| Vascular endothelium is a single layer of endothelial cells located between theblood and the vessel wall, which performs the capacity of permeability between bloodand tissues for exchange and selective. It can detect and identify the physical andchemical signals through the circulation, and then synthesis and release of varioustypes of bioactive molecules for response. Vascular endothelial is not only involved inhemostasis, coagulation and metabolism, also plays an important role in maintainingthe physiological activity of circulatory system, regulation of vascular tension andmaintain the balance of body fluid. Many high risk factors can cause endothelial cellinjury, including the changes in shear stress of blood flow, high cholesterol,hyperglycemia, hyperhomecysteinemia, exposure to the cold condition and so on. Theindury make vascular endothelial cells in a state of oxidative stress or inflammation,resulting in vascular endothelial cells dysfunction.Endothelial dysfunction is a risk factor in cardiovascular diseases, such asthrombosis, atherosclerosis and hypertension. The endothelium-dependentvasodilation response induced by acetylcholine is used to evaluate the state ofendothelial dysfunction. The multi wire myograph system is a pharmacologicaltechnical method that can evaluate vascular contraction and relaxation response invitro. Therefor, we use this method to evaluate the vascular tissue function or thecharacteristics of drug’s function in this study. Moreover we investigate the changesof vasoconstriction and vasodilatation under different conditions of temperature andthe protective effects of a new vasoactive drug HH103against endothelial injuryinduced by homocysteine. Our results suggest that the new drug HH103is effectivelyinvolved for antifreeze drugs and anti-atherosclerotic endothelial protective drug.Part One: The effects of several antihypertensives on mesenteric resistancearteries of Wistar ratsA promising candidate in this field is iptakalim (Ipt), a novel potassium (K+)channel opener that has been shown to have antihypertensive action in hypertensive animals. Notably, a number of preclinical investigations confirmed that Ipt improvesendothelial dysfunction, protects against hypertensive target organ damage, andameliorates the state of insulin resistance associated with hypertension. Despiteintense research, no clinical evidence is available concerning its efficacy, safety andtolerability. The clinical outcome of Ipt is attracting increasing attention in the field ofcardiovascular drug discovery worldwide. The data from this study suggest that Ipthighly selectively dilates resistance arteries (50-300μm in lumen diameter) and thesize is correlated with the sensitivity to Ipt, with a smaller artery diameter showing astronger dilatation. In addition, Ipt induced increasingly potent relaxation withgradually elevated perfusion pressure in the arterioles, but it did not influence thevascular tone under physiological perfusion pressure.The effects of Ipt on pressurized mesenteric arteries with lumen diameters in therange of50-500m were examined. Under the perfusion pressure of80mmHg, Iptinduced potent relaxation in resistance arteries (lumen diameters in the range of50~300m). Interestingly, Ipt induced weak relaxation in large arteries with lumendiameters over300m, and it almost failed to relax the larger arteries with lumendiameters over400m, suggesting its high selectivity for resistance arteries and thatsmaller arteries are more sensitive to Ipt, which is entirely different from theantihypertensive tested such as potassium channel openers pinacidil and diazoxide,calcium channel blockers amlodipine and nifedipine, α1adrenergic receptorantagonists prazosin and terazosin, angiotensin Ⅱ receptor antagonists losartan andvalsartan, as well as endothelin receptor antagonist bosentan. Interestingly, Ipt had nodilating effects under physiological pressure of20mmHg, which is also entirelydifferent from the antihypertensive drugs tested.Part Two: Pharmacological characteristies of anovel chemical entity HH103thattargeting NNMR protects against high homocysteine-induced endothelial cellinjuryHyperhomecysteinemia is related to a variety of cardiovascular andcerebrovascular diseases. Therefor the concentration of homocysteine in blood hasbecome a new independent risk factor for atherosclerosis and thrombotic diseases.The endothelial dysfunction induced by high homocysteine is a risk factor leading tocardiovascular diseases, such as hypertension, atherosclerosis,thrombosis and so on.Our previous study showed that the activation of endothelial cells of non-neuronalmuscarinic receptor can effectively delay the development of atherosclerosis. NNMR agonist arecoline can promote the release of nitric oxide by increasing eNOSexpression, which can delay the development of atherosclerosis. We take arecoline asa structure nucleus to designe and synthesize a new structure drug HH103. Its effectsof dilation of blood vessels and antagonism homocysteine injury were studied.The arterial rings’experiment in vitro showed that HH103has a better effect thanarecoline in vasodilation. Both of the two vasodilator effect wereendothelium-dependent. While, the muscarinic receptor antagonist atropine could’tantagonizes HH103vasodilator effect, while the arecoline effect was fully abolished.We also found that HH103could’t induces the contraction of isolated guinea pigileum in the previous study, indicating that the new compound does not activatemuscarinic receptors. By adding L-NAME or Indo we discovered that HH103andarecoline are subject to certain inhibited. After incubation jointly by both blockers thevasodilation has no difference in removal of endothelium, which suggested that thevasodilation induced by HH103and arecoline were only related with the release ofnitric oxide and PGI2.Homocysteine could weaken the acetylcholine-induced endothelium-dependentvasodilation response, while the endothelium-independent relaxation induced bysodium nitroprusside was regular. After pre-incubation with HH103, thevasodilatation induced by homocysteine is attenuated. In arterial rings pre-treatedwith indomethacin or L-NAME, the HH103increased the ACh-induced relaxationthat inhibited by homocysteine was attenuated. In addition, in endothelial cellproliferation experiments, HH103’s endothelium protect effect was also proved.What’s more, HH103could stimulate endothelial cells to release nitric oxidedose-dependently as well as decrease carrageenan-induced mice tail arterythrombosis.In summary, the new compound HH103, which was derivatives of arecoline anddesigned arecoline as original nucleus has shown a better effect on vasodilation andits effect is not achieved by activating the muscarinic receptor. In addition, HH103can protect the endothelium from injury induced by homocysteine and has significantantithrombotic effect. These pharmacological effects are related with stimulation ofendothelial cells, which release signal molecule nitric oxide and PGI2. Part Three: The effect of hypothermia on the vasoconstriction and vasodilatationand concerned with vasoactive drugsTwo distinct mechanisms are apparently responsible for cold injury:(1) cellulardeath occurring at the time of exposure to the cold, and (2) deterioration and necrosisattributable to progressive dermal ischemia. The endothelial cell damage caused bycold exposure will induce the inflammatory reactions and the formation of blood clotswhich can obstruct the microcirculation leading to progressive dermal ischemia. Asthe cold injury is more likely irreversible, we try to prevent endothelial cells fromdamage under the conditions of extreme cold. By studying the changes ofvasoconstriction and vasodilatation under different temperature conditions and theprotective effects of some medicines against endothelial injury induced byhypothermia, we can find a new method to protect vascular endothelial, improveblood circulation, reduce the feasibility of cold injury occurred.In the arterial rings’ experiment in vitro, we found that the hypothermia couldenhanced the dose-dependent construction induced by phenylephrine in mice tailartery and exposure to hypothermia also resulted in increasing of sodiumnitroprusside-induced relaxation. The vascular function of constriction was attenuatedby hypothermia, while the relaxation was increased. Compared with the normal group,prazosin and anisodamine were more effective in relax tail arterial rings at8℃with alower effective concentration. In concluding, we suspected that such drugs taken asthe prevention of drug would have little effect under normal body temperature whilethey can be more effective in the condition of low temperature.In this study, we found that low temperatures will cause theendothelium-dependent vasodilation response attenuated, which result in injury ofregulatory function. After incubation with alpha tocopherol, the vascular relaxationresponses to ACh increased in an endothelium-dependent manner, when comparedwith the hypothermia-treated group. But chlorhexidine did not behave the similareffect. Alpha tocopherol could prevent the hypothermia-induced decrease in vascularendothelial cells while the chlorhexidine show the converse effect. Also becausechlorhexidine has some damage in endothelial function we should pay attention to thedrug concentration in using, avoiding the endothelial injury due to permeabilitychanges.Our results suggest that exposure to the cold condition has causedvasoconstriction and vasodilatation, and the function of endothelial cells has changed in the same way. We develop strategy for prevention method of cold injury anddevelop highly potent antifreeze drugs. |
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