Current understanding of dystrophin-related muscular dystrophy and therapeutic challenges ahead

Objective To review the recent research progress in dystrophin-related muscular dystrophy includes X-linked hereditary Duchenne and Becker muscular dystrophies （DMD and BMD）. Data sources Information included in this article was identified by searches of PUBMED and other online resources using the key terms DMD, dystrophin, mutations, animal models, pathophysiology, gene expression, stem cells, gene therapy, cell therapy, and pharmacological. Study selection Mainly original milestone articles and timely reviews written by major pioneer investigators of the field were selected. Results The key issues related to the genetic basis and pathophysiological factors of the diseases were critically addressed. The availabilities and advantages of various animal models for the diseases were described. Major molecular and cellular therapeutic approaches were also discussed, many of which have indeed exhibited some success in pre-clinical studies but at the same time encountered a number of technical hurdles, including the efficient and systemic delivery of a functional gene and myogenic precursor/stem cells to repair genetic defects. Conclusions Further understanding of pathophysiological mechanisms at molecular levels and regenerative properites of myogenic precursor/stem cells will promote the development of multiple therapeutic strategies. The combined use of multiple strategies may represent the major challenge as well as the greatest hope for the therapy of these diseases in coming years.     

Fluvastatin prevents renal injury and expression of lactin-like exidized low-density lipoprotein receptor-1 in rabbits with hypercholesterolemia

Background Lipid abnormalities are often complicated by renal dysfunction. 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are the first-line choice for lowering cholesterol levels. The present study was designed to investigate whether statins could prevent and invert the development of renal injury in cholesterol-fed rabbits and to find the possible mechanism of their effects by detecting gene and protein expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in the renal artery.Methods Twenty-four male New Zealand white rabbits were divided into three groups: (1) control group, regular granules chow; (2) HC-diet group, granules chow with 1% cholesterol and 5% lard oil; and (3) fluvastatin group, 1% cholesterol and 5% lard oil diet plus fluvastatin ［10 mg·kg（-1）·d（-1）］ . After 16 weeks, serum total cholesterol (TC), low-density lipoprotein(LDL) and creatinine (Cr) levels were measured. Renal hemodynamics and function, mainly including glomerular filtration rate (GFR) in vivo were quantified using 99mTc-DTPA single photon emission computed tomograph (99mTc-DTPA SPECT). The thickness of the renal artery intima was quantitated in HE-stained segments by histomorphometry. Gene expression of LOX-1 in the renal artery was examined by semi-quantitative RT-PCR and its protein expression was evaluated by immunohistochemistry.Results High cholesterol diet induced hypercholesterolemia (HC) complicated by renal dysfunction with increased levels of serum lipid and Cr, decreased GFR and delayed excretion and extensively thickened renal arterial intima in the HC-diet group. Rabbits in the control group showed a minimal LOX-1 expression (mRNA and protein) in the endothelium and neointima of the renal artery. Intimal proliferation of the renal artery in the HC-diet group was associated with a marked increase of LOX-1 expression (protein and mRNA). Treatment with fluvastatin improved renal function, attenuated intimal proliferation of the renal artery and markedly decreased the enhanced LOX-1 expression in the endothelium and neointima of the renal artery in rabbits. Conclusions Fuvastatin treatment could prevent the development of renal injury in patients with HC and early atherosclerosis (AS). This beneficial effect might be mediated by its pleiotropic effects including a decrease in total cholesterol exposure level and prevention of LOX-1 expression in atherosclerotic arteries.     

Regularity of hypoxia inducible factor 1 alpha expression in acute myocardial ischaemia in rats

Acute myocardial ischaemia is a common acute .disease and a common cause of sudden death.However, it is difficult to diagnose in patients who died within 6 hours after the onset of myocardial ischaemia. The occurrence of sudden cardiac death often has pathological basis of primary heart diseases, which may lead to a series of changes in metabolism and gene expression.1 Recent research found that hypoxia inducible factor 1 alpha （HIF-1α） is a sensitive marker of hypoxia; its gene expression is upregulated within several minutes after acute myocardial ischaemia, followed by the upregulation of its protein and its expression will remain high if the inducement continues. Its expression in nonhypoxic cardiac muscle is very low. This characteristic may be used to differentiate hypoxic factors from nonhypoxic factors, and help to judge the cause of death. This study explored the expression of HIF-1α in hypoxic cardiac muscle by establishing an acute myocardial ischaemia model in mice, and observed its dynamic changes to provide reference for analysing causes of death within 48 hours after death.     

Inhibitory Effect of Tissue Transglutaminase （tTG） Antisense Oligodeoxynucleotides on tTG Expression in Cultured Bovine Trabecular Meshwork Cells

To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides （tTG-ASDON） on tTG expression in cultured bovine trabecular meshwork cells （BTMCs） in vitro and explore a new treatment alternative for primary open angle glaucoma （POAG）, the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON1 and tTCr-ASDON2 were significantly decreased as compared with that of the controls （P〈0.05）. On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.     

Expression of hepatocyte growth factor and its receptor c-Met in lens-induced myopia in guinea pigs

Background Myopia is a common disorder and the incidence has increased yearly,but its pathogenesis remains unclear.The aim of this study was to investigate the possible role of hepatocyte growth factor （HGF） and its receptor c-Met in the development of lens-induced myopia in guinea pigs.Methods Sixty one-week-old guinea pigs were chosen.The right eyes were treated with-10.0 diopters （D） lenses as the lens-induced myopia group; the left eyes remained untreated as the control group.Six weeks later,refractive status and axial length were determined by streak retinoscopy and A-scan ultrasonography,respectively.The guinea pigs were killed and both eyes collected.Morphological changes were observed by hematoxylin and eosin staining.The expression levels of HGF,c-Met,and matrix metalloproteinase 2 （MMP-2） mRNA and protein in the posterior sclera were analyzed by RT-PCR and Western blotting,respectively.Results The lens-induced myopia group became myopic with a significant increase in axial length and a significant decrease in refraction.Compared with the control group,the posterior retina and sclera were thinner in the lens-induced myopia group.The expression levels of HGF and MMP-2 mRNA and protein and of phosphorylated c-Met protein were significantly higher in the posterior sclera of the lens-induced myopia group than in the control group （all P ＜0.05）.In the lens-induced myopia group,the expression level of MMP-2 in the posterior sclera positively correlated with the expression level of HGF （r=0.902,P ＜0.05） and phosphorylated c-Met （r=0.885,P ＜0.05）.Conclusion HGF/c-Met might play a role in the development of lens-induced myopia in guinea pigs by upregulating the expression of MMP-2.     

Expressions of farnesoid X receptor and myeloid cell leukemia sequence 1 protein are associated with poor prognosis in patients with gallbladder cancer

Background Farnesoid X receptor （FXR） regulates tumorigenesis, but its clinical significance in gallbladder cancer （GBC） remains unclear. This study investigated its clinical and prognostic significance in GBC patients, as well as its association with the anti-apoptotic protein, myeloid cell leukemia sequence 1 （MCL1） protein. Methods FXR and MCL1 expression in 42 primary GBC and 15 normal gallbladder tissues were analyzed by immunohistochemistry. The patients and samples were collected from Ren Ji Hospital from January 2005 to December 2010. Their association with clinicopathologic factors and prognosis, as well as the correlation between FXR and MCL1 protein expression were analyzed by statistical analyses. Results Compared with normal gallbladder tissues, FXR expression was decreased and MCL1 expression was increased in GBC, during progression of tumor node metastasis （TNM） stage. The Kaplan-Meier survival analysis showed that FXR low-expression and MCL1 over-expression were significantly associated with overall poor survival. Furthermore, multivariate analysis showed that FXR and MCL1 are both prognostic factors for GBC patients. FXR low-expression was significantly correlated with MCL1 over-expression. Conclusion FXR might be a new molecular marker to predict the prognosis of patients with GBC and a novel therapeutic target. Chin Med J 2014;127 （14）： 2637-2642     

Human bone morphogenetic protein-2 gene transfer induces human mesenchymal stem cell proliferation and differentiation in vitro

To identify eukaryotie expression veetor of human bone morphogenetie protein 2 peDNA3/BMP2, verify its expression in transfected hulnan mesenchynlal stem cells(hMSCs) and the effect on hMSCs differentiation. Methods: The BMP2 gene was cloned into a eukaryotic expression veetor pcDNA3. Transfeeted the reeombinant into hMSCs by liposome, Lmmunnohistochemistry and it situ hybridization methods were used to identify the expression of BMP2 mRNA and protein; ALP and Von Kossa stains were performed to identify the BMP2 gene differentiated effect on the hMSCs. Resuits: The pcDNA3/BMP2 fragments were as large as theory. BMP2 mRNA and protein were espressed and synthesized both in 48 h and 4 weeks after transfeetion, the ALP and Ca deposit eshibitiio, which marked the osteogenie lineage of hMSCs, were enhanced and Sped. Conclusion: Transfeetion of pcDNA3/BMP2 is able to provide transient and persistent expressionin hMSCs, and promote the MSCs differentiation to osleogenic lineage.     

Effects of Tanshinone ⅡA on Transforming Growth Factor β1-Smads Signal Pathway in Renal Interstitial Fibroblasts of Rats

The effects of tanshinone ⅡA （TSN） on transforming growth factor β1 （TGFβ1） signal transduction in renal interstitial fibroblasts of rats were studied in order to investigate its mechanism in prevention of renal interstitial fibrosis. Rat renal fibroblasts of the line NRK/49F were cultured in vitro, stimulated with 5 ng/mL TGFβ1 and pretreated with 10-6, 10-5, 10-4 mol/L TSN respectively. The mRNA levels of fibronectin （FN） were examined by RT-PCR. The protein expression of FN and Smads was detected by Western blot. TGFβ1 induced the expression of FN mRNA and Smads in a time-dependent manner in a certain range. Compared with pre-stimulation, the FN mRNA and protein levels were increased by 1.1 times and 1.5 times respectively （P〈0.01, P〈0.01）, and the protein expression of phosphorylated Smad2/3 （p-Smad2/3） increased by 7 times at the end of TGFβ1 stimulation （P〈0.01）. TSN pretreatment may down-regulate the FN and p-Smad2/3 expression in a dose-dependent manner. 10-6 mol/L TSN pretreatment had no effect on the FN and p-Smad2/3 expression （both P〉0.05）. After pretreatment with 10-5 and 10-4 mol/L TSN, the FN mRNA levels were decreased by 28.1% and 43.8% respectively （P〈0.05, P〈0.01）, the FN protein levels were decreased by 40% and 44% respectively （P〈0.05, P〈0.05）, and the p-Smad2/3 protein expression were decreased by 40% and 65% respectively （P〈0.05, P〈0.01）. The inhibitory effect of TSN on renal interstitial fibrosis may be related to its blocking effect on TGFβ1-Smads signal pathway in renal intersti- tial fibroblasts.     

体外研究人LOX-1受体功能的细胞模型建立

目的研究LOX—1受体在早期动脉粥样硬化中的作用,建立体外LOX-1高表达细胞模型,用于探讨LOX-1与Ox—LDL的相互作用及参与泡沫细胞形成的机制。方法根据人类cDNA文库设计引物,采用巢式PCR技术用人单核细胞转化的巨噬细胞中扩增出LOX-1完整编码区,克隆至pEGFP—C1重组真核表达载体,经测序证实后,用脂质体转染法转染至293T细胞,采用RT—PCR鉴定外源基因的表达,倒置荧光显微镜检测质粒转染效率和融合蛋白的细胞膜上定位情况,流式细胞技术检测EGFP—LOX-1融合蛋白表达情况以及其对Ox—LDL的结合能力。结果pEGFP-LOX—1重建质粒经测序证实克隆的LOX—1序列完整,插入方向正确,转染后实验组LOX—1 mRNA大量表达,融合蛋白正确定位在细胞膜表面,并显示结合配体Dil—Ox—LDL的功能提高到6．3倍。表明所克隆的LOX-1受体具有特异性结合Ox—LDL的功能,从而建立了高表达LOX-1受体的细胞。结论成功克隆人类LOX-1基因,使其在293T细胞中高度表达并获得了相应的功能,为进一步研究LOX-1提供有利工具。     

血凝素样氧化低密度脂蛋白受体与内皮细胞p38丝裂素活化蛋白激酶关系的研究

目的探讨血凝素样氧化低密度脂蛋白受体1(LOX-1)在氧化低密度脂蛋白(ox-LDL)活化人脐静脉内皮细胞p38丝裂素活化蛋白激酶(p38MAPK)信号通路中的作用。方法培养人脐静脉内皮细胞(HUVEC),用不同浓度的ox-LDL孵育,通过W estern印迹检测LOX-1、p38MAPK蛋白水平,逆转录聚合酶链法观察LOX-1 mRNA表达,并以LOX-1特异阻断性抗体(JTX92)预处理内皮细胞,检测p38MAPK蛋白水平。四氮唑蓝法观察ox-LDL对细胞活力的影响。结果ox-LDL引起内皮细胞形态结构的改变,而且抑制内皮细胞的生长(P<0.05);ox-LDL呈浓度依赖性地上调LOX-1蛋白和mRNA表达(均P<0.05),并呈浓度依赖性激活p38MAPK通路,不同浓度ox-LDL(25μg/m l,50μg/m l,100μg/m l)组p-p38MAPK蛋白质表达水平均明显高于对照组(48±7,79±15,113±14 vs 24±5,P<0.01);JTX92+ox-LDL(100μg/m l)组p-p38MAPK蛋白水平明显低于ox-LDL(100μg/m l)组,(58±13 vs 113±14,P<0.01)。结论ox-LDL通过LOX-1途径激活p38MAPK信号通路,LOX-1上调是ox-LDL致动脉粥样硬化的重要环节。     

Intervention of Tongxinluo Capsule (通心络胶囊) against Vascular Lesion of Atherosclerosis and Its Effect on Lectin-like Oxidized Low Density Lipoprotein Receptor-1 Expression in Rabbits

Lectin-like oxidized low-density lipoprotein(ox-LDL) receptor-1 (LOX-1) is a newly identifiedreceptor for cytotoxic ox-LDL in endothelial cellsand plays an i mportant role in the occurrence anddevelopment of atherosclerosis ( AS)(1). Clinicalstudies have showed that Tongxinluo capsule (通心络胶囊,TXL) could alleviate the symptoms of angi-na and myocardial ischemia in patients with coro-nary heart disease.But there werefewstudies con-cerning the effect or therapeutic mechanism of itspreve…     

P21在单侧输尿管梗阻大鼠肾间质纤维化中的表达及依那普利对其表达的调节

目的：观察黄花倒水莲皂苷C抑制氧化型低密度脂蛋白（oxidized low density lipopprotein，ox-LDL）诱导的人脐静脉内皮细胞血凝素样氧化型低密度脂蛋白受体（oxidized low desity lipoprotein receptor-1，LOX-1）表达的作用。方法：培养人脐静脉内皮细胞株（HUVEC-12），随机分为：对照组、ox-LDL（50mg／L）组和氧化型低密度脂蛋白（50mg／L）＋黄花倒水莲皂苷C（1，3，10μmol／L）组，通过RTPCR和Western blot蛋白印迹分析检测LOX-1 mRNA和蛋白的表达。结果：ox-LDL上调LOX-1 mRNA和蛋白的表达，黄花倒水莲皂苷C明显抑制ox-LDL诱导的LOX-1 mRNA和蛋白的表达，且呈浓度依赖性。结论：黄花倒水莲皂苷C明显抑制ox-LDL诱导的内皮细胞LOX-1 mRNA和蛋白的表达。     

厄贝沙坦对巨噬细胞凝集素样氧化低密度脂蛋白受体(LOX-1)基因及蛋白表达的影响

目的研究不同浓度厄贝沙坦对血管紧张素Ⅱ(AngⅡ)诱导的人单核/巨噬细胞系(THP-1)凝集素样氧化低密度脂蛋白受体(LOX-1)mRNA和蛋白表达的影响,并探讨其可能的机制。方法 THP-1细胞经0.16μmoL/L佛波酯诱导分化后,将细胞分为3组:对照组、AngⅡ组、厄贝沙坦干预组,干预组分别加入不同浓度厄贝沙坦孵育2 h后,再加入AngⅡ1×10-6moL/L孵育24 h,用荧光定量PCR和细胞酶联免疫法检测(LOX-1)mRNA和蛋白表达。结果与对照组比较,Ang II可明显上调THP1细胞(LOX-1)mRNA和蛋白表达,差异有统计学意义(P<0.05)。不同浓度厄贝沙坦均能抑制(LOX-1)蛋白表达(P<0.05),并且随着厄贝沙坦浓度降低,抑制作用减低,即两者呈浓度依赖性。结论厄贝沙坦以浓度依赖方式抑制AngⅡ诱导的THP1细胞(LOX-1)mRNA和蛋白表达,减少巨噬细胞通过(LOX-1)途径的氧化低密度脂蛋白(oxLDL)摄入,影响泡沫细胞的发生、发展,发挥其抗动脉粥样硬化(AS)作用。     

银杏黄酮苷元对内皮细胞植物血凝素样低密度脂蛋白受体-1表达的影响

目的：探讨银杏黄酮苷元对氧化低密度脂蛋白（oxidized low density lipoprotein,ox-LDL）诱导脐静脉内皮细胞株ECV304植物血凝素样低密度脂蛋白受体-1（Lectin-like oxidized low density lipoprotein receptor-1,LOX-1）表达的调节作用。方法：应用逆转录聚合酶链式反应、SP免疫组织化学法,探讨ox-LDL诱导下内皮细胞表达LOX-1及银杏黄酮苷元的干预作用。结果：6.25~25 mg/L银杏黄酮苷元与脐静脉内皮细胞株ECV304共同培养6~48 h可显著抑制ox-LDL诱导的内皮细胞LOX-1mRNA和蛋白表达（P〈0.05）,具有浓度、时间效应关系（P〈0.05）;LOX-1拮抗剂聚肌苷酸可抑制ox-LDL诱导的内皮细胞LOX-1mRNA和蛋白表达（P〈0.05）。结论：ox-LDL可能通过LOX-1导致内皮功能障碍,银杏黄酮苷元可显著抑制ox-LDL诱导的内皮细胞LOX-1表达,这可能是其抗动脉粥样硬化的机制之一。     

Intervention of Tongxinluo capsule against vascular lesion of atherosclerosis and its effect on lectin-like oxidized low density lipoprotein receptor-1 expression in rabbits

Objective: To investigate the prevention by Tongxinluo capsule (TXL) of vascular lesions and its effect on the levels of protein and gene expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) of vascular wall in rabbits with atherosclerosis (AS), and to explore its possible mechanism against AS.Methods: AS models were established by feeding New Zealand white rabbits with high-cholesterol diet, and 24 immature rabbits were randomly divided into the control group, model group and treated group (treated with TXL capsule). The indexes of total cholesterol (TO and low density lipoprotein (LDL) levels were measured at the 16th week. The intima thickness and the plaque area of abdominal aorta were quantitatively analyzed by pathological morphological analysis, the expression of macrophage and smooth muscle cell (SMC) in intima were detected by immunohistochemical method and histologic segments were stained by Hematoxilin-Eosin (HE) to identify the degree of atherosclerotic lesion in the model group and the prevention by TXL. The LOX-1 gene and protein expression in abdominal aorta was detected by semi-quantitative RT-PCR and immunohistochemistry, respectively.Results: In the model group, the levels of TC and LDL were significantly elevated, aortic intima thickened extensively, the intima area enhanced, and macrophages expression increased; the levels of LOX-1 gene and protein expression was up-regulated in endothelium and neo-intima of the abdominal aorta. The treatment with TXL reduced blood lipids, attenuated arterial intimai proliferation, markedly inhibited the expression of macrophage and excessively expressed the level of LOX-1.Conclusion: TXL has an inhibitory effect on blood lipids, and it can prevent the occurrence of vascular lesion and cure its development, and its protection against AS was possibly associated with a crucial endothelial protective action through lowering the expression of LOX-1 in vascular walls. Supported by the State Natural Science Foundation of China (No. 30371496).     

阿托伐他汀对人脐静脉内皮细胞Lox-1、TNF-α及ICAM-1表达的影响

目的研究阿托伐他汀(ATV)对脂多糖(LPS)诱导的人脐静脉内皮细胞血凝素样氧化低密度脂蛋白受体-1(LOX-1)、肿瘤坏死因子-α(TNF-α)及细胞间黏附分子(ICAM-1)表达的影响,探讨ATV防治动脉粥样硬化的机制及其抗炎作用。方法体外培养人脐静脉内皮细胞,用LPS刺激人脐静脉内皮细胞后,加入ATV干预24h,收集细胞,用荧光定量PCR方法测定LOX-1、TNF-α及ICAM-1mRNA的表达;用Western blotting法测定LOX-1、TNF-α及ICAM-1蛋白表达。结果用LPS刺激人脐静脉内皮细胞后,引起LOX-1、TNF-α及ICAM-1的高表达,与空白对照组比较差异有统计学意义(P<0.01),用ATV干预以后显著抑制LOX-1、TNF-α及ICAM-1的表达,与模型组比较差异有统计学意义(P<0.01)。结论 ATV可抑制LOX-1、TNF-α及ICAM-1的表达,这可能是其发挥抗动脉粥样硬化作用及抗炎作用的机制之一。     

凝集素样氧化型低密度脂蛋白受体-1在C反应蛋白诱导人脐静脉内皮细胞组织因子表达中的作用

目的研究凝集素样氧化型低密度脂蛋白受体-1(lectin-type oxidized LDL receptor1,LOX-1)在C-反应蛋白(c-reactive protein,CRP)诱导人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)组织因子(tissue factor,TF)表达中的作用。方法用人重组质粒pTracer CMV2-CRP转染HUVECs细胞,分正常对照组、空质粒GFP组和CRP-GFP组(n=3),RT-PCR、Western blot检测TF、LOX-1 mRNA和蛋白表达。Western blot检测各组P-ERK1/2、T-ERK1/2、P-JNK、T-JNK蛋白表达。CRP-GFP组分别加LOX-1阻断剂、ERK1/2阻断剂(U0126)、JNK阻断剂(SP600125),Westernblot检测TF蛋白。结果用表达人CRP的质粒转染HUVECs显著增加LOX-1 mRNA和蛋白表达[(0.70±0.03)vs(0.24±0.01),(0.661 6±0.020 0)vs(0.255 8±0.040 0),P<0.05]、TF的mRNA和蛋白表达[(0.442±0.040)vs(0.146 0±0.030 0),(0.727 4±0.020 0)vs(0.217 4±0.020 0),P<0.05]。CRP启动了ERK1/2 MAPK,未启动JNKMAPK。CRP上调TF表达被ERK1/2阻断剂而非JNK阻断剂阻断.抗LOX-1的中和抗体显著减少CRP诱导的TF mRNA和蛋白表达[(0.264 0±0.030 0)vs(0.442 0±0.040 0),(0.324 4±0.040 0)vs(0.727 4±0.020 0),P<0.05]。结论CRP诱导HUVECs细胞LOX-1表达上调,并通过LOX-1受体这一新途径上调TF表达,且CRP通过ERK1/2 MAPK信号途径非JNK MAPK途径上调了HUVEC细胞TF的表达。