| Endothelium-derived hyperpolarizing factor （EDHF） is the third relaxing factor derived from vascular endothelium besides NO and PGI2. The phenomena of EDHF have been demonstrated in many blood vessels of different species, but until now the chemical nature of EDHF, especially in cerebral artery, is still unknown. Our previous studies indicated that the EDHF responses may be related with endogenous hydrogen sulfide （H2S）.Hyperin （Hyp）, a flavonol compound extracted from Abelmoschus manihot L. Medic, a Chinese herb, is reported to have the significant protective effect against cerebral ischemia-reperfusion injury in rat. In this study, the relaxation effect of Hyp in cerebral basilar artery （CBA） in rat was studied, and the protective mechanism of Hyp against cerebral ischemia-reperfusion injury was explored, especially, the relation between the effect of Hyp and EDHF or some ion channel was examined.Purpose:1. To examine the relaxant effects of rat CBA in vitro to Hyp and its mechanism.2. To investigate the relation between effect of Hyp against cerebral ischemia-reperfusion injury and EDHF or some ion channels in isolated artery vascular smooth muscle cells （VSMC）.Methods:1. Isolated rat CBA segments were suspended in baths containing PSS solution, and the diameter was measured. The relaxation effect of Hyp or NaHS or L-Cys was observed, the effect of co-application of N-nitro-L-arginine-methyl-ester （ L-NAME, an inhibitor of nitric oxide synthase） and indomethacin （ Indo, an inhibitor of prostaglandin synthases） on the relaxation of Hyp as well as the effect of DL-propargylglycine （PPG）, an inhibitor of cystathionine-γ-lyase （CSE, a synthase of the endogenous H2S） or tetraethylammonium（ TEA, an inhibiter of Kca） on Hyp-induced non NO, non PGI2 relaxation was studied.2. Transmembrane potentials were recorded to evaluate the effect of hyperpolarization. In the absence or presence of L-NAME+Indo, the hyperpolarization effects of Hyp, NaHS, and L-Cys were tested. And the effect of PPG or TEA on the Hyp induced non NO, non PGI2 hyperpolarization was also observed.3. Whole-cell patch clamp recording was used to examine the outward potassium current in VSMC of rat CBA.4. Cerebral ischemia reperfusion injury was induced by 4-vessle occlusion （4-VO） in rat. Electroencephalograph （EEG） was recorded. HE stain was used to observe the brain pathologic changes. The LDH in serum and the cerebrum MDA content were measured.5. The CBA endothelial cells of rat subjected to cerebral ischemia reperfusion were obtained by using immune magnetic cell separation method. The separated endothelial cells were used to detect the expression of cystathionine-γ-lyase （CSE）, and the effect of Hyp was also evaluated. The H2S content in the cerebrum was measured by ELIASA mothod.Results:1. In the rat CBA, preconstricted by 30 mmol/L KCl in vitro, Hyp (10-6-10-4mol/L) induced a concentration-dependent relaxation, and the effects was remarkably attenuated by removal of the vascular endothelium. 10-6-10-4mol/L Hyp also elicited marked and dose-dependent hyperpolarization on VSMC of rat CBA. 2. 3×10-5 mol/L L-NAME and 10-5 mol/L Indo could partly inhibit the relaxation of Hyp to the rat CBA, but there were still significant relaxation effect （P<0.01 vs vehicle）. In the presence of L-NAME and Indo, there also left marked hyperpolarition effects of VSMC of rat CBA. This Hyp-induced relaxation or hyperpolarization in the presence of L-NAME and Indo could be markedly inhibited by 1mmol/L TEA, a blocker of Ca2+-activated potassium channel, these results suggested that Hyp may evoke EDHF responses in rat CBA.3. 10-4 mol/L PPG, the inhibitor of CSE, could markedly inhibit Hyp-induced EDHF-mediated relaxation and hyperpolarization. 10-6-10-4mol/L L-Cys or 10-5-10-3 mol/L NaHS could induce a concentration-dependent relaxation in KCl preconstricted endothelium-intact rat CBA. NaHS and L-Cys had marked hyperpolarization effect in VSMC of rat CBA.4. At holding potential of -60 mV, Hyp (10-6-10-4mol/L) have no remarkably effects on calcium-activated potassium currents （ IKCa ） in VSMC of rat CBA. Nevertheless NaHS(10-4 mol/L ) activated IKCa from 33.62±2.00 pA / pF to 59.36±11.39 pA/ pF（n = 6 cells from 6 rats, P < 0.01）. L-Cys (10-6-10-4mol/L ) activated IKCa from 26.79±5.26 pA / pF to 42.34±8.34 pA/ pF （ n = 6 cells from 6 rats, P < 0.01） , respectively.5. On 4-VO model in rats, Hyp （25, 50, 100 mg/kg） significantly inhibited ischemia–reperfusion-induced the decrease of EEG amplitude （p<0.05 or p<0.01）.6. Hyp （25, 50, 100mg/kg） could significantly improve brain pathologic changes, and inhibit the release of LDH and serum MDA content （p<0.05, p<0.01）.7. Globe cerebral ischemia-reperfusion induced the decrease of expression of CSEmRNA in cerebral arteriesend othelial cells; Hyp （50, 100 mg/kg） could significantly inhibit the decrease of expression of CSEmRNA. 8. In the range of 25 to 100mg/kg, Hyp markedly inhibited the decrease of H2S in rat brain subjected to cerebral ischemia reperfusion.Conclusions:1. Hyp produces significant hyperpolarization in VSMC of rat CBA, and induces endothelium-dependent and endothelium-independent vasorelaxation in rat CBA; Endogenous H2S may be involved in EDHF-mediated hyperpolarization and relaxation to Hyp.2. Hyp (10-6-10-4mol/L) has no direct effects on IKca in VSMCs. Nevertheless NaHS (10-6-10-4mol/L) and L-Cys (10-6-10-4mol/L) could markedly activate Kca channel and enhance IKca .3. The result that Hyp has remarkable protective effects on global cerebral ischemia reperfusion injury in rats, and markedly up-regulated CSEmRNA expression in endothelial cell of rat cerebral arteries supposed the mechanism may be related to endogenous H2S. . |
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