MnTMPyP Inhibits Paraquat Induced Pulmonary Epithelial-like Cell Injury By Inhibiting Oxidative Stress

1 IntroductionParaquat?PQ;1,1?-dimethyl-4-4?-bipyridinium dichloride?is a strong herbicide that has been widely used in the agricultural field since 1960.However,PQ results in high toxicity after being absorbed via gastrointestinal ingestion,inhalation,or skin contact in both animals and humans.Due to a lack of antidotes,the mortality rate attribute to PQ poisoning remains extremely high.Accidental or intentional ingestion of PQ has led to thousands of deaths in Asia,with a mortality rate of 60-70%.The lung is the primary target organ of PQ poisoning.PQ can cause pulmonary edema,hemorrhage and exudation,as well as inflammatory cell infiltration of lung interstitial and alveoli,fibroblast proliferation,and excessive deposition of collagen,all of which results in lung and bronchi epithelial cell damage and subsequent pulmonary fibrosis.The typical clinical manifestation of PQ poisoning is termed"paraquat lung".Early manifestation is acute respiratory distress syndrome,while irreversible pulmonary interstitial fibrosis manifests at later stages,at which point a majority of patients suffer respiratory failure and death.The exact mechanism by which PQ poisoning causes lung injury remains unclear.However,many studies have confirmed that PQ-induced redox reactions produce large amounts of oxygen free radicals that cause lung injury.Reactive oxygen species?ROS?oxidize unsaturated fatty acids on various biofilms to trigger endoplasmic reticulum?ER?stress and apoptosis cascades,which exacerbate the lung injury.Currently,there is no available effective antidote for PQ poisoning,although many studies have focused on reducing ROS production,eliminating generated ROS,and reducing inflammatory responses against PQ-induced lung toxicity However,these treatments have been disappointing and mortality still remains high.Thus,there is an urgent need to develop new effective treatments.Studies have confirmed that PQ-induced oxidative stress and increased intracellular mitochondrial permeability lead to manganese-superoxide dismutase?Mn-SOD?inactivation or decreased activity.MnTMPyP,a mimic of superoxide dismutase,can penetrate cells and effectively scavenge superoxide ions.MnTMPyP can inhibit the production of free radicals and dopaminergic neurodegeneration induced by lipopolysaccharide.MnTMPyP inhibited the activation of Caspase-3 by reducing the expression of pro-apoptotic protein Bax by scavenging free radicals in renal ischemia-reperfusion injury model.This study was designed to investigate whether MnTMPyP could alleviate the injury of lung epithelial cells induced by PQ.2 Materials and Methods2.1 MaterialsCells lines:human lung epithelial-like cell A549.2.2 Experimental grouping2.2.1 Establish PQ-induced apoptosis of A549 cell model and detect related indicators of mitochondrial apoptosis pathway.Experimental grouping:control group?equal amount of culture medium?,MnTMPyP 10?M group,PQ 750?M group,PQ+MnTMPyP group.After 24h treatment,related indicators were detected.2.2.2 MnTMPyP inhibits PQ induced apoptosis in A549 cells by inhibiting mitochondrial interaction pathway.Experimental grouping:control group?equal amount of culture medium?,MnTMPyP 10?M group,PQ 750?M group,PQ+MnTMPyP group.After 24h treatment,related indicators were detected.2.2.3 MnTMPyP attenuated PQ induced apoptosis of A549 cells by inhibiting endoplasmic reticulum stress pathwayExperimental grouping:control group?equal amount of culture medium?,MnTMPyP 10?M group,PQ 750?M group,PQ+MnTMPyP group.After 24h treatment,related indicators were detected.2.3 Methods2.3.1 Establishment of PQ-induced A549 cells apoptosis model and detection of related indicators of mitochondrial apoptosis pathway.?1?A549 cells were cultured and treated with PQ 750?M.?2?Pretreatment of A549 cells with MnTMPyP 10?M.?3?MTT assay was used to detect the activity of A549 cells.?4?The fluorescence intensity of mitochondrial membrane potential of A549 cells was observed under fluorescence microscope after staining with rhodamine Rh123,and the fluorescence intensity of mitochondrial membrane was detected by flow cytometry.?5?Annexin V was used to detect apoptosis rate.?6?Caspase activity assay kit was used to detect Caspase-3 activity in A549 cells.?7?After DCFH-DA staining,the fluorescence intensity of reactive oxygen species?ROS?in A549 cells was observed under fluorescence microscope,and the level of ROS in A549 cells was detected by flow cytometry.?8?The Ca2+level in cells was detected by Fluo-3/AM.?9?Detection of Glutathione reductase activity in cells.?10?The expression of Bcl-2,Bax,Grp78,CHOP and beta-actin protein were detected by Western blot.2.3.2 MnTMPyP attenuated the apoptosis of PQ induced A549 cells by inhibiting mitochondrial apoptosis pathway.?1?Mitochondrial membrane potential,apoptosis rate,intracellular reactive oxygen species?ROS?and caspase-3 activity were detected after pretreatment with MnTMPyP.?2?After MnTMPyP pretreatment,Western blot was used to detect the changes of Bax,Bcl-2 and beta-actin protein expression.2.3.4 MnTMPyP attenuated PQ induced apoptosis in A549 cells by inhibiting endoplasmic reticulum stress pathway?1?After MnTMPyP pretreatment,Reactive oxygen species,Ca²⁺level and glutathione reductase activity were detected.?2?After MnTMPyP pretreatment,Western blot was used to detect the changes of Grp78,CHOP and beta-actin protein expression.3 Results3.1 Establish the model of PQ-induced A549 cell apoptosis and detection of related indicators of mitochondrial apoptosis pathway.?1?PQ has obvious cytotoxicity to A549 cells and significantly reduces cell activity.?2?After MnTMPyP pretreatment,the cell viability of A549 cells increased significantly compared with that of PQ group.?3?The fluorescence intensity of mitochondrial membrane potential of A549 cells was observed under fluorescence microscope after staining with rhodamine Rh123,and the fluorescence intensity of mitochondrial membrane potential of A549 cells was detected by flow cytometry.The results showed that the fluorescence intensity of mitochondrial membrane potential of PQ group was significantly lower than that of control group.?4?Apoptosis rate was detected by Annexin V,and the apoptotic rate in PQ group was significantly higher than that in control group.?5?Caspase-3 activity of A549 cells was detected by Caspase activity assay kit.The results showed that the activity of Caspase-3 in PQ group was significantly higher than that in control group.?6?After DCFH-DA staining,the fluorescence intensity of reactive oxygen species?ROS?in A549 cells was observed under fluorescence microscope,and the ROS level in A549 cells also detected with flow cytometry.The results showed that the ROS level in PQ group was significantly higher than that in control group.?7?Fluo-3/AM method was used to detect the intracellular Ca²⁺level.The results showed that the intracellular Ca²⁺level in PQ group was significantly higher than that in control group.?8?Intracellular glutathione reductase activity test showed that the activity of glutathione reductase in PQ group was significantly lower than that in control group.?9?Western blot results showed that the expression of Bcl-2 protein in PQ group was significantly lower than that in control group,and the expression of Bax,Grp78 and CHOP protein was significantly increased.3.2 MnTMPyP attenuated the apoptosis of PQ induced A549 cells by inhibiting mitochondrial apoptosis pathway.?1?Pretreatment with MnTMPyP significantly decreased the apoptosis rate induced by PQ and the damage of mitochondrial membrane potential,decreased the production of reactive oxygen species and inhibited the activity of Caspase-3.?2?After pretreatment with MnTMPyP,Western blot analysis showed that the expression of Bcl-2 protein increased significantly and the expression of BAX protein decreased significantly.3.3 MnTMPyP attenuated the apoptosis of PQ induced A549 cells by inhibiting endoplasmic reticulum stress pathway.?1?Pretreatment with MnTMPyP significantly decreased the intracellular Ca²⁺level induced by PQ,and the intracellular glutathione activity was detected.?2?After pretreatment with MnTMPyP,Western blot analysis showed that the expression of Grp78 and CHOP protein was significantly lower than that of PQ group.4 Conclusions?1?PQ induces A549 cell apoptosis by inducing mitochondrial apoptosis pathway.?2?PQ induces apoptosis of A549 cells by inducing endoplasmic reticulum stress pathway.?3?MnTMPyP attenuated the apoptosis of A549 cells by inhibiting mitochondrial apoptosis pathway.?4?MnTMPyP attenuated the apoptosis of A549 cells by inhibiting endoplasmic reticulum stress pathway.