| High-dose chemotherapy drugs are often used in clinical treatment of various tumors,so that some patients' testicular spermatogonial stem cells are seriously damaged or even exhausted,resulting in the formation of non-obstructive azoospermia.Platinum-based chemotherapeutics are also widely used clinically.There is increasing evidence that exposure to platinum-based chemotherapeutic drugs induces oxidative stress in the testes of rats and mice,eventually leading to oxidative damage to the testes and impaired spermatogenesis.However,the specific regulatory mechanism of testicular oxidative stress induced by platinum-based chemotherapy drugs and how to improve testicular oxidative damage caused by platinum-based chemotherapy drugs are not very clear.Melatonin is a neurohormone synthesized by the pineal gland.Melatonin can not only scavenge oxygen free radicals,but also regulate the activity of antioxidant enzymes through its receptors,thereby regulating apoptosis.However,there is no research on the molecular mechanism of melatonin and its receptors on the oxidative damage of testis caused by platinum chemotherapy drugs.Based on the above contents,this study systematically expounds the possible mechanism of melatonin receptor activation in improving the oxidative damage of testis caused by platinum-based chemotherapy drugs in the mode of "population-animal-cell":1.To clarify the effect of platinum-based chemotherapy drugs on oxidative stress levels in male tumor patients and melatonin receptors in testis;2.To investigate the protective effect of melatonin on oxidative damage of mouse testis and Leydig cells induced by cisplatin;3.To analyze the molecular mechanism of melatonin improving cisplatin-induced oxidative damage of Leydig cells by activating MT1/MT2 mediated SIRT1/Nrf2 signaling pathway.Part ?Effects of platinum chemotherapy drugs on peripheral blood sex hormone,oxidative stress and testicular MT1/MT2 in patients with gastrointestinal tumorsObjective:To investigate the effects of platinum chemotherapy drugs(cisplatin or oxaliplatin)on the levels of testicular MT1/MT2,peripheral blood sex hormone and oxidative stress in male patients with malignant gastrointestinal tumors.Methods:the peripheral blood of 30 normal healthy men,30 male patients with gastrointestinal tumors diagnosed for the first time and 30 male patients with gastrointestinal tumors treated with chemotherapy drugs(cisplatin or oxaliplatin)twice or more were collected.The supernatant was centrifuged and the levels of gonadotropin releasing hormone(GnRH),follicle stimulating hormone(FSH),luteinizing hormone(LH)and testosterone(T)in peripheral blood were detected by ELISA.Oxidative stress in serum were detected by colorimetry:lipid oxidation(MDA),superoxide dismutase activity(SOD)and total antioxidant capacity(T-AOC).The testicular tissue of 3 patients with obstructive azoospermia(OA)was retrievaled as normal control,and the testicular tissue of 3 patients with non-obstructive azoospermia caused by platinum-based chemotherapy drugs by testicular puncture.HE and TUNEL staining were used to observe the morphological changes and apoptosis of testicular tissue.Immunofluorescence double labeling method was used to detect the localization and expression of Leydig cells and melatonin receptor 1(MT1)/melatonin receptor 2(MT2).Results:compared with the control group,there were no significant changes in the levels of gonadal hormone and oxidative stress in the peripheral blood of male patients with gastrointestinal tumors who were not treated with platinum-based chemotherapy drugs.However,in the platinum-based chemotherapy drugs treatment group,the levels of GnRH remained unchanged,but the levels of FSH and LH increased,the levels of testosterone decreased,the indicators of oxidative stress MDA increased,SOD activity and T-AOC decreased.HE staining showed that no spermatogenic cells were found in the testis of patients with non-obstructive azoospermia caused by platinum-based chemotherapy drugs,only testicular Leydig cells and testicular Sertoli cells.TUNEL staining also showed DNA breakage and increased apoptosis in testicular tissue of platinum-based chemotherapy drugs patients.Immunofluorescence showed that MT1 and MT2 were highly expressed in Leydig cells of testis in patients with OA and platinum-based chemotherapy drugs patients.Compared with patients with OA,the expression of MT1/MT2 in Leydig cells of testis in patients with platinum-based chemotherapy drugs was lower.Conclusions:platinum chemotherapy drugs(cisplatin or oxaliplatin)can disorder gonadal hormone secretion,cause oxidative stress,down-regulate the expression of MT1 and MT2 in Leydig cells of testis and increase the apoptosis of testicular cells.Part ?Melatonin up-regulates SIRT1/Nrf2 signaling pathway to alleviate the damage of cisplatin to mouse testis and Leydig cellsObjective:To investigate the effects of cisplatin on testicular injury and oxidative stress in mice,whether MT1/MT2 is highly expressed in testicular stromal cells,and whether melatonin upregulates MT1/MT2 and SIRT1/Nrf2 antioxidant signals,so as to alleviate the damage of cisplatin to testicular and testicular stromal cells in mice.Methods:(1)C57BL/6J mice were randomly divided into 8 groups:control group d0(C d0),model group d0(CP d0),control group d8(C d8),model group d8(CP d8),control group d17(C d17),model group d17(CP d17),control group d34(C d34),model group d34(CP d34).The model group was given intraperitoneal continuous injection of cisplatin(CP)for 4 days,and recovered on day 0,day 8,day 17 and day 34.The control group was given intraperitoneal injection of the same amount of normal saline.(2)Mice were randomly divided into 4 groups:control group(control),model group(CP),melatonin+model group(CP+MT)and melatonin group(MT).The model group was given intraperitoneal continuous injection of cisplatin(CP)for 4 days and the same amount of normal saline for 17 days.The model group+melatonin(CP+MT)was given intraperitoneal injection of melatonin first and then cisplatin(CP)at an interval of 12 hours.Cisplatin was injected for 4 days and melatonin was injected for 21 days.The melatonin group(MT)was given intraperitoneal injection of melatonin for 21 days.The changes of body weight of mice were recorded every day.Peripheral blood,testis and epididymis were collected.The levels of testosterone were detected by ELISA.The indexes of oxidative stress in testis were detected by colorimetry:MDA,SOD and T-AOC.The morphological changes of epididymis and testis were observed by HE staining,the apoptosis of testis was observed by TUNEL staining,and the number of Leydig cells was counted by immunohistochemistry.ITRAQ proteomics was used to analyze the differential proteins of C d17 and CP d17.The localization and expression of MT1/MT2 in Leydig cells were detected by immunofluorescence double labeling.Western blot was used to detect the expression of antioxidant stress related signal SIRT1/Nrf2 protein.Results:compared with the control group,the weight of mice in the model group began to decline after continuous intraperitoneal injection of CP for 4 days,and recovered for half a spermatogenic cycle(17 days)and one spermatogenic cycle(34 days),and the weight could not completely return to the state of the control group.In the model group,the size of testis and epididymis decreased,and the testis in CP d17 group was the smallest.He staining showed that in the model group,the number and concentration of sperm in epididymal canal gradually decreased,and the gap between seminiferous tubules in testicular tissue became larger.Even the seminiferous tubules in CP d17 group showed vacuole-like structure,no sperm was found in CP d34 group,and testicular stromal cells were damaged.In the model group,the levels of testosterone decreased,the index of oxidative stress MDA increased,and the activity of SOD and T-AOC decreased.ITRAQ proteomics showed that 110 proteins between C d17 and CP d17 were related to oxidative stress,in which SIRT1/Nrf2 signal was the central region of the protein interacting with MT1.WB results also showed that the antioxidant stress related signal SIRT1/Nrf2 decreased in the model group.The volume of testis and epididymis in CP+MT group were larger than that in CP Group.The number and concentration of sperm in epididymal canal increased,the gap between seminiferous tubules in testicular tissue became smaller,the apoptosis of cells decreased,and the number of Leydig cells increased;The levels of testosterone increased,the index of oxidative stress MDA decreased,and the activity of SOD and T-AOC increased;The expression of MT1/MT2,melatonin synthase AANAT and ASMT,and the expression of antioxidant stress related signal SIRT1/Nrf2 increased.Conclusions:Cisplatin can cause oxidative damage to mouse testis and down-regulate the expression of MT1/MT2 and antioxidant signal SIRT1/Nrf2 in testis.Melatonin can effectively alleviate the oxidative damage of mouse testis caused by cisplatin,up-regulate the expression of MT1/MT2 and antioxidant signal SIRT1/Nrf2 in testicular stromal cells.Part ?Melatonin regulates SIRT1/Nrf2 signaling pathway through MT1/MT2 to improve the oxidative damage of cisplatin to Leydig cellsObjective:To investigate whether melatonin can regulate SIRT1/Nrf2 antioxidant signal through activating MT1/MT2 to alleviate the damage of cisplatin to Leydig cells.Methods:Mouse primary Leydig cells were extracted and cultured.The proliferation toxicity of cisplatin at different concentrations and at different times on mouse primary Leydig cells were observed by CCK-8 method.The effect of cisplatin on apoptosis of primary Leydig cells were detected by flow cytometry.WB was used to detect the effects of cisplatin on the expression of MT1/MT2 and antioxidant stress related signal SIRT1/Nrf2 protein in primary Leydig cells.After removing cisplatin,the proliferation toxicity of Leydig cells were detected,and the content of ROS in primary Leydig cells were detected by DCFH-DA fluorescent probe;And adding melatonin receptor agonists or inhibitors,WB detected the expression of MT1/MT2 and antioxidant stress-related signal SIRT1/Nrf2 protein.Results:Different concentrations of cisplatin decreased the activity of primary Leydig cells,the expression of MT1/MT2 and the expression of antioxidant stress related signal SIRT1/Nrf2.Compared with cisplatin treatment group,after the removal of cisplatin,the viability of primary Leydig cells could not be restored,the secretion of testosterone also decreased,the level of ROS increased,the expression of MT1/MT2 decreased,and the expression of antioxidant stress related signal SIRT1/Nrf2 further decreased.However,melatonin can activate the expression of MT1/MT2 and alleviate the decrease of SIRT1/Nrf2 signal caused by cisplatin.Melatonin receptor inhibitors exacerbated the decreased expression of MT1/MT2 and SIRT1/Nrf2 signals induced by cisplatin.Conclusions:Melatonin can effectively alleviate the oxidative damage of Leydig cells induced by cisplatin by activating MT1/MT2 to regulate SIRT1/Nrf2 antioxidant signal. |
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