Chronic Pulmonary Toxicity Of Methamphetamine: Oxidative Remodeling Of Pulmonary Arteries

Objective:The abuse of methamphetamine?MA?has become a serious global public and social problem.MA is widely distributed in many organs in body,and long-term abuse leads to damage to tissues and organs such as heart,brain and lungs.It is reported that MA has a high uptake in the body and the lung is one of the important target organs of the distribution of MA.In 2013,the Fifth Global Conference on Pulmonary Hypertension made a new classification of pulmonary hypertension that MA abuse was included as a potential risk factor in drug-or toxin-induced PAH.Our previous studies have found that long-term or high-dose MA can induce abnormalities in lung structure and function,including the pathological changes in lung inflammation and remodeling,but whether MA induces pulmonary artery remodeling and its mechanism remains unclear.Studies have shown that excessive oxidative stress is associated with the pathogenesis of cardiopulmonary disease.Oxidative stress refers to the production of excessive reactive oxygen species?ROS?in cells,which imbalances the antioxidant response and oxidative stress.ROS are involved in intracellular reactions that can cause oxidative damage in molecules,cells and tissues.Nrf2?NF-E2-related factor 2?as an endogenous antioxidant protein and maintains the balance of redox reactions in the body effectively.Nrf2 is expressed in various organs in the body,especially in skin,lungs which are exposed to the air.Under physiological conditions,Nrf2 is linked to Keap1?Kelch-like ECH2-related protein 1?in the cytoplasm to form a complex.When oxidative stress occurs,Nrf2 dissociates from Keap1,migrates into the nucleus and binds to the ARE?antioxidant response element?sequence.ARE is a specific DNA promoter binding sequence that can induce the expression of antioxidant enzymes such as HO-1.Nrf2-mediated signaling plays an important role in endogenous antioxidant stress.In oxidative stress,redox-sensitive transcription factors such as Nrf2 can be activated by ROS.Therefore,oxidation and antioxidant imbalances are caused by excessive production of ROS.It has been reported that ROS is involved in the regulation of cell differentiation,proliferation,senescence and apoptosis.However,there is no report of ROS involved in MA-induced pulmonary vascular remodeling.Therefore,the purpose of this study was:?1?whether MA stimulates the proliferation of Pulmonary artery smooth muscle cells?PASMCs?;?2?whether MA induces excessive production of ROS by PASMCs;?3?whether MA induces pulmonary vascular remodeling is related to ROS-mediated redox imbalance.Methods:Twenty male Wister rats?200 g±10 g?were randomly divided into control group and MA group,with 10 rats in each group.Rats in the MA group were given methamphetamine?10 mg/kg,intraperitoneal injection,once/day?for one week,then a daily dose of 1 mg/kg was added every week until the 6th week,the daily dose was increased to 15 mg/kg?intraperitoneal injection,once/day?.Rats in the control group were given an equal volume of 0.9%saline solution?intraperitoneal injection,once/day?.Six weeks later,all surviving rats were dissected out of lung tissue and stored in a-80°C refrigerator.Two groups of rats were immersed in 4%paraformaldehyde in water to form a wax block,and HE staining was performed on lung tissue sections.The pulmonary aorta was isolated from 7-day-old male Wistar rats,and the pulmonary aorta was cultured in DMEM high glucose medium containing 30%fetal bovine serum?FBS?and contained 5%CO₂ and 100%relative.The humidified atmosphere was humidified at 37°C until the pulmonary aortic smooth muscle cells were cultured.The MA was dissolved in PBS to prepare a 1 g/ml stock solution and diluted to 1 mg/ml,10 mg/ml,100 mg/ml working solution.Cells were treated with 0,0.1 mM,0.5 mM,1 mM and 5 mM MA and subsequent experiments were performed after 24 hours,36 hours and 48 hours,respectively.Nrf2,SOD2,GCS,BAX,Caspase-3,Bcl2 and PCNA were detected by Western blot.The thickness of the inner wall of pulmonary artery was observed by HE staining;reactive oxygen species was detected by flow cytometry;Nrf2 nuclear translocation was detected by immunofluorescence;cell count was performed by CCK-8.Result:The results of this study indicate that prolonged exposure to methamphetamine reduces the percentage of weight gain in rats and promotes pulmonary wall hypertrophy.In PASMCs,MA increased the expression of proliferation factor PCNA and anti-apoptotic factor Bcl-2,and decreased the expression of apoptosis proteins BAX and Caspase 3 in a dose-dependent manner at all time points?24h,36h,48h??0.1 mM,0.5 mM,1 mM,5 mM?.The effect of MA on the number of pulmonary artery smooth muscle cells was also found to be dose dependent and time dependent.Further studies showed that MA inhibited Nrf2-mediated oxidative stress by down-regulating Nrf2,GCS and up-regulating SOD2 at 48 h and 5 mM.MA induced ROS production in PASMC at all time points in a dose-dependent manner.Linear regression analysis showed that the relative levels of ROS were positively correlated with the proliferation of PASMCs.Conclusion:MA promotes the proliferation of PASMCs,induces excessive ROS production in PASMCs,inhibits Nrf2-mediated oxidative stress,leads to imbalance of redox system,and induces oxidative remodeling of pulmonary arteries.