Study On Vasodilating Effect Of Guanxinning Tablet And Its Mechanism

Objective The vasodilating effect of Guanxinning tablet?GXN?and its mechanism were studied by isolated thoracic aorta and vascular endothelial cell test,which could provide reference for clinical application of GXN.Methods?1?Using the isolated rabbit thoracic aortic rings to establish isolated vascular circulation perfusion model,to observe the vasodilatin effects of GXN vasoconstriction induced by neo-synephrine?PE?,noradrenaline tartrate?NE?,potassium chloride?KCl?and calcium chloride?CaCl₂?on vascular ring,and the intracellular and extracellular calcium contraction induced by PE were studied.EffectsofGXNvasoconstrictionpreconditioningofNOSinhibitor Nw-nitro-L-arginnine-methyl-ester?L-NAME?,guanylate cyclase inhibitor methylene blue?MB?,cyclooxygenase inhibitor indomethacin?Indo?,adenylate cyclase inhibitor SQ-22536,PKA inhibitor Rp-cAMPS,?receptor blocker metoprolol,M receptor blocker Atropine,Diltiazone hydrochloride,K_ATPTP channel inhibitor glibenclamide?Gli?,K_iRchannelinhibitorbariumchloride?BaCl₂?,K_Vchannelinhibitor4-aminopyridine?4-AP?and K_Caa channel inhibitor Tetraethylamine?TEA?.?2?The effects of GXN on proliferation of vascular endothelial cells were detected by MTT assay,and the suitable concentration of H₂O₂ was screened by different concentrations of H₂O₂ induced oxidative damage of human umbilical vein endothelial cells?HUVECs?.The oxidative damage model of HUVECs was induced by H₂O₂concentration,and GXN intervention was carried out,at the same time,intervention in normal HUVECs were also carried out.The supernatant was taken to detect NO concentration,and cell protein was extracted.The expression of Akt/eNOS and CaMK II/eNOS was detected by Western blotting method.The changes of calcium fluorescence value of endothelial cells were detected by GXN intervention with Fluo-4AM calcium fluorescent staining agent loaded on endothelial cells.Results In vitro vascular test results,?1?GXN had no effect on the basal tension of vessel that is endothelially removed or intact?P>0.05?.The vasodilation effect of0.25-0.8mg/mL GXN on PE precontracted vascular rings was dose-dependent?P<0.05,P<0.01?,and the relaxation effect of intact endothelial rings was stronger.0.25-0.8mg/mL GXN had a significant relaxation effect on NE precontracted vascular rings?P<0.05 P<0.01?,2-8 mg/mL GXN relaxes the KCl precontracted vascular rings in a dose-dependent manner?P<0.05,P<0.01?,2-8 mg/mL GXN significantly alleviated the contraction of vascular smooth muscle induced by CaCl₂?P<0.05,P<0.01?.?2?Pretreatment with 10^-4mol/L L-NAME,10^-55 mol/L MB,10^-66 mol/L Indomethacin,10^-7mol/L Wortmannin,10^-55 mol/L SQ-22536,5×10-⁴ mol/L Rp-cAMPS,10^-66 mol/L Atropine could partially block the vasodilation of GXN?P<0.05,P<0.01?,but 10^-6mol/L Metoprolol could not.?3?1mg/mL,2 mg/mL and 4 mg/mL GXN could significantly reduce the contraction amplitude of intracellular Ca²⁺?P<0.01?.At the same time,2mg/mL and 4 mg/mL of GXN could significantly reduce the contractile amplitude of extracellular Ca²⁺?P<0.05?.Pretreatment with 10^-55 mol/L Dil could partially inhibit the vasodilation of GXN?P<0.05,P<0.01?.?4?After pretreatment with 10^-55 mol/L Gli?10^-33 mol/L TEA,the vascular response of GXN could be blocked?P<0.05?,but after pretreatment with 10^-33 mol/L BaCl₂ and 10^-3mol/L 4-AP,the vasodilator response of GXN could not be blocked?P>0.05?.?5?The contents of NO and cGMP in vascular rings were increased by 2 mg/mL and 4 mg/mL of GXN after incubation for45 min.Vascular endothelial cell test results,?1?2mg/mL and 4mg/m L GXN significantly increased the NO level of the supernatant of normal endothelial cells and the H₂O₂ injured endothelial cells?P<0.05,P<0.01?.?2?GXN had no significant effect on the phosphorylation level of Akt of normal and model group?P>0.05?,but GXN can increase the level phosphorylation level of CaMK II and eNOS of normal and model group.0.5 mg/mL and 1 mg/mL GXN significantly increased the level of p-eNOS in normal endothelial cells?P<0.05,P<0.01?,and 0.25 mg/m L,0.5 mg/m L and 1 mg/mL GXN significantly increased the level of p-eNOS/Enos in the model group?P<0.05?,and 0.25 mg/mL,0.5 mg/mL and 1 mg/mL GXN can increase the level of p-CaMK II in normal and model group?P<0.05,P<0.01?.?3?0.25 mg/mL,0.5mg/mL and 1 mg/mL GXN can significantly decrease the level of Ca²⁺in endothelial cells?P<0.05,P<0.01?.Conclusions The results of isolated rabbit vascular experiments showed that GXN had a significant vasodilator effect.It may be involved in promoting the release of NO from endothelial cells,activating the NO-sGC-cGMP pathway,activating cyclooxygenase and activating the AC-cAMP-PKA pathway,and activating M receptor to inhibit the extracellular Ca²⁺influx and intracellular Ca²⁺release,which is related to the potassium channels such as K_ATPTP and K_Ca.The results of cell experiments confirmed that GXN can dilate blood vessels by promoting the release of NO,and its mechanism may be related to the activation of CaMK II/eNOS pathway.