Study On Male Reproductive Damage And Its Mechanism Caused By Polystyrene Microplastics

BackgroundMicroplastics are plastic debris contaminants with a particle size of less than 5 mm.Microplastics mainly come from plastic products.Over the past few decades,global plastic production has increased from 2 million tons in 1950 to 380 million tons in 2015.The production of microplastics is expected to reach 33 billion tons by 2050.Due to its stable physical and chemical properties,plastics are widely used in all aspects of life.The wide use of microplastics has increased human exposure.Human intake of microplastics is mainly from food,drinking water,and atmospheric environment.At present,microplastics have also been found in many aquatic products and salt.In addition,traces of microplastics were also found in agricultural and sideline products and baby milk powder.Microplastics have been detected in human feces and body fluids,and even in the placenta of pregnant women.Infertility is a very common disease at present.There are 70 million people affected by infertility in the world.According to WHO estimates,about 9%of couples in the world face the problem of infertility,of which 50%are caused by men,which makes male infertility a major scientific problem to be solved urgently all over the world,and abnormal spermatogenesis is also the most common cause of male infertility.Nearly 80%of human diseases in the world are related to environmental risk factors,and environmental pollutants are also one of the important factors affecting global reproductive problems.Microplastics have been proved to have potential reproductive toxicity,which can induce oxidative damage of aquatic gametes and abnormal embryonic development.However,there are few studies on reproductive toxicity in mammals.Mammalian and human genes have high homology.Therefore,taking mammalian mice as the research object to study the effect of microplastics on the reproductive system of male mice and its mechanism will help us understand the effect of microplastics on male infertility.Research ObjectivePrevious studies have found that microplastics can cause damage to gametes and embryos in aquatic organisms and inflammatory changes in liver and intestine in mice,and found that such damage is closely related to the inflammatory response and ROS caused by microplastics.Therefore,this study took ICR mice as the research object,PS-MPS as the experimental material,and the inflammatory changes caused by PS-MPS as the entry point to study the effects of PS-MPS on the reproductive system of mice and its toxic mechanism,providing a new research idea for the study of male infertility.Research Methods1.Experimental scheme:(1)firstly,the animal model of abnormal spermatogenesis in mice caused by polystyrene microplastics(PS-MPS)was established:the experimental animals were fed adaptively for 7 days and randomly divided into four dose groups:control group,low,medium and high dose group.The corresponding exposure doses of microplastics were 0 μg/L,100 μg/L,1000 μg/L and 10 mg/L respectively.Mice were exposed to PS-MPS by drinking freely for 35 days.During the exposure process,PS-MPS suspension was changed regularly and water bottles were shaken regularly to ensure the uniformity of PS-MPS suspension in water bottles.The mice were weighed on day 1,8,15,22,29 and 36 of exposure,and the experiment was terminated on day 36.The mice samples were collected for detection of reproductive injury related indicators.(2)High dose was selected as the exposure dose of the time series model,and mice were exposed for 35 days.Six observation endpoints were set.The experiment was terminated at 0w,1w,2w,3w,4w and 5w,respectively,and relevant samples were collected for the detection of inflammatory factors,apoptosis factors and other indicators.(3)High dose was selected as the exposure dose of the time series model,and mice were exposed.Six observation endpoints were set,and the experiment was terminated at 0h,6h,12h,24h,48h and 72h respectively so as to collect testicular tissue and detect the ROS level in testicular tissue.(4)Cell damage model was established by co-culture of GC-2 cells and PS-MPS:GC-2 cells were co-cultured with PS-MPS,and cellular proteins were collected for relevant detection at seven time points:0h,6h,12h,18h,24h,36h,48h after exposure to PS-MPS.(5)Establishment of DNA damage model of GC-2 cells induced by PS-MPS:GC-2 cells were pretreated with ML385 and PDTC and co-cultured with PS-MPS.24h later,cells were collected for apoptosis detection,and cell proteins were collected to detect DNA damage-related proteins.2.Statistical methods:Excel software was used to input the original data,GraphPad Prism software was used to plot the experimental results,and SPSS22.0 was used for statistical analysis of data between variables.P<0.05 indicates statistically significant differences.The results are finally expressed as mean±standard deviation.The homogeneity test and normality test of variance were carried out before the comparison between the dose groups of each model.For continuous variables with normal distribution,the difference between groups was analyzed by analysis of variance,and the difference between the two groups was analyzed by Dunnett’s T and Bonferroni.Research results1.PS-MPS causes reproductive damage in male mice1.1 Systemic toxicity:after exposure to different doses of PS-MPS,the body weight of mice in each group did not change significantly,the liver,kidney and testicular coefficients of mice decreased,and the fat coefficient increased;1.2 Manifestations of reproductive injury(1)Sperm damage:after exposure to different doses of PS-MPS,the live sperm count of mice decreased significantly and the sperm deformity increased.The types of sperm deformity were mainly double tail deformity,neck enlargement and uncinate deformity;(2)Pathological damage:After HE staining of mouse testicular tissue sections,it was found that the germ cells in each layer of testicular spermatogenic tubule were disordered,with cell rupture,nuclear pyknosis and reduced sperm count in the tubule.(3)Testicular cell apoptosis:① pathological TUNEL staining showed testicular cell apoptosis;② The detection of apoptotic proteins showed that the proapoptotic protein Bax increased significantly after exposure to PS-MPS,the expression of anti-apoptotic protein BCL2 decreased significantly after exposure to PS-MPS and the ratio of Bax/Bcl2 increased significantly after exposure to PS-MPS.2.Mechanism of reproductive injury in mice induced by PS-MPS2.1 ROS production in mouse testicular tissue cells induced by PS-MPS exposureThe level of ROS in mouse testis increased after PS-MPS intervention.During the 72 hours study,the level of ROS did not increase significantly within 72 hours,but began to increase significantly at 72 hours.In order to study the changes of ROS during PS-MPS exposure,the detection models at different time points in the spermatogenic cycle were established.The results showed that the level of ROS in mouse testis increased significantly at 1 w after PS-MPS exposure,and the level of ROS continued to be higher than that in the control group with the extension of exposure time.2.2 Inflammatory reaction of mouse testis caused by PS-MPS exposure①The inflammatory pathway in mouse testis changes,which was induced by PS-MPS.The expression level of inflammatory factor IL-1β,IL-6,and TNFa in mouse testicular increased significantly after exposure to PS-MPS,and the pathway protein NF-κB regulates inflammatory changes.The expression of NF-κB related protein increased,while the expression of Nrf2 related protein decreased;It is speculated that this pathway may be involved in the apoptosis of testicular cells induced by PS-MPS;②Apoptosis decreased after treatment with PDTC,an NF-κB inhibitor,and increased after treatment with ML385,an Nrf2 inhibitor,suggesting that passage of PS-MPS through Nrf2/HO-1/NF-κB inflammatory pathway lead to testicular cell apoptosis;③In the animal model exposed to PS-MPS for one spermatogenic cycle,the inflammatory factor TNF α And p-NF-KBp65 changed first.And with the extension of exposure time the expression of TNFα,IL-1β,p-NF-KBp65 increased significantly and peaked at 3w and 4w.IL-6 increased significantly at 3w and reached the peak at 4w.The results of cell experiment showed that after exposure to PS-MPS,The expression level of TNF α and IL-1β at 6h was significantly higher than that in the control group,and reached the peak at 18h and 36h respectively;p-NF-κBp65 decreased significantly at 12h and reached the peak at 18h,and after 18h,there was no significant difference with the control group;as a sensitive inflammatory factor in PS-MPPS induced testicular inflammation in mice,TNFαand IL-1β it may be involved in the regulation of Nrf2/HO-1/NF-κB inflammatory pathway.2.3 PS-MPS exposure affects androgen synthesis and damages blood testis barrier① After exposure to PS-MPS,the expression levels of testosterone,FSH and LH in serum of mice decreased;② The expression of steroid hormone synthase and androgen receptor in testis decreased;③ The expression of testicular tight junction protein decreased and the blood testicular barrier was damaged;④ It was found that the expression of tight junction molecule Occludin decreased gradually with the extension of exposure time.There was a significant decrease in the first week after exposure to PS-MPS,and with the extension of exposure time,the expression of Occludin reached the lowest value at 3w and 4w.Caludin-11 molecule began to decrease at 2w after exposure to PS-MPS and reached the lowest level at 5w;⑤ Relationship between blood testis barrier injury and inflammatory factors:with the increase of inflammatory factor expression,the expression of tight junction molecules Occludin and caludin-11 decreased gradually.PS-MPS exposure affects the remodeling of testicular blood testicular barrier by changing hormone levels and inflammatory response.2.4 DNA damage of testicular tissue cells after PS-MPS exposure① PS-MPS exposure can cause DNA damage in mouse testicular tissue;② PS-MPS exposure can lead to an increased expression of DNA damage proteins y-H2AX and 8-OHdG,resulting in the increased expression of p53 protein involved in regulating DNA repair;The expression of DNA damage protein decreased after treatment with NF-κB inhibitor PDTC in ER5,while increased after treatment with Nrf2 inhibitor ML385,suggesting that the cellular DNA damage induced by PS-MPS is related to the Nrf2/HO-1/NF-κB inflammatory pathway;③ The relationship between ROS and DNA damage detected by animal time series model shows that PS-MPS can lead to the continuous increase of ROS level in testicular tissue,while the expression of y-H2AX and P53 is significantly increased,suggesting that DNA damage is related to ROS.④ The results of animal time series models show that PS-MPS can lead to the continuous increase of inflammatory factor level in testicular tissue,while the expression of y-H2AX and p53 increased significantly,suggesting that DNA damage is closely related to inflammatory response.Research Conclusion1.Exposure to PS-MPS can mediate testicular inflammation in ICR mice through NF-κB/Nrf2/HO-1 pathway,resulting in testicular cell apoptosis and male reproductive injury.2.PS-MPS exposure can lead to DNA damage in testicular tissue and germ cells of ICR mice through inflammatory response mediated by NF-κB/Nrf2/HO-1 pathway,resulting in male reproductive damage.3.PS-MPS can reduce the level of serum sex hormones,inhibit the expression of hormone receptors and induce inflammatory response,resulting in the destruction of the integrity of the blood testosterone barrier in mice and the occurrence of male reproductive damage.4.Male reproductive injury caused by PS-MPS may be the result of testicular inflammation,DNA damage,blood testosterone barrier damage and hormone synthesis disorder involved in inflammation.