| Background:Paraquat(PQ)is a widely used herbicide worldwide that can cause acute oxidative lung injuries with high mortality.The nuclear factor erythroid 2-related factor 2(Nrf2)/antioxidant response element(ARE)pathway is essential for cellular defense against oxidative stress.Sulforaphane(SFN)is common isothiocyanate with multiple health-promoting potentials including antioxidative effects.Objective:This study aimed to investigate the regulatory role of Nrf2/ARE signaling pathway and the therapeutic effects of SFN on the PQ-induced lung injury in rats.Methods:First of all,this study intends to adopt normal healthy rats to set up paraquat poisoning model which was established by intraperitoneal injection of toxicants.We use the model to study the dynamic expression and distribution of Nrf2/ARE signaling pathway in rats after acute paraquat poisoning.Secondly,the lung protective effect and its regulation mechanism of Nrf2/ARE signaling pathway in rats after acute paraquat poisoning were investigated.Finally,the protective mechanism of SFN,a drug targeting this pathway,against acute paraquat poisoning was further validated.Therefore,this study was divided into three parts.The first part was the study on dynamic expression of lung tissue after acute paraquat poisoning in rats,the second part was the role of the Nrf2/ARE signaling pathway in the lung injury caused by paraquat poisoning,and the third part was the molecular mechanism study on the treatment of acute paraquat poisoning in rats with sulforaphane.In the first part,the rats were randomly divided into control group(n=30)and PQ group(n=30).The rats were given a single intraperitoneal injection of PQ(40 mg/kg),and the rats in the control group were given the same amount of saline.Six rats were randomly selected and killed at 12h,24h,48h,72h and 120h after injection of PQ.The lung tissues of the rats were separated for subsequent experiments.The specific methods are as follows:(1)Some lung tissues were taken out and fixed with paraformaldehyde for paraffin embedding and sectioning,followed by pathological examination.The pathological changes of lung tissues at different time points after exposure were observed by HE staining.(2)Some lung tissues were taken out to detect the changes of wet/dry(W/D)and observe the pulmonary edema at different time points after exposure.(3)Blood samples were taken from anterior before the rats were sacrificed.After centrifugation,the serum levels of MDA and SOD were measured to determine the changes of redox balance in vivo at different time.(4)Bronchoalveolar lavage fluid(BALF)was used to detect the levels of TNF and IL-1 beta in the left lung and the inflammatory reaction in lung tissue at different time after exposure.(5)The expression of Nrf2 and HO-1 protein in lung tissues at different points after exposure was detected by immunohistochemistry.(6)The expression of Nrf2 and HO-1 mRNA in lung tissues of rats at different time after exposure was monitored.(7)The expression of Nrf2 and HO-1 proteins in lung tissues at different time after exposure was detected by Western-blot.In the second part,rats were randomly divided into four groups:control group(n?6),PQ group(n = 6),Nrf agonist + PQ group(n = 6)and Nrf inhibitor + PQ group(n = 6).PQ group rats were given intraperitoneal injection of a single dose of PQ(40 mg/kg),Nrf agonist + PQ group rats were given intravenous injection of Nrf agonist(30 mg/kg/day)72 hours before PQ injection,Nrf inhibitor + PQ group rats were given intravenous injection of Nrf inhibitor(80 mg/kg/day)72 hours before PQ injection,control group rats were given intraperitoneal injection of the same amount of normal saline.The rats were killed at 48h after injection of PQ.Lung tissue from rats was used for subsequent experiments.The specific operation method is the same as the first part.In the third part,rats were randomly divided into four groups:control group(n =6),PQ group(n = 6),PQ + SFN group(n = 6)and PQ + saline group(n = 6).PQ group rats were given a single dose of PQ(40 mg/kg),the control group was injected with the same amount of normal saline,PQ + SFN group and PQ + normal saline group were injected with a single dose of SFN(5 mg/kg)and the same volume of normal saline 30 minutes after intraperitoneal injection of PQ.The rats were killed at 48h after injection of PQ and the lung tissue was isolated for subsequent experiments.The specific operation method is the same as the first part.Results:Part one:1.In the control group,the alveoli were intact and there was no infiltration of immune cells.On the contrary,the alveolar membrane structure of PQ group rats was destroyed,the epithelial cells were swollen,the basement membrane was thickened,and the alveoli were filled with a large number of immune cells and red blood cells.2.The lung W/D ratio of PQ group was significantly higher than that of control group(P<0.05)from 24 hours after PQ injection,and with the longer of the PQ injection time,the lung W/D ratio was getting higher.3.The content of MDA in serum of PQ group was significantly higher than that of control group(P<0.05),and with the longer of the PQ injection time,the content of MDA was getting higher;the activity of SOD in serum of PQ group was significantly lower than that of control group(P<0.05),and with the longer of the PQ injection time,the activity of SOD was getting lower.4.ELISA showed that the expression levels of TNF-a and IL-1 beta in lung tissue of PQ group were significantly higher than those of control group(P<0.05),and with the longer of the PQ injection time,the expression levels of TNF-a and IL-1 beta were getting higher.5.RT-PCR showed that the expression levels of Nrf2 and HO-1 mRNA in lung tissues of PQ rats were significantly increased(P<0.05),and with the longer of the PQ injection time,the expression levels of Nrf2 and HO-1 mRNA were getting higher.6.Immunohistochemistry showed that the expression of Nrf2 and HO-1 protein in lung tissue of PQ group was significantly higher than that of control group(P<0.05),and with the longer of the PQ injection time,the expression of Nrf2 and HO-1 protein were getting higher.7.Western-blot results showed that the expression of Nrf2 and HO-1 protein in lung tissue of PQ group was significantly higher than that of control group(P<0.05),and with the longer of the PQ injection time,the expression of Nrf2 and HO-1 protein were getting higher.Part two:1.In the Nrf agonist +PQ group,the alveoli were relatively intact,with a small number of immune cells infiltrating.In PQ group and Nrf inhibitor + PQ group,a large number of immune cells and red blood cells were found in the alveoli,and the alveolar membrane structure was destroyed.2.Compared with PQ group and Nrf inhibitor+PQ group,the W/D ratio of lung in Nrf agonist+PQ group was significantly lower than that in PQ group and Nrf inhibitor+PQ group,and the difference was statistically significant(P<0.05).3.Compared with Nrf agonist + PQ group,the expression levels of TNF-a and IL-1 beta in lung tissue of PQ group and Nrf inhibitor + PQ group were significantly higher than those of Nrf agonist + PQ group(P<0.05).4.Compared with the Nrf agonist + PQ group,the serum MDA levels in PQ group and Nrf inhibitor + PQ group were significantly higher(P<0.05).Compared with the Nrf agonist + PQ group,the serum SOD activities in PQ group and Nrf inhibitor + PQ group were significantly lower(P<0.05).5.RT-PCR showed that the expression of Nrf2 and HO-1 mRNA in lung tissue of Nrf agonist+PQ group was significantly higher than that of PQ group(P<0.05).6.Immunohistochemical results showed that the expression of Nrf2 and HO-1 in lung tissue of Nrf agonist + PQ group was significantly higher than that of PQ group(P<0.05).7.Western-blot results showed that the expression of Nrf2 and HO-1 protein in lung tissue of Nrf agonist + PQ group was significantly higher than that of PQ group(P<0.05).Part three:1.There were a lot of immune cells in the alveoli of PQ group and PQ + saline group.The alveolar membrane structure was destroyed and the alveoli were filled with a large number of red blood cells.The alveolar structural integrity of PQ + SFN group rats was significantly improved,and the content of immune cells was significantly reduced compared with PQ group.2.ELISA showed that the expression levels of TNF-a and IL-1 beta in lung tissue of PQ+SFN group were significantly lower than those of PQ group and PQ+saline group(P<0.05).3.ELISA showed that the level of 8-OhdG in lung tissue of PQ+SFN group was significantly lower than that of PQ+ saline group(P<0.05).4.The content of MDA of PQ + SFN group in serum was significantly lower than that in PQ group and PQ + saline group(P<0.05),and the content of SOD in serum was significantly higher than that in PQ group and PQ + saline group(P<0.05).5.RT-PCR showed that the expression levels of Nrf2,HO-1 and NQO1 mRNA in lung tissue of PQ+SFN group were significantly higher than those of PQ group and PQ+saline group(P<0.05).6.Western-blot and immunohistochemistry showed that the expression of Nrf2,HO-1 and NQOl protein in lung tissue of PQ + SFN group was significantly higher than that of PQ group and PQ + saline group(P<0.05).7.Flow cytometry showed that the expression of Nrf2,HO-1 and NQO1 protein in lung tissue of PQ + SFN group was significantly higher than that of PQ group and PQ+ saline group(P<0.05).Conclusions:In the first part of study,we confirmed the acute PQ poisoning will activate the body's inflammatory response,promote the immune cell infiltration,mediate the lung tissue excess expression of TNF a and IL-1?,destroy the redox balance in rats.During this process,the expression of Nrf2 and HO-1 increased significantly with the activation of Nrf2/ARE signaling pathway.In the second part of the study,we demonstrated that Nrf/ARE signaling pathway can inhibit the pneumonia induced by PQ poisoning,inhibit the release of immune factors,and maintain the redox balance in rats.In the third part of the study,we demonstrated that SFN treatment can inhibit oxidative stress injury induced by PQ toxicity by activating Nrf2/ARE signaling pathway.Our study has confirmed the important regulatory role of the Nrf2/ARE signaling pathway in the defense against PQ-induced acute lung injury,which is triggered to gradually reduce the intracellular oxidative stress after PQ exposure_SFN treatment can inhibit oxidative damage induced by PQ toxicity by activating Nrf2/ARE signaling pathway.The findings have revealed a molecular pathway for the molecular mechanism of SFN intervention in PQ lung injury,and provided a theoretical basis for the clinical application of SFN in the treatment of the urgent condition. |
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