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.     

Diabetes- and angiotensin II-induced cardiac endoplasmic reticulum stress and cell death: metallothionein protection

We have shown cardiac protection by metallothionein (MT) in the development of diabetic cardiomyopathy (DCM) via suppression of cardiac cell death in cardiac-specific MT-overexpressing transgenic (MT-TG) mice. The present study was undertaken to define whether diabetes can induce cardiac endoplasmic reticulum (ER) stress and whether MT can prevent cardiac cell death via attenuating ER stress. Diabetes was induced by streptozotocin in both MT-TG and wild-type (WT) mice. Two weeks, and 2 and 5 months after diabetes onset, cardiac ER stress was detected by expression of ER chaperones, and apoptosis was detected by CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) and cleaved caspase-3 and caspase-12. Cardiac apoptosis in the WT diabetic mice, but not in MT-TG diabetic mice, was significantly increased 2 weeks after diabetes onset. In parallel with apoptotic effect, significant up-regulation of the ER chaperones, including glucose-regulated protein (GRP)78 and GRP94, cleaved ATF6 and phosporylated eIF2α, in the hearts of WT, but not MT-TG diabetic mice. Infusion of angiotensin II (Ang II) also significantly induced ER stress and apoptosis in the hearts of WT, but not in MT-TG mice. Direct administration of chemical ER stress activator tunicamycin significantly increased cardiac cell death only in WT mice. Pre-treatment with antioxidants completely prevented Ang II-induced ER stress and apoptosis in the cultured cardiac cells. These results suggest that ER stress exists in the diabetic heart, which may cause the cardiac cell death. MT prevents both diabetes- and Ang II-induced cardiac ER stress and associated cell death most likely via its antioxidant action, which may be responsible for MT's prevention of DCM.     

白芍总苷对心肌缺血再灌注大鼠内质网应激因子CHOP、GRP78、GRP94的表达及凋亡的影响

目的:研究白芍总苷(TGP)对心肌缺血再灌注(I/R)大鼠内质网应激因子CCAAT/增强子结合蛋白的同源蛋白(CHOP)、葡萄糖调节蛋白78(GRP78)、葡萄糖调节蛋白94(GRP94)表达及凋亡的影响。方法:选择健康清洁级SD大鼠75只,根据随机数字表法分成5组,每组15只,分别记为假手术组、I/R组、50 mg/kg TGP组、100 mg/kg TGP组以及200 mg/kg TGP组。检测并对比各组大鼠CHOP、GRP78、GRP94水平,对比分析各组大鼠心肌I/R指标、梗死面积率以及心肌细胞的凋亡率。结果:I/R组和TGP各组的CHOP、GRP78及GRP94水平均明显高于假手术组,且TGP各组较I/R组明显更低(均P0.05)。100 mg/kg和200 mg/kg TGP组的CHOP、GRP78及GRP94水平均明显低于50 mg/kg TGP组,且200 mg/kg TGP组较100 mg/kg TGP组明显更低(均P0.05)。I/R组和TGP各组缺血30 min的T波改变、再灌注120 min的T波改变及LVEDP水平均明显高于假手术组,LVSP、+dp/dtmax及-dp/dtmax水平均明显低于假手术组(均P0.05)。与I/R组相比,TGP组缺血30 min的T波改变、再灌注120 min的T波改变及LVEDP水平呈剂量依赖型下降,而LVSP、+dp/dtmax及-dp/dtmax水平呈剂量依赖型上升(均P0.05)。I/R组和TGP各组的梗死面积率和心肌细胞的凋亡率均明显高于假手术组(均P0.05)。与I/R组相比,TGP组的梗死面积率和心肌细胞的凋亡率水平呈剂量依赖型下降(均P0.05)。结论:应用TGP能够明显降低MIRI大鼠内质网应激因子CHOP、GRP78、GRP94的表达,调节心肌缺血和再灌注相关标志物或临床参数的水平,显著减少心肌缺血和再灌注所致的心肌梗死面积率及细胞凋亡率。     

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.     

The protective effect of curcumin on cardiac markers and fibrosis in abemaciclib-induced cardiac damage in rats

Abemaciclib (ABE) is a cyclin-dependent kinase inhibitor used in combination with an antiestrogen in the treatment of breast cancer. In addition to the important therapeutic properties of this drug, its side effects are not fully known. In this study, we aimed to investigate the protective effect of curcumin (CUR) on cardiac damage caused by ABE administration. Forty rats were equally divided into control, dimethyl sulfoxide (150 µL), CUR (30 mg/kg/day), ABE (26 mg/kg/day), and ABE + CUR (26 mg/kg/day ABE and 30mg/kg/day CUR) groups (n = 8). Injections were administered daily for 28 days. Troponin-I, total cholesterol, and creatine kinase myocardial band (CK-MB) levels and cardiac fibrosis were higher in the ABE group than in the control group (p < 0.05), and were lower in the ABE + CUR group than in the ABE group (p < 0.05). The results showed that ABE administration can cause cardiac damage and increase cardiac fibrosis. However, they showed that coadministration of CUR with ABE could suppress increases in CK-MB, troponin-I, and total cholesterol levels and also cardiac fibrosis associated with cardiac damage. Therefore, we can infer that the subsequent administration of CUR ABE treatment can be used as a therapeutic strategy for preventing cardiac damage.     

Selenium Deficiency Is Associated with Endoplasmic Reticulum Stress in a Rat Model of Cardiac Malfunction

The relationship between selenium (Se) deficiency-induced cardiac malfunction and endoplasmic reticulum (ER) stress is poorly understood. In the present study, 18 weaning Sprague Dawley rats were randomly fed with three different Se diets, and myocardial glutathione peroxidase (GPx) activity was measured by an enzyme activity assay. Cardiac function was evaluated by hemodynamic parameters. ER stress markers immunoglobulin-binding protein (BiP)/glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) were detected by western blotting. Our data showed that myocardial GPx activity and cardiac function were conspicuously impaired in Se-deficient rats. Expression of GRP78 and CHOP was significantly upregulated by treatment of Se deficiency. Improvements in myocardial GPx activity and cardiac function, as well as decreases in expression of GRP78 and CHOP, were observed after Se supplementation. Consequently, our data show that ER stress was involved in Se deficiency-induced cardiac dysfunction.     

Androgen receptor gene knockout male mice exhibit impaired cardiac growth and exacerbation of angiotensin II-induced cardiac fibrosis

Androgen has anabolic effects on cardiac myocytes and has been shown to enhance left ventricular enlargement and function. However, the physiological and patho-physiological roles of androgen in cardiac growth and cardiac stress-induced remodeling remains unclear. We aimed to clarify whether the androgen-nuclear androgen receptor (AR) system contributes to the cardiac growth and angiotensin II (Ang II)-stimulated cardiac remodeling by using systemic AR-null male mice. AR knock-out (ARKO) male mice, at 25 weeks of age, and age-matched wild-type (WT) male mice were treated with or without Ang II stimulation (2.0 mg/kg/day) for 2 weeks. ARKO mice with or without Ang II stimulation showed a significant reduction in the heart-to-body weight ratio compared with those of WT mice. In addition, echocardiographic analysis demonstrated impairments of both the concentric hypertrophic response and left ventricular function in Ang II-stimulated ARKO mice. Western blot analysis of the myocardium revealed that activation of extracellular signal-regulated kinases (ERK) 1/2 and ERK5 by Ang II stimulation were lower in ARKO mice than those of WT mice. Ang II stimulation caused more prominent cardiac fibrosis in ARKO mice than in WT mice with enhanced expression of types I and III collagen and transforming growth factor-beta1 genes and with increased Smad2 activation. These results suggest that, in male mice, the androgen-AR system participates in normal cardiac growth and modulates cardiac adaptive hypertrophy and fibrosis during the process of cardiac remodeling under hypertrophic stress.     

转录因子GADDl53/CHOP的激活与糖尿病大鼠肾组织固有细胞凋亡的关系初探

目的检测内质网应激(endoplasmic reticulum stress,ERS)标志蛋白:葡萄糖调节蛋白(GRP78/Bip)、转录因子GADDl53/CHOP在糖尿病大鼠肾脏细胞中表达及其与肾脏固有细胞凋亡之间的关系,初步探讨ERS在糖尿病肾损害中的作用及机制。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、GADDl53/CHOP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、GAD-Dl53/CHOP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,糖尿病大鼠较正常组的肾细胞凋亡率明显升高,GRP78、GADDl53/CHOP表达明显增加。结论糖尿病肾损害过程中,ERS被诱导并可能通过激活转录因子GADDl53/CHOP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。     

Insulin induces chaperone and CHOP gene expressions in adipocytes

Adipocyte secretes bioactive proteins called adipocytokines, and biosynthesis of secretory proteins requires molecular chaperones and folding enzymes in endoplasmic reticulum (ER). ER chaperones are known to be induced by unfolded protein response (UPR) and growth factors, however, it has not been determined how ER chaperones expression is regulated in adipocytes. Here we show that insulin treatment induced GRP78 and ERO1L mRNA levels in 3T3-L1 adipocytes. Insulin also upregulated CHOP mRNA levels, but did not induce phosphorylation of eIF2α. Pretreatment with insulin protected 3T3-L1 adipocytes against thapsigargin-mediated phosphorylation of eIF2α but did not against DTT-mediated one. In vivo mice study showed that GRP78 and CHOP expressions were regulated by feeding conditions. These results suggest that insulin signaling is important to induce mRNA expressions of GRP78 and CHOP, and may have a protective role against UPR.     

拉西地平对内质网应激导致左室重塑的保护作用

目的:研究在高温高湿应激状态下拉西地平对葡萄糖调节蛋白(glucose-regulated protein78,GRP78)和C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白(C/EBP-homologous protein,CHOP)在大鼠心肌中表达及对心室重塑的影响。方法:将30只雄性Sprague-Dawly(SD)大鼠随机分为对照组、高温高湿组、拉西地平干预组,每组10只。喂养6周后颈动脉插管测定平均动脉压及心率。B超检测左室形态结构。免疫组化法检测大鼠心肌GRP78及CHOP蛋白及表达水平。结果:高温高湿组的大鼠平均动脉压(MBP)、隔厚度(IVST)、左室后壁厚度(LPWT)、左室重量指数(LVWI),GRP78及CHOP蛋白表达水平与对照组相比均有显著升高(p<0.01),拉西地平干预组能显著降低大鼠平均动脉压(MBP)、室间隔厚度(IVST)、左室重量指数(LVWI),GRP78及CHOP蛋白的表达水平(p<0.05)。结论:内质网应激可能参与了高温高湿诱导的左室重构;拉西地平可能通过降低GRP78及CHOP的表达干预了ERS介导的心肌肥厚通路,从而改善心脏功能。     

Scoparone alleviates Ang II-induced pathological myocardial hypertrophy in mice by inhibiting oxidative stress

Long-term poorly controlled myocardial hypertrophy often leads to heart failure and sudden death. Activation of ras-related C3 botulinum toxin substrate 1 (RAC1) by angiotensin II (Ang II) plays a pivotal role in myocardial hypertrophy. Previous studies have demonstrated that scoparone (SCO) has beneficial effects on hypertension and extracellular matrix remodelling. However, the function of SCO on Ang II-mediated myocardial hypertrophy remains unknown. In our study, a mouse model of myocardial hypertrophy was established by Ang II infusion (2 mg/kg/day) for 4 weeks, and SCO (60 mg/kg bodyweight) was administered by gavage daily. In vitro experiments were also performed. Our results showed that SCO could alleviate Ang II infusion-induced cardiac hypertrophy and fibrosis in mice. In vitro, SCO treatment blocks Ang II-induced cardiomyocyte hypertrophy, cardiac fibroblast collagen synthesis and differentiation to myofibroblasts. Meanwhile, we found that SCO treatment blocked Ang II-induced oxidative stress in cardiomyocytes and cardiac fibroblasts by inhibiting RAC1-GTP and total RAC1 in vivo and in vitro. Furthermore, reactive oxygen species (ROS) burst by overexpression of RAC1 completely abolished SCO-mediated protection in cardiomyocytes and cardiac fibroblasts in vitro. In conclusion, SCO, an antioxidant, may attenuate Ang II-induced myocardial hypertrophy by suppressing of RAC1 mediated oxidative stress.     

Beneficial effects of angiotensin II receptor blocker,olmesartan, in limiting the cardiotoxic effect of daunorubicin in rats

Abstract

The aim was to evaluate the role of the combination of olmesartan, an angiotensin II (Ang II) receptor blocker (ARB), with daunorubicin (DNR) in reducing cardiac toxicity in rats. DNR was administered at a dose of 3 mg/kg/day every other day for 12 days. Olmesartan was administered orally every day for 12 days. Rats treated with DNR alone showed cardiac toxicity as evidenced by worsening cardiac function, elevation of malondialdehyde level in heart tissue and decreased in the level of total glutathione peroxidase activity; treatment with ARB reversed these changes. Furthermore, ARB treatment down-regulated matrix metalloproteinase-2 expression, myocardial expression of Ang II, attenuated the increased protein expressions of p67^phox and Nox4 and reduced oxidative stress-induced DNA damage evaluated by expression of 8-hydroxydeoxyguanosine. In conclusion, the result demonstrated that Ang II and oxidative stress play a key role in anthracycline-induced cardiotoxicity and that treatment with ARB will be beneficial against DNR-induced cardiotoxicity.     

Sexual Dimorphism in Urinary Angiotensinogen Excretion During Chronic Angiotensin II−Salt Hypertension

BackgroundThe intrarenal renin−angiotensin system contributes to hypertension by regulating sodium and water reabsorption throughout the nephron. Sex differences in the intrarenal components of the renin−angiotensin system have been involved in the greater incidence of high blood pressure and progression to kidney damage in males than females.ObjectiveThis study investigated whether there is a sex difference in the intrarenal gene expression and urinary excretion of angiotensinogen (AGT) during angiotensin II (Ang II)−dependent hypertension and high-salt (HS) diet.MethodsMale and female Sprague-Dawley rats were divided into 5 groups for each sex: Normal-salt control, HS diet (8% NaCl), Ang II−infused (80 ng/min), Ang II−infused plus HS diet, and Ang II−infused plus HS diet and treatment with the Ang II receptor blocker, candesartan (25 mg/L in the drinking water). Rats were evaluated for systolic blood pressure (SBP), kidney AGT mRNA expression, urinary AGT excretion, and proteinuria at different time points during a 14-day protocol.ResultsBoth male and female rats exhibited similar increases in urinary AGT, with increases in SBP during chronic Ang II infusion. HS diet greatly exacerbated the urinary AGT excretion in Ang II−infused rats; males had a 9-fold increase over Ang II alone and females had a 2.5-fold increase. Male rats displayed salt-sensitive SBP increases during Ang II infusion and HS diet, and female rats did not. In the kidney cortex, males displayed greater AGT gene expression than females during all treatments. During Ang II infusion, both sexes exhibited increases in AGT gene message compared with same-sex controls. In addition, HS diet combined with Ang II infusion exacerbated the proteinuria in both sexes. Concomitant Ang II receptor blocker treatment during Ang II infusion and HS diet decreased SBP and urinary AGT similarly in both sexes; however, the decrease in proteinuria was greater in the females.ConclusionDuring Ang II−dependent hypertension and HS diet, higher intrarenal renin-angiotensin system activation in males, as reflected by higher AGT gene expression and urinary excretion, indicates a mechanism for greater progression of high blood pressure and might explain the sex disparity in development of salt-sensitive hypertension.     

Differential Responses of Hepatic Endoplasmic Reticulum Stress and Inflammation in Diet-Induced Obese Rats with High-Fat Diet Rich in Lard Oil or Soybean Oil

Scopes

To investigate the effects of high-fat diet enriched with lard oil or soybean oil on liver endoplasmic reticulum (ER) stress and inflammation markers in diet-induced obese (DIO) rats and estimate the influence of following low-fat diet feeding.

Methods and Results

Male SD rats were fed with standard low-fat diet (LF, n = 10) and two isoenergentic high-fat diets enriched with lard (HL, n = 45) or soybean oil (HS, n = 45) respectively for 10 weeks. Then DIO rats from HL and HS were fed either high-fat diet continuously (HL/HL, HS/HS) or switched to low-fat diet (HL/LF, HS/LF) for another 8 weeks. Rats in control group were maintained with low-fat diet. Body fat, serum insulin level, HOMA-IR and ectopic lipid deposition in liver were increased in HL/HL and HS/HS compared to control, but increased to a greater extent in HL/HL compared to HS/HS. Markers of ER stress including PERK and CHOP protein expression and phosphorylation of eIF2α were significantly elevated in HL/HL group while phosphorylation of IRE1α and GRP78 protein expression were suppressed in both HL/HL and HS/HS. Besides, inflammatory signals (OPN, TLR2, TLR4 and TNF-α) expressions significantly increased in HL/HL compared to others. Switching to low-fat diet reduced liver fat deposition, HOMA-IR, mRNA expression of TLR4, TNF-α, PERK in both HL/LF and HS/LF, but only decreased protein expression of OPN, PERK and CHOP in HL/LF group. In addition, HL/LF and HS/LF exhibited decreased phosphorylation of eIF2α and increased phosphorylation of IRE1α and GRP78 protein expression when compared with HL/HL and HS/HS respectively.

Conclusions

Lard oil was more deleterious in insulin resistance and hepatic steatosis via promoting ER stress and inflammation responses in DIO rats, which may be attributed to the enrichment of saturated fatty acid. Low-fat diet was confirmed to be useful in recovering from impaired insulin sensitivity and liver fat deposition in this study.     

Suppression of ADAM8 attenuates angiotensin II-induced cardiac fibrosis and endothelial-mesenchymal transition via inhibiting TGF-β1/Smad2/Smad3 pathways

Endothelial-to-mesenchymal transition (EndMT) is involved in cardiac fibrosis induced by angiotensin II (Ang II). A disintegrin and metalloproteinase 8 (ADAM8), a member of ADAMs family, participates in cell adhesion, proteolysis and various signaling. However, its effects on the development of cardiac fibrosis remain completely unknown. This study aimed to reveal whether ADAM8 aggravates cardiac fibrosis induced by Ang II in vivo and in vitro. The C57BL/6J mice or cardiac endothelial cells were subjected to Ang II infusion to induce fibrosis. The results showed that systolic blood pressure and diastolic blood pressure were significantly increased under Ang II infusion, and ADAM8 was up-regulated. ADAM8 inhibition attenuated Ang II-induced cardiac dysfunction. ADAM8 knockdown suppressed Ang II-induced cardiac fibrosis as evidenced by the down-regulation of CTGF, collagen I, and collagen III. In addition, the endothelial marker (VE-cadherin) was decreased, whilst mesenchymal markers (α-SMA and FSP1) were increased following Ang II infusion. However, ADAM8 repression inhibited Ang II-induced EndMT. Moreover, ADAM8 silencing repressed the activation of TGF-β1/Smad2/Smad3 pathways. Consistent with the results in vivo, we also found the inhibitory effects of ADAM8 inhibition on EndMT in vitro. All data suggest that ADAM8 promotes Ang II-induced cardiac fibrosis and EndMT via activating TGF-β1/Smad2/Smad3 pathways.     

Induction of apoptosis by cigarette smoke via ROS-dependent endoplasmic reticulum stress and CCAAT/enhancer-binding protein-homologous protein (CHOP)

In this report, we investigated a role of endoplasmic reticulum (ER) stress in cigarette smoke (CS)-induced apoptosis of human bronchial epithelial cells (hBEC). Exposure of hBEC to CS or CS extract (CSE) caused expression of endogenous ER stress markers GRP78 and CHOP and induction of apoptosis evidenced by nuclear condensation, membrane blebbing, and activation of caspase-3 and caspase-4. In vivo exposure of mice to CS also caused induction of GRP78 and CHOP in the lung. Attenuation of ER stress by overexpression of ER chaperone GRP78 or ORP150 significantly attenuated CSE-triggered apoptosis. Exposure of hBEC to CSE caused generation of reactive oxygen species, and treatment with antioxidants inhibited CSE-induced apoptosis. Interestingly, antioxidants including a scavenger of O(2)(*-) blunted induction of CHOP by CSE without affecting the level of GRP78, and dominant-negative inhibition of CHOP abolished CSE-induced apoptosis. Furthermore, a generator of O(2)(*-) selectively induced CHOP and apoptosis in hBEC. Our results revealed that: (1) CS induces ER stress in vitro and in vivo, (2) ER stress mediates CS-triggered apoptosis downstream of oxidative stress, (3) CS-initiated apoptosis is caused through oxidative stress-dependent induction of CHOP, (4) O(2)(*-) may play a dominant role in this process, and (5) oxidative stress-independent induction of GRP78 counterbalances the proapoptotic action of CHOP.