Study On Male Reproductive Toxicity Of Nano-nickel And Preliminary Study On The Mechanism Of Mitochondrial Division And Autophagy

As a kind of metal nanomaterial,nano-nickel is used in biomedicine,catalysts,supercapacitors and other fields due to its unique physical and chemical properties,and the health risks caused by nano-nickel have also attracted great attention.Studies have reported that at least 30 million men suffer from infertility worldwide.The decline of human semen quality and male reproductive health have raised issues of great concern in the field of public health.Fertility is largely affected by environmental factors.Nano-nickel can enter the human body through the respiratory tract and digestive tract,and a number of epidemiological studies have confirmed that it can cause adverse effects on human health.Therefore,it is necessary to study the male reproductive toxicity caused by nano-nickel by gastric gavage and tracheal instillation.Micron nickel is a necessary micron-level reference for studying the toxicity of nano-nickel,and the introduction of nickel chloride can be used to compare nanoparticles and ions.In addition,our previous studies also have verified that nano-nickel can damage male reproductive organs,causing germ cell toxicity and inducing cell apoptosis,which involves the mitochondria-mediated Caspase-9/Caspase-3 apoptotic signaling pathway.Since mitochondrial fusion and mitochondrial division are closely related to apoptosis via a complex interaction between mitochondrial autophagy and apoptosis.Therefore,we speculate that mitochondrial division and mitochondrial autophagy play important roles in the process of nano-nickel-induced germ cell apoptosis.Based on the above background,this study aims to explore the difference between intragastric administration and tracheal instillation in reproductive toxicity of nano-nickel,and to preliminarily explore how dynamin-related protein 1(Drp1)-mediated mitochondrial division and PTEN induced putative kinase 1(Pink1)-mediated mitochondrial autophagy regulate cell apoptosis during the process of nano-nickel-induced male reproductive toxicity.1.Study on the reproductive toxicity of nano-nickel to male mice(1)Characterization of nano-nickel materialsThe Malvern laser particle size analyzer was used to detect the hydrated particle size and Zeta potential of nano-nickel.The results showed that the hydrated particle size ranged from 200 nm to 1000 nm,and the Zeta potential distribution was around 0 m V.Scanning electron microscopy and transmission electron microscopy were used to detect the morphology and size of nano-nickel,respectively.The results showed that the nano-nickel particles were spherical and regular in shape,the particle size distribution ranged from 30 nm to 100 nm,and there was obvious agglomeration among the particles.(2)Study on the reproductive toxicity of nano-nickel to male mice by gavageBALB/c male mice were selected as the research object,and then divided into normal saline,low dose(5 mg/kg),middle dose(15 mg/kg),high dose(45 mg/kg)nano-nickel groups,and micron nickel(45 mg/kg)and nickel chloride(45 mg/kg)groups referring to the groups of early mouse experiments of the research group.After preparation for the dispersions of nano-nickel,micron nickel and nickel chloride in normal saline,each mouse was administered by gavage in accordance with the principle of 0.1 ml/10 g once a week for 28 days.The serum was collected to detect reproductive hormones,and the testis and epididymis were collected to calculate the testicular organ coefficient and to detect sperm abnormality rate,testicular pathological morphology,and the key proteins of mitochondrial division and mitochondrial autophagy.The results showed that there were no significant differences in the body weight and testicular organ coefficients among different groups(P>0.05).The serum levels of reproductive hormone testosterone(T),follicle-stimulating hormone(FSH)and luteinizing hormone(LH)were lower in the treatment groups than those in control group,and the difference was statistically significant(P<0.05).Compared with control group,the rate of sperm deformity of the treatment groups increased obviously(P<0.05),however,there were no statistical differences in both micron nickel and nickel chloride groups in comparison with high dose nano-nickel group(P>0.05).The results of hematoxylin-eosin stained testicular tissues in control group showed a distinct graded layer structure of the seminiferous tubules fulfilled with tight and organized spermatogenic cells at different stages.However,in each of the treatment groups,the space between seminiferous tubules increased,and the number of sperm,spermatogenic cells and interstitial cells all decreased.Notably,rupture of the basement membrane of seminiferous tubules was observed in high dose nano-nickel group,especially a more obvious damage of the seminiferous tubules in micron nickel group.Furthermore,the expression levels of Drp1,Pink1 and Parkin in the testis tissue in the treatment groups were higher than those in the control group(P<0.05).(3)Study on the reproductive toxicity of nano-nickel to male mice by intratracheal instillationBALB/c male mice were selected,and the dose setting and grouping method were the same as the gavage,namely the normal saline,low dose,middle dose and high dose nano-nickel,micro nickel and nickel chloride groups.Similarly,each mouse was administered with 40 μl poison solution each time by intratracheal instillation according to the principle of 0.1 ml/10 g for each one.After the experiment,the serum,alveolar lavage fluid,testis and epididymis were collected for testing.The results indicated that there were no significant differences in the body weight and testicular organ coefficients in each group(P>0.05).Compared with control group,the serum levels of reproductive hormone T,FSH and LH were lower in the treatment groups,and there were significant differences(P<0.05).Interestingly,the levels of reproductive hormones in nickel chloride group were higher than those in high dose nano-nickel group(P<0.05).The levels of inflammatory factors TNF-α,IL-6 and IL-1β in serum and alveolar lavage fluid increased obviously,and the increase was more significant in low dose nano-nickel group than that in other groups(P<0.05).The rate of sperm deformity of the treatment groups increased with an increase in the nano-nickel dose(P<0.05),but there were no marked differences in both micro nickel and nickel chloride groups compared with high dose nano-nickel group(P>0.05).The main pathological changes of testis tissue are similar to those of intragastric administration,the spermatogenic cells were distributed loosely and disorderly,and part of them shed into the tubules,in addition,the number of mesenchymal cells,spermatogenic cells and sperm all decreased.In comparison to control group,the results of TUNEL assay demonstrated that the positive apoptotic cells in the testis tissue in the treatment groups increased significantly.Furthermore,the expressions of Drp1,Pink1 and Parkin in testis tissues in the treatment groups were also up-regulated in comparison with control group(P<0.05).2.Study on the regulatory mechanism of mitochondrial division and autophagy in the male reproductive toxicity of nano-nickel(1)Cytotoxicity caused by nano-nickelThe dispersions of nano-nickel,micron nickel and nickel chloride were prepared with complete culture solution,respectively.Then mouse spermatogonia(GC-1 cells)were exposed to blank control,low dose(25 μg/ml),middle dose(50 μg/ml),high dose(100 μg/ml)nano-nickel groups,as well as micron nickel(100 μg/ml)and nickel chloride(100 μg/ml)groups with the same concentration of high dose nano-nickel group.After incubation for 24 h,CCK-8 assay and flow cytometry assay were used to detect cell viability and apoptosis rate,respectively.The results showed that with the increasing exposure dose,the cell survival rate decreased in a dose-dependent manner(P<0.05).Of note,the cell survival rate in micron nickel group was significantly lower than that in high dose nano-nickel group(P<0.05),conversely,that in nickel chloride group was slightly higher compared with high dose nano-nickel group(P>0.05).The total apoptosis rate increased in a dose-dependent manner(P<0.05).At the same time,total apoptosis rate in micron nickel group was higher than high dose nano-nickel group,while that in nickel chloride group was lower compared with high dose nano-nickel group(P<0.05).(2)Mitochondrial damage caused by nano-nickelAfter incubation with different concentrations of nano-nickel,mitochondrial damage was evaluated in GC-1 cells by a series of indicators including reactive oxygen species(ROS),adenosine triphosphate(ATP),mitochondrial membrane potential(MMP),mitochondrial autophagosome and mitochondrial division and mitochondrial autophagy key proteins Drp1,Pink1 and Parkin.The results showed that ROS increased with the increasing concentration of nano-nickel,ATP content decreased in a dose-dependent manner(P<0.05),MMP decreased and mitochondrial autophagosomes formed.Except for the decreased expression level of Bcl-2,the expression levels of Drp1,Pink1 and Parkin,as well as the cell apoptosis-related proteins Bax,Caspase-9 and Caspase-3 increased significantly,and the ratio of Bax/Bcl-2 increased in a dose-dependent manner(P<0.05).(3)Study on the regulatory mechanism of mitochondrial division and mitochondrial autophagyBased on the results of our previous cell experiments and animal experiments,GC-1 cells were used as the research object again,during which the mitochondrial division inhibitor Mdivi-1 was added to further explore the role and regulatory mechanism of Drp1-mediated mitochondrial division and Pink1-mediated mitochondrial autophagy in the male reproductive toxicity induced by nano-nickel.The results of CCK-8 assay confirmed that when the concentration of nano-nickel was 100 μg/ml,the cell survival rate was about 60%,and when the concentration of Mdivi-1 was 10 μmol/l,the cell survival rate was the highest in exposure to nano-nickel.Therefore,the treatment concentration of 100 μg/ml nano-nickel and the inhibitory concentration of 10 μmol/l Mdivi-1 were selected for subsequent experiments.The blank control group,inhibitor group,nano-nickel group,and combination of nano-nickel with inhibitor group were set up,respectively.Following the addition of Mdivi-1,the results of a series of cell experiments showed that compared with control group,the cell viability remarkably increased(P<0.05),total apoptosis rate significantly reduced(P<0.05),ROS level reduced,and the levels of ATP content and MMP increased in the combination of nano-nickel with inhibitor group.Additionally,except for an increased level of Bcl-2,the expression levels of Drp1,Pink1,Parkin,Bax,Caspase-9 and Caspase-3 proteins all decreased,and the ratio of Bax/Bcl-2 decreased(P<0.05).In summary,nano-nickel can cause reproductive toxicity in male mice through intragastric administration and tracheal instillation.The toxicity of nano-nickel in vivo is greater than that of micron nickel,and the toxicity of micron nickel is greater than that of nickel chloride.In addition,mitochondrial division and autophagy are involved in the reproductive toxicity process.Cell experiments found that nano-nickel can also induce GC-1 cell apoptosis and cause mitochondrial damage.Furthermore,it was also found that nano-nickel may induce apoptosis of spermatogenic cells by promoting mitochondrial division and autophagy,which preliminarily proved the mechanism of mitochondrial division and autophagy.In exposure to these particles,Drp1-mediated mitochondrial division increases rapidly in the cells,causing ROS accumulation,ATP content reduction and MMP decline,and inducing the mitochondrial apoptosis pathway regulated by Caspase-9 and Caspase-3,subsequently leading to cell apoptosis.Simultaneously,the increase of mitochondrial division can activate the Pink1-mediated mitochondrial autophagy pathway.However,the mechanism of mitochondrial autophagy promoting or protecting cell apoptosis is still unclear,and we will further explore it in the future.