| ?Objective?To investigate the protective effect and its possible mechanism of hesperetin which lung damage induced by paraquat in rats by detecting the changes of oxidative stress,cytokines and key protein expression of TGF-?1/Smad signaling pathway in rats with paraquat-poisoning.?Methods?140 Wistar male rats were randomly divided into normal control group,hesperetin control group,paraquat model group,pirfenidone control group,small,medium and large dose of hesperetin treatment group,20 rats in each group.All groups received a single intragastric administration of 50mg/kg paraquat except for the normal control group and the hesperetin control group.establishment of an animal model of rat lung fibrosis induced by paraquat.After 24 hours,the rats in each group were given drug intervention once daily.10 rats were randomly sacrificed respectively at 7th day and 28 th day after exposure to paraquat.Three of the rats were randomly selected for hematoxylin-eosin and masson staining with their lung tissues and to observe and compare the degree of pulmonary alveolitis and pulmonary fibrosis in each group at different time points.The content of hydroxyproline(HYP)in lung tissue homogenate of rats at different time points was determined by alkaline hydrolysis method,and to evaluate the deposition of collagen fibers in lung tissue.The level of malondialdehyde(MDA)and the activities of superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GSH-PX)and total antioxidant capacity(T-AOC)were detected in lung tissue of rats at different time points in all groups with each index kit.The levels of transforming growth factor-?(TGF-?1),tumor necrosis factor-?(TNF-?),interleukin-4(IL-4),interleukin-10(IL-10),interleukin-1?(IL-1?),interferon-?(IFN-?)in rat lung tissue at different time points were detected by enzyme-linked immunosorbent assay(ELISA).The mRNA and protein expressions of Smad2/3,p-Smad2/3 and Smad7 in lung tissue of rats in different groups at different time points after exposure to the paraquat were measured by real-time fluorescence quantitative(RT-PCR)and Western-blot.SPSS 22.0 statistical software was used to analyze the measurement data and pathological semi-quantitative data results.?Results?1.General condition of animalsThe rats in the negative control group and the hesperetin control group had normal diet and activity,and the hair was smooth.The rats in the model group had reduced the diet,the hair was dry and sparse,the mental state was depressed.Some rats developed diarrhea,and the body weight was slower than that in the negative control group.Some rats had dyspnea and accompanied with wet gong sound,and one died on the second day after modeling.The rats in the pirfenidone control group had normal diets,good hair,and good weight gain.One rat died on the 11 th day after the model was established.In the low-dose hesperetin treatment group,the hair of the rats was slightly dry,and the diet was decreased compared with the negative control group,and the weight gain was slowely.The rats in the middle-dose and the high-dose hesperetin group had fine hair,the diet and weight gain were normal,and the symptoms of dyspnea were alleviated and their mental state was good.2.Histopathological examinationAfter 7 days of exposure to paraquat,pulmonary histopathological examination of rats revealed that the infiltration of inflammatory cells dominated by neutrophils emerged in paraquat model group and a part of the organizational structure was destroyed.After treatment with pirfenidone and hesperetin,the degree of inflammatory infiltration was reduced and the alveolar structure was more complete in the lungs of rats.After treatment with pifenidone and hesperidin,the degree of inflammatory infiltration of lung tissue was decreased,and thealveolar structure was more complete in rats.After 28 days of paraquat exposure,the alveolitis of lung tissue decreased,the deposition of collagen fibers increased,and the degree of pulmonary fibrosis increased in the model group.The degree of pulmonary fibrosis in rats was reduced after treatment with pifenidone and hesperidin.The results of semi-quantitative analysis of alveolitis in rats showed that the alveolitis scores in the paraquat model group were significantly higher than those in the normal control group(P<0.01),and the control group at the 7d and 28 d treatment groups were all dose-treated.The scores of alveolitis in the group were lower than those in the model group(P<0.05,P<0.01)after treatment with pirfenidone and hesperetin.The scores of pulmonary fibrosis on the 28 th day in the paraquat model group was significantly higher than that in the normal control group(P<0.01),and in the pirfenidone control group and the dose of hesperetin treatment groups,the pulmonary fibrosis scores was reduced with varying degrees(P <0.05,P <0.01)compared with paraquat model group at the 28 th day.3.The changes of MDA,SOD,CAT,GSH-PX and T-AOC levels in lung tissue of rats in each groupThere was no significant difference in the level of oxidative indexes between the negative control groups and the hesperetin control groups(P>0.05).Compared with the negative control group,the level of MDA in the lung tissue of the model group was significantly increased after the exposure of paraquat(P<0.01),the activities of SOD,CAT,and GSH-PX decreased significantly(P<0.05).The MDA levels were significantly decreased after intervention with pirfenidone and hesperetin(P<0.05,P<0.01),and the activities of SOD,CAT,and GSH-PX was increased with varying degrees(P<0.05,P<0.01).4.The changes of TGF-?1?IL-1??IL-4?IL-10?TNF-? and IFN-? levels in lung tissue of rats in each groupThere was no significant difference in the level of inflammatory indexes between the negative control group and the hesperetin control group(P>0.05).Compared with the negative control group,the levels of TGF-?1,IL-1?,TNF-?,IL-4 and IL-10 were significantly increased in the lung tissue of the model group rats after the paraquat exposure(P<0.01),and there was no significant difference in IFN-? levels(P>0.05).After the intervention of pirfenidone and hesperetin,the level of inflammatory factors was significantlylower than that of the model group(P<0.05,P<0.01),and the level of anti-inflammatory cytokines was increased(P<0.05,P<0.01).5.The changes of Smad2/3 and Smad7 mRNA expression in lung tissue of rats in each groupThere was no significant difference in the level of indicators between the negative control group and the hesperetin control group(P>0.05).Compared with the negative control group,the expression of Smad2/3 mRNA in lung tissue of model group rats were significantly increased after paraquat exposure(P<0.05,P<0.01),while Smad7 mRNA expression levels had a decreasing trend,and the differences were more obvious at the fibrosis stage(P<0.05).The expression of Smad2/3 mRNA were significantly lower than those in the model group after intervention with pirfenidone and hesperetin(P<0.05,P<0.01).The levels of Smad7 mRNA expression were significantly elevated than those of the model group(P < 0.01).6.The changes of Smad2/3,p-Smad2/3 and Smad7 protein expression in lung tissue of rats in each groupThe expression of Smad2 and P-Smad2/3 protein in lung tissue of hesperetin control rats at 7d and 28 d after exposure to poisoning were not statistically different from that of negative control group(P>0.05).The expression of Smad3 protein in lung tissue of the hesperetin control group rats at the 7th day after exposure was higher than that of the negative control group(P<0.05),and the expression level of Smad3 protein in the lung tissue of rats in hesperetin control group at the 28 th day after poisoning was lower than that in the negative control group(P<0.05).The expression of Smad7 protein in lung tissue of hesperetin control group rats was significantly higher than that of the negative control group rats at two time points after poisoning(P<0.05,P<0.01).The expression of Smad2/3 and p-Smad2/3 protein in lung tissue of paraquat model group rats were higher than that of negative control group(P<0.01),and the expression level of Smad7 protein was lower than that of negative control group(P< 0.01);At the 7th day after treatment,the expression of Smad2/3 and p-Smad2/3protein in the lungs of the pirfenidone control group and hesperidin treatment groups' rats were decreased compared with the model group(P<0.05,P<0.01).The expression of Smad7 protein was significantly increased compared with paraquat model group at the 7th day after treatment(P<0.01).The expression of Smad7 protein in the lung tissue of the pirfenidone control group rats on the 28 th day was significantly higher than that of the paraquat modelgroup(P<0.01),while the Smad7 protein expression level in the rats lung tissue of the hesperetin-treated groups have no significant difference compared with the paraquat model group(P>0.05).?Conclusions?1.Paraquat poisoning can lead to imbalance of oxidative and antioxidant status in rats,which in turn can damage lung tissue.Hesperetin can inhibit the level of oxidative factors and increase the level of antioxidant factors in lung tissues of the rats poisoned by paraquat,thereby reducing the damage of paraquat on lung tissues.2.Hesperetin can inhibit the release of inflammatory factors and promote the secretion of anti-inflammatory factors in lung tissues of the rats poisoned by paraquat,thereby reducing the damage of paraquat on lung tissues.3.Hesperetin can inhibit the synthesis and secretion of TGF-?1 which as a fibrogenic factor,decrease the expression of Smad2/3 mRNA and protein and p-Smad2/3 protein,increase the expression of Smad7 mRNA and protein in the lung tissues of the rats poisoned by paraquat,and thus block the TGF-?1/Smad signaling pathway and to alleviate lung injury.4.Combined with lung histopathology studies,it was found that lung tissues injury in rats poisoned by paraquat was reduced with increasing dose of hesperetin,suggesting that the hesperetin hopefully to be a new drug for the treatment of paraquat poisoning,providing a reliable pharmacological basis for future guidance on the development and application of hesperetin. |
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