The Mitochondrial Apoptosis And Molecular Mechanism Of Fibrosis Induced By Coal-fired Air Pollutants

China is in the high levels of coal production and consumption.Sulfur dioxide?SO₂?,polycyclic aromatic hydrocarbons?PAHs?and fine particulate matter(PM_2.5),produced by incomplete combustion of fossil fuels,are typical coal-fired air pollutants.Coal-fired air pollutants are the main factors to affect China's air condition,which can cause cardiopulmonary system injury that are closely related to mitochondrial apoptosis and fibrosis.Mitochondrial apoptosis is involved in the pathophysiological processes of various diseases,mainly including the participation of anti-apoptotic family Bcl-2 and the accumulation and activation of p53.Mitochondrial dysfunction and oxidative stress are the main causes for mitochondrial apoptosis.The previous researches demonstrated that SO₂ or PAHs exposure lead to mitochondrial dysfunction in mice,increased ROS production,and mitochondrial apoptosis pathway.However,studies on the co-effects of SO₂ and PAHs to mitochondrial apoptosis have rarely been reported.In addition,fibrosis can be induced by apoptosis of epithelial cells,inflammatory response and oxidative stresss.Epidemiological investigations indicated that PM_2.5 inhalation is associated with increased susceptibility to CVD in sensitive populations.Fibrosis is an early symptom of CVD.PM_2.5 can affect the cardiac function and increase the risk of cardiovascular diseases through the lungblood system and fibrogenesis.However,the above researches are mostly limited to pathological research and lack the explore of pathogenesis.Therefore,in this thesis we intended to analyze the mitochondrial damage and apoptosis and the molecular mechanism of cardiopulmonary fibrosis induced by coal-fires air pollutants in vivo and in vitro.The results showed that:?1?SO₂ derivative NaHSO₃?0,10,30,100 ?M,24h?significantly upregulated ROS production in rat primary cardiomyocytes,the induced oxidative stress caused decreased mitochondrial membrane potential and inhibition of mitochondrial respiratory chain complexes IV and V,resulting in decreased ATP content and mitochondrial dysfunction.In addition,the antioxidant NALC can effectively alleviate the above effects induced by NaHSO₃ and reverse mitochondrial function and structural disorders.?2?Co-exposure of 7mg/m3 SO₂ dynamic inhalation?6 h/day,4 weeks?and 40 mg/kg b.w.BaP intraperitoneal injection?the first 5 days?induced cardiopulmonary mitochondrial apoptosis pathway in C57BL/6 mice.Decreased expression of the respiratory chain complex subunits and related regulatory genes lead to mitochondrial DNA damage.The impaired mitochondrial function then activated the apoptotic pathway,which was characterized by decreased expression in anti-apoptotic gene bcl-2,increased pro-apoptotic bax expression and accumulation of the tumor suppressor gene p53.?3?Combined exposure of SO₂ and BaP respectively induced proapoptotic and anti-apoptotic pathways at different post-exposure levels.Based on the above mice model,we revealed that 1 d post-exposure of SO₂ and BaP caused the mitochondrial apoptosis pathway in mice liver.The decrease in the expression of complex enzyme subunits and mitochondrial membrane potential resulted in mitochondrial disorder in structure and function.Furthermore,SO₂ and BaP down-regulated bcl-2 and up-regulated bax expression in m RNA level,induced accumulation and phosphorylation of p53,and ultimately activaed caspase-3,triggering apoptosis.However,after a 13-week post-exposure,it had been demonstrated that anti-apoptotic pathway was caused by SO₂ and BaP with bcl-2 overexpression and p53 inhibition.Antiapoptotic signals may be an escape state for cell apoptosis and are signs of precancerous lesions.?4?Apoptosis of lung epithelial cells contributes to inflammation and fibrosis.Based on the co-exposure mice modle of SO₂ and BaP,cell experiments in vitro revealed the synergistic effects of SO₂ and BaP in cellular metabolic activity by fitting curves.Co-treatment with SO₂ and BaP induced pulmonary fibrosis,including pathological changes and up-regulation of fibrosis marker proteins.The results in vivo and in vitro showed that SO₂ and BaP inhibited the expression of mir-30c-1-3p and promoted the TGF?R2 expression.The dual luciferase reporter assay confirmed that there were binding sites for mir-30c-1-3p in the 3'-UTR region of TGF?R2.?5?3 mg/kg b.w.PM_2.5 exposure in mice?once every other day,oropharyngeal aspiration,4 weeks?significantly induced the release of IL-1? and TNF-? in mouse lungs,serum and hearts.The experiments in vivo and in H9C2 cell(3 ?g/m L PM_2.5)showed that PM_2.5 exposure induced ROS accumulation and resulted in oxidative stress.In addition,the inflammatory response and oxidative stress lead to the release of the pro-fibrotic cytokine TGF?.The effects in turn induced the redox pathway involved in NOX4,activated the MAPK,caused myocardial fibrosis,and lead to cardiac dysfunction.Treatment with related inhibitors or si RNAs can effectively reverse the fibrosis induced by PM_2.5.The results revealed that TGF?/NOX4/ROS/p38 MAPK may be involved in the myocardial fibrosis after PM_2.5 exposure.Taken together,the thesis demonstrated that coal-fired air pollutants can induce mitochondrial damage and apoptosis in mice,whereas pro-apoptotic and anti-apoptotic pathways respectively were induced by SO₂ and BaP at different post-exposure levels in liver.In vitro and in vivo,the results showed the upstream events of the traditional TGF? pathway in the mechanism of cardiopulmonary fibrosis induced by coal-fired air pollutants from the aspect of micro RNA and dual luciferase assay veritied the findings.The current studies not only provide evidences for mitochondrial damage by coal-fired air pollutants,but also establish molecular biomarkers for the prevention and prognosis of cardiopulmonary diseases in susceptible populations.