Role And Mechanism Of Reactive Oxygen Species-mediated Apoptosis In Abnormal Heart Development Induced By Nickel Chloride Exposure In Juvenile Zebrafish

BackgroundNickel is an abundant metal in the earth’s crust and is widely present in occupations and the environment.Studies have shown that nickel and nickel compounds can accumulate in several vital organs,including the liver,kidneys,lungs,cardiovascular and blood,and may increase the risk of heart disease in offspring.Ni Cl₂has been shown to have cardiotoxic effects in rodents,including heart damage and myocardial injury.The specific mechanism of action of nickel leading to abnormal heart development has not been elucidated.ObjectiveIn this study,we used juvenile zebrafish as a model to confirm that the environmental carcinogen nickel chloride induced abnormal cardiac development in juvenile zebrafish,to investigate the possible effects on cardiac development through oxidative stress,apoptosis and Wnt signaling pathway,and to explore the protective role of NAC and its molecular mechanism,to provide theoretical and experimental basis for the development of prevention and control measures and strategies for the hazards of nickel chloride exposure.Research Methods1.In a study to confirm that nickel chloride exposure induces abnormal heart development in zebrafish larvae,zebrafish embryos were exposed to nickel chloride solutions at 2.5 mg/L,5 mg/L,10 mg/L and 15 mg/L for 4-6 h after fertilization,and zebrafish were placed under a somatoscope at 24 hpf,48 hpf,72 hpf,96 hpf and 120 hpf to record and calculate mortality,at 72 hpf,the heart morphology of the juvenile fish was observed under the somatoscope and photographed.The 1-min heart rate of juveniles was calculated;the body length of juveniles was calculated.HE staining was performed to detect pathological changes in juvenile hearts.Total RNA was extracted using 3-day-old juvenile fish,and q RT-PCR was used to detect the expression of cardiac development genes in juvenile fish.2.In exploring the specific mechanism of action in the abnormal heart development of zebrafish caused by nickel chloride,reactive oxygen levels in juvenile fish at 72 hpf were measured by DCFH-DA,and oxidative stress products and antioxidant enzymes were measured in juvenile fish.As well as apoptosis and apoptotic gene expression in juvenile fish heart cells,the expression levels of Wnt signaling pathway genes,and the expression levels of Wnt signaling pathway genes and apoptotic genes after the addition of Wnt activator BML-284.3.To investigate the protective effect of N-acetylcysteine on abnormal cardiac development induced by nickel chloride exposure in zebrafish,survival,hatching,malformation and morphological changes were recorded,and hatching,mortality and malformation rates were calculated.The heartbeats of juvenile fish were also recorded and statistically analyzed.ROS levels in juvenile fish and apoptosis of cardiomyocytes in the heart region of zebrafish embryos were detected.q RT-PCR was used to detect the expression levels of genes,such as cardiac development genes,apoptotic genes and Wnt signaling pathway.Results1.When zebrafish embryos were exposed to different concentrations of nickel chloride,mortality was positively correlated with time and increased in a dose-dependent manner with increasing dose.Nickel chloride significantly reduced the heart rate and body length of zebrafish embryos and showed a dose-effect relationship with the exposure concentration.At 72 hpf,the heart development phenotype of zebrafish juveniles suggested that the juvenile cardiac fraction showed different degrees of pericardial edema with increasing nickel chloride exposure concentration.The atrioventricular distance became larger,and cardiac cyclization was disrupted in juvenile fish;HE results showed that cardiomyocytes were reduced and the heart was linearized in juvenile fish after nickel chloride exposure.The expression levels of cardiac development genes were also significantly reduced in the exposed group.2.Nickel chloride exposure led to oxidative stress with a gradual increase in ROS fluorescence intensity.MDA concentration,CAT activity and SOD activity increased significantly in a dose-dependent manner,while GSH concentration decreased significantly in a dose-dependent manner.Nickel chloride exposure led to oxidative stress.3.Nickel chloride exposure induced apoptosis in embryonic hearts,and apoptotic cells in the heart increased with increasing concentrations of nickel chloride.q RT-PCR detected upregulated expression of some apoptosis-related genes,such as p53,bcl2,and bax,which indicated that nickel chloride induced apoptosis in the heart by activating the expression of apoptotic genes.4.The expression of genes in the Wnt signaling pathway was examined by q RT-PCR,and the results showed that nickel chloride exposure downregulated Wnt signaling pathway genes.Addition of BML-284,an activator of the Wnt signaling pathway,detected by q RT-PCR,upregulated the expression levels of Wnt signaling pathway molecules and target genes compared to embryos exposed with nickel chloride alone.5.Embryos co-treated with nickel chloride and BML-284 attenuated apoptosis of cardiomyocytes after acridine orange staining.The expression levels of apoptosis-related genes such as p53,bcl2,and bax were down-regulated after Wnt agonist treatment compared to embryos exposed with nickel chloride alone,indicating a reversal of nickel chloride-induced cardiotoxicity.6.NAC reduced nickel chloride-induced ROS production and restored oxidative stress-related enzyme activity.NAC supplementation reversed the decrease in hatching rate and increase in mortality caused by nickel chloride,reduced pericardial edema caused by nickel chloride,and significantly reduced the increase in malformation rate caused by nickel chloride.7.Supplementation with NAC reversed the decrease in heart rate induced by nickel chloride.NAC attenuated the nickel chloride-induced cardiotoxicity and the expression of cardiac development genes largely reversed to normal,which was protective against the developmental cardiotoxicity induced by nickel chloride exposure.8.NAC inhibited nickel exposure-induced apoptosis and attenuated cardiomyocyte apoptosis.The expression levels of apoptosis-related genes bcl2 and bax were also restored.NAC reversed the downregulation of Wnt signaling pathway molecules and target genes induced by nickel chloride exposure.ConclusionIn summary,the basal data,heart rate,pericardial edema,pathological changes,and cardiac development-related gene expression confirmed that nickel chloride exposure caused abnormal cardiac development in juvenile zebrafish.NAC had a protective effect on the abnormal cardiac development induced by nickel chloride exposure in juvenile zebrafish.