肝脏缺血再灌注时MIP-1α和HIF-1α表达的时空相关性分析和意义探讨
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摘要
肝脏是机体最大的实质性器官,接受25%的心脏输出血液,具有门静脉和肝动脉双重血供,供血量非常丰富。自1908年Pringle报道在肝切除术中应用控制肝十二指肠韧带的止血方法以来,肝脏对缺血耐受的时限及肝内不同细胞群体对缺氧的耐受性差别受到普遍关注,至今仍有较大争论。肝脏缺血再灌注常见于创伤、休克、及多种复杂肝脏手术,特别是用Pringle手法阻断肝血流切除大的肝内病灶和肝移植手术。在肝脏血供恢复后,肝脏受到进一步的“打击”,发生缺血再灌注损伤(hepatic ischemical reperfusion injury, HIRl)。HIRI发生机制复杂,炎症反应被认为是HIRI的实质。肝脏缺血再灌注时肝细胞发生代谢功能障碍,各种炎性因子生成,大量氧自由基生成,同时门静脉系统淤血,肠粘膜屏障功能障碍。再灌注后含有毒素的门静脉血流入肝产生损害作用,激活免疫细胞分泌大量炎性介质,炎性介质反向促进免疫细胞的激活。炎性介质和缺氧状态作用于肝脏细胞并可能产生复杂的病理生理过程。本文主要通过观察肝缺血再灌注不同时限大鼠肝脏和小肠的病理形态学变化及检测肝脏及小肠标本中的巨噬细胞炎性蛋白(macrophage inflamationg protein-1 a,MIP-1α)、缺氧诱导因子1 a (hypoxia inducible factor-1 a, HIF-1 a)的表达变化,探讨其相关性,并对二者在肝缺血再灌注损伤发生中的意义及相关机制进行探讨。
目的
通过建立SD大鼠肝脏缺血-再灌注损伤模型,观察大鼠肝脏缺血45min后再灌注不同时限肝脏及小肠的病理形态学变化,免疫组织化学方法检测MIP-1α和HIF-1a在肝脏及小肠组织中的表达情况,观察MIP-1a和HIF-1a在HIRI时表达的时空分布特点,进一步了解炎性损伤及氧化应激在肝脏缺血及再灌注时对肝脏缺血所致的消化道淤血性损伤以及后者对肝脏的再灌注损伤时发挥的作用。
方法
1.实验分组成年健康SD雄性大鼠55只,随机分为三组：正常组(n=5)、缺血再灌注组(n=25)、假手术组(n=25),缺血再灌注组和假手术组又根据缺血再灌注后不同时限点(0h、3h、12h、24h、72 h)随机分为5个亚组,每组5只。
2.动物模型建立术前禁食12h,自由饮水；正常组：处死后进腹直接取材。缺血再灌注组：术前处理同正常组,行腹腔内注射麻醉(3%戊巴比妥钠40mg/kg),取上腹3 cm正中切口,进入腹腔游离并用无损伤动脉钳夹闭肝蒂45min后放开动脉钳,再灌注0h组进腹后直接取材,其余大鼠切口给予缝合,继续饲养,于各再灌注时限点(3h、12h、24h、72h)取材。假手术组：术前处理及麻醉方法同缺血再灌注组,进入腹腔后仅游离肝蒂但不阻断,取材方法及时限点同缺血再灌注组。
3.观察指标与方法①光镜下病理变化观察：10%福尔马林固定大鼠肝脏及小肠标本(上段空肠组织长约5cm),梯度酒精脱水,石蜡包埋后,常规连续切片。切片行HE常规染色。②超微病理变化观察：肝脏及小肠电镜标本取材后放于3%戊二醛中固定20min修块取材,SPURR低粘度包埋剂浸透包埋聚合。组织标本行扫描电镜观察。③MIP-1α和HIF-1a表达检测——免疫组织化学SABC法：检测试剂盒购于武汉博士德生物制品公司。MIP-1a免疫组化结果以细胞浆内出现棕黄色颗粒为阳性表达。HIF-1a阳性判断标准为胞质棕黄色颗粒沉着,少量核染色。每张切片取10个200倍视野运用HPIAS-1000高清晰彩色病理图像分析系统行免疫组化染色阳性反应产物定量分析,记录阳性单位(positive unit, PU),并取其平均值,以PU值表示阳性产物表达的多少。
4.统计处理所有计量数据均以平均值±标准差(x±s)表示,计算基本统计量,所有实验组用析因设计的方差分析比较主效应和交互效应后,多重比较采取LSD法进行检验,P<0.05表示结果具有统计学意义。双变量相关分析(Bivariate)两变量间相关性。采用SPSS13.0统计软件进行统计分析,P<0.05代表有统计学意义。
结果
1.HE染色光镜下观察：正常组及假手术组的肝脏结构基本完整。缺血45min再灌注即时可见肝细胞轻度水肿,肝窦间隙开始狭窄；3h时可见肝细胞肿胀,胞质半透明、疏松呈网状；12h时肝细胞呈弥漫性肿大,水样变,肝小叶内可见点状坏死与嗜酸性小体形成；24h时肝细胞损伤最重,可见肝小叶变形,肝板破坏,部分出现片状坏死；72h时可见肝窦间隙狭窄减轻,肝小叶重构。正常组小肠结构完整,再灌注0h可见小肠上皮下间隙扩大,毛细血管充血,上皮与固有层出现分离,腺体轻度受损；3h腺体受损明显,伴部分顶部绒毛脱落；12h上皮与固有层分离明显,大部分腺体受损,毛细血管开始扩张；24h绒毛明显脱落,固有层部分消化、分解；72h小肠结构虽仍有部分受损,但基本恢复正常。
2.超微病理观察：电镜下正常对照组及假手术组肝脏及小肠组织超微结构形态基本正常；肝脏缺血45min再灌注即时电镜下即可见到线粒体及内质网轻度肿胀；3h时可见线粒体嵴变短,肿胀加重,核染色质浓缩边集；12h时可见细胞核皱缩,糖原消失；24h时可见少数肝细胞固缩死亡及新生的肝细胞；72h时肝细胞超微结构基本恢复正常,但线粒体仍稍肿胀。肠粘膜上皮细胞3h见腺体轻度受损,线粒体嵴变粗；12h时表现为线粒体明显肿胀,细胞核固缩；24h时线粒体嵴溶解呈泡状,严重呈散乱絮状；72h时小肠细胞超微结构基本恢复正常。
3. MIP-1α在正常组及假手术组肝脏及小肠组织中未见明显表达,两组差异无统计学意义(P>0.05)。再灌注0小时在肝血窦及中央静脉周围组织中可见表达,随再灌注时间的延长,阳性表达逐渐增多,在再灌注12h时达到峰值,随后逐渐下降,至72h下降明显但仍高于再灌注初期。在小肠组织,MIP-1α再灌注初期可见表达,至24h达到高峰,72h下降至正常水平。MIP-1α在小肠组织中的表达主要集中在固有层,肌层血管周围亦可见其表达。再灌注组MIP-1α的表达与正常组及假手术组相比有统计学意义(P<0.05)。
4. HIF-1α在正常组与假手术组各时限点肝脏及小肠内未见明显表达,两组差异无统计学意义(P>0.05)。缺血再灌注组SD大鼠肝脏再灌注0h时汇管区微血管、微胆管周围细胞浆中可见棕黄色阳性表达,12h时肝细胞内广泛阳性表达。随再灌注时间延长,阳性表达细胞逐渐减少,至72h基本恢复正常。小肠组织阳性表达主要集中在肠粘膜细胞,再灌注初期即开始表达,随时间延长阳性表达逐渐增多,至24h达峰值,72h降至再灌注初期水平。再灌注组HIF-1α的表达与正常组及假手术组相比有统计学意义(P<0.05)。
5.缺血-再灌注组,肝脏组织中MIP-1α、HIF-1α呈显著正相关关系(r。=0.72 P<0.05)；小肠组织中MIP-1α、HIF-1α呈显著正相关关系(r。=0.97P<0.05)。
结论
各种原因引起的一定程度的肝脏缺血当再灌注期血供恢复后,肝脏细胞功能代谢障碍及组织结构破坏进一步加重,在这一过程中大量炎性细胞浸润、激活释放炎性介质参与组织损伤。HIF-1α在多种组织缺氧应激状态下表达,并通过调节其他细胞分子的表达而对组织细胞发挥作用。MIP-1α属于CC趋化因子一员,肝脏Kuffer细胞可以分泌MIP-1α并引起炎性细胞的聚集,促进组织炎性损伤。
HIF-1α与目的基因结合后,调节相关基因的表达,使细胞对缺氧的适应能力增强,其表达增强可能与多种疾病的细胞凋亡损伤有关。本实验中,在正常组及假手术组大鼠肝脏及小肠组织中HIF-1a表达细微。缺血再灌注后,HIF-1α在肝脏及小肠组织均有明显表达。HIF-1a的首先表达部位可能与肝窦间隙、微胆管、中央静脉区组织细胞血供较差,氧浓度较低,阻断肝血流后缺血缺氧性损伤最先累及该区域有关。而在小肠组织中,肠粘膜细胞微绒毛处的血供最差,最易引起缺血缺氧性损害,故成为HIF-1a最先表达的部位。
HIF-1a和MIP-1a表达的正相关性表明,HIF-1a作为转录调节因子可能通过促进MIP-1a等趋化因子的表达从而增强炎症反应过程,HIF-1a可能是MIP-1α表达的重要调节因子。有研究表明HIF-1α可使细胞自发激活上调炎性蛋白表达而增强上皮炎性反应,并参与了骨髓细胞的增殖分化过程。MIP-1a等趋化因子的表达增强使炎性细胞向炎性损伤部位聚集,炎性细胞与内皮细胞粘附导致微血管出现无复流现象,进一步加重组织缺氧。HIF-1a与MIP-1a在小肠组织与肝脏组织中表达峰值时相及表达恢复情况的差异原因,考虑可能因为门静脉系统淤血造成胃肠道循环系统微小血栓形成,在肝门开放后,胃肠道继续存在部分缺血缺氧情况,从而影响胃肠道炎性损伤的恢复所致。
本研究结果表明肝脏的缺血不仅可造成肝脏实质性及非实质性细胞的自身损伤,而且可造成消化道的损伤。在再灌注初期,肝脏血流再通后各种途径产生的氧自由基对肝细胞损伤起主要作用。在小肠组织中,随着肝门阻断时间延长,门静脉系统淤血程度的增加造成消化道缺氧；小肠组织有氧代谢障碍产生的活性氧簇使小肠组织受损,活性氧簇通过脂质过氧化、促进中性粒细胞聚集及抑制线粒体功能加速细胞能量的消耗等作用导致小肠粘膜细胞损伤。此外,消化道的淤血性损伤也可能是造成肝脏复流后再灌注损伤的重要原因之一,因为消化道产生的氧自由基及各种炎性介质可随着再灌注后的门静脉血流进入肝脏,进而加重肝脏的损伤；二者互为因果,造成肝脏的缺血再灌注损伤和机体整体的氧化应激反应。
本研究通过对肝脏缺血再灌注不同时相肝脏与小肠病理形态变化及同期HIF-1α、MIP-1a表达变化分析,提示：肝脏缺血再灌注可以引起小肠组织的损伤；肝脏及小肠组织的损伤与炎性细胞聚集及缺氧应激状态有关。低氧环境及炎性反应共同促成肝脏缺血再灌注时肝脏及消化道应激反应,其中HIF-1a和MIP-1a发挥了重要作用。因而减轻肝脏组织细胞的缺血缺氧和炎性反应状态可作为改善肝缺血再灌注损伤的重要环节。
Liver is the largest parenchymatous organ in the body. It has a reach blood supply, accept 25% of the cardiac output, portal vein and hepatic artery both supply blood to it. The time limit of hepatic ischemia and the differences of different intrahepatic cell groups in tolerance to hypoxia are widly concerned, since Pringle has reported the method of control the hepatoduodenal ligament to stop bleeding during the periods of hepatic surgery in 1908. Hepatic ischemia is common in trauma, shock, and many liver surgeries, especially for the resection of large intrahepatic lesions and liver transplant. After the reperfusion of the liver, a further attack arrives, and then hepatic ischemia-reperfusion injury(HIRI) will happen. The mechanisms of HIRI are complex, inflammatory response is considered to be the substance of HIRI. Liver metabolic dysfunction occurs, and a variety of inflammatory factors and oxygen free radicals are also generated during the period of HIR. At the same time, intestinal barrier dysfunction comes because of the congestion of the portal vein system. The portal vein with toxins cause a damage to the liver. A large number of inflammatory mediators are secreted by activate immune cells, while inflammatory mediators reverse to boost the immune cells activation. Inflammatory mediators and hypoxia act on liver cells may lead to a complex pathophysiological process. But the relationship of the inflammatory mediators and the hypoxic state in HIRI and the pathological changes of the gastrointestinal tract are seldom studied. In this paper, the pathological changes of the liver and intestin will be observed, the expressions of macrophage inflammatory protein-1a(MIP-la) and hypoxia inducible factor-la (HIF-1a) will be detected, and their significance and relevant mechanisms will be analyzed.
Objective
The hepatic ischemia-reperfusion injury model of SD rat was established to observe liver and small intestine pathological changes at different reperfusion time points after hepatic ischemia for 45min, the expressions of MIP-la and HIF-la in the liver and small intestine tissues were detected by immunohistochemistry. The role of MIP-la and HIF-la in HIRI was disscused for further understanding the effect of inflammatory mediators and oxidative stress in liver and gastrointestinal congestive injury after hepatic ischemia-reperfusion.
Methords
1. Grouping
55 healthy adult male SD rats were randomly divided into three groups:normal control group (CO,n=5), ischemia-reperfusion group (IR,n=25), sham-operated group (SO,n= 25), then ischemia-reperfusion group and sham-operated group were randomly divided into five sub-groups according to different reperfusion time points (0h,3h,12h,24h,72h).
2. Animals
Fasting for 12h, free drinking of water before operation. Took samples soon after broken neck executed in CO group. The preparation of preoperation in IR group was the same as normal group, took the middle abdominal incision about 3cm into the abdominal after intraperitoneal injection anesthesia (3%pentobarbital sodium 40mg/kg), then dissociated and blocked hepatic pedicle by non-invasive artery clamp for 45min, and released it. Directly took samples at Oh subgroup, but sew up the wound of rats to continue feeding in other subgroups, and took samples at the reperfusion time points(3h,12h,24h,72h). Preoperative treatment and anesthesia methords were as the same as the ischemia-reperfusion group in SO group, dissociated but not blocked hepatic pedicle, took samples at different time points.
3. Detection mothods and indexes
①The changes of pathology under light microscope:Specimens of rat liver and small intestine (about 5cm up-jejunum organization) were fixed by 10% formalin, dehydrated by gradient ethanol, embed by paraffin, then routine sected and observed by rutine HE staining.②The ultrastructure changes under electron microscopy:The samples of liver and small intestine which detected by electron microscope were put into 3% glutaraldehyde, fixed for 20min, then embedd by SPURR medium. Specimens were observed by scanning electron microscope.③Immunohistochemistry methods to detected the expressions of MIP-la and HIF-la.:Test kit purchased from Wuhan Boster Biological Products Company. The appearation of brown-yellow granules in cytoplasm was considered as the standard of MIP-la positive expression; the criterion of HIF-la positive expression was the appearation of cytoplasmic brown granules or a small amount of nuclear staining.10 visions (200/H) of each slice were taken for the quantitative analysis of the positive products(using HPIAS-1000 high-resolution color pathological image analysis system), the positive units were recorded (positive unit, PU), and the average level of PU represented the amount of positive products expression.
4. Statistical analysis
All data were recorded as x±s, basic statistics were calculated. Factorial design ANOVE was used to analyze the main effects and interaction of all groups. The difference of every groups was tested by LSD method. Bivariate correlation was used to analyze (Bivariate) the correlation between two variables. SPSS13.0 statistical software was used for statistical analysis, P<0.05 represents statistical significance.
Results
1. The observation of HE staining under light microscope:The structure of the liver was basically integrity in CO and SO groups. Hepatocytes apparent mild edema and sinusoidal gap began to narrow at 45min time limit; swollen hepatocytes,. translucent cytoplasm, and slacked reticular appeared at 3h; diffuse swollen hepatocytes, spotty necrosis with acidophilic body formation could be seen at 12h within the hepatic lobules; the hepatocytes were seriously damaged at 24h, reflceted as the distorted hepatic lobules and the damaged liver panels with portion of necrosis; but the narrow of sinusoidal space reduced and hepatic lobule reconstructed at 72h after reperfusion. The structure of the small intestine was integrity in the CO group. The expanded subepithelial spaces, congested capillaries, separated epitheliums and lamina proprias, mild impairment glands all could be seen at the reperfusion time point of Oh; but glands damaged significantly at 3h, accompanied with some of the top hair off. The epitheliums obviously separated with lamina propriasand most of the glands damaged, capillaries also began to expand at 12h; obviously villus shedding appeared at 24h, part of the lamina propria digested and decomposited. Although there was still some structural damage at 72h of the small intestine, but the structure basically restored to normal.
2. The changes of ultrastructures under electron microscopy:The ultrastructures of the liver and small intestine were basically integrity in CO and SO groups. Mitochondria and endoplasmic reticulums slightly swollen at the beginning of the reperfusion; mitochondria turned shorter, intumesce got worse, nuclear chromatin condensation and margination were also detected at 3h after reperfusion; nucleus shrinked, glycogen disappeared at 12h after reperfusion; pyknosis death and neonatal hepotocytes could be seen at 24h after reperfusion; the ultrastructure of liver cells basicly restored to normal at 72h after reperfusion, but the mitochondrions were still slightly swollen. Gland of intestine epithelial cells could be seen mild impairment, and mitochondrial cristae turned thicker at 3h after reperfusion; significiently swollen mitochondria and agglutinated nuclears could be seen at 12h after reperfusion; mitochondrial cristaes were bubbly dissoluted, even flocculent scattered at 24h after reperfusion; but the ultrastructure of small intestine cells basicly returned to normal at 72h after reperfusion.
3. There were also no significant expressions of MIP-la in the liver and small intestine tissues between CO and SO group of all time points, no significant difference between the two gurops (P> 0.05). Positive expressions could be seen at sinusoids and central veins in liver at the begining of the reperfusion, then gradually increased with the reperfusion time prolonged, peak at 12h after reperfusion. The positive expression numbers were significantly declined but still high at 72h time point. In the small intestine tissues, the positive expressions of MIP-1a appeared since the early reperfusion, reached the peak at 24h after reperfusion. Despite decline, but the number of positive expressions still high at 72h after reperfusion. The MIP-la expressed mainly in lamina propria, its expression could also be seen at muscular layers around vascular. The expressions of MIP-la in IR group were statistically significant compared with CO and SO group.
4. There were no significant expressions of HIF-la in the liver and small intestine tissues within CO and SO groups at all time points, no significant difference between the two groups (P> 0.05). The positive expressions at cytoplasm could be seen at periportal capillaries, micro-duct in IR at Oh group, and wildly expressed at 12h after reperfusion. With the reperfusion time extending, the expressions of positive cells gradually reduced, and basically back to the normal level at 72h after reperfusion. In the tissues of small intestine, HIF-la expressed at the beginning of reperfusion and concentrated in the mucosal cells. They gradually increased with the time, reached the peak at 24h after reperfusion, and reduced to the initial level at 72h after reperfusion. The expressions of HIF-1a in IR group were statistically significant compared with the CO and SO group.
5. There was a significant positive correlation between MIP-la and HIF-la in the liver tissues(rp=0.72 P<0.05) and small intestine tissues(rp=0.97 P<0.05).
Conclusions
The structures and cell functions of liver are damaged and aggravated, when the blood supply of liver restored after hepatic ischemia. In this process a large number of inflammatory cells infiltrate and release inflammatory mediators to involve in tissue injury. HIF-la expresses in a variety of organizations under the state of hypoxic stress and plays a role on tissue cells by regulating the expression of other molecules. MIP-1a belongs to CC chemokine family, which can be secreted by liver Kuffer cells, cause an accumulation of inflammatory cells, and promote the inflammatory injury of tissues.
HIF-la can combine and regulate the expression of related genes, make an enhanced adaptability to huyoxia state. Its enhanced expression may be connected with a variety of cells apoptosis. In this experiment, the expressions of HIF-la were fine in CO and SO groups in liver and small intestine tissues. After ischemia-reperfusion, the expressions of HIF-1a were increased. The first expression site of HIF-la may be related to the poor blood supply and hypoxia state of liver sinusoidal space, micro-duct, central venous. While in the small intestine, the mucosal cells microvilli have the worst blood supply, and most vulnerable to the injury which caused by hypoxic-ischemic, so they becomes the first expression place of HIF-1a.
The positive correlation between HIF-1a and MIP-1a indicated that HIF-1a may be an important expression regulator of MIP-la, it can promote the expression of MIP-la, then enhance the process of inflammatory reaction. Studies have been shown that HIF-1a can increase the spontaneous expression of inflammatory proteins and enhance the dermatitis reaction, and involved in the differentiation process of bone marrow cells. The difference expression peak phase of HIF-la and MIP-la at the small intestine and liver tissues may because of the formation of microthrombus in gastrointestinal tract micro-circulatory system. So after the reperfusion of hepatic portal, there is still some ischemic and hypoxic conditions, which affect the recover of the gastrointestinal tract inflammatory injury.
The results indicated that the hepatic ischemia cause not only the liver substantive and non-parenchymal cells injury, but also gastrointestinal system injury. At the beginning of the reperfusion, oxygen free radicals play an important role in the injury of live. The mionectic state of gastrointestinal was aggravated by the congestion of portal vein, with the time of hepatic pedical blocking extending. Oxyradicals produced by small intestine brought about the small intestinal hypoxia and inflammatory injury by promote lipid peroxidation, neutrophil aggregation and inhibit mitochondrial function to accelerate cellular energy consumption. Furthermore, the congestive injury of digestive tract may be also an important reason for the injury of liver after hepatic reperfusion. Because oxyradical and various inflammatory mediators produced by digestive system may increase the injury of liver after hepatic reperfusion. The injury of liver and gastrointestin reinforce each other resulting in HIRI and oxidative stress in the body.
The pathological changes of liver and small intestine at different phases of HIRI and the variational expressions of HIF-la/MIP-la suggest that:hepatic ischemia and reperfusion can cause intestinal injury; liver and small intestine injury induced by the liver ischemia and reperfusion relate with inflammatory cell accumulation and hypoxic stress. Hypoxia and inflammatory reactions collaborative contribute to the liver and gastrointestine stress response. HIF-la and MIP-la play an important role in HIRI. It is may be an useful method to mitigate HIRI by improving the state of hypoxia stress and inflammation response.
目的
通过建立SD大鼠肝脏缺血-再灌注损伤模型,观察大鼠肝脏缺血45min后再灌注不同时限肝脏及小肠的病理形态学变化,免疫组织化学方法检测MIP-1α和HIF-1a在肝脏及小肠组织中的表达情况,观察MIP-1a和HIF-1a在HIRI时表达的时空分布特点,进一步了解炎性损伤及氧化应激在肝脏缺血及再灌注时对肝脏缺血所致的消化道淤血性损伤以及后者对肝脏的再灌注损伤时发挥的作用。
方法
1.实验分组成年健康SD雄性大鼠55只,随机分为三组：正常组(n=5)、缺血再灌注组(n=25)、假手术组(n=25),缺血再灌注组和假手术组又根据缺血再灌注后不同时限点(0h、3h、12h、24h、72 h)随机分为5个亚组,每组5只。
2.动物模型建立术前禁食12h,自由饮水；正常组：处死后进腹直接取材。缺血再灌注组：术前处理同正常组,行腹腔内注射麻醉(3%戊巴比妥钠40mg/kg),取上腹3 cm正中切口,进入腹腔游离并用无损伤动脉钳夹闭肝蒂45min后放开动脉钳,再灌注0h组进腹后直接取材,其余大鼠切口给予缝合,继续饲养,于各再灌注时限点(3h、12h、24h、72h)取材。假手术组：术前处理及麻醉方法同缺血再灌注组,进入腹腔后仅游离肝蒂但不阻断,取材方法及时限点同缺血再灌注组。
3.观察指标与方法①光镜下病理变化观察：10%福尔马林固定大鼠肝脏及小肠标本(上段空肠组织长约5cm),梯度酒精脱水,石蜡包埋后,常规连续切片。切片行HE常规染色。②超微病理变化观察：肝脏及小肠电镜标本取材后放于3%戊二醛中固定20min修块取材,SPURR低粘度包埋剂浸透包埋聚合。组织标本行扫描电镜观察。③MIP-1α和HIF-1a表达检测——免疫组织化学SABC法：检测试剂盒购于武汉博士德生物制品公司。MIP-1a免疫组化结果以细胞浆内出现棕黄色颗粒为阳性表达。HIF-1a阳性判断标准为胞质棕黄色颗粒沉着,少量核染色。每张切片取10个200倍视野运用HPIAS-1000高清晰彩色病理图像分析系统行免疫组化染色阳性反应产物定量分析,记录阳性单位(positive unit, PU),并取其平均值,以PU值表示阳性产物表达的多少。
4.统计处理所有计量数据均以平均值±标准差(x±s)表示,计算基本统计量,所有实验组用析因设计的方差分析比较主效应和交互效应后,多重比较采取LSD法进行检验,P<0.05表示结果具有统计学意义。双变量相关分析(Bivariate)两变量间相关性。采用SPSS13.0统计软件进行统计分析,P<0.05代表有统计学意义。
结果
1.HE染色光镜下观察：正常组及假手术组的肝脏结构基本完整。缺血45min再灌注即时可见肝细胞轻度水肿,肝窦间隙开始狭窄；3h时可见肝细胞肿胀,胞质半透明、疏松呈网状；12h时肝细胞呈弥漫性肿大,水样变,肝小叶内可见点状坏死与嗜酸性小体形成；24h时肝细胞损伤最重,可见肝小叶变形,肝板破坏,部分出现片状坏死；72h时可见肝窦间隙狭窄减轻,肝小叶重构。正常组小肠结构完整,再灌注0h可见小肠上皮下间隙扩大,毛细血管充血,上皮与固有层出现分离,腺体轻度受损；3h腺体受损明显,伴部分顶部绒毛脱落；12h上皮与固有层分离明显,大部分腺体受损,毛细血管开始扩张；24h绒毛明显脱落,固有层部分消化、分解；72h小肠结构虽仍有部分受损,但基本恢复正常。
2.超微病理观察：电镜下正常对照组及假手术组肝脏及小肠组织超微结构形态基本正常；肝脏缺血45min再灌注即时电镜下即可见到线粒体及内质网轻度肿胀；3h时可见线粒体嵴变短,肿胀加重,核染色质浓缩边集；12h时可见细胞核皱缩,糖原消失；24h时可见少数肝细胞固缩死亡及新生的肝细胞；72h时肝细胞超微结构基本恢复正常,但线粒体仍稍肿胀。肠粘膜上皮细胞3h见腺体轻度受损,线粒体嵴变粗；12h时表现为线粒体明显肿胀,细胞核固缩；24h时线粒体嵴溶解呈泡状,严重呈散乱絮状；72h时小肠细胞超微结构基本恢复正常。
3. MIP-1α在正常组及假手术组肝脏及小肠组织中未见明显表达,两组差异无统计学意义(P>0.05)。再灌注0小时在肝血窦及中央静脉周围组织中可见表达,随再灌注时间的延长,阳性表达逐渐增多,在再灌注12h时达到峰值,随后逐渐下降,至72h下降明显但仍高于再灌注初期。在小肠组织,MIP-1α再灌注初期可见表达,至24h达到高峰,72h下降至正常水平。MIP-1α在小肠组织中的表达主要集中在固有层,肌层血管周围亦可见其表达。再灌注组MIP-1α的表达与正常组及假手术组相比有统计学意义(P<0.05)。
4. HIF-1α在正常组与假手术组各时限点肝脏及小肠内未见明显表达,两组差异无统计学意义(P>0.05)。缺血再灌注组SD大鼠肝脏再灌注0h时汇管区微血管、微胆管周围细胞浆中可见棕黄色阳性表达,12h时肝细胞内广泛阳性表达。随再灌注时间延长,阳性表达细胞逐渐减少,至72h基本恢复正常。小肠组织阳性表达主要集中在肠粘膜细胞,再灌注初期即开始表达,随时间延长阳性表达逐渐增多,至24h达峰值,72h降至再灌注初期水平。再灌注组HIF-1α的表达与正常组及假手术组相比有统计学意义(P<0.05)。
5.缺血-再灌注组,肝脏组织中MIP-1α、HIF-1α呈显著正相关关系(r。=0.72 P<0.05)；小肠组织中MIP-1α、HIF-1α呈显著正相关关系(r。=0.97P<0.05)。
结论
各种原因引起的一定程度的肝脏缺血当再灌注期血供恢复后,肝脏细胞功能代谢障碍及组织结构破坏进一步加重,在这一过程中大量炎性细胞浸润、激活释放炎性介质参与组织损伤。HIF-1α在多种组织缺氧应激状态下表达,并通过调节其他细胞分子的表达而对组织细胞发挥作用。MIP-1α属于CC趋化因子一员,肝脏Kuffer细胞可以分泌MIP-1α并引起炎性细胞的聚集,促进组织炎性损伤。
HIF-1α与目的基因结合后,调节相关基因的表达,使细胞对缺氧的适应能力增强,其表达增强可能与多种疾病的细胞凋亡损伤有关。本实验中,在正常组及假手术组大鼠肝脏及小肠组织中HIF-1a表达细微。缺血再灌注后,HIF-1α在肝脏及小肠组织均有明显表达。HIF-1a的首先表达部位可能与肝窦间隙、微胆管、中央静脉区组织细胞血供较差,氧浓度较低,阻断肝血流后缺血缺氧性损伤最先累及该区域有关。而在小肠组织中,肠粘膜细胞微绒毛处的血供最差,最易引起缺血缺氧性损害,故成为HIF-1a最先表达的部位。
HIF-1a和MIP-1a表达的正相关性表明,HIF-1a作为转录调节因子可能通过促进MIP-1a等趋化因子的表达从而增强炎症反应过程,HIF-1a可能是MIP-1α表达的重要调节因子。有研究表明HIF-1α可使细胞自发激活上调炎性蛋白表达而增强上皮炎性反应,并参与了骨髓细胞的增殖分化过程。MIP-1a等趋化因子的表达增强使炎性细胞向炎性损伤部位聚集,炎性细胞与内皮细胞粘附导致微血管出现无复流现象,进一步加重组织缺氧。HIF-1a与MIP-1a在小肠组织与肝脏组织中表达峰值时相及表达恢复情况的差异原因,考虑可能因为门静脉系统淤血造成胃肠道循环系统微小血栓形成,在肝门开放后,胃肠道继续存在部分缺血缺氧情况,从而影响胃肠道炎性损伤的恢复所致。
本研究结果表明肝脏的缺血不仅可造成肝脏实质性及非实质性细胞的自身损伤,而且可造成消化道的损伤。在再灌注初期,肝脏血流再通后各种途径产生的氧自由基对肝细胞损伤起主要作用。在小肠组织中,随着肝门阻断时间延长,门静脉系统淤血程度的增加造成消化道缺氧；小肠组织有氧代谢障碍产生的活性氧簇使小肠组织受损,活性氧簇通过脂质过氧化、促进中性粒细胞聚集及抑制线粒体功能加速细胞能量的消耗等作用导致小肠粘膜细胞损伤。此外,消化道的淤血性损伤也可能是造成肝脏复流后再灌注损伤的重要原因之一,因为消化道产生的氧自由基及各种炎性介质可随着再灌注后的门静脉血流进入肝脏,进而加重肝脏的损伤；二者互为因果,造成肝脏的缺血再灌注损伤和机体整体的氧化应激反应。
本研究通过对肝脏缺血再灌注不同时相肝脏与小肠病理形态变化及同期HIF-1α、MIP-1a表达变化分析,提示：肝脏缺血再灌注可以引起小肠组织的损伤；肝脏及小肠组织的损伤与炎性细胞聚集及缺氧应激状态有关。低氧环境及炎性反应共同促成肝脏缺血再灌注时肝脏及消化道应激反应,其中HIF-1a和MIP-1a发挥了重要作用。因而减轻肝脏组织细胞的缺血缺氧和炎性反应状态可作为改善肝缺血再灌注损伤的重要环节。
Liver is the largest parenchymatous organ in the body. It has a reach blood supply, accept 25% of the cardiac output, portal vein and hepatic artery both supply blood to it. The time limit of hepatic ischemia and the differences of different intrahepatic cell groups in tolerance to hypoxia are widly concerned, since Pringle has reported the method of control the hepatoduodenal ligament to stop bleeding during the periods of hepatic surgery in 1908. Hepatic ischemia is common in trauma, shock, and many liver surgeries, especially for the resection of large intrahepatic lesions and liver transplant. After the reperfusion of the liver, a further attack arrives, and then hepatic ischemia-reperfusion injury(HIRI) will happen. The mechanisms of HIRI are complex, inflammatory response is considered to be the substance of HIRI. Liver metabolic dysfunction occurs, and a variety of inflammatory factors and oxygen free radicals are also generated during the period of HIR. At the same time, intestinal barrier dysfunction comes because of the congestion of the portal vein system. The portal vein with toxins cause a damage to the liver. A large number of inflammatory mediators are secreted by activate immune cells, while inflammatory mediators reverse to boost the immune cells activation. Inflammatory mediators and hypoxia act on liver cells may lead to a complex pathophysiological process. But the relationship of the inflammatory mediators and the hypoxic state in HIRI and the pathological changes of the gastrointestinal tract are seldom studied. In this paper, the pathological changes of the liver and intestin will be observed, the expressions of macrophage inflammatory protein-1a(MIP-la) and hypoxia inducible factor-la (HIF-1a) will be detected, and their significance and relevant mechanisms will be analyzed.
Objective
The hepatic ischemia-reperfusion injury model of SD rat was established to observe liver and small intestine pathological changes at different reperfusion time points after hepatic ischemia for 45min, the expressions of MIP-la and HIF-la in the liver and small intestine tissues were detected by immunohistochemistry. The role of MIP-la and HIF-la in HIRI was disscused for further understanding the effect of inflammatory mediators and oxidative stress in liver and gastrointestinal congestive injury after hepatic ischemia-reperfusion.
Methords
1. Grouping
55 healthy adult male SD rats were randomly divided into three groups:normal control group (CO,n=5), ischemia-reperfusion group (IR,n=25), sham-operated group (SO,n= 25), then ischemia-reperfusion group and sham-operated group were randomly divided into five sub-groups according to different reperfusion time points (0h,3h,12h,24h,72h).
2. Animals
Fasting for 12h, free drinking of water before operation. Took samples soon after broken neck executed in CO group. The preparation of preoperation in IR group was the same as normal group, took the middle abdominal incision about 3cm into the abdominal after intraperitoneal injection anesthesia (3%pentobarbital sodium 40mg/kg), then dissociated and blocked hepatic pedicle by non-invasive artery clamp for 45min, and released it. Directly took samples at Oh subgroup, but sew up the wound of rats to continue feeding in other subgroups, and took samples at the reperfusion time points(3h,12h,24h,72h). Preoperative treatment and anesthesia methords were as the same as the ischemia-reperfusion group in SO group, dissociated but not blocked hepatic pedicle, took samples at different time points.
3. Detection mothods and indexes
①The changes of pathology under light microscope:Specimens of rat liver and small intestine (about 5cm up-jejunum organization) were fixed by 10% formalin, dehydrated by gradient ethanol, embed by paraffin, then routine sected and observed by rutine HE staining.②The ultrastructure changes under electron microscopy:The samples of liver and small intestine which detected by electron microscope were put into 3% glutaraldehyde, fixed for 20min, then embedd by SPURR medium. Specimens were observed by scanning electron microscope.③Immunohistochemistry methods to detected the expressions of MIP-la and HIF-la.:Test kit purchased from Wuhan Boster Biological Products Company. The appearation of brown-yellow granules in cytoplasm was considered as the standard of MIP-la positive expression; the criterion of HIF-la positive expression was the appearation of cytoplasmic brown granules or a small amount of nuclear staining.10 visions (200/H) of each slice were taken for the quantitative analysis of the positive products(using HPIAS-1000 high-resolution color pathological image analysis system), the positive units were recorded (positive unit, PU), and the average level of PU represented the amount of positive products expression.
4. Statistical analysis
All data were recorded as x±s, basic statistics were calculated. Factorial design ANOVE was used to analyze the main effects and interaction of all groups. The difference of every groups was tested by LSD method. Bivariate correlation was used to analyze (Bivariate) the correlation between two variables. SPSS13.0 statistical software was used for statistical analysis, P<0.05 represents statistical significance.
Results
1. The observation of HE staining under light microscope:The structure of the liver was basically integrity in CO and SO groups. Hepatocytes apparent mild edema and sinusoidal gap began to narrow at 45min time limit; swollen hepatocytes,. translucent cytoplasm, and slacked reticular appeared at 3h; diffuse swollen hepatocytes, spotty necrosis with acidophilic body formation could be seen at 12h within the hepatic lobules; the hepatocytes were seriously damaged at 24h, reflceted as the distorted hepatic lobules and the damaged liver panels with portion of necrosis; but the narrow of sinusoidal space reduced and hepatic lobule reconstructed at 72h after reperfusion. The structure of the small intestine was integrity in the CO group. The expanded subepithelial spaces, congested capillaries, separated epitheliums and lamina proprias, mild impairment glands all could be seen at the reperfusion time point of Oh; but glands damaged significantly at 3h, accompanied with some of the top hair off. The epitheliums obviously separated with lamina propriasand most of the glands damaged, capillaries also began to expand at 12h; obviously villus shedding appeared at 24h, part of the lamina propria digested and decomposited. Although there was still some structural damage at 72h of the small intestine, but the structure basically restored to normal.
2. The changes of ultrastructures under electron microscopy:The ultrastructures of the liver and small intestine were basically integrity in CO and SO groups. Mitochondria and endoplasmic reticulums slightly swollen at the beginning of the reperfusion; mitochondria turned shorter, intumesce got worse, nuclear chromatin condensation and margination were also detected at 3h after reperfusion; nucleus shrinked, glycogen disappeared at 12h after reperfusion; pyknosis death and neonatal hepotocytes could be seen at 24h after reperfusion; the ultrastructure of liver cells basicly restored to normal at 72h after reperfusion, but the mitochondrions were still slightly swollen. Gland of intestine epithelial cells could be seen mild impairment, and mitochondrial cristae turned thicker at 3h after reperfusion; significiently swollen mitochondria and agglutinated nuclears could be seen at 12h after reperfusion; mitochondrial cristaes were bubbly dissoluted, even flocculent scattered at 24h after reperfusion; but the ultrastructure of small intestine cells basicly returned to normal at 72h after reperfusion.
3. There were also no significant expressions of MIP-la in the liver and small intestine tissues between CO and SO group of all time points, no significant difference between the two gurops (P> 0.05). Positive expressions could be seen at sinusoids and central veins in liver at the begining of the reperfusion, then gradually increased with the reperfusion time prolonged, peak at 12h after reperfusion. The positive expression numbers were significantly declined but still high at 72h time point. In the small intestine tissues, the positive expressions of MIP-1a appeared since the early reperfusion, reached the peak at 24h after reperfusion. Despite decline, but the number of positive expressions still high at 72h after reperfusion. The MIP-la expressed mainly in lamina propria, its expression could also be seen at muscular layers around vascular. The expressions of MIP-la in IR group were statistically significant compared with CO and SO group.
4. There were no significant expressions of HIF-la in the liver and small intestine tissues within CO and SO groups at all time points, no significant difference between the two groups (P> 0.05). The positive expressions at cytoplasm could be seen at periportal capillaries, micro-duct in IR at Oh group, and wildly expressed at 12h after reperfusion. With the reperfusion time extending, the expressions of positive cells gradually reduced, and basically back to the normal level at 72h after reperfusion. In the tissues of small intestine, HIF-la expressed at the beginning of reperfusion and concentrated in the mucosal cells. They gradually increased with the time, reached the peak at 24h after reperfusion, and reduced to the initial level at 72h after reperfusion. The expressions of HIF-1a in IR group were statistically significant compared with the CO and SO group.
5. There was a significant positive correlation between MIP-la and HIF-la in the liver tissues(rp=0.72 P<0.05) and small intestine tissues(rp=0.97 P<0.05).
Conclusions
The structures and cell functions of liver are damaged and aggravated, when the blood supply of liver restored after hepatic ischemia. In this process a large number of inflammatory cells infiltrate and release inflammatory mediators to involve in tissue injury. HIF-la expresses in a variety of organizations under the state of hypoxic stress and plays a role on tissue cells by regulating the expression of other molecules. MIP-1a belongs to CC chemokine family, which can be secreted by liver Kuffer cells, cause an accumulation of inflammatory cells, and promote the inflammatory injury of tissues.
HIF-la can combine and regulate the expression of related genes, make an enhanced adaptability to huyoxia state. Its enhanced expression may be connected with a variety of cells apoptosis. In this experiment, the expressions of HIF-la were fine in CO and SO groups in liver and small intestine tissues. After ischemia-reperfusion, the expressions of HIF-1a were increased. The first expression site of HIF-la may be related to the poor blood supply and hypoxia state of liver sinusoidal space, micro-duct, central venous. While in the small intestine, the mucosal cells microvilli have the worst blood supply, and most vulnerable to the injury which caused by hypoxic-ischemic, so they becomes the first expression place of HIF-1a.
The positive correlation between HIF-1a and MIP-1a indicated that HIF-1a may be an important expression regulator of MIP-la, it can promote the expression of MIP-la, then enhance the process of inflammatory reaction. Studies have been shown that HIF-1a can increase the spontaneous expression of inflammatory proteins and enhance the dermatitis reaction, and involved in the differentiation process of bone marrow cells. The difference expression peak phase of HIF-la and MIP-la at the small intestine and liver tissues may because of the formation of microthrombus in gastrointestinal tract micro-circulatory system. So after the reperfusion of hepatic portal, there is still some ischemic and hypoxic conditions, which affect the recover of the gastrointestinal tract inflammatory injury.
The results indicated that the hepatic ischemia cause not only the liver substantive and non-parenchymal cells injury, but also gastrointestinal system injury. At the beginning of the reperfusion, oxygen free radicals play an important role in the injury of live. The mionectic state of gastrointestinal was aggravated by the congestion of portal vein, with the time of hepatic pedical blocking extending. Oxyradicals produced by small intestine brought about the small intestinal hypoxia and inflammatory injury by promote lipid peroxidation, neutrophil aggregation and inhibit mitochondrial function to accelerate cellular energy consumption. Furthermore, the congestive injury of digestive tract may be also an important reason for the injury of liver after hepatic reperfusion. Because oxyradical and various inflammatory mediators produced by digestive system may increase the injury of liver after hepatic reperfusion. The injury of liver and gastrointestin reinforce each other resulting in HIRI and oxidative stress in the body.
The pathological changes of liver and small intestine at different phases of HIRI and the variational expressions of HIF-la/MIP-la suggest that:hepatic ischemia and reperfusion can cause intestinal injury; liver and small intestine injury induced by the liver ischemia and reperfusion relate with inflammatory cell accumulation and hypoxic stress. Hypoxia and inflammatory reactions collaborative contribute to the liver and gastrointestine stress response. HIF-la and MIP-la play an important role in HIRI. It is may be an useful method to mitigate HIRI by improving the state of hypoxia stress and inflammation response.
引文
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[3]Scarpino S, Stoppacciaro A,Ballerini F, et al. Papillary carcinoma of the thyroid:hepatocyte growth factor (HGF) stimulates tumor cells to release chemokines active in recruiting dendritic cells[J]. Am J Pathol, 2000,156(3):831-837.
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[5]周道远,李幼姬,梁鸣等.MIP-1a在狼疮肾炎患者肾组织的表达及意义中国免疫学杂志[J],2002,18(10)：724-726.
[6]Zang YC, Samanta AK, Halder JB, et al. Aberrant T cell migration toward RANTES and MIP-1 a in patients with multiple sclerosis:overexpression of chemokine receptor CCR5 [J] Brain,2000,123(9):1874-1882.
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[9]Day YJ, Marshall MA., Huang L, et al. Protection from ischemic liver injury by activation of A2A adenosine receptors during reperfusion:inhibition of chemokine induction[J]. Am J Physiol Gastrointest Liver Physiol. 2004,288(2):285-293.
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[11]Hsieh,CH; Frink,M; Hsieh,YC, et al.The Role of MIP-1{alpha} in the Development of Systemic Inflammatory Response and Organ Hemorrhage Injury following Trauma[J]. J Immunol,2008,181(4):2806-2812.
[12]邵堂雷蔡伟耀杨卫平等.供肝冷热缺血移植后损伤与炎症细胞的关系[J].中华肝脏病杂志,2002,10(6)：455-458.
[13]Kaplan AP,Kuna P,Reddigari SR.Chemokines and the allergic response[J]. Exp Dermatol.1995,4(2):260-265.
[14]He S,Cao Q,Yoneyama H,etal. MIP-3alpha and MIP-lalpha rapidly mobilize dendritic cell precursors into the peripheral blood.J Leukoc Biol,2008,84(6):1549-1556.
[15]Seki E, De Minicis S, Gwak GY,et al. CCR1 and CCR5 promote hepatic fibrosis in mice. The Journal of clinical investigation[J].2009,119(7):1858-1870.
[16]Wang HK, Park UJ, Kim SY, et al.Free radical production in CA1 neurons induces MIP-1 alpha expression, microglia recruitment, and delayed neuronal death after transient forebrain ischemia[J]. J Neurosci,2008,28(7):1721-1727.
[17]Donghoon Y, Pastore YD, Vladimir D, et al.HIF-la-deficiency results in dysregulated EPO signaling and iron homeostasis in mouse development[J]. J Biol Chem.2006,281(35):25703-25711.
[18]Bel EL,Klimova TA,Eisenbart J,et al.Mitochondrial reactive oxygen species trigger hypoxia-inducible factor-dependent cxtension of the replicative life span during hypoxia[J].Mol Cell Biol,2007,27(2):5737-5745.
[19]Guzy RD, Hoyos B, Robin Er,et al. Mitochondrial complexIII is required for hypoxia-induced ROS production and cellular oxy-gensensing[J]. Cell Metab,2005,1(6):401-408.
[20]Schroedl C,McClintock DS,Budinger GR,et al. Hypoxic but not anoxic stabilization of HIF-1 alpha requires mitochondrial reactive oxygen species[J]. Am J Physiol,2002,283(5):922-L931.
[21]Bell EL, Chande NS. Mitochondrial oxygen sensing:regulation of hypoxia inducible factor by mitochondrial generated reactive oxygen species [J]. Essays Biochem.2007,43:17-27.
[22]Bell EL,Klimova TA,Eisenbart J,et al..The Qosite of the mitochondrial complexIII is required for the transduction of hypoxic signaling via reactive oxygen species production[J].J Cell Biol,2007,177(6):1029-1036.
[23]Chandel NS, McClintock DS, Feliciano CE, et al. Reactive oxygen species generated at mitochondrial complexIII stabilize hypoxia inducible factor-1 alpha during hypoxia:a mechanism of 02 sensing[J]. J Biol Chem,2000,275(33):25130-25138.
[24]Vaupel P. The Role of Hypoxia-Induced Factors in Tumor Progression[J]. Oncologist,2004,9:10-17.
[25]Genbacev O, Zhou Y, Ludlow JW, et al. Regulation of human placental development by oxygen tension[J]. Science,1997;277(5332):1669-1672.
[26]Dery MA, Michaud MD, Richard DE. Hypoxia-inducible factor 1: regulation by hypoxic and non-hypoxic activators [J]. Int J Biochem Cell Bio,2005,37(3):535-540.
[27]LaderouteKR,AminK, Calaoagan JM, et al.5'-AMP-activated protein kinase (AMPK) is induced-by low-oxygen and glucose deprivation conditions found in solid tumormicroenvironments [J].Mol Cell Biol,2006,26(14):5336-5347.
[28]Zagzag D, Zhong H, Scalzitti JM, et al. Expression of hypoxia-inducible factor 1a in brain tumors:association with angiogenesis, invasion, and progression [J]. Cancer,2000,88(11):2606-2018.
[29]Cramer T, Yamanishi Y, Clausen BE, et al.HIF-la is essential for myeloid cell-mediated inflammation [J]. Cell,2003,112(5):645-657.
[30]Scortegagna M,Cataisson C,Martin RJ,et al. HIF-lalpha regulates epithelial inflammation by cell autonomous NFkappaB activation and paracrine stromal remodeling[J]. Blood,2008.111(7):3343-3354.
[31]Brouwer E, Gouw AS, Posthumus MD, et al. Hypoxia inducible factor-1-alpha (HIF-lalpha) is related to both angiogenesis and inflammation in rheumatoid arthritis [J]. Clin Exp Rheumatol,2009,27(6):945-951.
[32]Chang Y, Hsiao G, Chen SH, et al. Tetramethylpyrazine suppresses HIF-lalpha, TNF-alpha, and activated caspase-3 expression in middle cerebral artery occlusion-induced brain ischemia in rats[J]. Acta Pharmacol Sin,2007,28(3):327-333.
[33]Chen C,Hu Q,Yan J,et al. Multiple effects of 2ME2 and D609 on the cortical expression of HIF-lalpha and apoptotic genes in a middle cerebral artery occlusion-induced focal ischemia rat model[J].J Neurochem,2007,102(6):1831-1841.
[34]Baranova O, Miranda LF, Pichiule P, et al. Neuron-specific inactivation of the hypoxia inducible factor 1 alpha increases brain injury in a mouse model of transient focal cerebral ischemia[J]. J Neurosci,2007,27(23):6320-6332.
[35]Helton R, Cui J, Scheel JR,et al. Brain-specific knock-out of hypoxia-inducible factor-1 alpha reduces rather than increases hypoxic-ischemic damage[J]. J Neurosci 2005,25(16):4099-4107.
[36]Rajesh M, Pan H, Mukhopadhyay P,et al.Pivotal Advance:Cannabinoid-2 receptor agonist HU-308 protects against hepatic ischemia/reperfusion injury by attenuating oxidative stress, inflammatory response, and apoptosis[J]. J Leukoc Biol 2007,82(6):1382-1389.
[1]He S,Cao Q,Yoneyama H,etal. MIP-3alpha and MIP-1alpha rapidly mobilize dendritic cell precursors into the peripheral blood.J Leukoc Biol,2008,84(6):1549-1556.
[2]Donghoon Y, Pastore YD, Vladimir D, et al.HIF-1α-deficiency results in dysregulated EPO signaling and iron homeostasis in mouse development[J]. J Biol Chem.2006,281(35):25703-25711.
[3]Semenza GL, Nejfelt MK, Chi SM, et al. Hypoxia-inducible nuclear factors bind to an enhancer element located 3'to the human erythropoietin gene[J]. Proc Natl Acad Sci USA,1991,88(13):5680-5684.
[4]Chun YS, Kim MS, Park JW. Oxygen-dependent and-independent regulation of HIF-1alpha[J]. J Korean Med Sci.,2002,17(5):581-588.
[5]Ivan M, Kondo K, Yang H, et al. HIFalpha targeted for VHL-mediated destruction by proline hydroxylation:implications for O2 sensing[J]. Science,2001,292 (5516)::464-468.
[6]Chen W, Jadhav V, Tang J, et al. HIF-1α inhibition ameliorates neonatal brain injury in a rat pup hypoxic-ischemic model. Neurobiol Dis,2008,31(3): 433-441.
[7]Krick S, Eul BG, Hanze J, et al. Role of hypoxia inducible factor-1α in hypoxia induced apoptosis of primary alveolar epithelial type Ⅱ cells[J]. Am J Resp Cell Molec Bio,2005,32(5):395-403.
[8]Chen C, Hu Q, Yan J, et al.Early inhibition of HIF-1alpha with small interfering RNA reduces ischemic-reperfused brain injury in rats. Neurobiol Dis.2009,33(3):509-517.
[9]Scortegagna M, Cataisson C, Martin RJ, et al. HIF-1alpha regulates epithelial inflammation by cell autonomous NFkappaB activation and paracrine stromal remodeling. Blood,2008,111(7):3343-3354.
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