Expression of Bcl-2, Bax and Caspase-3 in Nerve Tissues of Rats Chronically Exposed to 2,5-hexanedione

Occupational exposure and experimental intoxication with n-hexane or its metabolite 2,5-hexanedione (HD) produce a central-peripheral neuropathy. However, the mechanism remains unknown. We hypothesized that HD affected the expression of Bcl-2, Bax and Caspase-3 in the central nervous system (CNS) and the peripheral nervous system (PNS). Male adult Wistar rats were administered by intraperitoneal injection at a dosage of 200 or 400 mg/kg HD, five days per week for 8 weeks. Samples of the cerebral cortex, cerebellum, spinal cord and sciatic nerves were collected and examined for Bcl-2, Bax and Caspase-3 expression using Western blotting. Subchronic exposure to HD resulted in significantly increased expression of both anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax and Caspase-3 in cerebral cortex and cerebellum, which exhibited a dose-dependent pattern. Though little change was detected in spinal cord, our results showed that the expression of Bcl-2, Bax and Caspase-3 was markedly enhanced in the sciatic nerves. These findings suggested that the changes of apoptosis-related protein level in rat nerve tissues were associated with the intoxication of HD, which might be involved in early molecular regulatory mechanism of apoptosis in the HD-induced neuropathy.     

SP600125对大鼠肺缺血/再灌注损伤的保护作用及机制

目的:探讨SP600125-c-Jun氨基末端激酶(JNK)特异性抑制剂对大鼠肺缺血/再灌注损伤的保护作用及机制。方法:复制在体大鼠原位单肺缺血/再灌注模型,随机分3组(n=10):假手术对照组(Control组)、缺血再灌注组(I/R组)与缺血再灌注+SP600125干预组(SP600125组)。实验结束时取肺组织测湿/干重比(W/D)、肺泡损伤率(IAR);采用蛋白印迹法检测肺组织磷酸化JNK(p-JNK)、JNK蛋白的表达;免疫组化法检测肺组织Bcl-2、Bax、Caspase-3蛋白的表达;原位末端标记法检测肺组织细胞凋亡指数(AI);电镜观察肺组织超微结构的改变。结果:SP600125组肺组织p-JNK、Bax、caspase-3的蛋白表达显著低于I/R组(均P<0.01),Bcl-2的蛋白表达及Bcl-2/Bax的比值显著高于I/R组(均P<0.01),AI、W/D及IAR显著低于I/R组(均P<0.01),肺组织超微结构损伤不同程度减轻。结论:SP600125可能通过抑制JNK信号通路,上调Bcl-2/Bax的比值减少caspase-3依赖性的肺细胞凋亡,从而减轻肺缺血/再灌注损伤。     

Oxysophoridine Protects Against Focal Cerebral Ischemic Injury by Inhibiting Oxidative Stress and Apoptosis in Mice

Our previous studies have demonstrated that oxysophoridine (OSR) has protective effects on cerebral neurons damage in vitro induced by oxygen and glucose deprivation. In this study, we further investigated whether OSR could reduce ischemic cerebral injury in vivo and its possible mechanism. Male Institute of cancer research mice were intraperitoneally injected with OSR (62.5, 125 and 250 mg/kg) for seven successive days, then subjected to brain ischemia induced by the model of middle cerebral artery occlusion. After reperfusion, neurological scores and infarct volume were estimated. Morphological examination of tissues was performed. Apoptotic neurons were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining. Oxidative stress levels were assessed by measurement of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels. The expression of various apoptotic markers as Caspase-3, Bax and Bcl-2 were investigated by immunohistochemistry and Western-blot analysis. OSR pretreatment groups significantly reduced infract volume and neurological deficit scores. OSR decreased the percentage of apoptotic neurons, relieved neuronal morphological damage. Moreover, OSR markedly decreased MDA content, and increased SOD, GSH-Px activities. Administration of OSR (250 mg/kg) significantly suppressed overexpression of Caspase-3 and Bax, and increased Bcl-2 expression. These findings indicate that OSR has a protective effect on focal cerebral ischemic injury through antioxidant and anti-apoptotic mechanisms.     

Smac调控Caspase-3对耐药肺腺癌细胞株生物活性及相关信号的研究

摘要 目的：探讨Smac基因调控Caspase-3表达对紫杉醇耐药肺腺癌细胞株生物活性及经典凋亡信号通路的作用机制。方法：取构建好的耐药A549细胞,将其分为A549细胞（LC）组、A549细胞+Smac-NC（SN）组、A549细胞+Smac抑制剂（SI）组、A549细胞+Smac激动剂（SM）组、A549细胞+Caspase-3-NC（CN）组、A549细胞+Caspase-3抑制剂（CI）组、A549细胞+Caspase-3激动剂（CM）组、A549细胞+Smac激动剂+Caspase-3激动剂（MM）组;Real-time PCR法检测正常肺上皮细胞及4种肺腺癌细胞系中Smac、Caspase-3表达水平,将阴性对照、Smac、Caspase-3类似物转染至紫杉醇耐药肺腺癌细胞株,MTT法检测细胞增殖,流式细胞仪检测细胞凋亡,免疫印迹法检测经典凋亡信号通路表达,并分析Smac与Caspase-3的相关性。结果：肺腺癌细胞系中的Smac、Caspase-3 mRNA表达量显著低于正常肺上皮细胞系BEAS-2B（P＜0.05）,其中A549的Smac、Caspase-3 mRNA值最小（P＜0.05）,因此选取其作为此次实验细胞;LC组与SN组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异（P＞0.05）,与SN组相比,SI组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显降低（P＜0.05）,增殖率、Bcl-2表达明显升高（P＜0.05）,与SI组相比,SM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高（P＜0.05）,增殖率、Bcl-2表达明显降低（P＜0.05）;LC组与CN组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异（P＞0.05）,与CN组相比,CI组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显降低（P＜0.05）,增殖率、Bcl-2表达明显升高（P＜0.05）,与CI组相比,CM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高（P＜0.05）,增殖率、Bcl-2表达明显降低（P＜0.05）;SM组与CM组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异（P＞0.05）,与CM组相比,MM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高（P＜0.05）,增殖率、Bcl-2表达明显降低（P＜0.05）;Smac与Caspase-3呈现正相关（r=0.470,P=0.002）,组间具有显著差异。结论：Smac基因可显著改善紫杉醇耐药肺腺癌细胞株细胞生物活性,并激活经典凋亡信号通路,其作用机制可能与调控Caspase-3表达有关。     

The protective role of verbenalin in rat model of focal cerebral ischemia reperfusion

To investigate the protective mechanism of verbenalin on cerebral ischemia-reperfusion injury. Middle cerebral artery occlusion in the left hemisphere was induced in rats by filament insertion, and rat model of focal cerebral ischemia-reperfusion was established. The high, medium and low dose of verbenalin groups were injected in the tail vein of corresponding drugs 10?min before reperfusion, and submitted for 22?h of reperfusion after the operation. Mortality rate was then calculated, and neurological deficits of rats were scored. The serum of rats was got to determine the S-100β protein level, and the brain tissue was removed to determine the levels of Bax, Bcl-2, Caspase-3 and ATPase. TTC staining was performed on the brain tissue to calculate the percentage of cerebral infarct size. Changes in brain tissue morphology were observed. Rat model of focal cerebral ischemia-reperfusion was successfully replicated. In groups that have taken different doses of verbenalin, the mortality rate, neurological deficit score and the percentage of cerebral infarction size were significantly reduced, and the levels of Bax, Caspase-3, S-100β level of the serum in the brain tissue were also significantly reduced. Increases in the levels of Bcl-2 and ATPase in brain tissue and improvement of pathological damage of hippocampus and cortex were observed. Verbenalin can inhibit the expression of apoptosis genes, promote the expression of anti-apoptosis genes, improve brain microcirculation and energy metabolism, hence reducing cerebral ischemia-reperfusion injury.     

Sevoflurane reduces ischemic brain injury in rats with diet and streptozotocin-induced diabetes

Abstract

To investigate the role of S100B, oxidative stress and the apoptosis signaling pathways in the sevoflurane induced neuroprotective effect on stroke. The brain injury, molecular and cellular damage, and functional recovery were investigated upon ischemic brain injury followed by sevoflurane treatment. Longa rodent stroke scales was used to quantify neurological deficits. TTC staining was used to measure infarct volume of brain tissue. Absolute brain water content was measured by wet/dry weight method. The neuronal morphological change was assessed by H and E staining. The spatial learning and memory ability were measured by water maze test. Serum proteins including S100B, GSH-PX, SOD, Bcl-2, Bax, Caspase-3 were measured by ELISA. The level of NOS and NO in serum was determined by colorimetric method. Compared with control, the serum proteins including S100B, Bax, NO, Caspase-3, and NOS activity in cerebral infarction rats increased significantly while SOD, GSH-PX, and Bcl-2 decreased significantly. Diabetic mellitus complicated with cerebral infarction rats showed more dramatic increase for S100B, Bax, NO, Caspase-3, and NOS activity and dramatic decrease for SOD, GSH-PX, and Bcl-2. Interestingly, sevoflurane reduced the changes significantly. The S100B level positively correlated with brain damage, NO, Bax, caspase-3, and NOS activity but negatively correlated with SOD, Bax, and GSH-PX. Brain damage in sevoflurane groups decreased while behavior outcomes including Longa neurologic score, learning, and memory increased significantly. The neuroprotective effect of sevoflurane is associated with defense mechanisms against free radical-induced oxidative stress and inhibition of apoptosis. S100B protein correlated with oxidative stress and the apoptosis signaling pathways.     

Effects of Panax notoginseng saponins on pneumocyte apoptosis and c-Jun N-terminal kinase in lung ischemia/reperfusion injury

The aim of the present study is to investigate the effects of Panax notoginseng saponins (PNS) on pneumocyte apoptosis and apoptosis-related protein, as well as c-Jun N-terminal kinase (JNK) in lung ischemia/reperfusion (I/R) injury. Thirty Wistar rats were randomly divided into control group, I/R group and PNS group. The unilateral lung I/R model was replicated by obstruction of left lung hilus for 30 min and reperfusion for 120 min in vivo. The rats in PNS group were given intraperitoneal injection of PNS at 60 min before ischemia and 10 min before reperfusion. Some lung tissues sampled at the end of the experiment were assayed for wet/dry weight ratio (W/T). The expressions of phosphorylated JNK (p-JNK) and JNK protein were detected by Western blot. The expressions of Bcl-2, Bax and Caspase-3 protein were detected by immunocytochemistry techniques. The pneumocyte apoptotic index (AI) was detected by terminal deoxynuleotidy1 transferase mediated dUTP nick end labeling (TUNEL). The morphological and ultrastructure changes were observed under light microscope and electron microscope, and the injured alveolus rate (IAR) was counted as well. The results showed that compared to control group, I/R group showed increased expressions of p-JNK, Bcl-2, Bax and Caspase-3 protein (all P < 0.01), decreased ratio of Bcl-2/Bax (P < 0.05), and increased values of AI, W/T and IAR (all P < 0.01). Moreover, light microscope and electron microscope showed serious morphological and ultrastructure injury in I/R group. Compared to I/R group, PNS group showed markedly decreased expressions of p-JNK, Bax and Caspase-3 protein (all P < 0.01), increased expression of Bcl-2 protein and ratio of Bcl-2/Bax (both P < 0.01), and lower values of AI, W/T and IAR (all P < 0.01). Meanwhile, light morphological and ultrastructure injury was found to be alleviated in PNS group. These results suggest that PNS can protect lung tissue from I/R injury, and the mechanism may correlate with suppressing JNK signal pathway, up-regulating the ratio of Bcl-2/Bax which results in inhibition of Caspase-3 dependent apoptosis.     

Germacrone attenuates cerebral ischemia/reperfusion injury in rats via antioxidative and antiapoptotic mechanisms

Germacrone (GM) is an anti-inflammatory compound extracted from Rhizoma curcuma. Here, we strived to investigate the neuroprotective effects of GM in rat models of transient middle cerebral artery occlusion/reperfusion injury. Rats immediately after cerebral ischemia were intraperitoneally injected with GM at doses of 5, 10, and 20 mg/kg. After 1 day of reperfusion, the water content in the brain, infarct volume, and neurological deficits were assessed. Hippocampus neurons were histopathologically examined by hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Activities of glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-PX) in brain tissue were detected. Real-time PCR and Western blotting were utilized to quantify the expression of apoptosis markers, such as caspase-3, Bax, and Bcl-2. The content of phospho-Akt (p-Akt) was also measured using Western blotting. GM treatment markedly decreased the brain water content, infarct volume and the neurological deficits, which was corroborated by attenuated histopathologic change. MDA levels were reduced and activities of GSH, SOD, and GSH-PX were elevated after GM treatment. Caspase-3 and Bax were decreased, and Bcl-2 was increased at both messenger RNA and protein levels by GM treatment. The p-Akt expression was increased by GM. Our data indicated that the neuroprotective effects of GM may attenuate the injuries from cerebral ischemia/reperfusion in rats through antioxidative and antiapoptotic mechanisms.     

镉诱导小鼠睾丸细胞凋亡与Caspase-3、Bcl-2和Bax mRNA的表达

一个月大雄性小鼠24只,随机分为6组,用30 μmol/kg CdCl2作用小鼠睾丸不同的时间(3 h、6 h、12 h、18 h、24 h)后,利用DNA电泳、免疫组化和半定量RT-PCR技术,分析生殖细胞凋亡过程中三种关键物质Caspase-3、Bcl-2、Bax蛋白和mRNA的表达量变化.结果显示:1) DNA各组 (除对照组外)均出现不同程度断裂.2)Caspase-3蛋白表达量一直上升,与对照组相比差异极显著;Bax蛋白在12 h前一直上升,与对照组相比差异显著,12 h后又开始下降,且与对照组相比无显著差异;Bcl-2蛋白在下降,与对照组相比差异显著.3)RT-PCR结果显示Caspase-3基因表达量减少;Bax基因表达量逐渐上升;Bcl-2基因表达量波动很大.综上所述,Caspase-3、Bcl-2和Bax三个基因可能参与了镉应激状态下小鼠睾丸组织细胞的凋亡过程.     

Role of microRNA-195 in cardiomyocyte apoptosis induced by myocardial ischaemia–reperfusion injury

This study aims to investigate microRNA-195 (miR-195) expression in myocardial ischaemia–reperfusion (I/R) injury and the roles of miR-195 in cardiomyocyte apoptosis though targeting Bcl-2. A mouse model of I/R injury was established. MiR-195 expression levels were detected by real-time quantitative PCR (qPCR), and the cardiomyocyte apoptosis was detected by TUNEL assay. After cardiomyocytes isolated from neonatal rats and transfected with miR-195 mimic or inhibitor, the hypoxia/reoxygenation (H/R) injury model was established. Cardiomyocyte apoptosis and mitochondrial membrane potential were evaluated using flow cytometry. Bcl-2 and Bax mRNA expressions were detected by RT-PCR. Bcl-2, Bax and cytochrome c (Cyt-c) protein levels were determined by Western blot. Caspase-3 and caspase-9 activities were assessed by luciferase assay. Compared with the sham group, miR-195 expression levels and rate of cardiomyocyte apoptosis increased significantly in I/R group (both P<0.05). Compared to H/R + negative control (NC) group, rate of cardiomyocyte apoptosis increased in H/R + miR-195 mimic group while decreased in H/R + miR-195 inhibitor group (both P<0.05). MiR-195 knockdown alleviated the loss of mitochondrial membrane potential (P<0.05). MiR-195 overexpression decreased Bcl-2 mRNA and protein expression, increased BaxmRNA and protein expression, Cyt-c protein expression and caspase-3 and caspase-9 activities (all P<0.05). While, downregulated MiR-195 increased Bcl-2 mRNA and protein expression, decreased Bax mRNA and protein expression, Cyt-c protein expression and caspase-3 and caspase-9 activities (all P<0.05). Our study identified that miR-195 expression was upregulated in myocardial I/R injury, and miR-195 overexpression may promote cardiomyocyte apoptosis by targeting Bcl-2 and inducing mitochondrial apoptotic pathway.     

20(R)-Ginsenoside Rg3 protects SH-SY5Y cells against apoptosis induced by oxygen and glucose deprivation/reperfusion

As shown in our previous studies, 20(R)-ginsenoside Rg3 [20(R)-Rg3] exerts a neuroprotective effect on a rat model of transient focal cerebral ischemia, and the mechanism through which it decreases the mRNA expression of calpain I and caspase-3 has been delineated. However, researchers do not know whether 20(R)-Rg3 exhibits a neuroprotective effect following oxygen-glucose deprivation and reperfusion (OGD/R) injury in vitro. In the present study, 20(R)-Rg3 increased cell viability, decreased the LDH leakage rate, and inhibited the apoptosis rate in a concentration-dependent manner. In addition, 20(R)-Rg3 markedly decreased cleaved caspase-3 protein expression. Furthermore, 20(R)-Rg3 significantly decreased the Bax mRNA and protein levels and increased the levels of Bcl-2 mRNA and protein, subsequently decreasing the Bax/Bcl-2 protein ratio. Based on these findings, 20(R)-Rg3 exerts a neuroprotective effect against OGD/R-induced apoptosis.     

绞股蓝总皂苷对光老化人皮肤成纤维细胞凋亡及Caspase-3信号通路的影响

目的:探讨绞股蓝总皂苷(Gyp)对光老化人皮肤成纤维细胞(HSF细胞)凋亡以及Caspase-3信号通路的影响。方法:分别以80、160、320 mg/d剂量的Gyp生理盐水溶液灌胃大鼠7d后取血并分离血清,长波紫外线(UVA)照射方法(照射剂量36 J/cm3)构建光老化HSF细胞模型以得到低剂量组、中剂量组、高剂量组,同时以空白对照组(未照射细胞)、UVA模型组、正常组为对照。UVA诱导的细胞活性氧表达采用二氯荧光素(DCF)法测定,细胞凋亡情况采用TUNEL法测定,HSF细胞活性采用四甲基偶氮唑盐微量酶反应比色法(MTT法)测定,Bax、Bcl-2、Caspase-3基因和蛋白表达分别采用反转录-聚合酶链反应(RT-PCR)和Western-blotting方法进行测定。结果:与空白对照组比较,其余5组的OD值、HSF细胞凋亡数、活性氧(平均荧光强度)、活性氧水平、Bax、Bcl-2、Caspase-3 mRNA及蛋白表达水平差异具有统计学意义(P0.05);与UVA模型组和正常组比较,低、中、高剂量组OD值、HSF细胞凋亡数、活性氧(平均荧光强度)、活性氧水平、Bax、Bcl-2、Caspase-3 mRNA及蛋白表达水平差异具有统计学意义(P0.05);低、中、高剂量组随着剂量增加OD值、Bcl-2mRNA和蛋白表达水平逐渐升高,细胞凋亡数、活性氧(平均荧光强度)、活性氧水平、Bax、Caspase-3 mRNA和蛋白表达水平逐渐降低(P0.05)。结论:Gyp通过抑制细胞内活性氧的产生以及Bax的表达,以及激活Bcl-2、Caspase-3信号通路而逆转UVA诱导的HSF细胞凋亡,进而延缓HSF细胞的光老化现象。     

下调TLR4对LPS诱导的支气管上皮16HBE细胞损伤的影响及其机制

目的探讨TLR4对脂多糖（LPS）诱导的支气管上皮16HBE细胞损伤的影响及其机制。 方法将3条siRNA-TLR4-1、siRNA-TLR4-2和siRNA-TLR4-3转染至16HBE细胞中,筛选出最佳的siRNA-TLR4干扰序列进行实验。实验分为对照组（未处理）、LPS组（给予50 μg/ml LPS处理）、LPS+siNC组（转染siRNA-NC后给予50 μg/ml LPS处理）和LPS+siTLR4组（分别转染设计的3条siRNA-TLR4后给予50 μg/ml LPS处理）,采用RT-PCR检测TLR4、IL-6和TNF-α mRNA的表达,MTT法检测细胞活力,流式细胞仪检测细胞凋亡率,Western Blot检测Bcl-2、Bax、Cleaved Caspase-3、NF-κB p65和IκBα蛋白的表达。多组间比较使用单因素方差分析,组间多重比较采用SNK-q,两组间比较采用独立样本t检验。 结果LPS组与LPS+siTLR4-2组TLR4 mRNA（2.05±0.12,3.28±0.15）和蛋白表达（0.38±0.03,0.77±0.05）比较,差异具有统计学意义（t?= 11.091,11.585,P均< 0.001）;LPS组与LPS+siTLR4-3组TLR4 mRNA（1.39±0.09,3.28±0.15）和蛋白表达（0.31±0.02,0.77±0.05）比较,差异具有统计学意义（t?= 20.857,12.650,P均?< 0.001）且siRNA-TLR4-3的效果最为显著。与对照组相比,LPS组、LPS+siNC组和LPS+siTLR4组细胞中IL-6 mRNA（11.42±1.05,11.38±1.34,6.22±0.35,0.97±0.06,F?= 98.803,P均< 0.01）、TNF-α mRNA（15.76±1.12,15.69±0.87,7.54±0.41,1.03±0.09,F?= 278.064,P均< 0.01）、Cleaved Caspase-3蛋白（0.75±0.06,0.77±0.05,0.38±0.03,0.13±0.02,F?= 154.851,P均< 0.01）、Bax蛋白（0.48±0.05,0.52±0.05,0.33±0.02,0.11±0.02,F?= 71.310,P均< 0.01）、NF-κBp65蛋白（0.64±0.05,0.67±0.05,0.45±0.04,0.28±0.02,F?= 56.571,P?均?< 0.01）的表达水平和细胞凋亡率[（21.36±2.85）﹪,（20.94±3.22）﹪,（13.08±1.16）?﹪,（7.25±1.28）﹪,F?= 25.685,P均< 0.01]均明显升高,而细胞活力（0.53±0.04,0.51±0.04,0.78±0.06,1.15±0.08,F?= 80.811,P均< 0.01）和Bcl-2蛋白（0.28±0.03,0.25±0.03,0.40±0.03,0.69±0.06,F?= 76.762,P均< 0.01）、IκBα蛋白（0.38±0.03,0.35±0.03,0.44±0.03,0.72±0.06,F?= 53.635,P均< 0.01）的表达水平均明显降低;与LPS组相比,LPS+siTLR4组中IL-6 mRNA、TNF-α mRNA、Cleaved Caspase-3蛋白、Bax蛋白、NF-κBp65蛋白的表达水平和细胞凋亡率均明显降低,差异有统计学意义（t = 8.138,11.937,9.553,4.825,5.140,4.661,P均< 0.01）,而细胞活力和Bcl-2蛋白、IκBα蛋白的表达水平均明显升高,差异有统计学意义（t = 6.005,4.899,3.674,P < 0.05）,而LPS组和LPS+siNC组间差异无统计学意义（P > 0.05）。 结论下调TLR4可通过抑制NF-κB通路激活抑制LPS诱导的细胞凋亡和炎症反应减轻16HBE细胞损伤。     

通窍明目IV号对青光眼模型大鼠视神经保护的机制研究

目的:观察不同浓度通窍明目Ⅳ号对青光眼模型大鼠凋亡相关蛋白表达的干预作用。方法:将42只SD大鼠随机分为空白组,模型组,通窍明目4号低、中、高剂量组和神经营养剂组,共计6组,每组7只。经眼球前房角膜缘注射卡波姆复制高眼压模型,成模21天后灌胃给药,给药期4周。采用HE染色观察视网膜形态及视神经节胞、免疫组化法测定BCL-2、Bax、P53蛋白表达,用Western Blot法测定Caspase-3、Caspase-9蛋白表达。结果:与空白组对比,模型组视网膜Bax与p53阳性表达显著升高(P<0.01);与模型组比较,神经营养剂组与通窍明目Ⅳ号高、中剂量组视网膜Bax与p53蛋白的阳性表达显著降低(P<0.01)。与空白组比较,模型组视网膜Bcl-2阳性表达显著降低(P<0.01);与模型组比较,神经营养剂组与通窍明目Ⅳ号各剂量组视网膜Bcl-2的阳性表达有不同程度的提高。模型组大鼠caspase-3、caspase-9的蛋白表达水平均高于空白组(P<0.01);与模型组比较,神经营养剂组与通窍明目Ⅳ号各剂量组大鼠视网膜Caspase3及caspase-9表达水平明显降低(P<0.01)。结论:通窍明目Ⅳ号可能通过下调Bax、P53表达,促进Bcl-2表达升高、抑制Caspase-3、Caspase-9激活凋亡途径,从而对青光眼视神经起到保护作用。     

下肢骨骼肌缺血后处理对兔缺血/再灌注心肌坏死和凋亡的影响

目的:探讨在体情况下,骨骼肌缺血后处理对兔缺血/再灌注心肌坏死和凋亡的影响。方法:新西兰大白兔36只,随机分成3组(每组随机选取6只进行梗死范围的测定,另外6只进行凋亡测定):①假手术组(Sham组);②缺血/再灌注组(I/R组);③远端后处理组(RPostC组)。在缺血前、后及再灌注60 min、120 min分别抽血测定肌酸激酶(CK),乳酸脱氢酶(LDH)的活性。采用伊文思兰(evans blue)和三苯基氯化四氮唑(TTC)染色方法确定心肌缺血区范围以及心肌坏死区范围。用Tunel法检测兔心肌缺血区细胞凋亡情况,免疫组织化学方法检测心肌缺血区蛋白caspase-3、Bcl-2及Bax的表达。结果:RPostC组心肌坏死程度、再灌注末CK活性较I/R组明显减低。RPostC组缺血区心肌Tunel阳性指数显著低于I/R组(21.79%±1.07%vs35.81%±1.10%,P<0.05)。而RPostC组缺血区心肌细胞caspase-3阳性指数显著低于I/R组(25.03%±1.16%vs39%±2.43%,P<0.05)。与Sham组比较,I/R组及RPostC组Bax蛋白表达指数、Bcl-2蛋白表达指数均升高;但RPostC组的Bax/Bcl-2比值降低,而I/R组的Bax/Bcl-2比值升高。与I/R组相比较,RPostC组Bax蛋白表达指数及Bax/Bcl-2比值显著降低,Bcl-2表达指数显著升高,差异均有统计学意义。结论:远端后处理能够明显的减少缺血/再灌注心肌细胞的坏死和凋亡,其减轻心肌细胞凋亡的机制可能与抑制促凋亡基因caspase-3的活化及Bcl-2表达的上调有关。     

Engeletin alleviates cerebral ischemia reperfusion-induced neuroinflammation via the HMGB1/TLR4/NF-κB network

High-mobility group box1 (HMGB1) induces inflammatory injury, and emerging reports suggest that it is critical for brain ischemia reperfusion. Engeletin, a natural Smilax glabra rhizomilax derivative, is reported to possess anti-inflammatory activity. Herein, we examined the mechanism of engeletin-mediated neuroprotection in rats having transient middle cerebral artery occlusion (tMCAO) against cerebral ischemia reperfusion injury. Male SD rats were induced using a 1.5 h tMCAO, following by reperfusion for 22.5 h. Engeletin (15, 30 or 60 mg/kg) was intravenously administered immediately following 0.5 h of ischemia. Based on our results, engeletin, in a dose-dependent fashion, reduced neurological deficits, infarct size, histopathological alterations, brain edema and inflammatory factors, namely, circulating IL-1β, TNF-α, IL-6 and IFN-γ. Furthermore, engeletin treatment markedly reduced neuronal apoptosis, which, in turn, elevated Bcl-2 protein levels, while suppressing Bax and Cleaved Caspase-3 protein levels. Meanwhile, engeletin significantly reduces overall expressions of HMGB1, TLR4, and NF-κB and attenuated nuclear transfer of nuclear factor kappa B (NF-κB) p65 in ischemic cortical tissue. In conclusion, engeletin strongly prevents focal cerebral ischemia via suppression of the HMGB1/TLR4/NF-κB inflammatory network.     

人参归脾丸对脾不统血证大鼠肝损伤的保护作用及其机制*

目的:观察人参归脾丸对脾不统血证大鼠的治疗作用及其对肝脏的影响。方法:40只SPF级雄性SD大鼠随机分为正常对照组(n=10)和实验组(n=30),前42 d,实验组每日游泳30 min,同时饮食一天禁食两天(饮食失节),第43～72日,在游泳力竭加饮食失节的基础上,每日皮下注射低分子肝素钙诱导出血,构建脾不统血证大鼠模型;将成功建立模型的30只大鼠随机分为3组(n=10):模型对照组(MD)、自然复健组(NR)、归脾丸组(GP)。第73～102日,模型组继续施加处理因素(游泳力竭、饮食失节及注射低分子肝素钙溶液),自然复健组和归脾丸组停止施加处理因素,归脾丸组每日灌胃人参归脾丸溶液(0.2 g/ml ,10 ml/(kg·d)),自然复健组灌胃等量生理盐水;第103日后,取血和肝脏,检测血清中各组大鼠丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、总胆红素(TBil)、总蛋白(TP)水平,检测血中红细胞(RBC)数量、血红蛋白(HGB)含量和红细胞压积(HCT),ELISA试剂盒检测大鼠血清中 IL-6和TNF-α 含量,Western blot检测各组大鼠肝组织细胞凋亡相关蛋白Bcl-2、Bax、Caspase3蛋白水平,荧光定量PCR法检测大鼠肝组织细胞凋亡相关蛋白Bcl-2、Bax、Caspase3 mRNA表达水平。结果:与正常对照组相比,模型组大鼠血清ALT、AST和TBil水平均显著升高,TP水平明显降低(P＜ 0.01),血RBC数量、HGB含量和HCT均明显降低(P＜0.01),血清IL-6和TNF-α含量均显著升高(P＜0.05),肝组织Bcl-2 蛋白及mRNA水平均明显降低、Bax和Caspase3 蛋白及mRNA水平均显著升高(P＜0.01);与模型组相比,自然复健组和归脾丸组大鼠血清ALT、AST和TBil水平均明显降低,TP水平显著升高(P＜0.01),血RBC数量、HGB含量和HCT均明显升高(P＜0.01),血清IL-6和TNF-α含量均明显降低(P＜0.05),Bcl-2 蛋白及mRNA水平升高,Bax和Caspase3 蛋白及mRNA 水平降低(P＜0.05);与自然复健组相比,归脾丸组血清ALT、AST和TBil均明显降低,TP含量明显升高(P＜0.01),血RBC数量明显升高(P＜0.05),肝脏Bcl-2蛋白和mRNA水平显著升高(P＜ 0.05),Caspase3蛋白水平明显降低(P＜0.05),Bax mRNA表达显著降低(P＜0.05)。结论:人参归脾丸对脾不统血证大鼠肝损伤具有保护作用,其机制可能与其抑制肝脏细胞凋亡、促炎细胞因子释放有关。     

Cold-inducible RNA-binding protein mediates neuroinflammation in cerebral ischemia

Background

Neuroinflammation is a key cascade after cerebral ischemia. Excessive production of proinflammatory mediators in ischemia exacerbates brain injury. Cold-inducible RNA-binding protein (CIRP) is a newly discovered proinflammatory mediator that can be released into the circulation during hemorrhage or septic shock. Here, we examine the involvement of CIRP in brain injury during ischemic stroke.

Methods

Stroke was induced by middle cerebral artery occlusion (MCAO). In vitro hypoxia was conducted in a hypoxia chamber containing 1% oxygen. CIRP and tumor necrosis factor-α (TNF-α) levels were assessed by RT-PCR and Western blot analysis.

Results

CIRP is elevated along with an upregulation of TNF-α expression in mouse brain after MCAO. In CIRP-deficient mice, the brain infarct volume, induction of TNF-α, and activation of microglia are markedly reduced after MCAO. Using microglial BV2 cells, we demonstrate that hypoxia induces the expression, translocation, and release of CIRP, which is associated with an increase of TNF-α levels. Addition of recombinant murine (rm) CIRP directly induces TNF-α release from BV2 cells and such induction is inhibited by neutralizing antisera to CIRP. Moreover, rmCIRP activates the NF-κB signaling pathway in BV2 cells. The conditioned medium from BV2 cells exposed to hypoxia triggers the apoptotic cascade by increasing caspase activity and decreasing Bcl-2 expression in neural SH-SY5Y cells, which is inhibited by antisera to CIRP.

Conclusion

Extracellular CIRP is a detrimental factor in stimulating inflammation to cause neuronal damage in cerebral ischemia.

General significance

Development of an anti-CIRP therapy may benefit patients with brain ischemia.