Quantitative analysis of effects of salvianic acid a combined with hydroxy safflower yellow a on rat endothelial cells after hypoxic injury using the combination index method

Abstract Cerebrovascular disease is the second most serious disease in the world. It has the features of high morbidity, high mortality and recurrence rate. Numerous research on the compatibility of Chinese medicine with effective ingredients of cerebral ischemia has been made during the past decades. The purpose of this study is to quantitatively analyze the combined pharmacological effect of effective ingredients in Danshen and Honghua (Dan Hong) on rat microvascular endothelial cells after gradually oxygen-glucose deprivation. The experimental concentration range for the compatibility of two effective ingredients were determined in the preliminary experiments by Cell Counting kit-8 (CCK-8) method. Drugs were added to rat brain microvascular endothelial cells at a non-toxic dose level. After that, the cells were cultured for 12 h, and placed in a hypoxic environment. Finally, the cell survival rate was used as a measure of drug effect. In order to determine synergism or antagonism, the combination index (CI)-isobologram method was performed to analyze the data from the experiments. Based on this theory, the potencies of each drug and the shapes of their does-effect curves are both taken into account. The results show that the synergism or the antagonism between two effective ingredients compatibility change with different proportion and dosage. Furthermore, it can be seen from the results of these experiments that when these drugs are used in combination, the dosage required to achieve the same therapeutic effects is greatly reduced compared with the case of single one. It is worth mentioning that our experiments also prove that the median-effect equation and the CI method can be applied in the field of traditional Chinese medicine.

Effects of Shenfu Yixin Decoction on Reactive Oxygen Species and Energy Metabolism of Primary Hypoxic Cardiomyocytes / 中国药房

OBJECTIVE： To observe the effects of Shenfu yixin decoction on reactive oxygen species （ROS） and energy metabolism in primary hypoxic cardiomyocytes. METHODS： After isolation， culture and identification， primary cardiomyocytes of neonatal SD rats were randomly divided into normal group， model group， positive control group （coenzyme Q10， 0.1 mmol/L） and Shenfu yixin decoction low-dose and high-dose groups （0.25， 0.5 mg/mL）. Except for normal group， other groups were cultured with 5％O2， 5％CO2 and 90％N2 for 6 h to induce hypoxic injury model. After 6 hours of hypoxia， ROS contents in cardiomyocytes and mitochondria of each group were detected by ROS probe and flow cytometry. Luciferase luminescence and Western blotting were used to detect ATP content and CK protein expression of each group. Transmission electron microscope was used to observe ultrastructure of cardiomyocytes in each group. RESULTS： Compared with normal group， the expression of ROS in primary hypoxic cardiomyocytes and mitochondria as well as the content of ROS were increased significantly， while the content of ATP and expression levels of CK protein were decreased significantly （P＜0.05）； there were swelling of endoplasmic reticulum and mitochondria， dissolution or even disappearance of mitochondrial ridge， obvious cardiomyocytes injury. Compared with model group， the expression of ROS in primary hypoxic cardiomyocytes and mitochondria of administration groups， the contents of ROS in primary hypoxic cardiomyocytes of positive control group and Shenfu yixin decoction high-dose group as well as the content of ROS in primary hypoxic cardiomyocytes mitochondria of administration groups were all decreased significantly， while ATP contents in primary hypoxic cardiomyocytes of positive control group and Shenfu yixin decoction high-dose group as well as expression levels of CK protein in primary hypoxic cardiomyocytes of administration groups were all increased significantly （P＜0.05）. The primary hypoxic cardiomyocytes injury was relieved significantly in positive control group and Shenfu yixin decoction high-dose group. CONCLUSIONS： Shenfu yixin decoction can improve primary hypoxic cardiomyocytes， down-regulate the expression of ROS in cardiomyocytes and mitochondria and also improve its energy metabolism.

Protective effect of seven kinds of phenolic acids of total salvianolic acids components on human umbilical vein endothelial cells with hypoxic injury / 中国中药杂志

In this paper, human umbilical vein endothelial cells (HUVEC) hypoxic injury models were established by sodium dithionite (Na2S2O4). With the protective effects of total salvianolic acids components (TSAC) against oxidative damage of HUVEC as a starting point, cellviability was measured by MTT colorimetric method； intracellular superoxide dismutase (SOD) activity, malondialdehyde (MDA), lactatedehydrogenase (LDH) level, nitric oxide (NO) level, interleukin6 (IL-6), and human tumor necrosis factor α (TNF-α) expression were measured by kits, to investigate the effect of seven kinds of phenolic acids of TSAC on Na2S2O4-induced HUVEC hypoxic injury. Based on the "component structure" theory, the contribution of the main components in TSAC for the protective effect on hypoxic injury of HUVEC was studied. The results showed that salvianolic acid B, posrnarinic acid A, salvianolic acid A, lactic acid, and lithospermic acid in TSAC play a larger role in protective effect of hypoxic injury of HUVEC. These five components were administered in combinations respectively, and it was concluded that the four components including salvianolic acid B, posrnarinic acid A, salvianolic acid A, lactic acid could be used as the representative components of TSAC.

Effect of oroxylin A on blood-brain barrier permeability induced by hypoxic injury in vitro and its mechanism / 中草药

Objective: To study the protective effect of oroxylin A on blood-brain barrier (BBB) permeability induced by hypoxic injury and its possible mechanism. Methods: The BBB model in vitro was established by primarily culturing brain microvascular endothelial cells (BMEC), and the hypoxic injury was induced. To observe the changes of BBB permeability, fluorospectrophotometry was used to detect the fluorescence intensity of oroxylin A on fluorescein isothiocyanate (FITC)-dextran in BMEC. The effects of oroxylin A on the mRNA expression of ZO-1 and claudin-1 were assayed by RT-PCR, and the effect on the ZO-1 protein expression was measured by Cell-Based ELISA. Results: Hypoxia significantly increased FITC-dextran permeability, decreased the expression of ZO-1 mRNA and protein, while oroxylin A could antagonize these effects caused by hypoxia. Conclusion: Oroxylin A has a protective effect on BBB permeability induced by hypoxia in vitro, and the mechanism is involved in the regulation of ZO-1 expression.

Evaluation of the Antenatal Fetal Brain Metabolites using Proton Magnetic Resonance Spectroscopy / 대한주산의학회잡지

OBJECTIVE:The purpose of this study was to compare the sensitivity of proton Magnetic Resonance Spectrography (MRS) for estimating absolute metabolite concentrations and ratio of fetal brains. METHODS:Between September 2005 and August 2006, our study was prospective single center trial and included 39 healthy women (Group 1: fetuses with risk factor of fetal distress or hypoxic damage [n=15], Group 2: fetal CNS anomalies on ultrasound [n=12], Group 3: normal fetuses [n=12]). We quantified resonances for the main proton MRS-detectable brain and calculated metabolite ratios of the three groups. We compared the obtained metabolite levels of the three groups with electronic fetal cardiotocography, Doppler ultrasound examination, Apgar score, and umbilical artery blood gas analysis. RESULTS:Abnormal amniotic fluid, abnormal Doppler studies, and abnormal cardiotocograms were significantly more prevalent in Group 1 compared with those of Group 2 and 3. In Group 1, choline (Cho) levels (7.86+/-3.51mmol/L) were significantly higher than in Group 2 or 3 (p=0.024). The ratios of N-acetylasparate (NAA)/creatinine-phosphocreatine (Cr) and Cho/Cr were increased whereas the ratios of NAA/Cho, lactate (Lac)/Cho, Lac/NAA, and Lac/Cr were decreased; however, there was no statistical significance. In patients who have oligohydramnios and absence of umbilical diastolic flow, choline and N-acetylasparate levels were significantly elevated (p<0.05, p<0.05, respectively). But, MRS metabolites and ratios showed no significant differences for low Apgar scores, umbilical arterial academia, uterine artery notching, maternal blood pressure or abnormal fetal cardiotocograms. CONCLUSION:This study demonstrates the possibility of performing proton MRS to assess the metabolic information of the fetal brain. Further technical progress may be useful of improving the degree of detection of hypoxic changes or an impending hypoxic state for prenatal diagnosis.

Effects of microtubule disassembly on hypoxic injury of cultured cardiomyocytes / 第三军医大学学报

Objective To investigate whether microtubule disassembly plays an important role in the pathogenesis of the opening of mitochondria permeability transition pore (MPTP) in hypoxic cardiomyocytes and the decrease of its activity, resulting in its hypoxic injury. Methods Neonatal rat cardiomyocytes in primary culture were randomized as normoxia group (A), hypoxic group (B), normoxia treated with microtubule destabilizing agent (Colchicine) group (C), hypoxia treated with microtubule stabilizing agent (Taxol) group (D). At 0.5, 1, 3, 6, 12 h after treatment, polymeric tubulin was detected by immunofluorescence and Western blotting, mitochondria permeability transition pore (MPTP) open by coloading with calcein AM and cobalt chloride, and the activity of cells by measuring the mitochondrial-dependent reduction of MTT to formazan. Results Early microtubule disassembly, MPTP open and activity decrease of cardiomyocytes in both groups B and C were observed at 0.5 h after treatment. These phenomena all became more and more significant with the prolongation of treatment. However, microtubule disassembly, MPTP open and activity decrease of cardiomyocytes of group D were significantly lower than those of group B. Conclusion Microtubule disassembly happened at 0.5 h after hypoxic treatment. Microtubule stabling agent Taxol and destabilizing agent Colchicine can regulate microtubule integrity efficiently. The microtubule damage plays an important role in the hypoxic injury of cardiomyocytes.

Effect of anti-digoxin antiserum on endogenous digitalis-like factor and membrane ATPase activity in hypoxic injury myocardium / 中国临床药理学与治疗学

To evaluate the protective effect of anti-digoxin antiserum on hypoxic injury myocardium and its mechanism.Methods It was observed that different concentration anti-digoxin antiserum effect on endogenous digitalis-like factor and cell membrane ATPase activity in hypoxic myocardium model.Results The level of endogenous digitalis-like factor was remarkably higher,cell membrane ATPase activity were remarkably lower in hypoxic group than those of normal group;anti-digoxin antiserum can resume membrane ATPase activity.Conclusion Rise of endogenous digitalis-like factor was basic of molecular biology of myocardial damage during myocardial hypoxia.Anti-digoxin antiserum has lightened myocardial injury and has protective effect on hypoxic myocardium by against effect of endogenous digitalis-like factor.

Effect of anti-digoxin antiserum on endogenous digitalis-like factorand membrane ATPase activity in hypoxic injury myocardium / 中国临床药理学与治疗学

Aim To evaluate the protective effect of anti_digoxin antiserum on hypoxic injury myocardium and its mechanism. Methods It was observed that different concentration anti_digoxin antiserum effect on endogenous digitalis_like factor and cell membrane ATPase activity in hypoxic myocardium model. Results The level of endogenous digitalis_like factor was remarkably higher, cell membrane ATPase activity were remarkably lower in hypoxic group than those of normal group; anti_digoxin antiserum can resume membrane ATPase activity.Conclusion Rise of endogenous digitalis_like factor was basic of molecular biology of myocardial damage during myocardial hypoxia. Anti_digoxin antiserum has lightened myocardial injury and has protective effect on hypoxic myocardium by against effect of endogenous digitalis_like factor.

Neuroprotective Effects of BAPTA-AM: A Dose-response Study and Estimation of Therapeutic Window

In central neurons, an excessive or sustained rise in the concentration of free cytoplasmic Ca2+ ions([Ca2+]i) after hypoxia may promote rapid neurodegeneration both in vitro and in vivo. Treating cells with Ca2+ chelating agents may prevent or delay a loss of cellular Ca2+ homeostasis after hypoxic injury and thus constitute an effective strategy for minimizing neuronal damage. Cell-permeant Ca2+ chelators such as 1,2-bis-(2-aminophenoxy) ethrane -N,N,N',N' -tetraacetic acid acetoxymethyl ester(BAPTA-AM) have shown evidence of neuroprotective effect against hypoxic neuronal injury. This study was designed to examine dose response and to estimate therapeutic window of BAPTA-AM for the recovery from hypoxia in vitro. Electrophysiological studies were made in CA1 neurons in rat hippocampal slices which were superfused with artificial cerebrospinal fluid(ASCF) in tissue chamber. Hypoxia was induced by replacement of 95% N2+5% CO2 from 95% O2+5% CO2 for 20min. Recovery from hypoxic injury was evaluated by using a percentage recovery of population spike. BAPTA-AM in concentration of 1, 10 and 50micrometer were administered to the artificial cerebrospinal fluid(ASCF) for 2 hours prior to hypoxia, simultaneous with hypoxia and after hypoxia. The experimental specimens were divided to seven groups and each group was compared to control ASCF group. Recovery of population spike after hypoxia was about 70% in control ASCF group, which was mild type hypoxic injury. BAPTA-AM in 10 micrometer concentration, when given just prior to hypoxia, enhanced recovery of poppulation spikes at 15 and 30min following reoxygenation(p<0.05), in comparison with control ASCF. BAPTA-AM had no neuroprotective acitvity when given after the onset of hypoxia. Also, BAPTA-AM in 1 and 50 micrometer concentration did not accentuate recovery of population spike after hypoxia. Dose response curve was inverted U-shape and the response was maximun in 10 micrometer concentration of BAPTA-AM.