Study On The Mechanism Of COPD Induced By PM_2.5 Based On The ROS-NEAT1-PINK1 Signaling Pathway

BackgroundChronic Obstructive Pulmonary Disease(COPD)is a common chronic respiratory disease with high morbidity and mortality,which has become a global public health problem.Except for cigarette smoke(CS),fine particulate matter(PM2.5)is the second major risk factor for COPD.Recent studies have shown that PM2.5 can induce toxic effects by inflammation infiltration,oxidative stress,cell apoptosis,autophagy and so on,but what role of PM2.5-induced mitophagy disorder in the occurrence and development of COPD has not been fully elucidated.In recent years,our country pays more attention to prevention,control and treatment of COPD.There are some important research programs to do,such as how to decrease the impact of environmental risk factors in the occurrence and development of COPD,how to early diagnosis and treatment of COPD and how to improve patient prognosis and life quality.Therefore,there are positive clinical and public health value to find a new,efficient and specific molecular target for the prevention and control of COPD.More studies have shown that long non-coding RNAs(lncRNA)play important biological roles in the occurrence and development of chronic pulmonary diseases.However,the application value of lncRNA in the clinical diagnosis,treatment,and prognosis of COPD has not yet been clear,which needs to be clarified through evidencebased medicine.Environmental risk factors may cause differential expression of lncRNA,and affect the occurrence and development of COPD.LncRNA NEAT1 plays a vital role in cancer,neurodegenerative diseases and COPD.Studies have shown that NEA T1 is upregulated in COPD patients,but the underlay regulation mechanism of NEAT1 in the occurrence and development of COPD has not been fully studied.To sum up,both in vivo and in vitro,this study try to research the epigenetic regulation and underlay mechanism of NEAT1 in the occurrence and development of COPD,which will help clarify the pathophysiological mechanism of COPD and provide new research direction and ideas for the diagnosis,treatment and prognosis of COPD.Object1.To study potential value of lncRNA in the early diagnosis of COPD.2.To observe biological toxicity of PM2.5 and CS by building COPD animal model,and clarify the expression of NEAT1 and detect change of PINK 1/Parkin-mediated mitophagy.3.To research biological toxicity in lung epithelial cells exposed by PM2.5 and CS and observe the expression of reactive oxygen species(ROS),NEAT1 expression and PINK 1/Parkin-mediated mitophagy.4.To explore the underlay mechanism of ROS,NEAT1 and PINK 1/Parkin-mediated mitophagy in lung epithelial cell exposed by PM2.5 and CS.Methods1.Pubmed,Embase,Cochrane Central Register of Controlled Trials(CENTRAL),Chinese biomedical literature database(CBM),CNKI,WangFang Data,CQVIP were searched for all researches of lncRNA for diagnosis of COPD,with the last articles up to Jan.2021.According to the inclusion and exclusion criteria,literature screening and data extraction are performed,QUADAS-2 checklist was used for quality evaluation,and diagnostic Meta-analysis performed by R language(version 4.0).The primary outcomes were sensitivity(SEN),specificity(SPE)and diagnostic odds ratio(DOR),then drawed Summary receiver operating characteristic(SROC),calculated the area under curve(AUC),performed subgroup analysis and Metaregression,,analyzed sensitivity and publication bias.2.Wistar rats(male,6-8 w,180±20 g)were selected for establishing COPD animal models through PM2.5 and CS chronic exposure,and divided them into PM2.5 exposure group(PMM),CS exposure group(CSM)and control group(Ctrl).After 90 days’ exposure,arterial blood gas test,pulmonary function,inflammation cytokines and anti-oxidative stress indicators in serum and bronchoalveolar lavage fluid(BALF),HE staining,immunohistochemistry(IHC)analysis,TUNEL observation and transmission electron microscope(TEM)observation would been executed.Furthermore,high-throughput sequencing of lncRNAs in the 3 groups of animal models was performed.LncRNA NEAT1 and lncRNAs selected for mitochondrial dysfunction and inflammatory response were validated by qRT-PCR,and the expression of proteins related to mitophagy,such as LC3B,p62,PINK1,Parkin,Mfn2 were detected by Western Blotting(WB).3.Two lung epithelial cells(16HBE,A549)were used to study biological toxicity of PM2.5 and CS.Firstly,the concentration of PM2.5 suspension(PMS)and cigarette smoke extract(CSE)and exposure time were confirmed by CCK8 assay.After PMS and CSE exposure,cell apoptosis and inflammatory cytokines were detected by flow cytometry,then ROS was observed under fluorescence microscopy and analyzed by flow cytometry,anti-oxidative stress indicators such as GSH,T-AOC,CAT and SOD also were detected.Changes of mitochondrial membrane potential(MMP)was tested by JC-1 assay and MitoTracker and LysoTracker double staining was observed under fluorescence microscopy,NEAT1 expression was detected by qRTPCR and the expression of proteins including PINK1,Parkin,LC3B,p62 and Mfn2 detected by WB.4.Firstly,the cells pretreated with NAC and MitoQ and exposed by PMS and CSE.Secondly,the cells were executed by SiRNA NEAT1 transfection and were exposed by PMS and CSE.Thirdly,cell apoptosis,inflammatory cytokines,ROS,oxidative stress indicators,MMP and mitophagy,NEAT1 expression,the expression of proteins related to mitophagy,which are LC3B,p62,PINK1,Parkin and Mfn2,were performed.the mechanism of ROS-NEAT1-PINK 1 signaling pathway was demonstrated in lung epithelial cells exposed by PMS and CSE.Results1.7 articles were included,and 8 lncRNAs were found for diagnosis of COPD,of which 6 lncRNAs were up-regulated(NEAT1,NR-026690,ENST00000447867,PACER,MALAT1,PVT1)and 2 lncRNAs were down-regulated(ANRIL,lnc-IL7R).The results of Meta-analysis showed that SEN was 0.73(95%CI[0.68,0.77]),SPE was 0.71(95%CI[0.38,0.91])and DOR was 5.605(95%)CI[1.815,17.309]in COPD vs.healthy controls(NC).Comparison of AECOPD and NC,it showed that SEN was 0.93(95%CI[0.86,0.97]),SPE was 0.72(95%CI[0.54,0.85])and DOR was 35.028(95%CI[12.404,98.914]).Comparison of AECOPD and COPD,it showed that SEN was 0.72(95%CI[0.55,0.85]),SPE was 0.78(95%CI[0.65,0.88])and DOR was 9.214(95%CI[6.754,12.569]).The results of Subgroup analysis and Meta-regression showed that research results were correlated with the specimens and detection methods(P<0.05).Sensitivity analysis indicated that there was no substantial change to the results of Meta-analysis whether any study was excluded.Publication bias of selected studies about AECOPD vs.NC and AECOPD vs.COPD were small,but COPD vs.NC was big.2.The COPD animal model was successfully constructed by chronic PM2.5 and CS exposure.Compared with the Ctrl group,the body weight of the PMM and CSM group decreased significantly(P<0.05).The results of arterial blood gas test shown that value of pH,blood oxygen saturation and oxygen partial pressure in the PMM and CSM group were lower than that of the Ctrl group,while the carbon dioxide partial pressure increased(P<0.05).Pulmonary function of the CSM and PMM group were reduced compared with the Ctrl group,The levels of IL6,Il8,and TNF-a in serum and BALF of the PMM and CSM group rose compared with the Ctrl group(P<0.05),anti-oxidative stress indicators including GSH,T-AOC,and CAT decreased in the PMM and CSM group(P<0.05),HE staining showed emphysemalike forms in the lung tissues of the PMM and CSM group,IHC analysis showed the expression of MUC5AC,PINK1 and cell counts of LC3B increased(P<0.05)and Parkin expression decreased(P<0.05).TUNEL staining showed increased cell apoptosis(P<0.05).TEM showed that mitochondria damages and autophagosomes in the PMM and CSM group.NEAT1 expression in the PMM and CSM were higher than the Ctrl group(P<0.05).Compared with the Ctrl group,the ratio of LC3 Ⅱ/Ⅰ,PINK1 expression increased(P<0.05)and Parkin and Mfn2 expression decressed(P<0.05)in the PMM and CSM group.3.High-throughput sequencing of lung tissue of 3 rats model indicated there were 727 differential expressed lncRNAs and 2508 dysregulated mRNAs in the comparison of PMM,CSM and Ctrl group.We identified 123 and 444 lncRNAs were significantly up-regulated and down-regulated in PMM versus Ctrl,while 621 and 1178 mRNAs were up-regulated and down-regulated respectively.Meanwhile,81 and 340 lncRNAs were consistently up-regulated and down-regulated respectively in CSM versus Ctrl,while 408 and 931 mRNAs were up-regulated and down-regulated.GO and KEGG analysis suggested that COPD was related with immune responds,cell Metabolism,mitochondrial dysfunction and so on.Then 7 dysregulated lncRNAs related to inflammatory response and mitochandrial dysfunction to validated by qRTPCR.4.Two Lung epithelial cells(16HBE,A549)were exposed with 10%CSE and 200μg/mL PMS for 24 h.Compared with the control group(CON),the cells showed obvious cell apoptosis(P<0.05),IL6 and IL8 in cell culture medium increased(P<0.05);ROS generation increased(P<0.05),GSH and AOC decreased(P<0.05),MMP reduced(P<0.05)and mitophagy disorders;increased expression of NEAT1(P<0.05);ratio of LC3 Ⅱ/Ⅰ and PINK1 expression up-regulated(P<0.05).When the cells pretreated by 5 mM NAC,4 μM MitoQ,Si-NEAT1 transfection were exposed by PMS and CSE,compared with 10%CSE and 200 μg/mL PMS exposure directly,cell apoptosis relatively reduced(P<0.05),inflammatory cytokines relatively decreased(P<0.05),the production of ROS relatively reduced(P<0.05),the reduction of MMP lessened(P<0.05),the expression of NEAT1 relatively decreased(P<0.05),and the ratio of LC3 Ⅱ/Ⅰ,the expression of PINK1 relatively decreased(P<0.05).Conclusions1.LncRNA can be used as an auxiliary biomarker for the diagnosis of COPD.2.PM2.5,CS can cause COPD-like changes such as emphysema,inflammation infiltration,oxidative stress disorder,and up-regulation expression of NEAT1,PINK 1/Parkin-mediated mitophagy disorders in vivo.3.PM2.5,CS can cause differences in the expression of lncRNA in lung tissues of animal models,and may play an regulatory role in the processes of cell differentiation,immune metabolism,and mitophagy.4.Both 200 μg/mL PMS and 10%CSE exposure could decrease the cell validity,increase cell apoptosis,increase production of ROS,increase mitochondrial depolarization,up-regulated NEAT1 and PINK1/Parkin-mediated mitophagy disorder in lung epithelial cells(16HBE,A549).5.5mM NAC and 4μM MitoQ pretreated can reduce the toxicity of lung epithelial cells exposed by 200 μg/mL PMS and 10%CSE,decreased ROS generation,lessen mitochondrial depolarization,reduce NEAT1 and PINK1 expression,and lessen PINK 1/Parkin-mediated mitophagy disorder.6.Knockdown of NEAT1 can reduce the toxicity of lung epithelial cells exposed by 200μg/mL PMS and 10%CSE,decreased ROS generation,lessen mitochondrial depolarization,reduce NEAT1 and PINK1 expression,and lessen PINK1/Parkinmediated mitophagy disorder.7.ROS-NEAT1-PINK1 signaling pathway plays an important role in mitophagy disorder induced by PMS and CSE.