Effect Of Sulforaphane On Hexavalent Chromium-Induced Lung Injury In Rats

Chromium is an internationally recognized carcinogen,and the high concentration exposure of chromium in the working environment poses a great threat to the global professionals,which can cause serious lung toxicity.Chromium is a common environmental pollutant.Every year,more than 170000 tons of chromium containing waste are discharged into the natural environment,which has high toxicity in natural water and other resources.The World Health Organization and the European Union stipulate that the chromium content shall not exceed 50?g/L of domestic water.In nature,chromium exists in the form of trivalent chromium and hexavalent chromium.People pay more attention to hexavalent chromium,which is more toxic.Hexavalent chromium is usually soluble chromate or dichromate,which has strong toxicity to livestock and poultry,and can cause mutation and cancer.Hexavalent chromium will be reduced to pentavalent chromium in the body,which will produce a lot of reactive oxygen species,destroy the body's oxidation balance and cause oxidative stress.Long term exposure to chromium has been reported t o induce histopathological changes in the lungs.Chromium poisoning occurs in livestock and poultry through exposure to chromium polluted drinking water,feed or environment.However,up to now,the lung toxicity caused by chromium is lack of effective the rapeutic drugs.Therefore,it is of great significance to study the mechanism of lung toxicity induced by chromium exposure and the effective therapeutic drugs.Sulforaphane?SFN?is a natural isothiocyanate compound,which has attracted more and more attention due to its unique biological function.SFN exists in cruciferous vegetables,including cabbage,broccoli and Brussels sprouts,and has biological characteristics of antioxidant,anti-inflammatory,antibacterial and anti-tumor effects.SFN is an indirect antioxidant,which can induce phase II detoxification enzyme and antioxidant gene.The potential mechanism of SFN on the lung toxicity induced by potassium dichromate?K₂Cr₂O₇?,a hexavalent chromium compound,was studied.In vivo experiments were carried out from the following two aspects:healthy 6-8-week-old male Wistar rats weighing 170±10 g.The first part is the establishment of the lung injury model of rats induced by K₂Cr₂O₇.Rats were randomly divided into 4 groups?n=7?:Control group,K₂Cr₂O₇ low,medium and high dose group?2,4 and 6 mg/kg K₂Cr₂O₇ were injected intraperitoneally respectively?,and the test period was 35 days.Blood and tissue samples were collected for the following tests:detection of hematology and oxidative stress indexes.The second part is the experiment of SFN intervention on K₂Cr₂O₇-induced lung injury in rats.Rats were randomly divided into 4 groups?n=7?:Control group,K₂Cr₂O₇ group,K₂Cr₂O₇+SFN group and SFN group,with a cycle of 35 days.The rats in the control group were injected with sterile saline intraperitoneally and subcutaneously every day;the rats in the K ₂Cr₂O₇ group?4 mg/kg K₂Cr₂O₇intraperitoneal injection,saline subcutaneous injection?;the rats in the K ₂Cr₂O₇+SFN group?4mg/kg K₂Cr₂O₇ intraperitoneal injection,4 mg/kg SFN subcutaneous injection?;the rats in the SFN group?saline intraperitoneal injection,4 mg/kg SFN subcutaneous injection?.Blood and tissue samples were collected for the following tests:hematology test;oxidative stress r elated indicators test;lung tissue hematoxylin eosin staining pathological observation;in situ end labeling method to determine the rate of apoptosis;fluorescent quantitative PCR and immunoblotting method to verify the signal pathway.The mouse alveolar type II epithelial cell line?MLE-12?was divided into four groups:control group,K₂Cr₂O₇ group?1?g/m L?,K₂Cr₂O₇ group?1?g/m L?+SFN group?0.1?M?and K₂Cr₂O₇group?1?g/m L?+SFN group?0.1?M?+MK-2206 2HCl group?10?M?.MK-2206 2HCl was used as a specific inhibitor of protein kinase B?Akt?.The effect of MK-2206 2HCl on the results of K₂Cr₂O₇+SFN combined administration group was compared in vitro,which was verified by Akt/GSK-3?/Fyn signaling pathway.MLE-12 cell survival rate,ROS level,scratch test,mitochondrial membrane potential test and western blot were used to verify the signal pathway.The results of the first part of the in vivo experiment showed that compared with the control group,the number of WBC,RBC and oxidative stress indexes in the low,medium and high dose K₂Cr₂O₇ group showed dose-dependent changes,indicating that K₂Cr₂O₇ could induce lung injury in rats in a dose-dependent manner.The second part of the results showed that compared with the control group,the number of WBC in K₂Cr₂O₇ group was significantly increased and RBC was significantly reduced;the MDA content of oxidative stress index was significantly increased and the SOD activity and GSH content were significantly reduced;histopathological observation showed that K₂Cr₂O₇ group caused serious lung injury;K₂Cr₂O₇ group significantly enhanced lung inflammation and activated inflammation related signal pathway;K ₂Cr₂O₇ group significantly increased Strengthen the apoptotic rate of lung and activate the apoptotic signal pathway.The results of K₂Cr₂O₇+SFN group showed that SFN could treat K₂Cr₂O₇-induced lung injury.Western blot analysis of Akt,GSK-3?,and nuclear factor E2 related protein showed that compared with control group,the expression of Akt,Nrf2,HO-1 and NQO1 in K₂Cr₂O₇ group decreased significantly,the activity of GSK-3?and the accumulation of Fyn increased significantly;compared with K₂Cr₂O₇ group,the expression of Akt,Nrf2,HO-1 and NQO1 in the lung of K₂Cr₂O₇+SFN group increased significantly,the activity of GSK-3?and the accumulation of Fyn decreased significantly.Therefore,SFN activated Akt and Nrf2,inhibited GSK-3?activity and Fyn nuclear translocation.The results of in vitro experiments showed that SFN reduced the decrease of cell viability and migration,the increase of ROS level and mitochondrial membrane potential induced by K₂Cr₂O₇.After pretreatment with MK-22062 HCl?Akt specific inhibitor?,MLE-12 cells in K₂Cr₂O₇+SFN group were inhibited to increase survival rate and decrea se ROS level.Meanwhile,Akt,Nrf2,HO-1 and NQO1 expression in lung of K₂Cr₂O₇+SFN group were inhibited to increase significantly,GSK-3?activity and Fyn accumulation were significantly reduced,and the therapeutic effect of SFN was significantly reduced.In conclusion,SFN can reduce the oxidative stress induced by K ₂Cr₂O₇ by activating Akt/GSK-3?/Fyn signaling pathway,thus alleviating the lung injury induced by K₂Cr₂O₇ and protecting the lung,which indicates that SFN has good therapeutic effect and po tential application value on K₂Cr₂O₇ induced lung injury.