Study On The Mechanism Of Hyperoside In The Treatment Of Oligoasthenozoospermia Based On Keap1/Nrf2/HO-1 Pathway

Objective:Observation of the protective effect of Hyperoside（Hyp）on oxidative damage in mouse primary spermatocytes（GC-2）model and the mouse model of cyclophosphamide-induced hypospermia（OA）,and to analyze its molecular mechanism from the perspective of Keap1/Nrf2/HO-1 signaling pathway,so as to provide experimental basis for the further development and utilization of Hyp.Methods:Section one:Study on the protective effect and mechanism of Hyp on oxidative damage in GC-2 cells based on Keap1/Nrf2/HO-1 pathway.GC-2 cells with a growth density of 80%-90%were randomly divided into normal group,model group,2.5 mmol/L nitrogen acetylcysteine group（positive group,NAC）and 50,100 and 200μmol/L gibberellin group after 48 h incubation in the NAC and gibberellin groups at the dose,all groups except the normal group were treated with 150μmol/L H₂O₂treatment for 2 h,while the normal group was treated with equal volume of culture medium.Cell viability was measured by CCK-8 method,apoptosis level by flow cytometry,superoxide dismutase（SOD）,glutathione peroxidase（GSH-PX）,catalase（CAT）activity and malondialdehyde（MDA）content by ELISA,Nrf2,Keap1,HO-1 by Western blot and q PCR.The relative expression levels of Nrf2,Keap1,HO-1 and mRNA were measured by Western blot and RT-q PCR,and the nuclear translocation level of Nrf2 was detected by immunofluorescence.The second section:Study on the preventive and therapeutic effects and mechanism of Hyp on OA mice based on Keap1/Nrf2/HO-1 pathway.Sixty healthy male Kunming mice were randomly divided into normal group,model group,0.25 g/kg vitamin E group（positive group）,and 100,200 and 400mg/kg gentiside group.The vitamin E and gentiside groups were gavaged continuously for 4 weeks at the dose,while the normal group and the model group were given equal volumes of distilled water.At the end of the third week of administration,cyclophosphamide（80 mg/kg）was administered intraperitoneally for5 d to the model group,the vitamin E group and the gibberellin group,and an equal volume of saline was administered intraperitoneally to the normal group.Sperm quality（number,viability and viability）was measured by Makler sperm counting plate;structural changes in testicular tissues were measured by HE staining;ROS levels in serum and testicular tissues were measured by fluorescence spectrophotometer;GSH-PX,CAT,SOD activity and MDA levels in serum and testicular tissues were measured by ELISA;serum FSH,LH and T levels were measured by ELISA.Real-time PCR and Western blot were used to determine Keap1,Nrf2,HO-1 mRNA and protein expression respectively.Results:Section one:A model of oxidative damage in GC-2 cells could be successfully prepared by using 150μmol/L H₂O₂intervention for 2 h.Compared with the normal group,the proliferation rate and the antioxidant enzymes SOD,GSH and CAT activity of GC-2cells in the model group were significantly lower,and the MDA content,apoptosis rate,Keap1 mRNA and protein expression and Nrf2 mRNA expression were significantly higher（P<0.05）.Compared with the model group,the cell value-added rate and SOD,GSH-PX and CAT activity were significantly higher,and MDA content and apoptosis rate were significantly lower in the NAC and 200μmol/L gentianoside groups（P<0.05）;Keap1 mRNA expression was significantly lower and HO-1 mRNA expression was significantly higher in the 200μmol/L gentianoside group（P<0.01）.The protein expression of Nrf2 nuclear protein（NEs-Nrf2）and HO-1was significantly increased（P<0.05）and that of Keap1 was significantly decreased（P<0.01）in the 200μmol/L Hyperoside group;a significant nuclear translocation phenomenon was observed in the Hyperoside group（P<0.05）.The second section:Compared with normal group,the sperm quality of mice in model group was decreased,the number of spermatogenic cells at all levels of testis tissue was less,vacuolation occurred in the fine ductus seminis,the interstitial cells were seriously damaged,the sperm number in the lumen was decreased,the void was increased,the cell distance was increased,the activities of GSH-PX,CAT and SOD in serum and testis tissue were decreased,and the contents of MDA and ROS were increased.Serum levels of FSH and LH were significantly increased,while T levels were significantly decreased mRNA and protein expressions of Keap1 were significantly increased,with statistical significance（P<0.05）,while mRNA and protein expressions of Nrf2 and HO-1 were increased,with no statistical significance.Compared with model group,sperm quality of mice in Vit.E group and Hyperoside 400mg/kg group increased,spermatogenic cells of all levels increased,interstitial vacuoles of testis decreased,dispersion degree decreased,intracavitary sperm count increased,activities of GSH-PX,CAT and SOD in serum and testis tissue increased,and contents of MDA and ROS decreased.Serum FSH and LH levels were significantly decreased,while T levels were significantly increased,Keap1 mRNA and protein expressions were significantly decreased,and Nrf2 mRNA,NEs-Nrf2 protein,HO-1 mRNA and protein expressions were significantly increased,with statistical significance（P<0.05）.Conclusion:Hyp may play an antioxidant role by activating the Keap1/Nrf2/HO-1 pathway in vivo and in vitro,increase the level of GSH-PX,CAT,SOD,T,and reduce the content of MDA,ROS,FSH,LH,reduce the oxidative stress damage of testicular tissue and GC-2 cells,improve sperm quality,and thus treat OA.