,TLR9.We also knows that a mass of immune cells participates in decidualization.So,our study is to explore if Hmgb1 regulates immune response in decidualization.In summary,our study clarify that Hmgb1 regulates decidualization in nucleus,cytoplasm,respectively.It provides forceful and new evidences in decidualization studies.Methods:1.Establishment of the animal model:8-weeks-old adult female mice were mated with male for establishing a mouse pregnancy model.The day of finding the postcoital vaginal plug was designated as day 1(Day 1,D1).Pregnancy was confirmed by recovering embryos from the oviducts or uteri from D1 to D4.The implantation sites on D5 and D6 were visualized using caudal veins injections of 0.1 ml of 1% Chicago blue dye in saline.Tissues were collected on D1,D4,D5,D6 and D8.8-weeks-old adult female mice were mated with vasectomized males for pseudopregnancy.The first day of finding the postcoital vaginal plug was designated as psedopregnancy D1(PD1).Collect tissues from PD1,PD4,PD5,PD6,PD8.Pregnant mice in D3 were bilaterally ovariectomized.They were injected subcutaneously with progesterone 100 μl(1mg/0.1ml corn oil)from D4 to D7.Estradiol 100 μl(25ng/0.1ml corn oil)was given to progesterone-primed mouse to terminate delayed implatation on D7.The delayed group and the activation group were confirmed by flushing blastocysts from the uterine horn prior to implantation on D8.Artificial decidualization experiments were induced by infusing 10 μl corn oil into one uterine horn(Sigma)on day 4 of pseudopregnancy,while the other side of the uterine horn served as the control.The mouse uterus had a robust decidual response on D8 of pseudopregnancy after injection of intraluminal oil.Glycyrrhizia acid(GA)was dissolved in DMSO and injected in uteri horn with Oil(5μl)on day 4 of pseudopregnancy.DMSO was injected as a solvent control.Only Oil injection for artificially decidualization was a positive control.After mice were sacrificed at 10:00 on D8 of pseudopregnancy,the weight of uteri was measured.2.In-vitro induced decidualization was performed to detect the effect of Hmgb1 on decidualization.10 nM of oestradiol-17β(Sigma),1 μM of progesterone(Sigma)in DMEM/F-12 with 10% charcoalstripped fetal bovine serum were used to induce primary mESCs for in-vitro decidualization for 72 h.3.Detect the relashionship between Hmgb1 and decidualization:(1)Endometrial decidual detection: calculate the number and diameter of swelling globe of the uters in decidual period(D5-D8)in mice.Weigh the uterus of artifically induced decidualization model,detect decidualization marker dtprp mRNA levels by RT – qPCR in vivo and vitro artificially decidualization.(2)The expression of Hmgb1 in pregnancy: detect Hmgb1 expression on D1,D4,D5 IS,D5IIS,D6 IS,D6IIS,D8 by IHC,ISH,WB,RT-qPCR.(3)The expression of Hmgb1 in pseudopregnancy: detect Hmgb1 expression in PD1,PD4,PD5,PD6,PD8 by IHC,WB,RT-qPCR.(4)The expression of Hmgb1 in delayed implatation model: Hmgb1 expression in delayed groups and activated groups.(5)The expression of Hmgb1 in artificially induced decidualization in vivo: IHC,WB,RT-qPCR detect Hmgb1 expression in ID and Vehicle.(6)The expression of Hmgb1 in in vitro induced decidualization of primary endometrial stromal cells in vitro: identify cell purity using vimentin first antibody by immunofluorescence and the vitro decidualization model using decidual marker dtprp by RT-qPCR.Detect dtprp,hmgb1 mRNA levels in si-Nc+E2+P4 group and si-Hmgb1+E2+P4 group.4.Idetify decidualization by infusing GA in uteri horn: capture the morphology of uterus and weigh the uterus.5.Mitochondrial quality control:(1)Observation of mitochondrial morphology: mitochondria staining by Mitotracker Red and observation of mitochondrial fragment by laser confocal microscope.(2)Detection mitochondrial membrane potential change by flow cytometry to assess mitochondrial function.(3)Detection the production of ATP by Multiscan Spectrum to assess mitochondrial ATP production.(4)Detection the releases of ROS by flow cytometry to assess mitochondrial function.6.Detection cell autophagy:(1)Detect mitochondria-related factor and autophagy-related factor by Western Blot in artificially decidualization in vivo.(2)Detect mitochondria-related factor and autophagy-related factor by Western Blot and RT-qPCR between si-Nc+E2+P4 and si-Hmgb1+E2+P4 in artificially decidualization in vitro.(3)Detect mitochondria-related factor and autophagy-related factor by Western Blot and RT-qPCR in infusing glycyrrhizin and oil in uterui horn in vivo.(4)Detect autophagosome by Electron microscopy7.Detect differencial gene and cell mechanism of Hmgb1 influncing decidualization by RNA-seq.8.Data analysis by RNA-seq:(1)Analyze the differential genes about Hmgb1 regulating mitochondrial fuctions in decidualization.(2)Analyze the differential genes about Hmgb1 regulating ER/PR transcription factors in decidualization.(3)Analyze the differential genes about Hmgb1 regulating immune response in decidualization.Results:1.Results of Hmgb1 regulating decidualization:(1)The expression of Hmgb1 in pregnancy: IHC results showed that Hmgb1 had positive expression in cavity epithelium and glandular epithelium on D1 and D4.There were also a small part of stromal cells on D4.Hmgb1 had a high expression in primary decidual zone and secondary decidual zone on D5 IS,D6IS and D8 IS,wheras only in cavity epithelium and glandular epithelium on D5 IIS,D6IIS.ISH results about Hmgb1 expression and localization were in keeping with IHC results.Western Blot and RT-qPCR results showed that Hmgb1 highly expressed in embryo implantation site.(2)The expression of Hmgb1 in psedopregnancy: IHC results showed that Hmgb1 localized in cavity epithelium and glandular epithelium on PD1,PD4,PD5,PD6,PD8.Western Blot and RT-qPCR results show that Hmgb1 had a weak expression on PD5,PD6 and PD8 compared with D1 and D4.(3)The expression of Hmgb1 in delayed implantation model: IHC results showed that Hmgb1 had higher expression in activated groups compared with delayed groups.(4)The expression of Hmgb1 in artificially induced decidualization in vivo: successfully established aitificially induced decidualization model by detecting dtprp mRNA levels with RT-qPCR.Hmgb1 had a higher positive expression in decidualization compared with control.(5)The expression of Hmgb1 in artificially induced decidualization in primary endometrial stromal cells in vitro: Hmgb1 deficient by si-Hmgb1 knocking down inhibited decidualization.2.Impressed Hmgb1 activity deficient by infusing glycyrrhizia acid in uteri horn induced compromised decidualization.3.Hmgb1 regulated mitochondria quality control in decidualization(1)mitochondrial fragements were higher in si-Hmgb1 and si-Hmgb1+E2+P4 groups compared with si-Nc+E2+P4 by laser confocal microscope after Mitochondria staining using Mitotracker Red.(2)The levels of mitochondrial membrane potential were lower in si-Hmgb1 and si-Hmgb1+E2+P4 groups compared with si-Nc+E2+P4.(3)The levels of ATP production were lower in si-Hmgb1 and si-Hmgb1+E2+P4 groups compared with si-Nc+E2+P4.(4)The levels of ROS releases were higher in si-Hmgb1 and si-Hmgb1+E2+P4 groups compared with si-Nc+E2+P4.4.Hmgb1 regulated mitochondria quality control(1)Mitochondria-related protein Drp1,Slp2 and autophagy-related protein Lc3b-Ⅱ highly expressed in artificially induced decidualization by Western Blot detecting.(2)Mitochondria-related protein Slp2 and autophagy-related protein Lc3b-Ⅱ decreased in si-Hmgb1+E2+P4 group compared with si-Nc+E2+P4 group by Western Blot detecting.Mitochondria-related gene slp2,dnm1 l and autophagy-related gene map1lc3 b,atg5 decreased,whereas sqstm1 increased in si-Hmgb1+E2+P4 group compared with si-Nc+E2+P4 group by RT-qPCR.(3)Mitochondria-related protein Slp2 and autophagy-related protein Lc3b-Ⅱ decreased,whereas p62 incresed in GA+Oil injection group compared with Oil injection group by Western Blot detecting.Mitochondria-related gene slp2,dnm1 l and autophagy-related gene map1lc3 b,atg5 decreased,whereas sqstm1 increased in GA+Oil injection group compared with oil injection group by RT-qPCR.(4)Autophagosomes were found in oil injection group,however,none in oil+glycyrrhizin injection group.5.There were 29 differential genes ralated with mitochondria quality control in decidualization among 1505 differential genes by RNA-seq.There were 27 differential genes ralated with ER/PR binding in decidualization among 1505 differential genes by RNA-seq.There were 19 differential genes ralated with immune response in decidualization among 1505 differential genes by RNA-seq.Conclusions:1.Hmgb1 has spatial and temporal distributions regulating decidualization in pregnancy.Hmgb1 deficient inhibits decidualization to prevent normal embryo implantation.2.Hmgb1 in cytoplasm regulates mitochondria quality control by cell autophagy in decidualization.Hmgb1 deficient prevents decidualization by inhibiting cell autophagy and causing mitochondria morphology and function damage.3.Hmgb1 in nucleus regulates ER/PR binding genes to promoting cell proliferation and differentiation in decidualization.4.Hmgb1 regulates immune response to promoting decidualization.