Effect of Chaiqinchengqi decoction on sarco/endoplasmic reticulum Ca^(2+)-ATPase mRNA expression of pancreatic tissues in acute pancreatitis rats

AIM: To investigate the effect of Chaiqinchengqi decoction (CQCQD) on sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) mRNA expression of pancreatic tissues in acute pancreatitis (AP) rats. METHODS: Thirty Sprague-Dawley (SD) rats were randomized into control group, AP group and CQCQD group (n = 3 × 10). The rats in the CQCQD group were intragastrically administered with CQCQD (10 mL/kg every 2 h) after induction of AP by intraperitoneal injection of caerulein (50 μg/kg.h × 5) within 4 h. At 6 h after the induction of AP model, pancreatic tissues were collected for the pathological observation, mRNA extraction for determination of SERCA1 and SERCA2 mRNA expression or pancreatic acinar cell isolation for measurement of fluorescence intensity (FI) of intracellular calcium ion concentration [Ca2+]i. RESULTS: There was no expression of pancreatic SERCA1 mRNA in the control group and the AP group. The expression of pancreatic SERCA2 mRNA in the AP group was down-regulated (expression ratio = 0.536; P = 0.001) compared with the control group, while that in the CQCQD group was up-regulated (expression ratio= 2.00; P = 0.012) compared with AP group. The FI of intracellular [Ca2+] of pancreatic acinar cells in the AP group (138.2 ± 23.1) was higher than the C group (111.0 ± 18.4) and the CQCQD group (118.7 ± 15.2 ) (P < 0.05) and the pancreatic pathological score in the CQCQD group was lower than that in the AP group (5.7 ± 1.9 vs 9.2 ± 2.7, P < 0.05).CONCLUSION: CQCQD can up-regulate the expression of SERCA2 mRNA of pancreatic tissues, reduce intracellular calcium overload and relieve pancreatic tissue lesions.

The Effects of Etimicin and Gentamicin on Renal Endoplasmic Reticulum ^(45)Ca^(2+)-Uptake and Ca^(2+)-Mg^(2+)-ATPase Activity

Objective: To study the effects of etimicin (EM) and gentamicin (GM) on renal endoplasmic reticulum 45 Ca 2+ uptake and Ca 2+ Mg 2+ ATPase activity. Methods: Using 45 Ca 2+ incorporation technique and peacock blue spectrophotometry respectively. Results: EM and GM (≥3.4×10 4 mol·L 1 ) inhibited endoplasmic reticulum 45 Ca 2+ uptake (the rate of inhibition: ≥17.4% and ≥25.5%, respectively); EM and GM (3.4×10 2 mol·L 1 ) inhibited Ca 2+ Mg 2+ ATPase activity (the rate of inhibition: 24.2% and 29.2%, respectively). Conclusion: At high concentration, EM and GM elevated intracellular calcium which may be related to their nephrotoxicity.

Effects of combination of irbesartan and perindopril on calcineurin expression and sarcoplasmic reticulum Ca^(2+)-ATPase activity in rat cardiac pressure-overload hypertrophy

Aim： To observe effects of angiotensin （Ang） II receptor antagonist （ATI） irbesartan and angiotensin-converting enzyme （ACE） inhibitor perindopril on rat myocardium calcineurin expression and sarcoplasmic reticulum Ca^2＋-ATPase activity in the model of pressure-overload cardiac hypertrophy. Methods： Forty male adult Sprague Dawley rats were divided into 5 groups One group was treated by sham operation; four groups were myocardium hypertrophy cases caused by banding aortic above renal artery. Drugs were given one week after operation. Group 1： sham group, rats （n=8） were gavaged with normal saline 2 ml/（kg·d） （ig）; Group 2： control group, rats （n=8） were treated with normal saline 2 ml/（kg·d） （ig）; Group 3： rats （n=8） were given perindopril 2 mg/（kg·d） （ig）; Group 4： rats （n=8） were treated with irbesartan 20 mg/（kg·d） （ig）; Group 5： rats （n=8） were given irbesartan 20 mg/（kg·d） plus perindopril 2 mg/（kg·d） （ig）. Morphometric determination, calcineurin expression and sarcoplasmic reticulum Ca^2＋-ATPase activity were done at the end of 6 week of drug intervention. Expression of calcineurin in myocardium was detected by immunohistochemistry. Results： Left ventricular mass index （LVMI）, transverse diameter of myocardial cell （TDM）, calcineurin activity were remarkably decreased after drug intervention and this decrease was most remarkable in the combination drug therapy group. Sarcoplasmic reticulum Ca^2＋-ATPase activity was increased after drug intervention, especially in the combined drug therapy group. Calcineurin expression in myocardium was remarkably decreased after drug intervention. LVMI was positively correlated with TDM and calcineurin, negatively correlated with sarcoplasmic reticulum Ca^2＋-ATPase. Conclusion： These data suggest that irbesartan and perindopril inhibit cardiac hypertrophy through the increased activity of sarcoplasmic reticulum Ca^2＋-ATPase and decreased expression of calcineurin. Their combination had better effects on regressing of ventricular hypertrophy.

Ca^(2+)-ATPase参与植物耐盐性调控的研究进展

盐胁迫下细胞质Ca^(2+)浓度升高,细胞会激活Ca^(2+)调节的靶酶或者与Ca^(2+)高度亲和的受体蛋白,其中,与Ca^(2+)高度亲和的受体蛋白中,植物钙泵(Ca^(2+)-ATPase)是P型ATP酶,包含内质网Ca^(2+)-ATPases与质膜Ca^(2+)-ATPas‐es,通过主动运输将Ca^(2+)从细胞质转移到质外体或细胞器。大量研究表明,植物的耐盐性在很大程度上与其维持钙泵即Ca^(2+)-ATPase活性的能力有关。多种植物Ca^(2+)-ATPase对盐胁迫表现出敏感性,并受到外源Ca^(2+)的保护,表明外源钙处理与Ca^(2+)-ATPase活性可能在盐胁迫下的细胞内钙稳态和信号转导中起重要作用。该研究概述了植物Ca^(2+)-ATPase类型、结构与性质,亚细胞定位Ca^(2+)-ATPase及外源钙与亚细胞定位Ca^(2+)-ATPase参与植物耐盐调控研究进展,重点对质膜、液泡膜、核膜、内质网及高尔基体Ca^(2+)-ATPases参与植物耐盐调控的研究进展进行了综述,并提出展望。该研究为了解植物耐盐性生理及分子机制提供帮助,同时为作物耐盐栽培提供新思路。

Effects of fructose-1,6-diphosphate on concentration of calcium and activities of sarcoplosnic Ca^(2+)-ATPase in cardiomyocytes of Adriamycin-treated rats

Objective: To observe the effects of fructose-1,6-diphosphate (FDP) on serum levels of cardiac troponin 1 (cTnl) and creatine kinase-MB (CK-MB), as well as the concentration of calcium in cardiomyocytes (Myo[Ca2+]) and activity of sarcoplosnic Ca2+-ATPase (SRCa2+-ATPase) in Adriamycin (ADR)-treated rats. Methods: Rats were intraperitoneally injected with ADR (2.5 mg/kg every other day for 6 times) and then with different dosages of FDP (every other day for twenty-one times). Bi-antibodies sandwich Enzyme linked immune absorption assay (ELISA) was performed to detect serum level of cTnl. CK-MB was detected by monoclonal antibody, Myo[Ca2+] was detected by fluorescent spectrophotometry and the activity of SRCa2+-ATPase was detected by inorganic phosphate method. Results: FDP (300, 600, 1200 mg/kg) significantly reduced the serum levels of cTnl and CK-MB, while at the same time decreased calcium concentration and increased SRCa2+-ATPase activity in cardiomyocytes of ADR-treated rats (P<0.01). Conclusions: FDP might alleviate the cardiotoxic effects induced by ADR through decreasing calcium level as well as increasing SRCa2+-ATPase activity in cardiomyocytes.

Phospholamban Antisense RNA Improves SR Ca^(2+)-ATPase Activity and Left Ventricular Function in STZ-induced Diabetic Rats

Objective To study the effect of phospholamban antisense RNA （asPLB） on sarcoplasmic reticulum Ca2＋-ATPase activity and cardiac function in rats with diabetes mellitus （DM） mediated by recombinant adeno-associated virus （rAAV） vector. Methods Six weeks after the induction of DM by streptozotocin injected intraperitoneally, the rats were divided into three groups, namely： DM-rAAV-asPLB group, DM-saline group and DM group （control group）. The rats in the DM-rAAV-asPLB group were intramyocardially injected with rAAV-asPLB, the rats in the DM-saline group were injected with saline, and those in the control group did not receive any treatment. Six weeks after gene transfer, the expressions of PLB protein and PLB phosphorylation were detected by Western-blot, while the activity of sarcoplasmic reticulum （SR） Ca2＋-ATPase and left ventricular function were measured. Results The PLB protein expression level was significantly higher whereas the PLB phosphorylation, SR Ca2＋-ATPase activity and left ventricular function were significantly lower in the DM-saline group than in the control group. No significant difference was found in PLB protein expression level, PLB phosphorylation or SR Ca2＋-ATPase activity between the DM-rAAV-asPLB group and the control group. The left ventricular function in the DM-rAAV-asPLB group was poorer than in the control group and was better than in the DM-saline group. Conclusion rAAV-asPLB can down-regulate PLB protein expression and up-regulate phosphorylation and SR Ca2＋-ATPase activity, thus contributing to the improvement of in vivo ventricutar function. PLB left

内质网相关降解系统通过Ca^(2+)调控肝细胞线粒体能量代谢活性

肝细胞含有丰富的内质网和线粒体,两者以相互作用的方式调控肝细胞生命活动,但相互作用的机制尚未完全阐明。内质网中存在一套高度保守的蛋白质质量控制系统,即内质网相关降解(endoplasmic reticulum associated degradation,ERAD)。最新研究发现,ERAD可通过线粒体相关膜性结构(mitochondria associated membranes,MAMs)调控脂肪细胞线粒体功能,但在肝细胞中,关于ERAD与线粒体之间的相互作用及机制目前仍不明确。本研究选用HepG2作为细胞模型,通过化学药物诱导和基因敲除2种方法构建ERAD功能障碍肝细胞模型。采用荧光标记、流式分析和免疫印迹等技术手段,探究ERAD在肝细胞线粒体中的作用。结果发现,ERAD功能障碍会降低HepG2肝细胞线粒体膜电位和ATP水平(P<0.01);而且,ERAD功能障碍还会破坏HepG2细胞内质网与线粒体Ca^(2+)稳态,当减少内质网Ca^(2+)释放时,可改善线粒体能量代谢功能(P<0.05)。这些发现在ERAD功能障碍小鼠原代肝细胞中得到进一步验证。上述结果表明,ERAD在肝细胞内质网与线粒体相互作用中具有重要的调控作用,增强ERAD功能可能提升肝细胞线粒体活性,为线粒体功能障碍相关疾病提供新的研究思路。

当归补血汤通过调控Ca^(2+)-SERCA2a抑制肥厚型心肌病大鼠内质网应激反应改善心肌损伤

目的观察当归补血汤对肥厚型心肌病(HCM)大鼠心室重构及心功能的改善作用,初步探讨Ca^(2+)-SERCA2a在其中的调控机制,为临床中药复方防治HCM提供理论依据。方法 SPF级SD大鼠30只,随机分为假手术组(Sham组),肥厚性心肌病模型组(HCM组),肥厚性心肌病模型+当归补血汤干预组(DGBXT组),采用主动脉缩窄法建立HCM大鼠模型,超声心动图评价大鼠心脏结构及心功能变化,HE、Masson染色观察大鼠心肌组织病理学改变,ELISA检测氧化应激因子表达,免疫荧光观察心肌组织中ROS表达,Western Blot检测心肌组织中内质网应激相关蛋白GRP78、Caspase-12、SERCA2a表达。结果当归补血汤可以抑制大鼠心室重构,提高LVEF、LVFS水平,缓解心肌细胞损伤及心肌纤维,同时上调SOD、Cat、GRP78、SERCA2a表达,下调MDA、ROS、Caspase-12表达。结论当归补血汤可以显著改善HCM大鼠心室重构及心功能损伤,其作用机制可能与其调控Ca^(2+)-SERCA2a抑制HCM大鼠内质网应激反应有关。

线粒体及内质网对铅引起的[Ca^(2+)]_i升高的作用

目的 研究线粒体、内质网Ca2 + ATP酶以及Na+ 和K+ ATP酶活力的改变对铅引起的 [Ca2 + ] i 升高的调节作用 ,探讨铅引起的 [Ca2 + ] i 增高对学习记忆功能的影响。方法 Wistar大鼠神经肉瘤C6 细胞培养于含醋酸铅的培养液中 ,于不同时间终止染铅 ,检测 [Ca2 + ] i 及线粒体、内质网Ca2 + ATP酶、Na+ 和K+ ATP酶活力。结果 (1) 0 2 μmol L染铅组 :[Ca2 + ] i 轻度升高 ,线粒体Ca2 + ATP酶、Na+ 和K+ ATP酶活力升高 ,内质网酶活力略增高 ;(2 ) 1 0 μmol L染铅组 :[Ca2 + ] i 于染铅后 0 5h升至最高 ,以后逐渐降低 ,波动于 160～ 190nmol L之间 ;线粒体、内质网Ca2 + ATP酶、Na+ 和K+ ATP酶于染铅后 0 5h升至最高 (分别为未染铅前酶活力的 2 9 1、3 9 2、10 8和 19 8倍 ) ,2d后降至接近正常水平。结论(1)铅可使Wistar大鼠神经肉瘤C6 细胞Ca2 + 浓度及线粒体、内质网Ca2 + ATP酶、Na+ 和K+ ATP酶活力增高 ;(2 )当[Ca2 + ] i 增高不显著时以线粒体Ca2 + ATP酶、Na+ 和K+ ATP酶活力增高为主 ;当 [Ca2 + ] i 急剧升高时 ,线粒体、内质网Ca2 + ATP酶、Na+ 和K+ ATP酶活力均明显增高 ,尤其是线粒体Ca2 + ATP酶、Na+ 和K+ ATP酶活力增高更为显著。

药物诱导后的人大肠癌细胞的 [Ca^(2+)]_i 的变化

采用荧光分光光度计法检测维甲酸（ＲＡ）、１，２５（ＯＨ）２ＶＤ３及佛波酯（ＰＭＡ）诱导ＣＣＬ２２９细胞分化后［Ｃａ２＋］ｉ变化，并观察内质网（ＥＲ）特异的Ｃａ２＋－ＡＴＰａｓｅ抑制剂Ｔｈａｐｓｉｇａｒｇｉｎ（ＴＧ）、ＩＰ３受体抑制剂Ｈｅｐａｒｉｎ对ＲＡ诱导［Ｃａ２＋］ｉ变化的影响，从而探讨ＲＡ诱导［Ｃａ２＋］ｉ变化与ＥＲ的关系。结果显示：ＲＡ和１，２５（ＯＨ）２ＶＤ３在数秒内引起［Ｃａ２＋］ｉ显著升高。在ＥＧＴＡ和Ｖｅｒａｐａｍｉｌ预处理细胞条件下，ＴＧ不能抑制ＲＡ引起Ｃａ２＋从细胞内钙池中外流，ＲＡ作用后ＴＧ仍能升高［Ｃａ２＋］ｉ。另外，Ｈｅｐａｒｉｎ也不能完全抑制ＲＡ升高［Ｃａ２＋］ｉ。提示ＲＡ诱导大肠癌细胞升高［Ｃａ２＋］ｉ可能通过ＥＲ上ＩＰ３敏感性和非敏感性钙池，亦可能细胞内存在除ＥＲ外对ＲＡ敏感的钙池。

铅对大鼠神经肉瘤C_6细胞[Ca^(2+)]_i及内质网相关调节机制的影响

目的 :观察不同时间铅暴露后大鼠神经肉瘤 C6 细胞 [Ca2 +]i、内质网 Ca2 +- ATP酶、Na+,K+- ATP酶活性变化 ,探讨不同时间铅暴露对 [Ca2 +]i及内质网相关调节机制的影响。方法 :Wistar大鼠神经肉瘤 C6 细胞培养于含醋酸铅培养液中 ,分别于不同时间终止染铅 ,检测 [Ca2 +]i、内质网 Ca2 +- ATP酶、Na+,K+- ATP酶活性。结果 :( 1) 0 .2 μm ol/ L 染铅组 :[Ca2 +]i轻度升高 ,内质网 Ca2 +- ATP酶、Na+,K+- ATP酶活性缓慢增高 ,2 d达最高 (分别为未染铅前酶活性的 2 .9倍、5 .2倍 ) ;( 2 ) 1.0μmol/ L染铅组 :[Ca2 +]i 于染铅后 0 .5 h升至最高 ,以后逐渐降低 ,波动于 16 0～ 190 nm ol/ L 之间 ;内质网 Ca2 +- ATP酶、Na+,K+- ATP酶于染铅后 0 .5 h升至最高 (分别为未染铅前酶活性的 10 .8倍、19.8倍 ) ,两天后降至正常水平。结论 :铅可使 Wistar大鼠神经肉瘤 C6 细胞 [Ca2 +]i 及内质网Ca2 +- ATP酶、Na+,K+- ATP酶活性增高 ;内质网 Ca2 +- ATP酶、Na+,K+- ATP酶活性增高是急性铅暴露时维持[Ca2 +]i 相对稳定的重要机制。

爱大霉素和庆大霉素对胞浆Ca^(2+)影响的初步机制

目的 :探讨爱大霉素 (EM )和庆大霉素 (GM )对胞浆Ca2 +影响的初步机制。方法 :以Fura -2/AM为探针测定EM和GM在不同浓度时对LLC -PK1肾上皮细胞胞浆Ca2 +的影响 ,同位素示踪法测定EM和GM对线粒体Ca2 +摄取和内质网Ca2 +摄取的影响。结果 :EM和GM在1mmol/L时对胞浆Ca2 +浓度无显著影响 (P>0 05) ;在10mmol/L时可使胞浆Ca2 +显著上升 (P<0 01)。EM和GM在1mmol/L时显著促进线粒体Ca2 +摄取 (P<0 05) ;在10mmol/L时 ,二者显著抑制线粒体Ca2 +摄取。EM和GM在>3 4×10-1mmol/L时显著抑制内质网Ca2 +摄取 (P<0 05或P<0 01)。结论 :低浓度EM和GM未能引起胞浆Ca2 +升高 ,可能与其促进线粒体Ca2 +摄取与抑制内质网Ca2 +摄取相互平衡有关 ;高浓度EM和GM引起胞浆Ca2 +升高 ,可能与其均抑制线粒体和内质网Ca2

心肌肌浆网Ca^(2+)-ATP酶研究进展

在正常心肌舒张期,心肌肌浆网Ca2+-ATP酶(SERCA2a)将Ca2+从胞质中摄回肌浆网中,在心肌肥厚、心力衰竭等病理生理过程中有多条信号通路对SERCA2a进行调节,使其活性和表达量下调。通过转基因及药物治疗上调SERCA2a的表达,可能会成为心力衰竭等疾病治疗的新手段。

Total Glycosides of Ranunculus Japonius Prevent Hypertrophy in Cardiomyocytes via Alleviating Chronic Ca^(2+) Overload

Objective To evaluate the in vitro anti-hypertrophic effect of total Glycosides of Ranunculus Japonius （TGRJ）. Methods Neonatal rat cardiomyocytes were cultured and hypertrophy was induced by adminis- trating isoproterenol （ISO, 10 gmol/L） or angiotensin Ⅱ （AngⅡ, 1 gmol/L） for 48 hours. In the treatment groups, cells were pretreated with TGRJ （0.3 g/L） for 30 minutes prior to hypertrophic stimuli. The anti-hypertrophic effects of TGRJ were examined by measuring cell size, total protein content, and protein synthesis. Intracellular free Ca2＋ concentration （[Ca2＋]i） was evaluated using fluorescence dye Fura-2/AM. Sacroplasmic/endoplasmic reticulum Ca2＋ ATPase 2a （SERCA2a）, atrial natriuretic peptide （ANP）, B-type natriuretic peptide （BNP）, and beta-myosin heavy chain （β-MHC） protein expression levels were measured by Western blotting. SERCA2a activity was assayed by p-nitrophenal phosphate disodium salt hexahydrate method. Results Increased cell size, total protein content, and protein synthesis following ISO or Ang II stimulation were significantly inhibited by pretreatment with TGRJ （all P〈0.05）. This anti-hypertrophic effect of TGRJ was confirmed by its suppressing effect on elevated expression of the three hypertrophic related genetic markers, ANP, BNP, and ^-MHC. In addition, TGRJ inhibited ISO or Ang Ⅱ induced up-regulation of [Ca2＋] under chronic but not acute conditions. And ISO or Ang Ⅱ induced down-regulation of SERCA2a expression and activity was also effectively rectified byTGRJ pretreatment. Conclusions The results of present study suggested that TGRJ could prevent ISO or Ang Ⅱ induced cardiac hypertrophy through improving chronic [Ca2＋]i disorder, might via normalizing SERCA2a expression and activity.

Update on vascular endothelial Ca^(2+) signalling:A tale of ion channels,pumps and transporters

A monolayer of endothelial cells (ECs) lines the lumen of blood vessels and forms a multifunctional transducing organ that mediates a plethora of cardiovascular processes. The activation of ECs from as state of quiescence is, therefore, regarded among the early events leading to the onset and progression of potentially lethal diseases, such as hypertension, myocardial infarction, brain stroke, and tumor. Intracellular Ca2+ signals have long been know to play a central role in the complex network of signaling pathways regulating the endothelial functions. Notably, recent work has outlined how any change in the pattern of expression of endothelial channels, transporters and pumps involved in the modulation of intracellular Ca2+ levels may dramatically affect whole body homeostasis. Vascular ECs may react to both mechanical and chemical stimuli by generating a variety of intracellular Ca2+ signals, ranging from brief, localized Ca2+ pulses to prolonged Ca2+ oscillations engulfing the whole cytoplasm. The well-defined spatiotemporal profile of the subcellular Ca2+ signals elicited in ECs by specific extracellular inputs depends on the interaction between Ca2+ releasing channels, which arelocated both on the plasma membrane and in a number of intracellular organelles, and Ca2+ removing systems. The present article aims to summarize both the past and recent literature in the field to provide a clear-cut picture of our current knowledge on the molecular nature and the role played by the components of the Ca2+ machinery in vascular ECs under both physiological and pathological conditions.

SERCA2表达对哮喘大鼠气道平滑肌细胞分泌IL-8的影响

目的探讨哮喘大鼠气道平滑肌细胞(ASMCs)中肌浆/内质网Ca^2+-ATP酶(SERCA2)的表达对IL-8分泌的影响。方法建立卵清蛋白(OVA)诱导的哮喘大鼠模型,分离原代ASMCs,分别用qRT-PCR及Western blotting、Fura PE-3/AM和ELISA法检测各组ASMCs中SERCA2表达、细胞内Ca^2+浓度([Ca^2+]i)和IL-8,利用si RNA技术下调SERCA2、毒胡萝卜素(TSG)抑制SERCA2以及吡咯烷二硫氨基甲酸(PDTC)抑制NF-κB活化,检测不同状态下IL-8的变化。结果与正常组比较,哮喘组SERCA2的表达减少,经缓激肽(BK)刺激的钙瞬变峰值明显下降,需更长时间重新摄取钙使[Ca^2+]i恢复至基线水平,IL-8基础值和经IL-17刺激后的诱导值均增加(P<0.05或0.01)。无论有无IL-17刺激,基因下调组IL-8 m RNA表达和分泌均显著增加(均P<0.01),与哮喘对照组比较差异均无统计学意义(均P>0.05)。哮喘组IκBα磷酸化和NF-κB p65核转位均较正常组增加(P<0.05或0.01),基因下调组和TSG抑制组的NF-κB p65核转位亦增加(P<0.05或0.01),而使用PDTC后,基因下调组、哮喘对照组和正常对照组在IL-17刺激诱导下的IL-8 m RNA表达和分泌均减少(P<0.05或0.01)。结论哮喘大鼠ASMCs中SERCA2表达减少,导致钙稳态的改变,可能通过增强NF-κB活化来介导IL-8分泌亢进。

Ca2+ influx and ROS generation trigger CDDO-Me-induced dilation of the ER and apoptosis in breast cancer cells

OBJECTIVE The synthetic triterpenoid 2-cyano-3,12-dioxoolean-1,9(11)-dien-C28-methyl ester(CDDO-Me)is considered a promising anti-tumorigenic compound.In this study,we investigated the anti-cancer effect of CDDO-Me on breast cancer cells and its underlying mechanisms.METHODS To investigate the effect of CDDO-Me on various breast cancer cells,cell viability assay using calcein-AM and EthD-1 as well as MTT assay was performed.To clarify the origin of CDDO-Me-induced vacuoles,electron microscopy as well as fluorescence microscopy using YFP-ER or YFP-Mito construct was performed.To measure the changes in intracellular Ca2+and ROS levels,flow cytometry using Fluo-3 and H2DCF-DA was performed.RESULTS CDDO-Me treatment induces progressive ER-derived vacuolation and subsequent apoptosis in various breast cancer cells.CDDO-Me-induced increases in intracellular Ca2+ levels,reflecting influx from the extracellular milieu,make a critical contribution to ER-derived vacuolation and subsequent cell death.In parallel with increasing 2+ Calevels,CDDO-Me markedly increases the generation of reactive oxygen species(ROS).Interestingly,we found that there exists a reciprocal positive-regulatory loop between Ca2+ influx and ROS generation that triggers ER stress and ER dilation in response to CDDO-Me.CONCLUSION ER-derived vacuolation via Ca2+ influx and ROS generation is responsible for the potent anticancer effects of CDDOMe on breast cancer cells.

肌质网钙泵SERCA基因在中华绒螯蟹钙离子调控中的功能及其对蜕壳的影响

中华绒螯蟹的蜕壳周期中具有高度复杂的钙调节机制,本研究探索了水体中不同Ca2+浓度(40、80、160和320 mg/L)对中华绒螯蟹的Ca^(2+)信号转导的影响,并对其中的关键因子肌质网钙泵(SERCA)进行基因干扰以探索其功能。结果显示:①养殖水体高浓度Ca^(2+)可致Y器官和鳃组织中Ca^(2+)信号通路相关基因SERCA、PMCA、RyR和NCX相对表达量上升,眼柄中MIH表达量下降,血淋巴中Ca^(2+)与蜕皮激素水平上升,说明高浓度的Ca^(2+)可促进中华绒螯蟹蜕壳。②利用RNAi技术干扰SERCA基因表达,普通水体和加入320 mg/L Ca^(2+)的水体中中华绒螯蟹Y器官中PMCA和NCX基因表达量上升、RyR基因表达量下降,且细胞内Ca^(2+)浓度上升,表明SERCA基因在中华绒螯蟹维持细胞内Ca^(2+)浓度和细胞内钙稳态机制中发挥重要作用。③长期干扰SERCA基因的表达可延长中华绒螯蟹的蜕壳间隔,且降低增重率与存活率。然而,高钙环境下长期干扰SERCA基因,相对于普通水体,蜕壳间隔缩短,存活率降低。研究表明,SERCA基因可能是通过对Ca^(2+)信号的干扰而影响中华绒螯蟹的蜕壳。本实验将为中华绒螯蟹蜕壳机制的进一步研究提供基础参考。

Transmembrane Ca^(2+) gradient-mediated phosphatidylcholine modulating sarcoplasmic reticulum Ca^(2+)-ATPase

The sarcoplasmic reticulum (SR) Ca2+-ATPase was purified and reconstituted into the sealed phospholipids vesicles with or without transmembrane Ca2+ gradient. The role ofphospholipids, especially phosphatidylcholine(PC), in the modulation of Ca2+-ATPase by transmembrane Ca2+ gradient was investigated. The results are as follows, (i) Incubated with phospholiplds, the enzyme activity of the delipidated Ca2+-ATPase is inhibited by Ca2+ and the highest inhibition is observed in the presence of PC. (ii) When there exists a transmembrane Ca2+ gradient (higher Ca2+ concentration inside vesicles, 1 000μmol/L:50μmol/L, similar to the physiological condition), the inhibition of Ca2+-ATPase by transmembrane Ca2+ gradient can be only observed in the vesicles containing PC:PE, but not in those containing PS:PE or PG:PE. The highest inhibition is obtained at a 50.50 molar ratio of PC:PE. (iii) By comparing the effects of PC differing in acyl chains, higher inhibition of Ca2+-ATPase is observed in vesicles containing DPPC:PE and DOPC:PE, while no inhibition in DMPC:PE vesicles (iv) If the transmembrane Ca2+ gradient is in the inverse direction, the enzyme activity of Ca2+-ATPase is inhibited whenever reconstituted with acidic or neutral phospholipids.

黄芪总皂甙对病毒性心肌炎小鼠慢性期SERCA活性的影响

目的 探讨黄芪提取物 -黄芪总皂甙对柯萨奇病毒B3(CVB3)感染的病毒性心肌炎(VMC)小鼠慢性期心肌肌浆网钙泵 (SERCA)活性变化的影响。方法 将BALB/C小鼠分为正常组(31只 )、模型组 (36只 )及黄芪组 (2 6只 ) ,模型组及黄芪组每只小鼠以 2× 10 4 TCID50 CVB3(TCID50 为10 -5 83)腹腔注射感染。黄芪组每鼠每天腹腔注射 0 15ml黄芪总皂甙注射液 ,正常及模型组予同体积的生理盐水。 3个月后 ,观察各组小鼠死亡率及心肌病理改变 ,检测血清心肌肌钙蛋白I(cTnI)含量及心肌组织中SERCA活性。结果 (1)和正常组、黄芪组相比 ,模型组小鼠死亡率显著增高 (P <0 0 5～ 0 0 0 1)。 (2 )感染 3个月后模型组、黄芪组心肌肌钙蛋白I(cTnI)及心肌病理改变与正常组无显著性差异 (P >0 0 5 )。 (3)模型组心肌SERCA活性显著低于正常组及黄芪组 (P <0 0 1)。结论 黄芪总皂甙可以降低VMC小鼠的死亡率 。