| Objective1.To explore the effect of cyfluthrin on lung injury.2.To explore the effect of ALDH2 gene regulating c GAS-STING pathway in lung injury induced by cyfluthrin.Methods1.Forty SPF SD adult rats with the body weight of 180～ 400 g were randomly divided into 4 groups,namely solvent control group and cyfluthrin low-dose,medium-dose and high-dose groups,with 10 rats in each group(half male and half female).Each group was poisoned daily for 4 weeks.The change and trend of body weight and lung coefficient of rats were measured by weighing.The morphological changes of rat lung tissue were observed by HE staining.The ultrastructural changes of lung cells were observed by transmission electron microscopy.2.Cell experiments were also divided into control group and cyfluthrin low,medium and high dose groups.The cell concentration was determined by CCK8 method.Morphological changes of cyfluthrin infected cells were observed by light microscope.The migration ability of cyfluthrin infected cells was detected by scratch test.3.SOD activity and MDA content were detected by SOD and MDA kits in animal models and cell models.Western blot and q PCR were used to detect m RNA and protein expression levels of ALDH2,TNF-α,IL-6 and c GAS-STING pathway related factors c GAS,STING,NFκB and p-NFκB.4.It was overexpressed by ALDH2 activator Alda-1.Western blot and q PCR were used to detect the m RNA and protein expression levels of inflammatory cytokines IL-6,TNF-α and c GAS-STING pathway related factors in ALDH2 high-expression group.SOD and MDA expression levels of ALDH2 high expression group were detected by SOD and MDAkit.Results1.Changes in body mass and lung organ coefficients after 28 d exposure to cyfluthrinThe high-dose group gradually lost weight during the last week of exposure,from beginning to end.Compared with the control group,the average body weight of rats in the poisoned group was decreased,and the difference between the high-dose group and the control group was statistically significant(P<0.05).Compared with the control group,the lung organ coefficients in the medium-dose and high-dose groups were increased,and the difference was statistically significant(P<0.05).2.Changes of HE staining in lung tissue injury induced by cyfluthrin in ratsHE staining results showed that compared with the control group,with the increase of poison dose,the alveolar interstitial of rats in the low and medium dose groups was significantly widened,the alveolar wall was thickened,the lung tissue was congested,foam cells appeared,and the degree of fibrosis was increased,especially in the high dose group,the pulmonary tissue vascular wall was significantly thickened,the lumen was significantly narrowed,and the pulmonary muscle artery intima had obvious cellular hyperplasia.There was significant inflammatory cell infiltration.3.Changes of lung tissue injury induced by cyfluthrin in rats by transmission electron microscopyThe results of transmission electron microscopy showed that,compared with the control group,the mitochondrial crists of type II alveolar epithelial cells in the low-dose and medium-dose groups were blurred and the double-layer membrane structure disappeared with the increase of dose,especially in the high-dose group,the mitochondria showed obvious swelling and mitochondrial ridge fracture.Compared with the control group,the basal membrane of the low-dose,medium-dose and high-dose groups was blurred gradually with the increase of the dose,and the basal membrane of the high-dose group was dilated.Macrophages were observed in alveolar cavities in the medium and high dose groups,and the structure of macrophages in the medium dose group was more clear.In the high-dose group,endothelial cells proliferated in a vascular lumen of lung tissue.4.Changes in cell status and migration ability of A549 after cyfluthrin exposureIn the cell model,A549 cells in the control group had regular morphology,but compared with the control group,under the same culture conditions,the cell density of A549 cells in the infected group decreased with the increase of the infected concentration,and the irregular cell morphology changed more obviously with the increase of the infected concentration.The cell scratch test results showed that compared with the control group,the mobility of low-dose,medium-dose and high-dose groups decreased,the difference was statistically significant(P<0.05),and the mobility of A549 cells decreased more significantly with the increase of the concentration of poison.5.Effect of cyfluthrin poisoning on oxidative stress levelIn animal model and cell model,MDA content in low,medium and high dose groups increased in a dose-dependent manner compared with control group,with statistical significance(P<0.05).Compared with the control group,SOD activity in low,medium and high dose groups increased in a dose-dependent manner,with statistical significance(P<0.05).Compared with the control group,the protein expression and m RNA expression of ALDH2 in low-dose,medium-dose and high-dose groups decreased significantly with the increase of dose,except that in the cell model,the difference of ALDH2 protein expression was statistically significant between medium-dose and high-dose groups(P<0.05).There were statistically significant differences among low,medium and high dose groups(P<0.05).6.Effects of cyfluthrin on expression of inflammatory cytokines IL-6 and TNF-αIn the animal model,compared with the control group,the expressions of TNF-α and IL-6protein and m RNA were increased in the low-dose,medium-dose and high-dose groups;in the cell model,the expressions of IL-6 protein were significantly increased in the medium-dose and high-dose groups compared with the control group,with statistical significance(P<0.05).The m RNA expression level of IL-6 was significantly increased in low-dose,medium-dose and high-dose groups,with statistical significance(P<0.05),and the expressions of TNF-α protein and m RNA were significantly increased in low-dose,medium-dose and high-dose groups,with statistical significance(P<0.05).7.Expression results of cyfluthrin on c GAS-STING inflammatory pathway and related factorsIn animal models,compared with the control group,m RNA levels of inflammatory pathway related factors c GAS,STING and NFκB and protein expression levels of c GAS,STING and p-NFκB in lung tissue of the infected group were increased,and they all increased with the increase of the infected concentration.There was statistical significance in the expression of P-NF κB protein in the low-dose,medium-dose and high-dose groups(P<0.05)except that there was statistical significance in the expression of P-NF κB protein in the medium-dose and high-dose groups(P<0.05).In the cell model,the m RNA levels of inflammatory pathway related factors c GAS,STING and NFκB and the protein expression levels of c GAS,STING and p-NFκB in A549 cells of the infected group were increased,and they all increased with the increase of the infected concentration.Except for c GAS,STING and P-NF κB protein expression,there were statistically significant differences between medium-dose and high-dose groups(P<0.05),there were statistically significant differences between low-dose,medium-dose and high-dose groups(P<0.05).8.Effect of ALDH2 gene on cyfluthrin-induced lung injuryIn the cell model with high expression of ALDH2 gene,MDA content decreased and SOD content increased.The expression of inflammatory cytokines IL-6 and TNF-α decreased,and the level of inflammation decreased.The m RNA levels of inflammatory pathway related factors c GAS,STING and NFκB and the protein expression levels of c GAS,STING and p-NFκB were decreased.ConclusionsCyfluthrin can cause lung injury,and the mechanism may be that it induces oxidative stress and activates c GAS-STING pathway to produce inflammatory response.In cyfluthrin-induced lung injury,activation of ALDH2 gene can reduce oxidative stress,inhibit the activation of c GAS-STING pathway,and reduce inflammatory response. |
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