Effect Of Txnrd3 On Nickel Induced Lung Injury In Mice And Detoxification Effect Of Melatonin

Nickel is a heavy metal with strong toxicity.It harms the biological environment through atmospheric deposition,automobile manufacturing,military,electronic,pharmaceutical and electroless nickel plating materials.Environmental nickel exposure can lead to acute lung inflammation,pulmonary fibrosis,and other respiratory diseases.In models of colon cancer and liver fibrosis,Txnrd3 has been shown to have a protective effect on the body and is involved in sperm maturation,but the mechanism in lung tissue is unknown.The anti-inflammatory and antioxidant mechanisms of melatonin(Mel)are well understood.VEGF activation enhances vascular permeability,and abnormal activation is also associated with the development of pulmonary fibrosis.In this study,nickel exposure models were established in Wild-type and Txnrd3 knockout mice.We investigated the effects of nickel on the VEGF/VEGFR-2 pathway through oxidative stress and TNF-α/NF-κB,and Txnrd3 knock-out intensified the toxicity in mice.Melatonin can intervene in lung tissue damage caused by nickel exposure and play a protective role.In this study,160 8-week-old C57BL/6N male Wild-type mice and Txnrd3-/-mice with body weight between 25 g and 30 g were selected.We randomly divided the mice into eight groups of Wild-NC,Wild-Ni,Wild-Mel,Wild-Ni+Mel,Homozygote-NC,HomozygoteNi,Homozygote-Mel,and Homozygote-Ni+Mel,with 20 animals in each group.10mg/kg is the concentration of Ni and 2mg /kg is the concentration of Mel.Animals were given nickel chloride in the morning and melatonin in the evening by gavage.The follow-up experiment was performed after 21 days of intragastric administration according to the standard 0.1ml /10 g body weight.After nickel exposure model was established successfully,the lung tissue damage was observed by H&E staining and transmission electron microscopy.At the same time,q RT-PCR,Western Blot,malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT)and total antioxidant capacity(T-AOC)kits were used for evaluation,and genes related to inflammatory factors and VEGF were determined.To investigate the positive effects of melatonin and Txnrd3 in lung injury induced by nickel exposure in constructed knockout mice through VEGF/VEGFR-2 axis.The results are as follows:(1)H&E staining showed that nodal expansion structure of alveolar ducts was not abnormal in the Wild-NC group,and no abnormal thickening of alveolar septum was found.In Wild-Ni group,mononuclear macrophage hyperplasia,alveolar wall telangiectasis and local alveolar congestion were observed.In the Wild-Ni+Mel group,the hyperemia was reduced and no inflammatory cell infiltration was found.The alveolar structure of the Homozygote-NC group was intact and no obvious abnormality was observed.In the Homozygote-Ni group,protein fragments and epithelial cell shedding were observed in the bronchioles,with nearby inflammatory cell infiltration and occasional alveolar hemorrhage.Alveolar congestion was reduced in the Homozygote-Ni+Mel group with only local thickening of the alveolar septum.The results showed that nickel exposure had a damaging effect on mice,Txnrd3-/-mice were more damaged than Wild mice,and the structure was more disordered.(2)The results of electron microscopy indicated that the mitochondrial structure of the Wild-NC mice group was intact and the nucleus was not abnormal.In Wild-Ni group,the tight connections between capillary endothelial cells became shorter,the basement membrane dissolved,mitochondrial crists disappeared,and transparent vacuoles appeared in the inner cavity.The capillary endothelial cells in the Homozygote-NC group were closely connected and the basement membrane was relatively intact.The organelles of the Homozygote-Ni group were seriously damaged and a large amount of collagen fibers were accumulated.The results showed that nickel exposure could lead to increased permeability of pulmonary capillary endothelial cells through structural changes such as tight junction loss.Lung tissue cell damage was more pronounced in Txnrd3 knockout mice than in wild-type mice under Ni exposure.(3)The expression of antioxidant enzyme activity under nickel exposure,MDA was significantly increased in Wild-Ni group,Homozygote-NC and Homozygote-Ni group,while T-AOC,CAT and SOD were significantly decreased(p<0.05)compared with the Wild-NC group.Compared with Wild-Ni and Homozygote-Ni,MDA was significantly increased,while T-AOC,CAT and SOD were significantly decreased(p<0.05).Compared with Wild-Ni group,MDA was remarkably up-regulated and TAOC,CAT and SOD were remarkably down-regulated in Homozygote-Ni group(p<0.05).Compared with Ni group,MDA in Ni+Mel group was significantly decreased,while T-AOC,CAT and SOD in Ni+ MEL group were remarkably increased(p<0.05).There was no significant difference in MDA,TAOC,CAT and SOD between NC group and Mel group.This suggests that proper melatonin concentrations do not cause oxidative damage to the body.(4)The protein and mRNA expression levels of inflammation-related factors in mouse lung tissues were detected by Western Blot and q RT-PCR.Compared with Wild-NC group,The expression levels of i NOS,HO-1,IL-1β,COX-2,TNF-α and NF-κB protein in Wild-Ni group and Homozygote-Ni group were increased(p<0.05).The expression levels of i NOS,HO-1,IL-1β,TNF-α and NF-κB protein in Homozygote-NC group were significantly higher than those in Wild-NC group(p<0.05).Compared with Homozygote-Ni and homozygote-Ni,i NOS,HO-1,IL-1β,COX-2,TNF-α and NF-κB protein expressions were significantly up-regulated(p<0.05).Compared with Wild-Ni group,the expression levels of corresponding inflammatory factor proteins in the Homozygote-Ni group were significantly increased(p<0.05).Compared with Ni group,the expressions of i NOS,HO-1,IL-1β,COX-2,TNF-α and NF-κB proteins in the Ni+Mel group were significantly decreased(p<0.05).The results of q RT-PCR showed that the m RNA expressions of i NOS,HO-1,IL-1β,TNF-α,NF-ΚB,IL-2,IL-6,IL-10 in Wild-Ni group and Homozygote-Ni group were significantly increased compared with those in the Wild-NC group(p< 0.05).It shows the toxic effect of nickel.The levels of HO-1,IL-1β,TNF-α,NF-κB,IL-2,IL-6 and IL-10 in the Homozygote-NC group were significantly higher than those in the Wild-NC group(p< 0.05).Compared with the Homozygote-Ni group,the authors dealed so as to ensure the conclusion of these conclusions.i NOS,HO-1,IL-1β,TNF-α,NF-κB,IL-2,IL-6 and IL-10 were significantly increased(p<0.05).Compared with Ni group,the levels of i NOS,HO-1,IL-1β,TNF-α,NF-κB,IL-2,IL-6 and IL-10 in Ni+Mel group were significantly decreased(p<0.05).These results suggest that the TNF-α / NF-κB pathway may be activated by the body to cause inflammatory response,and Txnrd3 deficiency may aggravate the inflammatory response.(5)The protein and mRNA expressions of VEGF/VEGFR-2 pathway related factors in mouse lung tissues were detected by Western Blot and q RT-PCR.The differences between VEGFA,p-VEGFR-2and Src in Homozygote-NC group and Wild-NC group were statistically significant.VEGF,PVEGFR-2 and Src were significantly increased(p< 0.05).VEGF,P-VEGFR-2 and Src in Wild-Ni group were significantly higher than those in Wild-NC group,and VEGF,P-Veg FR-2 and Src in Homozygote-Ni group were significantly higher than those in Homozygote-NC group(p< 0.05).Moreover,compared with the Wild-Ni group,Homozygote-Ni increased significantly.Compared with Ni group,VEGF/VEGFR-2 related factors in Ni+Mel group were significantly down-regulated(p<0.05).The results of q RT-PCR showed that the levels of VEGF,VEGFR-2,Src and Rac in Wild-Ni and Homozygote-Ni groups were significantly higher than those in the corresponding NC group(p<0.05).VEGF,VEGFR-2,Src and Rac in the Homozygote-Ni and Wild-Ni groups were increased more significantly than those in the Wild-NC group(p< 0.05).Compared with Wild-Ni and Homozygote-Ni groups,VEGF-related factors were significantly down-regulated in the corresponding Ni+Mel group(p< 0.05).This indicated that VEGF activated VEGFR-2 and activated the downstream pathway under nickel exposure,and Txnrd3 knockout mice aggravated lung injury through VEGF/VEGFR2,resulting in increased vascular permeability and vascular endothelity-related pathologic reactions.In summary,our findings suggest that Ni affects pulmonary vascular permeability by generating inflammatory factors to activate the VEGF/VEGFR-2 pathway through oxidative stress,and Txnrd3 knockout aggravates lung injury after Ni exposure.Mel can significantly reduce oxidative stress and inflammation produced by Ni,and down-regulate VEGF/VEGFR-2.