Study The Effect Of Tadalafil To Mediate Calcium Influx By Regulating TRPM8 On The Treatment Of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension（PAH）is a pathologic condition which characterized by abnormal proliferation of pulmonary artery smooth muscle cells（PASMCs）and pulmonary vascular remodeling.The increased pulmonary vascular resistance may cause decompensation of right heart function,leading to right heart hypertrophy and even death.The disturbance of Ca²⁺homeostasis within PASMCs is the main factor for the pathological changes of PAH.It is well established that store-operated calcium entry（SOCE）and receptor-operated calcium entry（ROCE）are augmented in PASMCs during PAH.Moreover,the melastatin-related transient receptor potential 8（TRPM8）is significantly down-regulated,and activation of TRPM8 causes inhibition of SOCE function in PASMCs of PAH animal models.Tadalafil which a phosphodiesterase 5（PDE5）inhibitor is often used to the treatment of PAH,and can induce vasodilation by enhancing the concentration of cyclic guanosine phosphate（c GMP）in PASMCs.Studies have shown that the activation of c GMP signaling pathway could restrain the functions of SOCE and ROCE,but the mechanism of c GMP-induced vasodilation is still unclear.Therefore,in this study,we examined the interaction of c GMP and TRPM8 in regulating the functions of SOCE and ROCE in PASMCs of MCT-induced PAH rats.Firstly,we detected the alteration of TRPM8 and TRPC6 expression in PASMCs of MCT-induced PAH rats,as well as the alteration in SOCE and ROCE functions in PASMCs of PAH rats,and then observed the effect of PDE5inhibitor on the vasodilation effect caused by TRPM8 activation,to clarify the effects of tadalafil on TRPM8 expression and function,as well as the relationship between tadalafil and TRPM8 on inhibition of SOCE and ROCE functions,and to provide new scientific basis for elucidating the pathogenesis of PAH and develop novel therapeutic approaches.Objective:To observe the effects of tadalafil on expression and function of TRPM8 in PASMCs of MCT-induced PAH rats,and to explore the regulation of PDE5 inhibitors on TRPM8-mediated calcium influx during the development of PAH.Methods:（1）To construct animal model of MCT-induced PAH,and treat with tadalafil,and detect the right ventricular systolic pressure（RVSP）and right ventricle mass index（RVMI）;To observe and calculate the media wall thickness（WT）and thickness percentage（WT%）of pulmonary arterioles（50～150μm）;To detect the expression of TRPM8 and TRPC6 by real-time fluorescent quantitative PCR and Western blot;（2）To detect the effects of tadalafil on pulmonary arteries（PAs）contraction induced by Phen,ET-1,CPA and OAG by vascular ring tension detection technology in MCT-induced PAH rats;（3）To detect the effects of tadalafil on the vasodilation effect of TRPM8 activation by vascular ring tension detection technology.Results:（1）Compared with CON,the RVSP,RVMI,WT and WT%of PAH rats induced by MCT augment significantly,indicating that the model of PAH were successful.Compared with the MCT group,tadalafil treatment significantly inhibited the increases of RVSP,RVMI,WT and WT%in PAH rats,indicating that tadalafil has a therapeutic effect on PAH;Compared with CON,the expression of TRPM8 was down-regulated and the expression of TRPC6 was fartify in PAs of the MCT group,indicating that the expression of TRPM8 and TRPC6 channel proteins and their channel functions may have an opposite trend.Compared with the MCT group,tadalafil significantly aggrandize the expression of TRPM8,and descend the expression of TRPC6,indicating that PDE5 inhibitors may be involved in the regulation of calcium homeostasis in PASMCs by regulating the expression of TRPM8 and TRPC6,thereby playing an important role in the treatment of PAH.（2）Compared with CON,the contraction effects of Phen were significantly enhanced in MCT group.Compared with the MCT group,the contraction effects of Phen were significantly reduced in tadalafil group,further suggesting that tadalafil treatment may regulate the calcium Channel function in PASMCs;Compared with CON,the contractile effects of ET-1 were significantly enhanced in the MCT group,indicating that the non-voltage-dependent calcium channel activity were increased in PAH rats.Compared with the MCT group,the contraction effects of ET-1 were significantly reduced in tadalafil group,indicating that tadalafil treatment can inhibit the function of voltage-independent calcium channels;Compared with CON,the contraction effects of CPA and OAG were significantly enhanced in the MCT group,indicating that the functions of SOCE and ROCE were enhanced in PAH rats,and compared with the MCT group,the contraction effects of CPA and OAG were significantly reduced in tadalafil group,indicating that tadalafil treatment can inhibit the functions of SOCE and ROCE in PASMCs.Whatmore,compared with CPA,the inhibition of tadalafil on OAG is more significant,indicating that PDE5 inhibitors may inhibit the pulmonary vasoconstriction mainly by reducing the function of ROCE in PASMCs.（3）After icilin which the TRPM8 specific agonist was added,the contraction effects of ET-1,CPA and OAG were significantly relaxed,indicating that the activation of TRPM8could inhibit the functions of SOCE and ROCE;Compared with CON,the inhibition rates of TRPM8 activation on vasoconstriction mediated by ET-1,CPA and OAG were enhanced in MCT group,it may be related to the enhancement of SOCE and ROCE functions in PASMCs of PAH rats.Compared with the MCT group,tadalafil treatment enhanced the vasodilator effects of TRPM8 activation,suggesting that PDE5 inhibitors could up-regulate the inhibitory effects of TRPM8 on SOCE and ROCE functions.There may be a synergistic relationship between the activation of TRPM8 and c GMP signaling pathway;Similarly,the inhibition rate of icilin on OAG was significantly higher than that of CPA,again indicating that tadalafil mainly inhibits the pulmonary vasoconstriction by inhibiting the function of ROCE.Conclusion:Tadalafil treatment significantly up-regulates the expression of TRPM8 in PASMCs of PAH rats and enhances the vasodilator effects of TRPM8 activation;the activation of TRPM8 and c GMP signaling pathway may have a synergistic relationship in inhibiting ROCE and SOCE functions.