Occludin and connexin 43 expression contribute to the pathogenesis of traumatic brain edema

The experimental model of traumatic brain injury was established in Sprague-Dawley rats according to Feeney＇s free falling method. The brains were harvested at 2, 6 and 24 hours, and at 3 and 5 days after injury. Changes in brain water content were determined using the wet and dry weights. Our results showed that water content of tissue significantly increased after traumatic brain injury, and reached minimum at 24 hours. Hematoxylin-eosin staining revealed pathological impairment of brain tissue at each time point after injury, particularly at 3 days, with nerve cell edema, degenera- tion, and necrosis observed, and the apoptotic rate significantly increased. Immunohistochemistry and western blot analysis revealed that the expression of occludin at the injured site gradually de- creased as injury time advanced and reached a minimum at 3 days after injury; the expression of connexin 43 gradually increased as injury time advanced and reached a peak at 24 hours after in-jury. The experimental findings indicate that changes in occludin and connexin 43 expression were consistent with the development of brain edema, and may reflect the pathogenesis of brain injury.     

Buyang Huanwu Decoction regulates neural stem cell behavior in ischemic brain

The traditional Chinese medicine Buyang Huanwu Decoction has been shown to improve the neu-rological function of patients with stroke. However, the precise mechanisms underlying its effect remain poorly understood. In this study, we established a rat model of cerebral ischemia by middle cerebral artery occlusion and intragastrically administered 5 g/kg Buyang Huanwu Decoction, once per day, for 1, 7, 14 and 28 days after cerebral ischemia. Immunohistochemical staining revealed a number of cells positive for the neural stem cell marker nestin in the cerebral cortex, the subven-tricular zone and the ipsilateral hippocampal dentate gyrus in rat models of cerebral ischemia. Buyang Huanwu Decoction significantly increased the number of cells positive for 5-bromodeoxyuridine (BrdU), a cell proliferation-related marker, microtubule-associated protein-2, a marker of neuronal differentiation, and growth-associated protein 43, a marker of synaptic plasticity in the ischemic rat cerebral regions. The number of positive cells peaked at 14 and 28 days after intragastric administration of Buyang Huanwu Decoction. These findings suggest that Buyang Huanwu Decoction can promote the proliferation and differentiation of neural stem cells and hance synaptic plasticity in ischemic rat brain tissue.     

脑血管痉挛中缝隙连接蛋白Cx43磷酸化位点分析及S368位点与其关系的研究

目的 观察脑血管痉挛中缝隙连接蛋白Cx43的磷酸化位点及其S368位点磷酸化水平的变化,探讨其与脑血管痉挛的关系. 方法 新西兰大白兔78只按随机数字表法分为4组:正常对照组(n=6)、单纯脑池注血组(n=24)、甘珀酸(CBX)脑池处理组(n=24)和溶媒脑池处理组(n=24),后3组应用枕大池二次注血法建立兔蛛网膜下腔出血后脑血管痉挛模型及相应给药,并按1d、3d、7d、14d分成4亚组,每亚组6只.采用磷酸化蛋白富集试剂盒富集各组基底动脉中Cx43磷酸化总蛋白,再利用质谱技术鉴定出其磷酸化位点;应用Western blotting方法分析各组Cx43S368位点磷酸化水平的变化;通过数字减影血管造影技术(DSA)观察各组基底动脉直径变化情况. 结果 (1)质谱技术成功鉴定出Cx43的4个磷酸化位点,分别为Y265、S364、S365、S368.(2)Western blotting结果显示:正常对照组基底动脉Cx43 S368位点磷酸化水平较低(17.0％±2.3％);单纯脑池注血组及溶媒脑池处理组与正常对照组相比,Cx43 S368位点磷酸化水平在1d、3d、7d、14d各时间点均显著升高,差异有统计学意义(P＜0.05),且以7d表达最高,14d开始下降;CBX脑池处理组各时间点基底动脉Cx43 S368位点磷酸化水平显著低于单纯脑池注血组及溶媒脑池处理组,差异有统计学意义(P＜0.05).(3)DSA显示正常对照组第二次与首次造影基底动脉直径的百分比值平均为99.1％±1.3％,单纯脑池注血组、CBX脑池处理组、溶媒脑池处理组分别为66.1％±7.2％、91.3％±5.3％、63.7％±6.6％,CBX脑池处理组基底动脉直径显著狭窄于单纯脑池注血组,差异有统计学意义(P＜0.05). 结论 缝隙连接蛋白Cx43 S368位点磷酸化可能与脑血管痉挛密切相关,且其可能是CBX缓解脑血管痉挛的机制之一.     

A new target for treatment of cerebral ischemia/reperfusion injury

Rho kinase inhibitor fasudil hydrochloride has been shown to reduce cerebral vasospasm, to inhibit inflammation and apoptosis and to promote the recovery of neurological function. However, the effect of fasudil hydrochloride on claudin-5 protein expression has not been reported after cerebral ischemia/reperfusion. Therefore, this study sought to explore the effects of fasudil hydrochloride on blood-brain barrier permeability, growth-associated protein-43 and claudin-5 protein expression, and to further understand the neuroprotective effect of fasudil hydrochloride. A focal cerebral ischemia/reperfusion model was established using the intraluminal suture technique. Fasudil hydrochloride (15 mg/kg) was intraperitoneally injected once a day. Neurological deficit was evaluated using Longa’s method. Changes in permeability of blood-brain barrier were measured using Evans blue. Changes in RhoA, growth-associated protein-43 and claudin-5 protein expression were detected using immunohistochemistry and western blotting. Results revealed that fasudil hydrochloride noticeably contributed to the recovery of neurological function, improved the function of blood-brain barrier, inhibited RhoA protein expression, and upregulated growth-associated protein-43 and claudin-5 protein expression following cerebral ischemia/reperfusion. Results indicated that Rho kinase exhibits a certain effect on neurovascular damage following cerebral ischemia/reperfusion. Intervention targeted Rho kinase might be a new therapeutic target in the treatment of cerebral ischemia/reperfusion.     

Cyclic stretch influenced expression of membrane connexin 43 in human periodontal ligament cell

Objective

Periodontal ligament (PDL) cells play an important role in preserving periodontal homeostasis and periodontal remodelling in response to mechanical stimulations. Gap junction intercellular communication (GJIC) is essential for homeostasis and many other biological processes of multicellular organisms. While the role of GJIC in mechanotransduction of PDL cells remains largely unknown. In the present study, we examined the influence of cyclic stretch on the expression of membrane gap junction protein connexin 43 (Cx43) in cultured human PDL cells.

Design

Cultured human PDL cells were exposed to 1%, 10% and 20% stretch strains for 0.5 h, 1 h and 24 h. Then the membrane Cx43 protein expression was measured by flow cytometry and the Cx43 mRNA level was measured by real-time polymerase chain reaction.

Results

Half hour and 1 h cyclic stretches with strains up to 20% did not change the expression of membrane Cx43 protein, while 24 h cyclic stretches with 10% and 20% strains down-regulated the expression of membrane Cx43 protein in a strain magnitude-dependent manner. Furthermore, cyclic stretch also changed the Cx43 mRNA level and induced realignment in cells.

Conclusion

The present research provide the first evidence that cyclic stretch influenced the membrane Cx43 protein expression in cultured human PDL cells.     

拮抗P2X7受体在保护大鼠脑缺血/再灌注损伤中的作用

目的：探讨拮抗P2X7受体(P2X7 receptor,P2X7R)在保护大鼠脑缺血/再灌注(ischemia/reperfusion,I/R)损 伤中的作用及其机制。方法：采用改良线栓法建立大鼠大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)模型; 随后对其进行缺血2 h再灌注24 h处理,完成大鼠大脑局灶性脑I/R损伤模型的制备。采用Longa五分法对大鼠进行神 经行为学评分;2,3,5-氯化三苯基四氮唑(TTC)染色法检测大鼠大脑梗死体积的变化;Western印迹检测细胞外信号调 节激酶(extracellular signal-regulated kinase,ERK),磷酸化细胞外信号调节激酶(phosphorylation extracellular signal-regulated kinase,p-ERK),缝隙连接蛋白43(connexin 43,Cx43),Bax,Bcl-2及cleaved caspase-3蛋白表达的变化。结果：Longa五 分法与TTC染色法结果显示：与假手术组比较,I/R组大鼠神经行为学评分(P<0.05)和大脑梗死体积(P<0.01)均明显增 加。与I/R组大鼠相比,P2X7R拮抗剂明亮蓝(brilliant blue G,BBG)或ERK抑制剂PD98059均可明显降低大鼠神经行为 学评分(P<0.01)和大鼠大脑梗死体积(P<0.05)。在阻断P2X7R的基础上使用PD98059抑制ERK活性后,大鼠神经行为学 评分和大脑梗死体积进一步降低(P<0.05);Western印迹结果显示：BBG或PD98059均可降低p-ERK,Cx43,Bax/Bcl-2及 cleaved caspase-3蛋白表达量(P<0.05)。在阻断P2X7R的基础上使用PD98059抑制ERK活性后,p-ERK,Cx43,Bax/Bcl-2及 cleaved caspase-3等蛋白表达量进一步降低(P<0.05)。结论：拮抗P2X7R可减轻大鼠脑I/R损伤,其机制可能与抑制ERK 激活,进而降低Cx43和cleaved caspase-3蛋白表达及Bax/Bcl-2比值有关。     

环磷酰胺及长春新碱对睾丸支持细胞间隙连接蛋白表达的影响

目的：探讨环磷酰胺、长春新碱对睾丸支持细胞间隙连接蛋白（Cx）43表达的影响。方法：培养小鼠睾丸支持细胞,分别加入环磷酰胺（0、1、2、4、8μg/mL）和长春新碱（0.00、0.01、0.02、0.04、0.08μg/mL）行细胞毒染24 h,MTT法检查细胞毒性。选择4μg/mL环磷酰胺、0.04μg/mL长春新碱作用支持细胞6、12、24及48 h,结果行RT-PCR分析;选择同样浓度药物作用支持细胞24 h,并用免疫荧光染色检测培养的细胞中Cx43的表达情况。结果：环磷酰胺、长春新碱染毒细胞后,Cx43 mRNA水平开始随时间增加而逐渐下降（P〈0.05）,且随剂量增加Cx43表达强度逐渐减弱（P〈0.05）。结论：环磷酰胺及长春新碱对睾丸支持细胞有毒性,并随着药物浓度增大毒性增强,且环磷酰胺比长春新碱更明显。     

转移抑制因子1和钙激活蛋白43在食管鳞状细胞癌组织中的表达及其临床意义

目的：探讨转移抑制因子1（MTSS1）和钙激活蛋白43(Cap43)在食管鳞状细胞癌(鳞癌)组织中表达,阐明其与食管鳞癌临床病理特征间的关
系。方法：收集食管鳞癌标本80例和癌旁正常组织30例,采用免疫组织化学SP染色法和RT-PCR法检测食管鳞癌组织和正常组织中MTSS1、Cap43蛋白和mRNA的表达情况,分析MTSS1和Cap43表达与食管鳞癌临床病理特征之间的关系,分析两者在食管鳞癌组织中表达的相关性。结果：免疫组织化学SP染色法,MTSS1和C
ap43在癌细胞的胞浆/细胞膜呈现棕黄色,MTSS1在正常组织和食管鳞癌组织中的阳性表达率分别为83.3%（25/30）和21.3%（17/80）,Cap43在正常组织和食管癌组织中的阳性表达率分别为16.7%（5/30）和76.3%（61/80）,组间比较差异均有统计学意义（P<0.01）。RT-PCR法检测,MTSS1 mRNA在正常组织和食管鳞癌组织中表达水平分别为0.703±0.085和0.295±0.065,而Cap43 mRNA在正常组织和食管鳞癌组织中的表达水平分别为0.236±0.052和0.693±0.078,组间比较差异均有统计学意义(P<0.01)。食管鳞癌组织中MTSS1和Cap43的表达与食管鳞癌的浸润深度、淋巴结转移和临床TNM分期有关联（P<0.05）,而与食管鳞癌患者的性别、年龄、肿瘤大小和组织类型均无关联（P>0.05）;MTSS1与Cap43的表达呈负相关关系（r＝－0.457,P<0.05）。结论：食管鳞癌组织中MTSS1的低表达和Cap43的高表达可能促进
了肿瘤的浸润和转移,二者之间的平衡失调可能是食管鳞癌侵袭和转移的重要机制之一。     

ALA-PDT对皮肤鳞状细胞癌A431细胞Caspase-3及Caspase-7表达的影响

目的观察盐酸氨酮戊酸散(ALA)-光动力疗法(PDT)对皮肤鳞状细胞癌A431细胞中Caspase-3及Caspase-7表达的影响。方法体外培养A431细胞,应用M TT法检测不同ALA浓度、不同培养时间、不同PDT光照剂量以及单独加药和单独光照对A431细胞增殖与凋亡的影响,并设Hacat作为对照;应用实时荧光定量PCR及Western blotting检测A431细胞及Hacat细胞中Caspase-3及Caspase-7 mRNA和蛋白的表达水平。结果A431细胞中Caspase-3及Caspase-7 mRNA和蛋白的表达量低于Hacat细胞,其表达强弱差异有统计学意义(P<0.05)。ALA-PDT对A431细胞的增殖具有一定的抑制作用,并引起Caspase-3及Caspase-7 mRNA和蛋白的表达量升高(P<0.01)。当光照剂量为80 J/cm2时,Caspase-3、Caspase-7 mRNA及蛋白的表达量均达到峰值。结论ALA-PDT对皮肤鳞状细胞癌A431细胞的增殖抑制可能是通过诱导Caspase-3及Caspase-7的表达,引起细胞的凋亡从而发挥作用。     

Effects of repetitive magnetic stimulation on motor function and GAP43 and 5-HT expression in rats with spinal cord injury

ObjectivesSpinal cord injury (SCI) is a disabling central nervous system disorder. This study aimed to explore the effects of repetitive trans-spinal magnetic stimulation (rTSMS) of different spinal cord segments on movement function and growth-associated protein-43 (GAP43) and 5-hydroxytryptamine (5-HT) expression in rats after acute SCI and to preliminarily discuss the optimal rTSMS treatment site to provide a theoretical foundation and experimental evidence for clinical application of rTSMS in SCI.MethodsA rat T10 laminectomy SCI model produced by transient application of an aneurysm clip was used in the study. The rats were divided into group A (sham surgery), group B (acute SCI without stimulation), group C (T6 segment stimulation), group D (T10 segment stimulation), and group E (L2 segment stimulation).ResultsIn vivo magnetic stimulation protected motor function, alleviated myelin sheath damage, decreased NgR and Nogo-A expression levels, increased GAP43 and 5-HT expression levels, and inhibited terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells and apoptosis-related protein expression in rats at 8 weeks after the surgery.ConclusionsThis study suggests that rTSMS can promote GAP43 and 5-HT expression and axonal regeneration in the spinal cord, which is beneficial to motor function recovery after acute SCI.