P53 Inhibitor Pifithrin-? Attenuates Paraquat-induced Apoptosis In Human Lung Epithelial-like Cells A549 Through Mitochondrial Apoptosis Pathway And Mitophagy Pathway

1 IntroductionParaquat(PQ;1,1?-dimethyl-4,4?-bipyridinium dichloride)is a non-selective and contact herbicide.The number of PQ poisoning cases involving PQ suicide or accidental exposure to PQ has increased in Asia,especially in China.A lot of studies have investigated PQ poisoning in vivo or in vitro,although the molecular mechanism underlying the pathogenesis of PQ-induced lung injury is not fully understood.Therefore,determining the distinct molecular mechanism(s)underlying PQ-induced lung alveolar epithelial cell damage could provide useful information and advances in our ability to control unwanted side effects following exposure to PQ.PQ can induce apoptosis in vivo or in vitro.The goal and function of apoptosis is to remove damaged or abnormal cells and maintain homeostasis.However,abnormal apoptosis is involved in many diseases.The increase of apoptosis of alveolar epithelial cells in the early stage of PQ poisoning is the key event of subsequent lung injury and interstitial fibrosis.It has previously been demonstrated that PQ attacks chloroplasts in green plants to interfere with the vital process of photosynthesis.Meanwhile,in mammals,PQ targets mitochondria,the organelles that produce ATP and regulate cell fate by coordinating cell survival and cell death-related signaling pathways.Research demonstrated that the toxic effects of PQ on the cells are achieved by damaging the mitochondria.Mitochondrial dysfunction can directly induce apoptosis.Damaged mitochondria are removed via a process known as mitophagy.Mitophagy involves hydrolytic degradation of mitochondria by the lysosome.The timely removal of damaged mitochondria contributes to the recovery of cellular homeostasis and avoids further cells necrosis or apoptosis.P53 play important roles in the intrinsic signal transduction pathway of apoptosis.Inresponse to toxic stress,the expression of P53 increase in cytoplasm and P53 regulates the expression of Bcl-2 family proteins via transcription-dependent pathways and it also acts directly on mitochondria-mediated cell apoptosis.P53 has been reported that inhabit Parkin-mediated mitophagy and disturb the clearance of damaged mitochondria by interaction with mitophagy pathway protein Parkin.In this study,human lung epithelial-like A549 cells were selected as an in vitro model to study the PQ-induced lung injury and to observe whether the apoptotic process involves mitochondrial apoptotic pathway and mitophagy pathway.Further we explore whether PINK1/Parkin-mediated mitophagy play a protective role and whether P53 inhibitor pifithrin-?(PFT-?)can attenuate the activity of mitochondrial apoptotic signaling pathway and promote PINK1/Parkin pathway,ameliorate mitochondrial dysfunction and decrease apoptosis.2 Materials and Methods2.1 MaterialsCells lines: human lung epithelial-like cells A549.2.2 Experimental grouping2.2.1 Establishment of PQ-induced A549 cells apoptosis model and detection of related indicators of mitochondrial apoptosis pathway.Experimental grouping: control group(equal PBS),PQ50?M group,PQ100?M group,PQ150?M group,PQ200?M group.After the treatment of 24 h and 48 h,related indicators were detected.2.2.2 PFT-? attenuated PQ induced apoptosis in A549 cells through mitochondrial apoptosis pathway.Experimental grouping: control group(equal 0.1% DMSO),PFT-?(30?M)group,PQ(200?M)group,PFT-?(30?M)+PQ(200?M)group.After the treatment of 24 h,related indicators were detected.2.2.3 Detection of mitophagy pathway associated with PQ-induced apoptosis of A549 cells.Experimental grouping: control group(equal PBS),PQ50?M group,PQ100?M group,PQ150?M group,PQ200?M group.After the treatment of 24 h,related indicators were detected.2.2.4 The Role of PINK1 / Parkin pathway in PQ-induced apoptosis of A549 cells.Experimental grouping: control group(equal PBS),siRNA group(Parkin siRNA transfection),PQ(200?M)group,PQ(200?M)+siRNA(Parkin siRNA transfection)group.After the treatment of 24 h,related indicators were detected.2.2.5 PFT-? attenuated PQ induced apoptosis in A549 cells through mitophagy pathway.Experimental grouping: control group(equal 0.1% DMSO),PFT-?(30?M)group,PQ(200?M)group,PFT-?(30?M)+PQ(200?M)group.After the treatment of 24 h,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 different concentrations of PQ.(2)The cell survival rate was assessed by MTT method.(3)A549 cells were stained with Hoechst 33258 and observed by fluorescence microscopy.These cells were also subjected to an apoptosis detection kit to measure rates of apoptosis.(4)Rh123 fluorescent staining was performed to evaluate mitochondrial membrane potential.Both fluorescence microscopy and flow cytometry were performed to examine the intensity of fluorescence.(5)The activities of Caspase-3 and-9 were assayed by spectrophotometric method.(6)The protein expression of Bax,Bak,Bcl-2,Bcl-XL and ?-actin were detected by Western blot.2.3.2 PFT-? attenuated PQ induced apoptosis in A549 cells through mitochondrial apoptosis pathway.(1)The protein expression of P53 was detected by Western blot.(2)The A549 cells were preconditioned with PFT-?.Then apoptosis rate,mitochondrial transmembrane potential,Caspase-3 and Caspase-9 activation were detected.(3)The A549 cells were preconditioned with PFT-?.The protein expression of Bax,Bcl-2 and ?-actin were detected by Western blot.2.3.3 Detection of mitophagy pathway associated with PQ-induced apoptosis of A549 cells.(1)A549 cells were stained with MTG and LTR and investigated mitophagy by fluorescence microscopy.(2)The protein expression of PINK1,Parkin,P62,LC3 B,COXIV and ?-actin were detected by Western blot.2.3.4 The Role of PINK1 / Parkin pathway in PQ-induced apoptosis of A549 cells.Silenced the Parkin gene of the A549 cells with si RNA interference method and the expression of Parkin protein was detected by Western blot.The apoptosis rate and Caspase-3 /-9 activities were further examined.2.3.5 PFT-? attenuated PQ induced apoptosis in A549 cells through mitophagy pathway.The A549 cells were preconditioned with PFT-?.Then the protein expression of PINK1,Parkin,P62,LC3 B,COXIV and ?-actin were detected by Western blot.3 Results3.1 Establishment of PQ-induced A549 cells apoptosis model and detection of related indicators of mitochondrial apoptosis pathway.(1)Obvious cytotoxicity was observed for the A549 cells that were treated with PQ,and cell viability was reduced in a time-and concentration-dependent manner.(2)Condensation and lysis of nucleus and the formation of apoptotic bodies were observed after Hoechst 33258 staining.The rate of apoptosis increased after PQ treatment,and these were also in a time-and concentration-dependent manner.(3)The change of mitochondrial membrane potential was observed by fluorescence microscopy and flow cytometry.The results showed that the mitochondrial membrane potential was decreased in a time-and concentration-dependent manner.(4)After PQ treatment,the levels of active Caspase-3 and Caspase-9 increased in a time-and concentration-dependent manner.(5)The results of Western blot showed that the expression of Bax and Bak significantly increased after PQ treatment.The expressions of Bcl-2 and Bcl-XL were significantly decreased.3.2 PFT-? attenuated PQ induced apoptosis in A549 cells through mitochondrial apoptosis pathway.(1)The results of Western blot showed that the expression of P53 significantly increased after PQ treatment.(2)Pretreatment with PFT-? significantly decreased the apoptotic rate,attenuated the mitochondrial transmembrane potential disruption and inhabited the activities of both Caspase-3 and Caspase-9.(3)Pretreatment with PFT-? significantly decreased the ratio of relative protein level of Bax / Bcl-2.3.3 Detection of mitophagy pathway associated with PQ-induced apoptosis of A549 cells.(1)Co-staining with MTG and LTR in A549 cells and then by fluorescence microscopy revealed a significant enhancement of mitochondrial autophagy after PQ treatment.(2)The results of Western blot showed that the expression of PINK1,Total Parkin,Mito Parkin and LC3-II / LC3-I significantly increased after PQ treatment.The expressions of P62 were significantly decreased.3.4 The Role of PINK1 / Parkin pathway in PQ-induced apoptosis of A549 cells.Pretreatment of cells with Parkin-specific siRNA followed by PQ treatment resulted in a higher rate of apoptosis and a significant increase in Caspase-3 and Caspase-9activity compared with PQ treatment.3.5 PFT-? attenuated PQ induced apoptosis in A549 cells through mitophagy pathway.Pretreatment with PFT-? followed by PQ treatment A549 cells,mitophagy-related protein mito Parkin and LC3-? / LC3-?expression increased.4 Conclusions(1)PQ induced A549 cell apoptosis by activating mitochondrial apoptotic pathway.(2)Mitophagy was also activated during PQ-induced A549 cells apoptosis.(3)PINK1 / Parkin-mediated mitophagy plays a direct protective role in PQ-induced A549 cells damage.(4)P53 inhibitor PFT-? attenuated PQ induced apoptosis in A549 cells through mitochondrial apoptosis pathway and mitophagy pathway.