Molecular Mechanisms Related To Nickel Oxide Nanoparticles-induced Pulmonary Toxicity In Rats

Objectives: The aim of present study was to construct subchronic pulmonary injury model in rats via intratracheal instillation of nano Ni O,and explore its molecular mecha nisms through oxidative stress,activation of NF-κB and TGF-β1/Smad pathway in lung tissue.Methods: A total of forty male and healthy Wistar rats(200~240 g)were randomized into control group(normal saline),nano Ni O(0.015,0.06 and 0.24 mg/kg b.w),micro Ni O(0.24 mg/kg b.w)to recive intratracheal instillation twice a week for six weeks.Lung tissue were collected after exsanguination via heart at the end of exposure.(1)The body weight and food intake were recorded to caculate the food utilization efficiencies.The lung wet weight and lung coefficient to body weight were also evaluated.(2)The hematoxylin and eosin(HE)staining and Masson trichrome staining were performed to observe lung lesion and fibrosis respectively.(3)After the lung tissue were homogenized,8-hydroxyguanosine(8-OHd G),fibrosis indicator of transforming growth factor-beta 1(TGF-β1),inflammatory cytokines and chemokines including interleukin-6(IL-6),IL-10,tumor necrosis factor-alpha(TNF-α),cytokine-induced neutrophil chemoattractants-1(CINC-1),CINC-2αβ and CINC-3 were detected by enzyme linked immunosorbent assay(ELISA),but the indicators of oxidative stress such as hydroxy radical(·OH),lipid peroxidation(LPO),catalase(CAT),glutathione peroxidase(GSH-Px)and total antioxidant capacity(T-AOC)were measured by biochemical method.(4)RT-q PCR were performed to quantify the relative m RNA expression of heme oxygenase-1(HO-1),metallothionein-1(MT-1),nuclearfactor-κB(NF-κB),inhibitor of κB kinase-alpha(IKK-α),nuclear factor-inducing kinase(NIK),T-box expressed in T cells(T-bet),GATA-3,TGF-β1,Smad2,Smad4,Ecadherin,alpha-smooth muscle actin(α-SMA),Vimentin,matrix metalloproteinases-9(MMP-9),tissue inhibitor of metalloproteinases-1(TIMP-1)and connective tissue growth factor(CTGF)in lung tissue.(5)Western blot were used to detect the protein contents of collagen type I alpha 1(COL1A1),collagen type III alpha 1(COL3A1),NF-κB,IKK-α,NIK,T-bet and GATA-3.Results:(1)Compared to control group,the body weight and food utilization efficiencies decreased in 0.06 and 0.24 mg/kg nano Ni O groups at 5 and 6 weeks,and the lung wet weight and lung coefficient to body weight increased after 0.24 mg/kg nano Ni O exposure(p<0.05).(2)The HE staining of lung tissue appeared inflammatory infiltration,widened alveolar septum,and focal particles deposition in gaps and the alveolar cavity in 0.24 mg/kg nano Ni O group.(3)After nano Ni O exposure,lung tissue with the Masson trichrome staining showed obvious fibrotic injury and collagen deposition in alveolar septum and cavities obviously.Meanwhile,the hydroxyproline content of lung tissue increased,as well as the relative protein expression of COL1A1 and COL3A1 upregulated,especially in 0.24 mg/kg nano Ni O group.(4)In 0.24 mg/kg nano Ni O group,the levels of pro-inflammatory cytokines NO,i NOS,TNF-α and IL-6,as well as chemokines CINC-1,CINC-2αβ and CINC-3 were higher than control group,while the anti-inflammatory cytokine IL-10 was lower than control group(p<0.05).(5)Compared to the control group,the ·OH,LPO and 8-OHd G of lung tissue increased,while the CAT,GSH-Px and T-AOC decerased in 0.24 mg/kg nano Ni O group(p<0.05).(6)In 0.24 mg/kg nano Ni O group,the levels of m RNA and protein of NF-κB,IKK-α and NIK were higher than control group(p<0.05).(7)Comapred to the control group,the m RNA and protein content of T-bet decreased,while the m RNA and protein expression of GATA-3 increased in 0.24 mg/kg nano Ni O group(p<0.05).(8)Compared to the control group,the TGF-β1 protein content and m RNA expression increased,as well as the upregulated gene expression of Smad2,Smad4,α-SMA,Vimentin,MMP-9,TIMP-1 and CTGF,while the Ecadherin gene expression downregulated in 0.24 mg/kg nano Ni O group(p<0.05).Conclusions: The lung injury model was constructed by intratracheal instillation of nano Ni O in rats,and its molecular mechanisms could be related to the oxidative stress,the activated NF-κB and TGF-β1/Smad signaling pathway in lung tissue.