18β-Glycyrrhetinic Acid Improves Cardiac Diastolic Function by Attenuating Intracellular Calcium Overload

Summary:Ranolazine,a late sodium current inhibitor,has been demonstrated to be effective on heart failure.18B-glycyrrhetinic acid(18β-GA)has the similar inhibitory effect on late sodium currents.However,its effect on diastolic function is still unknown.This study aimed to determine whether 18β-GA can improve the diastolic function and to explore the underlying mechanisms.Eighty male Sprague Dawley(SD)rats of Langendorff model were randomly divided into the following groups:group A,normal cardiac perfusion group;group B,ischemia-reperfusion group;group C,ischemia-reperfusion with anemoniasulcata toxinⅡ(ATX-Ⅱ);group D,ranolazine group;and group E,18β-GA group with four different concentrations.Furthermore,a pressure-overloaded rat model induced by trans-aortic constriction(TAC)was established.Echocardiography and hemodynamics were used to evaluate diastolic function at 14th day after TAC.Changes of free intracellular calcium(Ca27)concentration was indirectly detected by laser scanning confocal microscope to confirm the inhibition of late sodium currents.With the intervention of ATX-Ⅱon ischemia reperfusion injury group,5 umol/L ranolazine,and 5,10,20,40μmol/L 18β-GA could improve ATX-I-induced cardiac diastolic dysfunction.630 mg/kg glycyrrhizin tablets could improve cardiac diastolic function in the pressure-overloaded rats.18B-GA and ranolazine had similar effects on reducing the free calcium in cardiomyocytes.The study demonstrates that 18B-GA and glycyrrhizin could improve diastolic dysfunction induced by ischemia-reperfusion injury in Langendorff-perfused rat hearts and pressure-overloaded rats.The mechanism may be attributed to the inhibition of enhanced late sodium currents.

Effects of Cornus Officinal Is Total Glycoside on Cardiomyocyte Apoptosis and Calcium Overload after Hypoxia/Reoxygenation Injury in Rats

Objective: To investigate the mechanism of Cornus officinalis Total Glycosides (COTG) on myocardial protection, by studying effects of COTG on cardiomyocyte apoptosis induced by hypoxia/reoxygenation and calcium concentration in rats. Methods: The myocardial cells of born 1-3d SD rats were isolated by enzyme digestion, cultured for 3 days. Cells were divided into five groups: Control group, H/R group, Cornus officinalis Total Glycosides low-dose group (LDG), Cornus officinalis Total Glycosides middle-dose group (MDG) and Cornus officinalis Total Glycosides high-dose group (HDG). Three drug groups were pretreated with different doses of Cornus officinalis Total Glycosides before hypoxia/reoxygenation treatment. The apoptotic rate was determined by flow cytometry assay, the intracellular free calcium concentration was examined by flow cytometry, and the ultrastructure of myocardial cells was observed under transmission electron microscope. Results: The results revealed that Cornus officinalis Total Glycosides pretreatment decreased apoptosis rate, but the effect of lower dosage is not significant. Furthermore, Cornus officinalis Total Glycosides can attenuate mitochondrial calcium overload, improve mitochondrial morphology and inhibit cardiomyocyte apoptosis caused by H/R. Conclusion: Cornus officinalis Total Glycosides pretreatment can inhibit cardiomyocyte apoptosis and calcium overload during H/R injury. However, the underlying mechanisms require us to further study.

SENP2-mediated SERCA2a deSUMOylation increases calcium overload in cardiomyocytes to aggravate myocardial ischemia/reperfusion injury

Background:Sarcoplasmic reticulum calcium ATPase 2a(SERCA2a)is a key protein that maintains myocardial Ca2+homeostasis.The present study aimed to investigate the mechanism underlying the SERCA2a-SUMOylation(small ubiquitinlike modifier)process after ischemia/reperfusion injury(I/RI)in vitro and in vivo.Methods:Calcium transient and systolic/diastolic function of cardiomyocytes isolated from Serca2a knockout(KO)and wildtype mice with I/RI were compared.SUMO-relevant protein expression and localization were detected by quantitative real-time PCR(RT-qPCR),Western blotting,and immunofluorescence in vitro and in vivo.Serca2a-SUMOylation,infarct size,and cardiac function of Senp1 or Senp2 overexpressed/suppressed adenovirus infected cardiomyocytes,were detected by immunoprecipitation,triphenyltetrazolium chloride(TTC)-Evans blue staining,and echocardiography respectively.Results:The results showed that the changes of Fura-2 fluorescence intensity and contraction amplitude of cardiomyocytes decreased in the I/RI groups and were further reduced in the Serca2a KO+I/RI groups.Senp1 and Senp2 messenger ribose nucleic acid(mRNA)and protein expression levels in vivo and in cardiomyocytes were highest at 6 h and declined at 12 h after I/RI.However,the highest levels in HL-1 cells were recorded at 12 h.Senp2 expression increased in the cytoplasm,unlike that of Senp1.Inhibition of Senp2 protein reversed the I/RI-induced Serca2a-SUMOylation decline,reduced the infarction area,and improved cardiac function,while inhibition of Senp1 protein could not restore the above indicators.Conclusion:I/RI activated Senp1 and Senp2 protein expression,which promoted Serca2a-deSUMOylation,while inhibition of Senp2 expression reversed Serca2a-SUMOylation and improved cardiac function.

Blocking Cyclic Adenosine Diphosphate Ribose-mediated Calcium Overload Attenuates Sepsis-induced Acute Lung Injury in Rats

Background： Acute lung injury （ALI） is a common complication of sepsis that is associated with high mortality, lntracellular Ca^2＋ overload plays an important role in the pathophysiology of sepsis-induced ALl, and cyclic adenosine diphosphate ribose （cADPR） is an important regulator of intracellular Ca^2＋ mobilization. The cluster of differentiation 38 （CD38）/cADPR pathway has been found to play roles in multiple inflammatory processes but its role in sepsis-induced ALl is still unknown. This study aimed to investigate whether the CD38/cADPR signaling pathway is activated in sepsis-induced ALl and whether blocking cADPR-mediated calcium overload attenuates ALl. Methods： Septic rat models were established by cecal ligation and puncture （CLP）. Rats were divided into the sham group, the CLP group, and the CLP＋ 8-bromo-cyclic adenosine diphosphate ribose （8-Br-cADPR） group. Nicotinamide adenine dinucleotide （NAD＋）, cADPR, CD38, and intracellular Ca^2＋ levels in the lung tissues were measured at 6, 12, 24, and 48 h after CLP surgery. Lung histologic injury, tumor necrosis factor （TNF）-a, malondialdehyde （MDA） levels, and superoxide dismutase （SOD） activities were measured. Results： NAD＋, cADPR, CD38, and intracellular Ca-＋ levels in the lungs of septic rats increased significantly at 24 h after CLP surgery. Treatment with 8-Br-cADPR, a specific inhibitor of cADPR, significantly reduced intracellular Ca^2＋ levels （P = 0.007）, attenuated lung histological injury （P = 0.023）, reduced TNF-a and MDA levels （P 〈 0.001 and P = 0.002, respectively） and recovered SOD activity （P = 0.031） in the lungs of septic rats. Conclusions： The CD38/cADPR pathway is activated in the lungs of septic rats, and blocking cADPR-mediated calcium overload with 8-Br-cADPR protects against sepsis-induced ALl.

Extract of Paris polyphylla Simth Protects Cardiomyocytes from Anoxia-Reoxia Injury through Inhibition of Calcium Overload

Objective：To assess any direct effect of extract of Paris polyphylla Simth（EPPS）,a Chinese plant,on a cardiomyocyte subject to ischemia-reperfusion injury and to further elucidate its protective effect against myocardium ischemia on the cellular level.Methods：Neonatal rat cardiomyocytes were isolated and subjected to an anoxia-reoxia injury simulating the ischemia-reperfusion injury in vivo in the presence or absence of EPPS or diltizem,a positive control.The lactate dehydrogenase（LDH） activities in culture supematants and cell viabilities were analyzed using the enzymatic reaction kinetics monitoring-method and MTT method, respectively.Free intracellular calcium concentrations and activities of Na~＋-K~＋ ATPase and Ca^（2＋） ATPase in cells were also measured with laser confocal microscopy and the inorganic phosphorus-transformation method,respectively.Results：In cardiomyocytes subject to anoxia-reoxia injury,EPPS at 50-400 mg/L showed a concentration-dependent inhibition on LDH leakage and maintenance of cell viability,and the effect was significant at 275 and 400 mg/L（both P0.01）.In addition,EPPS at 275 and 400 mg/L significantly inhibited the increase in intracellular free calcium（both P0.01） as well as decreased the activities of Na~＋-K~＋ ATPase and Ca^（2＋） ATPase（P0.01,P0.05）.Conclusions：EPPS prevents anoxia-reoxia injury in neonatal rat cardiomyocytes in vitro by preservation of Na~＋-K~＋ ATPase and Ca^（2＋） ATPase activities and inhibition of calcium overload.The direct protective effect on cardiomyocytes may be one of the key mechanisms that underlie the potential therapeutic benefit of EPPS against myocardium ischemia.

miRNA-208a-3p过表达致慢性心衰大鼠心肌细胞线粒体钙超载和功能障碍的机制研究

目的观察miRNA-208a-3p在慢性心力衰竭大鼠心肌中的表达水平,探讨其在线粒体钙稳态和线粒体功能方面的调节机制。方法35只健康SD大鼠,随机分为模型组(n=20)和对照组(n=15),模型组采用腹主动脉直径缩窄法建立慢性心衰模型,对照组行假手术。通过心功能和组织病理学检测评价模型,测定心肌miR-208a-3p表达,心肌线粒体去乙酰化酶3(SIRT3)蛋白和NADH脱氢酶亚基1(ND1)蛋白表达、线粒体Ca2+水平、心肌细胞活性氧(ROS)生成。结果模型组大鼠心肌miR-208a-3p表达水平显著高于对照组(P<0.05),SIRT3蛋白表达显著低于对照组(P<0.001),且miR-208a-3p与SIRT3表达呈显著负相关;模型组ND1蛋白表达显著低于对照组(P<0.05),且ND1与SIRT3表达呈显著正相关;模型组心肌细胞线粒体内Ca2+水平显著高于对照组(P<0.05),心肌细胞ROS生成也显著高于对照组(P<0.05)。结论慢性心衰心肌组织miR-208a-3p过度表达与SIRT3/ND1活性降低相关,抑制线粒体呼吸链活性,此外,心肌细胞出现线粒体钙超载和ROS生成增加,进一步加剧线粒体呼吸功能障碍,是慢性心衰线粒体功能障碍的重要机制。

乌头碱配伍甘草次酸前后对衰竭心肌细胞影响的比较研究

目的比较乌头碱配伍甘草次酸前后对衰竭心肌细胞的影响。方法将H9c2细胞随机分为9组,即空白组,模型组,甘草次酸组,30、120、480μmol·L^(-1)乌头碱组,30μmol·L^(-1)联用组(30μmol·L^(-1)乌头碱+30μmol·L^(-1)甘草次酸)、120μmol·L^(-1)联用组(120μmol·L^(-1)乌头碱+30μmol·L^(-1)甘草次酸)、480μmol·L^(-1)联用组(480μmol·L^(-1)乌头碱+30μmol·L^(-1)甘草次酸)。除空白组外,各组均以阿霉素法构建衰竭H9c2心肌细胞模型。造模成功后,空白组和模型组给予DMEM,其余各组给予相应药物干预24 h。显微镜观察细胞形态和线粒体微观结构,CCK-8法测定细胞存活率,ELISA检测乳酸脱氢酶(lactate dehydrogenase,LDH)、Na+-K+-ATP酶、Ca^(2+)-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶、超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)、过氧化氢酶(catalase,CAT)、谷胱甘肽(glutathione,GSH)、ATP,荧光探针法测定活性氧(reactive oxygen species,ROS)、膜电位、胞内及线粒体内Ca^(2+)浓度。蛋白免疫印迹法(western blot,WB)检测AMPK通路和CaMKⅡ通路蛋白。结果与模型组比较,120、480μmol·L^(-1)乌头碱降低细胞存活率,升高MDA含量(P<0.01);480μmol·L^(-1)乌头碱破坏线粒体结构;30、120、480μmol·L^(-1)乌头碱升高ROS含量,降低SOD、CAT、GSH酶活性,降低线粒体膜电位和ATP生成,升高胞内及线粒体内钙离子浓度,降低Na+-K+-ATP酶、Ca^(2+)-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶活性(P<0.05或P<0.01);480μmol·L^(-1)乌头碱抑制AMPK磷酸化,降低SERCA2a和PGC-1α蛋白表达,促进RyR2蛋白表达(P<0.05或P<0.01)。与单用乌头碱比较,配伍甘草次酸改善其线粒体结构损伤,降低ROS含量,升高SOD、CAT、Na+-K+-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶活性,降低胞内及线粒体内的钙离子浓度(P<0.05或P<0.01);30、120μmol·L^(-1)联用组升高线粒体膜电位(P<0.05或P<0.01);120、480μmol·L^(-1)联用组降低MDA含量,提高GSH、Ca^(2+)-ATP酶活性,升高ATP含量(P<0.05或P<0.01);480μmol·L^(-1)联用组可抑制CaMKⅡ磷酸化,升高PGC-1α蛋白表达(P<0.05或P<0.01)。结论甘草次酸可拮抗乌头碱对衰竭心肌细胞的线粒体毒性,作用环节可能与缓解氧化应激和钙超载,增加线粒体产能有关。

CDC25C表达下调的黑色素瘤细胞线粒体应激反应和线粒体途径凋亡情况观察

目的探讨细胞分裂周期蛋白25C(CDC25C)对黑色素瘤B16细胞线粒体应激反应和线粒体途径凋亡的影响。方法取黑色素瘤B16细胞进行培养,将细胞随机分为转染组、转染对照组、空白对照组。转染组转染CDC25C低表达慢病毒质粒,转染对照组转染慢病毒空质粒,空白对照组正常培养。利用Rhod-2/AM与MitoSOX荧光探针检测细胞线粒体内Ca^(2+)浓度及线粒体活性氧(ROS),RT-qPCR法和Western blotting法分别检测细胞线粒体应激反应相关分子ClpP、LONP1及线粒体途径凋亡相关分子Caspase-9、Caspase-3 mRNA和蛋白表达。结果与转染对照组和空白对照组相比,转染组细胞线粒体Ca^(2+)及ROS含量增加,ClpP、LONP1、Caspase-9、Caspase-3 mRNA及蛋白表达水平升高(P<0.05或<0.01)。结论CDC25C表达下调激活黑色素瘤B16细胞发生线粒体应激反应,并诱导线粒体途径的细胞凋亡。

基于线粒体钙稳态探讨血府逐瘀汤对冠心病血瘀证模型大鼠的作用机制

目的基于线粒体钙稳态探讨血府逐瘀汤治疗冠心病血瘀证的作用机制。方法150只SD大鼠随机均分为正常组、假手术组(只穿线不结扎)、模型组、阿托伐他汀钙组(0.90 mg/kg·d,以下简称他汀组)以及血府逐瘀汤低、中、高剂量组[3.51、7.02、14.04 g/(kg·d),以下简称低、中、高剂量组],每组6只。除正常组和假手术组外,其他组均通过结扎大鼠冠状动脉左前降支制备冠心病血瘀证模型,造模成功24 h后灌胃给药,连续灌胃7 d,取各组大鼠左室缺血区心肌组织。HE染色观察样本坏死病变情况;线粒体钙离子探针检测线粒体内钙离子的荧光强度;定磷法检测心肌细胞Ca^(2+)-Mg^(2+)-ATP酶活性;免疫组织化学检测心肌组织线粒体钙单向转运蛋白(mitochondrial calcium uniporter,MCU)、心肌肌浆网钙离子ATP酶(sarco/sndoplasmic reticulum Ca^(2+)-ATPase 2a,SERCA2a)、瞬时受体电位通道3(transient receptor potential canonical 3,TRPC3)、钙释放激活钙通道1(calcium release-activated calcium channel protein 1,ORAI1)、半胱氨酸(cysteinyl aspartate specific proteinase,Caspase)-3、Caspase-9的蛋白表达。结果与正常组相比,模型组大鼠心肌组织可见细胞排列紊乱,横纹模糊,大量心肌细胞坏死,线粒体Ca^(2+)平均荧光强度、MCU、TRPC3、ORAI1、Caspase-3、Caspase-9的AOD值明显升高(P<0.01),Ca^(2+)-Mg^(2+)-ATP酶活性、SERCA2a的AOD值明显下降(P<0.01);与模型组相比,他汀组以及血府逐瘀汤各剂量组,心肌细胞水肿程度减轻,炎性细胞浸润减少,细胞排列相对紧密,细胞病理损伤程度有所缓解,他汀组以及血府逐瘀汤各剂量组的SERCA2a的AOD值显著上升(P<0.01),线粒体Ca^(2+)平均荧光强度、MCU、TRPC3、ORAI1、Caspase-9的AOD值显著下降(P<0.05,P<0.01),血府逐瘀汤各剂量组的Ca^(2+)-Mg^(2+)-ATP酶活性显著上升(P<0.05,P<0.01)以及Caspase-3的AOD值显著下降(P<0.05,P<0.01)。结论血府逐瘀汤可以调控冠心病血瘀证模型大鼠心肌线粒体钙离子转运,调节心肌细胞线粒体钙稳态,可能与上调SERCA2a的蛋白表达,下调MCU、TRPC3、ORAI1、Caspase-3、Caspase-9的蛋白表达相关。

下调GSK3β通过抑制ITPR1-GRP75-VDAC1复合体功能减轻衰老肾小管上皮细胞缺氧/复氧损伤

目的探讨糖原合成酶激酶3β(GSK3β)对衰老小鼠原代肾小管上皮细胞(RTEC)缺氧/复氧(H/R)损伤的影响及其调控机制。方法将RTEC分成为Young组即正常生长的年轻RTEC、Old组即使用Etoposide诱导的衰老RTEC、Old+Ad-shNC+H/R组即使用Etoposide诱导衰老再转染腺病毒阴性对照(AdshNC)后进行H/R处理,Old+Ad-shGSK3β+H/R组即使用Etoposide诱导衰老后再转染靶向沉默GSK3β的短发夹RNA腺病毒(Ad-shGSK3β)后进行H/R处理。采用流式细胞术检测各组细胞凋亡水平和线粒体活性氧水平,采用免疫荧光染色法检测各组钙离子水平,采用蛋白质印迹法检测各组GSK3β、线粒体相关的内质网膜(MAM)相关蛋白肌醇1,4,5-三磷酸受体1(ITPR1)、电压依赖性阴离子通道1(VDAC1)、葡萄糖调节蛋白75(GRP75)表达及磷酸化水平,采用免疫共沉淀分析GSK3β与MAM相关蛋白的相互作用。结果与Young组比较,Old组细胞凋亡水平、线粒体活性氧水平及线粒体钙离子水平均较高;与Old组比较,Old+AdshNC+H/R组细胞凋亡水平、线粒体活性氧水平及线粒体钙离子水平均较高;与Old+Ad-shNC+H/R组比较,Old+Ad-shGSK3β+H/R组细胞凋亡水平、线粒体活性氧水平及线粒体钙离子水平均较低,差异均有统计学意义(均为P<0.05)。与Young组比较,Old组ITPR1、GRP75和GSK3β总蛋白表达增多,ITPR1和GRP75磷酸化水平升高,而VDAC1总蛋白和磷酸化水平均下降;与Old组比较,Old+Ad-shNC+H/R组GSK3β蛋白表达不变,ITPR1和GRP75总蛋白和磷酸化水平升高,VDAC1总蛋白表达不变,磷酸化水平增高;与Old+AdshNC+H/R组比较,Old+Ad-shGSK3β+H/R组GSK3β蛋白表达减少,ITPR1、GRP75和VDAC1总蛋白表达不变,磷酸化水平均下降。免疫共沉淀结果显示,GSK3β能够与ITPR1、GRP75和VDAC1蛋白发生相互作用。结论GSK3β在衰老RTEC中表达升高,抑制GSK3β表达能够降低ITPR1-GRP75-VDAC1复合体磷酸化水平,限制线粒体钙离子超负荷,保护线粒体功能,减少再灌注时细胞损伤。

下调XBP1s通过抑制ITPR介导的线粒体功能障碍改善肾小管上皮细胞缺氧/复氧损伤

目的 探讨剪接型X盒结合蛋白1(XBP1s)对小鼠肾小管上皮细胞缺氧/复氧(H/R)损伤的影响及其作用机制。方法 将小鼠肾小管上皮细胞分为腺病毒阴性对照组(Ad-shNC组)、靶向沉默XBP1s腺病毒组(Ad-shXBP1s组)、Ad-shNC+H/R组、Ad-shXBP1s+H/R组。检测各组细胞凋亡水平、线粒体活性氧活性、线粒体膜电位及线粒体钙离子水平。使用染色质免疫共沉淀测序(ChIP-seq)分析XBP1s调控肌醇1,4,5-三磷酸受体(ITPR)家族的结合位点。检测各组XBP1s和ITPR家族信使RNA(mRNA)和蛋白表达水平。结果 与AdshNC组比较,Ad-shNC+H/R组细胞凋亡水平更高,线粒体活性氧水平升高,线粒体膜电位降低,线粒体钙离子水平升高;与Ad-shNC+H/R组比较,Ad-shXBP1s+H/R组细胞凋亡水平较低,线粒体活性氧水平下降,线粒体膜电位升高,线粒体钙离子水平降低(均为P<0.05)。与Ad-shNC组比较,Ad-shXBP1s组XBP1s、ITPR1、ITPR2和ITPR3 mRNA和蛋白相对表达量降低(均为P<0.05)。与Ad-shNC组相比,Ad-shNC+H/R组XBP1s、ITPR1、ITPR2和ITPR3蛋白相对表达量升高;与Ad-shNC+H/R组相比,Ad-shXBP1s+H/R组XBP1s、ITPR1、ITPR2和ITPR3蛋白相对表达量下降(均为P<0.05)。ChIP-seq结果显示,XBP1s能够结合ITPR1的启动子和外显子、ITPR2外显子和ITPR3外显子。结论 XBP1s可能通过直接调控ITPR转录和翻译而影响线粒体相关的内质网膜结构功能,下调XBP1s能够抑制ITPR表达,改善线粒体损伤。

腺泡细胞钙离子超载与急性胰腺炎关系的研究进展

急性胰腺炎(AP)是消化科的常见的疾病类型之一,大多数患者病情轻微,但约25%的患者会发展为重症胰腺炎,其中重症胰腺炎的致死率则高达30%~50%。国内诸多对AP病因病机的基础研究显示,使用雨蛙素法、无胆盐乙硫氨酸食物诱导法、胆胰管逆行牛黄胆酸钠注射法诱导的AP动物模型中腺泡细胞内钙离子浓度均持续增高,进而引起胞内钙离子依赖性的酶原颗粒提前活化、空泡形成最终导致细胞凋亡,以上结论均提示腺泡细胞钙离子超载在AP的发生发展中起到至关重要的作用,而针对AP的治疗目前临床上缺乏高效且具有针对性的靶向药物,故探索维持细胞内钙稳态的机制及腺泡细胞钙超载所引起的病理过程,可为研究AP的直接靶向治疗药物提供新思路。该文就细胞内钙稳态与调控、腺泡细胞钙超载的发生、Ca2+超载在AP发病机制中的作用、腺泡细胞Ca2+超载的临床诊治方法的研究进展作一综述。

大豆异黄酮通过抑制Wnt/Ca2+信号通路减轻大鼠脑缺血再灌注引起的钙超载

目的 探讨大豆异黄酮(SI)减轻脑缺血再灌注(I/R)引起钙超载的作用机制。方法 将48只SD大鼠采用随机数法分为4组:假手术组(Sham组)、脑缺血再灌注模型组(I/R组)、病毒空载组(NC组)、Frizzled-2敲低组(Knock down组),12只/组。采用线栓法堵塞大脑中动脉2 h,再灌注24 h构建I/R模型。采用Western blot验证病毒敲低效率并检测Wnt/Ca2+信号通路相关蛋白Wnt5a、Frizzled-2和P-CaMKⅡ的变化。采用钙含量显色法检测各组缺血半暗带(IP)区钙离子浓度变化,HE检测各组IP区组织结构变化。另将72只SD大鼠采用随机数法分为3组:Sham组、I/R组、大豆异黄酮预处理组(SI组),24只/组。采用多普勒血流仪检测局部脑血流变化,TTC染色检测脑梗死体积,HE和尼氏染色检测IP区组织变化、免疫荧光检测ROS、Ca2+和细胞凋亡水平、流式检测细胞钙离子浓度,试剂盒检测血清MDA和SOD水平,Western blotting和免疫组化检测IP区Wnt5a、Frizzled-2和P-CaMKⅡ蛋白表达。结果 与I/R组比较,Knock down组钙离子浓度(P<0.001)、Wnt5a(P<0.05)、Frizzled-2(P<0.05)和P-CaMKⅡ(P<0.001)表达水平均降低;与I/R组比较,SI组钙离子浓度(P<0.05)、ROS和MDA水平(P<0.001)、细胞凋亡程度(P<0.001)、脑梗死体积(P<0.001)、Wnt5a、Frizzled-2和P-CaMKⅡ表达水平(P<0.05)均降低,SOD水平升高(P<0.001)。结论大豆异黄酮可能通过抑制Wnt/Ca2+信号通路减轻大鼠脑缺血再灌注引起的钙超载。

压电型机械门控离子通道组件1在大鼠压力性损伤中的作用机制

背景:压力性损伤的发生机制复杂,哪些因素在其发生中起到核心作用,这些因素具体又是如何发生的尚不完全清楚。目的:探讨压电型机械门控离子通道组件1(piezo-type mechanosensitive ion channel component 1,PIEZO1)与压力性损伤发生的关系。方法:①细胞实验:采用不同浓度的PIEZO1激动剂Yoda1干预人永生化角质形成细胞(HaCaT),检测细胞活性、钙离子内流及PIEZO1、凋亡相关蛋白的表达。②动物实验:采用随机数字表法将12只SD大鼠随机分为4组,每组3只:对照组大鼠不进行任何处理,1,2,3 mm组分别采用厚度1,2,3 mm的磁铁在大鼠背部两侧压迫1 h,建立压力性损伤模型,造模后切除全部创面组织,分别进行苏木精-伊红、Masson、免疫荧光染色及Western blot检测。结果与结论:①细胞实验:活/死细胞染色结果显示,随着Yoda1浓度(0,2.5,5,10μmol/L)的升高,HaCaT细胞凋亡增加;随着Yoda1浓度(0,5,10μmol/L)的升高,HaCaT细胞钙离子内流增加,并且随着处理时间的延长,钙离子内流增多;Western blot检测结果显示,与对照组(0μmol/L Yoda1干预)比较,5,10μmol/L Yoda1干预可提升HaCaT细胞中BAX、TG2、PIEZO1蛋白的表达,降低Bcl-2蛋白的表达;②动物实验:苏木精-伊红与Masson染色结果显示,3个实验组压迫部位皮肤结构被破坏,皮下脂肪液化坏死,胶原蛋白稀疏且排列紊乱,并且随着磁铁厚度(压力)的增加,压迫部位皮肤组织结构破坏程度加重;免疫荧光染色与Western blot检测结果显示,与对照组比较,3个实验组大鼠BAX、TG2、Yap1与PIEZO1蛋白表达升高,Bcl-2蛋白表达降低,并且随着磁铁厚度(压力)的增加,相关蛋白表达值改变更加明显;③结果表明:皮肤受压会激活PIEZO1使钙离子大量内流,随着所受压力的不断增加,最终导致钙超载后的细胞凋亡。

黄芪甲苷在N-甲基-D-天冬氨酸受体介导的神经元兴奋性损伤中的保护作用

目的探讨黄芪甲苷(astragalosideⅣ)对N-甲基-D-天冬氨酸受体(N-methyl-D-aspartic acid receptor,NMDAR)介导的神经元兴奋性损伤的保护作用。方法选择新生0~1 d的C57/BL6J小鼠,分离海马区神经元进行原代细胞培养,将培养的神经元随机分为对照组、N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid,NMDA)损伤模型组、氧-糖剥夺(oxygen and glucose deprivation,OGD)模型组、NMDAR抑制剂氯胺酮组及黄芪甲苷组。采用Hoechst-33342染色观察各组神经元死亡情况,采用ELISA法检测各组神经元乳酸脱氢酶(lactate dehydrogenase,LDH)释放情况;采用Ca^(2+)成像观察不同条件下〔加入NMDA;NMDAR抑制剂APV(100μmol/L)预处理+NMDA;黄芪甲苷(100μmol/L)预处理+NMDA;无Ca^(2+)+NMDA〕神经元内钙离子浓度;采用Western blot测定各组神经元活化型半胱氨酸天冬氨酸蛋白水解酶-3(cysteinyl aspartate specific proteinase-3,Caspase-3)的表达。另选择出生后4~6周的C57BL/6雄性小鼠,制备包含海马的冠状脑切片,使用电压钳观察黄芪甲苷应用前后突触后膜电流的变化。结果与应用黄芪甲苷(100μmol/L)前比较,应用黄芪甲苷(100μmol/L)后海马CA1区锥体细胞微小兴奋性突触后电流的平均峰值降低〔(9.96±0.43)pA比(12.63±0.45)pA;t=3.741,P<0.05〕,平均频率〔(0.52±0.03)Hz比(0.68±0.05)Hz;t=2.933,P<0.05〕下降;与NMDA损伤模型组相比,黄芪甲苷组神经元凋亡率降低,LDH释放减少,活化型Caspase-3表达减低,细胞内钙离子内流减轻(均P<0.05);在体外OGD模型实验中,黄芪甲苷亦表现出同样的神经元保护作用。结论黄芪甲苷在NMDAR介导的神经元兴奋性损伤中具有保护作用,其作用机制与抑制NMDAR有关。

Liraglutide directly protects cardiomyocytes against reperfusion injury possibly via modulation of intracellular calcium homeostasis

Liraglutide 是为与类型 2 糖尿病 mellitus 对待病人的象 glucagon 一样 peptide-1 受体收缩筋。我们的以前的研究证明了 liraglutide 通过与经历经皮的冠的干预的尖锐心肌的梗塞在病人改进 endothelial 功能保护心脏的功能。现在的学习将调查 liraglutide 是否能对灌注 injury.MethodsIn vitro 实验在 cardiomyocytes 上执行直接保护的效果用 H9C2 房间和新生的老鼠被执行经历模拟 hypoxia/reoxygenation (H/R ) 的室的 cadiomyocytes 感应。Cardiomyocytes apoptosis 被荧光 TUNEL 检测。Mitochondrial 膜潜力(m) 和细胞内部的反应的氧种类(ROS ) 被 JC-1 和 DHE 分别地估计。Fura-2/AM 被用来测量细胞内部的 Ca 2+ 集中和钙短暂。染色的 Immunofluorescence 被用来估计 sarcoplasmic 蜂窝胃 Ca 2+-ATPase (SERCA2a ) 的表示水平。在 vivo 实验， SERCA2a 的心肌的 apoptosis 和表示被比色的 TUNEL 并且由染色的 immunofluorescence 检测， respectively.ResultsIn vitro liraglutide 对 H/R 禁止了 cardiomyotes apoptosis。cardiomyocytes 的 m 比 H/R 组在 liraglutide 组是更高的。H/R 在被 liraglutide 稀释的 H9C2 房间增加了 ROS 生产。Liraglutide 显著地降低了超载和与 H/R 相比短暂的改进的钙组织的 Ca 2+ 。染色结果的 Immunofluorescence 证明 liraglutide 支持了在 H/R 组被减少的 SERCA2a 表示。在 ischemia/reperfusion 老鼠心， apoptosis 显著地被稀释， SERCA2a 表示被 liraglutide 与 H/R group.ConclusionsLiraglutide 相比增加能直接通过细胞内部的钙动态平衡的调整可能保护 cardiomyocytes 免于是的灌注损害。

Calcium signaling of pancreatic acinar cells in the pathogenesis of pancreatitis

Pancreatitis is an increasingly common and sometimes severe disease that lacks a specific therapy.The pathogenesis of pancreatitis is still not well understood.Calcium(Ca2+)is a versatile carrier of signals regulating many aspects of cellular activity and plays a central role in controlling digestive enzyme secretion in pancreatic acinar cells.Ca2+overload is a key early event and is crucial in the pathogenesis of many diseases.In pancreatic acinar cells,pathological Ca2+signaling(stimulated by bile,alcohol metabolites and othercauses)is a key contributor to the initiation of cell injury due to prolonged and global Ca2+elevation that results in trypsin activation,vacuolization and necrosis,all of which are crucial in the development of pancreatitis.Increased release of Ca2+from stores in the intracellular endoplasmic reticulum and/or increased Ca2+entry through the plasma membrane are causes of such cell damage.Failed mitochondrial adenosine triphosphate(ATP)production reduces re-uptake and extrusion of Ca2+by the sarco/endoplasmic reticulum Ca2+-activated ATPase and plasma membrane Ca2+-ATPase pumps,which contribute to Ca2+overload.Current findings have provided further insight into the roles and mechanisms of abnormal pancreatic acinar Ca2+signals in pancreatitis.The lack of available specific treatments is therefore an objective of ongoing research.Research is currently underway to establish the mechanisms and interactions of Ca2+signals in the pathogenesis of pancreatitis.

Plasma membrane calcium pump regulation by metabolic stress

The plasma membrane Ca2+-ATPase(PMCA)is an ATPdriven pump that is critical for the maintenance of low resting[Ca2+]i in all eukaryotic cells.Metabolic stress, either due to inhibition of mitochondrial or glycolytic metabolism,has the capacity to cause ATP depletion and thus inhibit PMCA activity.This has potentially fatal consequences,particularly for non-excitable cells in which the PMCA is the major Ca2+efflux pathway.This is because inhibition of the PMCA inevitably leads to cytosolic Ca2+ overload and the consequent cell death.However,the relationship between metabolic stress,ATP depletion and inhibition of the PMCA is not as simple as one would have originally predicted.There is increasing evidence that metabolic stress can lead to the inhibition of PMCA activity independent of ATP or prior to substantial ATP depletion.In particular,there is evidence that the PMCA has its own glycolytic ATP supply that can fuel the PMCA in the face of impaired mitochondrial function.Moreover, membrane phospholipids,mitochondrial membrane potential,caspase/calpain cleavage and oxidative stress have all been implicated in metabolic stress-induced inhibition of the PMCA.The major focus of this review is to challenge the conventional view of ATP-dependent regulation of the PMCA and bring together some of the alternative or additional mechanisms by which metabolic stress impairs PMCA activity resulting in cytosolic Ca2+ overload and cytotoxicity.

Adipose mesenchymal stem cell-derived extracellular vesicles reduce glutamate-induced excitotoxicity in the retina

Adipose mesenchymal stem cells(ADSCs)have protective effects against glutamate-induced excitotoxicity,but ADSCs are limited in use for treatment of optic nerve injury.Studies have shown that the extracellular vesicles(EVs)secreted by ADSCs(ADSC-EVs)not only have the function of ADSCs,but also have unique advantages including non-immunogenicity,low probability of abnormal growth,and easy access to target cells.In the present study,we showed that intravitreal injection of ADSC-EVs substantially reduced glutamate-induced damage to retinal morphology and electroretinography.In addition,R28 cell pretreatment with ADSC-EVs before injury inhibited glutamate-induced overload of intracellular calcium,downregulation ofα-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor(AMPAR)subunit GluA2,and phosphorylation of GluA2 and protein kinase C alpha in vitro.A protein kinase C alpha agonist,12-O-tetradecanoylphorbol 13-acetate,inhibited the neuroprotective effects of ADSC-EVs on glutamate-induced R28 cells.These findings suggest that ADSCEVs ameliorate glutamate-induced excitotoxicity in the retina through inhibiting protein kinase C alpha activation.

Calcium channels and iron uptake into the heart

Iron overload can lead to iron deposits in many tissues,particularly in the heart.It has also been shown to be associated with elevated oxidative stress in tissues.Elevated cardiac iron deposits can lead to iron overload cardiomyopathy,a condition which provokes mortality due to heart failure in iron-overloaded patients.Currently,the mechanism of iron uptake into cardiomyocytes is still not clearly understood.Growing evidence suggests L-type Ca2+channels(LTCCs)as a possible pathway for ferrous iron(Fe2+)uptake into cardiomyocytes under iron overload conditions.Nevertheless,controversy still exists since some findings on pharmacological interventions and those using different cell types do not support LTCC’s role as a portal for iron uptake in cardiac cells.Recently,T-type Ca2+channels (TTCC)have been shown to play an important role in the diseased heart.Although TTCC and iron uptake in cardiomyocytes has not been investigated greatly,a recent finding indicated that TTCC could be an important portal in thalassemic hearts.In this review,comprehensive findings collected from previous studies as well as a discussion of the controversy regarding iron uptake mechanisms into cardiomyocytes via calcium channels are presented with the hope that understanding the cellular iron uptake mechanism in cardiomyocytes will lead to improved treatment and prevention strategies,particularly in iron-overloaded patients.