Effects Of Docosahexaenoic Acid Against Paraquat Induced Pulmonary Injury And The Underlying Mechanisms Exploration

ObjectiveParaquat is a non-selective contact herbicide of the dipyridinyl class. After its introduction, the use of paraquat (PQ) rapidly expanded worldwide. However, with the widely use of paraquat, the number of poisoned people is increasing by accident or suicide. The lung is the primary target organ of PQ. The acute inflammation and following lung fibrosis in later period are major causes of morbidity and mortality induced by PQ. There is no effective therapeutic remedy for PQ induced pulmonary injury so far, and it’s mortality rates is high ranging from50%-90%. In recent years, although substantial research efforts have been made on it, the exactly underlying mechanisms still remain unclear.Docosahexaenoic acid (DHA) is an omega-3(n-3) long chain polyunsaturated fatty acid s, derived from fish oil or microalgal. Previous research indicated that DHA is important for nervous system function, also benefits for prevention and treatment a number of diseases including hypertension, atherosclerosis, cancer and so on. More recently, DHA is increasingly being recognized as important modulators of multiple biological pathways that affect health and disease. Growing epidemiological evidence indicates that DHA can exhibit protective and anti-inflammatory activities. Newly identified lipid mediators produced from DHA have inflammation-dampening effects in models of inflammation. A line of studies have shown that supplementary DHA is associated with the alleviation of some tissues fibrosis. Therefore, we hypothesis that DHA may exert protective bioactivity through these pathways for PQ induced pulmonary injury.For studies evaluating the effects of DHA in vivo, we developed the acute and chronic PQ poisoning animal model respectively, the histopathological changes and biochemical parameters of lung were determined with or without DHA administration. Methods1. Acute lung injury mouse modelMale healthy KM mice were randomized divided into3groups with24in each, i.e. control group, PQ model group, DHA treated group. The mice of DHA treated group received500mg/kw.bw DHA respectively by gavage, while the mice in control and PQ model groups received equivalent corn oil. On day8of DHA treatment, a single dose of50mg/kg.bw paraquat was given to mice of PQ model and DHA treated groups, whereas control mice received saline. After7days of PQ treatment, the mice were initially anesthetized with an intraperitoneal injection of20%urethane, and the lungs were removed and then stored at-80℃until analyzed. The lungs were homogenized, and levels of the myeloperoxidase (MPO), malondialdehyde (MDA) and reduced glutathione (GSH) were determined by spectrophotometric method. The protein adduct levels of4-hydroxynonenal (4-HNE) and MDA in mice lung were also investigated by western blotting.2. The pulmonary fibrosis modelForty male Wistar rats, weighted200-220g, were randomized divided into4groups with10in each, i.e. control group, PQ model group, DHA treated group, and DHA control group. The rats of DHA treated and DHA control groups received500mg/kw.bw DHA respectively by gavage, while rats in control and PQ model groups received equivalent corn oil. On day8of DHA treatment, a single dose of50mg/kg.bw paraquat was given to rats of PQ model group, DHA treated group, whereas control and DHA control rats received saline. After35days of PQ treatment, the rats were sacrificed and the lungs were removed. Pulmonary wet-to-dry weight ratio and hydroxyproline (HYP) concentrations were quantified in conjunction with histopathological examination by HE and Masson Stain to determine the fibrotic changes in rat lung. The protein levels of Smad7and SnoN were determined by immunohistochemistry and Western blotting respectively.Results1. The protective effects of DHA against acute lung injury induced by PQ1.1Effect of DHA on mice mortality induced by PQAfter7days of single50mg/kg.bw PQ treatment, the mice mortality of PQ model and DHA treated groups were58.3%,33.3%respectively. There were significant differences between the two groups. So, DHA pretreatment for7days could significantly attenuate the acute lung damage by PQ.1.2Effect of DHA on mice organ coefficient of the lungAfter7days of50mg/kg.bw PQ treatment, the mice were sacrificed. Postmortem examination revealed that the lungs of model group mice were dark red and larger, characterized by hyperemia, edema and haemorrhage in alveoli or bronchioli relative to the control mice lung. While, the conditions of DHA treated group mice lungs were much better than that of model group mice, with light hyperaemia, oedema. The lung coefficients of three groups were (0.574±0.085)%,(1.021±0.683)%,(0.710±0.20)%, respectively. Compared with the control group, the lung coefficient of PQ treated mice was increased by77.87%(P<0.01). DHA pretreatment for7days effectively attenuated the hyperemia, edema of acute lung injury, and the lung coefficient was significant lower than that of the model group (P<0.05).1.3Effect of DHA on the levels of MPO, MDA, GSH in lungThe MPO content in model group which compared with the control group, was increased by20.6%(P<0.01). While, compared with the model group, the MPO content in DHA treated group were significantly lower (P<0.05), and had no differences with the control group.Results showed that PQ treatment resulted in lipid peroxidation of lung. Compared with the control group, the MDA content of lungs in model group was increased by21.7%(P<0.01). And compared with the model group, the MDA content in DHA treated group were decreased by14.4%(P<0.01). DHA pretreatment for7days could effectively decrease the level of MDA.After PQ administration, the GSH content of lung in model group was decreased by14.2%(P<0.01), compared to control group. The GSH content in DHA treated group were significantly higher (P<0.05) than those of model group, and had no differences with the control group.1.6Effects of DHA on the protein adducts level of4-HNE and MDAThe results of western blotting showed that compared with control group, the4-HNE protein adduct levels of the model group was significantly increased by43.97%(P<0.05), while those of the DHA treated group was only incresed by3.16%, which had no significant differences with the control group (P>0.05). And had significant differences with the model group (P<0.05). Compared with control group, the MDA protein adduct levels of the model group was significantly increased by43.39%(P<0.05), which of the DHA treated group was increased by16.97%. DHA control group had no significant differences with the control group (P>0.05). So, DHA pretreatment for7days could effectively reduce the production of oxidation products4-HNE and MDA protein adduct.2. The protective effects of DHA on paraquat-induced pulmonary fibrosis in rats2.1The general conditions and body weightAfter PQ treatment, rats showed slow-moving, poor coordination, dingy coat colour, decrease into appetite, retardation of growth rate and so on.28days later, the body weight of rats in model group was significantly lower than that in the control group (P<0.05), DHA treated group and DHA control group had no significant differences with the control group.2.2Pathological changes were observed by HE stainingAfter HE staining, changes in lung were observed under light microscope. The lungs of the control group rats had clear structure, with thin alveolar walls and no infiltration of inflammatory cells. While the alveolar walls of the model group rats were thickened and filled with inflammatory cells. The pulmonary architecture was damaged and some were collapsed. The lungs of the DHA treated group rats had slight pathologic changes. DHA control group had no significant differences with the control group.2.3The fibrosis change of lung2.3.1Wet-to-dry weight ratioCompared with the control group, the w/d of lung in model group was decreased significantly (P<0.05); while, compared with the model group, the w/d of DHA treated and DHA control group mice lungs were significantly increased (P<0.05), and had no significant differences with the control group.2.3.2The HYP contentAfter35days of single50mg/kg.bw PQ treatment, the HYP content in model group was increased18.3%than that of the control group (P<0.05). Compared with the model group, the HYP content of DHA treated and DHA control group were significantly decreased (P<0.05).2.3.3Masson staining The results of masson staining showed that the alveolar walls of the control group were thin and filled with less blue collagen, no collagen deposited in alveolar space, and had clear structure. The alveolar walls of the model group were were thick, less alveolar space, partial consol, and filled with much collagen. The DHA treated lungs had no obvious collagen deposited and consol, and the structure is complete. DHA control group had no significant differences with the control group.2.4Effects of DHA on the protein levels of Smad7and SnoN2.4.1Immunohistochemical AnalysesIn the control group rats, the predominant cells in lung tissues that expressed Smad7were alveolar and epithelial cells. The positive staining of control group was intensively distributed, while the positive expression of the model group was remarkably decreased. Compared with the model group, the expression of Smad7of the DHA treated group was increased. Quantitative image analysis showed that compared with the control group, the model group’s integrated optical density (IOD) was decreased by63%(P<0.01). While compared with the model group, the DHA treated group was increased by112.7%(P<0.01). DHA control group had no significant differences with the control group (P>0.05).The positive expression of SnoN in control group rats lung was evenly distributed, while the positive expression of the model group was unevenly and decreased. Compared with the model group, the expression of SnoN of the DHA treated group was increased. Quantitative image analysis showed that compared with the control group, the model group’s IOD was decreased by41%(P<0.01). Compared with the model group, the DHA treated group was increased by42.4%(P<0.01). DHA control group had no significant differences with the control group (P>0.05).2.4.2Western blotting AnalysesWestern blotting results showed that compared with control group, the Smad7and SnoN protein levels of the model group were significantly decreased (P<0.05, P<0.01), while those of the DHA treated group were no significantly decreased (P>0.05). So DHA pretreatment for7days could effectively inhibit the decrease expression of the Smad7and SnoN. DHA control group had no significant differences with the control group (P>0.05). Conclusion:1. DHA (500mg/kg.bw) could effectively attenuate PQ (50mg/kg.bw) induced acute lung injury in mice.2. DHA could significantly reduce the protein adduct levels of4-HNE and MDA induced by PQ, thus the effects against PQ induced acute lung injury may be attributed to its antioxidative activities.3. DHA (500mg/kg.bw) could effectively ameliorate chronic pulmonary fibrosis of rats induced by PQ (50mg/kg.bw).4. DHA could significantly increase the expression of Smad7and SnoN.The anti-lung fibrosis capacity of DHA might be associated with the up-regulating of Smad7and SnoN, which were two important fators in PQ induced pulmonary fibrosis.