Changes in the localization of ileal P-glycoprotein induced by intestinal ischemia/reperfusion

P-glycoprotein is one of the most important transporters in the ATP binding cassette transporter. Moreover, it is well known that the efficacy of immunosuppressants, which are used after organ transplantation, is controlled by P-glycoprotein (P-gp). We investigated how ischemia/reperfusion (I/R), which occurs after transplantation, influences the expression level and function of P-gp. To clarify the influence of intestinal I/R on the localization of P-gp, an intestinal ischemia model was produced using a spring scale and surgical sutures for 1 h, followed by reperfusion for 24 h. The expression levels of mRNA and protein of P-gp were examined. The protein expression levels of P-gp in ileal homogenate and the brush border membrane (BBM) were significantly decreased until 3 h after reperfusion. While the protein expression level of P-gp in homogenate showed a tendency to increase, that in the BBM continued to significantly decrease until 24 h after reperfusion. In contrast, the protein expression level of P-gp in the basolateral membrane (BLM) increased significantly until 24 h after reperfusion. While no significant change in multidrug resistance (mdr)-1a mRNA was found, the levels of mdr-1b and mdr-2 significantly increased during intestinal I/R. In addition, the levels of inflammatory cytokines mRNA and nitric oxide (NO) also significantly increased. It was shown that mdr-1b and mdr-2 mRNA strongly participate in the recovery of P-gp protein level after intestinal I/R. We detected the abnormal localization of P-gp in the ileal membrane during intestinal I/R, suggesting NO and/or inflammatory cytokines participate in the abnormal localization of P-gp.     

Toxicological effects of nickel chloride on the cytokine mRNA expression and protein levels in intestinal mucosal immunity of broilers

The purpose of this study was to examine the toxicological effects of nickel chloride (NiCl₂; 300, 600, and 900 mg kg^?1 diet) on the cytokine mRNA expression and protein levels in the intestinal mucosa and cecal tonsil, and on the ileac and cecal tonsil T cells in broilers by the methods of qRT‐PCR, flow cytometry and ELISA for 42 days. Results showed that the IL‐2, IL‐6, IL‐10, IL‐17, IFN‐γ, and TNF‐α (LITAF) cytokine mRNA expression and protein levels were lower (P < 0.05 or P < 0.01) and the percentages of T‐cell subsets were also lower in the 300, 600, and 900 mg kg^?1 groups than in the control group. It was concluded that dietary NiCl₂ in excess of 300 mg kg^?1 could reduce cytokine mRNA expression and protein levels in the intestinal mucosa and cecal tonsil, and the percentages of ileac and cecal tonsil T‐cell subsets. Decreasing in cytokine mRNA expression and protein levels of intestinal mucosa and cecal tonsil induced by NiCl₂ was closely related to the reduction of T‐cell population. Thus, the abnormal expression of these cytokines impacts the intestinal mucosal immune function by the pathways of reducing of lymphocyte population and activation. Also, this study first proved that NiCl₂ at higher levels has the toxicological effects on intestinal mucosal immunity. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1309–1321, 2015.     

Absorption and metabolism of albendazole after intestinal ischemia/reperfusion.

Pathophysiological processes involving inflammatory response may affect absorption and biotransformation of some drugs, modifying their pharmacokinetic behaviour. Ischemia/reperfusion (I/R) injury has been used as a model for inflammatory processes. The aim of this work was to study the effect of intestinal I/R injury on the absorption and metabolism processes of one orally administered drug, albendazole that is anthelmintic drug, it undergoes intestinal bioconversion into albendazole sulfoxide by two enzymatic systems, cytochromes P450 (CYP450) and flavin-containing monooxygenase (FMO). Male Wistar rats were used to study the influence of I/R in the intestinal absorption and metabolism of albendazole, after 60 min of mesenteric occlusion and 30 min of reperfusion. The intestinal studies were performed in microsomal, and everted ring incubations. During in situ studies, the I/R group had faster disappearance of albendazole from the lumen. In addition, albendazole only appeared in blood samples of the I/R group, while albendazole sulfoxide appeared in both samples and was higher in the control group. These findings are supported by significant reductions of albendazole sulfoxide formation in intestinal everted ring assays and in microsomal incubations after the I/R process. Both metabolizing systems, CYP4503A and FMO, were affected by I/R. Our data indicate that I/R injury, considered as an inflammatory model, reduces absorption and metabolism processes of albendazole.     

大鼠局灶性脑缺血/再灌注后NF-kB表达和细胞凋亡

目的:研究脑缺血再灌注后大鼠脑组织半暗区核因子(nuclear factor kappa B,NF-kB)基因表达的变化与神经细胞凋亡的关系。方法:用插线法制作大鼠大脑中动脉(M CAO)栓塞/再灌注模型,原位杂交法检测大鼠脑重度缺血(3h)及再灌注损伤不同时间点(2、3d)半暗区NF-kB表达;TUNEL法测定凋亡细胞。结果:脑缺血再灌注后,缺血半暗区NF-kB的表达明显升高(P<0.01)。凋亡细胞数量亦显著增加(P<0.01)。细胞凋亡的时程变化与NF-kB的表达基本一致。结论:局灶性重度脑缺血再灌注后可引起NF-K b表达的增加,参与缺血再灌注神经细胞损伤机制。     

Effects of nitric oxide on mucosal barrier dysfunction during early phase of intestinal ischemia/reperfusion

Ischemia/reperfusion (I/R) injury must be overcome for successful small intestinal transplantation. During intestinal I/R, the expression level of nitric oxide (NO) is increased, and vermiculation of the mucosal tract is induced by NO. Although NO has many beneficial effects on intestinal I/R injury, its role in intestinal I/R injury is controversial. Therefore, in the present study, we examined changes in the tight junctions (TJ) and P-glycoprotein (P-gp) by aminoguanidine (AG), which can be considered a selective inducible NO synthase inhibitor during intestinal I/R, to clarify the effect of NO on mucosal barrier dysfunction during intestinal I/R. A mucosal lesion was induced by intestinal I/R. The protein expression levels of the claudin family organizing TJ and P-gp, were decreased, and their functions were also decreased. Through the inhibition of NO generation by AG in the above mucosal lesion, TJ and P-gp dysfunction was significantly inhibited. NO participated in opening TJ and decreasing P-gp function and expression induced during intestinal I/R. Therefore, it is important to consider the level of NO generation in the ileal mucosa in drug therapy for intestinal I/R injury.     

Attenuation of contractile dysfunction by atorvastatin after intestinal ischemia reperfusion injury in rats

Growing number of studies implicate that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, have beneficial effects on ischemia/reperfusion injury that are unrelated to their cholesterol-lowering action. In the present study, we aimed to evaluate possible effects of atorvastatin on oxidative stress, neutrophil accumulation, and contractile response of terminal ileum segments in rats subjected to intestinal ischemia/reperfusion. Intestinal ischemia/reperfusion model was generated by clamping the superior mesenteric artery for 30 min followed by reperfusion for 3 h. Oral administration of atorvastatin at a dose of 10 mg/kg/day lasted 3 days just before induction of intestinal ischemia. At the end of reperfusion period, terminal ileum samples were removed to determine the concentrations of malondialdehyde, reduced glutathione, and myeloperoxidase. Samples were collected also to assess histopathological alterations and contractile response to agonists. Ischemia/reperfusion significantly decreased contractile responses, and this decrease was attenuated by atorvastatin. Pretreatment with atorvastatin caused remarkable decrease in both oxidative stress and neutrophil accumulation. Atorvastatin appeared to be restoring amount of reduced glutathione back to about control level. Furthermore, the pretreatment lowered mucosal damage at histopathological level. Our results suggested that pretreatment with atorvastatin attenuated intestinal muscle dysfunction associated with ischemia/reperfusion. This remarkable effect of atorvastatin is accomplished at least by decreasing oxidative stress and neutrophil accumulation as well as preventing the depletion of reduced glutathione.     

Melatonin alleviates intestinal injury,neuroinflammation and cognitive dysfunction caused by intestinal ischemia/reperfusion

Intestinal ischemia/reperfusion (I/R) can cause multiple organ damage with extremely high morbidity and mortality. Melatonin has anti-inflammatory, anti-oxidative and anti-apoptotic effects against various diseases. This study aimed to explore whether melatonin had a protective effect against intestinal I/R-induced neuroinflammation and cognitive dysfunction, and investigate its potential mechanisms. In this study, melatonin was administered to the rats with intestinal I/R, then histological changes in intestine and brain (frontal cortex and hippocampal CA1 area) tissues and cognitive function were detected, respectively. The encephaledema and blood–brain barrier (BBB) permeability were observed. Moreover, the alterations of proinflammatory factors (tumor necrosis factor-α, interleukin-6 and interleukin-1β), oxidative response (malondialdehyde, superoxide dismutase, and reactive oxygen species), apoptosis and proteins associated with inflammation, including Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (Myd88) and phosphorylated nuclear factor kappa beta (NF-κB), and apoptosis (cleaved caspase-3) in brain tissues were examined. Furthermore, the expressions of TLR4, Myd88, and microglial activity were observed by multiple immunofluorescence staining. The results showed that intestinal I/R-induced abnormal neurobehavior and cerebral damage were ameliorated after melatonin treatment, which were demonstrated by improved cognitive dysfunction and aggravated histology. Furthermore, melatonin decreased the levels of proinflammatory factors and oxidative stress in plasma, intestine and brain tissues, attenuated apoptotic cell, and inhibited the expressions of related proteins and the immunoreactivity of TLR4 or Myd88 in microglia in brain tissues. These findings showed that melatonin might relieve neuroinflammation and cognitive dysfunction caused by intestinal I/R, which could be, at least partially, related to the inhibition of the TLR4/Myd88 signaling in microglia.     

Changes and mechanisms of protein-tyrosine kinase and protein-tyrosine phosphatase activities after brain ischemia/reperfusion

AIM: To study the changes and mechanisms of protein-tyrosine kinase (PTK) and protein-tyrosine phosphatase (PTP) activities in the hippocamal synaptosome following cerebral ischemia/reperfusion (I/R) in gerbil. METHODS: Transient (15 min) global ischemia was produced by bilateral carotid artery occlusion. Total PTK and PTP activities were measured by [r-32P] incorporation and colorimetric analysis, respectively. Src and proline-rich tyrosine kinase2 (PYK2) activities were measured by immunoprecipitation and [r-32P] incorporation. RESULTS: Total PTK activity increased significantly after I/R, but the PTP activity did not change. The Src activity was much higher than PYK2 activity in sham-operated controls. I/R mainly caused a pronounced increase in Src activity, but not PYK2 activity. The increase in Src activity had no relation to the expression of Src protein. Administration of ketamine (KT) or nifedipine (ND) 20 min before ischemia caused a decrease in total PTK and Src activities, and no change in the PYK2 and PTP activities. CONCLUSION: The increase in PTK activity caused by I/R may be mainly due to the increase in Src activity. This increase in Src activity has no relation to the expression of Src protein. But it is related to the activation of NMDA (N-methyl-D-aspartate) receptor (NR) and L-type voltage-gated calcium channel (L-type VGCC). In other words, the increase in total PTK and Src activities induced by I/R may be mediated via NR and L-type VGCC. The PTP activity did not change during I/R.     

雷公藤内酯醇抑制大鼠脑局灶性缺血再灌注后脑组织IL-1βmRNA表达的研究

目的　观察雷公藤内酯醇 (Triptolide ,TL)对局灶性脑缺血再灌注大鼠脑组织内IL 1βmRNA水平变化的影响 ,探讨雷公藤内酯醇对大鼠局灶性缺血再灌注脑损伤保护作用的机制。方法　建立大鼠局灶性脑缺血再灌注模型 ,观察应用雷公藤内酯醇 (0 2和 0 4mg·kg-1)对大鼠脑组织微血管内附壁中性粒细胞计数 ,以及神经功能受损程度的影响。应用RT PCR技术 ,检测大中动脉闭塞侧脑组织IL 1βmRNA表达水平的变化情况。结果　与对照组比较 ,雷公藤内酯醇两治疗组大脑中动脉闭塞侧微血管内附壁中性粒细胞计数、脑组织IL 1βmRNA水平均明显下降。神经功能受损程度也明显改善。结论　雷公藤内酯醇具有明显抑制缺血再灌注大鼠脑组织内IL 1βmRNA表达、降低IL 1β含量和抑制白细胞浸润的作用。这可能是雷公藤内酯醇对大鼠缺血再灌注脑损伤保护作用的机制之一     

Leptin attenuates cerebral ischemia/reperfusion injury partially by CGRP expression

Ischemic stroke is a medical emergency triggered by a rapid reduction in blood supply to localized portions of the brain, usually because of thrombosis or embolism, which leads to neuronal dysfunction and death in the affected brain areas. Leptin is generally considered to be a strong and quick stress mediator after injuries. However, whether and how peripherally administered leptin performs neuroprotective potency in cerebral stroke has not been fully investigated. It has been reported that CGRP(8-37), an antagonist of the CGRP receptor, could reverse the protective effect of leptin on rats with CIP (caerulein-induced pancreatitis). However, the question remains: are leptin and CGRP associated in cerebral ischemia/reperfusion injury? The present study attempted to evaluate the relationship between CGRP expression and leptin neuroprotective effects (1mg/kg in 200 μL normal saline, i.p.) on focal cerebral ischemia/reperfusion injury in mice and the protective effect of leptin (500 μg/L) on neurons during hypoxia/reoxygenation injury. Peripheral administration of leptin alleviated injury-evoked brain damage by promoting CGRP expression, improving regional cerebral blood flow, and reducing local infarct volume and neurological deficits. Furthermore, leptin also promoted bcl-2 expression and suppressed caspase-3 in vivo and vitro after injury. Administration of CGRP(8-37) (4 × 10(-8)mol/L) partly abolished the beneficial effects of leptin, and restored the normal expression levels of bcl-2 and caspase-3 in neurons, which indicated that leptin-induced protection of neurons was correlated with release of CGRP. These results indicate that the neuroprotective effect of leptin against cerebral ischemia/reperfusion injury may be strongly relevant to the increase of CGRP expression.     

七氟醚预处理通过升高血浆脂联素水平保护心肌缺血/再灌注损伤

目的探讨脂联素是否参与七氟醚预处理对心肌缺血/再灌注(myocardial ischemia-reperfusion,MI/R)损伤的保护作用。方法将C57小鼠随机分为4组,每组10只:Sham-Control(假手术组)、MI/R-control(模型组)、MI/R-Sevopre(预处理组)、APN-KO-MI/R-Sevopre(敲基因预处理组),MI/R造模术后24 h检测各组小鼠血浆脂联素水平;检测各组小鼠24 h左心室收缩末期内径,舒张末期内径以及射血分数;Tunel染色观察各组心肌细胞形态和凋亡情况;伊文思蓝-TTC染色观察心梗面积。结果相比于假手术组,模型组心功能受损,射血分数降低,舒张末期内径以及收缩末期内径增加,同时,模型组心肌梗死面积显著增加,凋亡细胞增多,并且血浆脂连素水平降低;相比于模型组,七氟醚预处理组小鼠心功能改善,心肌梗死面积降低,凋亡细胞减少,血浆脂连素水平升高;敲基因预处理组小鼠相比于预处理组,七氟醚预处理改善MI/R损伤作用降低。结论七氟醚预处理通过升高血浆脂连素水平保护MI/R损伤。     

茶多酚对大鼠肠缺血再灌注肠损伤的保护作用

目的:研究茶多酚(TP)对大鼠肠缺血再灌注(I/R)所致肠损伤的保护作用及其可能的作用机制,为其临床新用途提供实验依据。方法:大鼠随机分为肠I/R损伤对照组、假手术组及TP给药组(100,50,25和12.5 mg.kg-1),通过夹闭肠系膜上动脉1 h、再灌注2 h,建立肠I/R损伤模型。于缺血前20 m in舌下静脉注射药物,假手术组仅分离、不夹闭肠系膜上动脉。再灌2 h后,各组取血及中段小肠组织,测定血清及小肠组织中超氧化物歧化酶(SOD)、丙二醛(MDA)和一氧化氮(NO)含量,光镜下观察小肠组织形态学改变。结果:与假手术组相比,肠I/R损伤对照组血清及小肠组织中的SOD活力降低,MDA和NO含量升高,镜检发现小肠有明显组织形态学损伤。与肠I/R损伤对照组相比,TP组呈剂量依赖性增强SOD活力,减少MDA及NO含量,减轻小肠组织形态学损伤。结论:TP对肠I/R所致肠急性损伤有显著的及剂量依赖性的保护作用,可能与其自由基清除作用有关。     

血管内皮生长因子165通过抑制钙敏感性受体减少缺血/再灌注心肌细胞凋亡

目的:探讨血管内皮生长因子165(vascu-lar endothelial growth factor 165,VEGF165)在缺血/再灌注损伤中的抗细胞凋亡作用及与钙敏感性受体(calciumsensing receptor,CaSR)表达下调的关系。方法:新生鼠心肌细胞在模拟心肌缺血状态下孵育2 h,然后在标准培养液中再培养24 h,从而建立一个模拟心肌缺血/再灌注模型。通过末端脱氧核苷酰基转移酶介导性dUTP切口末端标记(TUNEL法)检测心肌细胞凋亡。CaSR mRNA表达通过逆转录聚合酶链反应(RT-PCR)测定。通过免疫印迹法(Westernblot)测定促凋亡蛋白Bax、抗凋亡蛋白Bcl-2含量。结果:模拟的缺血/再灌注后CaSR mRNA的表达(I/R组:2.6±0.4;对照组:1.0±0.3,P<0.01)和TUNEL阳性细胞增加(I/R组:15.3%±2.5%;对照组:2.9%±1.4%,P<0.01)。GdCl3、CaSR mRNA的表达(GdCl3组:4.5±0.6;I/R组:2.6±0.4,P<0.01)及TUNEL阳性细胞进一步增加(GdCl3组:25.4%±2.6%;I/R组:15.3%±2.5%,P<0.01),同时上调了Bax表达(GdCl3组:1.93±0.28;I/R组:1.50±0.21,P<0.01),下调了Bcl-2的表达(GdCl3组:0.82±0.18;I/R组:1.71±0.30,P<0.01)。VEGF165组Bax表达减少(GdCl3+VEGF165组:1.12±0.23;GdCl3组:1.93±0.28,P<0.05)和Bcl-2的表达增加(GdCl3+VEGF165组:2.56±0.54;GdCl3组:0.82±0.18,P<0.05),TUNEL阳性细胞减少(GdCl3+VEGF165组:11.8%±1.9%;GdCl3组:25.4%±2.6%,P<0.05),CaSR mRNA的表达下调(GdCl3+VEGF165组:1.5±0.4;GdCl3组:4.5±0.6,P<0.01)。结论:VEGF165通过抑制CaSR,促进Bcl-2和抑制Bax的表达来减少缺血/再灌注诱导的心肌细胞凋亡。     

鼠脑缺血再灌注后脑组织内P27、c-fos蛋白的表达及DWI观察

目的探讨大鼠脑缺血再灌注后脑内P27、c-fos蛋白阳性表达分布的规律,并应用磁共振弥散加权成像（magnetic resonance diffusion-weighted imaging,DWI）技术进行相关弥散加权成像研究.方法采用右侧大脑中动脉脑缺血模型,SD健康大鼠随机分为三组：A组,缺血30 min再灌注60 min;B组,缺血60 min再灌注60 min;C组,缺血60 min,非再通组.模型成功行DWI成像.免疫组化技术观察P27、c-fos蛋白脑组织内表达结果.结果缺血及再灌注各组中脑内P27蛋白未见免疫阳性细胞表达;在缺血侧皮层和基底节区可见c-fos阳性表达.15 min即可在DWI上观察到缺血区高信号表现.结论P27蛋白是一种非特异性的细胞周期素依赖酶抑制剂.它在大鼠脑组织中的表达低下,推测可能与神经元缺血后易引起程序性死亡的结局有关系.即早基因c-fos参与缺血性细胞损伤的发生.DWI对缺血有极好的成像敏感性.     

银杏叶提取物(GBE)对大鼠局灶性脑缺血后细胞间黏附分子-1及其mRNA表达的影响

目的　探讨银杏叶提取物对鼠脑缺血后细胞间黏附分子 1(ICAM 1)及其mRNA表达的影响。方法　建立大鼠大脑中动脉闭塞 /再灌注模型 ,应用RT PCR及免疫组织化学的方法 ,观察各实验组鼠脑缺血后细胞间黏附分子 1及其mRNA的表达。结果　ICAM 1及其mRNA在假手术组鼠脑组织呈低表达 ;缺血 90min再灌 2 4h脑组织ICAM 1及其mRNA表达较前显著增高 (P 0 .0 1) ;银杏叶提取物治疗组于相同时限ICAM 1及其mRNA的表达与缺血再灌注组相比较显著下调 (P 0 .0 1)。结论　银杏叶提取物能显著下调ICAM 1及其mRNA的表达 ,减轻脑缺血后炎性病理损害 ,具有明显的缺血脑保护作用。     

四逆汤对大鼠肠缺血再灌注后肠粘膜细胞凋亡的影响及神经酰胺机制

目的　研究四逆汤(SND)对大鼠肠缺血再灌注 (II/R后肠粘膜细胞凋亡的影响,并从神经酰胺信使通路探讨其抗凋亡机制。方法　24只SD大鼠随机分为 3组(n=8):对照组(仅分离不阻断肠系膜上动脉)、模型组 (阻断肠系膜上动脉 1h后再灌注 3h)、SND组(每天 3g·kg-1 SND灌胃,连续 3天后手术)。取回肠末端组织行电镜检查;检测肠粘膜组织SOD活性、MDA及神经酰胺含量的变化,TUNEL法检测肠粘膜上皮细胞的凋亡指数,RT PCR法分析肠粘膜组织鞘磷脂酶(SMase)基因表达的变化。结果　模型组电镜下可发现许多典型凋亡的上皮细胞,凋亡指数达 30 82%6 34%,SND预处理后凋亡指数为 14 91%±5 40%,明显低于模型组(P<0 01);模型组SOD活性明显低于对照组 (<0 01),MDA与神经酰胺含量以及SMase的基因表达明显高于对照组(P<0 01),且神经酰胺含量与凋亡指数具有良好的正相关 (r=0 852,P<0 01),与SOD活性具有负相关(r= -0 775,P<0 01 )。SND预处理能明显增强SOD活性,降低肠粘膜组织MDA及神经酰胺含量,减少肠粘膜组织SMase的基因表达(P<0 01)。SND组凋亡指数与神经酰胺的含量亦呈显著的正相关 (r=0 832,P<0 01 )。结论　SND具有抗II/R后肠粘膜细胞凋亡的作用,其作用与它清楚氧自由基、抑制SMase的基因表达、减少神经酰胺的生成有关。     

姜黄素对大鼠肠缺血再灌注后肠黏膜细胞凋亡及GRP78表达的影响

目的 观察姜黄素对大鼠肠缺血再灌注后肠黏膜细胞凋亡及GRP78表达的影响。方法 30只雄性SD大鼠随机分为假手术组、模型组、姜黄素组。采用无创性动脉夹夹闭肠系膜上动脉1 h后实施再灌注来建立肠缺血再灌注模型,姜黄素组在手术前5 d开始每天按200 mg/kg ig给药1次,假手术组仅分离肠系膜上动脉,不夹闭。采用HE染色观察肠黏膜损伤情况并进行病理评分;TUNEl法观察肠黏膜细胞凋亡情况并计算凋亡指数;Western blotting检测再灌注损伤3 h时肠黏膜GRP78及Caspase-12蛋白表达。结果 模型组肠黏膜损伤严重,绒毛上皮成块脱落,固有层破坏、溃疡,腺体破坏严重;姜黄素组肠黏膜绒毛上皮仅部分脱落,腺体排列紊乱,与模型组比较,病理评分显著降低（P<0.05）;模型组肠黏膜隐窝可见大量凋亡阳性细胞;与模型组比较,姜黄素组肠黏膜隐窝上皮细胞凋亡情况明显减轻,凋亡指数显著下降（P<0.05）;与模型组比较,姜黄素组GRP78蛋白表达量显著增加（P<0.05）,Caspase-12蛋白表达量显著下降（P<0.05）。结论 姜黄素有可能通过上调GRP78表达,减少内质网应激所致肠黏膜细胞凋亡,来减轻肠黏膜的再灌注损伤,达到保护肠黏膜作用。