Exploration Of The Protective Effect And Mechanism Of Nano-selenium On Oxidative Stress-induced Male Reproductive Injury Based On Pre-pregnancy Family

【Background】According to the survey in China,most respondents are willing to have a second child,including a large number of couples over 35 years old.With the release of the two-child policy,the prevention and control of birth defects has become more difficult and important.The influence of preconception risk factors on fertility,pregnancy rate,live birth rate and the genetics of offspring is increasingly receiving attention.However,alcohol acts on spermatogenic cells and supporting cells at all levels by destroying the blood-testosterone barrier,causing structural damage and functional changes of cells.The key mechanism lies in protein degradation,lipid peroxidation and DNA damage caused by intracellular oxidative stress injury induced cell apoptosis,but the specific molecular mechanism is not clear.Selenium is essential for sperm production in male animals,and the mitochondrial outer membrane of mammalian sperm is rich in cystine and most selenium proteins.After being absorbed into the duodenum,selenium enters the body to participate in the synthesis of glutathione peroxidase GPx and other selenase related enzymes.Its important role in regulating antioxidant defense mechanisms and other basic biological pathways as well as REDOX sensitive transcription factors is beyond doubt.Many studies have shown that testicular tissue contains high concentration of selenium,mainly in the form of glutathione peroxidase4（GPx4）.GPx4 prevents oxidative damage of spermatocytes DNA at various stages by scavenging free radicals,and protects the structural and functional integrity of cells and cell membranes.Recent literature has reported that oxidative stress intake affects GPx4 levels to some extent.Irvine et al.moderately increased dietary selenium content in adult males,and found that GPx activity significantly increased and male fertility significantly improved,but the specific mechanism of action was not clear.Compared with other selenides,nano-selenium is more useful in the treatment of cancer because of its higher biological absorption and utilization.Recent literature reports suggest that nano-selenium plays an important role in maintaining the normal structure and function of male reproductive organs,and can protect against the reproductive toxicity of male mice induced by nickel,cadmium and the antitumor drug cisplatin,which leads to testicular injury and apoptosis in rats.However,the detailed mechanism of action has not been clarified.【Objective】To explore the protective effect and mechanism of nano-selenium on oxidative stress-induced male reproductive injury based on pre-pregnancy family cohort【Experimental methods】（1）By establishing a pre-pregnancy cohort of a certain size,the study group intended to evaluate the correlation between trace element concentration and reproductive damage in 53 clinical azoospermia patients by measuring different semen quality and selenoprotein concentration in males.Se NPs@LNT,Se NPs@PEG,Se NPs@CS and three kinds of organic selenium,inorganic selenium 1μM,2μM,4μM,8μM,16μM,32μM mouse spermatogonial stem cell GC1 cells,respectively.The cytotoxicity of selenium was observed in mouse spermatogonocyte GC2-spg cells and mouse testicular interstitial cell TM3 cells at 24to 72 h.CCK-8 was used to measure cell viability.（2）GC2-spg cells were randomly divided into control group,H₂O₂ group,Se NPs group and H₂O₂+Se NPs group.H₂O₂+Se NPs group:Se NPs 4μM pretreatment for 12 h,then H₂O₂ 600μM treatment for 8 h.Cell viability and mitochondrial membrane potential were determined by CCK-8 and JC-1 methods.Reactive oxygen species（ROS）,superoxide dismutase（SOD）activities and total glutathione peroxidase（GSH）activities were measured with each kit.The expression levels of signaling pathway protein and selenoprotein were detected by Western blot.（3）TM3 cells were randomly divided into control group,H₂O₂ group,Se NPs@LNT group and H₂O₂+Se NPs@LNT group.H₂O₂+Se NPs@LNT group:Se NPs 4μM pretreatment for 12 h,then H₂O₂ 600μM treatment for 8 h.Cell viability was determined by CCK-8 method,mitochondrial membrane potential was determined by JC-1 method,MDA content and glutathione peroxidase（GPx）activity were determined,TUNEL staining was used to observe the apoptosis of testicular cells in each group,and semi-quantitative analysis was performed.The protein expression levels of Bak,Bcl-2,Cytochrome c,Caspase 9 and Caspase 3 in apoptotic pathways were detected by Western blot.The correlation between alcohol concentration and oxidative stress and the effect of nano-selenium supplementation on reducing oxidative stress and anti-apoptosis were further evaluated.【Results】（1）The family birth cohort before pregnancy was established,and the model for analyzing the influence of various influencing factors on male fertility was successfully constructed;The correlation between trace elements and semen quality was analyzed.The results indicated that the decrease of selenium content was associated with the decrease of semen quality（P<0.05）.Se NPs@LNT,Se NPs@PEG,Se NPs@CS showed the maximum cell viability at 4μM,compared with the control group in 0-32μM,three kinds of organic selenium and inorganic selenium showed the maximum cell viability at 4μM.However,cell viability decreased to cell death in the range of 8μM-32μM（all P<0.001）.（2）GC2-spg cells were treated with 0,125,250,500,600,700,800 and 1000μM of hydrogen peroxide for 8 h.With the increase of hydrogen peroxide concentration,cell viability decreased gradually.When hydrogen peroxide concentration was 600μM,cell viability decreased to 76.07%（P<0.001）.The difference was statistically significant.In order to ensure oxidative damage and avoid the decline of cell vitality to irreversible damage,an appropriate hydrogen peroxide concentration of 600μM and treatment time of 8 h were selected as oxidative stress model for follow-up experiments.We treated normal control group and oxidative stress model group with 4μM nano-selenium for 12 h,and the cell viability of H₂O₂group decreased to 72.09%,the difference was statistically significant（P<0.001）,Se NPs@LNT+H₂O₂group cell viability was 88.02%,compared with H₂O₂ group,the difference was statistically significant（P<0.05）.The cell vitality of Se NPs@LNT treatment group was 40%,which was statistically significant compared with H₂O₂group and Se NPs@LNT+H₂O₂ group.Compared with the H₂O₂ group,the Se NPs@LNT+H₂O₂ group showed significant red fluorescence,that is,higher mitochondrial membrane potential level.Through fusion fluorescence,we can observe that the ratio of red to green fluorescence in Se NPs@LNT treatment group is higher than that in oxidative stress group.The intracellular ROS content of Se NPs@LNT group was significantly decreased,and the green fluorescence intensity of Se NPs@LNT+H₂O₂ group was significantly decreased compared with H₂O₂ group.The average GPx activity was 32.76（m U/mgprot）in control group and 12.70（m U/mgprot）in oxidative stress group.The highest GPX activity was found in Se NPs@LNT group（362.05）（P<0.001）.Compared with H₂O₂ group alone,glutathione peroxidase activity in Se NPs@LNT+H₂O₂ and Se NPs@LNT groups was significantly increased,with P<0.001.The mean SOD activity of the control group was 1.32（m U/mgprot）,and that of the hydrogen peroxide group was 0.60（m U/mgprot）（P<0.01）,the mean value of SOD activity in Se NPs@LNT+H₂O₂ group was 1.68（m U/mgprot）,and that in Se NPs@LNT group was 3.25（m U/mgprot）（P<0.001）.With the intervention of nano-selenium,the expression of p53 was significantly decreased,the expression level of GPx1 was increased,and the expressions of DNA damage-related proteins and apoptosis-related proteins in oxidative stress group were higher than those in other groups.The expression of Caspase 3 was decreased and the expression level of ERK was lower than that of other groups.（3）TM3 cells were treated with concentration gradients of 0,125,250,500,600,700,800 and 1000μM hydrogen peroxide for 8 h.When the concentration was 800μM,the cell viability was 58.81%（P<0.0001）,the difference was statistically significant.The concentration of 800μM was selected as the concentration of hydrogen peroxide treatment according to the IC50 standard.The cell vitality in the oxidative stress group was 62.71%.The cell viability of H₂O₂+Se NPs group was97.79%,which was significantly different from that of H₂O₂ group（P<0.0001）The comparative difference was statistically significant.The cell vitality of Se NPs group was 108.56%,which was statistically significant compared with oxidative stress group and H₂O₂+Se NPs group.In the nano-Se treatment group,the membrane potential level was higher,JC-1 gathered in the mitochondrial matrix to form a polymer,and the red fluorescence level was stronger.In the oxidative stress group,the level of JC-1 monomer was higher,and green fluorescence could be seen under fluorescence microscope.The quantitative results of intracellular reactive oxygen species（ROS）and fluorescence results showed that the intracellular active oxygen content in the oxygenated response group was 162.59%（P<0.0001）,the content of intracellular reactive oxygen species in H₂O₂+Se NPs group was 65.00%（P<0.0001）.There was no significant increase in reactive oxygen species in control group and nano selenium treatment group.Under fluorescence microscope,the number of cell necrosis and apoptotic cells increased in oxidative stress group,and the degree of necrosis decreased after nano selenium treatment.No obvious cell apoptosis and necrosis were observed in the control group and nano selenium group.Oxidative stress caused decreased expression of STAT-3 and C3 protein,and increased expression of P-P53 protein.Se NPs@LNT enhances the expression of JAK2 and decreases the expression of AKT.【Conclusion】1.The family birth cohort before pregnancy was established,and the model for analyzing the influence of various influencing factors on male fertility was successfully constructed.The correlation between trace elements and semen quality of men was analyzed,indicating that the decrease of trace elements content and the decrease of semen quality of men were epidemiological factors.Compared with organic selenium and inorganic selenium,three nano-selenium drugs have low biotoxicity,which can be used as a good drug for male reproductive diseases.2.Nano-selenium can reduce the level of active oxygen and apoptosis necrosis of germ cells.It can increase the activity of cell oxidase,regulate the reduction of mitochondrial membrane potential,and reduce the expression of a series of oxidative stress-related proteins,which has a good anti-apoptotic necrosis effect.