人口老龄化已成为全球共同面临的问题。伴随生殖衰老所发生的性激素分泌水平及其调节机制的改变，不仅导致生殖功能的减退, 也和衰老相关的疾病密切相关，因此减缓生殖器官的衰老或让生殖器官老而不衰会改善很多老年疾病。近年来，间充质干细胞已广泛用于各种组织器官的再生研究，但其在延缓衰老方面的研究刚刚起步。间充质干细胞(mesenchymal stem cell; MSCs)是否可以延缓生殖器官衰老有待深入研究，辅酶Q10（coenzyme Q10，CoQ10）和脱氢表雄酮（dehydroepiandrosterone，DHEA）是卵巢调理药物，其单独或者与MSCs共同给予是否可以增强生殖器官的抗衰老力尚不明确。在此，我们预探索人脐带间充质干细胞（human umbilical cord bloodmesenchymal stem cells，hUCMSCs）或/和CoQ10、DHEA对抗生殖器官衰老的作用及其可能的机制。

我们选用20月龄的雌性C57BL/6J小鼠作为自然衰老动物模型，基于延缓衰老的两个重要因素抗氧化和激素补充开展研究，分为hUCMSCs或/和CoQ10、DHEA两种治疗方案。尾静脉注射1×10⁷/kg体重的hUCMSCs，每两周移植一次，共进行三次移植。给予0.6 mg/ml的CoQ10或0.15 mg/ml 的DHEA，每天6-10 ml，连续2个月。第一个方案实验分组为Control组、hUCMSCs组、CoQ10组和hUCMSCs+CoQ10组，第二个方案实验分组为Control组、hUCMSCs组、DHEA组和hUCMSCs+DHEA组。通过观察动物体态外观，利用避暗实验和动物跑步台实验做行为学检测以分析衰老鼠的学习记忆和运动能力，苏木素-伊红（Hematoxylin-Eosin，HE）染色法进行卵巢组织的卵泡计数和子宫形态观察及腺体数目统计，马松（Masson）染色分析子宫组织胶原容积分数，双抗体夹心酶联免疫法和/或荧光实时定量聚合酶链式反应（Quantitative Real-Time Polymerase Chain Reaction，qRT-PCR）法检测相关激素指标、炎症相关因子、纤维化相关因子、细胞焦亡因子、氧化应激相关分子的水平。此外，我们以生殖系统相关细胞：人绒毛膜滋养层细胞（HTR8/SVneo细胞）、人子宫内膜癌细胞（RL95-2细胞）、人子宫腺癌细胞（HEC1A细胞）和中国仓鼠卵巢细胞(CHO细胞)为研究对象，利用过氧化氢（hydrogen peroxide，H₂O₂）损伤处理建立细胞衰老模型。用qRT-PCR法检测自然衰老小鼠子宫、卵巢和体外过氧化氢诱导的衰老细胞中AKT-mTOR信号通路关键分子的表达来探究 对生殖细胞衰老发挥重要作用的分子。

结果显示给予hUCMSCs或/和CoQ10、DHEA能明显改善老年小鼠的脱毛现象和动物精神状态，动物死亡率也有所降低，尤其是CoQ10，但是对体重没有明显影响。避暗实验和跑步机实验结果提示hUCMSCs或/和CoQ10、DHEA可增强动物的记忆能力、运能能力和耐力。组织HE染色显示hUCMSCs或/和CoQ10、DHEA能增加子宫腺体的数量和卵巢成熟卵泡的数量。这些结果显示了hUCMSCs或/和CoQ10、DHEA能够改善自然衰老小鼠的身体机能，延缓子宫和卵巢组织的老化。

探讨hUCMSCs或/和CoQ10、DHEA延缓生殖器官衰老的可能原因，我们可以看到hUCMSCs或/和CoQ10、DHEA可以抵抗衰老导致血清中FSH和LH水平升高、E2和AMH水平降低的激素变化。组织纤维化是器官老化损伤的一个重要特征，我们利用Masson染色检测了组织纤维化情况，发现hUCMSCs和CoQ10协同作用可以明显抑制子宫组织的纤维化，降低促纤维化因子Smad-3的表达。而hUCMSCs和DHEA协同抑制纤维化的作用不明显。检测外周血清中促炎性细胞因子的变化显示DHEA或/和CoQ10协同hUCMSCs可以促进促炎性细胞因子的表达，但qRT-PCR检测子宫和卵巢组织中促炎性因子的表达发现CoQ10或DHEA可以抑制其在组织中的表达， DHEA或/和CoQ10协同hUCMSCs作用结果与二者单独作用结果相反。检测外周血中抗炎性细胞因子的变化显示hUCMSCs单独或联合CoQ10、DHEA可以抑制TGF-b和IL-10、促进IL-13和IL-14等抗炎性细胞因子的表达，但qRT-PCR检测子宫和卵巢组织中抗炎性因子的表达发现DHEA或/和CoQ10协同hUCMSCs可以促进TGF-b、IL-13的表达。这些结果提示了炎症反应参与了DHEA或/和CoQ10协同hUCMSCs修复老化子宫内膜和老化卵巢的功能。我们检测了细胞焦亡相关蛋白的表达，研究发现外周血清中hUCMSCs可以升高Caspase-1的水平，降低Caspase-4的水平； hUCMSCs与CoQ10联合可以升高Caspase-1的水平。hUCMSCs与CoQ10联合可以降低子宫组织中GSDMD的水平。这些结果提示细胞焦亡参与了炎症反应过程。我们也检测了子宫内膜标志物的变化，结果发现hUCMSCs和/或CoQ10、DHEA对子宫内膜上皮细胞标志物Pan-Keratin和子宫内膜基质细胞标志物Vimentin的表达无显著性影响。检测AKT-mTOR信号通路分子发现hUCMSCs和/或与CoQ10、DHEA可以促进AKT、PDK-1、SGK1、PKC-α、PRAS40、HIF-3α等的表达。我们实验室构建了HIF3α的过表达载体，筛选了有效的HIF-3α siRNA，并检测了其功能，发现HIF-3α的表达水平与其对细胞增殖、迁移和浸润的影响呈正向相关。衰老细胞模型研究结果发现HIF-3α的表达在HTR8/SVneo细胞和RL95-2细胞衰老状态中上调，但在CHO细胞和HEC1A细胞衰老状态中下调。

综上所述，我们的研究发现hUCMSCs或/和CoQ10、DHEA能够改善自然衰老小鼠的身体机能，延缓子宫和卵巢组织的老化。纤维化、炎症反应、细胞焦亡等参与了hUCMSCs或/和CoQ10、DHEA延缓子宫组织老化的过程，其可能通过AKT-mTOR信号通路发挥作用。

Population aging is a common problem facing the world today. 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 and not aging 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 at its 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 - β 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 - β 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- α、 PRAS40、HIF-3 α and so on. The effective HIF-3α 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-3α 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.