Experimental Research On Activated Effect Of Astragaloside Ⅳ On Vessel

Objectives:To observe the antagonized effect of astragaloside IV on rat thoracic aortic rings at resting tension or precontracted by different stimulus, and further analyze its possible mechanism of vasorelaxant action; and to primarily explore the selective regulatory-effect of astragaloside IV on rat ceberal rings, coronary rings, renal rings and uterine spiral rings.Methods:1. Research on mechanism and effect of astragaloside Ⅳ on rat thoracic aortic ringsThe isolated thoracic rings were suspended in the tissue perfusion model in vitro and the change of isometric tension was collected and analyzed by PowerLab. The effect of astragaloside IV on the vascular rings at resting state and precontracted by potassium chloride (KC1) or phenylephrine (PHE) were observed. The endothelium-denuded arterial rings pre-contracted by PHE were treated with astragaloside IV to investigate the relationship between endothelial cells and relaxant effect of astragaloside IV. The nitric oxide (NO) synthase (NOS) inhibitor NG-nitro-L-arginine methylester (L-NAME), cyclooxygenase (COX) inhibitor indomethacin or potassium (K+) channels inhibitors were used to study their influence on astragaloside Ⅳ-induced vasorelaxation. U-46619, intracellular or extracellular calcium (Ca) was acted as agonists to observe if contractile response was affected by astragaloside IV which preincubated the vascular rings. Finally, the interfered effect of astragaloside IV and nifedipine by pre-incubation were compared in arterial rings which were contracted by KCl or PHE.2. Primary research on selective regulatory-effect of astragaloside IV on rat vascular rings from different organsThe effect of astragaloside IV on rat ceberal rings, coronary rings, renal rings and uterine spiral rings which were at the basic state or were precontracted with KCl were observed with the method of using record and analysis system of PowerLab and DMT.Results:1. Effect of astragaloside Ⅳ on endothelium-intact thoracic rings at resting tensionThe cumulative addition of astragaloside IV (1×10-4-3×10-1,g/L) exhibited a slightly vasomotor action on rat thoracic aortic rings at resting tension (P>0.05, compared with control).2. Effect of astragaloside Ⅳ on KCl or PHE-induced contraction in endothelium-intact thoracic ringsAfter adding astragaloside IV from 1 ×10-4 g/L to 3 ×10-1 g/L, it produced a concentration-dependent relaxation in endothelium-intact arterial rings precontracted by KCl (60 mmol/L) or PHE (1μmol/L). In aortic rings precontracted by KC1, astragaloside IV(300 mg/L)-induced the maximal relaxation magnitude (Emax) were 57.60%±9.55%, and the sensitivity (pD2) were 0.79±0.18. While in aortic rings pre-contracted by PHE, astragaloside IV (300 mg/L)-induced ax were 100.01%±9.64%, and pD2 were 1.71±0.27.In addition, treatment arterial rings with 100 mg/L of astragaloside IV, it made the concentration-contraction curve for KC1 (10-90 mmol/L) or for PHE (1×10-9-3×10-5 mmol/L) to right nonparallel. Their maximal contraction altitude (Emax) were both of declined from 53.88±10.19 mg/mm2 to 38.42±8.08 mg/mm2 for KCl (P<0.01), decreasing by 31.94%±5.60%, and from 31.26±7.60 mg/mm2 to 11.85±2.87 mg/mm2 for PHE (P<0.01), decreasing by 63.66%±12.16%. Their pD2 were also both of declined from 1.54±0.05 to 1.47±0.06 for KCl (P<0.05), and from 7.38±0.07 to 7.26±0.10 for PHE (P<0.01). These results suggest that astragaloside IV exerts vascular relaxation by inhibition of voltage dependent Ca2+ channels (VDC) and receptor operated Ca2+channels (ROC).3. Effect of astragaloside Ⅳ on U46619-induced contraction in endothelium-intact thoracic ringsPretreatment endothelium-intact aortic rings with 100 mg/L of astragaloside IV had no significantly inhibitory influence on contraction induced by cumulative concentration of U46619 (P>0.05, compared with control), a thromboxane A2 (TXA2) analogue, at the dose of from 1×10-9 mol/L to 1×10-6mol/L. For example, the Emax were only reduced from 45.36±9.46 mg/mm2 to 44.58±9.55 mg/mm2 (P>0.05, compared with control); the pD2 values were only reduced from 8.38±0.32 to 8.22±0.37 (P> 0.05, compared with control), suggesting that astragaloside Ⅳ may fail to antagonize TXA2-induced vascular contraction.4. Effect of astragaloside Ⅳ on PHE-induced contraction in thoracic rings with denuded endotheliumTo investigate whether the role of endothelium in dilating vessel or not, the experiments on effect of astragaloside Ⅳ on the both endothelium-intact and -denuded aortic rings were performed. The results had showed that, all doses of astragaloside Ⅳ (1×10-4-3 ×10-1g/L) concentration-dependently relaxed the endothelium-intact and -denuded rings precontracted by PHE (1μmol/L). In the denuded aortic rings, the Emax (82.81%±3.63%, P<0.01) were attenuated for astragaloside IV, decreasing by 16.95%±7.92%,but the 1.45±0.51 of pD>2(P>0.05) showed no statistically significance when compared with that in intact endothelial rings, suggesting that the pathways of astragaloside IV dilating vessel are both endothelium-dependent and endothelium-independent.5. Effects of NO or PGI2 on astragaloside Ⅳ-induced relaxation in endothelium-intact aortic rings pre-contracted by PHEIn endothelium-intact thoracic aortic rings precontracted with PHE (1μmol/L), pre-incubation with nitric oxide synthase (NOS) inhibitor L-NAME (1×10-4mol/L) significantly attenuated the relaxant effect of cumulative concentration of astragaloside Ⅳ (1×10-1-3×10-1g/L), the Emax were 66.75%±11.05%(P<0.01, compared with control), decreasing by 33.26%±12.62%; while pre-incubation with cyclooxygenase (COX) inhibitor indomethacin (1×10-5 mol/L) did not influence the relaxant action of all doses of astragaloside Ⅳ (P>0.05, compared with control), and the Emax were 97.00%±3.32%(P<0.01, compared with control), decreasing by 7.00%±12.27%. the pD2 were 1.17±0.30 (P<0.01, compared with control) and 1.47±0.28 (P>0.05, compared with control), respectively, for two cases. The results indicate that vasorelaxant effect of astragaloside Ⅳ perhaps be involved in NO instead of prostanoids (PGI2).6. Effects of potassium channels activation on astragaloside Ⅳ-induced relaxation in endothelium-intact aortic rings pre-contracted by KClThis study observed the possible relationship between potassium channels and effect of astragaloside Ⅳ, the results had showed that, in endothelium-intact thoracic aortic rings depolarized by KCl (60 mmol/L), respective successing pretreatment with Ca+-activated K+ (KCa) channels inhibitor TEA (1×10-2 mol/L), voltage-dependent K+(Kv) channels inhibitor 4-AP (1×10-3 mol/L), ATP-sensitive K+(KATp) channels inhibitor glibenclamide (1×10-5 mol/L), and inward rectifier K+ (KIR) channels inhibitor BaCl2 (1×10-4mol/L), TEA and 4-AP remarkably weakened relaxant effect of astragaloside IV at the dose of from 1×10-4 g/L to 3×10-1g/L, the Emax were 29.31%±10.61%(P<0.01, compared with control) and 34.00%±9.20%(P<0.01, compared with control), decreasing by 24.37%±12.60% and 22.17%±12.94%, respectively; glibenclamide had no influence on vasorelaxation of all doses of astragaloside IV (1×10-4-3×10-1,g/L), the Emax were 48.58%±10.81%(P >0.05, compared with control), decreasing by 11.09%±5.84%; BaCl2 (P < 0.01, compared with control) also inhibited vasorelaxation of astragaloside IV (3×10-3-1×10-1 g/L) to some extent, but the Emax (54.94%±13.97%, P>0.05) and the pD2 (0.73±0.07, P>0.05) had no difference when compared with control. These findings indicate astragaloside IV-induced vasorelaxant response may be related to the openness of KCa, KV, and partial KIR channels except KATP channels.7. Effect of extra-calcium inflow and intra-calcium release on astragaloside IV-induced relaxation in endothelium-intact aortic rings in Ca2+-free solutionIn Ca2+-free solution, pre-incubation arterial rings with 100 mg/L of AS IV significantly attenuated PHE (1 μmol/L)-induced transient contraction, and the Emax values were declined from 13.27±6.41 mg/mm2 to 6.91±1.98 mg/mm2 (P<0.05), decreasing by 42.63%±19.29%. On the plateau tension obtained in rings, exogenous addition of CaCl2 at 3 mmol/L, the aortic rings appeared contraction once again, and the Emax were declined from 1.89±1.26 mg/mm2 to 1.65±1.12 mg/mm2 (P>0.05), decreasing by 17.05%±3.45%, suggesting that astragaloside IV can inhibit release of storage Ca2+ from sarcoplasmic reticulum.8. Comparison of inhibitory effects of astragaloside IV or nifedipine on KCl or PHE- induced contractions in endothelium-intact aortic ringsPretreatment aortic rings with astragaloside Ⅳ(100 mg/L), nifedipine (100 mg/L), astragaloside Ⅳ (100 mg/L) plus nifedipine (100 mg/L), respectively, all inhibited contractile response to KCl (60 mmol/L) or PHE (1 μmol/L) when compare to control (P<0.01 in all, compared with control for KCl or PHE), respectively. But astragaloside IV manifested a more potent effect on blocking PHE-induced contraction than that on blocking KCl-induced contraction (KCl 36.06±9.00 mg/mm2, PHE 11.57±2.65 mg/mm2, P<0.01), nifedipine manifested a more potent effect on blocking KCl-induced contraction than that on blocking PHE-induced contraction (KCl 3.89±1.51 mg/mm2, PHE 6.92±0.38 mg/mm2, P<0.01). And AS IV plus nifedipine also inhibited the contraction induce by KCl (60 mmol/L) or PHE (1 μmol/L) in the rings (P<0.01 and P<0.01, compared with control for KCl or PHE), and the action for KC1 was similar to that for PHE(KCl 3.56±2.89 mg/mm2, PHE 5.02±3.79 mg/mm2, P> 0.05). Moreover, the combination of astragaloside Ⅳ and nifedipine manifested a synergic additive effect on inhibiting contraction induce by KCl (P<0.01, compared with astragaloside Ⅳ; P>0.05, compared with nifedipine) or PHE (P<0.01, compared with astragaloside Ⅳ; P>0.05, compared with nifedipine). So there is a suggestion that the mode of action to astragaloside Ⅳ block both VDC and ROC to cause relaxation of vessel.9. The effect of astragaloside Ⅳ on rat different organ-vascular ringsAdding cumulative concentration astragaloside Ⅳ (1×10-4-3×10-1g/L) had no effect on rat ceberal rings, coronary rings, renal rings and uterine spiral rings when compared with corresponding vehicle control (P>0.05 in all).10. The effect of astragaloside Ⅳ on rat different organ-vascular rings precontracted with KClThe results showed that astragaloside Ⅳ (1×10-4-3×10-1,g/L) could concentration-dependently relax ceberal rings, coronary rings, and uterine spiral rings except renal rings, the Emax were 92.73%±13.29%(P<0.01), 86.78%±1.63%(P< 0.01),92.62%±7.99%(P< 0.01) and 40.04%±0.23%(P> 0.05), respectively.Conclusions:Astragaloside IV serves for vasorelaxant effect through nitric oxide (NO)-base endothelium-depenendent and endothelium-independent pathways. Its cellular mechanism may be involved in decreasing extracellular Ca2+ influx through blockade of both VDC and ROC, inhibiting intracellular Ca2+ release, and promoting K+ efflux through activation upon Kv, Kca and partial KIR; and astragaloside IV and ferulic acid had selectively regulatory effect on rat different organ-vascular rings.