The Protective Effect Of 3,4,5,6-tetrahydroxyxanthone On Ischemic Myocardium And Endothelial Cells

Part â…  3,4,5,6-tetrahydroxyxanthone protects myocardium against ischemia-reperfusion injury in ratsBACKGROUNDPrevious investigations have sugested that the pathogenesis of myocardial ischemia-reperfusion injury is due to stimulation of reactive oxygen species (ROS) generation and Ca²⁺ overloding, and some drugs have been shown to protect against ischemia-reperfusion injury in animals. Recently, myocardial reperfusion injury has been considered to be a inflammatory reaction, and multiple sytokines have been involved in the development of ischemia-reperfusion injury. There is substantial evidence that the tumor necrosis factor-alpha (TNF-α) in the myocardium is increased during ischemia-reperfusion, and that anti-TNF-a antibody improves the recovery of cardiac functional during reperfusion, suggesting that TNF-a may be an important factor contributing myocardial ischemia-reperfusion injury.Xanthone, a kind of ployphenolic compounds that commonly occur in plants, has extensive pharmacological actions. Previous investigations have shown that some xanthones have a protection of the ischemic myocardium and anti-inflammation. Recently, it was reported that protective effects of some xanthones on endothelial cells injury wasrelated to reduction of TNF-a level in cultured human endothelial cells. Since myocardial reperfusion injury involves inflammatory reaction and some xanthones have anti-inflammation properties, we examined whether the protective effect of S^^^^tetrahydroxyxanthone, a synthetic xanthone, on myocardial injury induced by ischeamia-reperfusion is related to reduction of TNF-a in rats.METHODSIschemia-reperfusion injury was induced by 30 min of globalischemia and 30 min of reperfusion in isolated rat hearts or 30 min coronary artery occlusion and 120 min reperfusion in vivo, respectively. Heart rate, coronary flow (CF), left ventricular pressure (LVP), and its first derivative (±dp/dtmax) were recorded, and the activity of creatine kinase (CK) in coronary effluent and TNF-a content in myocardial tissues were measured in vitro. The activity of serum CK, the level of TNF-a and interleukin-6 (IL-6), and myocardial infarct size were measured in vivo. All hearts had an initial stabilization period for 20 min. In the control group, hearts were perfused with Krebs-Henseleit solution throughout the experiment. The ischemia-reperfusion group experienced 30-min global ischemia and 30-min reperfusion. For 3,4,5,6-tetrahydroxyxanthones, verapamil and vehicle groups, hearts were perfused with 3,4,5,6-tetrahydroxyxanthone (30, 100 or 300 jiM),verapamil (10 |xg/L) or vehicle of xanthone (1 ml/L) for 10 min before ischemia, and then the drugs remained in the perfusion throughout the remainder of the experiment.The second series of experiments was designed to further examine the protection effect of 3,4,5,6-tetrahydroxyxanthone on the ischemic myocardium in vivo. The sham group underwent the same procedure but without clampping of the coronary artery. The ischemia-reperfusion group, rats were subjected to 30 min of coronary artery occlusion followed by 120 min of reperfusion. For 3,4,5,6-tetrahydroxyxanthones, verapamil and vehicle groups, the rats were treated with 3,4,5,6-tetrahydroxyxanthone (0.5 or 1.0 mg/kg, i.v.), verapamil (1.0 mg/kg, i.v.) or vehicle of xanthone (1 ml/kg) through inferior lingual vein 5 min before ischemia.RESULTS3,4,5,6-tetrahydroxyxanthone (30, 100 or 300 ^M) caused asignificant improvement of cardiac function (LVP and ±dp/dtmax) and a decrease in the release of CK in coronary effluent as well as the level of TNF-a in myocardial tissues in vitro. 3,4,5,6-tetrahydroxyxanthone (0.5 or 1.0 mg/kg) also markedly decreased infarct size and the release of CK and TNF-a, and increased serum IL-6 level in vivo.CONCLUSIONThese results suggest that 3,4,5,6-tetrahydroxyxanthone possesses aprotective effect on myocardial ischemia-reperfusion injury, and that the protective effects of 3,4,5,6-tetrahydroxyxanthone may be related to inhibition of TNF-a production and stimulation of IL-6 generation by antioxidation.Part â…¡ The protection of 3,4,5,6-tetrahydroxyxanthone against damages of endothelial cells induced by high glucoseBACKGROUNDDecreases in endothelial dysfunction were found in diabetic animals and patients, and chronic hyperglycemia is regarded as an important cause of endothelial dysfunction. It has been reported that in vitro acute hyperglycemia attenuates endothelium-dependent vasodilation. In cultured endothelial cells, high glucose caused cell injury and dead.Apoptosis, one form of cell death, is an active process which regulated by multiple genes. It was reported that retinal microvascular cells were accelerated death in diabetic animals and patients. In cultured vascular endothelial cells, high glucose induced cell apoptosis. These results suggest that endothelial cells apoptosis induced by high glucose plays an important role in early events of the pathogenesis of diabetic vascular complications.Recent studies have demonstrated that high glucose-induced impairment of endothelium-dependent relaxation and endothelial cell apoptosis is mediated by reactive oxygen species (ROS), and some antioxidants have been shown to attenuate high glucose-induced human endothelial cell apoptosis and improve endothelium-dependent relaxation via inhibition of ROS generation, suggesting that ROS mayplay a pivotal role in apoptosis induced by high glucose.Xanthones, a kind of ployphenolic compounds that commonly occur in plants, have widely been synthesized. Previous investigations have shown that a great number of xanthones have antioxidation. Recently, it was reported that protective effects of some xanthones on ox-LDL and LPC induced endothelium injury was related to inhibition of lipid peroxidation. It has been reported that some xanthones have hypoglycemic activity. According to facilitation of ROS on endothelial dysfunction and cell apoptosis, and antioxidative properties of xanthones, in the present study we tested the protective effect of 3,4,5,6-tetrahydroxyxanthone, a synthetic xanthone derivative, on high glucose-induced endothelium injury and its mechanism.METHODSThe aortic rings obtained from the rat were exposured to highglucose (25 mM) for 24 h or endothelial cells were incubated with high glucose (30 mM) for 48 h, respectively. Endothelium-dependent relaxation was measured. Cell viability was assayed by MTT method. Lactate dehydrogenase (LDH) activity, malondialdehyde (MDA) and nitric oxide (NO) content in the medium were measured. DNA fragmental ladder was visualized by agarose gel electrophoresis. The percentage of DNA fragmentation was assayed by Burton's method. Thecell cycle, the percentage of cell apoptosis and the expression of Bcl-2 protein of human umbilical vein endothelial cell (HUVEC) were analyzed using flow cytometric techniques. The concentration of glucose in the control was 5.5 mM. Mannitol (19.5 or 24.5 mM) was used as the osmotic control of high glucose (25 or 30 mM). For 3,4,5,6-tetrahydroxyxanthone and probucol, blood vessel or endothelial cells were treated with 3,4,5,6-tetrahydroxyxanthone at the concentration of 1, 3, 10 \iM or probucol at the concentration of 10 \iM before treatment with high glucose.RESULTSExposure of aorta rings to high glucose (25 mM) for 24 h caused amarked decrease in endotheliun-dependent relaxation. Pretreatment with 3,4,5,6-tetrahydroxyxanthone (1, 3 or 10 \iM) or probucol (10 \iM) significantly improved endotheliun-dependent relaxation in a dose-dependent manner. Exposure of HUVEC to high glucose (30 mM) for 48 h caused a marked increase in LDH release and MDA content in the medium and induced the apoptosis and the expression of Bcl-2 protein of HUVEC. Pretreatment with 3,4,5,6-tetrahydroxyxanthone (1, 3 or 10 |xM) or probucol (10 \iM) significantly decreased the level of LDH and MDA in the medium, reduced the apoptosis, and increased the expression of Bcl-2 protein in HUVEC.CONCLUSIONThese results suggest that 3,4,5,6-tetrahydroxyxanthone possesses aprotective effect on endothelial cells, and that the protective effects of 3,4,5,6-tetrahydroxyxanthone are due to inhibition of cell apoptosis by increasing expression of Bcl-2 protein in HUVEC.