Myocardial fibrosis is a major pathogenic factor contributing to cardiac remodeling and heart failure. Recent research has indicated that micro RNAs play a crucial role in the progression of cardiac fibrosis. Bone morphogenetic protein and activin membrane-bound inhibitor(BAMBI) have been shown to alleviate myocardial fibrosis by inhibiting the transforming growth factor β1(TGF-β1) signaling pathway. Therefore, the current study aimed to elucidate the post-transcriptional regulation of BAMBI by miR-19a-3p and its role in TGF-β1-induced cardiac fibroblast activation. We found that transverse aortic constriction induced both myocardial interstitial and perivascular collagen deposition. Quantitative reverse transcription-PCR(q RT-PCR) analysis showed that the expression level of miR-19a-3p was increased in the myocardial tissues of cardiac fibrosis, and TGF-β1 induced an upregulation in miR-19a-3p expression in cardiac fibroblasts. The dual-luciferase reporter assay and q RT-PCR verified that miR-19a-3p directly bound to the 3 ′ untranslated regions of BAMBI m RNA, thereby reducing BAMBI expression and diminishing its ability to inhibit the TGF-β1 signaling pathway. Furthermore,overexpression of miR-19a-3p mimic increased the activation of TGF-β1/SMAD2/3 pathway signaling,promoting cardiac fibroblast activation. However, this activation was blocked by BAMBI overexpression. These findings imply that miR-19a-3p enhances the activation of TGF-β1/SMAD2/3 by inhibiting BAMBI, further boosting the activation of cardiac fibroblasts and contributing to myocardial fibrosis.

目的:鉴定成年小鼠Toll途径进化保守信号介导因子(evolutionarily conserved signaling intermediate in Toll path-ways,ECSIT)的3'非翻译区(3'-untranslatedregion,3'-UTR)序列,并在细胞中验证非编码RNA对ECSIT表达的影响.方法:采用RACE技术克隆得到小鼠ECSIT3'-UTR序列,并与基因组数据库进行比对;预测ECSIT3'-UTR可能结合的微小RNA(mi-croRNA,miRNA),并针对ECSIT的全长序列设计小干扰RNA(siRNA);通过Western blot分别检测使用不同miRNA和siRNA干扰后,细胞中ECSIT的表达.结果:成功鉴定了346bp的小鼠ECSIT3'-UTR,与NCBI上序列一致率达到99％;在细胞中miR-7-5p和siRNA1、2可以干扰ECSIT的表达.结论:成功鉴定得到成年小鼠心脏ECSIT mRNA3'-UTR序列,该非编码区可作为非编码RNA的调控区域,为进一步从分子水平探明ECSIT在小鼠生长发育及疾病中的作用提供科学依据.

目的:构建线粒体靶向过表达ECSIT转基因小鼠,并对其进行鉴定和心功能分析,建立ECSIT基因相关功能研究模型动物.方法:构建过表达打靶载体pCAG-OTCL-ECSIT-3Xflag-BPA,采用电转导方法将线性化打靶载体转入胚胎干细胞(ES细胞);将含有过表达载体的ES细胞进行囊胚腔注射,并将嵌合囊胚移植至代孕小鼠体内,繁殖嵌合体小鼠.嵌合体小鼠和C57BL/6J鼠交配繁殖出杂合子,PCR筛选阳性过表达小鼠.采用小动物超声分析线粒体靶向过表达ECSIT小鼠的心功能.结果:成功构建了线粒体靶向过表达ECSIT载体pCAG-OTCL-ECSIT-3Xflag-BPA,经PCR鉴定为阳性;完成线粒体靶向过表达ECSIT基因打靶及囊胚注射,经PCR鉴定为阳性;分别提取转基因小鼠心肌组织线粒体和胞浆蛋白,检测发现线粒体特异性过表达ECSIT.8周龄转基因小鼠心功能与同龄野生型小鼠无明显差异.结论:成功构建出线粒体靶向过表达ECSIT小鼠;线粒体过表达ECSIT对8周龄小鼠心功能无明显影响.

目的:研究Pellino1蛋白翻译后修饰——小泛素相关修饰物(small ubiquitin-related modifier,SUMO)修饰对肿瘤坏死因子受体相关分子-6 (TNF receptor associated factor 6,TRAF6)介导的核因子κB (nuclear factor κB,NF-κB)信号转导的影响.方法:构建Pellino1 SUMO修饰位点突变的质粒,与TRAF6、泛素(ubiquitin,Ub)质粒共转染至HEK-293细胞中,荧光显微镜观察转染效率,免疫共沉淀的方法检测突变型Pellino1与TRAF6的相互结合作用;同时,检测Pellino1和TRAF6的泛素化修饰水平.采用脂多糖(lipopolysaccharide,LPS)诱导炎症反应,分提胞浆胞核蛋白,Western blot检测核因子κB的抑制蛋白α(inhibitor of NF-κBα,IκBα)的磷酸化和NF-κB P65的核转位,分析Pellino1蛋白SUMO修饰突变对TRAF6介导的NF-κB信号通路的影响.结果:与野生型Pellino1相比,SUMO修饰突变型Pellino1不仅增加Pellino1的自身泛素化修饰水平,而且可增加其与TRAF6的相互结合作用,并增强TRAF6的泛素化修饰;LPS刺激可增加IκBα的磷酸化和NF-κB P65的核转位,转染使Pellino1 SUMO修饰突变高表达可明显增强LPS诱导的NF-κB信号激活.结论:Pellino1 SUMO修饰突变,可通过增加Pellino1的自身泛素化修饰及其与TRAF6的结合,增强TRAF6的泛素化,最终促进TRAF6介导的NF-κB信号通路的激活.

目的:研究缺氧/复氧（hypoxia/reoxygenation,H/R）刺激是否可通过激活程序性坏死信号通路诱导心肌细胞坏死。方法 :体外培养新生大鼠原代心肌细胞,采用缺氧2 h复氧4 h的方法复制心肌细胞损伤模型,碘化丙腚（propidium iodide,PI）染色检测心肌细胞坏死情况,Western blot和免疫共沉淀方法检测程序性坏死信号通路中受体相互作用蛋白1（receptor interacting protein-1,RIP1）、受体相互作用蛋白3（receptor interacting protein-3,RIP3）的蛋白表达水平和RIP1/RIP3复合物Ⅱ形成情况,以及RIP1、RIP3的泛素化水平。结果:与对照组相比,缺氧/复氧组心肌细胞坏死数量明显增多,RIP1、RIP3蛋白表达水平显著升高,RIP1/RIP3复合物Ⅱ的形成增多,且RIP1与RIP3的泛素化水平也有所增加。结论:缺氧复氧可诱导心肌细胞坏死,其机制可能与激活程序性坏死RIP1/RIP3信号通路有关。

目的:构建Peli1蛋白小类泛素修饰因子(small ubiquitin-like modifier,SUMO)修饰位点突变体,并进行鉴定.方法:提取C57BL/6J小鼠心肌组织RNA,逆转录成cDNA,采用分子克隆的方法,克隆小鼠Peli1基因,构建真核表达载体;进一步采用点突变的方法,设计特异性引物,构建Peli15个SUMO修饰位点的突变体.结果:在构建小鼠Peli1真核表达质粒的基础上,分子测序显示成功构建Peli1 SUMO修饰位点突变体,Western blotting检测突变体蛋白可以体外表达.结论:成功构建Peli1蛋白SUMO修饰位点突变体,为进一步探讨Peli1蛋白功能奠定了基础.

目的:采用超声多普勒的方法评价小鼠主动脉弓缩窄模型建立的成败及程度,比较主动脉弓缩窄程度对心脏形态及功能影响的差异.方法:60只雄性C57BL/6小鼠随机分成假手术组(sham组)20只,26G缩窄针主动脉弓缩窄组(TAC26G组)20只,27G缩窄针主动脉弓缩窄组(TAC27G组)20只,采用超声多普勒的方法检测小鼠双侧颈动脉流速,通过右侧颈动脉(rightcarotid arteries,RCA)流速与左侧颈动脉(left carotid arteries,LCA)流速之比(RCA/LCA)来评价主动脉弓缩窄术的成败及程度;术后2周进行超声心动图检测,并对各组心脏进行拍照,计算心重/体重及左室重/胫骨长度,比较主动脉弓不同缩窄程度对心肌肥大形成的严重程度以及心脏功能的影响.结果:与sham组相比,TAC批组及TAC27G组RCA/LCA均显著上升(P＜0.05),TAC27G组的RCA/LCA为8～10,较之TAC26G组(RCA/LCA为5～7)具有显著性差异(P＜0.05).术后2周,TAC26G组表现为向心性肥大,与sham组相比,舒张期室间隔厚度(IVSd)、舒张期后壁厚度(LVPWd)、射血分数(EF)、短轴缩短率(FS)均显著上升(P＜0.05),左室舒张末期内径(LVIDd)显著下降(P＜ 0.05);TAC27G组表现为离心性肥大,与sham组相比,IVSd、LVPWd、LVIDd均显著上升(P＜0.05),EF及FS均显著下降(P＜0.05).术后2周,与sham组相比,TAC26G及TAC27G组心重/体重及左室重/胫骨长度均显著上升(P＜0.05),TAC27G组上述两个肥大指标较之TAC26G组明显增加(P＜0.05).结论:采用超声多普勒检测RCA/LCA在实验早期即可准确判断主动脉弓缩窄术的成败及程度,对压力超负荷心肌肥大及心力衰竭相关研究动物模型的选择具有重要的指导作用.

Objective:To investigate the effect of 17β-estradiol(E2) on cultured cardiomyocyte hypertrophy induced by mechanical stretch.Methods:Cardiomyocytes from neonatal rats were cultured in vitro and cardiomyocyte hypertrophy was induced by mechanical stretch.The effect of E2 on cardiomyocyte hypertrophy was evaluated by the surface areas of cardiomyocytes using immunocytochmistry and the β-MHC protein levels detected by Western blot.The integrin β1 protein levels were determined by Western blot.Results:Mechanical stretch for 24 h significantly increased cell surface areas and β-MHC protein levels,which suggested that mechanical stretch is a direct stimulus for isolated cardiomyocytes to develop hypertrophy.Mechanical stretch also significantly increased the expression of integrin β1 protein in cultured cardiomyocytes.Pretreatment with 100 nmol/L E2 for 30 min significantly attenuated the increases in cell surface areas and β-MHC protein levels induced by mechanical stretch which were attenuated by estrogen receptor antagonist ICI182780.Furthermore,E2 also decreased the levels of integrin β1 protein induced by mechanical stretch,and this was also attenuated by ICI182780.Conclusion:E2 prevents cardiomyocyte hypertrophy and the increase of integrin β1 protein induced by mechanical stretch in vitro.

Activation of NF-κB contributes to cardiac hypertrophy and the interleukin-1 receptor (IL-1R)-mediated MyD88-dependent signaling pathway predominately activates NF-κB. Recent studies have shown that the TIR/BB-Loop mimetic (AS-1) disrupted the interaction of MyD88 with the IL-1R, resulting in blunting of NF-κB activation. We have examined the effects of AS-1 on the IL-1β-induced hypertrophic response using cultured neonatal cardiac myocytes in vitro and transverse aortic constriction (TAC) pressure overload-induced cardiac hypertrophy in vivo. Neonatal cardiac myocytes were treated with AS-1 15 min prior to IL-1β stimulation for 24 h. AS-1 treatment significantly attenuated IL-1β-induced hypertrophic responses of cardiac myocytes. In vivo experiments showed that AS-1 administration prevented cardiac hypertrophy and dysfunction induced by pressure overload. AS-1 administration disrupted the interaction of IL-1R with MyD88 in the pressure overloaded hearts and prevented activation of NF-κB. In addition, AS-1 prevented increases in activation of the MAPK pathway (p38 and p-ERK) in TAC-induced hypertrophic hearts. Our data suggest that the IL-1R-mediated MyD88-dependent signaling pathway plays a role in the development of cardiac hypertrophy and AS-1 attenuation of cardiac hypertrophy is mediated by blocking the interaction between IL-1R and MyD88, resulting in decreased NF-κB binding activity and decreased MAPK activation.

We examined the role of Tollip in the hypertrophic response of cardiomyocytes. C57BL/6 mice were subjected to transverse aortic constriction (TAC) for 2 weeks and age-matched sham surgical operated mice served as control. TAC significantly reduced the association of Tollip with IRAK-1 by 66.4 percent and increased NF-kappaB binding activity by 86.5 percent and the levels of phospho-p38 by 114.6 percent in the myocardium compared with sham control, respectively. In vitro experiments showed that IL-1beta stimulation also significantly reduced the association of Tollip with IRAK-1 and increased NF-kappaB binding activity in neonatal cardiomyocytes. Tollip overexpression by transfection of cardiac myocytes significantly attenuated the IL-1beta-induced hypertrophic response of cardiac myocytes as evidenced by reduced cell size (16.4 percent) and decreased ANP expression (33.3 percent). Overexpression of Tollip also reduced NF-kappaB binding activity by 30.7 percent and phospho-p38 by 47.1 percent, respectively. The results suggest that Tollip could be a negative regulator during the development of cardiac hypertrophy. The negative regulation of cardiac hypertrophy by Tollip may involve downregulation of the MyD88-dependent NF-kappaB activation pathway.

Cardiac fibroblasts play an important role in myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix proteins. Estrogen has been reported to have a number of cardioprotective properties. However, it is unclear whether estrogen affects cardiac fibroblast differentiation. The purpose of the present study was to investigate the effect of estrogen on angiotensin II-induced cardiac fibroblast proliferation and differentiation. Cardiac fibroblasts were stimulated with angiotensin II (1 microM) in the presence or absence of 17beta-estradiol (100 nM). Pretreatment of cardiac fibroblasts with 17beta-estradiol significantly inhibited angiotensin II-induced cardiac fibroblast proliferation and differentiation (indicated by a reduction in alpha-smooth muscle actin (alpha-SMA) expression) by 25% and 20%. Pretreatment of 17beta-estradiol significantly reduced angiotensin II-increased levels of phospho-p38 mitogen-activated protein kinase (MAPK) by 40% and nuclear factor-kappaB (NF-kappaB) binding activity in cardiac fibroblasts by 55%. Our data suggests estrogen could have an anti-fibrotic effect through limiting cardiac fibroblast proliferation and differentiation, which are the critical steps in the pathogenesis of cardiac fibrosis.

This study was to examine the effect of estrogen on mechanical stretching-induced cardiac dysfunction in an isolated heart model. The isolated rat hearts were perfused via the Langendorff system and exposed to left ventricular stretching. One group hearts (n=6) were perfused with 17beta-estradiol (100nM) and the other group hearts (n=6) were perfused with estrogen plus its receptor antagonist ICI182,780 (1microM) before myocardial stretching was performed. Control hearts (n=6) were perfused with perfusion buffer. Cardiac functions were recorded. At the end of perfusion, the hearts were harvested and the levels of tumor necrosis factor-alpha (TNF-alpha), phospho-p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) binding activity were examined. Acute ventricular stretching resulted in significantly decrease in left ventricular developed pressure (LVDP) by 42.7%, maximal positive and negative values of the first derivative of pressure (+dP/dt and -dP/dt) by 43.2%, and 43.5%, respectively. The levels of TNF-alpha, phospho-p38 MAPK and NF-kappaB DNA binding activity were significantly increased following myocardial stretching. In 17beta-estradiol treated hearts, the myocardial functions were significantly improved. The levels of TNF-alpha, phospho-p38 MAPK, and NF-kappaB binding activity in myocardium were also significantly reduced by 35.7%, 56.9%, and 50%, respectively, compared with untreated stretched hearts. The beneficial effects of 17beta-estradiol on the stretched hearts were abolished by ICI182,780. The results suggest that pharmacological dose of 17beta-estradiol will attenuate stretching-induced cardiac dysfunction in an isolated heart model. The mechanisms could involve in blunting p38 MAPK and NF-kappaB signaling.

Aim: To investigate the effects of chronic ethanol intake on the locomotor activity and the levels of calcium/calmodulin-dependent protein kinase IV (CaM kinase IV) in the nucleus accumbens (NAc) of rats. Simultaneously, the effects of non-selective opioid antagonist (naloxone) on the CaM kinase IV expression in the NAc and ethanol consumption of rats were also observed. Methods: Ethanol was administered in drinking water at the concentrations of 6% ( v/v ), for 28 d. The locomotor activity of rats was investigated in the open-field apparatus. CaM kinase IV levels in the NAc were analyzed using Western blotting. Results: Rats consuming ethanol solution exhibited a significant decrease of ambulation activity, accompanied by a reduced frequency of explorative rearing in an open-field task on d 7 and d 14 of chronic ethanol ingestion, whereas presumed adaptation to the neurological effects of ethanol was observed on d 28. Chronic ethanol intake elicited a significant decrease of the CaM kinase IV expression in the nuclei, but not in the cytoplasm of the NAc on d 28. Naloxone treatment significantly attenuated ethanol intake of rats and antagonized the decrease of CaM kinase IV in the nuclei of NAc neurons. The cytosolic CaM kinase IV protein levels of the NAc also increased in rats exposed to ethanol plus naloxone. Conclusion: Chronic ethanol intake-induced changes in explorative behavior is mediated at least partly by changes in CaM kinase IV signaling in the nuclei of the NAc, and naloxone attenuates ethanol consumption through antagonizing the downregulation of CaM kinase IV in the NAc.

Objective To investigate the therapeutic effects of sinomenine on the activity of brain nuclear factor-κB (NF-κB) rat in experimental autoimmune encephalomyelitis (EAE). Methods EAE rats were induced by immunization of female Wistar rats with brain and spinal cord homogenate followed by treatment with or without sinomine. The severity of EAE rats was evaluated by clinical scores and histological examination. The activity of brain NF-κB and the plasma cortisol were determined by ELISA kits. Plasma prolactin was assessed by the RIA kit. Results The brain NF-κB binding activity was increased significantly in EAE rats. The treatment of sinomenine reduced the clinical scores of EAE significantly, decreased the number of inflammatory foci in the central nervous system, and inhibited the activation of brain NF-κB in EAE rats. Besides, the level of plasma cortisol was increased and the level of plasma prolactin was decreased after the treatment of sinomenine.Conclusion Inhibiting brain NF-κB binding activity could be a mechanism of sinomenine protecting of rats from EAE, and the inhibition of NF-κB is related to the alteration of cortisol and prolactin in vivo.

AIM: To further study the therapeutic effect of chitosan compound on rats of experimental fatty liver and it's mechanisms. METHODS: Male SD rats were treated with a small dosage of CCl 4 and fed by a high-lipid diet for 6 wk to induce fatty liver model. Then the therapeutic group was ig chitosan compound with 0.7g·kg -1·d -1 for 8 wk. The steatosis degree was determined by the contents of lipids in the rat liver, and the free fatty acid(FFA) of blood and liver, the expression of peroxisome proliferator activated receptor α(PPARα) were measured. RESULTS: Combined use of CCl 4 and high-lipid diet increased significantly the contents of triglyceride(TG), total cholesterol(TC) and FFA in liver or in serum and markedly decrease the expression of the PPARα mRNA. Compared with the model group, the chitosan compound could obviously decrease the contents of TG, TC, FFA in liver and FFA in serum, and increase the PPARα mRNA expression, respectively(P0.01). CONCLUTION: The results indicate that chitosan compound is effective in treating rat fatty liver by inducing PPARα mRNA expression and promoing liver intake and oxidation of fatty acid.

Experimental autoimmune encephalomyelitis (EAE) is a good model for human multiple sclerosis (MS) research. However, there are some defects in the traditional models. Here, we improved the model by using the human myelin basic protein (MBP) as antigen. EAE was induced by immunization of female Wistar rats with human MBP. Compared with the traditional models, the new model was evaluated by clinical signs to pathological changes. The immune state of the model was assessed by the lymphocyte infiltrative response and levels of TNF-alpha, IFN-gamma, IL-10. It was found that most of rats exhibited tail tone loss and hind-limb paralysis, also there were demyelination, infiltrative lymphocyte foci, "neuronophagia" in the cortex of cerebra and the white matter of spinal cords. PBMCs and spleen lymphocytes were strongly responsive to the stimulation of MBP and PHA. The levels of TNF-alpha and IFN-gamma were altered with the severity of EAE. In the remitting phase, IL-10 was increased significantly. This study demonstrates that the animal model of EAE induced by human MBP bears resemblance to the features of human multiple sclerosis and promises to be a better model than ever before for the study of MS.