Fasudil, a Rho-associated protein kinase inhibitor, attenuates retinal ischemia and reperfusion injury in rats

Abnormal activation of Rho kinase (ROCK) plays a vital role in the pathogenesis of ischemia/reperfusion (I/R)-induced retinal injury. The aim of this study was to investigate whether fasudil, a potent inhibitor of ROCK, has a protective effect on retinal I/R injury in rats and to explore the possible underlying mechanisms. Forty adult Sprague-Dawley rats were randomly assigned into sham, I/R injury model (I/R), model plus normal saline (control), and model plus fasudil (fasudil) groups. Rats in the control and fasudil groups were intravitreously injected with normal saline and fasudil, respectively, 5 min prior to the induction of ischemia. Retinal ischemia was induced by increasing the intraocular pressure to 100 mmHg for 60 min. Overall retinal thickness and retinal cell apoptosis was evaluated by histological analysis and the TUNEL assay, respectively. The protein expression of caspase-3 and the Bax/Bcl-2 mRNA ratio were also examined. Moreover, the retinal expression of inducible nitric oxide synthase (iNOS) was determined by immunohistochemical staining, quantitative real-time RT-PCR and Western blot analysis. Fasudil attenuated the I/R-induced apoptosis of retinal cells in the inner nuclear and ganglion cells of the rat retina. Fasudil significantly decreased the Bax/Bcl-2 mRNA ratio and the expression of caspase-3 and iNOS compared to the control group (P<0.05). Seven days after I/R, the overall retinal thickness in the fasudil group was significantly greater compared to that in the control group (P<0.05). In conclusion, fasudil can protect the rat retina from I/R injury by inhibiting apoptosis and iNOS expression, suggesting that fasudil may have a therapeutic potential for the prevention of retinal diseases associated with I/R.     

法舒地尔通过抑制兔尿道成纤维细胞Rho/ROCK通路激活减少尿道损伤后狭窄的发生

目的:研究法舒地尔对创伤后兔尿道狭窄发生的影响及机制,观察兔尿道成纤维细胞活力、迁移以及细胞外基质合成情况。方法:利用显微外科技术建立兔尿道创伤动物模型,分为5组:假手术组,手术组,不同剂量(3 mg/kg、10 mg/kg、30 mg/kg)法舒地尔组,术后3个月逆行尿道造影测量狭窄直径。从兔尿道瘢痕组织提取成纤维细胞进行传代培养,培养液中分别加入TGF-β1(10μg/L)和/或法舒地尔(12.5μmol/L,25μmol/L,50μmol/L)。MTT比色法检测细胞活力;用Transwell小室实验检测细胞迁移能力;Western blot法检测各组Rho相关激酶(ROCK)、α-平滑肌肌动蛋白(α-SMA)以及胶原蛋白Ⅰ和Ⅲ的表达情况。结果:法舒地尔显著减少尿道损伤后狭窄发生(P0.05)。随着法舒地尔浓度的增加,成纤维细胞的活力受到抑制,细胞迁移能力逐渐减弱,ROCK、α-SMA以及胶原蛋白Ⅰ和Ⅲ的表达受到抑制;法舒地尔对兔尿道成纤维细胞外基质及ROCK表达起抑制作用,并呈剂量依赖性(P0.05)。结论:法舒地尔可以通过下调TGF-β1诱导的兔尿道成纤维细胞Rho/ROCK通路,抑制细胞外基质形成,减少尿道损伤后狭窄的发生。     

Fasudil,an inhibitor of Rho-associated coiled-coil kinase,attenuates hyperoxia-induced pulmonary fibrosis in neonatal rats

Background: Oxygen therapy is important during the management of high-risk neonatal infants, such as those with preterm birth, low birth weight, and asphyxia. However, prolonged exposure to high oxygen concentrations can readily lead to diffuse nonspecific inflammation, which promotes airway remodeling and pulmonary fibrosis. The Rho/Rho-associated coiled-coil kinase (Rho/ROCK) signaling pathway plays an important role in numerous developmental and proliferative diseases. This study was performed to determine the efficacy of ROCK inhibitor fasudil in blocking the development of hyperoxia-induced lung injury and fibrosis in neonatal rats. Methods: Neonatal rats were randomly divided into four groups: air + saline group, air + fasudil group, hyperoxia + saline group, and hyperoxia + fasudil group. The hyperoxia + saline and Hyp + fasudil groups were exposed to 95% oxygen for 21 days and administered intraperitoneal saline or fasudil once daily. The air + saline and air + fasudil group were exposed to 21% oxygen (room air) and administered the same volume of intraperitoneal saline or fasudil. Results: Fasudil-treated rats exhibited improved histopathological changes and decreased lung hydroxyproline content. Fasudil attenuated the protein level of alpha-smooth muscle actin, transforming growth factor-β1, and connective tissue growth factor. Additionally, fasudil reduced the activation of ROCK1 and myosin phosphatase targeting subunit 1 protein in the Rho/ROCK signaling pathway. Conclusions: Fasudil may be a potentially effective therapeutic drug for hyperoxia-induced pulmonary fibrosis.     

子宫缺血再灌注损伤大鼠模型的建立

背景：子宫缺血再灌注损伤可导致能量代谢障碍、大量自由基产生及细胞凋亡等。 目的：建立一种简易、实用、可靠的子宫缺血再灌注损伤大鼠模型。 方法：将50只健康Wistar雌性大鼠随机等分为5组。子宫缺血组(共3组)开腹后采用线栓法分别阻断大鼠子宫动脉45 min、阻断大鼠腹腔动脉30 min或阻断大鼠腹腔动脉45 min进行子宫缺血处理;子宫缺血再灌注组开腹后采用线栓法阻断大鼠腹腔动脉30 min再灌注60 min;假手术组不阻断子宫供血动脉。 结果与结论：子宫动脉纤细,结扎过紧,子宫动脉常常断裂,结扎过松,不能够充分阻断血液,不适宜建立子宫缺血再灌注损伤模型。苏木精-伊红染色显示,阻断大鼠腹腔动脉30 min后子宫细胞及间质肿胀,肌纤维排列尚整齐;阻断大鼠腹腔动脉45 min及子宫缺血再灌注组的子宫细胞及间质明显水肿,渗出增加,肌纤维排列紊乱,有大量中性粒细胞浸润。分光光度计检测显示,阻断大鼠腹腔动脉45 min及子宫缺血再灌注组子宫丙二醛水平升高最明显 (P < 0.01)。提示采用线栓法结扎腹腔动脉30 min后进行再灌注可建立大鼠子宫缺血再灌注损伤模型。     

ROCK-I and ROCK-II cooperatively regulate closure of eyelid and ventral body wall in mouse embryo

Rho-associated kinase (ROCK) is a serine/threonine kinase working in the Rho signaling to actin cytoskeleton. We previously reported that loss of ROCK-I results in the eyelid open at birth (EOB) and omphalocele phenotype in mice, while loss of ROCK-II results in placental dysfunction leading to intrauterine growth retardation and fetal death. Here, we report that after backcross to the C57BL/6 N genetic background, ROCK-II knockout (KO) neonates are born also with open eyelid and umbilical hernia, a phenotype similar to that of ROCK-I KO mice. ROCK-II KO embryos show impaired extension of the eyelid epithelial sheet with disorganized actin bundles in the leading edge of the sheet. These results suggest that ROCK-I and ROCK-II cooperatively regulates the assembly of actin bundles essential for closure of the eyelid and ventral body wall in mouse embryos. Consistently, ROCK-I(+/-)ROCK-II(+/-) double heterozygous mice also show the EOB and omphalocele phenotype.     

硫化氢增加肝脏缺血再灌注损伤模型大鼠胰岛素样生长因子Ⅰ、 核转录因子κB、肿瘤坏死因子α的表达

背景：硫化氢气体是一种新型气体信号分子,在生理浓度下具有抑制Ca2+内流、开放KATP通道功能,氧化还原等明确特定的作用。 目的：观察硫化氢对大鼠肝脏缺血再灌注损伤胰岛素样生长因子Ⅰ、核转录因子κB及肿瘤坏死因子α表达水平的影响 方法：70只SD大鼠随机等分为假手术组、缺血20 min组,缺血20 min+灌注2,4 h组、硫氢化钠+缺血20 min组、硫氢化钠+缺血20 min再灌注2,4 h组。除假手术组外,其余各组大鼠通过Pringle法建立大鼠全肝缺血再灌注模型,在术前5 d中每天及术中向硫氢化钠+缺血20 min组、硫氢化钠+缺血20 min再灌注2,4 h组,大鼠腹腔注射56 μmol/kg 硫氢化钠。 结果与结论：肝脏缺血大鼠肝组织中胰岛素样生长因子Ⅰ 、核转录因子κB和肿瘤坏死因子α表达明显下降(P < 0.05),与缺血组比较,大鼠再灌注和腹腔注射硫化氢均能增加大鼠肝组织中胰岛素样生长因子Ⅰ 、硫化氢、核转录因子κB和肿瘤坏死因子α的表达(P < 0.05)。提示全肝缺血再灌注后可造成肝脏损伤,硫化氢增加肝脏缺血再灌注损伤模型大鼠胰岛素样生长因子Ⅰ、核转录因子κB、肿瘤坏死因子α的表达,肝脏损伤具有保护作用。     

晚期缺血预适应促进低氧诱导因子的表达减轻肾脏缺血再灌注损伤

目的: 探讨晚期缺血预适应对肾脏缺血再灌注损伤的作用,以及低氧诱导因子1α(HIF-1α)在其中的作用。方法: 将雄性C57/BL6N小鼠随机分为3组:假手术组(sham)、缺血再灌注组(IR)和缺血预适应组(IPC)。采用夹闭双侧肾蒂30 min后恢复灌注的方法建立肾缺血再灌注小鼠模型;缺血预适应组4 d前给予肾脏15 min预缺血。观察缺血预处理对再灌注后不同时点血肌酐(Scr)、尿素氮(BUN)、肾组织形态学和细胞凋亡的影响。采用免疫组化及Western印迹法,分析HIF-1α在肾组织的表达;采用实时定量RT-PCR法,检测血管内皮生长因子(VEGF)和葡萄糖转运子-1(Glut-1)的mRNA表达。结果: 再灌注24 h后,IPC组小管间质损伤程度较IR组显著减轻,Scr、BUN水平以及小管上皮细胞凋亡明显下降。IPC组的HIF-1α核内表达显著高于IR组,且HIF-1下游靶基因VEGF和Glut-1的mRNA表达亦显著增加。结论: 晚期缺血预适应能够显著改善缺血再灌注后肾脏的形态和功能,这种保护作用可能与促进低氧诱导因子高表达有关。     

灯盏花乙素干预缺血再灌注损伤人脐静脉内皮细胞血管内皮生长因子与蛋白激酶Cε的表达

背景：有关灯盏花乙素的研究多集中在神经细胞、神经元及平滑肌细胞上,但对血管内皮细胞的作用研究尚少。 目的：观察灯盏花乙素预处理后对人脐静脉内皮细胞缺血再灌注损伤后血管内皮生长因子与蛋白激酶Cε表达的影响。 方法：体外培养的人脐静脉内皮细胞,分别设立对照组,模型组和及灯盏花乙素高、中、低剂量组。将人脐静脉内皮细胞予缺氧缺糖3 h/复氧糖5或24 h;灯盏花乙素高、中、低剂量组分别加1×10^-5,1×10^-6,1×10^-7 mol/L灯盏花乙素孵育30 min,然后予缺血再灌注处理。实验结束后取细胞裂解液用Western blot检测血管内皮生长因子、蛋白激酶Cε的蛋白表达。 结果与结论：缺血3 h再灌注5及24 h,较对照组,模型组血管内皮生长因子的表达降低,细胞颗粒部分蛋白激酶Cε的表达明显增加。灯盏花乙素能促进缺血3 h再灌注5 h人脐静脉内皮细胞血管内皮生长因子的表达,尤其以灯盏花乙素高剂量和中剂量组明显,但对蛋白激酶Cε的表达没有影响。     

人参皂苷Rb1对大鼠脑缺血再灌注损伤中NAIP表达的影响

目的:观察人参皂苷Rb1对神经元凋亡抑制蛋白(neuronal apoptosis inhibitory protein,NAIP)在脑缺血再灌注损伤中表达的影响,探讨人参皂苷Rb1对缺血性脑病的防治机制.方法:线栓法阻断大鼠大脑中动脉2 h,再灌注3 h～5 d制备脑缺血再灌注模型,应用免疫组织化学方法标记NAIP阳性细胞,观察人参皂苷Rb1对NAIP阳性细胞数的影响.结果:缺血再灌注后,缺血半暗带内的NAIP阳性细胞表达增加,海马CA 1区等部位的NAIP阳性细胞数量也明显增加,而缺血中心区无NAIP阳性细胞的表达.实验对照组中NAIP阳性细胞数量随再灌注时间延长逐渐升高,12 h达到高峰,至5 d时NAIP阳性细胞数量表达低于正常水平.实验用药组中NAIP阳性细胞数量在2 d时达到高峰,且其高峰值明显高于实验对照组中的高峰值,后随再灌注时间的延长而逐渐下降,至5 d时仍处于较高水平.结论:脑缺血再灌注后,NAIP表达增加是脑组织对损伤的一种保护性反应,其对缺血区周围的神经细胞的保护作用更为明显.NAIP在脑组织中的表达具有一定的时间规律性,并且人参皂苷Rb1可能通过上调NAIP的表达发挥对受损脑组织的保护作用.     

高迁移率族蛋白B1在肾脏冷缺血再灌注损伤中的作用

背景：缺血再灌注损伤是临床器官移植不可避免的病理生理过程,冷缺血再灌注损伤具有研究肾移植更强的针对性。 目的：观察高迁移率族蛋白B1在大鼠肾脏冷缺血再灌注损伤中的作用。 方法：将SD大鼠随机分为假手术组、冷缺血再灌注组、丙酮酸乙酯(可显著抑制高迁移率族蛋白B1的合成与释放)治疗组。冷缺血再灌注组和丙酮酸乙酯治疗组制冷缺血再灌注模型前分别经阴茎背静脉注射林格液与丙酮酸乙酯,假手术组将腹腔打开后经阴茎背静脉注射林格液,45 min后关闭腹腔。 结果与结论：冷缺血再灌注组和丙酮酸乙酯治疗组各时间点肌酐、高迁移率族蛋白B1、肿瘤坏死因子α、核因子κB水平均显著高于假手术组(P < 0.01),其中冷缺血再灌注组上述指标高于丙酮酸乙酯治疗组(P < 0.01)。表明高迁移率族蛋白参与了肾移植冷缺血再灌注的病理过程,丙酮酸乙酯能够减轻肾脏冷缺血再灌注损伤。     

Transforming growth factor-β1 promotes homing of bone marrow mesenchymal stem cells in renal ischemia-reperfusion injury

Backgrounds: Acute ischemia reperfusion-induced kidney injury is a common cause of acute renal failure, and it is also an important cause of delayed recovery of transplanted kidney functions and even loss of function. However, there is no effective treatment method in clinical applications presently. Objective: The objective was to investigate effects of transforming growth factor-β1 on homing of bone marrow mesenchymal stem cells in renal ischemia-reperfusion injury. Methods: Effects of TGF-β1 over-expression in MSCs on expression of CXCR4 and chemotactic effect to SDF-1 were investigated by in vitro transmembrane chemotaxis. Anti-TGF-β1 antibody was incubated with ischemia reperfusion injury renal tissue homogenate and effects of anti-TGF-β1 antibody were observed. In addition, effects of TGF-β1 gene transfection and anti-CXCR4 antibody treatment in MSCs on expression of SDF-1/CXCR4 axis of renal tissues and damage repair were further explored. Results: Expression of TGF-β1 mRNA in the IRI group increased significantly, and MSCs transplantation could enhance expression of CXCR4 mRNA in rats of the IRI group, the expression of CXCR4 can be decreased by the anti-TGF-β1 antibody and the anti-CXCR4 antibody. TGF-β1 induced homing of MSCs in repair of renal ischemic reperfusion injury by regulating expression of CXCR4 on cell membranes. Blue fluorescence of DAPI-positive MSCs cells of renal parenchyma in the IRI+MSC group was enhanced significantly, which was significantly inhibited by anti-TGF-β1 and anti-CXCR4 antibody, and the inhibitory effect of anti-CXCR4 antibody was more obvious than that of anti-TGF-β1 antibody. Conclusion: Transforming growth factor-β1 promotes homing of bone marrow mesenchymal stem cells in renal ischemia-reperfusion injury, which will provide useful data on role of TGF-β1 in regulating SDF-1/CXCR4 axis-induced MSCs homing.     

热休克蛋白70在大鼠心肌缺血再灌注过程中的表达及其意义

目的 :探讨热休克蛋白 70 (HSP70 )在心肌缺血再灌注损伤中的表达及其意义。方法 :结扎 /松解SD大鼠左冠状动脉前降支 ,建立大鼠缺血 0min、3 0min ,再灌注 60min、12 0min动物模型。免疫组化技术和图像分析技术检测心肌细胞内HSP70、Bcl 2 /Bax基因的蛋白表达 ,硫代巴比妥酸 (thiobarbituricacid ,TBA)法测心肌组织丙二醛 (MDA)含量 ,优化紫外分光光度法测血清肌酸激酶 (CK)活性。结果 :( 1)HSP70于缺血 0min即有表达 ,再灌注 60min达高峰 ,之后下降。 ( 2 )Bcl 2蛋白再灌注组表达较缺血组明显降低 (P <0 .0 5 )。( 3 )Bax蛋白表达、MDA含量、CK活性于缺血 3 0min开始升高直至再灌注 12 0min。结论 :HSP70的异常表达与心肌缺血再灌注损伤的发生有关 ,且可能是缺血再灌注损伤细胞发展、恶化的重要标志。     

不同预缺血时间对肾小管间质纤维化的影响

目的：在建立缺血所诱发的肾小管间质纤维化大鼠模型基础上，探讨不同预缺血时间对肾脏纤维化的影响。方法:双侧肾蒂夹闭40 min后恢复灌注，制作缺血再灌注损伤模型，8 d前用同样方法分别造成肾脏预缺血10 min、20 min和30 min。预缺血后1 d、4 d、8 d和第2次缺血后5 周收集血样和肾脏标本。Masson特殊染色法观察小管间质纤维化程度；Western blotting法测定α-平滑肌肌动蛋白(α-SMA)、TGF-β1和磷酸化Smad2蛋白表达；免疫组织化学法观察肾脏α-SMA 和TGF-β1的分布。结果:术后5周单纯缺血再灌注组（I/R）和10 min 缺血预处理组（I-10/I）出现了相似程度的小管间质纤维化病变；30 min缺血预处理组（I-30/I）的肾脏纤维化明显加重，并伴有肾脏重量的增加、肾功能的减退和α-SMA、TGF-β1、phospho-Smad2蛋白表达进一步上调；相反，20 min缺血预处理组（I-20/I）的肾脏纤维化显著减轻甚至消失，与假手术对照组（sham）相比上述蛋白的表达无显著差异。结论:长时间的肾脏缺血可以引起小管间质纤维化，提前进行缺血预处理影响此病理改变，其作用结果与预缺血的时间长短密切相关，时间适中的预缺血能保护肾脏免于纤维化结局，其保护机制有待进一步研究。另外，肌成纤维细胞增多（α-SMA阳性）和TGF-β1/Smad信号通路激活可能参与缺血性小管间质纤维化病变过程。     

实验性脑缺血再灌流体感诱发电位变化的研究

目的：研究大鼠脑缺血再灌流体感诱发电位（ＳＥＰ）变化。方法：建立脑缺血再灌流动物模型,随机分成正常对照组９只,缺血和再灌流组２９只,在缺血１ｈ、２ｈ和再灌流１ｈ分别进行神经功能缺损评分,于缺血２ｈ再灌流１ｈ进行ＳＥＰ观察。结果：大鼠脑缺血时,神经功能缺损以轻、中度局灶性损害为主要表现,再灌流后变化不明显,只有１只体征加重。ＳＥＰ检测缺血、再灌流组与正常对照组比较,Ｐ１、Ｎ１峰值潜伏期延长,Ｐ１Ｎ１峰峰波幅降低（Ｐ＜００１）,缺血组与再灌流组比较,Ｐ１、Ｎ１峰值潜伏期、Ｐ１Ｎ１峰峰波幅没有显著差异（Ｐ＞００５）。结论：ＳＥＰ是研究大鼠脑缺血再灌流有效可靠的手段,它能较早反映脑缺血及大脑神经功能的改变,客观反映再灌流后神经功能有无恢复的情况     

Neuroprotective effects of olprinone after cerebral ischemia/reperfusion injury in rats

Olprinone hydrochloride, a specific phosphodiesterase III inhibitor, has anti-inflammatory effects in addition to its inotropic and vasodilatory effects. The purpose of this study was to examine the beneficial effects of olprinone on cerebral ischemia reperfusion injury. In the present study, we examined the detailed mechanisms underlying the inhibitory effects of olprinone on inflammatory and apoptotic responses induced by middle cerebral artery occlusion (MCAo) in rats. Focal cerebral ischemia was induced by transient MCAo in the right hemisphere, via the external carotid artery into the internal carotid to block the origin of the median carotid artery. The rats were subjected to artery occlusion (2 h) followed by reperfusion (22 h). Olprinone was administered 5 min before reperfusion. MCAo-induced cerebral ischemia was associated with an increase in inducible nitric oxide synthase expression, nitrotyrosine formation, as well as IL-1β expression and ICAM-1 expression in ischemic regions. Olprinone treatment showed marked reduction in infarct size compared with control rats. These expressions were markedly inhibited by olprinone treatment. We also demonstrated that olprinone reduces levels of apoptosis (TUNEL, Bax and Bcl-2 expression) resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Based on these findings we propose that olprinone would be useful in lowering the risk of damage or improving function in ischemia-reperfusion brain injury-related disorders.     

红花注射液对兔肺缺血/再灌注损伤时环氧酶基因表达的影响

目的：探讨红花注射液(SI)对肺缺血/再灌注损伤（PIRI）时环氧酶(COX)基因表达的影响。方法: 复制在体兔肺缺血/再灌注损伤模型。30只日本大耳兔，随机均分为3组：假手术组（S组）、缺血/再灌注组(I/R组)和缺血-再灌注+红花注射液组 (SI组)。实验结束时，取肺组织测湿干重比（W/D），计算肺泡损伤率(IAR)，电镜观察细胞超微结构改变。免疫组化法检测肺组织COX-1、COX-2蛋白表达；原位杂交法检测肺组织COX-1mRNA、COX-2mRNA表达的变化。结果：在肺小动脉内（外）膜、肺小静脉内膜、肺泡上皮及细支气管上皮，I/R组COX-2蛋白和COX-2mRNA表达皆显著高于SI组(均P<0.01)，COX-1蛋白和COX-1mRNA表达3组间无明显变化；I/R组和SI组的W/D与IAR均高于S组（P<0.05或P<0.01），但SI组显著低于I/R组（P<0.01）；I/R组肺组织的超微结构损伤严重，SI组损伤程度明显较轻。结论：肺缺血/再灌注可诱导肺组织COX-2的表达，SI可能通过下调肺组织COX-2蛋白及其基因的表达而减轻PIRI。     

Fasudil联合骨髓源神经干细胞对EAE小鼠的神经保护作用

目的:探讨Rho激酶抑制剂法舒地尔(fasudil)联合骨髓源神经干细胞(bone marrow-derived neural stem cells,BM-NSCs)对实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)小鼠的神经保护作用。方法:32只雌性C57BL/6小鼠(8~10周龄),用髓鞘少突胶质细胞糖蛋白35-55(MOG35-55)免疫,制备EAE,随机分为对照(dd H_2O)组、fasudil组、BM-NSCs组和fasudil+BM-NSCs组,并分别给予相应处理。免疫后检测小鼠临床症状和相关神经营养因子的表达,Image-Pro Plus 6.0软件进行阳性细胞计数,Graph Pad Prism 5软件统计分析。结果:与dd H_2O组比较,fasudil+BM-NSCs处理组明显延迟小鼠的平均起病时间,降低最高临床评分,并缓解EAE小鼠的临床症状;fasudil组、BM-NSCs组和fasudil+BM-NSCs组中脑源性神经营养因子、胶质细胞源性神经营养因子、神经生长因子、神经营养因子3和睫状神经营养因子阳性细胞数均有不同程度的增加,其中,fasudil+BM-NSCs组上述神经营养因子的表达明显多于dd H_2O组、fasudil组和BM-NSCs组(P0.01)。结论:Fasudil联合BM-NSCs通过协同和叠加效应促进神经营养因子表达,改善中枢神经系统微环境,发挥神经保护作用,从而缓解EAE的临床症状。     

法舒地尔和RhoA沉默对许旺细胞增殖的影响

背景：许旺细胞在周围神经损伤和修复再生过程中发挥重要作用。 目的：观察Rho激酶抑制剂法舒地尔和RNAi介导的RhoA基因沉默对大鼠许旺细胞增殖的影响。 方法：体外培养Wistar大鼠许旺细胞,分6组干预：对照组,5,10,15,20 μmol/L 法苏地尔组,siRNA沉默RhoA基因组。处理后第3天,RT-PCR、Western blot检测各组许旺细胞RhoA基因和蛋白的表达。连续5 d用细胞计数法测定生长曲线。应用MTT比色法观察许旺细胞增殖情况;采用流式细胞术测定许旺细胞周期分布的变化。 结果与结论：法苏地尔浓度增加到20 μmol/L时对细胞的作用并非随浓度的增加而增强,与15 μmol/L组的差异无显著性意义(P > 0.05)。Rho激酶抑制剂法舒地尔和RNAi介导的RhoA基因沉默在体外实验均能促进许旺细胞,法舒地尔最佳作用浓度为15 μmol/L,沉默RhoA基因效果较好。     

利多卡因对兔骨骼肌缺血-再灌注损伤骨骼肌存活率及超微结构的影响

目的研究利多卡因全身用药对家兔骨骼肌缺血-再灌注损伤骨骼肌存活率及超微结果的影响。方法雄性新西兰兔12只,体重2.5~3kg,随机分为4组,分别记为A1、A2、B1、B2,每组3只。兔右下肢股三角区脱毛,手术显露股血管,切断股神经,无损伤血管夹阻断股动、静脉血流,右大腿中部用止血带捆扎,缺血3小时后松开血管夹和止血带,再灌注3小时。A1组和A2组兔于缺血前5分钟先经耳缘静脉分别推注利多卡因(2mg/Kg)和等体积的生理盐水,后分别持续经耳缘静脉滴注利多卡因(5ug/Kg/min)和等体积的生理盐水;B1组和B2组兔于再灌注前5分钟按照上述方法给药,在缺血0、3小时,再灌注3小时时分别取胫骨前肌、小腿三头肌,MTT法检测肌肉坏死率,取胫骨前肌透射电镜下观察不同时间的肌细胞超微结构的变化。结果与缺血前给予生理盐水组比较,缺血前给予利多卡因能够显著增加兔骨骼肌缺血3小时和再灌3小时时的存活率;与再灌注前给予生理盐水组比较,再灌注前给予利多卡因能够显著增加兔骨骼肌再灌注3小时时的存活率;缺血前给予利多卡因与再灌注前给予利多卡因均能显著增加兔骨骼肌再灌注3小时时的存活率,但是两种给药方式对骨骼肌存活率的影响无显著性差异。结论缺血前给予利多卡因能够显著改善兔的缺血和再灌注损伤,而再灌注前给予利多卡因能够显著改善兔的再灌注损伤。虽然缺血前给予利多卡因与再灌注前给予利多卡因均能改善兔的再灌注损伤,但是这两种给药放式改善骨骼肌再灌注损伤的效果无显著性差异。     

七氟烷对心肌缺血再灌注内皮细胞细胞间黏附分子-1、血管细胞黏附分子-1和E-选择素表达的影响

目的： 探讨七氟烷对心肌缺血再灌注内皮细胞促炎作用的细胞间黏附分子-1（ ICAM-1）、血管细胞黏附 分子-1（ VCAM-1）和E- 选择素表达的影响。方法 ：在大鼠心肌缺血再灌注模型的基础上,将大鼠随机分为假 手术组（ 对照组）、缺血再灌注损伤组（ I/R 组）和七氟烷组;观察各组大鼠手术前、缺血15 min 和再灌注4 h 的 心率、平均动脉压和心率-收缩压乘积（ RPP ）;免疫组织化学法检测心肌组织中CD68+ 巨噬细胞数目、内皮细胞 ICAM-1、VCAM-1 和E- 选择素的表达;TUNEL 染色法检测凋亡细胞的比例。结果：缺血15 min 时,I/R 组和 七氟烷组平均动脉压和RPP 均显著下降;再灌注4 h 时,七氟烷组平均动脉压和RPP 均有所上升,相对于I/R 组, 差异具有统计学意义;与对照组比较,I/R 组内皮细胞ICAM-1、VCAM-1 和E- 选择素的表达均显著升高,七氟 烷则能够有效抑制I/R 引起的内皮细胞促炎分子的表达;对照组CD68+ 巨噬细胞为5.83 个/ 高倍镜视野（ HPF）, I/R 组数目为55.67 个/HPF,两组间差异具有统计学意义;七氟烷能够显著减少心组织内巨噬细胞的浸润,与I/R 组比较,降低了66.46%;TUNEL 染色结果显示对照组心肌细胞凋亡率2.20%,I/R 组为28.63%,两组间差异明显; 七氟烷能够显著降低心肌细胞的凋亡,相对于I/R 组,降低了51.76%。结论：七氟烷可降低缺血再灌注损伤后内 皮细胞表面促炎分子的表达,减少心肌组织巨噬细胞浸润和细胞凋亡。