| Objective:Female follicles are constantly reduced and depleted with the increase of age,in addition to affecting female reproductive life span,they will also bring many perimenopausal clinical symptoms,such as hot flashes,anxiety,night sweats and menstrual changes,which seriously affect women’s quality of life and mental and physical health.At present,the main means of clinical treatment for ovarian aging is hormone supplementation therapy,but its side effects are attracting more and more attention,including uterine bleeding,thrombosis,endometrial cancer,breast cancer and so on.Melatonin(MT)is an endogenous indexing hormone secreted by the human body,which is common in the human body and can enter the mitochondria to clear free radicals.Exogenous melatonin can also easily enter the mitochondria to improve oxidative stress,with anti-aging,anti-inflammation and immune enhancement effects on the body.It has been reported that MT can delay ovarian aging,but the specific mechanism is completely clear,and whether it is related to ovarian reproductive stem cells is not reported.This study intends to observe the protective and rescue effect of MT on chemotherapeutic ovarian aging through regular and continuous intraperitoneal injection.Methods:Kunming mice were divided into CON group(Control group),ovarian mold aging group(phosphoramide-white elimination safety,Cyclophosphamide-Busulfan,CY-BUS group),melatonin group(MT group),and melatonin treatment group(CYBUS+MT group),After drug administration,ovarian function and ovarian aging were detected by measuring ovarian index,follicle number,serum estrogen level,and expression level of reproductive stem cell markers,The mechanism of melatonin on ovarian aging was analyzed by measuring the polarization status of ovarian macrophages,serum immunokines,inflammatory factors and HIF-1-NF B-VEGF proteins or/and genes.Results:(1)Compared with the CON group,The ovarian structure and morphology of the CY-BUS group were destroyed,and the number of follicles at all levels decreased;Mice body weight,ovarian weight,and ovarian index were decreased;Hormones,AMH,and E2 content decreased by 10.5%and 22.6%(p<0.05),FSH,LH,and progesterone content increased,respectively;Cytokines such as IL-6,IL-4 and TNFincreased by 15.1%,19.8%and 23.9%(p<0.05),IL-10 decreased by 4.5%(p<0.001),and IL-6 and TNF-increased by 12.64 and 8.47 times,respectively(p<0.001);The content of antioxidant SOD2 and its gene expression decreased by 11.5%and 48.71%,and the content of ROS,8-OHdG and 4-HNE increased by 16.0%,16.6%and 23.1%,respectively(p<0.05);In the HIF-1-NF B-VEGF pathway,HIF-1,p-NF B,and VEGF mRNA and protein expression were significantly increased;CY-BUS promotes ovarian M1-type macrophage polarization,Inhibition of the M2-type macrophage poles;The expression ratio of ovarian reproductive stem cell marker OCT4 to cell proliferation marker BrdU was decreased.All of the above differences were statistically significant,with p<0.05.(2)Compared with CY-BUS group,the number of follicles(primitive,primary,secondary,and sinus)increased in CY-BUS+MT group;E2 content increased by 31.3%(p<0.001),FSH and progesterone decreased by 14.0%and 21.0%,was statistically significant;AMH and LH were not statistically significant;mouse weight,ovarian weight and ovarian index were still lower than the CON group.(3)Compared with the CY-BUS group,the content of IL-6 and TNF-in the CYBUS+MT group decreased to 152.93pg/mL and 52.77pg/mL and gene expression,respectively,with a statistically significant difference.The IL-10 content increased significantly to the 1123.87pg/mL(p<0.001),and the IL-4 and TGF-1 were not statistically significant.(4)Compared with the CY-BUS group,the ROS content of the CY-BUS+MT group decreased by 11.0%,and the SOD2 content and gene expression increased by 12.3%and 69.6%,respectively,showing a significant difference.(5)Compared with CY-BUS group,CY-BUS+MT group can reverse the increase of HIF-1,p-NF B,VEGF mRNA and protein expression in mouse ovaries caused by CY-BUS,with a statistically significant difference.(6)Compared with the CY-BUS group,the expression ratio of the ovarian reproductive stem cell marker OCT4 and the cell proliferation marker BrdU expression ratio in the CY-BUS+MT group was significantly increased.(7)Compared with the CY-BUS group,the CY-BUS+MT group stimulated ovarian M2 type macrophage polarization,increased the proportion of M2 type macrophages,and inhibited M1 type macrophages,with a statistically significant difference.Conclusions:Melatonin can rescue ovarian senescence caused by chemotherapeutic drugs,and its mechanism may be related to the regulation of HIF-1-NF B-VEGF pathway affecting macrophage polarization,and thus regulating the function of ovarian reproductive stem cells. |
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