Protective Effect Of Polysaccharides From Ostrea Rivularis Gould Against Oxidative Damage Via Regulating Autophagy

Background:In modern society,due to the influence of multiple unfavorable factors（deterioration of the living environment,poor human lifestyles,aging of the population,etc.）,the male reproductive system is directly or indirectly exposed to the reactive oxygen species produced in the environment,and is subjected to oxidative stress.It causes male reproductive system spermatogenesis disorders,which is one of the reasons why the incidence of male infertility is increasing year by year.Previous studies had shown that polysaccharides from Ostrea rivularis Gould（ORP）had antioxidant activity and possessed a certain protective effect on the testis.But the corresponding mechanism is still not completely clear.Objective:In order to clarify the protective effect of ORP on the male reproductive system and its effect on autophagy,experiments in vivo were conducted to explore the adverse effects of cyclophosphamide（CTX）on the oxidative stress damage of the mouse reproductive system.Moreover,we also operated in vitro experiments to explore the effect of ORP on oxidative stress damage of TM4 cells and its mechanism.Methods:1.One hundred and eight male BALB/c mice at the age of 6～8 weeks were randomly divided into 6 groups:normal group,model group（intraperitoneal injection of 75 mg/kg CTX）,and positive drug group（intraperitoneal injection of 75 mg/kg CTX）,intragastric administration of 5 mg/mL VE),ORP high-dose group（intraperitoneal injection of 75 mg/kg CTX,intragastric administration of 200 mg/kg）,ORP medium-dose group（intraperitoneal injection of 75 mg/kg CTX,Oral administration 100 mg/kg）,ORP low-dose group（intraperitoneal injection of 75 mg/kg CTX,oral administration of 50 mg/kg）.The mice were intraperitoneally injected with cyclophosphamide injection once every 4 days to establish a mouse oxidative stress model from the day after the mice were grouped,and the mice were dissected 7,14,and 19 days after the administration.Record the weight of mice and the weight of testis,spleen,thymus,and detect the index of each tissue.Sperm parameters were recorded by an inverted optical microscope.The H&E staining method was appllied to determine the pathological changes of testis and spleen.ELISA kit were used to determine the content of SOD,GSH-Px and testosterone.Transmission electron microscopy was used to observe the level of autophagosomes in testicular tissue.2.Different concentrations of H₂O₂ were applied to induce oxidative stress damage in TM4 cells,and the best modeling concentration was selected by the CCK-8 method.TM4 cells were treated by H₂O₂ with different concentrations of ORP,and CCK8 method was used to detect cell viability.TM4 cells were divided into blank group,model group（400μM H₂O₂）,and positive drug group（2 μg/mL VE+400 μM H₂O₂）,High-dose group（200μg/mL ORP+400 μM H₂O₂）,medium-dose group（100 μg/mL ORP+400 μM H₂O₂）,low-dose group（50 μg/mL ORP+400 μM H₂O₂）,high-dose-3MA Group（200 μg/mL ORP+400 μM H₂O₂+3-MA）,3MA group（100μg/mL ORP+400 μM H₂O₂+3-MA）.TM4 cells growth status of each group was observed under an optical microscope.The ROS level of testicular was measured by DCFH-DA fluorescent probe.The content of CAT and MDA was measured by ELISA kit.The expression of protein Cleaved Caspase-3,Bax,Bcl-2 in TM4 cells were measured by Western blot.Also the expression of protein p62,beclinl,and the ratio of LC3Ⅱ/Ⅰ.Immunofluorescence technology to detect changes in the expression of key autophagy proteins LC3B and p62.Transmission electron microscopy was used to observe the level of autophagosomes in TM4 cells.Results:1.Pharmacodynamic study of polysaccharides from Ostrea rivularis Gould on CTX-induced reproductive oxidative stress injury in BALB/c mice.The results showed that ORP had a protective effect on the oxidative stress damage of the mice reproductive system caused by CTX:after CTX induction,mice in the model group lost weight;testis index,spleen index,and thymus index were decreased（P<0.05）;testis and spleen tissues were significantly affected;sperm count and sperm survival rate were decreased（P<0.05）;sperm deformity rate were increased（P<0.05）;content of testosterone were significantly decreased（P<0.05）.CTX could also significantly reduce the content of SOD and GSH-Px in BALB/c mice（P<0.05）,and increase the autophagy level of testicular cells,suggesting that CTX could cause oxidative stress damage to the reproductive system of mice.As the modeling time increases,CTX damages the mice more severely.After ORP intervention,the testis index,spleen index,and thymus index of mice increased（P<0.05）;the lesions of the testis and spleen tissue were alleviated;the sperm count and sperm survival rate increased（P<0.05）;and the sperm deformity rate decreased（P<0.05）;testosterone content was significantly increased（P<0.05）;SOD,GSH-Px content in mouse serum was significantly increased（P<0.05）;transmission electron microscope observed that the level of autophagy in mouse testicular cells was significant decreased after ORP intervention.Compared with the normal group,there was still a certain degree of autophagy.It showed that ORP could improve the antioxidant capacity of mice,enhance the immune function of mice,alleviate the excessive autophagy of testicular cells caused by CTX in mice,and protect the reproductive system of mice.2.Study on the mechanism of polysaccharides from Ostrea rivularis Gould in regulating autophagy against oxidative stress damage of TM4 cells in vitro.The effects of ORP on oxidative stress damage of TM4 cells treated with H₂O₂ to induces oxidative stress were investigated through ORP intervention at different concentrations.At the same time,3-M A was added as control treatment to study the effect of changes in autophagy levels on the antioxidant protection of ORP.The results showed that compared with the model group,cells morphology was significantly improved after ORP intervention,cell damage was alleviated,intracellular ROS levels were significantly reduced（P<0.05）,CAT content increased significantly（P<0.05）,MDA content decreased（P<0.05）,the expression of pro-apoptotic proteins Cleaved Caspase-3 and Bax decreased in the cells,and the expression of anti-apoptotic protein Bcl-2 increased（P<0.05）.At the same time,the ratio of autophagy-related proteins LC3Ⅱ/LC3-Ⅰ and the expression of beclin1 decreased（P<0.05）,the expression of p62 increased（P<0.05）were also.The above results indicated that over-inhibition of cells could aggravate cellular oxidative stress damage and inhibit the protective effect of ORP on TM4 cells,while ORP could alleviate H₂O₂-induced oxidative stress damage and autophagy levels in TM4 cells,but still retain a certain amount of autophagy in the cell to maintain normal physiological activities.Conclusion:1.CTX could reduce the antioxidant capacity of the testis,cause oxidative stress damage and nduce excessive autophagy,while ORP could effectively alleviate the testicular toxicity of CTX in the body,protect the normal secretory function of the testis,and improve the immunity of mice.The corresponding mechanism might be to reduce the level of autophagy in the cell to increase the antioxidant capacity of testicular tissue.2.H₂O₂ could directly cause oxidative stress damage,induce apoptosis and autophagy to TM4 cells.Adding ORP could inhibit cell autophagy and oxidative stress damage caused by H₂O₂ and enhance the antioxidant capacity of TM4 cells.3-MA autophagy inhibitors could inhibit cell autophagy,and excessive inhibition of autophagy will aggravate cell oxidative stress damage.3.ORP could significantly inhibit the excessive autophagy induced by CTX and H₂O₂,but there was still a certain amount of autophagy in the cell to assist the body in clearing the level of ROS and antagonize cell apoptosis,so as to protect TM4 cells from H₂O₂ oxidative damage.