Experimental Study Of Umbilical Cord Mesenchymal Stem Cells Or/and CoQ10 And DHEA Against Reproductive Organ Aging

Population aging has become a global common challenge.The changes of sex hormone secretion level and its regulatory mechanism accompanied with reproductive aging not only lead to the decline of reproductive function,but also are closely related to aging related diseases.Therefore,slowing down the aging of reproductive organs or keeping them old will improve many elderly diseases.In recent years,mesenchymal stem cells have been widely used in the study of regeneration of various tissues and organs,but the research on delaying aging is just beginning.Mesenchymal stem cells(MSCs;Whether MSCs can delay the aging of reproductive organs needs to be further studied.Coenzyme Q10(CoQ10)and dehydroepiandrosterone(DHEA)are ovarian conditioning drugs.Whether they can enhance the anti-aging of reproductive organs alone or in combination with MSCs has not been reported.Here,we explore the anti-aging effects of hUCMSCs and/or CoQ10,DHEA and their possible mechanisms.We selected 20 month old female C57BL/6J mice as the natural aging animal model,based on the two important factors of anti-aging,antioxidant and hormone supplement,and divided them into hUCMSCs or/and CoQ10,DHEA two kinds of treatment.Intravenous injection of 1×107/kg were transplanted every two weeks for three times.The animals were given 0.6 mg/ml CoQ10 or 0.15 mg/ml DHEA,6-10 ml daily for 2 months.The first protocol was divided into control group,hUCMSCs group,CoQ10 group and hUCMSCs+CoQ10 group.The second protocol was divided into control group,hUCMSCs group,DHEA group and hUCMSCs+DHEA group.The ability of learning,memory and movement of aging mice was analyzed by observing the appearance of the animal body,using the dark avoidance test and animal running platform test.Hematoxylin eosin(HE)staining method was used to count the follicles in ovarian tissue,observe the uterine morphology and count the number of glands.Masson staining was used to analyze the collagen volume fraction in uterine tissue,Double antibody sandwich enzyme-linked immunosorbent assay(ELISA)and/or quantitative real-time polymerase chain reaction(qRT-PCR)were used to detect the levels of related hormone indexes,inflammation related factors,fibrosis related factors,cytokine and oxidative stress related molecules.In addition,we used human chorionic trophoblast cells(HTR8/SVneo cells),human endometrial cancer cells(RL95-2 cells),human endometrial cancer cells(HEC1A cells)and Chinese hamster ovary cells(CHO cells)to establish cell aging models.qRT-PCR was used to detect the expression of key molecules of AKT-mTOR signaling pathway in uterus,ovary and senescent cells induced by hydrogen peroxide in order to explore the molecules that play an important role in germ cell senescence.The results showed that hUCMSCs or/and CoQ10,DHEA could significantly improve the depilation and mental state of the aged mice,and reduce the mortality of the animals,especially CoQ10,but had no significant effect on the body weight.The results of dark avoidance test and treadmill test suggest that hUCMSCs or/and CoQ10,DHEA can enhance the memory ability,transport ability and endurance of animals.He staining showed that hUCMSCs or/and CoQ10,DHEA could increase the number of glands and mature follicles.These results indicate that hUCMSCs or/and CoQ10,DHEA can improve the physical function of natural aging mice and delay the aging of uterus and ovary.We can see that hUCMSCs or/and CoQ10,DHEA can resist the hormonal changes that lead to the increase of serum FSH and LH levels and the decrease of E2 and AMH levels.Tissue fibrosis is an important feature of organ aging injury.We used Masson staining to detect tissue fibrosis,and found that hUCMSCs and CoQ10 can significantly inhibit uterine fibrosis and reduce the expression of Pro fibrogenic factor Smad-3.The synergistic effect of hUCMSCs and DHEA on fibrosis was not obvious.The changes of proinflammatory cytokines in peripheral serum showed that DHEA or/and CoQ10 could promote the expression of proinflammatory cytokines in hUCMSCs,but QRT PCR showed that CoQ10 or DHEA could inhibit the expression of proinflammatory cytokines in uterine and ovarian tissues.The synergistic effect of DHEA or/and CoQ10 on hUCMSCs was contrary to that of the two alone.Detection of anti-inflammatory cytokines in peripheral blood showed that hUCMSCs alone or combined with CoQ10 and DHEA could inhibit the expression of TGF-? and IL-10,and promote the expression of IL-13 and IL-14,but qRT-PCR detection of anti-inflammatory cytokines in uterus and ovary showed that DHEA or/and CoQ10 combined with hUCMSCs could promote the expression of TGF-? and IL-13.These results suggest that inflammatory response is involved in the repair of aged endometrium and ovary by DHEA or/and CoQ10 in combination with hUCMSCs.We detected the expression of pyrolytic protein and found that hUCMSCs in peripheral serum could increase the level of caspase-1 and decrease the level of caspase-4;HUCMSCs combined with CoQ10 can increase the level of Caspase-1.HUCMSCs combined with CoQ10 can reduce the level of GSDMD in uterine tissue,and hUCMSCs combined with DHEA can promote the expression of GSDMD.These results suggest that pyrocytosis is involved in the inflammatory process.We also detected the changes of endometrial markers,and found that hUCMSCs and/or CoQ10,DHEA had no significant effect on the expression of endometrial epithelial cell marker pan keratin and endometrial stromal cell marker vimentin.Detection of Akt mTOR signaling pathway molecules found that hUCMSCs and/or with CoQ10,DHEA can promote AKT,PDK-1,SGK1,PKC-??PRAS40?HIF-3 ? and so on.The effective HIF-3? was screened and detected.There was a positive correlation between the expression level of VEGF and its effect on cell proliferation,migration and invasion.The results of aging cell model showed that HIF-3? in HTR8/SVneo cells and RL95-2 cells,but down regulated in CHO and HEC1A cells.In conclusion,our study found that hUCMSCs or/and CoQ10,DHEA can improve the physical function of natural aging mice and delay the aging of uterus and ovary.Fibrosis,inflammation and pyroptosis are involved in the process of hUCMSCs or/and CoQ10,DHEA delaying the aging of uterine tissue,which may play a role through Akt mTOR signaling pathway.