复方三七川芎软胶囊对原代心肌细胞过氧化氢损伤的保护作用

采用体外培养幼鼠心肌细胞过氧化氢损伤模型,检测细胞培养液中乳酸脱氢酶(LDH)、心肌细胞线粒体琥珀酸脱氢酶(MSD)、超氧化物歧化酶(SOD)的活性和丙二醛(MDA)含量,并用流式细胞仪及DNA电泳检测细胞凋亡,观察复方三七川芎软胶囊135,67.5,13.5 mg/L预处理组对过氧化氢损伤的原代培养心肌细胞的保护作用。结果:乳鼠心肌细胞经0.1mmol/L H2O2损伤后LDH、MSD、SOD、MDA、心肌细胞凋亡率均与对照组有显著性差异(P<0.01),135,67.5 mg/L预处理组与模型组比较LDH活性降低、MSD和SOD活性提高、心肌细胞凋亡率降低,差异显著(P<0.05)。     

Multi-parameter in vitro toxicity testing of crizotinib,sunitinib, erlotinib,and nilotinib in human cardiomyocytes

Tyrosine kinase inhibitors (TKi) have greatly improved the treatment and prognosis of multiple cancer types. However, unexpected cardiotoxicity has arisen in a subset of patients treated with these agents that was not wholly predicted by pre-clinical testing, which centers around animal toxicity studies and inhibition of the human Ether-à-go-go-Related Gene (hERG) channel. Therefore, we sought to determine whether a multi-parameter test panel assessing the effect of drug treatment on cellular, molecular, and electrophysiological endpoints could accurately predict cardiotoxicity. We examined how 4 FDA-approved TKi agents impacted cell viability, apoptosis, reactive oxygen species (ROS) generation, metabolic status, impedance, and ion channel function in human cardiomyocytes. The 3 drugs clinically associated with severe cardiac adverse events (crizotinib, sunitinib, nilotinib) all proved to be cardiotoxic in our in vitro tests while the relatively cardiac-safe drug erlotinib showed only minor changes in cardiac cell health. Crizotinib, an ALK/MET inhibitor, led to increased ROS production, caspase activation, cholesterol accumulation, disruption in cardiac cell beat rate, and blockage of ion channels. The multi-targeted TKi sunitinib showed decreased cardiomyocyte viability, AMPK inhibition, increased lipid accumulation, disrupted beat pattern, and hERG block. Nilotinib, a second generation Bcr-Abl inhibitor, led to increased ROS generation, caspase activation, hERG block, and an arrhythmic beat pattern. Thus, each drug showed a unique toxicity profile that may reflect the multiple mechanisms leading to cardiotoxicity. This study demonstrates that a multi-parameter approach can provide a robust characterization of drug-induced cardiomyocyte damage that can be leveraged to improve drug safety during early phase development.     

PKCɛ mediates serine phosphorylation of connexin43 induced by lysophosphatidylcholine in neonatal rat cardiomyocytes

Lysophosphatidylcholine (LPC) is a potent pro-arrhythmic derivative of the membrane phosphotidylcholine, which is accumulated in heart tissues during cardiac ischemia. However, the cellular mechanism underlying LPC-induced cardiomyocyte damage remains to be elucidated. This study focuses on the effects of LPC on cardiomyocyte gap junction. At 30 μM, LPC decreased the spontaneous contraction rates of cardiomyocytes, and caused arrhythmic contraction without affecting cell viability. Connexin43 (Cx43) was seen as large plaques at cell junctions in control cells, whereas upon LPC treatment, the intensity of Cx43 staining was decreased in a concentration-sensitive manner and Cx43 staining appeared as tiny dots at cell junctions with a corresponding increase in cytoplasmic punctate staining. This distributional change of Cx43 was accompanied by an impairment of the gap junction intercellular communication (GJIC). Further, LPC treatment induced protein kinase C (PKC) activation, and PKC-dependent Cx43 phosphorylation at serine (Ser) 368. Pre-treatment with a specific PKC? inhibitor, eV1-2, prevented the LPC-induced Cx43 phosphorylation at Ser368 and the loss of Cx43 from gap junctions, both of which may disturb GJIC functions. Furthermore, siRNA knockdown of PKC? in H9c2 cells prevented LPC-induced serine phosphorylation of Cx43, confirming the role of PKC? in Cx43 serine phosphorylation. Double labeling immunofluorescence showed that LPC increased the colocalization of Cx43 with ubiquitin, and pretreatment with MG132 effectively prevented LPC-induced gap junction disassembly. LPC increased the ubiquitination of Cx43, which was blocked by eV1-2 pretreatment, suggesting that LPC accelerated the intracellular degradation of Cx43 via the ubiquitin-proteasomal pathway. It can be concluded that LPC destroyed the structure and function of gap junctions via PKC?-mediated serine phosphorylation of Cx43. PKC? inhibitors might therefore be effective in prevention of LPC-related diseases.     

丹参酮ⅡA通过AMPK介导的自噬减轻阿霉素所致H9c2心肌细胞损伤

目的:探讨丹参酮ⅡA对阿霉素(又称多柔比星)所致大鼠H9c2心肌细胞损伤的影响和机制。方法:以H9c2细胞为研究对象,在有或无AMPK抑制剂dorsomorphin处理下,采用丹参酮ⅡA和(或)阿霉素处理H9c2细胞,应用CCK-8法测定细胞活力,LDH法测定细胞损伤情况,免疫荧光实验分析细胞自噬情况,Western blot检测细胞AMPK的活化情况。结果:与对照组相比,阿霉素处理后H9c2细胞的活力减弱,LDH释放增多,自噬增加,AMPK的活化受抑制(P0.05);与阿霉素组相比,加用丹参酮ⅡA联合处理能部分恢复H9c2细胞的活力,减少LDH释放,进一步增加自噬,促进AMPK活化(P0.05);AMPK抑制剂dorsomorphin处理后,丹参酮ⅡA恢复H9c2细胞活力、减少LDH释放和促进自噬的作用减弱(P0.05)。结论:丹参酮ⅡA能减轻阿霉素所致H9c2心肌细胞的损伤,其机制可能与激活AMPK介导的自噬有关。本研究为临床上应用丹参酮ⅡA防治阿霉素心肌损伤提供实验基础和理论依据。     

miR-93-3p alleviates lipopolysaccharide-induced inflammation and apoptosis in H9c2 cardiomyocytes by inhibiting toll-like receptor 4

Background

miR-93 is recently recognized to perform anti-inflammatory action in the pathological process of cardiomyocytes dysfunction. However, it remains unclear whether miR-93-3p involves in lipopolysaccharide (LPS)-induced inflammation and apoptosis in H9c2 cells. The present study aimed to investigate the functions of miR-93-3p and its target, toll-like receptor 4 (TLR4), in LPS-stimulated cardiomyocytes.

自噬标志物LC3的节律表达破坏参与β1-AA诱导的H9c2大鼠心肌细胞死亡

目的:探讨β₁-肾上腺素受体(β₁-AR)自身抗体(β₁-AA)对大鼠心肌细胞自噬标志物微管相关蛋白1轻链3(LC3)节律表达的影响及其在心肌细胞死亡中的作用。方法:实验材料为Sprague-Dawley(SD)大鼠和H9c2大鼠心肌细胞。将SD大鼠随机分为免疫组(β₁-AR组)和对照(control)组,每组6只;将H9c2细胞随机分为control组、β₁-AA组、慢病毒(LV)-NC组和LV-shPer2组(n=6);合成β₁-AR细胞外第二环抗原肽段,用于主动免疫大鼠,并使用亲和层析法从大鼠血清中提纯β₁-AA;β₁-AA处理H9c2细胞24 h后使用CCK-8法检测细胞活力;用地塞米松同步化细胞后,再给予β₁-AA处理,采用real-time PCR及Western blot法检测LC3的表达情况,采用Western blot法检测生物钟蛋白Per2的表达情况,使用JTK_CYCLE算法分析昼夜节律参数;用LV-shPer2感染H9c2细胞以破坏LC3的节律表达,进而采用CCK-8法检测细胞活力。结果:β₁-AR组大鼠血清中β₁-AA的A值与control组相比显著升高(P<0.05)。β₁-AA组H9c2细胞的活力显著低于control组(P<0.05)。β₁-AA可破坏H9c2细胞LC3和Per2的节律表达(JTK_CYCLE P<0.05)。通过LV-shPer2干扰Per2基因而破坏H9c2细胞LC3节律表达(JTK_CYCLE P<0.05)后,细胞活力显著降低(P<0.05)。结论:β₁-AA破坏H9c2大鼠心肌细胞自噬标志物LC3的节律表达,从而促进细胞死亡。     

A novel dihydropyridine with 3-aryl meta-hydroxyl substitution blocks L-type calcium channels in rat cardiomyocytes

Rationale

Dihydropyridines are widely used for the treatment of several cardiac diseases due to their blocking activity on L-type Ca^2 + channels and their renowned antioxidant properties.

Methods

We synthesized six novel dihydropyridine molecules and performed docking studies on the binding site of the L-type Ca^2 + channel. We used biochemical techniques on isolated adult rat cardiomyocytes to assess the efficacy of these molecules on their Ca^2 + channel-blocking activity and antioxidant properties. The Ca^2 + channel-blocking activity was evaluated by confocal microscopy on fluo-3AM loaded cardiomyocytes, as well as using patch clamp experiments. Antioxidant properties were evaluated by flow cytometry using the ROS sensitive dye 1,2,3 DHR.

Results

Our docking studies show that a novel compound with 3-OH substitution inserts into the active binding site of the L-type Ca^2 + channel previously described for nitrendipine. In biochemical assays, the novel meta-OH group in the aryl in C4 showed a high blocking effect on L-type Ca^2 + channel as opposed to para-substituted compounds. In the tests we performed, none of the molecules showed antioxidant properties.

Conclusions

Only substitutions in C2, C3 and C5 of the aryl ring render dihydropyridine compounds with the capacity of blocking LTCC. Based on our docking studies, we postulate that the antioxidant activity requires a larger group than the meta-OH substitution in C2, C3 or C5 of the dihydropyridine ring.     

钙激活氯通道ANO1在小鼠心肌细胞的表达及其功能鉴定

目的:探讨钙激活氯通道蛋白anoctamin 1(ANO1)在小鼠原代培养心肌细胞中的表达及其功能特性。方法:采用胰酶与胶原酶共同消化,联合2次差速贴壁法获得C57BL/6小鼠原代心肌细胞;并用免疫荧光染色法检测α-横纹肌肌动蛋白,以鉴定心肌细胞纯度;应用RT-PCR检测小鼠心肌细胞ANO1 mRNA的表达;免疫印迹检测ANO1蛋白在小鼠心肌细胞的表达情况;应用荧光淬灭动力学实验检测ANO1钙激活氯通道的功能特性。结果:RT-PCR结果表明原代培养的小鼠心肌细胞表达ANO1 mRNA。免疫印迹实验结果显示原代培养的小鼠心肌细胞表达ANO1蛋白。荧光淬灭动力学实验证实表达于小鼠心肌细胞的ANO1具有钙激活氯通道典型的阴离子转运功能特性。结论:ANO1在小鼠心肌细胞中有明确表达,并具有钙激活氯离子通道特性,提示ANO1是钙激活氯通道的分子基础。     

p300介导组蛋白乙酰化修饰下调PPAR-γ致妊娠期糖尿病小鼠子代心肌细胞糖脂代谢紊乱

目的:阐明妊娠期糖尿病(GDM)对子代心肌细胞糖脂代谢的影响,并探讨其可能的调控机制。方法:雌性昆明小鼠于妊娠中期给予腹腔注射链脲佐菌素(30 mg/kg)建立GDM模型,另设对照(control)组。分娩后F1代饲养至8周,测定随机血糖和空腹血脂等相关指标。利用CO2窒息处死F1代实验小鼠,剖开胸腔后分离心脏组织,用于后续实验。q PCR检测p300及p300/CBP相关因子(PCAF)的mRNA表达水平,q PCR及Western blot检测过氧化物酶体增殖物激活受体γ(PPAR-γ)、葡萄糖转运蛋白4(GLUT-4)及中链酰基辅酶A脱氧酶(MCAD)的mRNA和蛋白表达水平,染色质免疫共沉淀(Ch IP)结合q PCR检测p300与PPAR-γ启动子结合水平及PPAR-γ启动子区域组蛋白H3的乙酰化水平。结果:F1代小鼠血糖和总胆固醇轻度升高(P0.05),甘油三酯、高密度及低密度脂蛋白无明显改变;心肌组织中p300、PPAR-γ、GLUT-4及MCAD表达明显降低(P0.05),PCAF的表达两组间差异无统计学显著性;p300与PPAR-γ启动子结合水平和PPAR-γ启动子区域组蛋白H3的乙酰化水平均明显下降(P0.05)。结论:GDM子代小鼠中p300通过介导组蛋白乙酰化修饰而下调PPAR-γ表达,可能引起心肌细胞糖脂代谢紊乱。     

程序化细胞死亡因子5对缺氧/复氧诱导的心肌细胞凋亡和自噬的影响及机制

目的:探讨程序化细胞死亡因子5(PDCD5)对缺氧/复氧(H/R)诱导的心肌细胞凋亡和自噬的影响及其作用机制。方法:以H9c2心肌细胞系为研究对象,建立H/R损伤模型,采用RNA干扰方法抑制PDCD5的表达,MTT法检测心肌细胞的存活率,TUNEL显色法检测细胞凋亡率,RT-qPCR和Western blot法分别检测mRNA和蛋白的表达水平。结果:H/R损伤的H9c2心肌细胞中,PDCD5表达水平升高,同时细胞的存活率降低,细胞凋亡率和自噬水平增加,而PDCD5沉默能够增加细胞存活率,同时减少细胞凋亡率,促进Bax表达且抑制抑Bcl-2、LC3-Ⅱ/LC3-Ⅰ及自噬相关蛋白beclin-1的表达。此外,沉默PDCD5可通过降低p-P65的蛋白水平抑制NF-κB通路。结论:沉默PDCD5通过阻断NF-κB信号通路而抑制H/R损伤诱导的心肌细胞凋亡和自噬,从而保护心肌细胞。