Herba Cistanche extract enhances mitochondrial glutathione status and respiration in rat hearts,with possible induction of uncoupling proteins

Herba Cistanche, a Chinese herb derived from the whole plant of Cistanche deserticola Y.C. Ma (Orobanchaceae), has been shown to enhance mitochondrial ATP generation and to protect against myocardial ischemia/reperfusion (I/R) injury ex vivo in rats. To define the role of mitochondria in the cardioprotective action of Herba Cistanche, we investigated the effect of Herba Cistanche treatment on mitochondrial glutathione status and functional parameters in rat hearts. Treatment with a methanol extract of Herba Cistanche enhanced mitochondrial glutathione status, decreased mitochondrial Ca²⁺ content, and increased mitochondrial membrane potential. In addition, an increase in State 4 respiration, indicative of uncoupled respiration, was observed in mitochondria isolated from Herba Cistanche-treated rat hearts. The enhancement of mitochondrial glutathione status and functional ability, as well as the putative induction of uncoupling proteins, may be related to cardioprotection afforded by Herba Cistanche treatment protecting against I/R injury.     

全国肉苁蓉饮片专项抽验情况分析与研究

目的：通过全国中药材饮片专项抽验工作,了解肉苁蓉饮片市场整体质量现状及存在的问题,为相关部门实施监管措施提供技术支持和依据,完善肉苁蓉检验标准,保障公众用药安全。方法：从全国30个省级行政区抽取肉苁蓉饮片样品,按照《中华人民共和国药典》2015年版一部肉苁蓉项下规定开展检验工作,出具检验报告,分析检验数据以评价肉苁蓉饮片市场情况。针对检验中发现的问题,采用实地调研、生药学鉴定等技术开展探索性研究。结果与结论：本次抽验的肉苁蓉样品共计171批次,按现行质量标准检验合格159批,不合格12批,均为【性状】不合格,经探索性研究鉴定不合格样品为伪品沙苁蓉片或掺有沙苁蓉片的肉苁蓉饮片样品。     

基于体外药效学结合分子对接技术研究穿山龙抗心肌缺血的作用机制

目的 通过液质联用技术、体外药效学及分子对接技术探究穿山龙抗心肌缺血的物质基础和作用机制。方法 利用UPLC-Q TOF-MS技术确定穿山龙醇提物中的主要成分,比较不同质量浓度（40、10、4、0.4、0.004 mg/mL）的穿山龙醇提物对H9c2心肌细胞缺氧/复氧（H/R）损伤后细胞活力的影响。应用AutoDock软件对潜在药效物质和关键靶点进行分子对接,相关结果采用Cytoscape 3.6.1进行构图和网络拓扑结构分析。结果 从穿山龙的醇提物中鉴定出7个化学成分,并证明穿山龙醇提物对H/R损伤H9c2心肌细胞具有一定的保护作用。筛选出5种（薯蓣皂苷元、薯蓣皂苷、对羟基苄基酒石酸、原薯蓣皂苷、薯蓣次皂苷A）与抗心肌缺血相关的活性成分,5种活性成分的分子对接结果显示潜在的药效物质与关键靶点内皮一氧化氮合酶（NOS）2、NOS3、环氧化酶/细胞色素C氧化酶多肽Ⅱ(COX2)、热休克蛋白（HSP70）对接结果能量低于-8 kcal/mol。结论 穿山龙中的甾体皂苷类成分可能是其治疗心肌缺血的物质基础,穿山龙治疗心肌缺血是多成分与多靶点相互作用的结果。     

Methylamine irisolidone,a novel compound,increases total ATPase activity and inhibits apoptosis in vivo and in vitro

We propose to further research the protective effect of MMI on myocardium ischemic rat model and H9c2 cells that underwent cell apoptosis induced by hypoxia. We established the myocardium ischemic rat model via the cardiac surgical procedures in vivo and treated the model rats with different concentration of MMI. In vitro, with the pretreatment of MMI for 12 h in the model of Na₂S₂O₄-induced hypoxia injury, the H9c2 cells viability was determined by MTT assay. We found that MMI had significantly improved cardiac function of the myocardial ischemia, and significantly decreased the reactive oxygen species level. The expression of P53, Bcl-2, Bax, and caspase-9 was also induced by MMI. In vitro study revealed a concentration-dependent increase in cell viability associated with MMI pretreatment. Annexin V-FITC and PI staining results showed that MMI had a preventive effect on hypoxia-induced apoptosis in H9c2 cells. MMI also inhibited the mitochondrial membrane potential decrease and increased total ATPase activity during hypoxia in H9c2 cells. In conclusion, MMI can enhance the cardiac function in myocardial ischemic rat and increase cell viability and attenuate the apoptosis in H9c2 cells induced by hypoxia, which was associated with inhibiting MMP decreasion and increasing total ATPase activity.     

制草乌配方颗粒与饮片煎剂对H9c2心肌细胞毒性的比较研究

目的 比较相同生药量浓度下制草乌中药配方颗粒与饮片煎剂对H9c2心肌细胞毒性的差异。方法 以不同浓度制草乌配方颗粒与同批次原料饮片煎剂作用于H9c2心肌细胞,MTT法检测细胞活性;Hoechest 33258荧光染色法检测细胞核形态并半定量统计分析;乳酸脱氢酶（lactate dehydrogenase,LDH）试剂盒测定细胞LDH释放率。结果 H9c2细胞在制草乌饮片煎剂的干预下,高剂量组细胞活力下降,LDH释放率提高,且呈浓度依赖,较配方颗粒组有显著性差异;Hoechst 33258荧光染色下可见细胞核固缩碎裂等,相同浓度下饮片煎剂组荧光强度强于配方颗粒组（P<0.05）。结论 在相同生药浓度下,制草乌中药配方颗粒对H9c2心肌细胞的毒性显著小于制草乌饮片煎剂。     

鸭跖草总黄酮的大孔树脂纯化工艺及抗氧化活性研究

【】目的：研究鸭跖草(Commelina communis L.)总黄酮的大孔树脂纯化工艺及其抗氧化活性。方法：以静态饱和吸附量和解析率为指标,对3种大孔树脂(D101,NKA-9,AB-8)进行筛选,并以回收率为指标,通过选用L₉(3₄)正交表设计实验,确立纯化总黄酮的最佳条件;以V_C为对照品,测定鸭跖草总黄酮清除1,1-二苯基-2-苦肼基自由基(DPPH)、超氧阴离子(O_2- )和羟基自由基(.OH)的抗氧化活性。结果：AB-8纯化鸭跖草总黄酮效果最好,优于D101和NKA-9,最佳纯化工艺：上梯液质量浓度为1.482mg/ml,淋洗pH为4,乙醇洗脱液体积分数70％,洗脱流速为2ml/min;鸭跖草总黄酮对3种自由基的清除效率与浓度呈正相关,清除DPPH、超氧阴离子自由基、羟自由基的半数抑制浓度(IC₅₀)分别为0.0170mg/ml、0.0055mg/ml、1.9327mg/ml,对DPPH和羟自由基的清除作用弱于Vc,而对超氧阴离子的清除作用强于Vc,其中鸭跖草对超氧阴离子自由基的清除能力最强。结论：大孔树脂纯化鸭跖草总黄酮的效果显著,鸭跖草黄酮类化合物具有较强的抗氧化活性,值得进一步开发。     

NDUFA4L2 protects against ischaemia/reperfusion‐induced cardiomyocyte apoptosis and mitochondrial dysfunction by inhibiting complex I

Myocardial ischaemia/reperfusion (I/R) injury may cause the apoptosis of cardiomyocytes as well as mitochondrial dysfunction. The aims of the present study were to investigate whether NADH dehydrogenase 1 alpha subcomplex subunit 4‐like 2 (NDUFA4L2) on myocardial ischaemia‐reperfusion (I/R) injury and the underlying molecular mechanism. The hypoxia‐reperfusion (H/R) model was established in vitro using H9c2 cells to simulate I/R injury. NDUFA4L2 and complex I expression levels were detected using RT‐PCR and western blot. The apoptosis of H9c2 cells was evaluated by flow cytometry and the expression of Bax and Bcl‐2 was detected by western blot. The mitochondrial function was assessed by ATP concentration, mPTP opening and cytochrome c (cyto C) expression. Our data indicated that NDUFA4L2 expression was significantly down‐regulated in myocardial H/R injury. Overexpression of NDUFA4L2 led to a dramatic prevention of H/R‐induced apoptosis accompanied by a decrease in the expression of Bax and an increase in the expression of Bcl‐2. Meanwhile, augmentation of NDUFA4L2 dramatically prevented mitochondrial dysfunction caused by H/R as reflecting in the increased ATP concentration, delayed mPTP opening, as well as down‐regulated cyto C expression. Moreover, complex I activation was heightened and negatively regulated by NDUFA4L2. Silencing complex I conspicuously attenuated cell apoptosis and mitochondrial dysfunction. Taken together, our findings demonstrated that NDUFA4L2 protects against H/R injury by preventing myocardium apoptosis and mitochondrial dysfunction via the complex I, and may be a potential therapeutic approach for attenuating myocardial I/R injury.     

Isolation and characterization of α-(1→6)-glucans from Cistanche deserticola

Three unique polysaccharides (1–3) have been obtained from the 0.5 M NaOH extract of the stem of Cistanche deserticola Y. C. Ma. The results of methylation analysis, partial acid hydrolysis, ¹³C, ¹H NMR, ¹H–¹H COSY, HMQC and HMBC spectroscopic analyses indicate that they are all composed of glucose, having a backbone of α-(1 → 6)-glucan, and have different molecular weights. Their structures differ from that of linear starch.     

Protective effects of Sesamum indicum extract against oxidative stress induced by vanadium on isolated rat hepatocytes

Vanadium toxicity is a challenging problem to human and animal health with no entirely understanding cytotoxic mechanisms. Previous studies in vanadium toxicity showed involvement of oxidative stress in isolated liver hepatocytes and mitochondria via increasing of ROS formation, release of cytochrome c and ATP depletion after incubation with different concentrations (25–200 µM). Therefore, we aimed to investigate the protective effects of Sesamum indicum seed extract (100–300 μg/mL) against oxidative stress induced by vanadium on isolated rat hepatocytes. Our results showed that quite similar to Alpha‐tocopherol (100 µM), different concentrations of extract (100–300 μg/mL) protected the isolated hepatocyte against all oxidative stress/cytotoxicity markers induced by vanadium in including cell lysis, ROS generation, mitochondrial membrane potential decrease and lysosomal membrane damage. Besides, vanadium induced mitochondrial/lysosomal toxic interaction and vanadium reductive activation mediated by glutathione in vanadium toxicity was significantly (P < 0.05) ameliorated by Sesamum indicum extracts. These findings suggested a hepato‐protective role for extracts against liver injury resulted from vanadium toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 979–985, 2016.     

Reactive oxygen species-independent rapid initiation of mitochondrial apoptotic pathway by chelerythrine

Chelerythrine, formerly identified as a protein kinase C inhibitor, has also been shown to inhibit the anti-apoptotic Bcl-2 family proteins. However, recent studies have now demonstrated that chelerythrine can induce the loss of mitochondrial membrane potential (ΔΨ_m), a membrane permeability transition (MPT), and the subsequent activation of the mitochondrial apoptotic pathway, even in the cells deficient in Bax and Bak. This suggests the existence of an alternative Bax/Bak-independent pathway for apoptosis. The generation of reactive oxygen species (ROS) from the mitochondrial electron transport chain (ETC) is also implicated in the cytotoxity elicited by chelerythrine. In our current study, we show that chelerythrine induces the rapid apoptotic death of H9c2 cardiomyocyte-derived cells within 8 min of treatment. The proteolytic activation of caspase9 and caspase3, crucial mediators of the mitochondrial apoptotic pathway, are also observed within 6 min of exposure to this drug. The generation of ROS is detected but at only marginal levels in the treated cells. The inhibition of the mitochondrial ETC by rotenone and malonate had almost no effects on ROS generation but in both cases effectively inhibited both cell death and the caspase activation induced by chelerythrine. Hence, chelerythrine initiates the rapid mitochondrial apoptotic death of H9c2 cardiomyoblastoma cells in a manner that is likely independent of the generation of ROS from mitochondria.     

The cardioprotective efficacy of TVP1022 in a rat model of ischaemia/reperfusion

BACKGROUND AND PURPOSE

Because myocardial infarction is a major cause of morbidity and mortality worldwide, protecting the heart from the ischaemia and reperfusion (I/R) damage is the focus of intense research. Based on our in vitro findings showing that TVP1022 (the S-enantiomer of rasagiline, an anti-Parkinsonian drug) possesses cardioprotective effects, in the present study we investigated the hypothesis that TVP1022 can attenuate myocardial damage in an I/R model in rats.

EXPERIMENTAL APPROACH

The model consisted of 30-min occlusion of the left anterior descending artery followed by 4 or 24 h reperfusion. In addition, we investigated the possible mechanisms of cardioprotection in H9c2 cells and neonatal rat ventricular myocytes (NRVM) exposed to oxidative stress induced by H₂O₂.

KEY RESULTS

TVP1022 (20 and 40 mg·kg⁻¹) administered 5 min before reperfusion followed by an additional dose 4 h after reperfusion reduced the infarct size and attenuated the decline in ventricular function. TVP1022 also attenuated I/R-induced deterioration in cardiac mitochondrial integrity evaluated by mitochondrial swelling capacity. In vitro, using H9c2 cells and NRVM, TVP1022 attenuated both serum free- and H₂O₂-induced damage, preserved mitochondrial membrane potential and Bcl-2 levels, inhibited mitochondrial cytochrome c release and the increase in cleaved caspase 9 and 3 levels, and enhanced the phosphorylation of protein kinase C and glycogen synthase kinase-3β.

CONCLUSIONS AND IMPLICATIONS

TVP1022 provided cardioprotection in a model of myocardial infarction, and therefore should be considered as a novel adjunctive therapy for attenuating myocardial damage resulting from I/R injuries.     

血根碱对脂多糖致H9c2细胞氧化损伤的改善作用

目的 观察血根碱（sanguinarine,SAN）对脂多糖（lipopolysaccharide,LPS）诱导的H9c2细胞氧化应激的影响,并对其与HO-1/NOX2途径之间的关系进行探讨。方法 不同因素干预H9c2细胞后,采用细胞活性试剂盒检测不同浓度SAN和/或LPS对H9c2细胞生存的影响;酶标仪及荧光显微镜检测SAN对LPS诱导细胞产生活性氧（reactive oxygen species,ROS）的影响; Real time RT-PCR检测SAN对LPS诱导的NOX2、P47PHOX、HO-1、SOD1、SOD2基因表达的影响; MDA检测试剂盒检测MDA的变化。结果 单用SAN对H9c2细胞活性无影响,LPS可显著降低细胞活性,而SAN与LPS共同干预则可呈浓度依赖性地提高细胞活性,降低ROS产生。LPS处理12 h上调H9c2细胞NOX2、p47phox mRNA表达、下调HO-1 mRNA表达; SAN预处理后,细胞内NOX2、p47phox mRNA下调、HO-1 mRNA上调,而SAN与锌原卟啉IX预处理则可逆转这种现象。SAN上调SOD1及SOD2 mRNA表达,降低MDA产生。结论 SAN可通过活化HO-1/NOX2途径,抑制ROS的产生,从而使H9c2细胞免受LPS介导的损伤,提高细胞活力。     

肉苁蓉的研究进展

近年来,肉苁蓉以其独特的医疗及保健功效在治疗疾病、保障健康中发挥着重要的作用。传统医学认为肉苁蓉具有补肾阳、益精血、润肠通便的作用,现代药理研究证明肉苁蓉及其有效成分具有抗疲劳、抗衰老、抗肿瘤、增强机体免疫力等药理作用,备受人们的关注,成为医药领域研究的热点之一。从肉苁蓉化学成分、提取方法、质量控制、药理作用等方面进行综述,为广泛深入研究该植物资源及临床应用提供参考。     

逍遥散对抑郁大鼠运动能力和肝脏线粒体的影响

目的 研究逍遥散对抑郁大鼠运动能力及肝脏线粒体结构和功能的影响。方法 将SD大鼠随机分为对照组、模型组、文拉法辛（给予盐酸文拉法辛胶囊35mg·kg^-1）组和逍遥散（生药21.2g·kg^-1）组,除对照组外,采用慢性不可预知温和应激（CUMS）建立抑郁模型,于造模同时ig给药,连续28d,每周称体质量。造模结束后进行旷场实验、糖水偏好实验、爬杆实验和转棒实验行为学测试;通过透射电镜观察肝脏线粒体超微结构;通过试剂盒测定血清睾酮、皮质酮水平和线粒体腺嘌呤核苷三磷酸（ATP）和线粒体呼吸链复合体（MRCC）I、III、IV、V水平。结果 与对照组比较,模型组大鼠体质量增长缓慢（P＜0.01）,糖水偏爱率、旷场实验的穿越格数及直立次数、爬杆实验得分和转棒实验在棒时间均显著下降（P＜0.05、0.01）;血清皮质酮水平显著增加（P＜0.05）,睾酮水平和睾酮-皮质酮比值（T/C）显著降低（P＜0.05、0.01）;大鼠肝脏线粒体数目减少,外膜模糊,基质疏松;肝脏线粒体ATP水平及MRCCⅠ、Ⅲ、Ⅳ、Ⅴ活性降低（P＜0.05、0.01）。与模型组比较,逍遥散组大鼠体质量显著增加（P＜0.05）,旷场实验穿越格数和直立次数增加（P＜0.05、0.01）,糖水偏好率显著增高（P＜0.05）,爬杆实验得分显著增加（P＜0.01）,转棒实验的在棒时间显著延长（P＜0.05）;血清睾酮水平显著升高（P＜0.05）,皮质酮水平显著降低（P＜0.05）,T/C显著升高（P＜0.01）;大鼠肝脏线粒体嵴清晰,结构较为完整;肝脏线粒体ATP水平和MRCCⅠ、Ⅴ活性显著增加（P＜0.05）。结论 逍遥散能够显著改善CUMS大鼠肝脏线粒体形态和功能,提高其运动能力,发挥抗抑郁作用。     

Effects of Khaya senegalensis Purinergic Transmission in the Rat Bladder

Abstract

The effects of a methanol extract of the stembark of Khaya senegalensis (Meliaceae) on smooth muscle of rat bladder have been investigated in vitro. The extract showed a dual agonist action. At concentrations lower than 1 × 10^?5 g/ml, the extract produced a dose-dependent relaxation of the rat bladder while greater extract concentrations produced a dose-dependent contraction. The relaxation was mediated by stimulation of adrenergic receptors and also by depression of the bladder by a direct action. The contraction was eliminated by 1 × 10^?5 g/ml quinacrine, indicating it was due to purinoc-eptor stimulation.     

羌活与宽叶羌活的GC/HPLC指纹图谱研究

目的：分别建立羌活与宽叶羌活的GC指纹图谱、HPLC指纹图谱,为羌活的挥发性及非挥发性成分质量评价与控制提供有效可靠方法。方法：采用GC、HPLC色谱法分别建立羌活与宽叶羌活的指纹图谱,通过相似度评价软件,综合GC、HPLC指纹图谱相似度结果对222批样品的挥发性及非挥发性成分进行质量分析。结果：建立的GC指纹图谱结果显示,羌活共有峰有10个,宽叶羌活共有峰有12个,二者共有成分有9个。HPLC指纹图谱结果显示,羌活共有峰有9个,宽叶羌活共有峰有7个,二者共有成分6个。其中,羌活醇与异欧前胡素在羌活和宽叶羌活中的含量存在明显差异。GC和HPLC指纹图谱结果均显示,222批样品中有7批伪品,有21批宽叶羌活样品,有118批羌活样品,有76批含有两种基原羌活的样品,二者结果一致。结论：建立的气相及液相指纹图谱能有效区分羌活的基原与真伪,并能够有效表征药材的质量优劣,可为羌活的整体质量评价及控制提供依据。     

Diazinon impairs bioenergetics and induces membrane permeability transition on mitochondria isolated from rat liver

ABSTRACT

Diazinon (DZN) is a broad-spectrum insecticide extensively used to control pests in crops and animals. Several investigators demonstrated that DZN produced tissue toxicity especially to the liver. In addition, the mitochondrion was implicated in DZN-induced toxicity, but the precise role of this organelle remains to be determined. The aim of this study was thus to examine the effects of DZN (50 to 150 μM) on the bioenergetics and mitochondrial permeability transition (MPT) associated processes in isolated rat liver mitochondria. DZN inhibited state-3 respiration in mitochondria energized with glutamate plus malate, substrates of complex I, and succinate, substrate of complex II of the respiratory chain and decreased the mitochondrial membrane potential resulting in inhibition of ATP synthesis. MPT was estimated by the extent of mitochondrial swelling, in the presence of 10 µM Ca²⁺. DZN elicited MPT in a concentration-dependent manner, via a mechanism sensitive to cyclosporine A, EGTA, ruthenium red and N-ethylmaleimide, which was associated with mitochondrial Ca²⁺ efflux and cytochrome c release. DZN did not result in hydrogen peroxide accumulation or glutathione oxidation, but this insecticide oxidized endogenous NAD(P)H and protein thiol groups. Data suggest the involvement of mitochondria, via apoptosis, in the hepatic cytotoxicity attributed to DZN.     

Analgesic and Anti-inflammatory Activities of Phyllanthus debilis. Whole Plant

Abstract

A petroleum ether extract of Phyllanthus debelis. Klein. ex Willd whole plant was subjected to analgesic and anti-inflammatory screening using various animal models. The extract exhibited significant anti-inflammatory activities in the acute carrageenan-induced rat paw edema and the chronic granuloma pouch models. However, it was devoid of analgesic activity in the tail-flick model.     

Mitochondrial impairment and cytotoxicity effects induced by the marine epibenthic dinoflagellate Coolia malayensis

Mitochondria was used to clarify the effects of Coolia malayensis strain UNR-02 crude extract by studying mitochondrial membrane potential (ΔΨ_m) generation and the fluctuations of ΔΨ_m associated with the induction of mitochondrial permeability transition (MPT). The cytoxicity of C. malayensis was also determined using both HepG2 and H9c2(2−1) cells. C. malayensis extract significantly depressed the oxidative phosphorylation efficiency, as was inferred from the perturbations in ΔΨ_m and in the phosphorylative cycle induced by ADP. Increased susceptibility to Ca²⁺-induced MPT was also observed. At the cellular level, the extract significantly decreased cell mass of both cell lines.