Study On Mitochondria Injury Of Testicular Tissue Cells Induced By Polystyrene Microplastics

SignificanceFertility is the core issue of the family and social development,which not only affects the current generation,but also affects the next generation.Infertility has become one of the important problems in society and public health,and it is also the third biggest disease that seriously endangers human health after tumor and cardiovascular and cerebrovascular diseases.In recent years,the number of sperm per milliliter of semen in men has decreased year by year,and male infertility has become a major problem affecting China’s national economy and people’s livelihood and social harmony.Studies have found that environmental factors can lead to male reproductive damage.Microplastics(MPS)are plastic particles with a particle size of less than 5 mm that are derived from the degradation of plastic products to form smaller microplastic fragments or particles.MPS not only exists in the natural environment,such as oceans,lakes,rivers,etc.,but also exists in human production and life,such as fruits,vegetables,drinking water,etc.MPS is even found in human feces,blood and placenta.The existence of MPS has been found in human body,but the potential harm of MPS to human body has not been reported.Among them,reproductive function is more sensitive to environmental poisons and is crucial to the survival and healthy development of species.At the same time,the production and consumption of plastic products are also increasing year by year,greatly increasing the chances of human exposure to microplastics.Therefore,we carried out relevant studies on MPS on the male reproductive system.In addition,we also made long-term observations on whether the effects of MPS on the reproductive system are recoverable,which can provide a basis for population prevention and control,and has strong public health significance.ObjectiveFocusing on the prominent public health issues related to the potential threat of microplastics to human health in the environment,the research project focused on the sensitive and far-reaching reproductive system by constructing cell and animal models of mammalian Polystyrene microplastics(PS-MPS),styrene microplastics,and styrene microplastics.To investigate the effect of PS-MPS on mitochondrial damage of testicular tissue cells,the role of oxidative damage in mitochondrial damage of testicular tissue cells caused by PS-MPS,and the repeatability of mitochondrial damage of testicular tissue cells caused by PS-MPS under natural conditions.The results of this study can provide scientific basis for further interpreting the male reproductive toxicity of PS-MPS and its mechanism of toxic action,evaluating the long-term effects of reproductive toxicity of PS-MPS and revealing the potential health hazards of PS-MPS to mammalian reproductive system.MethodsⅠ In Vitro experiment(1)Cell experimental model:Mouse spermatocyte line GC-2 was divided into control group and poisoned group.The poisoned group was exposed to PS-MPS for 6 h,12 h,18 h and 24 h,while the poisoned group was divided into different concentrations of PS-MPS for 24 h,and the damage of spermatocyte mitochondria under different concentrations and different observation endpoints of PS-MPS was observed.(2)Experimental techniques and observation indexes:①Western blotting techniques were used to detect the expression levels of mitochondrial kinetic homeostasis related proteins(Drpl and OPA1),mitochondrial autophagy(LC3B),mitochondrial autophagy regulatory proteins(PINK1,Parkin),and SOD2 of cells in each group;②Real-time fluorescent PCR was used to detect mRNA levels of related genes and mitochondrial DNA copy number;③Cell survival,ROS,mitochondrial membrane potential,ATP and MDA levels were detected by relevant kits;④ Ordinary PCR:detection of mitochondrial DNA integrity;⑤Transmission electron microscopy:observe the ultrastructure of cells and mitochondria.Ⅱ In vivo experiment(1)Animal model:Control group and dose group were set in the experiment.Male mice were exposed to PS-MPS for 35 days(one spermatogenic cycle)to construct male reproductive toxicity model of male mice exposed to PS-MPS.After exposure,some mice were randomly selected to observe mitochondrial damage in sperm and testicular tissue of mice.After the end of exposure,the remaining mice were kept for observation,and the reproductive toxicity and mitochondrial damage of mice were detected on the day 71(recovery of one spermatogenic cycle)and the day 106(recovery of two spermatogenic cycles),and the reproducibility of mitochondrial damage and reproductive toxicity under natural conditions was analyzed.In addition,five observation endpoints were set at day 8,day 15,day 22,day 29 and day 36,as well as day 2,day 4,day 6 and day 8,respectively,to observe the oxidative damage and mitochondrial damage of testicular tissue under different observation endpoints.(2)Experimental techniques and observation indexes:①HE staining was used to observe the pathology of testis;②The expression levels of mitochondrial kinetic homeostasis related proteins(Drp1,OPA1),mitochondrial autophagy(LC3B),and mitochondrial autophagy regulatory proteins(PINK1,Parkin)in testicular tissues of each group were detected by western blotting technique;③Real-time PCR was used to detect mRNA levels and mitochondrial DNA copy numbers of related genes;④Sperm analysis system:analysis and detection of sperm activity,movement;⑤Diff fast staining:detection of sperm malformation;⑥The levels of ROS,mitochondrial membrane potential,ATP and MDA in testicular tissue were detected by relevant kits;⑦Ordinary PCR:detection of mitochondrial DNA integrity of mouse testicular tissue;⑧Transmission electron microscopy:The ultrastructure of testicular tissue and mitochondria was observed.Ⅲ Statistical analysisSPSS 21.0 software was used for statistical analysis.The average level of two independent samples conforming to normal distribution and homogeneity of variance was compared by the t test of independent samples.Two independent samples Wilcoxon rank-sum test were used when normal distribution or variance was not obeyed.When comparing multiple groups,the homogeneity test of variances was performed first.If the variances were homogeneous,the One-Way ANOVA test was used,and then the Turkey multiple comparison test of the multi-group comparison was performed.If the variances are not homogeneous,the Kruskal-Wallis H test is compared with multiple independent samples,and the Nemenyi non-parametric test is used for ppair comparison.The changes of body weight and food intake were analyzed by repeated measurement ANOVA.When P<0.05,the difference was considered statistically significant.Results1.PS-MPS exposure caused mitochondrial damage in GC-2 cells(1)Mitochondrial autophagy of GC-2 cells induced by PS-MPS exposure:After PS-MPS exposure,mitochondria of GC-2 cells contracted under transmission electron microscopy,increased matrix density,more autophagic lysosomes,and LC3BII relative protein expression in GC-2 cells increased.(2)PS-MPS exposure caused mitochondrial structure and function damage in GC-2 cells:①The content of mitochondrial autophagy regulation protein increased:the transcription and expression levels of PINK1 and Parkin genes increased in GC-2 cells after PS-MPS exposure;②Decreased mitochondrial membrane potential:the green fluorescence intensity of GC-2 cells decreased after staining in the exposed group;③Mitochondrial dynamic homeostasis imbalance:The transcription and expression levels of the mitochondrial fusion gene OPA1 and the division gene Drpl in GC-2 cells increased after PS-MPS exposure;④Changes in mitochondrial genetic characteristics:the mitochondrial DNA integrity of GC-2 cells decreased after exposure to PS-MPS,and there was no significant difference in mitochondrial DNA copy number;⑤Decreased mitochondrial function:ATP content decreased in the exposed group.(3)Mitochondrial damage in GC-2 cells exposed to PS-MPS at different concentrations:With the increase of PS-MPS concentration,the mRNA of PINK1 did not change,but when the concentration was 100 μg/mL,the mRNA of PINK1 began to increase,and the ATP content began to decrease at 50 μg/mL,reaching the lowest at 100 μg/mL.2.PS-MPS exposure caused mitochondrial damage in mouse testicular tissue(1)PS-MPS exposed mice testicular mitochondrial structure and function damage:①mitochondrial membrane potential decreased;②Mitochondrial dynamic homeostasis imbalance:the contents of mitochondrial fission protein Drpl and fusion protein OPA1 decreased in the exposed group;③Changes in mitochondrial genetic characteristics:the integrity and copy number of mitochondrial genes in exposed group were decreased;④The levels of mitochondrial autophagy regulatory proteins(PINK1 and Parkin)and autophagy protein LC3BⅡ were decreased;⑤Mitochondrial ATP content decreased.(2)PS-MPS exposure caused structural damage to testicular tissue of mice:①HE staining showed that spermatogenic tubule cells were disordered and spermatogenic cells at all levels were reduced in the exposed group.②Transmission electron microscopy:After exposure of PS-MPS,the mitochondria of testicular tissue were severely swollen and stroma dissolved under transmission electron microscopy.Autophagosomes and autophagolysosomes were isolated.(3)The sperm quality of mice exposed to PS-MPS decreased:Compared with the control group,the sperm motility of mice exposed to PS-MPS decreased by about 9.6%,the forward motility rate decreased by about 11.5%,and the malformation rate increased by about 12.8%.(4)General toxicity of mice exposed to PS-MPS:Within 35 days of exposure,there was no statistical difference in the weight,food intake,organ weight and coefficient of mice in the exposed group and the control group over time.3.The role of oxidative damage in mitochondrial damage of mouse testicular tissue cells induced by PS-MPS3.1 Role of oxidative damage in mitochondrial damage of mouse testicular tissue induced by PS-MPS(1)Time series analysis of mitochondrial damage in testicular tissue after PS-MPS exposure:The mitochondrial damage proteins decreased with the increase of exposure weeks,and the decrease of PINK1 protein was obvious.ATP content decreased gradually with the extension of exposure weeks.(2)Changes in testicular tissue oxidation levels caused by PS-MPS exposure:ROS and MDA levels in testicular tissue of mice in the exposed group were increased.(3)The relationship between mitochondrial damage and oxidation in testicular tissue caused by PS-MPS exposure:①The relationship between mitochondrial structural damage and oxidation:With the extension of exposure time,ROS levels gradually increased,reaching the highest in the second week and maintaining.The expression level of PINK1 protein reached the highest on the fifth day of exposure,and began to decrease after one week.;②Relationship between mitochondrial function damage and oxidation:With the extension of exposure time,ROS levels gradually increased,reaching the highest in the second week and maintaining.ATP content showed a downward trend and reached the lowest level in the fourth week.3.2 Role of oxidative damage in mitochondrial damage of GC-2 cells induced by PS-MPS(1)Changes in the oxidation level of GC-2 cells after PS-MS exposure:Through time series analysis,the ROS level began to increase at 18 h,the MDA content gradually increased at 6 h and reached the maximum at 24 h,and the expression of SOD2 protein increased at 6 h,reached the highest at 12 h,and then began to decline.(2)The relationship between mitochondrial damage and oxidation level of GC-2 cells:①Mitochondrial function damage and oxidation level:Cell oxidation gradually increased,reached the highest at 12 h,and then remained stable,while ATP content decreased significantly at 6 h,and then remained at a low level;②Mitochondrial structural damage and oxidation levels:Cell oxidation gradually increased,reaching the highest at 12 h,and then remained stable,while PINK1 transcription did not change significantly at 6 h,but gradually increased after 12 h.(3)Effect of antioxidant NAC on mitochondrial damage:After NAC treatment,MDA and mRNA levels of PINK 1 in exposed group were still higher than those in control group,but lower than those in exposed group without NAC treatment.After NAC treatment,the ATP content of the microplastic exposed group was still lower than that of the control group,but higher than that of the non-NAC exposed group.4.Long-term effect of mitochondrial damage in mouse testicular tissue caused by PS-MPS(1)Changes of mitochondrial structure and function in testicular tissue after the first recovery period:①The mitochondrial membrane potential returned to normal level;②Mitochondrial kinetic homeostasis(expression levels of fission protein Drpl and fusion protein OPA1)returned to normal levels;③Mitochondrial genetic characteristics(mitochondrial genome integrity and copy number)returned to normal levels;④Mitochondrial autophagy regulatory proteins(PINK1 and Parkin)and mitochondrial autophagy protein(LC3BⅡ)recovered to normal levels;⑤Mitochondrial ATP recovered to normal levels.(2)Changes in testicular tissue structure:After the first recovery period,the results of HE staining and transmission electron microscopy showed that the testicular tissue returned to normal structure.(3)Changes in testicular tissue oxidation levels:Testicular tissue oxidation(ROS and MDA)returned to normal levels after the first recovery period.(4)Recovery of sperm quality:After the end of the first recovery period,the sperm activity of mice in the exposed group was still lower than that of the control group,the forward movement rate was no different from that of the control group,and the malformation rate was higher than that of the control group;After the second recovery period,there was no statistical difference in the above sperm quality indexes compared with the control group.(5)General situation of animals in the recovery period:In the two recovery periods after exposure,there was no statistical difference in the changes of body weight,food intake,and organ coefficient of mice in the exposure group and the control group over time.Conclusion1.PS-MPS can destroy the mitochondrial structure of GC-2 cells and mouse testicular tissue,reduce ATP content,reduce mitochondrial membrane potential,cause changes in mitochondrial genome genetic characteristics,lead to the imbalance of mitochondrial dynamic homeostasis,and cause mitochondrial autophagy changes.2.Oxidative damage may be an important cause of mitochondrial damage in testicular tissue cells caused by PS-MPS.3.The damage of PS-MPS to mouse sperm and mitochondria at a certain concentration can be recovered in two spermatogenic cycles after stopping exposure.Mitochondrial damage recovers after one spermatogenic cycle,whereas sperm damage takes longer spermatogenic cycles to recover.