The Role Of Mitochondrial Reactive Oxygen Species Regulated By PARP1/Sirt1 In Paraquat-Induced Apoptosis In Lung Epithelial Cells

Background:Paraquat（1,1’-dimethyl-4,4’-bipyridine dichloride,PQ）is a widely used herbicide in the world.Although its water formulation has been banned in China,PQ remaining in the environment has many opportunities to enter the human body,which can cause damage.Due to its low price and effective herbicidal property,it is still illegally used in agriculture.Clinically,PQ poisoning cases are also common with poor prognosis by accidental or intentional administration of PQ.At present,the mechanism underlying PQ poisoning is still unclear,and there is no specific antidote.In addition,PQ is a typical free radical inducer and commonly used in medical research to establish oxidative stress-related damage models.Exploring new molecular targets and signaling pathways,and finding effective intervention by in-depth study of PQ poisoning mechanism not only have significance for clinical PQ treatment,but also provide insights into the study of the biological effects of reactive oxygen species（ROS）.Lung is the main target organ of PQ toxicity.PQ selectively accumulates in type II alveolar epithelial cells through the polyamine uptake system,which often leads to severe lung injury.At present,the mechanism of lung injury caused by PQ is unclear.Many studies have found that PQ-induced lung damage is associated with apoptosis,DNA damage,intracellular calcium overload,oxidative stress and mitochondrial damage.Mitochondria are involved in various processes such as intracellular substance metabolism,ATP production and apoptosis.In the process of ATP production,a small amount of electrons in mitochondrial electron transport chain inevitably"leak out"and react with oxygen to produce superoxide anion,namely mitochondrial reactive oxygen species（mtROS）.Although the source of PQ-induced ROS is controversial,it is generally considered to be derived from mtROS.It has been confirmed that PQ can cause damage to DNA structures by generating large amounts of ROS.Poly（ADP-ribose）polymerase（PARP）is an evolutionarily conserved ribozyme with multiple biological functions.PARPs are widely involved in DNA repair,transcriptional regulation,oxidative stress-mediated cell death,metabolism and immune regulation.PARP1 is the first member to be discovered and the most important subtype of the PARP family,and its main biochemical function is to modify proteins by poly-ADP ribosylation（PAR）to repair damaged DNA.Although PARP1-mediated PAR is involved in DNA repair,in some cases,PARP1 overactivation can mediate cell death.The issues have yet to be elucidated that whether PQ exposure leads to overactivation of PARP1 and increased PAR modification,or whether PARP1 activation is involved in PQ-induced oxidative stress and apoptosis.Then if pharmacological inhibition of PARP1 by specific inhibitor can protect against PQ-induced cell damage,what the mechanism is.Sirt1 is a NAD-dependent histone deacetylase in cells,which is involved in the regulation of multiple cellular responses,such as the regulation of intracellular redox homeostasis.Studies have suggested that PQ can down-regulate Sirt1,and inhibition of Sirt1can promote PQ-induced ROS production.Overexpression of Sirt1 can attenuate PQ toxicity by inhibiting oxidative stress.However,it is unclear which mechanism underlying PQ-mediated inhibition of Sirt1.Since PARP1 can suppress Sirt1,whether the inhibition of Sirt1 by PQ is related to PARP1 remains to be studied.Chlorogenic acid（CA）is an important dietary polyphenol with a variety of important pharmacological effects.Recent studies suggest that the mechanism by which CA plays pharmacological protective roles may be related to Sirt1 activation.Whether CA can attenuate PQ-induced apoptosis by increasing Sirt1 levels still needs further study.Based on the above studies,our scientific hypothesis is that in PQ-induced lung injury model,PQ exposure leads to severe DNA damage in lung epithelial cells,causing excessive activation of PARP1,which in turn inhibits Sirt1 to induce mtROS production,mitochondrial dysfunction,oxidative stress and apoptosis,eventually leading to the development of lung injury.Objective:1.To determine the dose-effect relationship of PQ-induced mitochondrial dysfunction,oxidative stress and apoptosis in lung epithelial cells.2.To study the role and mechanism of PARP1 in PQ-induced apoptosis in A549 cells.3.To investigate the role of Sirt1 in PARP1-mediated apoptosis in PQ-exposed A549cells.4.To clarify the role of mtROS in the regulation of PQ-induced apoptosis by the PARP1/Sirt1 pathway.5.To investigate the protective effect of CA（potential agonist of Sirt1）on PQ-induced A549 cell apoptosis and its mechanism.Methods:1.The establishment of cell model of PQ-induced cell damage in vitro:Human lung type II epithelial cell line A549 was selected to conduct experiments.After treatment with PQ at concentrations of 0,20,40,80,160 and 320μM,cell viability was measured by PrestoBlue^?reagent.Cell morphology was observed under light microscope.Tunel staining and AnnexinV/PI method were used to assess apoptosis.Intracellular Ca²⁺level was measured by Fluo4 probe.Intracellular O2^-·,H2O2 and mtROS levels were detected by DHE,DCFH-DA and MitoSOX,respectively.Mitochondrial membrane potential was measured by JC-1 probes to assess mitochondrial function.A representative cell model of PQ-induced cell damage was determined based on the above results.2.The role of PARP1 in PQ-induced apoptosis:PARP1 inhibitor AG14361（AG）was employed as a treatment method for PQ-induced cell injury and apoptosis.The potentially protective dose of AG was screened by cell viability assay.The function of PARP1 was evaluated by western blot analysis of PAR modification.The effect of AG on PQ-induced apoptosis was evaluated by measuring apoptosis,and the intracellular Ca²⁺level was detected to assess the effect of AG on calcium overload induced by PQ.The levels of O2^-·,H2O2 and mtROS were examined to evaluate the effect of AG on oxidative stress induced by PQ.Mitochondrial membrane potential was measured by rhodamine 123 and JC-1 probes to assess mitochondrial function,and the submicroscopic structure of mitochondria was observed by transmission electron microscopy to evaluate the damage of mitochondrial structure.In addition,in order to study post-treatment effect of AG,after cells were treated with PQ for 12 h,AG was then added for another 12 h,and the above indicators were assessed.3.The role of Sirt1 in PARP1-mediated apoptosis in PQ-exposed A549 cells:Negative control cells（ShNC）and Sirt1-silenced cells（ShSirt1）were constructed by lentiviral infection,respectively.The silencing efficiency of Sirt1 was verified by western blot.To study the role of Sirt1 in PQ-induced cell damage and apoptosis,after treatment with PQ for24 h in ShNC and ShSirt1 cells,apoptosis and mtROS content were examined.Sirt1 level was detected after treatment with AG in PQ-exposed cells.In order to investigate the role of Sirt1 in the protective effect of AG in PQ-induced damage and apoptosis,some parameters such as cell viability,apoptosis,intracellular Ca²⁺content,H2O2 and mtROS levels and mitochondrial membrane potential were measured after AG treatment in PQ-exposed ShNC and ShSirt1 cells.4.The role and regulation mechanism of mtROS in PQ-induced apoptosis:The mitochondria-targeted antioxidant MitoTEMPO was used as an intervention for PQ-induced cell damage and apoptosis,and the potentially protective concentration of MitoTEMPO was screened by cell viability assay.Apoptosis was measured to evaluate the effect of MitoTEMPO on PQ-induced apoptosis.The intracellular Ca²⁺level was detected to assess the effect of MitoTEMPO on PQ-induced calcium overload.The levels of intracellular O2^-·,H2O2 and mtROS were examined respectively to determine the effect of MitoTEMPO on oxidative stress induced by PQ.Mitochondrial membrane potential was detected to assess mitochondrial function,and the submicroscopic structure of mitochondria was observed by transmission electron microscopy to assess the damage of mitochondrial structure.In addition,in order to study the post-treatment effect of MitoTEMPO,cells were treated with PQ for 12 h and then MitoTEMPO was added in the presence of PQ.Another 12 h later,the changes of the above indicators were assessed.In order to further study the mechanism underlying regulation of mtROS in PQ-induced apoptosis,PQ-exposed ShNC and ShSirt1cells were treated with AG and/or MitoTEMPO,respectively.Apoptosis and intracellular Ca²⁺content were measured.The levels of intracellular O2^-·,H2O2 and mtROS were detected,and mitochondrial membrane potential was examined.5.Protective effect of CA（a potential agonist of Sirt1）on PQ-induced apoptosis and its mechanism:The maximum no-damage dose of CA was screened by cell viability assay.After pretreatment with CA for 24 h,cells were exposed to PQ,and the protective effect of CA was determined by cell viability assay.Apoptosis and intracellular Ca²⁺content were measured.Intracellular O2^-·and mtROS levels was assessed.GSH content was detected by mBCl probe.The submicroscopic structure of mitochondria was observed by transmission electron microscope.The number of mitochondria was evaluated by MitoTrackerRed staining,and the intracellular ATP level was determined by CellTiter-Glo^?kit.The protein contents of cytochrome c,Bax,cleaved Caspase 9 and 3,Nrf2,SOD1,SOD2 and Sirt1 were assessed by western blot.In order to determine the effect of CA on Sirt1 content,cells were treated with CA for 0,6,12 and 24 h,and then Sirt1 level was evaluated by western blot.In addition,in order to elucidate the function of Sirt1,ShNC and ShSirt1 cells were pretreated with CA for 24 h and then exposed to PQ to detect the changes of apoptosis and ATP level.Results:1.A cell model of PQ exposure was successfully established.PQ caused cell damage with dose-effect relationship.PQ of≥40μM decreased cell viability and increased intracellular O₂^-·,H₂O₂ and mtROS levels;PQ of≥80μM caused apoptosis and reduced mitochondrial membrane potential;Cell damage emerged under light microscope after PQ of≥160μM,and PQ of≥160μM also made Tunel staining positive and induced intracellular Ca²⁺overload.Based on the above results,160μM PQ for 24 h was representative for establishing PQ-induced cell damage model.2.PARP1 plays a key role in PQ-induced apoptosis.Inhibition of PARP1 by AG attenuated PQ-induced cell viability damage and PAR modification,and decreased Tunel-positive cells and apoptosis,as well as mitigated PQ-induced intracellular Ca²⁺overload and mtROS production,and improved PQ-resulted damage to mitochondrial function and structure.In addition,post-treatment with AG also improved the above indicators to a certain extent.However,AG had no effect on PQ-induced intracellular O2^-·production,and further increased intracellular H2O2 level induced by PQ.3.Sirt1 plays an important role in PARP1-mediated apoptosis in PQ-exposed cells.In this study,we found that PQ down-regulated Sirt1 expression.Silencing Sirt1 increased the sensitivity of PQ to apoptosis and the content of mtROS induced by PQ.After silencing Sirt1,the protective effects on cell viability and apoptosis by AG disappeared;the inhibitory effect on PQ-induced Ca²⁺overload decreased;both promotion of H2O2 level and suppression of mtROS content were abolished.Finally,the protective effect of AG on PQ-resulted mitochondrial damage was reversed in Sirt1 silencing cells.4.MtROS plays a key role in PQ-induced apoptosis.MitoTEMPO inhibited PQ-induced cell viability reduction,apoptosis,intracellular Ca²⁺overload,as well as the production of O2^-·,H2O2 or mtROS,mitochondrial membrane potential depolarization and damage of mitochondrial structure.Additionally,post-treatment with MitoTEMPO also improved above most indicators to a certain extent.In Sirt1-silenced cells,PQ-induced apoptosis,calcium overload,and the induction of intracelluar O2^-·,H2O2 or mtROS were all alleviated by MitoTEMPO,and mitochondrial membrane potential was protected against PQ insults.5.CA has a significant protective effect on PQ-induced apoptosis by activating Sirt1.CA increased Sirt1 level,and inhibited the decrease of Sirt1 level induced by PQ.In PQ-exposed cells,CA increased the expressions of antioxidant-related proteins,enhanced GSH content,decreased the levels of O2^-·and mtROS,mitigated PQ-induced reduction of mitochondrial number and mitochondrial structure damage,improved mitochondrial function,inhibited Ca²⁺overload,and reduced the levels of apoptosis-related proteins,finally exerting a protective effect against PQ-induced apoptosis.After Sirt1 was silenced,CA didn’t inhibit PQ-induced mitochondrial dysfunction and cell apoptosis.Conclusions:1.PQ induced oxidative stress,mitochondrial damage and apoptosis with a dose-effect relationship in lung epithelial cells.2.PQ induced PARP1 overactivation and PAR modification,and PARP1 was involved in PQ-induced apoptosis.3.PQ down-regulated Sirt1 level,and silencing Sirt1 promoted apoptosis in PQ-exposed cells.Sirt1 may be an important downstream molecule of PARP1 which mediated PQ-induced apoptosis.4.Overproduction of mtROS is the main cause of oxidative stress and apoptosis induced by PQ.PARP1 and Sirt1 play key roles in positive and negative regulation of mtROS production,respectively.5.CA may protect mitochondrial function and reduce oxidative stress by increasing Sirt1 level,and ultimately inhibit PQ-induced apoptosis.6.PARP1/Sirt1/mtROS pathway was found to be key molecular target in PQ poisoning.PARP1 inhibitor AG,Sirt1 agonist CA and mtROS scavenger MitoTEMPO could be potential therapeutic drugs for PQ poisoning.