The Mechanism Of Tetrahydroxystilbene Glucoside In Vasomotor Dysfunction Induced By Homocysteine

1 Background Polygonum Multiflorum is a traditional restorative Chinese medicine,the description of the 2015 Pharmacopoeia is: Buganshen,Yijingxue,Wuxufa,Qiangjingu,Huazhuojiangzhi.And combined with modern pharmacological research findings were that it reduces blood lipids,cholesterol and blood pressure,and improves coronary sclerosis etc.However the material basis of the efficacy and mechanism of Polygonum Multiflorum is still not clear at present.The preliminary studies was that 2,3,5,4'-Tetrahydroxystilbene-2-O-?-D-Glucoside(TSG)-one of the markers in the Polygonum Multiflorum can anti-atherosclerosis and anti-hypertension by anti-inflammatory,anti-oxidation,suppression on platelet aggregation,protection on vascular endothelial cell and suppression on phenotype switch of vascular smooth muscle cells.But,its mechanism are scarce in reports.Besides,hyperhomocysteinemia(HHcy)has been shown positive correlation with the morbidity of angiocardiopathy as hypertension and atherosclerosis.The increasing homocysteine(Hcy)in blood(over 15?mol/L)have direct damage on vascular endothelium and smooth muscle,and induce vascular remodeling and vasomotor dysfunction of smooth muscle.HHcy has become a new and one of the independent risk factors that induce angiocardiopathy,and it is still unclear whether effects of Polygonum Multiflorum on coronary sclerosis and reduction of blood pressure have a correlation with the reduction of Hcy content.This research mainly focuses on three aspects: The mark ingredients in Polygonum multiflorum—TSG was proved for significant vasodilatation effect;It was made clear that TSG could reduce the release of endothelial cell TXA2 by inhibiting the cox-2 activity of vascular endothelial cells,causing vascular dilatation.And On the other hand,TSG may also directly act on the upper voltage-dependent K+ channels of vascular smooth muscle cells and block Ca2+ release and external Ca2+ flow in cells to play a role in Shu vascular.It is further proved that TSG can reduce the expression of abnormal ETB receptor of vascular smooth muscle cells induced by HHcy by inhibiting the MAPK/NF-?B/ETB signal axis,and play a role in reducing HHcy hypertension with diastolic blood vessels.2 Methods 2.1 The vascular ring tension of superior mesenteric artery in rats was recorded in vitro by microvascular tension detection system.To observe the effects of the extract from Polygonum Multiflorum(PME,TSG content was 71.2%)and TSG of high purity(TSG content was 98.1%)on vasomotion of superior mesenteric artery of rat in basic status;Detection of vasomotion rate of artery by PME induced by vasoconstrictor substance hyperkalemia and PE were detected with complete endothelia and without endothelia;Detection of vasomotion rate of artery by TSG induced by vasoconstrictor substance hyperkalemia,PE and 5-HT were with complete endothelia,and use vascular pressure diameter determination system to observe the change of outer diameter of artery in real time.2.2 Further observation of the role of endothelial cells in TSG diastolic superior mesenteric artery by microvascular tension detector in vitro.Using endothelial diastolic vascular pathway and vascular smooth muscle cells K+ and Ca2+ ion channel antagonists to detect vascular ring tension and observe the effect of TSG on diastolic blood vessels through endothelial cells and vascular smooth muscle cells.The acute vasoconstriction model was reproduced by intravenous injection of pituitary posterior Yesu in mice,and the changes of the average blood flow of the total left carotid artery were monitored in real time,and the concentrations of TXA2,PGI2,cox-1 and cox-2 in the blood of each group of mice were determined by ELISA.2.3 Organ culture in vitro was selected,the diastolic effect of TSG on vascular smooth muscle under the action of ET-1 and S6 c of vasoconstriction and endothelin receptor agonists was observed by microvascular tension determination system.The expression of vascular smooth muscle ETA,ETB receptor and the MAPK signaling pathway-related protein were detected by Western Blot.2.4 Animal model of HHcy mouse was established with successive TSG administration for four weeks,and changes of the mouse's blood pressure were monitored by the analytical system of non-invasive blood pressure measurement;the concentration of Hcy,Ang II,ET-1,AD,NE and 5-HT were detected by the ELISA;Histopathologic examination on aorta and arteriole was carried out with HE,masson and resorcinol rosein stain,observe structural changes of vessels,smooth muscle fibers and collagen sedimentation;The expression of vascular smooth muscle ETA,ETB receptor and the MAPK signaling pathway-related protein and changes of its phosphorylation were detected by Western Blot3 Results 3.1 Polygonum chinensis Extract--PME(styrene glycosides content was 71.2%) can be concentrated depending on the way diastolic PE or KCl pre-constricted superior mesenteric artery in rats,and the diastolic effect of PME on superior mesenteric artery in rats with PE(10?M)or high potassium(60m M)pre-shrinkage was significantly stronger than that of superior mesenteric artery in rats except endothelium;The diastolic effect of TSG(two styrene glycosides was 98.1%)on superior mesenteric artery in rats with KCl,PE or 5-HT pre-contraction was more obvious.Both had no obvious contractile or diastolic effect on the superior mesenteric artery vascular ring(complete endothelium)in the basic state,and the vasoconstriction ability did not change significantly before and after the experiment,so it was speculated that TSG had no direct toxicity to blood vessels.3.2 The diastolic effect of TSG on the superior mesenteric artery in rats with high potassium,PE or 5-HT pre-contraction was significantly stronger than that of the blood vessels that removed the endothelium.L-NAME and ODQ are not able to affect the diastolic vascular action of TSG.It is suggested that TSG induced vasodilation without nitric oxide.However,TSG induced vasodilation can be inhibited by Indo,and selective cox-2 inhibitors nimesulide significantly enhance the vasodilation of TSG.At the same time,in the overall experiment: TSG oral administration with 80 and 160mg/kg can reduce the average blood flow caused by the vasoconstriction of mouse caused by posterior pituitary lutein,which indirectly reflects the effect of TSG.TSG can also reduce the concentration of TXA2 and cox-2 in serum of model mouse.It is suggested that TSG can inhibit the activity of cox-2,reduce the TXA2 level of blood vessel and increase the effect of vasodilation;In addition,4-AP inhibits vascular diastolic induced by TSG,while Gli,Bacl2 and TEA are not inhibited,suggesting that TSG salbutamol action is related to voltage-sensitive K+ channels.Finally,it was also found that TSG could inhibit Ca2+ release and K+ induced extracellular Ca2+ internal flow induced by PE in the muscular pulp of vascular smooth muscle cells.3.3 The sensitivity of vascular smooth muscle with the shrinkage vessels(KCl,PE or 5-HT),ETA receptor agonist ET-1 and ETB receptor agonist S6 c was significantly increased in vitro cultured 24 h,and upregulated the ETA and ETB receptors in vascular smooth muscle.TSG incubation can significantly inhibit the contractile effect of the above substances on vascular smooth muscle and reduce the expression of the ETB receptor;With Hcy incubation,the sensitivity of shrinkage vessels,and S6 c increased significantly,and Hcy could lead to upregulated the abnormal ETB receptors in vascular smooth muscle.TSG and Hcy incubation can significantly reduce the expression of the abnormal ETB receptor,and have an antagonistic effect on the vascular smooth muscle contraction induced by the shrinkage of vascular substances and S6 c.But have no effect on the expression of ETA receptor;TSG can reduce the expression of MAPK signal pathway related protein ERK1/2 and JNK caused by culture in vitro.After hcy induced 24 h in vitro,the expression of vascular smooth muscle ERK1/2 protein increased significantly,but the expression of JNK and P38 protein was not obvious.After TSG and Hcy Co-incubation,TSG can significantly reduce the expression of ERK1/2 and JNK protein in vascular smooth muscle,but it has no effect on the expression of P38 protein.3.4 TSG can reduce blood pressure in HHcy mice,reduce the concentration of Ang ? and ET-1 in the blood of model mice,and have protective effect on aortic and injury caused by HHcy.The aortic collagen fiber deposition can obviously reduce the arrangement rule of elastic fibers,the fracture condition is alleviated,and the collagen fiber deposition decreases.And the arteriole collagen fiber was obviously reduced,the elastic fiber increased greatly,and the deposition of collagen fiber decreased obviously.TSG can significantly reduce the expression of vascular smooth muscle p65 protein in HHcy mouse,inhibit HHcy induced the increase of p65 protein phosphorylation,and then inhibit MAPK signaling pathway related protein ERK1/2,JNK protein expression,and inhibit ERK1/2,JNK,P38 protein phosphorylation blocking the activate of the MAPK signal pathway.Thus,the ETB receptor was mediated by lowering the abnormal expression in vascular smooth muscle to mediate the diastolic.4 Conclusion This study determined that TSG is the main effective constituent for arteriole relaxation in PME.It might suppress the activity of cox-2 and reduce release of endothelial cell TXA2 via endothelium pathway on one hand,and might block the voltage-dependence K+ channel on vascular smooth muscle cells,intracellular Ca2+ release and extracellular Ca2+ influx.TSG shows good antagonism against HHcy.In vitro TSG down-regulation on ETB receptor level and reduced the reactivity of vascular smooth muscle to vasoconstrictor substance via suppression on ERK1/2 and JNK protein expression.In vivo TSG can reduce the increasing concentration of Ang ? and ET-1,protect vessels and reverse vascular remodeling,have down-regulation on ETB receptor expression,and decreased the increasing blood pressure induced by HHcy through suppression on the signaling pathway of MAPK/NF-?B/ETB.