Study On The Mechanism Of Action Of ATF3 In Regulating The Apoptosis Of Viginal Fibroblasts And ECM Degradation By P38/Nrf2 Signaling Pathway In Pelvic Organ Prolapse

Objective:Pelvic organ prolapse（POP）is a kind of disease in which the function of the pelvic floor support structure declines,and the pelvic organs and surrounding tissues decline from the normal position,resulting in changes in defecation,urination and sexual function.Most of POP occurs in postmenopausal women,while a small part occurs in premenopausal women.With the aging of the population,the incidence of POP increases year by year.Although POP is not life-threatening,it seriously affects the quality of life.Its pathogenesis is not clear and needs to be discussed.The occurrence of POP is a multi-factorial and multi-stage pathological process,which is related to the destruction of the structure and function of the pelvic floor support system.The occurrence of POP is related to oxidative stress,apoptosis and ECM generation.The P38 pathway plays an important role in apoptosis,inflammation and oxidative stress.Nrf2 is a key transcription factor regulating oxidative stress in cells.Under normal circumstances,Keap1 binds to Nrf2 in the cytoplasm.When cells are stimulated by external stimuli,the combination of Nrf2 and Keap1 is inhibited,and Nrf2 is released and incorporated into the nucleus to activate downstream gene transcription and play physiological functions.KEGG and protein interaction network analysis was performed by comparing differentially expressed genes in POP and non-POP tissue samples.ATF3 expression was abnormally upregulated in the vaginal anterior wall tissue of POP（GSE53868）,and ATF3 was located at a key hub in the protein interaction network.However,there are few studies on whether ATF3 can regulate the pathological progression of POP.Then how does ATF3 play a role?what is its role in the occurrence and development of POP,and its relationship with the P38/Nrf2 pathway?Based on this,the following experiments were designed and completed to further explore the molecular pathogenesis of POP.Methods:The anterior vaginal wall tissues of 30 patients with POP and non-POP controls were collected,and the expression of ATF3 in the anterior vaginal wall tissues was detected by real-time PCR and immunohistochemistry.The anterior vaginal wall tissues of 3 patients with POP and non-POP controls were respectively used for primary culture and purification of fibroblasts by adherent method and differential time adherent method.The 0,2 and 4 generations of vaginal fibroblasts were reserved,and the 4generations of vaginal fibroblasts were identified by immunofluorescence method to detect the expression of Vimentin（fibroblast labeling）,Cytokeratin（epithelial cell labeling）and Desmin（smooth muscle cell labeling）.The induced cell injury model was treated with H₂O₂,and cell apoptosis was detected by TUNEL staining.ROS production was detected by flow cytometry.Collagen I and Collagen III expressions were detected by real-time PCR.Sh RNA targeting ATF3 and its control sequence were constructed by cell transfection technology,and adenovirus was packaged.Real-time PCR was used to detect The expression of ATF3 in cells.Western blot was used to detect the expression of ATF3,Bax,Bcl2,cleaved-caspase3,cleaved-caspase9.Cell activity was detected by MTT.Cell cycle was detected by flow cytometry.TUNEL staining was used to detect cell apoptosis.Mitochondrial membrane potential was detected by JC-1 staining.Flow cytometry was used to detect ROS production.The expression of 8-OHd G and 4-HNE wasdetected by immunofluorescence.Theα-SMA content was detected by immunofluorescence.Western blot was used to detect the expression of Mature-MMP2,Mature-MMP9,TIMP2 and TGF-β1.Collagen I,Collagen III and Elastin expressions in cells were detected by real-time PCR.The nucleation of Nrf2 was verified by immunofluorescence.Western blot was used to verify the expression of P38,P-P38（Thr180/Tyr182）,Nrf2（nuclear）and HO-1 in cells.After treatment with 0.6 m M H₂O₂and/or 10μM P38 inhibitor（SB203580）and/or 20μM Nrf2 agonist（TBHQ）for 24h,cells were collected and apoptosis was detected by TUNEL staining.ROS production in cells was detected by flow cytometry.Western Blot was used to detect the expression of Bcl2,Cleaved Caspase3,α-SMA,Collagen I.Results:Immunofluorescence showed that the expression of ATF3 in POP group was significantly higher than that in control group.The cell oxidative damage model was established by H₂O₂.Compared with the Control group,Collagen I and Collagen III expression levels were lower in POP group.Collagen I and Collagen III levels in Control+H₂O₂group were lower than those in Control group.Collagen I and Collagen III levels in POP+H₂O₂group were lower than those in POP group.By constructing cell oxidative damage model,it was found that after H₂O₂treatment,cell apoptosis and ROS levels increased in both POP group and control group,but the levels of apoptosis and ROS in POP group were higher than those in control group,and Collagen I and Collagen III expressions decreased in POP group and control group after H₂O₂treatment.Collagen I and Collagen III expression levels in POP group were lower than those in control group.After H₂O₂treatment,the active expression level of POP group cells was low,and while adenovirus interfered with targeted silencing of ATF3,the active expression level of POP group cells was increased.At the same time,flow cytometry showed that the number of cells entering G1 phase decreased and the cell cycle changed in POP group after H₂O₂treatment after targeted silencing ATF3.TUNEL staining showed a significant decrease in apoptosis after targeted silencing of ATF3.Flow cytometry showed that the ratio of red-green fluorescence in the POP+H₂O₂group was significantly lower than that in the Control group.When adenovirus interfered with the targeted silencing of ATF3,the ratio of red-green fluorescence in the POP+H₂O₂group was significantly higher than that in the Control group.The expression levels of Bax,cleaved-caspase3,cleaved-caspase9were higher in the POP group than in the control group,and the expression levels of Bcl2were lower than in the control group.After H₂O₂treatment,the expression of reactive oxygen species increased in POP group,and when adenovirus interfered with targeted silencing of ATF3,the expression of reactive oxygen species decreased.After H₂O₂treatment,the expressions of 8-OHd G and 4-HNE in POP group were up-regulated,and when adenovirus interfered with targeted silencing of ATF3,the expressions of 8-OHd G and 4-HNE were significantly decreased.After adding SB203580,the expression level of reactive oxygen species in POP group increased again,but decreased after adding TBHQ.Immunofluorescence assay showed that theα-SMA level of POP cells was down-regulated after H₂O₂treatment,and theα-SMA level of POP cells was up-regulated after targeted silencing of ATF3.Western blotting analysis showed that the expression levels of MMP2 and MMP9 were higher in POP group,while the expression levels of TIMP2 and TGF-β1 were lower.When adenovirus interference targets ATF3 silencing.The expression levels of MMP2 and MMP9 were down-regulated,while the expression levels of TIMP2 and TGF-β1 were up-regulated.PCR showed that collagen I,Collagen III and Elastin expression levels of POP cells decreased after H₂O₂treatment,and ECM level was up-regulated after adenovirus interference and targeted silencing of ATF3.Immunofluorescence experiments showed that Nrf2 entered the nucleus of POP fibroblasts after ATF3 knockdown,and the levels of P38,P-P38,HO-1 and Nrf2increased.Activation of P38 signaling pathway promotes Nrf2 entry into the nucleus,and then promotes HO-1 transcription and alleviates oxidative stress.Silencing ATF3 can alleviate oxidative stress and inhibit cell apoptosis.SB203580 is an inhibitor of P38,thereby blocking the expression of P38 signaling pathway,and TBHQ is an Nrf2 entry agonist.ATF3 silencing promoted p38 signaling pathway and Collagen I expression was up-regulated.cleaved-caspase3 expression was up-regulated and Bcl2 expression was down-regulated when SB203580 was added.Collagen I expression was down-regulated;After TBHQ was added,Nrf2 was promoted into the nucleus,cleaved-caspase3expression was down-regulated,Collagen I expression was up-regulated,and Collagen I expression was up-regulated.The level of ROS was detected by flow cytometry,and the patients in POP+H₂O₂group were used as the control group.After adenovirus interference with targeted silencing of ATF3,ROS was down-regulated;after SB203580was added,ROS was up-regulated;after TBHQ was added,ROS was down-regulated.Conclusion:The expression of ATF3 in POP group was significantly higher than that in control group.The occurrence of POP was related to oxidative stress and degradation of ECM,which weakened the strength of pelvic floor support structure.ATF3 regulates the apoptosis,oxidative stress and ECM production of vaginal fibroblasts.H₂O₂treatment simulated cell damage model caused by oxidative stress,adenovirus interference with targeted silencing of ATF3 increased cell activity,changed cell cycle,and reduced apoptosis and mitochondrial depolarization.Bax,cleaved-caspase3,cleaved-caspase9,8-OHd G,4-HNE were down-regulated,while Bcl2,Collagen I,Collagen III,Elastin were up-regulated;MMP2 and MMP9 were down-regulated,while TIMP2 and TGF-β1 were up-regulated.Targeted silencing of ATF3 promotes the transduction of P38/Nrf2/HO-1signaling pathway and regulates apoptosis,oxidative stress and ECM production,thus playing a therapeutic role on POP.Using The P38 inhibitor SB203580 and Nrf2 nuclear agonist TBHQ,it was confirmed that knocking ATF3 could alleviate the oxidative stress damage caused by H₂O₂in fibroblasts through the P38/Nrf2/HO-1 signaling pathway.In order to provide ideas for exploring the pathogenesis of POP,the p38/Nrf2/HO-1signaling pathway after down-regulated ATF3 inhibits the apoptosis,oxidative stress and degradation of ECM in vaginal fibroblasts.It provides a theoretical basis for us to better explore the molecular mechanism of POP.