Significance Of Autophagy And Apoptosis Induced By ERS In The Pathogenesis Of Menstruation In Mice Menstrual-like Model And Its Regulation Of Progesterone

Objective: To detect the expression of autophagy related signaling pathway PERK/e IF2?/ATF4 and apoptosis-related signaling molecules CHOP and Caspase12 induced by endoplasmic reticulum stress(ERS)in the endometrium tissue of mouse menstrual like model,and the role of progesterone regulation.Methods: 80 healthy C57BL/6J female mice aged 8-10 weeks were subjected to bilateral ovariectomy was performed under ether anesthesia.A week later,mice were injected with quantitative estrogen and progesterone in the abdomen subcutaneously,on the seventh day,the progesterone(P)implant was inserted subcutaneously into the back of each mouse.On the ninth day,20?L filtered arachis oil was injected into the bilateral uterine cavity in each mouse to induce decidualization.After 49 hours,the progestterone implants were removed and this time point is regarded as 0h,50 mice were sacrificed at 0h,8h,12 h,16h,and 24 h(10 mice per time point),and their bilateral uterine tissue ware harvested for further analysis.Another 30 mice established a menstrual model of progesterone-replanted mice based on the mouse menstrual-like model: 10 mice were returned progesterone implant 12 h after progesterone withdrawal and were sacrificed at 16 h,10 mice were returned progesterone implant 12 h after progesterone withdrawal and then sacrificed mice at 24 h,10 mice were returned progesterone implant 16 h after progesterone withdrawal and then sacrificed mice at 24 h,and labeled as 1216 h,1224h,1624 h.The expression and localization of t-PERK/p-PERK,t-e IF2?/p-e IF2?,ATF4,CHOP and Caspase12 proteins in mouse endometrial tissues were detected by Western blot and immunohistochemistry,and the m RNA expression of the above signal molecules was detected by real-time PCR.Results: 1.The expression of autophagy related signaling pathway PERK/e IF2? /ATF4 and apoptosis-related signal molecules CHOP and Caspase12 induced by ERS in the endometrium tissue of mouse menstrual model.Autophagy related signaling pathways t-PERK and t-e IF2? proteins showed no significant differential expression in mouse uterus at various time points after progesterone withdrawal(P>0.05).The expression of PERK/e IF2? m RNA and p-PERK/p-e IF2? proteins increased gradually at 0 h after progesterone withdrawal,the peak value was reached at 12h(P<0.05),then it gradually decreases to the lowest value after 24 hours(P<0.05).The expression trends of ATF4 m RNA and ATF4 protein was consistent with that of PERK/e IF2 m RNA and p-PERK/p-e IF2? proteins.The results of immunohistochemistry showed that: the distribution of p-PERK,p-e IF2? and ATF4 proteins in endometrial tissues has very similar characteristics.Positive staining mainly occurs in stromal cells,glandular epithelial cells and luminal epithelial cells with sufficient decidualization of the endometrium,and is also slightly expressed in the surrounding deciduation zone and basal layer.The expression of CHOP/Caspase12 m RNA and CHOP/Caspase12 proteins increased gradually at 0h after progesterone withdrawal.From 12 h,the expression was significantly enhanced,with the prolongation of progesterone withdrawal time,reaching the highest value at 24 h after progesterone withdrawal.The results of immunohistochemistry showed that: the CHOP/Caspase12 proteins is mainly found in the peripheral region of decidualized stromal cells that are about to fall off or have fallen off.2.The influence of progesterone returning in the early stages of menstruation on the expression of t-PERK/p-PERK,t-e IF2? /p-e IF2?,ATF4,CHOP and Caspase12 in endometrial tissues.The expression levels of t-PERK and t-e IF2? protein in mouse uterine tissue were almost equal in the 12 h group,1216 h group and 16 h group(P>0.05).The expression levels of PERK/e IF2? m RNA and p-PERK/p-e IF2? proteins were not significantly different between 12 h group and 1216 h group,but significantly higher than16 h group(P<0.05).The expression trends of ATF4 m RNA and protein were consistent with that of PERK/e IF2? m RNA and p-PERK/p-e IF2? proteins.The expression levels of CHOP/Caspase12 m RNA and proteins were not significantly different between the 12 h group and the 1216 h group,but were significantly lower than 16 h group(P<0.05).3.The influence of progesterone returning in the late stages of menstruation on the expression of t-PERK/p-PERK,t-e IF2?/p-e IF2?,ATF4,CHOP and Caspase12 in endometrial tissues.The expression levels of t-perk and t-e IF2? protein in mouse uterine tissue were almost equal in the 1224 h group,1624 h group and 24 h group(P>0.05).The expression levels of PERK/e IF2? m RNA and p-PERK/p-e IF2? proteins in the 1624 h group was significantly lower than that in the 1224 h group(P<0.05),and significantly higher than that in the 24 h group(P<0.05).The expression trends of ATF4 m RNA and protein were consistent with that of PERK/e IF2 m RNA and proteins.The expression levels of CHOP/Caspase12 m RNA and proteins in the 1624 h group were significantly higher than that in the 1224 h group(P<0.05),and significantly lower than that in the 24 h group(P<0.05).Conclusion:1.From 0 to 12 hours,mouse uterine ischemia and hypoxia induced by progesterone withdrawal lead to endoplasmic reticulum stress in endometrial stromal cells,enhancing autophagy by promoting PERK/e IF2?/ATF4 expression,and maintaining the survival of endometrial stromal cells,the ERS response was further aggravated with the prolonged progesterone withdrawal time.At this time,ERS turned to induce the expression of CHOP and Caspase12,key members of the apoptotic signaling pathway in endometrial stromal cells,and promotes stromal cell apoptosis,and thus the autophagy of endometrial cells induced by PERK/e IF2?/ATF4 signaling pathway was weakened.2.Returning of progesterone before the critical period of menstruation(12h),not only effectively maintain the high expression level of PERK/e IF2? /ATF4 signaling pathway,but also block the expression of CHOP and Caspase12 in apoptotic signaling pathway therefore effectively blocks the occurrence of menstruation.And this can only be partially achieved by progesterone returning after the critical period of menstruation(16h).