Erythropoietin Attenuates Cardiac Dysfunction in Rats by Inhibiting Endoplasmic Reticulum Stress-Induced Diabetic Cardiomyopathy

Purpose

Enhanced endoplasmic reticulum (ER) stress and down-regulated SERCA2a expression play crucial roles in diabetes. We aimed to verify whether erythropoietin (EPO) attenuates cardiac dysfunction by suppressing ER stress in diabetic rats.

Methods

Forty male SD rats were randomly divided into four groups: control, EPO-treated control, vehicle-treated diabetic, and EPO-treated diabetic groups. The animals in the EPO-treated control and diabetic groups were administered recombinant human EPO (1000 U/kg body weight) once per week for 12 weeks. RT-PCR and Western blotting assays were performed to detect the expression of 78-kDa glucose-regulated protein precursor (GRP78) and sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a). We cultured neonatal rat cardiomyocytes and investigated the protective effects of EPO against high glucose (HG)-induced apoptosis. Intracellular calcium levels were measured through confocal microscopy.

Results

We observed increased myocardial GRP78 expression and decreased myocardial SERCA2a expression in diabetic rats. EPO prevented the changes in GRP78, SERCA2a expression and cardiac dysfunction in diabetic rats. The levels of GRP78 protein were significantly reduced in EPO-treated diabetic rats compared with vehicle-treated diabetic rats (GRP78 protein 0.09 ± 0.03 vs. 0.54 ± 0.04, P < 0.01). The levels of the SERCA2a proteins were significantly increased in EPO-treated diabetic rats compared with vehicle-treated diabetic rats (SERCA2a protein 0.60 ± 0.05 vs. 0.13 ± 0.04, P < 0.01). A reduction in apoptosis was observed in the cardiomyocytes treated with 20 U/mL EPO compared with the cardiomyocytes cultured under HG conditions (apoptosis rate 18.9 ± 1.94 vs. 37.9 ± 1.59%, P < 0.01).

Conclusions

This study demonstrates that EPO treatment improved the parameters of cardiac function following HG-induced injury by suppressing ER stress and inducing SERCA2a expression.

     

Sparstolonin B Attenuates Hypoxia-Induced Apoptosis,Necrosis and Inflammation in Cultured Rat Left Ventricular Tissue Slices

Purpose

Ischemia/reperfusion results in tissue damage, a rapid increase in cytokines and chemokines and inflammatory cell infiltration. Herein we investigated the ability of a selective TLR2/4 antagonist, Sparstolonin B (SsnB), to protect rat cultured left ventricular tissue (LV) slices from hypoxic injury by inhibiting the myocardial inflammatory response independent of inflammatory cell infiltration.

Methods and Results

Media Lactate dehydrogenase (LDH) levels were measured to reflect hypoxia-induced cytotoxicity and cell injury with and without SsnB. Incubation with SsnB (15 and 30 μM) significantly reduced by 20 and 40 %, respectively, the amount of LDH released from the hypoxic LV slices. TUNEL staining showed that SsnB significantly attenuated the levels of hypoxia-induced apoptotic cells from 61.5?±?4.0 to 27.0?±?2.1 (15 μM SsnB) and 23.5?±?2.2 (30 μM SsnB) cells/unit area. Similarly, the Periodic Acid-Schiff (PAS) staining of ischemic areas in untreated hypoxic LV slices was increased 17 fold from 0.26?±?0.09 to 4.41?±?0.43 %, while in hypoxic slices incubated with 15 and 30 μM of SsnB, the PAS positive ischemic areas were increased by only 6.4 fold to 1.66?±?0.39 % and 3.8 fold to 1.00?±?0.22 %, respectively. Rt-PCR confirmed that MCP1 and IL-6 expression during hypoxia was elevated by 2 and 4 fold, respectively, while their up-regulation was significantly inhibited (i.e., <0.7 fold increase) by SsnB.

Conclusion

The selective TLR2/4 antagonist, Sparstolonin B, can substantially protect LV myocardium via its ability to inhibit injury resulting from hypoxic myocardial-generated inflammation. Accordingly SsnB has potential as a therapeutic agent for the attenuation of myocardial ischemia-reperfusion injury.     

Z-LLY-FMK Attenuates Intestinal Apoptosis After Bile Duct Ligation in Rats

Apoptosis is an important process in a wide variety of different biological systems. In addition to caspases, recently, calpains, another family of proteases, have been found to be involved in apoptosis of many cell systems. This study is designed with the aims to evaluate the possible effect of Z-LLY-FMK (a calpain inhibitor) on intestine apoptosis after bile duct ligation in rats. Male Sprague–Dawley rats weighing 250–300 g were randomized to five groups (n = 6 in each group). Group 1 (Control: C) underwent Sham operation and were simultaneously treated with the same amount of normal saline. Group 2 (Control with DMSO: CDMSO) underwent Sham operation and were simultaneously treated with the same amount of dimethylsulfoxide (DMSO). Group 3 (Obstructive jaundice: OB) underwent common bile duct ligation without any other manipulation. Group 4 (Obstructive jaundice with Z-LLY-FMK: OBZLLY) underwent common bile duct ligation and were simultaneously treated with Z-LLY-FMK (dissolved in DMSO). Group 5 (Obstructive jaundice with ZFA-FMK: OBZFA) underwent common bile duct ligation and were simultaneously treated with ZFA-FMK (dissolved in DMSO). After 3 days, intestine tissue was harvested for apoptosis measurements. There was no significant difference between Sham operation group (C) and Sham operation with DMSO group (CDMSO) either in jejunum (P = 0.924) or in ileum (P = 0.996). When compared to Sham operation group (C), increased intestine apoptosis occurred in either jejunum (P < 0.001) or in ileum (P < 0.001) after common bile duct ligation (OB). After administration of Z-LLY-FMK (OBZLLY), the increased intestine apoptosis after common bile duct ligation (OB) was significantly diminished either in jejunum or in ileum (P < 0.001 and P < 0.001). Moreover, administration of ZFA (OBZFA) failed to show the same phenomenon in either jejunum (P = 0.993) or ileum (P = 0.485). There was a significant difference in intestine apoptosis in either jejunum (P < 0.001) or in ileum (P < 0.001) between OBZLLY group and OBZFA group. Significantly increased intestine apoptosis occurred after common bile duct ligation. The administration of Z-LLY-FMK could effectively diminish the intestine apoptosis after common bile duct ligation, whereas the administration of ZFA-FMK failed to show the same effect.     

Tetradecylthioacetic Acid Attenuates Inflammation and Has Antioxidative Potential During Experimental Colitis in Rats

Background

The fatty acid analogue tetradecylthioacetic acid (TTA) is a moderate pan-activator of peroxisome proliferator-activated receptors (PPARs), and has in previous studies showed potential as an antioxidant and anti-inflammatory agent, both through PPAR and non-PPAR mediated mechanisms.

Aims

This study aimed to determine whether TTA could alleviate dextran sulfate sodium (DSS)-induced colitis in rats.

Methods

Male Wistar rats were fed a control diet (control- and DSS-group) or a diet supplemented with 0.4 % TTA (TTA + DSS-group) for 30 days, and DSS was added to the drinking water the last 7 days. Ultrasound measurements were performed at day 29. At day 30, rats were sacrificed and the distal colon was removed for histological evaluation and measurement of cytokine levels, oxidative damage, and gene expression.

Results

The disease activity index was not improved in the TTA + DSS-group compared to the DSS-group. However, ultrasound measurements showed a significantly reduced colonic wall thickening in the TTA + DSS-group. TNF-α, IL-1β, and IL-6 were reduced at the protein and mRNA level in the TTA + DSS-group. Moreover, TTA-treated rats demonstrated reduced colonic oxidative damage, while inducible nitric oxide synthase 2 mRNA expression was elevated in both the DSS- and TTA + DSS-groups. PPARγ signaling may be involved in the anti-inflammatory response to TTA, as Pparg mRNA expression was significantly upregulated in colon.

Conclusions

This study demonstrated that the pan-PPAR agonist TTA reduced colonic oxidative damage and cytokine levels in a rat model of colitis, and its potential to ameliorate colitis should be further explored.     

沉默信息调节因子蛋白家族、衰老与糖尿病心肌病

糖尿病心肌病是除外冠状动脉疾病或高血压等糖尿病继发性损害之外独立存在的特异性心肌损害,是糖尿病患者发展为心力衰竭的重要原因之一。衰老和代谢异常之间的关系密切,而研究证实沉默信息调节因子蛋白家族可通过去乙酰化作用调节寿命及代谢。现主要以沉默信息调节因子蛋白家族、衰老及糖尿病心肌病三者之间关系的研究进展展开本综述,以进一步阐明糖尿病心肌病的发病机制及沉默信息调节因子基因作为治疗靶点的潜能。     

Advanced Interfere Treatment of Diabetic Cardiomyopathy Rats by aFGF-Loaded Heparin-Modified Microbubbles and UTMD Technique

This study aims to investigate the preclinical performance and mechanism of a novel strategy of aFGF-loaded heparin-modified microbubbles (aFGF-HMB) combined with ultrasound-targeted microbubble destruction (UTMD) technique for diabetic cardiomyopathy (DCM) prevention. Type 1 diabetic rats were induced by streptozotocin. Twelve weeks after intervention, indexes from transthoracic echocardiography and cardiac catheterization showed that the left ventricular function in the aFGF-HMB/UTMD group was significantly improved compared with diabetes control (DM). From Picrosirius Red staining and TUNEL staining, the aFGF-HMB/UTMD group showed significant difference from the other groups. The cardiac collagen volume fraction (CVF) and myocardial cell apoptosis index (AI) in aFGF-HMB/UTMD group decreased to 7.2 % and 7.11 % respectively, compared with the DM group (CVF = 24.5 % and AI =20.3 % respectively). The results of myocardial microvascular density (MCD) also proved the strongest inhibition of aFGF-HMB/UTMD group on DCM progress. CD31 staining of aFGF-HMB/UTMD group reached 22 n/hrp, much higher than that of DM group (9 n/hrp). These results confirmed that the abnormalities including left ventricular dysfunction, myocardial fibrosis, cardiomyocytes apoptosis and microvascular rarefaction could be suppressed by twice weekly aFGF treatments for 12 consecutive weeks (free aFGF or aFGF-HMB+/?UTMD), with the strongest improvements observed in the aFGF-HMB/UTMD group (P < 0.05 vs free aFGF or aFGF-HMB). Western blot analyses of heart tissue further revealed the highest aFGF, anti-apoptosis protein (Bcl-2), VEGF-C, pAkt, pFoxo-3a levels and strongest reduction in pro-apoptosis proteins (Bax) level in aFGF-HMB/UTMD group. Overall, aFGF-HMB combined with UTMD technique might be developed as an effective strategy to prevent DCM in future clinical therapy.     

黄芩苷对脂多糖诱导的巨噬细胞Toll样受体4表达的影响

目的 研究黄芩苷对脂多糖诱导的小鼠巨噬细胞Toll样受体4和其下游信号分子抑制蛋白kB以及效应分子肿瘤坏死因子α表达的影响.方法 分别用脂多糖(终浓度1 mg/L)或脂多糖+黄芩苷(终浓度10、50和100 μmol/L)处理生长良好的小鼠巨噬细胞RAW264.7,逆转录聚合酶链反应和Western Blot检测细胞Toll样受体4表达情况和抑制蛋白kB含量变化,酶联免疫吸附法检测细胞上清液中肿瘤坏死因子α的浓度.结果 脂多糖刺激RAW264.7细胞可导致Toll样受体4表达增高,促进抑制蛋白kB降解,上调肿瘤坏死因子α表达;黄芩苷预处理能降低脂多糖诱导的Toll样受体4表达增高,降低抑制蛋白kB降解,下调肿瘤坏死因子α分泌.结论 黄芩苷可通过抑制Toll样受体4表达和降低抑制蛋白kB降解,影响Toll样受体4/抑制蛋白kB-核因子kB炎症信号途径,阻碍炎症因子肿瘤坏死因子α的生成,发挥抗炎作用,这可能是其抗动脉粥样硬化的作用机制之一.     

糖尿病心肌病患者与抗G-蛋白偶联型受体血管紧张素Ⅱ1型受体和肾上腺素能α1受体自身抗体的初步研究

目的:探讨抗G-蛋白偶联型受体血管紧张素Ⅱl型受体(AT1受体)和肾上腺素能α1受体(α1受体)自身抗体是否与糖尿病心肌病发病有关.方法:以合成的AT1与α1受体多肽片段为抗原,应用酶联免疫吸附测定技术,检测196例住院及门诊体检者血清中抗G-蛋白偶联型受体AT1和α1受体自身抗体,其中糖尿病心肌病患者46例(糖尿病心肌病组),2型糖尿病52例(糖尿病组),高血压无靶器官受损患者58例(高血压组)及正常对照组40例.结果:糖尿病心肌病组抗AT1受体自体抗体阳性率和抗α1受体自身抗体阳性率,明显高于糖尿病组、高血压组以及正常对照组,有显著性差异(P均＜0.05～0.01).结论:免疫学机制可能参与糖尿病心肌病病理生理过程.     

怡心饮对糖尿病心脏病大鼠心肌CTGF-mRNA和MCP-1mRNA表达的影响

目的观察怡心饮对糖尿病心脏病大鼠心脏单核趋化因子1(MCP-1)和结缔组织生长因子(CTGT)mRNA表达的影响。方法运用高糖高脂饲料加链脲佐菌素腹腔注射建立大鼠2型糖尿病心肌病模型,分为怡心饮高剂量组、怡心饮低剂量组、生脉饮对照组、正常组、模型组。灌胃治疗8周后,观察糖尿病心肌病大鼠的心脏单核趋化因子1和结缔组织生长因子mRNA的表达,以及血清结缔组织生长因子的变化,分析其减轻大鼠心肌病变的机制。结果怡心饮能明显降低大鼠心肌单核趋化因子1和结缔组织生长因子mRNA表达,降低大鼠血清结缔组织生长因子含量。结论怡心饮对糖尿病大鼠心肌病变有一定的改善作用,其作用机制可能与降低单核趋化因子1和结缔组织生长因子mRNA的表达有关。     

血管紧张素Ⅱ-1型受体拮抗剂对心肌梗死大鼠骨桥蛋白表达及胶原沉积的影响

目的:探讨血管紧张素Ⅱ-1型受体拮抗剂对心肌梗死大鼠骨桥蛋白(OPN)的表达及心肌间质胶原沉积的影响。方法:将心肌梗死后24小时存活大鼠随机分为两组:盐水组(16只,5ml/d),厄贝沙坦组[17只,45mg/(kg·d)];另设假手术组(15只)作对照。分别于心肌梗死后4周:导管法测定左心室有创血流动力学及心功能;组织学方法检测非梗死区胶原纤维沉积和心肌细胞横径;Western blot法检测心肌组织骨桥蛋白表达。结果:盐水组与厄贝沙坦组大鼠梗死面积相似,无显著性差异(P>0.05);假手术组大鼠心肌组织Western blot法未检测到骨桥蛋白表达,盐水组大鼠心肌组织有大量骨桥蛋白表达,该上调的蛋白能被厄贝沙坦治疗显著抑制(P<0.01)。与假手术组相比,所有心肌梗死大鼠均出现显著的心肌间质纤维沉积,左心室相对重量增大,非梗死区心肌细胞横径增加,均有显著性差异(P均<0.01);与盐水组相比,厄贝沙坦组心肌间质纤维沉积减轻,左心室相对重量及非梗死区心肌细胞横径降低,均有显著性差异(P均<0.01)。与假手术组相比,所有心肌梗死大鼠在4周后均表现出左心室收缩压和左心室压力最大上升和下降速率显著下降,左心室舒张末压显著上升,均有显著性差异(P均<0.01),提示了显著的左心室收缩和舒张功能不全;与盐水组相比,厄贝沙坦组大鼠心功能显著改善,均有显著性差异(P均<0.01)。结论:心肌梗死后大鼠心肌组织出现大量骨桥蛋白表达,厄贝沙坦治疗显著抑制心肌梗死大鼠骨桥蛋白的表达,并能改善心肌的纤维化,改善心脏功能。     

The Expression of Protease-Activated Receptor 2 and 4 in the Colon of Irritable Bowel Syndrome Patients

Background  

Irritable bowel syndrome (IBS) pathophysiology has not been fully understood. Abnormalities of serine proteases have been identified in IBS patients. In addition, protease-activated receptors (PAR) activation interferes with several components of the pathogenesis of IBS, so, evaluating the PAR expression in IBS patients may contribute to understanding the pathogenesis of the disease.     

柯萨奇-腺病毒受体在扩张型心肌病心肌中的表达及意义

目的探讨柯萨奇-腺病毒受体(CAR)在扩张型心肌病(DCM)心肌组织中的表达及其在DCM发病中的意义.方法利用多聚酶链反应(PCR)技术检测20例原位异体心脏移植的DCM受体心肌组织以及5例正常对照心肌组织中柯萨奇B组病毒(CVB)、腺病毒(ADV)基因,采用免疫组化及逆转录多聚酶链反应(RT-PCR)检测心肌柯萨奇-腺病毒受体mRNA及蛋白表达水平.结果20例DCM心肌标本中,柯萨奇B组病毒阳性7例(柯萨奇病毒组),腺病毒阳性5例(腺病毒组);其余8例柯萨奇B组病毒、腺病毒均阴性(其它组),5例正常对照柯萨奇B组病毒、腺病毒也均为阴性.正常对照和其它组心肌中均无柯萨奇-腺病毒受体mRNA及蛋白表达,而在柯萨奇病毒组和腺病毒组其表达水平明显增加,但两组之间比较,差异无显著性.结论柯萨奇-腺病毒受体表达上调可能是导致柯萨奇病毒或腺病毒易感性增加,促使病毒性心肌炎向扩张型心肌病转化的重要原因.     

Elimination of Neutrophils by Apoptosis During the Resolution of Acute Pulmonary Inflammation in Rats

We evaluated the apoptosis of neutrophils during the resolution of acute pulmonary inflammation induced by exposure to ozone. The inflammatory response was assessed in rat lungs 0, 1, 3, and 7 days after 4-h exposure to air or 2 ppm ozone. Analysis of bronchoalveolar lavage fluid demonstrated significant increases in albumin concentrations on days 0 and 1 and in the number of lavageable neutrophils on days 0, 1, and 3, indicating the presence of acute pulmonary inflammation. These parameters returned to control values on day 7, which suggests that the acute pulmonary inflammation induced by ozone was reversible. On days 1 and 3, but not on day 0, the neutrophils showed morphologic evidence of apoptosis. Based on morphologic analysis, the proportion of apoptotic neutrophils was 23.3 ± 2.2% on day 1 and 55.7 ± 3.2% on day 3. Terminal deoxynucleotidyl transferase-mediated dUTP end labeling (TUNEL), in contrast, revealed that the proportion of apoptotic cells was 59.7 ± 9.1% on day 1 and 68.0 ± 4.3% on day 3. On day 3, light microscopy and electron microscopy demonstrated engulfment of the neutrophils by macrophages. These findings indicate that the apoptosis of neutrophils followed by their engulfment by macrophages contributes to the clearance of neutrophils from the sites of inflammation. Moreover, TUNEL detected apoptotic neutrophils with greater sensitivity compared with morphologic assessment. Accepted for publication: 4 June 1997     

Up-Regulated Expression of Advanced Glycation End-Products and Their Receptor in the Small Intestine and Colon of Diabetic Rats

Background and Aims  

Gastrointestinal disorders and symptoms are common in diabetic patients. Advanced glycation end-products (AGEs) and their receptor (RAGE) have been proposed as an important pathological mechanism underlying diabetic complications, such as diabetic cardiopathy, retinopathy, nephropathy, etc. The aims were to study the distribution of AGE and RAGE in the normal and diabetic small intestine and colon in rats and the possible relationship between AGEs/RAGE and diabetes-induced intestinal structural remodeling.     

四氯化碳诱导大鼠肝纤维化过程中TLR4的作用机制研究

目的 动态观察Kupffer细胞(KCs) Toll样受体4(TLR4)及其蛋白在四氯化碳(CCl4)致大鼠肝纤维化过程中的表达及作用.方法 用40％四氯化碳花生油溶液皮下注射建立肝纤维化模型,于第0、4、6、8、10周收集血液和肝脏组织.检测血清内毒素、Ⅳ型胶原(collagenⅣ,CⅣ)、转化生长因子β1(TGF-β1)水平,并检测KCsTLR4 mRNA表达水平,免疫组化SP法检测肝脏TLR4蛋白、核因子κB(nuclear factor-κB,NF-κB)蛋白和组织金属蛋白酶组织抑制剂-1(TIMP-1)蛋白表达.结果 大鼠4、6、8、10周组肝组织TLR4蛋白、NF-κB蛋白、纤维化积分和KCs TLR4 mRNA以及血清内毒素、TGF-β1、CⅣ型胶原水平明显升高,同0周组比较差异有统计学意义(P＜0.01).NF-κB蛋白、TLR4蛋白和KCs TLR4 mRNA在第10周时较第8周有轻度下降;肝KCs TLR4 mRNA的表达与血浆内毒素水平呈正相关(r=0.845,P＜0.01),与血清TGF-β1水平成正相关(r=0.665,P＜0.01).结论 在CCl4诱导的肝纤维化过程中,内毒素可上调Kupffer细胞TLR4的表达,其可能为通过TLR4信号途径在肝纤维化中起重要作用.     

Growth Hormone-Releasing Hormone Receptor Antagonist Modulates Lung Inflammation and Fibrosis due to Bleomycin

Lung - Growth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that a GHRH receptor (GHRH-R) antagonist, MIA-602, would...     

Angiotensin II Participates in Hepatic Inflammation and Fibrosis through MCP-1 Expression

In this study, we assessed the hypothesis that angiotensin (Ang) II could modulate inflammatory cell recruitment into the liver through hepatic expression of monocyte chemoattractant protein (MCP)-1 during liver injury. For in vivo study, Ang II type 1a knockout (AT1a KO) mice and wild-type (WT) mice were treated with CCl₄ for 4 weeks. After CCl₄ treatment, AT1a KO mice showed lower expression of MCP-1 and fewer CD68-positive cells in the liver compared with WT mice. For in vitro study, Ang II was added to LI90 cells. Ang II enhanced MCP-1 mRNA together with RhoA mRNA and also induced secretion of MCP-1 into the culture medium. This change was strongly blocked by Y-27632, a specific Rho-kinase inhibitor. These results suggest that Ang II modulates hepatic inflammation via production of MCP-1 by hepatic stellate cells, and the effect of Ang II on MCP-1 production is, at least partly, mediated by the Rho/Rho-kinase pathway.     

Ethanol Attenuates Spatial Memory Deficits and Increases mGlu1a Receptor Expression in the Hippocampus of Rats Exposed to Prenatal Stress

Background: Although it is generally believed that chronic ethanol consumption impairs learning and memory, results obtained in experimental animals are not univocal, and there are conditions in which ethanol paradoxically improves cognitive functions. In the present work, we investigated the effects of prenatal stress and of chronic ethanol exposure during adulthood on spatial memory in rats. Methods: Rats were subjected to a prenatal stress delivered as 3 daily 45‐minute sections of restraint stress to the mothers during the last 10 days of pregnancy (PRS rats). After 7 months of ethanol exposure (ethanol 10%, oral intake), memory performances were evaluated in a spatial discrimination test in control and PRS male rats. Then, the oxidative damages and the expression of metabotropic glutamate (mGlu) receptors were assessed in their hippocampus. Results: Chronic ethanol exposure resulted in a reduced performance in a spatial recognition task in control animals. Unexpectedly, however, the same treatment attenuated spatial memory deficits in rats that had been subjected to prenatal stress. This paradigm of ethanol administration did not produce detectable signs of oxidative damage in the hippocampus in either unstressed or PRS rats. Interestingly, ethanol intake resulted in differential effects in the expression of mGlu receptor subtypes implicated in mechanisms of learning and memory. In control rats, ethanol intake reduced mGlu2/3 and mGlu5 receptor levels in the hippocampus; in PRS rats, which exhibited a constitutive reduction in the levels of these mGlu receptor subtypes, ethanol increased the expression of mGlu1a receptors but did not change the expression of mGlu2/3 or mGlu5 receptors. Conclusion: Our findings support the idea that stress‐related events occurring before birth have long‐lasting effects on brain function and behavior, and suggest that the impact of ethanol on cognition is not only dose‐ and duration‐dependent, but also critically influenced by early life experiences.