Role of PERK pathway in morphine-induced myocardial protection of H9c2 cells

AIM: To explore whether morphine protects oxidative stress-damaged myocardial cells by inhibiting the PERK pathway to reduce endoplasmic reticulum stress and prevent mitochondrial permeability transition pore (mPTP) opening. METHODS: Rat myocardial H9c2 cells were cultured to establish an oxidative stress model, and then randomly divided into control group, H₂O₂ group, H₂O₂+morphine group, H₂O₂+morphine+PERK pathway inhibitor GSK2656157 group, morphine group and GSK2656157 group. Immunohistochemical method was used to detect the effects of morphine on expression of glucose-regulated protein (GRP) 78 and GRP94 induced by oxidative stress. The protein levels of PERK signaling pathway-related molecules were determined by Western blot. Confocal microscopy was used to observe the effects of morphine on mPTP opening and endoplasmic reticulum induced by oxidative stress. Cellular toxicity was detected by lactate dehydrogenase (LDH) kit and cell viability was measured by MTT assay. RESULTS: Compared with control group, GRP78 and GRP94 proteins in H₂O₂ group were strongly expressed, and the brown-yellow particles were significantly increased, but morphine significantly inhibited this process. Compared with control group, the phosphorylation of PERK was significantly reduced with GSK2656157 treatment at different concentrations, among which 2 μmol/L had the most significant effect (P < 0.05). Oxidative stress significantly increased the protein levels of GRP78, GRP94, p-PERK and CHOP, but significantly decreased p-GSK-3β level. These changes were inhibited by morphine, and the effects of morphine were further enhanced by GSK2656157 (P < 0.05). Compared with control group, oxidative stress significantly reduced the fluorescence intensity of mitochondrial TMRE and ER-Tracker Red. Morphine significantly inhibited this effect even when mitochondrial membrane potential was reduced, mPTP was open, and endoplasmic reticulum was damaged, while GSK2656157 further enhanced the effect of morphine (P < 0.05). Compared with control group, H₂O₂ significantly increased cellular toxicity and decreased the cell viability. Morphine inhibited this effect and GSK2656157 significantly enhanced the effect of morphine (P < 0.05). CONCLUSION: Morphine protects cardiac H9c2 cells under oxidative condition by inhibiting endoplasmic reticulum stress through PERK pathway and preventing the mPTP opening via GSK-3β inactivation.     

Mechanism of elemene enhancing radiosensitivity of human glioma U251 cells

AIM To investigate the effect of elemene on the radiosensitivity of human glioma U251 cells and its mechanism. METHODS The U251 cells were used as a glioma model in vitro, and were exposed to different concentrations of elemene and different doses of radiation. The cell viability was measured by MTT assay, the apoptosis and cell cycle distribution were analyzed by flow cytometry, and the related protein levels were determined by Western blot. RESULTS Elemene inhibited the viability of U251 cells in vitro and enhanced the radiosensitivity of the cells. The cells in radiotherapy combined with elemene group had higher rates of early apoptosis, secondary necrosis and total cell death than those in radiation group. Elemene induced G₂/M phase arrest in the U251 cells. Elemene reduced the protein expression of cell division cycle protein 2 （Cdc2）, which resulted in the decrease in cyclin B1 expression induced by radiotherapy, thereby inhibiting the formation of cyclin B-Cdc2 complex. Elemene reduced Cdc2 activity by inhibiting the phosphorylation of Cdc2 protein at threonine 161, thereby inducing G₂/M phase arrest in the cells. It also mediated apoptosis by down-regulating survivin expression. CONCLUSION Elemene may increase the sensitivity of U251 cells to radiotherapy by down-regulating Cdc2 protein, decreasing cyclin B1 expression, inhibiting the formation of cylcin B-Cdc2 complex and down-regulating the expression of survivin.     

ROCK promotes high glucose-induced cardiomyocyte apoptosis by inhi-biting PI3K/Akt signaling pathway

AIMTo investigate whether Rho-associated coiled-coil kinase (ROCK) is involved in high glucose-induced apoptosis of primary cardiomyocytes by regulating PI3K/Akt signaling pathway. METHODSPrimary Wistar rat cardiomyocytes were cultured and identified by α-sarcomeric actin (α-SCA) immunohistochemistry. Cardiomyocytes were treated with 5.5, 33 and 40 mmol/L glucose for 48 h. The cell viability was measured by MTT assay, and the mRNA expression of ROCK1 and ROCK2 in the cardiomyocytes was detected by RT-qPCR. Flow cytometry was used to analyze the apoptosis of the cardiomyocytes. The protein levels of ROCK1, ROCK2, cleaved caspase-3, Bcl-2, PI3K, Akt and p-Akt were determined by Western blot. In order to confirm the regulatory effect of ROCKs on PI3K/Akt signaling pathway, the cells were divided into control group (5.5 mmol/L glucose), high glucose group (33 mmol/L glucose) and high glucose+Y27632 (ROCK inhibitor) group. Western blot was used to detect the protein levels of ROCK1, ROCK2, PI3K, Akt and p-Akt. RESULTSAfter 48 h of high glucose exposure, the values of relative cell viability in 33 and 40 mmol/L glucose groups were (79.71±2.43)% and (68.41±7.49)%, respectively, both of which were significantly decreased compared with normal control group (P<0.05). After 48 h of high glucose exposure, the relative mRNA levels of ROCK1 and ROCK2 in 33 and 40 mmol/L glucose groups were significantly increased compared with normal control group (P<0.05). Compared with normal control group, the apoptotic rate in 33 and 40 mmol/L glucose groups was increased significantly (P<0.05). Compared with normal control group, the protein expression of ROCK1, ROCK2 and cleaved caspase-3 in 33 and 40 mmol/L glucose groups was increased (P<0.05), while the protein expression of Bcl-2 was decreased (P<0.05). No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed, while the protein level of p-Akt in 33 and 40 mmol/L glucose groups was decreased compared with normal control group (P<0.05). Compared with high glucose group, the expression of ROCK1 and ROCK2 was decreased in high glucose+Y27632 group. No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed. Compared with normal control group, the protein level of p-Akt in high glucose group was decreased, and the protein level of p-Akt in high glucose+Y27632 group was increased significantly compared with high glucose group. CONCLUSION Under high glucose environment, ROCK may reduce the level of p-Akt by inhibiting the PI3K/Akt signaling pathway， thus promoting the apoptosis of cardiomyocytes.     

Importance of mitochondrial calcium uniporter in high glucose-induced cardiac myocyte apoptosis

AIM: To investigate the role of mitochondrial calcium uniporter (MCU) in high glucose(HG)-induced apoptosis of cardiac myocytes. METHODS: Cardiac myocytes were exposed to normal glucose (5.5 mmol/L glucose+ 19.5 mmol/L mannitol), HG (25 mmol/L glucose), or HG combined with 5 μmol/L spermine for 72 h. Mitochondrial free Ca²⁺ concentration ([Ca²⁺]_m), MCU at mRNA and protein levels, pyruvate dehydrogenase (PDH) activity, mitochondrial membrane potential (Δψ_m), the levels of ATP and reactive oxygen species (ROS), and apoptosis were determined. RESULTS: The [Ca²⁺]_m, the mRNA and protein levels of MCU, PDH activity, ATP levels, and Δψ_m were reduced (P<0.05), while ROS content and the protein levels of caspase-9 and caspase-3 were increased in HG group (P<0.05). Adding 5 μmol/L spermine returned these parameters toward control levels (P<0.05). Moreover, apoptosis was reduced by adding spermine and HG treatment (P<0.05). CONCLUSION: HG-induced cardiac myocyte apoptosis may be associated with the decreased MCU expression and activity, abnormal mitochondrial Ca²⁺ handling, deviant mitochon-drial respiratory chain, and mitochondrial dysfunction.     

欧洲葡萄‘粉红亚都蜜’NAC基因DRL1负向调节植物抗旱性

以欧洲葡萄‘粉红亚都蜜’（Vitis vinifera‘Yatomo Rose’）为材料，利用荧光定量PCR技术和转基因技术研究葡萄NAC转录因子DRL1基因对逆境的响应。欧洲葡萄‘粉红亚都蜜’在激素和逆境胁迫下，DRL1表达呈下降趋势，其中以ABA和干旱胁迫处理最为显著。在ABA处理下DRL1转基因烟草株系种子萌发率和根长均高于野生型。干旱处理下，转基因植株对干旱的耐受性降低，同时胁迫相关基因NtLEA5、NtP5CR1、NtPSCS1、NtERD10C和NtDREB3的表达水平比野生型显著下降。此外，DRL1转基因烟草茎中柱发育受到抑制，尤其是导管横切面积仅为野生型的58%。以上结果表明，DRL1基因可能作为1个负向调节子参与植物的干旱胁迫。     

特早熟高糖猕猴桃新品种‘金美’

‘金美’是从云南野外美味猕猴桃资源中选育出的绿肉高糖新品种。果实为卵形，果面黄褐色硬毛，果形整齐；平均单果质量60 ~ 80 g。果肉绿色或黄绿色，质嫩多汁，风味浓甜。软熟果实含可溶性固形物24.1%，可溶性总糖16.2%，总酸1.25%，维生素C 1 180 mg • kg-1，钾 342 mg • kg-1，钙84.2 mg • kg-1。在武汉地区8月果实成熟，此时果实采后7 d开始软熟，1 ~ 2 ℃低温和95%相对湿度下可存放100 ~ 120 d。盛果期产量22 t • hm-2。     

晚熟苹果新品种‘华蜜’

苹果新品种‘华蜜’是以‘金冠’作母本，‘华富’作父本，杂交选育而成。果实圆锥形，果形指数1.1，平均单果质量220 g；果皮鲜红色，底色黄绿色，果面较光滑，果点中大；果肉较细，淡黄色，肉质松脆多汁，去皮硬度7.5 kg • cm-2，可溶性固形物含量17.5%，口感香甜，有香味，品质上等。果实生育期168 d左右，属晚熟品种，在辽宁省兴城地区10月下旬成熟，产量32 t • hm-2。     

不同种植方式对大跨度外覆盖大棚番茄群体微环境及植株生长与产量和品质的影响

以番茄品种罗拉为试材,设置5个处理,分别为:CK,定植密度33 300株·hm~(-2),留6穗果;T1,49 500株·hm~(-2),留4穗果;T2,66 000株·hm~(-2),留3穗果;T3,49 500株·hm~(-2),单株留2穗果与留6穗果相间;T4,66 000株·hm~(~(-2)),单株留2穗果与留4穗果相间,研究不同种植密度和留果方式对外保温覆盖大棚内番茄植株生长、产量、品质、群体微环境的影响。结果表明:T2和T1处理生育期平均温度、温度适宜度较高;T1和T3处理摘心前的叶片SPAD值显著高于对照;T1、T2处理的前期产量和总产量均显著高于对照;VC、可滴定酸、可溶性糖、番茄红素等果实品质以T1处理最优。综合产量、品质以及大棚内的温湿度和光照适宜度,T1处理种植风险较小,是外保温覆盖大棚中较为合适的种植方式。     

不同花色牡丹品种花瓣色素含量及成分分析

以分属白、粉、红3个色系的6个牡丹品种花瓣为试材,采用薄层层析色谱法(TLC)、紫外-可见分光光度计(UV)和高效液相色谱仪-二极管阵列检测器(HPLC-DAD)研究不同花色牡丹品种花瓣中的色素含量,并进行了定性分析。结果表明:粉色和红色系牡丹中检测出2种花青苷,分别是矢车菊素-3,5-二葡糖苷和芍药花素-3,5-二葡糖苷,白色系牡丹中未检测到花青苷类物质,其中矢车菊素-3,5-二葡糖苷在粉色系牡丹中含量较高,而红色系中总黄酮和总花青苷的含量最高。