| With the rapid development of nanotechnology,nanomaterial-related products have also been continuously developed and widely used in various fields.Nickle nanoparticles(Ni NPs)are products with many new characteristics,which include a high level of surface energy,high magnetism,low melting point,low burning point,and high surface area.These characteristics are only present at the nanoscale level and have thus led to its heightened experimentation and use in modern industries such as catalysts,sensors,and electronic applications.However,these characteristics of nickel particles may bring unique potential health hazards.The nano size of these particles renders them the ability to be easily transported into biological systems,thus raising the question of their effects on the susceptible system.Therefore,scientific research on the environmental and human health effects of Ni NPs are imperative.The male reproductive health has become one of the important problems caused by the current environmental pollution,which has attracted more and more attention and is also one of the hotspots in toxicology research.LncRNAs,which were defined as endogenously expressed RNA transcripts >200 nucleotides in length and not translated into proteins,were once considered to be transcriptional “noise.” Recently,increasing studies have revealed that lncRNAs play significant role in many biological processes,including cell differentiation,marker of cell fate.Our previous study found that nano-nickel has reproductive toxicity to both rats and nematodes,especially for male rat germ cells.Further research shows that nano-nickel can induce germ cell damage by inducing apoptosis.But so far,the mechanism of nanoparticle-induced germ cell damage is not clear,and the key molecular events involved in this process have not been explored yet.Therefore,this study selected SD rat sertoli-germ co-culture cells as the research object,through a series of studies to observe the role of Ni NPs on the apoptosis of this kind of cells,and to explore the function of lncRNA on key signaling pathways and regulatory mechanisms.1.Characterization of Ni NPs: SEM observation showed that the nickel nanoparticles were spherical and the particle size was between 30 nm and 100 nm and there was some agglomeration.TEM observation showed that the nickel nanoparticles were in a regular spherical shape with uneven particle size distribution.There was some agglomeration between 25 nm and 125 nm.In the dispersion(cell culture solution),the nickel nanoparticles with a concentration of 5 μg/ml have a particle size ranging from 260 nm to 725 nm with a peak at 444 nm;the nickel nanoparticles with a concentration of 12.5 μg/ml have a particle size ranging from 400 nm to 879 nm.The peak is at 522 nm.2.Ni NPs-induced sertoli-germ cells cytotoxicity in rats: After 24 h exposure to 25,50,100,200,400,800 μg/ml nickel nanoparticles solution,cells viability decreased in a dose-dependent manner;25,50,100,200 μg/ml nickel nanoparticles solution can induce cell morphology changes after 24 hours.It was found that the cells in the normal group were normal blue.The cells in the treated group were densely stained and white in color.As the dose increased,apoptotic cells increased.Apoptosis rate detection showed that compared with the control group,the nickel nanoparticles solutions(25、50、100、200、400 μg/ml)were exposed to a dose-dependent increase in apoptosis rate for 24 hours.3.High-throughput gene chip screening for nickel nanoparticles-induced difference of lncRNA in rat sertoli-germ cells: In this study,rat sertoli-germ cells treated with 100 μg/ml nickel nanoparticles were extracted total RNA.Apoptosis-related lncRNAs were screened by gene chips,and a lncRNA-miRNA-mRNA co-expression network was constructed.Combined with the degree of contribution in the co-expression network and the multiple of chip detection,six apoptosis-related lncRNAs were selected and tested by RT-qPCR.The expression level of LOC102551356 in the 100 μg/ml group was significantly up-regulated,which was consistent with the chip detection results.Based on the bioinformatics analysis of apoptosis-related lncRNA and the study of biological function,we speculated that LOC102551356 may affect the apoptosis of rat sertoli-germ cells by acting on the potential target gene IGF-BP3 to participate in the mitochondrial apoptosis pathway.4.Study on the biological function and regulatory mechanism of LOC102551356 in Ni NPs-induced apoptosis of rat sertoli-germ cells: By constructing LOC102551356 low expression adenovirus vector,the optimal MOI for rat sertoli-germ cells adenovirus transfection was studied.The best MOI for rat sertoli-germ cells adenovirus transfection was 300.LOC102551356 has an effect on mitochondrial membrane potential:The ratio of JC-1 polymer to JC-1 monomer was 100 μg/ml and the negative control group was lower than the blank control group,while the proportion of JC-1 polymer and JC-1 monomer in the LOC102551356 transfection group was also low but higher than 100 μg/ml exposure group and negative control.LOC102551356 has an effect on caspase activity: Compared with the blank control group,the activities of caspase3 and caspase9 in the 100 μg/ml group and the negative control group increased significantly,while the activities of the LOC102551356 caspase3 and caspase9 protein increased compared with the blank control group;LOC102551356 has an effect on the apoptosis of sertoli-germ cells: The apoptotic rate in the blank control group was 2.60±0.82%.The apoptotic rates in the 100 μg/ml treated group and the negative control group were 15.40±1.42% and 17.00±2.21%.The apoptotic rate was higher than that in the blank control group.The difference was statistically significant.The total apoptosis rate of LOC102551356 transfection group was 8.30%±1.57%,which was significantly higher than that of the blank control group.The difference was statistically significant.However,the total apoptotic rate of the LOC102551356 transfection group was lower than the 100 μg/ml group and the negative control group,and the difference was also statistically significant;The level of IGF-BP3 gene and protein expression were upregulated in both 100 μg/ml and negative control groups,suggesting that IGF-BP3 is associated with nano-nickel-induced apoptosis of rat sertoli-germ cells,whereas LOC102551356 transfection group IGF-BP3 There was no significant difference in gene level and protein level between the control group and blank control group,indicating that LOC102551356 played a targeted role in the expression of IGF-BP3 in the process of Ni NPs-induced apoptosis of rat spermatogenic cells,which in turn led to apoptosis occurs.RT-qPCR results showed that compared with the blank control group,Bax,Caspase3,and Caspase 9 gene levels were up-regulated and Bcl-2 gene levels were down-regulated in the 100 μg/ml and negative control groups,while Bax and Bcl-2 were detected in the LOC102551356 transfection group.There was no significant change in the levels of Caspase3 and Caspase9 genes;the relative ratio of Bax/Bcl-2 gene expression levels in the 100 μg/ml exposure group and the negative control group increased significantly,and the relative expression ratio of Bax/Bcl-2 gene expression in the LOC102551356 transfection group.The difference was not statistically significant.The results of WB experiments showed that compared with the blank control group,the expression of Bax,Caspase3,and Caspase9 protein in the 100 μg/ml group and the negative control group increased and the Bcl-2 protein expression level decreased;LOC102551356 transfection group Bax,Bcl-2.There was no significant difference in the expression levels of Caspase3 and Caspase9 protein between the control group and the blank control group.The relative ratio of Bax/Bcl-2 protein expression in the 100 μg/ml treatment group and the negative control group also increased significantly.The relative ratio of Bax/Bcl-2 protein expression in LOC102551356 transfection group was not statistically different from the blank control group.This shows that the mitochondrial pathway is indeed involved in the apoptosis of male germ cells caused by Ni NPs exposure,and exposure to Ni NPs will impair the integrity of the mitochondrial structure and function of the cells.At the same time,it is further proved that LOC102551356’s targeted regulation of IGF-BP3 expression will affect the mitochondrial-dependent apoptotic pathway,thus affecting the apoptosis results.In summary,nickel nanoparticles have a certain apoptosis effect on rat sertoligerm cells.Nickel nanoparticle-induced apoptosis in rat sertoli-germ cells involves LOC102551356,IGF-BP3 and mitochondrial apoptosis pathway.The specific mechanism may be: during the process of nickel nanoparticles-induced apoptosis in cocultured cells,the expression level of LOC102551356 is up-regulated,and LOC102551356 induces mitochondrial apoptosis pathway through targeted regulation of the target gene IGF-BP3 in the P53 signaling pathway in that way affects the occurrence and development of apoptosis. |
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