The Effects And Related Mechanisms Of Tetrahydrobiopterin On Myocardial Injury And Cardiac Dysfunction Through Oxidative Stress

Objective: Tetrahydrobiopterin?BH₄?is an essential cofactor of nitric oxide synthases?NOS?and is critical for the synthesis of nitric oxide?NO?.The aim of this study is to evaluate the effects on oxidative stress and molecular expression levels of NO/c GMP/PKG pathway in myocardium with BH₄ or /and nebivolol?N?treatment and confirm whether these therapeutic strategies can improve cardiac diastolic dysfunction in spontaneously hypertensive rats?SHRs?.We also confirm whether the combination treatment can exhibit synergistic effect on oxidative stressand preliminarily elucidate the underlining mechanism.Methods: Forty of 12 weeks-old male SHRs were randomly divided into five groups?n=8?,baseline group,placebo control group,BH₄?25mg/kg.d?treatment group,N?5mg/kg.d?treatment group,BH₄&N?BH₄ 25mg/kg.d+N 5mg/kg.d?treatment group and same age male normotensive WKY rats served as control group?WKYs,n=10?.Left ventricle function was evaluated by noninvasive echocardiographic assessment and invasive right carotid artery catheterization methods.Reactive oxygen species?ROS?in myocardium was determined by fluorescent molecular probe method.3-nitro tyrosine?3-NT?,nitric oxide?NO? and cyclic guanosine monophosphate?cGMP?concentrations in myocardium were measured by ELISA method.The activity of eNOS was detected by the rate of.¹⁴C L-arginine conversion into.¹⁴C L-citrulline.RT-PCR and western blot were used to determine expression of mRNA and protein of eNOS,phospholamban?PLN?,sarcoplasmic reticulum calcium pump 2 alpha?SERCA2??,as well as the expression level of cardiac beta 3 adrenergic receptor??₃-AR? and protein kinase G?PKG? in myocardium.Results: 1.Compared with same age male WKYs,12-week old SHRs exhibited cardiac diastolic dysfunction.The ratio of E/ E? significantly increased?P<0.01?,while the ratio of E? / A 'decreased significantly?P<0.01?.2.Compared with the control group,there was no significant difference on blood pressure and heart rate in BH₄ treatment group?P >0.05?.However,N treatment significantly reduced blood pressure and heart rate?P <0.01?.Although compared with the placebo control group andBH₄ treatment group,the heart rate and blood pressure of BH₄&N?BH₄ 25mg/kg.d+N 5mg/kg.d?treatment group decreased significantly?P <0.01?,compared with those in N treatment alone the heart rate and blood pressure had no significant difference?P >0.05?.Compared with the placebo control group,no matter BH₄ or N treatment alone and combination treatment,the left ventricular end diastolic pressure?LVEDP?,E/E ' all were reduced?P all <0.01?,and E'/ A' increased?P<0.01?.whereas the left ventricular relaxation time constant?tau?was shorten and LV-dP/dtmax/LVSP was more negative?P <0.01?.The combination treatment group?BH₄&N?indicated synergistic protective effects on diastolic function in some extents?P <0.05?.3.Compared with the placebo control group,after BH₄ and nebivolol treatment alone or combination,the expression of myocardial eNOS mRNA,eNOS dimer expression increased significantly and also NO production.The expression level of p-PLN/SERCA2 a,beta 3-AR,c GMP and PKG were all upregulated significantly?P<0.05 or P<0.01?.However,compared with placebo control group,in BH₄ and N treatment alone or combination group 3-NT and ROS concentration were downregulated in cardium?P<0.05 or P<0.01?.Notably,BH₄ and N combination had better cardioprotective effects than those monotherapies.Conclusions: BH₄ or N treatment alone can improve cardiac diastolic function by eNOS recoupling,increasing the activity of eNOS and NO production in myocardium.In addition to,nebivolol as an agonist for ?₃-AR and antagnist for ?₁-AR N also make eNOS recouple and reduce heart rate and blood pressure,resulted in benefit for diastolic dysfunction.Moreover,BH₄ combined N with the corresponding dose can exhibit synergistic protective effect on diastolic dysfunction by further improving BH₄/BH₂,reducing oxidative stress and increasing the eNOS activity in myocardium.its main effect is through eNOS recoupling and NO/c GMP/PKG pathway.Objective: The radiation induced X-heart disease is common in clinic,but its pathogenesis remains unclear and treatment method is still limited.The aim of this study is to explore the effect of BH₄ on radiation induced myocardial cell proliferation inhibition,apoptosis,oxidative stress and elucidate the underling mechanism.Methods: Clonogenic assays were performed to determine the effects of X-ray on H9c2 cells with or without BH₄ treatment.The concentrations of lactate dehydrogenase?LDH?,superoxide dismutase?SOD?and malondialdehyde?MDA?in H9c2 cells were measured to investigate the levels of oxidative stress.Hoechst 33342 staining was used to detect apoptosis.The cell cycle of H9c2 cells undergoing radiation with or without BH₄ treatment was detected using flow cytometry.The expression levels of proteins in the PI3K-Akt-p53 signaling pathway,inducible nitric oxide synthase?iNOS?and endothelial nitric oxide synthase?eNOS?were examined by Western blot method.Results: X-ray radiation significantly inhibited the proliferation of H9c2 cells in a dose-dependent manner?P<0.01?,whereas BH₄ treatment significantly reduced the X-ray radiation-induced proliferation inhibition in H9c2 cells?P<0.05 or P<0.01?.X-ray radiation induced LDH release,apoptosis and G₀/G₁ peak accumulation,as well as significantly increased the level of MDA and the production of NO,and decreased the level of SOD in H9c2 cells?P<0.05 or P<0.01?,whereas BH₄ treatment significantly reversed these processes.X-ray radiation significantly upregulated the expression levels of Bax,P53,and caspase-3 but downregulated the levels of PI3 K,Akt,phosphorylated Akt,and Bcl-2?P<0.05 or P<0.01?.In radiation damaged cells,BH₄ treatment significantly downregulated the expression levels of Bax,P53,and caspase-3 and upregulated the expression levels of PI3 K,Akt,phosphorylated Akt,and Bcl-2(P<0.05 or P<0.01.BH₄ enhanced the production of NO in 2 Gy and 4 Gy radiated groups by upregulating eNOS protein expression and downregulating iNOS protein expression.Conclusions: BH₄ treatment can protect against X-ray-induced cardiomyocyte injury,possibly by recoupling eNOS rather than iNOS.BH₄ treatment also decreased oxidative stress and apotosis in radiated H9c2 cells.