Ginsenoside Rg1 ameliorates diabetic cardiomyopathy by inhibiting endoplasmic reticulum stress‐induced apoptosis in a streptozotocin‐induced diabetes rat model

Ginsenoside Rg1 has been demonstrated to have cardiovascular protective effects. However, whether the cardioprotective effects of ginsenoside Rg1 are mediated by endoplasmic reticulum (ER) stress‐induced apoptosis remain unclear. In this study, among 80 male Wistar rats, 15 rats were randomly selected as controls; the remaining 65 rats received a diet rich in fat and sugar content for 4 weeks, followed by intraperitoneal injection of streptozotocin (STZ, 40 mg/kg) to establish a diabetes model. Seven days after STZ injection, 10 rats were randomly selected as diabetic model (DM) controls, 45 eligible diabetic rats were randomized to three treatment groups and administered ginsenoside Rg1 in a dosage of 10, 15 or 20 mg/kg/day, respectively. After 12 weeks of treatment, rats were killed and serum samples obtained to determine cardiac troponin (cTn)‐I. Myocardial tissues were harvested for morphological analysis to detect myocardial cell apoptosis, and to analyse protein expression of glucose‐regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and Caspase‐12. Treatment with ginsenoside Rg1 (10–20 mg/kg) significantly reduced serum cTnI levels compared with DM control group (all P < 0.01). Ginsenoside Rg1 (15 and 20 mg/kg) significantly reduced the percentage of apoptotic myocardial cells and improved the parameters of cardiac function. Haematoxylin and eosin and Masson staining indicated that ginsenoside Rg1 could attenuate myocardial lesions and myocardial collagen volume fraction. Additionally, ginsenoside Rg1 significantly reduced GRP78, CHOP, and cleaved Caspase‐12 protein expression in a dose‐dependent manner. These findings suggest that ginsenoside Rg1 appeared to ameliorate diabetic cardiomyopathy by inhibiting ER stress‐induced apoptosis in diabetic rats.     

Neuregulin‐1 protects myocardial cells against H2O2‐induced apoptosis by regulating endoplasmic reticulum stress

Neuregulin‐1 (NRG‐1) is a stress‐mediated growth factor secreted by cardiovascular endothelial cells and provides the protection to myocardial cells, but the underlying mechanisms are not fully understood. This study aimed to demonstrate that NRG‐1 protects myocardial cells exposed to oxidative damage by regulating endoplasmic reticulum (ER) stress. Neonatal rat cardiac myocytes (NRCMs) were isolated and treated with H₂O₂ as a cellular model of ER stress. NRCMs were pretreated with different concentrations of NRG‐1. We found that NRG‐1 increased the viability and reduced the apoptosis of NRCMs treated by H₂O₂. Moreover, NRG‐1 reduced lactate dehydrogenase level, increased superoxide dismutase activity and decreased malondialdehyde content in NRCMs treated by H₂O₂. Finally, we demonstrated that NRG‐1 alleviated ER stress and decreased CHOP and GRP78 protein levels in NRCMs treated by H₂O₂. Taken together, these data indicate that NRG‐1 relieves oxidative and ER stress in NRCMs and suggest that NRG‐1 is a promising agent for cardioprotection. Copyright © 2014 John Wiley & Sons, Ltd.     

Exercise protects against diabetic cardiomyopathy by the inhibition of the endoplasmic reticulum stress pathway in rats

To explore the protective effect of exercise training on the injury of myocardium tissues induced by streptozotocin (STZ) in diabetic rats and the relationship with endoplasmic reticulum stress (ERS), the male sprague-dawley (SD) rats were fed with high-fat and high-sugar diet for 4 weeks, followed by intraperitoneal injection of STZ, 40 mg/kg, to establish a diabetes model, and then 10 rats were randomly selected as diabetes mellitus (DM) controls and 20 eligible diabetic rats were randomized into two groups: low-intensity exercise training (n = 10) and high-intensity exercise training (n = 10). After 12 weeks of exercise training, rats were killed and serum samples were used to determine cardiac troponin-I (cTn-I). Myocardial tissues were sampled for morphological analysis to detect myocardial cell apoptosis, and to analyze protein expression of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and caspase-12. Different intensities (low and high) significantly reduced serum cTn-I levels compared with the DCM group (p < 0.01), and significantly reduced the percentage of apoptotic myocardial cells and improved the parameters of cardiac function. Hematoxylin and eosin and Masson staining indicated that exercise training could attenuate myocardial apoptosis. Additionally, exercise training significantly reduced GRP78, CHOP, and cleaved caspase-12 protein expression in an intensity-dependent manner. These findings suggest that exercise appeared to ameliorate diabetic cardiomyopathy by inhibiting endoplasmic reticulum stress-induced apoptosis in diabetic rats.     

氧化应激对原代培养乳鼠心房肌细胞中内质网应激及凋亡因子的影响

目的：研究氧化应激对原代培养乳鼠心房肌细胞凋亡、内质网应激及凋亡因子的影响。方法：实验分2组：对照组、氧化应激组。原代培养乳鼠心房肌细胞,氧化应激组在培养的原代心房肌细胞中加入终浓度为100μmol/L的H₂O₂培养2 h,检测氧化和抗氧化指标超氧化物歧化酶（SOD）活力、丙二醛（MDA）及还原型谷胱甘肽（GSH）含量;检测细胞凋亡、细胞GRP78、GRP94及chop、bax、bcl-2 mRNA表达。结果：与对照组相比较,氧化应激组心房肌细胞SOD活力和GSH含量下降、MDA含量增加（P < 0.01）,细胞凋亡增加（P < 0.01）,细胞GRP78、GRP94、chop、bax mRNA表达增加、bcl-2 mRNA表达减少（P < 0.01）。结论：氧化应激反应可能介导内质网应激反应并激活促凋亡因子表达,抑制抗凋亡因子表达,引起心房肌细胞凋亡增加。这可能与心房纤颤的发生有一定关联性。     

HMGB1 mediates hyperglycaemia‐induced cardiomyocyte apoptosis via ERK/Ets‐1 signalling pathway

Apoptosis is a key event involved in diabetic cardiomyopathy. The expression of high mobility group box 1 protein (HMGB1) is up‐regulated in diabetic mice. However, the molecular mechanism of high glucose (HG)‐induced cardiomyocyte apoptosis remains obscure. We aimed to determine the role of HMGB1 in HG‐induced apoptosis of cardiomyocytes. Treating neonatal primary cardiomyocytes with HG increased cell apoptosis, which was accompanied by elevated levels of HMGB1. Inhibition of HMGB1 by short‐hairpin RNA significantly decreased HG‐induced cell apoptosis by reducing caspase‐3 activation and ratio of Bcl2‐associated X protein to B‐cell lymphoma/leukemia‐2 (bax/bcl‐2). Furthermore, HG activated E26 transformation‐specific sequence‐1 (Ets‐1), and HMGB1 inhibition attenuated HG‐induced activation of Ets‐1 via extracellular signal‐regulated kinase 1/2 (ERK1/2) signalling. In addition, inhibition of Ets‐1 significantly decreased HG‐induced cardiomyocyte apoptosis. Similar results were observed in streptozotocin‐treated diabetic mice. Inhibition of HMGB1 by short‐hairpin RNA markedly decreased myocardial cell apoptosis and activation of ERK and Ets‐1 in diabetic mice. In conclusion, inhibition of HMGB1 may protect against hyperglycaemia‐induced cardiomyocyte apoptosis by down‐regulating ERK‐dependent activation of Ets‐1.     

Deficiency of CCAAT/enhancer‐binding protein homologous protein (CHOP) prevents diet‐induced aortic valve calcification in vivo

Aortic valve (AoV) calcification is common in aged populations. Its subsequent aortic stenosis has been linked with increased morbidity, but still has no effective pharmacological intervention. Our previous data show endoplasmic reticulum (ER) stress is involved in AoV calcification. Here, we investigated whether deficiency of ER stress downstream effector CCAAT/enhancer‐binding protein homology protein (CHOP) may prevent development of AoV calcification. AoV calcification was evaluated in Apoe^?/? mice (n = 10) or in mice with dual deficiencies of ApoE and CHOP (Apoe^?/?CHOP^?/?, n = 10) fed with Western diet for 24 weeks. Histological and echocardiographic analysis showed that genetic ablation of CHOP attenuated AoV calcification, pro‐calcification signaling activation, and apoptosis in the leaflets of Apoe^?/? mice. In cultured human aortic valvular interstitial cells (VIC), we found oxidized low‐density lipoprotein (oxLDL) promoted apoptosis and osteoblastic differentiation of VIC via CHOP activation. Using conditioned media (CM) from oxLDL‐treated VIC, we further identified that oxLDL triggered osteoblastic differentiation of VIC via paracrine pathway, while depletion of apoptotic bodies (ABs) in CM suppressed the effect. CM from oxLDL‐exposed CHOP‐silenced cells prevented osteoblastic differentiation of VIC, while depletion of ABs did not further enhance this protective effect. Overall, our study indicates that CHOP deficiency protects against Western diet‐induced AoV calcification in Apoe^?/? mice. CHOP deficiency prevents oxLDL‐induced VIC osteoblastic differentiation via preventing VIC‐derived ABs releasing.     

Chelerythrine induces apoptosis via ROS‐mediated endoplasmic reticulum stress and STAT3 pathways in human renal cell carcinoma

Renal cell carcinoma (RCC) is a heterogeneous histological disease and it is one of the most common kidney cancer. The treatment of RCC has been improved for the past few years, but its mortality still remains high. Chelerythrine (CHE) is a natural benzo[c]phenanthridine alkaloid and a widely used broad‐range protein kinase C inhibitor which has anti‐cancer effect on various types of human cancer cells. However, its effect on RCC has not been fully elucidated. In this study, we evaluated the effect and mechanism of CHE on RCC cells. Our study showed that CHE induced colony formation inhibition and G2/M cell cycle arrest in a dose‐dependent manner in RCC cells. In addition, CHE increased cellular ROS level, leading to endoplasmic reticulum (ER) stress, inactivating STAT3 activities and inducing apoptosis in RCC cells which were suppressed by NAC, a special ROS inhibitor. We further found that both knockdown of ATF4 protein and overexpression of STAT3 protein could reduce CHE‐induced apoptosis in Caki cells. These results demonstrated that the apoptosis induced by CHE was mediated by ROS‐caused ER stress and STAT3 inactivation. Collectively, our studies provided support for CHE as a potential new therapeutic agent for the management of RCC.     

Manganese activates autophagy to alleviate endoplasmic reticulum stress–induced apoptosis via PERK pathway

Overexposure to manganese (Mn) is neurotoxic. Our previous research has demonstrated that the interaction of endoplasmic reticulum (ER) stress and autophagy participates in the early stage of Mn‐mediated neurotoxicity in mouse. However, the mechanisms of ER stress signalling pathways in the initiation of autophagy remain confused. In the current study, we first validated that ER stress–mediated cell apoptosis is accompanied by autophagy in SH‐SY5Y cells. Then, we found that inhibiting ER stress with 4‐phenylbutyrate (4‐PBA) decreased ER stress–related protein expression and reduced cell apoptosis, whereas blocking autophagy with 3‐methyladenine (3‐MA) increased cell apoptosis. These data indicate that protective autophagy was activated to alleviate ER stress–mediated apoptosis. Knockdown of the protein kinase RNA‐like ER kinase (PERK) gene inhibited Mn‐induced autophagy and weakened the interaction between ATF4 and the LC3 promoter. Our results reveal a novel molecular mechanism in which ER stress may regulate autophagy via the PERK/eIF2α/ATF4 signalling pathway. Additionally, Mn may activate protective autophagy to alleviate ER stress–mediated apoptosis via the PERK/eIF2α/ATF4 signalling pathway in SH‐SY5Y cells.     

内质网应激及其特有凋亡途径Caspase-12与糖尿病大鼠肾组织固有细胞凋亡之间的关系

目的：检测内质网应激（ERS）标志蛋白在糖尿病大鼠肾组织的表达及其与细胞凋亡之间的关系。方法：单侧肾切除大鼠腹腔注射链脲佐菌素诱发糖尿病,于8周应用免疫组织化学检测GRP78、Caspase-12、PCNA的表达与定位,TUNEL染色检测细胞凋亡部位,流式细胞术检测细胞凋亡程度,并对GRP78、Caspase-12表达水平进行半定量分析,同时观察尿蛋白、BUN、尿肌酐等反应肾功能的相关指标。结果：建模8周,糖尿病组大鼠较对照组肾小球、肾小管凋亡细胞数明显增多,GRP78、Caspase-12表达显著增强。PCNA表达与对照组无明显差异。结论：糖尿病肾损害过程中,ERS被诱导并可能通过激活其特有的凋亡途径Caspase-12引起肾脏细胞过多丢失在糖尿病肾病的发病机制中起重要作用。     

Polystyrene nanoplastics aggravates lipopolysaccharide-induced apoptosis in mouse kidney cells by regulating IRE1/XBP1 endoplasmic reticulum stress pathway via oxidative stress

Nanoplastics (NPs) pollution poses a huge threat to the ecosystem and has become one of the environmental pollutants that have attracted much attention. There is increasing evidence that both oxidative stress and endoplasmic reticulum stress (ERS) are associated with polystyrene nanoplastics (PS-NPs) exposure. Lipopolysaccharide (LPS) has been shown to induce apoptotic damage in various tissues, but whether PS-NPs can aggravate LPS-induced apoptosis in mouse kidneys through oxidative stress-regulated inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) ERS pathway remains unclear. In this study, based on the establishment of in vitro and in vivo PS-NPs and LPS exposure models alone and in combination in mice and HEK293 cells, the effects and mechanisms of PS-NPs on LPS-induced renal cell apoptosis were investigated. The results showed that PS-NPs could aggravate LPS-induced apoptosis. PS-NPs/LPS can induce ERS through oxidative stress, activate the IRE1/XBP1 pathway, and promote the expression of apoptosis markers (Caspase-3 and Caspase-12). Kidney oxidative stress, ERS, and apoptosis in PS-NPs + LPS combined exposure group were more severe than those in the single exposure group. Interestingly, 4-phenylbutyric acid-treated HEK293 cells inhibited the expression of the IRE1/XBP1 ERS pathway and apoptotic factors in the PS-NPs + LPS combined exposure group. N-acetyl-L-cysteine effectively blocked the activation of the IRE1/XBP1 ERS pathway, suggesting that PS-NPs-induced oxidative stress is an early event that triggers ERS. Collectively, these results confirmed that PS-NPs aggravated LPS-induced apoptosis through the oxidative stress-induced IRE1/XBP1 ERS pathway. Our study provides new insights into the health threats of PS-NPs exposed to mammals and humans.     

Icariside II enhances cisplatin-induced apoptosis by promoting endoplasmic reticulum stress signalling in non-small cell lung cancer cells

Although cisplatin is the most effective first-line drug in the management of advanced non-small cell lung cancer (NSCLC), drug resistance remains a major clinical challenge. There is increasing evidence that icariside II (IS) exhibits antitumour activity in a variety of cancers. In the current study, we investigated the anticancer effects of icariside II combined with cisplatin and elucidated the underlying mechanism in NSCLC. Here, we showed that cotreatment with IS and cisplatin inhibited cell proliferation and induced cellular apoptosis. Using mRNA sequencing (mRNA-seq), we identified differentially expressed genes (DEGs) in which there was an enrichment in PERK-mediated unfolded protein response (UPR) signalling. The western blot results revealed that IS activated endoplasmic reticulum (ER) stress, including three branches of UPR signalling, PERK, IRE1 and ATF6, and the downstream PERK-eIF2α-ATF4-CHOP pathway, thus potentiating the apoptosis induced by cisplatin. In addition, the combination of IS with cisplatin significantly reduced xenograft tumour growth in C57BL/6 and BALB/c nude mice in vivo. Notably, the combination therapy displayed no evident toxicity. Taken together, IS enhances cisplatin-induced apoptosis partially by promoting ER stress signalling in NSCLC, suggesting that combination treatment with IS and cisplatin is a novel potential therapeutic strategy for NSCLC.     

内质网应激的细胞效应分子机制

内质网应激（endoplasmic reticulum stress,ERs）是内质网腔内错误折叠蛋白聚积的一种适应性反应,适度ERs通过激活未折叠蛋白反应起适应性的细胞保护作用,而过高和持久的ERs则通过诱导转录因子CHOP表达、激活caspase-12和c—Jun氨基末端激酶（JNK）等导致细胞凋亡。近年来,越来越多的研究提示内质网应激是神经退行性病变、2型糖尿病以及肥胖等疾病发生过程中的重要环节。对内质网应激的细胞效应分子机制进行综述。随着对ERs机制理解的深入,有可能会发现新的分子标志物或新的诊疗策略。     

病毒性心肌炎所致小鼠心力衰竭心肌组织内质网应激相关的凋亡关系的研究

目的：探讨病毒性心肌炎心力衰竭小鼠心肌组织内质网应激介导的凋亡途径。方法：40只雄性Balb／c小鼠分为病毒性心肌炎组和正常对照组（n=20）,病毒性心肌炎组应用柯萨奇B3病毒制作BALB／c小鼠病毒性心肌炎模型,观察小鼠的一般情况,7d行血流动力学检查后处死取心脏标本,用TUNEL法检测心肌细胞凋亡,RT-PCR检测心肌细胞内质网伴侣蛋白葡萄糖调节蛋白（GAP）78和GRP04的mRNA表达水平。结果：①与正常对照组相比,病毒性心肌炎组小鼠血流动力学指标明显降低（P〈0．01）;②TUNEL染色显示病毒性心肌炎心力衰竭小鼠心肌组织凋亡明显增多（P〈0．01）;③病毒性心肌炎组小鼠内质网伴侣蛋白GRP78和GRP94的mRNA表达水平均明显高于对照组（P〈0．01）。结论：病毒性心肌炎心力衰竭小鼠内质网应激可能介导了心肌细胞凋亡。     

Long non‐coding RNA MEG3 knockdown attenuates endoplasmic reticulum stress‐mediated apoptosis by targeting p53 following myocardial infarction

Mounting evidence has indicated that long non‐coding RNA maternally expressed gene 3 (lncRNA MEG3) regulates cell apoptosis, and is involved in a variety of diseases. However, its exact role in myocardial infarction (MI) has not been fully elucidated. In the present study, we firstly observed that the expression levels of the lncRNA MEG3 in infarct hearts and hypoxic neonatal mice ventricular myocytes (NMVMs) were up‐regulated by quantitative real‐time PCR (qRT‐PCR). Then, we knocked down lncRNA MEG3 by lentiviral delivery in the myocardial border region following multipoint injection. Following 28 days of MI, the lncRNA MEG3 knockdown mice indicated better cardiac function, and less cardiac remodelling by ultrasonic cardiogram and histological analysis. In addition, we indicated that lncRNA MEG3 knockdown reduced myocyte apoptosis and reactive oxygen species production in MI mice model and hypoxic NMVMs. Furthermore, we revealed that knockdown of lncRNA MEG3 protected against endoplasmic reticulum stress (ERS)‐mediated myocardial apoptosis including the induction of PERK‐eIF2α and caspase 12 pathways. At last, we provided evidence that p53 was identified as a protein target of lncRNA MEG3 to regulate NF‐κB‐ and ERS‐associated apoptosis. Taken collectively, our findings demonstrated that lncRNA MEG3 knockdown exerted cardioprotection by reducing ERS‐mediated apoptosis through targeting p53 post‐MI.     

内质网应激与心血管疾病研究概述

内质网应激是继死亡受体信号途径和线粒体途径之后新近发现的一条细胞凋亡通路,适度的应激可通过未折叠蛋白反应(UPR)产生细胞保护作用,但当应激过强或长时间不缓解时则会触发CHOP、ASK1/JNK及Caspases等通路诱导细胞凋亡。近年来研究发现内质网应激参与多种心血管疾病的发生发展,通过对相关通路的干预可以产生心肌细胞的保护作用,这有望成为防治心脏疾病的新靶点。     

缺血性心肌病大鼠心肌细胞自噬在心肌重塑中的作用*

目的:研究缺血性心肌病大鼠心肌细胞自噬在心肌重塑中的作用。方法:36 只雄性SD大鼠分为正常对照组、假手术组、缺血性心肌病组( n=12),3组大鼠术前行心脏彩超检查,正常对照组大鼠不进行处理;假手术组大鼠开胸后不结扎冠状动脉,关闭胸腔;缺血性心肌病组大鼠开胸结扎冠状动脉20 min后,解开结扎线行再灌注后关闭胸腔,3组大鼠术后4周行心脏彩超检查后处死大鼠取心脏行HE染色、masson染色,观察心肌病理改变,用Western blot技术检测各组大鼠心肌细胞GRP78、LC3-I、LC3-II、Beclin-I表达及LC3-II/LC3-I比值的变化。结果:与正常组及假手术组比较,缺血性心肌病大鼠心室扩大,EF值降低;心肌排列紊乱,心肌纤维化增加;线粒体空泡化严重;内质网应激关键蛋白GRP78上调;自噬相关蛋白LC3-I、LC3-II、Beclin-I及LC3-II/LC3-I比值增加。结论:缺血性心肌病大鼠心肌细胞中内质网应激和自噬可能在心肌重塑中具有重要作用。