五味子乙素对HK-2细胞缺氧损伤的保护作用

卢爱龙 1, 谭小月 2, 张勉之 3△, 吴银娜摘要： 目的 探讨五味子乙素 （Sch B） 对氯化钴 （CoCl2） 诱导的人类近端肾小管上皮 （HK-2） 细胞缺氧损伤的保护作用及其可能机制。方法 取离体培养 HK-2 细胞, 随机分为 4 组。对照 （C） 组： 细胞未经任何处理。CoCl2组 （化学乏氧组）： 加入 600 μmol/L 的 CoCl2 处理 24 h。Sch B 预保护（CoCl2+ Sch B）组： 分别加入终浓度为 1 μmol/L 和 10 μmol/L Sch B 预处理 2 h 后, 其余操作同 CoCl2 组。Sch B 组： 分别加入终浓度 1 μmol/L 和 10 μmol/L Sch B 处理 2 h。CCK-8 试剂盒检测各组细胞活性; AnnexinV-FITC/PI 双标记流式细胞仪检测各组细胞凋亡率; Western Blot 检测各组缺氧诱导因子-1α（HIF-1α）蛋白表达; RT-PCR 检测各组 HIF-1α和诱导型一氧化氮合酶（iNOS） mRNA 表达。结果 与对照组相比, CoCl2组细胞活性明显降低, 细胞凋亡率、 HIF-1α蛋白表达量和 iNOS mRNA 表达量显著增加, HIF-1α mRNA 表达量差异无统计学意义; Sch B 预保护组较 CoCl2组细胞活性显著增加, 细胞凋亡率、 HIF-1α蛋白表达量、 HIF-1α及 iNOS mRNA 表达量均显著减少; Sch B 组与对照组细胞活性、 细胞凋亡率差异无统计学意义, Sch B 组几乎不表达 HIF-1α蛋白。结论 Sch B 可能通过抑制 HIF-1α蛋白和 iNOS mRNA 的表达减少 HK-2 细胞的凋亡, 从而对 HK-2 细胞缺氧损伤起保护作用。     

介导缺氧诱导因子-1α表达的相关信号通路在类风湿性关节炎中的研究进展

类风湿性关节炎(rheumatoid arthritis, RA)是一种系统性自身免疫性疾病,其病情的发生发展与关节缺氧微环境密切相关。缺氧诱导因子1α(hypoxia-inducible factor-1α, HIF-1α)是调节细胞对缺氧反应的核转录因子,在促进滑膜炎症、血管新生及血管翳形成、软骨破坏及骨侵蚀方面发挥重要作用。HIF-1α的表达除受氧感受器调节外,还受多条信号转导途径的调控,包括PI3K/Akt、Ras-Raf-MEK-ERK、JAK/STAT、NF-κB等。该文就参与调控HIF-1α表达的信号通路进行概述,以期为治疗RA以及寻找新的改善病情药物提供新的思路和依据。     

缺氧诱导因子-1调控转录及相关转录抑制剂的研究进展

细胞低氧适应性反应可通过转录调节蛋白——缺氧诱导因子-1（HIF-1)和p300/CBP辅激动因子对低氧应答基因的转录激活介导。HIF-1是药学上重要的靶点，设计以HIF-1α/300复合物为靶点的小分子转录抑制剂，可用于心脑血管疾病或肿瘤的治疗。本文就HIF-1α与p300的相互作用，以及氧依赖的特殊氨基酸羟化对HIF-1α蛋白稳定性和转录激活调控的研究进展作一综述。     

缺氧诱导因子-1与宫颈癌关系的研究进展

缺氧诱导因子-1(hypoxia-inducible factor-1,HIF-1)就是一个在缺氧状态下重要的转录调节因子.HIF-1在人体各种肿瘤中的过度表达,提示其与肿瘤进展和转移密切相关[1].近年来研究显示HIF-1与宫颈癌的关系密切,因此,了解HIF-1在宫颈癌中的研究进展具有重要意义.1缺氧诱导因子-1的生物学特性1.1结构缺氧诱导因子l(hypoxia-inducible factor1,HIF-1)于1992年在低氧诱导促红细胞生成素(erythropoietin,EPO)基因的转录激活中发现,能特异性地结合于促红细胞生成素编码区下游的缺氧反应元件(hypoxia response element,HRE)的特异性DNA序列.HIF-1为异二聚体蛋白聚合物,由120KD的α亚基和91-94KD3亚基组成.HIF-1β在任何状态下都表达,而HIF-1α只在氧浓度低于6％时才表达,正常氧浓度下IF-1的半衰期＜1-2min[2],所以HIF-1α是HIF-1的功能亚单位.1.2机制HIF-1α的401 -603氨基酸残基位有一个氧依赖性降解区,对HIF-1α的稳定性起重要作用.     

缺氧诱导因子 1 在卵巢癌中的研究进展

1988年研究人员在研究低氧和低氧诱导的促红细胞生成素（EPO）基因表时发现了低氧对基因表达的作用,随后于1995年Wang等和Semenza等发现了缺氧诱导因子1（hypoxia-inducible factor-1,HIF-1）转录因子和低氧反应元件（HRE）。     

大鼠肺动脉平滑肌细胞原代培养及低氧对缺氧诱导因子- 1α的影响

目的获得活力稳定、高度纯化的体外培养大鼠血管平滑肌细胞,为肿瘤研究提供实验材料;并探讨低氧对抗肿瘤药物研究新靶点：缺氧诱导因子-1α（HIF-1α）的影响。方法无菌下取SD大鼠心肺组织,然后分离出肺动脉段,分离肺动脉中膜层,用组织块贴壁法培养,倒置相差显微镜观察细胞形态、普通免疫组织化学法（SP法）进行细胞鉴定。随后,将体外培养的大鼠PASMCs,设计常氧组、低氧组H2,H6,H12,H24,用Western blotting法检测HIF-1α的蛋白表达。结果用组织贴块法成功培养大鼠PASMCs,得到生长稳定、纯度高的PASMCs,且培养与纯化可以同时进行,可获得稳定传代的细胞;HIF-1α蛋白在常氧组有少量表达,在低氧条件下,其表达明显增加。结论成功建立体外大鼠肺动脉平滑肌细胞原代培养模型;低氧条件下促进其中HIF-1α的表达;这提示HIF-1α蛋白的表达可能是肿瘤细胞适应缺氧环境的重要原因。     

脂多糖对小胶质细胞血管内皮生长因子表达的影响

摘要： 目的 探讨脂多糖（LPS）是否能够通过低氧诱导因子-1α（HIF-1α）调节小胶质细胞血管内皮生长因子（VEGF） 的表达。方法 培养 BV2 小胶质细胞系, 分为 4 组： 对照组、 LPS （100 µg/L） 刺激组、 LRS 干预组 （LPS 100 µg/L及脂多糖拮抗剂 200 µg/L）, HIF-1α抑制剂干预组 （LPS 100 µg/L 及 FM19G11 10 mmol/L）。分别采用免疫荧光、 Western blot、 ELISA 检测 VEGF 和 HIF-1α 的表达情况。结果 与对照组比较, LPS 可显著增加 BV2 细胞 VEGF 表达, 脂多糖拮抗剂可明显抑制 LPS 的这一作用。HIF-1α的表达在 LPS 刺激 8 h 后明显增加。HIF-1α抑制剂 FM19G11 可减少 LPS 引起的 BV2 细胞 VEGF 表达增加。结论 LPS 促进小胶质细胞 VEGF 的表达, HIF-1α参与这一调节过程。     

缺氧诱导因子-1与血液肿瘤

缺氧诱导因子-1（HIF-1）是缺氧条件下广泛存在于哺乳动物和人体内的一种转录因子，通过上调一些与血管新生和糖酵解有关的基因来介导组织细胞对缺氧的适应，是一种重要的缺氧应答调控因子，其生物学效应由HIF-1亚基实现。HIF-1在维持氧稳定、细胞能量代谢、肿瘤血管形成、细胞增殖与凋亡等很多方面都起着重要作用。本文就HIF-1的生物学特性，HIF-1在血液肿瘤发生发展中的作用及其对预后的影响等研究进展作一综述。     

缺氧诱导因子-1α与妇科肿瘤

缺氧诱导因子-1α（HIF-1α）是一类重要的缺氧应答调控因子,与肿瘤的发生、发展密切相关。其在维持氧稳定、细胞的能量代谢和肿瘤血管生成以及转移、细胞增殖、凋亡等方面有重要的作用。HIF-1α已成为近年来在肿瘤学术研究方面的一个热点,现就HIF-1α与妇科肿瘤的关系作以概述。     

缺氧对食管癌细胞缺氧诱导因子-1α及己糖激酶表达的影响

目的探讨不同缺氧程度对食管癌细胞株TE1中缺氧诱导因子-1α（Hypoxia-inducible factor-1α,HIF-1α）及糖酵解关键酶表达的影响。方法 TE1细胞分别在正常氧分压和缺氧条件下培养,缺氧时间设定为6、12、24及48h,使用Western blot方法检测缺氧培养不同时间后细胞中HIF-1α及糖酵解关键酶己糖激酶II（Hexokinase-II,HK-II）的蛋白水平表达变化。结果常氧及缺氧条件下TE1细胞中HIF-1α及HK-II均有表达,缺氧后表达均较常氧培养时明显增强,且随缺氧时间不同而呈先增多后减少的动态变化。结论低氧能够增加食管癌细胞中HIF-1α及HK-II表达而促进糖酵解进程,联合抑制HIF-1α和糖酵解酶可能成为治疗食管癌的潜在的靶点。     

Attenuated expression and function of the RECK tumor suppressor under hypoxic conditions is mediated by the MAPK signaling pathways

Downregulation of the tumor suppressor, reversion-inducing cysteine-rich protein with Kazal motifs (RECK) has been reported under hypoxic conditions (Lee et al., 2010); however, the signaling pathways involved in this downregulation have not yet been identified. Hypoxia causes the silencing of RECK mRNA expression, but treatment with inhibitors of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated kinase (MAPK) (PD98059, SP600125, and SB203580 respectively) or their dominant negative mutants recovered RECK suppression induced by hypoxia as analyzed with semiquantitative RT-PCR analysis and a RECK promoter luciferase assay. Hypoxia increased phosphorylation of ERK1/2, JNK and p38 MAPKs. The activities of matrix metalloproteinase (MMP)-9 and MMP-2 were increased under hypoxic conditions but treatment with PD98059, SP600125 and SB203580 inhibited their activation in cancer cells, as seen by zymography. Moreover, treatment with the inhibitors blocked cancer cell migration induced by hypoxia in H-Ras transformed MCF10A mammary cells. RECK suppression under hypoxic conditions was inversely related to HIF-1α expression; however, treatment with PD98059, SP600125 and SB203580 did not influence binding of HIF-1α to the reverse hypoxia responsive element site of the RECK promoter in a DNA precipitation assay. These results suggest that the ERK, JNK and p38 MAPK signaling pathways are indirectly involved in RECK suppression but are not involved in the binding activity of HIF-1α to the reverse hypoxia responsive element site on the RECK promoter under hypoxic conditions.     

Bilobalide abates inflammation,insulin resistance and secretion of angiogenic factors induced by hypoxia in 3T3-L1 adipocytes by controlling NF-κB and JNK activation

Obesity leads to inflammation and insulin resistance in adipose tissue. Hypoxia, observed in obese adipose tissue is suggested as a major cause of inflammation and insulin resistance in obesity. However, the role of hypoxia in adipose tissue during obesity and insulin resistance was not well established. Here we mainly explored the crosstalk between hypoxia induced inflammation, and insulin resistance and also secretion of angiogenic factors in 3T3-L1 adipocytes and possible reversal with bilobalide. Hypoxia for 24 h significantly (P ≤ 0.05) increased the secretion of MCP-1 (4.59 fold), leptin (2.96 fold) and reduced adiponectin secretion (2.93 fold). In addition, the mRNA level of resistin (6.8 fold) and TLR4 receptors (8.8 fold) was upregulated in hypoxic adipocytes. The release of inflammatory cytokines and expression of TLR4 receptors led to activation of JNK and NF-κB signalling. We further investigated the effects of JNK and NF-κB activation on insulin signalling receptors. The present study showed increased (P ≤ 0.05) serine 307 phosphorylation of IRS-1 (1.9 fold) and decreased expression of IRS-2 (0.53 fold) in hypoxic group showing hypoxia induced impairment in insulin signalling. Hypoxia significantly (P ≤ 0.05) increased basal glucose uptake (3.3 fold) as well as GLUT-1 expression in adipocytes indicating GLUT-1 mediated glucose uptake. Hypoxia for 24 h significantly increased (P ≤ 0.05) the expression of angiogenic factors. Bilobalide protected adipocytes from hypoxia induced inflammation and insulin resistance mainly by reducing inflammatory adipokine secretion, improving adiponectin secretion, reducing NF-κB/JNK activation, and inhibiting serine phosphorylation of IRS-1 receptors of insulin signalling pathway.     

Amelioration of ER stress by 4-phenylbutyric acid reduces chronic hypoxia induced cardiac damage and improves hypoxic tolerance through upregulation of HIF-1α

While endoplasmic reticulum (ER) stress has been observed in several human diseases, few studies have reported the involvement of ER stress in chronic hypoxia (CH) induced cardiac damage. Hypoxia, such as that prevalent at high altitude (HA), forms the underlying cause of several maladies including cardiovascular diseases. While the role of hypoxia inducible factor-1 (HIF-1α) in the adaptive responses to hypoxia is known, the role of the unfolded protein response (UPR) is only recently being explored in the HA pathophysiologies. The present study investigates the effect of ER stress modulation on CH mediated injury and the cardioprotective action of 4-phenylbutyric acid (PBA) in enhancing survival response under hypoxia. Here, we observed that exposure of rats, for 1, 7 and 14 days CH to a simulated altitude of 7620 m, led to cardiac hypertrophy and significant protein oxidation. This induced the activation of UPR signaling mechanisms, mediated by PERK, IRE1α and ATF6. By 14 days, there was a marked upregulation of apoptosis, evident in increased CHOP and caspase-3/9 activity. PBA reduced CH induced right ventricular enlargement and apoptosis. Further, in contrast to tunicamycin, PBA considerably enhanced hypoxic tolerance. An elevation in the level of antioxidant enzymes, HIF-1α and its regulated proteins (HO-1, GLUT-1) was observed in the PBA administered animals, along with a concomitant suppression of UPR markers. Our study thus emphasizes upon the attenuation of ER stress by PBA as a mechanism to diminish CH induced cardiac injury and boost hypoxic survival, providing an insight into the novel relationship between the HIF-1α and UPR under hypoxia.     

Vascular Endothelial Growth Factor Signaling in Hypoxia and Inflammation

Infection, cancer and cardiovascular diseases are the major causes for morbidity and mortality in the United States according to the Center for Disease Control. The underlying etiology that contributes to the severity of these diseases is either hypoxia induced inflammation or inflammation resulting in hypoxia. Therefore, molecular mechanisms that regulate hypoxia-induced adaptive responses in cells are important areas of investigation. Oxygen availability is sensed by molecular switches which regulate synthesis and secretion of growth factors and inflammatory mediators. As a consequence, tissue microenvironment is altered by re-programming metabolic pathways, angiogenesis, vascular permeability, pH homeostasis to facilitate tissue remodeling. Hypoxia inducible factor (HIF) is the central mediator of hypoxic response. HIF regulates several hundred genes and vascular endothelial growth factor (VEGF) is one of the primary target genes. Understanding the regulation of HIF and its influence on inflammatory response offers unique opportunities for drug development to modulate inflammation and ischemia in pathological conditions.     

Hypoxia-induced iNOS expression in microglia is regulated by the PI3-kinase/Akt/mTOR signaling pathway and activation of hypoxia inducible factor-1alpha

Exposure to hypoxia induced microglia activation and animal studies have shown that neuronal cell death is correlated with microglial activation following cerebral ischemia. Thus, it is likely that toxic inflammatory mediators produced by activated microglia under hypoxic conditions may exacerbate neuronal injury following cerebral ischemia. The hypoxia-inducible factor-1 (HIF-1) is primarily involved in the sensing and adapting of cells to changes in the O(2) level, which is regulated by many physiological functions. However, the role of HIF-1 in microglia activation under hypoxia has not yet been defined. In the current work, we investigate the signaling pathways of HIF-1alpha involved in the regulation of hypoxia-induced overexpression of inducible NO synthase (iNOS) in microglia. Exposure of primary rat microglial cultures as well as established microglial cell line BV-2 to hypoxia induced the expression of iNOS, indicating that hypoxia could lead to the inflammatory activation of microglia. iNOS induction was accompanied with NO production. Moreover, the molecular analysis of these events indicated that iNOS expression was regulated by the phosphatidylinositol 3-kinase (PI3-kinase)/AKT/ mammalian target of rapamycin (mTOR) signaling pathway and activation of hypoxia inducible factor-1alpha (HIF-1alpha). Thus, during cerebral ischemia, hypoxia may not only directly damage neurons, but also promote neuronal injury indirectly via microglia activation. In this study, we demonstrated that hypoxia induced iNOS expression by regulation of HIF-1alpha in microglia.     

低氧诱导因子-2α基因沉默对骨肉瘤细胞株MG-63生物学行为的影响

【摘要】 目的 探讨低氧诱导因子-2（HIF-2α）在骨肉瘤形成过程中的作用以及HIF-2α基因沉默对低氧状态下骨肉瘤MG-63细胞的影响。 方法 小干扰RNA（siRNA）沉默HIF-2α基因,获得MG-63/siHIF-2α（siHIF-2α）,阴性对照为MG-63/scramble(NC)细胞,分为siHIF-2α组和NC组,MTS试剂检测细胞活性,划痕迁移试验检测细胞迁移能力,Western Blot检测HIF-2α、VEGF、p-Erk1/2及Mcl-1的表达,集落形成试验和裸鼠皮下移植瘤试验证实HIF-2α基因沉默对肿瘤生长的影响。 结果 低氧诱导HIF-2α蛋白表达。低氧12h和24h,siHIF-2α组细胞活性均低于NC组（P=0.0211, 0.0017）,siHIF-2α组相对划痕宽度均大于NC组(P=0.0024, <0.0001)。 1000-5000细胞种植数的siHIF-2α组的集落形成率均小于NC组(P=0.0027, 0.0038, 0.0063, 0.0276, 0.0062)。HIF-2α基因沉默抑制HIF-2α、VEGF、p-Erk1/2和Mcl-1的表达。裸鼠皮下移植瘤siHIF-2α组肿瘤体积和重量均小于NC组(P=0.006, <0.0001)。 结论 HIF-2α对骨肉瘤的存活、迁移及生长有促进作用,HIF-2α基因沉默可作为骨肉瘤的临床治疗的新策略。      

Casticin suppresses monoiodoacetic acid-induced knee osteoarthritis through inhibiting HIF-1α/NLRP3 inflammasome signaling

Knee osteoarthritis (KOA) is a disabling chronic inflammatory disease that is closely associated with synovium tissue hypoxia and synovial fibrosis. Casticin, a compound purified from the Chinese herb Viticis Fructus, has been proved effective in preventing inflammation and fibrosis in previous studies. However, the effect of casticin on synovial fibrosis in KOA is not clear. In present study, we aimed to investigate how did casticin affect synovial fibrosis on monoiodoacetic acid (MIA)-induced KOA in rats. The MIA-induced knee osteoarthritis model and lipopolysaccharide (LPS) stimulated primary synovial fibroblasts inflammation model were established. Pathological and morphological changes in synovial tissue were observed by H&E and sirius red staining. The hypoxia of synovium was detected by pimonidazole staining and immunohistochemistry of hypoxia-inducible factors 1α (HIF-1α). The levels of nucleotide oligomerization domain-like receptor protein 3 (NLRP3) inflammasome components, fibrogenic markers (TGF-β, COL1A1 and TIMP1) and inflammatory cytokines were examined by western blotting, qRT-PCR or ELISA in both KOA rat models and primary synovial fibroblasts. Our data suggested that casticin improved hypoxia and inflammation in synovium tissue, as well the synovial fibrosis in rats. Besides, casticin inhibited the activation of NLRP3 inflammasome in MIA-induced KOA rats and synovial fibroblasts. In conclusion, our findings demonstrated that casticin alleviated MIA-induced KOA by inhibiting of HIF-1α/NLRP3 inflammasome activation. Therefore, casticin could be a potential treatment strategy for KOA.