Study On The Intervention Effect And Mechanism Of Melatonin On Rat Lung Injury Induced By Hexavalent Chromium

Chromium is heavy metal in nature and an internationally recognized respiratory toxin and carcinogen.With the rapid development of industry and agriculture,the content of chromium in the air,soil,and water quality exceeds the standard,and it accumulates in crops and water,posing a serious threat to the safety of animal husbandry and animal-derived food.Chromium often exists in the form of compounds,among which hexavalent chromium is easily soluble in water,is more common in groundwater and surface water,and is more damaging to organisms.When hexavalent chromium enters the body of livestock and poultry,it can induce the body to produce a large amount of active oxygen,and promote the occurrence of oxidative stress,and further endanger health.Melatonin（MEL）is the main product of the pineal gland and is an endogenous antioxidant.MEL stimulates the synthesis of many antioxidant enzymes,including superoxide dismutase（SOD）and glutathione peroxidase,thereby scavenging free radicals and inhibiting lipid peroxidation and DNA damage.Endogenously synthesized or exogenously added MEL can regulate different transcription factors,and treat stress-related diseases by regulating the receptor proteins of these transcription factors.MEL has been shown to protect the lungs from the toxicity caused by nitrogen mustard gas,but it is not clear whether it has a similar protective effect on lung damage caused by chromium.This study aims to explore whether MEL has a therapeutic effect on potassium dichromate（K₂Cr₂O₇）-induced lung injury and its mechanism of action.Our study is divided into in vivo tests and in vitro tests.In vivo test:（1）Establish a model of lung injury caused by K₂Cr₂O₇.Twenty-eight male Wistar rats aged 6-8 weeks were selected and randomly divided into four groups（n=7）,Control group,K₂Cr₂O₇ low,medium,and high-dose groups,2,4,and 6 mg/kg K₂Cr₂O₇ were intraperitoneally injected respectively.After 35 days of continuous administration,blood and tissue samples of the rats were collected for routine blood examination and oxidative stress index detection.Hematoxylin-eosin staining method was used to observe the lungs tissue pathological changes.（2）Conduct MEL intervention K₂Cr₂O₇-induced lung toxicity test.Twenty-eight rats were randomly divided into four groups（n=7）,Control group,K₂Cr₂O₇ group,K₂Cr₂O₇+MEL group,and MEL group.Rats in the Control and MEL groups were intraperitoneally injected with normal saline,rats in the K₂Cr₂O₇ and K₂Cr₂O₇+MEL groups were intraperitoneally injected with 4 mg/kg K₂Cr₂O₇.One hour later,rats in the Control and K₂Cr₂O₇groups were injected subcutaneously with normal saline,in the MEL group and K₂Cr₂O₇+MEL group were injected subcutaneously with 20 mg/kg of MEL.After 35 days of continuous administration,blood and tissue samples of rats were collected for routine blood examination,oxidative stress index detection,and myeloperoxidase（MPO）activity detection.Hematoxylin-eosin staining method was used to observe the lungs tissue pathological changes.Terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling was used to detect the rate of lung cell apoptosis.In addition,Western blot and Quantitative real-time PCR（q PCR）were used to detect the related pathways of silent information regulator 1（Sirt1）,peroxisome proliferator-activated receptor-γcoactivator-1α（Pgc-1α）,and nuclear factor erythroid 2-related factor 2（Nrf2）and other related factors.In vitro test:MEL intervention K₂Cr₂O₇ induced MLE-12 cytotoxicity test.The mouse alveolar type II epithelial cell line（MLE-12）was used and divided into four groups,Control group,K₂Cr₂O₇ group,K₂Cr₂O₇+MEL group,and MEL group.The survival rate,migration rate,and mitochondrial membrane potential of MLE-12 cells were measured.Simultaneously,EX527,a specific inhibitor of Sirt1,was used to verify the level of reactive oxygen species（ROS）and the changes in related signaling pathway proteins.In vivo test results:（1）K₂Cr₂O₇-induced lung injury model test,compared with the control group,the number of white blood cell in the K₂Cr₂O₇ group gradually increased（p<0.05）,and the number of red blood cell and hemoglobin content gradually decreased（p<0.05）,the oxidative stress index malondialdehyde content increased significantly,SOD activity and glutathione content decreased significantly,obvious lesions were observed in the lung tissues of the K₂Cr₂O₇ group,indicating that the toxic effect of K₂Cr₂O₇ on rats gradually increases.（2）In the MEL intervention K₂Cr₂O₇-induced lung toxicity test,we found that the hematological changes,oxidative stress index changes and pathological changes in the K₂Cr₂O₇ group were the same as the results of（1）.In addition,compared with the control group,the MPO activity of the K₂Cr₂O₇ group was significantly increased,and the apoptosis rate of lung cells was significantly increased.However,in the K₂Cr₂O₇+MEL group,these changes were significantly alleviated.Based on this,it is determined that MEL can reduce the lung injury induced by K₂Cr₂O₇.Western blot and q PCR detection of Sirt1/Pgc-1α/Nrf2 pathway-related protein and gene levels also revealed that MEL reduced K₂Cr₂O₇-induced lung oxidative damage.In vitro test results:MEL alleviated the decrease in MLE-12 cell survival rate and cell migration rate and the increase in mitochondrial membrane potential caused by K₂Cr₂O₇.In addition,MEL can not only activate the Sirt1/Pgc-1α/Nrf2 signaling pathway,but also reduce the increase in ROS levels caused by K₂Cr₂O₇.However,after treatment with the Sirt1 inhibitor EX527,MEL cannot protect MLE-12 cells.In summary,MEL can reduce lung damage caused by K₂Cr₂O₇ by activating the Sirt1/Pgc-1α/Nrf2 signaling pathway.MEL has a good therapeutic effect and potential application value for lung toxicity caused by K₂Cr₂O₇.