Osthole Reprograms Metabolism To Alleviate Pulmonary Vascular Remodeling By The Regulation Of MiRNA-22

Background and purposePulmonary arterial hypertension is a malignant pulmonary vascular disease,which is characterized by pulmonary vascular remodeling.At present,single-target drug combination therapy of PAH is mainly aimed at pulmonary vasoconstriction with large side effects and expensive cost,and mortality rate is high due to drug withdrawal.Therefore,it is urgent to find safe and effective drugs for the treatment of PAH.Traditional Chinese medicine is characteristic with multi-ingredients and multi-target in the treatment of diseases.Osthole can effectively relieve pulmonary vascular remodeling,which is a candidate drug for the prevention and treatment of PAH.Although studies have reported that Osthole inhibits the proliferation of PASMCs by regulating inflammatory signaling pathways,metabolic reprogramming is very important pathological mechanism of PAH.However,the mechanism of Osthole on pulmonary vascular remodeling has not been revealed from metabolic regulation perspective.Therefore,integration PAH rats model with cells model,this study focused on analyzing the changes of metabolites,metabolic enzymes and genes in the serum,lung tissue and cells of PAH model before and after Osthole administration,and analyzed the metabolic targets,proteins and genes of Osthole modulation on pulmonary vascular remodeling thus to reveal the novel mechanism of Osthole.MethodsFirstly,male SD rats(200±20 g)were randomly divided into the following three groups,the Control group,the PAH model group and the Osthole treatment group(n =10).PAH model was established by intraperitoneal injection of MCT(60 mg/kg)in PAH model group and Osthole treatment group,and Osthole treatment group was administrated with Osthole(80 mg/kg)on the second day after model established.The Control group and PAH model group were given equal volume of solvent carboxymethyl cellulose water solution.Blood was collected at 28 days after model established,and the levels of serum triglyceride(TG),total cholesterol(TC)and high density lipoprotein cholesterol(HDL-C)were detected.Secondly,ELISA method to detect the expression level of testosterone(T),androgen receptor(AR),c GMP,ATP and decadienyl-L-carnitine(C10:2)in rat serum or lung tissue.Western Blot and q RT-PCR were used to detect miR-22-3p,Phosphodiesterase 5A(PDE-5A),Fatty acid translocase(CD36),Fatty acid synthase(FAS),Carnitine palmityl transferase1A(CPT1A),Phospholipase A2(PLA2),Hexokinase 2(HK2),Isocitrate dehydrogenase3α(IDH3α)and NADH dehydrogenase 1(ND1)expression levels.Then PASMCs were cultured in vitro,the optimal time and concentration of PDGF-BB to induce cell proliferation and the optimal concentration of Osthole to inhibit cell proliferation were screened with CCK-8.In addition,after exposed to miR-22-3p inhibitor,CCK-8 was used to detect cell viability.q RT-PCR,Western Blot and ELISA technology were used to detect the expression level of miR-22-3p,C10:2 and PCNA,CPT1 A,FAS and HK2 proteins.ResultsThe results of biochemical examination showed that the PAH model group developed insulin resistance,and TG and TC were increased significantly,and HDL-C was decreased significantly when compared with Control group,while Osthole can relieve dyslipidemia and insulin resistance.ELISA results showed the testosterone,AR and c GMP levels in PAH group were significantly reduced compared with Control group,and C10:2 was significantly higher than that in Control group,and Osthole can increase the testosterone,AR and c GMP levels and decrease C10:2expression.The expression of miR-22-3p,PDE-5A,CD36,PLA2,FAS,CPT1 A,and HK2 in lung tissue of PAH model group were increased significantly,while the expression levels of IDH3α,ND1 and ATP were decreased significantly when compared with Control group examined by Western Blot and q RT-PCR.Osthole administration reversed miR-22-3p and key metabolic enzymes to normal levels,and promoted ATP production.CCK-8 results showed that 20 ng/m L PDGF-BB and 24 hours were the most suitable dosage and time in inducing PASMCs proliferation,and100 nmol/L Osthole was the most suitable dosage in blocking PASMCs proliferation,and used in the following experiments.miR-22-3p inhibitor could significantly inhibit PASMCs proliferation and expression of PCNA,HK2,FAS,CPT1 A and C10:2.ConclusionThe present study is the first to identified miR-22-3p as a critical metabolic regulator involved in pulmonary vascular remodeling.We firstly found that osthole inhibited PDE-5,promote testosterone,reversed dysregulated serum lipid and C10:2in rats with PAH.Then,we confirmed osthole could reprogram energy metabolism to suppress cell proliferation by modulating miR-22-3p mediated metabolic enzymes and metabolite C10:2,thus to ameliorate pulmonary vascular remodeling.