The Protective Effect Of Paeonol Combined With Danshensu In The Cerebral Basilar Artery Of Diabetic Rats

Background：Diabetes mellitus （DM） causes multiple dysfunctions such as vascular dysfunction.Diabetic vascular complications refers to heart, brain and peripheral vascular disease,including coronary heart disease, stroke and intermittent claudication and lower limbgangrene, is a major cause of diabetic death and disability, accounting for60-70%ofcauses of death in diabetic patients. Diabetic patients with ischemic stroke incidence aretwo to four times that of the normal population. Modern medicine effective therapy fordiabetic vascular but has obvious side effects. Chinese medicine because of its lowtoxicity characteristics and multiple targets, in the treatment of diabetes vascular lesions isexpected to bring new breakthrough. Diabetes vascular lesions of traditional Chinesemedical science pathological mechanism is phlegm turbid and blood stasis block in themeridians and collaterals.In traditional Chinese medicine for activating blood circulationin treating diabetes vascular lesions is of great significance. Shuang-Dan prescription combines the use of Cortex Moutan （root bark of Paeoniasuffruticosa Andrew） and Radix Salviae miltiorrhizae （root and rhizome of Salviamiltiorrhiza Bunge）, which are famous herbs widely used in traditional Chinese medicine.In clinical practice, the Shuang-Dan prescription is often used for treating cerebrovascularand cardiovascular diseases.Paeonol （Pae,20-hydroxy-40-methoxyacetophenone） is a major phenolic componentin Cortex Moutan, whereas danshensu （DSS,3-（3,4-dihydroxyphenyl） lactic acid） is awater-soluble active component isolated from Radix Salviae miltiorrhizae. Similar to othernatural compounds, several studies showed that Pae and DSS elicit relaxation of isolatedrat aorta and protection of endothelial cells, respectively. We previously reported that thecombined use of Pae and DSS has synergistic protective effects on focal cerebralischemia-reperfusion injury in rats. Moreover, Pae combined with other hydrophilicphenolics of Radix Salviae miltiorrhizae could attenuate oxidative stress, protect vascularfunctions, and synergistically protect against myocardial ischemia in rabbits. Recently, wefound that the co-administration of DSS increases the concentration of Pae in heart andbrain tissues and increases pharmacological activity in treating cerebrovascular andcardiovascular diseases. However, the mechanism of the interactions of representativeactive components in the protection of vascular function is not well understood.Vascular dysfunctions are one of the major causes of morbidity and mortality inpatients with DM. Previous studies reported that forearm blood flow responsive toacetylcholineis reduced in type2diabetes, suggesting endothelial dysfunction. Moreover,vascular smooth muscle （VSMC） exhibits hyper-reactivity, hypertrophy, and apoptosis indiabetes. One of the pathogenesis of diabetic vascular dysfunction is oxygen derived freeradicals, which are significantly elevated under DM. Diabetic vascular dysfunction is alsorelated to increased Ca²⁺influx and inhibited vascular K+channels. Previous studiesshowed that the inhibition of vascular K+channels increases Ca²⁺influx, which leads todepolarization and vasoconstriction. Therefore, the aim of this study is to investigate theeffects of Pae+DSS on diabetes-induced dysfunction of cerebral arteries compared withthe individual effects of Pae or DSS. Methods：We assessed the role of the endothelium and smooth muscle in the responses to Pae+DSS, on cerebral artery （basal artery, Willis’ circle, and middle cerebral artery） fromdiabetic rats and on cultured endothelial/smooth muscle cells damaged by high glucose.Pae+DSS-induced effect was evaluated in vitro and in vivo. Experimental analyses wereperformed by using wire myograph, colorimetric method，PI-Hoechst dyeing，MTT andWestern blot, et al.Results：1. Pae+DSS Induced Relaxation in Rat Cerebral ArteryPae+DSS induced a strong relaxation on arterial rings obtained from rats in adose-dependent manner. The effect of Pae+DSS in endothelium-intact and endothelium-denuded rat arterial rings was investigated to identify the role of VSMC on Pae+DSSinduced vasorelaxation. The vascular relaxation induced by Pae+DSS was not abolishedin the endothelium removed rings of rats.2. Effect of Chronic Pae+DSS Administration on ACh Relaxation ResponseWhen the PE-induced contraction reached a plateau, ACh （109M to105M） wasadded cumulatively. The capability of the concentration-dependent relaxation induced byACh, which had a maximum response of105M, was significantly weaker in arterialsegments obtained from diabetic rats than those from normal rats. After chronicadministration of Pae+DSS, Pae, and DSS, the capability of ACh-induced relaxation inthe arterial segments of diabetic rats was enhanced significantly. In addition, the degree ofACh-induced relaxation in the DM+Pae+DSS group was stronger than that of Pae andDSS treated diabetic rats. No marked changes were observed concerning the degree ofACh-induced relaxation between the DM+Pae+DSS group and SDOS group.3. Effect of Chronic Pae+DSS Administration on Phenylephrine （PE） ContractionResponseWe also found that treatment with Pae+DSS significantly reduced the maximumcontraction of rings from diabetic rats. Meanwhile, the enhancement rates of contractileresponses to PE in all groups were significantly different in E+and E rings. Particularly, the enhancement rate of contractile responses in Pae+DSS treated diabetic rats was lowerthan that Pae and DSS treated diabetic rats. No marked changes were observed concerningthe degree of contractile responses to PE between the DM+Pae+DSS group and SDOSgroup.4. Effects of Pae+DSS on SOD Activities and TBARS Content in the CerebralArtery from Diabetic RatsThe enhanced generation of reactive oxygen species is induced by oxidative stress.The superoxide dismutase （SOD） activities and thiobarbituric acid reactive substances（TBARS） concentrations in the arterial tissues of all groups at the end of the study. Alltreated groups （Pae, DSS, and Pae+DSS） exhibited increased SOD activities anddecreased TBARS concentrations. Moreover, the Pae+DSS group exhibited morereduction in oxidative stress compared to Pae and DSS groups.5. Influence of Pae+DSS on CalciumIn Ca²⁺free solutions, pretreatment of Pae+DSS attenuated the CaCl₂inducedcontractions of denuded cerebral arteries from normal and diabetic rats.6. Influence of Pae+DSS on Potassium ChannelWe examined the transient vasoconstrictor PE response in the presence of thenon-selective K+channels blocker, tetraethylammonium （TEA）. In the presence of TEA,the vasorelaxation effect of Pae+DSS was partially inhibited when Pae+DSS was addedafter the PE contraction reached a plateau.7. Pae+DSS inhibited vascular endothelial cell apoptosis in high glucose.Cell apoptosis were attenuated in the Pae+DSS–pretreated cells.And, Pae+DSSprotects cells by the p-38signaling pathway.8. Pae+DSS can effectively enhance the protein expression of BK_Ca-β1in vascularsmooth muscle cells exposed to high glucose.Conclusions：1. Pae+DSS can attenuate endothelial dysfunction associated with diabetes andprotect high glucose-induced damage of endothelial cells.The p38MAPK pathway issuppressed for the protective effect of Pae+DSS.2. Pae+DSS can attenuate vascular smooth muscle dysfunction associated withdiabetes.The BK_Cais required for the protective effect of Pae+DSS.