Angiotensin II induces mitochondrial dysfunction and promotes apoptosis via JNK signalling pathway in primary mouse calvaria osteoblast

Objectives

This present study was designed to investigate the effects of Angiotensin II on mitochondrial functions, ROS generation and c-jun N-terminal kinases (JNK) signalling pathway-mediated cell apoptosis in mouse calvaria osteoblasts.

Methods

Calvaria osteoblast were isolated and cultured. The cells were separated into two groups—control and treated groups—where the latter was stimulated with angiotensin II (Ang II). Mitochondrial reactive oxygen species (ROS) and superoxide production were measured. Intracellular ATP levels were also detected. The cell proliferation rate was determined for the two groups. Protein production such as Anti-Bax, Bcl-2, COX IV and activation of c-jun N-terminal kinases signal (JNK) pathway was measured by enzyme-linked immunosorbent assay (ELISA) methods and Western blotting in this study.

Results

Ang II treated cells showed significantly higher levels of superoxide production compared to the control group (p < 0.05). Conversely, Ang II induced inhibitory effects on mitochondrial respiratory enzyme complexes, cause membrane potential dissipation, ATP loss and promote ROS generation, cell apoptosis in cultured osteoblasts. In addition, JNK phosphorylations were involved in activating the mitochondria-dependent apoptotic pathway following Ang II stimulation, as pre-treatment of JNK-specific inhibitor SP600125 could rescue osteoblast cells from apoptosis by enhancing the anti-apoptotic protein Bcl-2 expressions, suppressing the translocation of Bax from cytosol into mitochondria, blocking cytochrome C release and caspase-3 activation.

Conclusions

Ang II stimulates osteoblast apoptosis via suppression of the mitochondrial respiratory enzymes, membrane potential and cellular ATP productions. Clinical application with Ang II-stimulated osteoblast could be used for modelling or bone resorption in the oral region.     

临床路径管理实施过程中的障碍原因与处置对策研究

目的探讨临床路径管理实施过程中的障碍原因及处置对策。方法回顾性分析医院2013年8-12月进入临床路径管理的282例患者(病种为胆囊结石腹腔镜胆囊切除术、慢性鼻-鼻窦炎、2型糖尿病)的完整临床资料,按照是否顺利完成临床路径管理分为两组,观察组140例均顺利完成临床路径,对照组142例未顺利完成临床路径,统计分析临床路径实施障碍原因,总结处置对策。结果观察组患者年龄显著小于对照组,文化程度显著优于对照组,自费者显著高于对照组,2型糖尿病患者病程显著短于对照组,差异均具有统计学意义(均P<0.05);多因素分析显示,年龄大、文化程度低、医保就医及2型糖尿病病程长是临床路径管理实施障碍的原因,差异均具有统计学意义(均P<0.05)。结论针对年龄大、文化程度低、医保就医及2型糖尿病病程长等临床路径实施障碍原因,可加强健康教育、心理干预及康复指导,保证临床路径顺利实施。     

Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system

Aim:

To report our methods for expression and purification of α7 nicotinic acetylcholine receptor (α7-nAChR), a ligand-gated pentameric ion channel and an important drug target.

Methods:

α7-nAChRs of 10 different species were cloned into an inducible BacMam vector with an N-terminal tag of a tandem maltose-binding protein (MBP) and a TEV cleavage site. This α7-nAChR fusion receptor was expressed in mammalian HEK293F cells and detected by Western blot. The expression was scaled up to liters. The receptor was purified using amylose resin and size-exclusion chromatography. The quality of the purified receptor was assessed using SDS-PAGE gels, thermal stability analysis, and negative stain electron microscopy (EM). The expression construct was optimized through terminal truncations and site-directed mutagenesis.

Results:

Expression screening revealed that α7-nAChR from Taeniopygia guttata had the highest expression levels. The fusion receptor was expressed mostly on the cell surface, and it could be efficiently purified using one-step amylose affinity chromatography. One to two milligrams of the optimized α7-nAChR expression construct were purified from one liter of cell culture. The purified α7-nAChR samples displayed high thermal stability with a Tm of 60 °C, which was further enhanced by antagonist binding but decreased in the presence of agonist. EM analysis revealed ring-like structures with a central hydrophilic hole, which was consistent with the pentameric assembly of the α7-nAChR channel.

Conclusion:

We have established methods for crystallization scale expression and purification of α7-nAChR, which lays a foundation for high-resolution structural studies using X-ray crystallography or single particle cryo-EM analysis.     

玉竹多糖对衰老模型鼠免疫功能的影响

目的研究玉竹多糖对衰老模型鼠免疫功能的影响。方法制备D-半乳糖亚急性衰老模型小鼠，同时腹腔注射不同剂量的玉竹多糖。6W后，用MTT法测定脾淋巴细胞转化刺激指数（SI）；用免疫荧光法测定脾T淋巴细胞亚群；用PI染色法测脾淋巴细胞的凋亡率，观察其对免疫功能的影响。结果3种不同剂量的玉竹多糖均能提高脾B淋巴细胞转化SI；高剂量玉竹多糖能够提高脾T淋巴细胞转化SI；玉竹多糖能够显著提高衰老模型鼠的CD8^＋细胞数及降低CD4^＋／CD8^＋比值；高剂量玉竹多糖能够明显抑制脾淋巴细胞的凋亡。结论玉竹多糖可增强衰老模型小鼠的细胞及体液免疫功能。     

赵玉庸教授治疗无症状IgA肾病临床经验研究

无症状IgA肾病患者临床多见,西医无特殊办法,中医治疗有一定优势,赵玉庸教授根据多年临床经验,认为IgA肾病主要病机为脾肾亏虚,瘀血阻络,采用病证结合方法辨证治疗,包括结合IgA肾病临床特点治疗、结合IgA肾病病理改变治疗、基础方结合实验室检查加减治疗等方面,疗效较好,故进行总结研究,为临床治疗此类患者提供一些思路和方法。     

痔瘘洗必灵袋泡剂治疗血栓外痔30例

目的:观察痔瘘洗必灵袋泡剂熏洗坐浴治疗血栓外痔的临床疗效。方法:将58例血栓外痔患者随机分为治疗组30例和对照组28例,治疗组给予痔瘘洗必灵袋泡剂熏洗坐浴治疗,对照组给予1:5 000高锰酸钾熏洗坐浴治疗。结果:治疗后,治疗组疼痛评分优于对照组,差异均有统计学意义(P0.05);治疗组有效率96.7%,对照组有效率78.6%,治疗组明显优于对照组(P0.05)。结论:痔瘘洗必灵袋泡剂熏洗坐浴治疗血栓外痔有明显疗效。     

鞣花酸抗S180,H22肿瘤及其抑制新生血管机制探讨

目的: 观察鞣花酸对S180,H22荷瘤小鼠肿瘤生长及微血管生成的影响,以及对血小板衍生因子B(PDGFB),转录激活因子-3(STAT3)及磷酸化STAT3(p-STAT3)基因和蛋白表达的影响,探讨其抗血管生成作用可能的机制。方法: SPF级昆明种小鼠100只,建立S180,H22皮下荷瘤小鼠2种模型,分别随机分为模型组(0.5% CMC溶液)、环磷酰胺组(阳性药,20 μg·g^-1·d^-1)、鞣花酸高、中、低(200,100,50 μg·g^-1·d^-1)剂量组,每组10只,连续给药ig 10 d,观察鞣花酸对荷瘤小鼠肿瘤生长、体重、胸腺指数及脾脏指数,免疫组化法检测肿瘤微血管密度,PDGFB,STAT3及p-STAT3的表达情况。结果: 鞣花酸高、中、低剂量组对S180小鼠抑瘤率分别为35.3%,10.6%,5.6%,对H22小鼠抑瘤率分别为36.3%,38.8%,20.6%,其对小鼠体重无明显影响;与模型组比较,高剂量组对S180,H22小鼠脾脏指数较模型组明显上升(P<0.05),鞣花酸高、中剂量组能明显降低S180,H22小鼠肿瘤微血管密度(P<0.05),鞣花酸高、中、低剂量组在S180,H22 2种瘤体中PDGFB,STAT3和p-STAT3的表达明显降低。结论: 鞣花酸抑制S180,H22荷瘤小鼠肿瘤生长及微血管形成,其机制可能与下调肿瘤组织中PDGFB表达并抑制下游STAT3的蛋白表达及磷酸化有关。     

血管内皮细胞第Ⅷ因子在胃癌间质血管的研究

目的:应用血管内皮细胞单克隆第Ⅷ因子相关抗原对胃癌间质血管内皮细胞形态进行观察。方法:手术切除的120例标本应用免疫组织化学及荧光染色进行观察。均以正常羊血清替代第一抗体作空白对照。结果:对分布在胃癌间质中的各级血管内径进行测量,胃癌中心带、间带和外带间质血管内径均比无癌区带的血管内径大。结论:胃癌形成与血管内皮细胞增生是同步发生,甚至癌旁区带组织中血管内皮细胞已有增生。增生的内皮细胞阻塞血流,影响局部组织血液循环,抑制肿瘤间质血管内皮细胞增生是抗肿瘤治疗的重要手段。     

Effect of Chinese Patent Medicine Naodesheng against Repeated Transient Global Cerebral Ischemia in Mice

Objective To investigate the therapeutic effect and possible mechanisms of Chinese ptent medicine Naodesheng(NDS) on repeated transient global cerebral ischemia(GCI) in mice. Methods The repeated transient GCI mice were induced by bilateral carotid arteries ligation, and were randomly divided into model group, Sham group without arteries ligation, NDS groups(1.25 and 2.5 g/kg) and positive control(vinpocetine 3.1 mg/kg, VP) group. After oral administration once daily for successive 7 d, the transient GCI was induced. The degree of neurological deficit, histological changes, and neurons loss in the hippocampus were evaluated. In order to investigate the possible mechanisms, the oxidative stress and inflammatory factor were measured after 24 h of GCI. Comparison among multiple groups was performed with one-way analysis of variance(ANOVA). Results NDS could significantly alleviate the neurological function impairment, histological injury, and neurons loss, increase the superoxide dismutase(SOD) activity, decrease the content of malondialdehyde(MDA), and reduce inflammatory factor in the ischemic brain tissue. Conclusion NDS could significantly reduce brain injury induced by global ischemia, and its mechanism is closely associated with anti-oxidation and anti-inflammation.