Hypoxia,angiogenesis and liver fibrogenesis in the progression of chronic liver diseases

Angiogenesis is a dynamic,hypoxia-stimulated and growth factor-dependent process,and is currently referred to as the formation of new vessels from preexisting blood vessels.Experimental and clinical studies have unequivocally reported that hepatic angiogenesis,irrespective of aetiology,occurs in conditions of chronic liver diseases(CLDs) characterized by perpetuation of cell injury and death,inflammatory response and progressive fibrogenesis.Angiogenesis and related changes in liver vascular architecture,that in turn concur to increase vascular resistance and portal hypertension and to decrease parenchymal perfusion,have been proposed to favour fibrogenic progression of the disease towards the end-point of cirrhosis.Moreover,hepatic angiogenesis has also been proposed to modulate the genesis of portal-systemic shunts and increase splanchnic blood flow,thus potentially affecting complications of cirrhosis.Hepatic angiogenesis is also crucial for the growth and progression of hepatocellular carcinoma.Recent literature has identified a number of cellular and molecular mechanisms governing the cross-talk between angiogenesis and fibrogenesis,with a specifi c emphasis on the crucial role of hypoxic conditions and hepatic stellate cells,particularly when activated to the myofibroblast-like pro-fibrogenic.Experimental anti-angiogenic therapy has been proven to be effective in limiting the progression of CLDs in animal models.From a clinical point of view,anti-angiogenic therapy is currently emerging as a new pharmacologic intervention in patients with advanced fibrosis and cirrhosis.

Hypoxia and fatty liver

The liver is a central organ that metabolizes excessive nutrients for storage in the form of glycogen and lipids and supplies energy-producing substrates to the peripheral tissues to maintain their function,even under starved conditions.These processes require a considerable amount of oxygen,which causes a steep oxygen gradient throughout the hepatic lobules.Alcohol consumption and/or excessive food intake can alter the hepatic metabolic balance drastically,which can precipitate fatty liver disease,a major cause of chronic liver diseases worldwide,ranging from simple steatosis,through steatohepatitis and hepatic fibrosis,to liver cirrhosis.Altered hepatic metabolism and tissue remodeling in fatty liver disease further disrupt hepatic oxygen homeostasis,resulting in severe liver hypoxia.As master regulators of adaptive responses to hypoxic stress,hypoxia-inducible factors(HIFs) modulate various cellular and organ functions,including erythropoiesis,angiogenesis,metabolic demand,and cell survival,by activating their target genes during fetal development and also in many disease conditions such as cancer,heart failure,and diabetes.In the past decade,it has become clear that HIFs serve as key factors in the regulation of lipid metabolism and fatty liver formation.This review discusses the molecular mechanisms by which hypoxia and HIFs regulate lipid metabolism in the development and progression of fatty liver disease.

Is the hypoxia-inducible factor-1 alpha mRNA expression activated by ethanol-induced injury, the mechanism underlying alcoholic liver disease?

BACKGROUND: Excessive alcohol consumption can result in multiple organ injury, of which alcoholic liver disease (ALD) is the most common. With economic development and improvement of living standards, the incidence of diseases caused by alcohol abuse has been increasing in China, although its pathogenesis remains obscure. The aim of this study was to investigate the role of hypoxia in chronic ALD. METHODS: Twenty-eight male Sprague-Dawley rats were randomized into a control group (n=12) with a normal history and an experimental group (n=16) fed with 10 ml/ kg of 56% (vol/vol) ethanol once per day by gastric lavage for 24 weeks. At 24 weeks, blood samples were collected and then the rats were killed. Liver samples were frozen at -80 ℃ and used for RT-PCR; other liver samples were obtained for immunohistochemical staining. RESULTS: When the period of alcohol consumption increased, the positive rate of expression of hypoxia- inducible factor-1 alpha (HIF-1α) mRNA was more significantly elevated in the liver of the alcohol group than in the control group (P≤0.05). The HIF-1α protein located in the cytoplasm was seldom expressed in the control group, but significantly in the alcohol group (P≤0.01). CONCLUSION: HIF-1α mRNA expression was activated by ethanol-induced injury in this study, suggesting that hypoxia is involved in the underlying mechanism of ALD.

Hypoxia inducible factor-1αaccumulation in steatotic liver preservation:Role of nitric oxide

AIM:To examine the relevance of hypoxia inducible factor(HIF-1)and nitric oxide(NO)on the preservation of fatty liver against cold ischemia-reperfusion injury(IRI). METHODS:We used an isolated perfused rat liver model and we evaluated HIF-1αin steatotic and non-steatotic livers preserved for 24 h at 4℃in University of Wisconsin and IGL-1 solutions,and then subjected to 2 h of normothermic reperfusion.After normoxic reperfusion,liver enzymes,bile production,bromosulfophthalein clearance,as well as HIF-1αand NO[endothelial NO synthase(eNOS)activity and nitrites/nitrates]were also measured.Other factors associated with the higher susceptibility of steatotic livers to IRI,such as mitochondrial damage and vascular resistance were evaluated. RESULTS:A significant increase in HIF-1αwas found in steatotic and non-steatotic livers preserved in IGL-1 after cold storage.Livers preserved in IGL-1 showed a significant attenuation of liver injury and improvement in liver function parameters.These benefits were enhanced by the addition of trimetazidine(an antiischemic drug),which induces NO and eNOS activation, to IGL-1 solution.In normoxic reperfusion,the presence of NO favors HIF-1αaccumulation,promoting also the activation of other cytoprotective genes,such as hemeoxygenase-1. CONCLUSION:We found evidence for the role of the HIF-1α/NO system in fatty liver preservation,especially when IGL-1 solution is used.

Expression of Hypoxia-inducible Factor-1α in Liver Tumors after Transcatheter Arterial Embolization in an Animal Model

To examine the effect of transcatheter arterial embolization （TAE） of liver tumors on hypoxia-inducible factor-1α （HIF-1α） expression in the residual viable tumor, a total of 30 New Zealand White rabbits implanted with VX2 liver tumor were divided into 2 groups. TAE-treated group animals （n=15） were subjected to TAE with 150–250 μm polyvinyl alcohol particles. Control group animals （n=15） underwent sham embolization with distilled water. Six hours, 3 days or 7 days after TAE, the animals were sacrificed, and samples of tumor and adjacent normal liver tissue were harvested. Expression of HIF-1α protein was examined immunohistochemically. Real-time PCR was performed to examine the HIF-1α mRNA levels. Our results showed that HIF-1α protein was expressed in the VX2 tumors but not in the adjacent normal liver tissue. The HIF-1α-positive tumor cells were located predominantly at the periphery of necrotic tumor regions. The mean levels of HIF-1α protein were significantly higher in TAE-treated tumors than those in control tumors （P=0.002）. Among the three sacrificing time points, the difference in increase in HIF-1α protein was significant between the two groups at the sacrificing time point of 6 h and 3 days after TAE （P=0.020, P=0.031, respectively）, whereas no significant increase was noted 7 days after TAE （P=0.502）. In contrast, although HIF-1α mRNA was expressed in TAE-treated and control VX2 tumors, there existed no significant difference in the HIF-1α mRNA level between the two groups （P=0.372）. It is concluded that TAE of liver tumors increases the expression of HIF-1α at protein level in the residual viable tumor, which could be attributed to hypoxia generated by the procedure.

Cytoprotective role of heme oxygenase-1 and heme degradation derived end products in liver injury

The activation of heme oxygenase-1(HO-1) appears to be an endogenous defensive mechanism used by cells to reduce inflammation and tissue damage in a number of injury models. HO-1, a stress-responsive enzyme that catabolizes heme into carbon monoxide(CO), biliverdin and iron, has previously been shown to protect grafts from ischemia/reperfusion and rejection.In addition, the products of the HO-catalyzed reaction, particularly CO and biliverdin/bilirubin, have been shown to exert protective effects in the liver against a number of stimuli, as in chronic hepatitis C and in transplanted liver grafts. Furthermore, the induction of HO-1 expression can protect the liver against damage caused by a number of chemical compounds. More specifically, the CO derived from HO-1-mediated heme catabolism has been shown to be involved in the regulation of inflammation; furthermore, administration of low concentrations of exogenous CO has a protective effect against inflammation. Both murine and human HO-1 deficiencies have systemic manifestations associated with iron metabolism, such as hepatic overload(with signs of a chronic hepatitis) and iron deficiency anemia(with paradoxical increased levels of ferritin).Hypoxia induces HO-1 expression in multiple rodent,bovine and monkey cell lines, but interestingly, hypoxia represses expression of the human HO-1 gene in a variety of human cell types(endothelial cells, epithelial cells, T cells). These data suggest that HO-1 and CO are promising novel therapeutic molecules for patients with inflammatory diseases. In this review, we present what is currently known regarding the role of HO-1 in liver injuries and in particular, we focus on the implications of targeted induction of HO-1 as a potential therapeutic strategy to protect the liver against chemically induced injury.

Molecular mechanisms of liver preconditioning

Ischemia/reperfusion (I/R) injury still represents an important cause of morbidity following hepatic surgery and limits the use of marginal livers in hepatic transplantation. Transient blood flow interruption followed by reperfusion protects tissues against damage induced by subsequent I/R. This process known as ischemic pre-conditioning (IP) depends upon intrinsic cytoprotective systems whose activation can inhibit the progression of irreversible tissue damage. Compared to other organs,liver IP has additional features as it reduces inflammation and promotes hepatic regeneration. Our present understanding of the molecular mechanisms involved in liver IP is still largely incomplete. Experimental studies have shown that the protective effects of liver IP are triggered by the release of adenosine and nitric oxide and the subsequent activation of signal networks involving protein kinases such as phosphatidylinositol 3-kinase,protein kinase C δ/ε and p38 MAP kinase,and transcription factors such as signal transducer and activator of transcription 3,nuclear factor-κB and hypoxia-inducible factor 1. This article offers an overview of the molecular events underlying the preconditioning effects in the liver and points to the possibility of developing pharmacological approaches aimed at activating the intrinsic protective systems in patients undergoing liver surgery.

Regulation of heme oxygenase expression by alcohol,hypoxia and oxidative stress

AIM:To study the effect of both acute and chronic alcohol exposure on heme oxygenases(HOs) in the brain,liver and duodenum.METHODS:Wild-type C57BL/6 mice,heterozygous Sod2 knockout mice,which exhibit attenuated manganese superoxide dismutase activity,and liver-specific ARNT knockout mice were used to investigate the role of alcohol-induced oxidative stress and hypoxia.For acute alcohol exposure,ethanol was administered in the drinking water for 1 wk.Mice were pair-fed with regular or ethanol-containing Lieber De Carli liquid diets for 4 wk for chronic alcohol studies.HO expression was analyzed by real-time quantitative polymerase chain reaction and Western blotting.RESULTS:Chronic alcohol exposure downregulated HO-1 expression in the brain but upregulated it in the duodenum of wild-type mice.It did not alter liver HO-1 expression,nor HO-2 expression in the brain,liver or duodenum.In contrast,acute alcohol exposure decreased both liver HO-1 and HO-2 expression,and HO-2 expression in the duodenum of wild-type mice.The decrease in liver HO-1 expression was abolished in ARNT+/-mice.Sod2+/-mice with acute alcohol exposure did not exhibit any changes in liver HO-1 and HO-2 expression or in brain HO-2 expression.However,alcohol inhibited brain HO-1 and duodenal HO-2 but increased duodenal HO-1 expression in Sod2+/-mice.Collectively,these findings indicate that acute and chronic alcohol exposure regulates HO expression in a tissue-specific manner.Chronic alcohol exposure alters brain and duodenal,but not liver HO expression.However,acute alcohol exposure inhibits liver HO-1 and HO-2,and also duodenal HO-2 expression.CONCLUSION:The inhibition of liver HO expression by acute alcohol-induced hypoxia may play a role in the early phases of alcoholic liver disease progression.

A Correlative Study between CT Perfusion Parameters and Angiogenesis in Rabbit VX2 Liver Tumors

Objective: The purpose of this study was to evaluate the correlation between CT perfusion parameters and the hypoxia-inducible factor-1 alpha (HIF-1α), vascular en-dothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and microvessel density (MVD) marked by CD34 molecular of rabbit VX2 liver tumors and to investigate the value of CT perfusion imaging in evaluating tumor angiogenesis. Material and methods: Twenty-four cases of rabbit VX2 liver tumor were performed by CT perfusion scanning. Hepatic artery perfusion (HAP), portal vein perfusion (PVP), total hepatic blood flow (THBF) and hepatic perfusion index (HPI) were measured by perfusion software. HIF-1α, VEGF and MMP-2 expression and MVD were detected in the 24 rabbit VX2 liver tumor tissue samples using immunohistochemical method. The correlation between the HIF-1α, VEGF, MMP-2 expression and MVD and CT perfusion parameters were analyzed. Results: Correlation analysis revealed that the expression of HIF-1α, MMP-2, MVD were positively related to the HAP, THBF, HPI (p < 0.01), but no relations with PVP (p > 0.05);and correlation analysis revealed that the expression of VEGF was positively related to the HAP, HPI (p 0.05). There was a positive relationship between the expression of HIF-1α, VEGF, MMP-2 and MVD (p < 0.01). Conclusions: CT perfusion imaging can reflect the blood perfusion of the rabbit VX2 liver tumors and evaluate the information of angiogenesis about tumors.

3种高原裂腹鱼肝脏组织转录组比较分析

青藏高原低温、低氧的极端环境特征是生物适应性进化的天然实验室。裂腹鱼亚科鱼类广泛分布于青藏高原附近水域,在长期进化过程中适应了独特的高原环境。为揭示裂腹鱼适应高原环境的关键信号通路,以2种高海拔(>3000 m)的高原裂腹鱼[巨须裂腹鱼(Schizothorax macropogon)、拉萨裂腹鱼(S.waltoni)]和1种低海拔(<1000 m)的原始裂腹鱼[齐口裂腹鱼(S.prenanti)]为研究对象,对3种裂腹鱼的能量代谢主要组织—肝脏进行转录组测序比较分析。通过对高海拔与低海拔裂腹鱼肝脏转录组的比较,在2种高海拔裂腹鱼中找到共同差异变化的基因106个,其中66个基因表达上调,40个下调。GO和KEGG富集分析显示,氧化还原过程、血液发生相关途径、氨基酸的代谢和类固醇的生物合成是高海拔裂腹鱼应对高原环境的最重要途径。其中,氧化还原和血液生成相关基因(如:faxdc2、cpox、cyp51和NADH-cytochrome b5 reductase 2)的高表达可能在应对高原环境中发挥着重要作用。该研究为鱼类高原低氧适应的分子机制研究提供了新见解。

基于“厥阴伏邪”理论探讨加味连理汤抑制肝癌细胞HepG2浸润侵袭和血管生成的作用机制

目的基于“厥阴伏邪”理论探讨加味连理汤抑制肝癌细胞HepG2浸润侵袭和血管生成的作用机制。方法肝癌细胞HepG2体外培养,采用6孔培养板传代接种细胞,将细胞分为4组,空白组、阴性对照组、缺氧诱导因子(Hypoxia inducible factor,HIF)组与中药干预HIF组,其中阴性对照组造模前转染HIF阴性对照片段,HIF组转染HIF反义RNA-2,中药干预HIF组的干预药物为加味连理汤。细胞转染后24、36 h MTT法检测细胞增殖抑制作用,Transwell小室法检测细胞侵袭能力,流式细胞仪检测细胞凋亡情况,Western Blot法检测DEP结构域含有雷帕霉素靶蛋白相互作用蛋白(DEP domain containing mTOR-interae-ting protein,DEPTOR)、程序性细胞死亡4(Programmed cell death 4,PDCD4)及e-Jun(AP-1)蛋白表达水平。结果细胞转染后24、36 h HIF组与中药干预HIF组细胞增殖指数及侵袭指数均显著低于空白组、阴性对照组,细胞凋亡指数均显著高于空白组、阴性对照组,差异均有统计学意义(P<0.05),且中药干预HIF组细胞增殖指数及侵袭指数均显著低于HIF组,细胞凋亡指数均显著高于HIF组,差异均有统计学意义(P<0.05)。细胞转染后24、36 h,HIF组与中药干预HIF组DEPTOR蛋白表达水平显著低于空白组、阴性对照组,PDCD4及e-Jun(AP-1)蛋白表达水平均显著高于空白组、阴性对照组,差异均有统计学意义(P<0.05),且中药干预HIF组DEPTOR蛋白表达水平显著低于HIF组,PDCD4及e-Jun(AP-1)蛋白表达水平均显著高于HIF组,差异均有统计学意义(P<0.05)。结论加味连理汤温清并用,能够抑制肝癌细胞HepG2的增殖和侵袭,促进其凋亡,介导DEPTOR蛋白表达,激活PDCD4/e-Jun(AP-1)转录激活因子形成的信号轴,抑制血管生成。

贯叶金丝桃提取物对急性缺氧大鼠肝肾损伤的改善作用

【目的】研究贯叶金丝桃提取物(Hypericum perforatum extract,HPE)对急性缺氧大鼠肝肾损伤的改善作用。【方法】通过小鼠常压密闭实验模型初步筛选HPE(20%乙醇提取物、70%乙醇提取物和95%乙醇提取物)的抗缺氧活性组分。进一步建立大鼠低压低氧肝、肾损伤模型:将80只健康大鼠随机分为8组,分别为常氧空白组(NG),缺氧模型组(HG),HPE低、中、高剂量组(HPE-L、HPE-M、HPE-H,100、200、400 mg/kg),金丝桃苷低、高剂量组(HYP-L、HYP-H,50、100 mg/kg)和醋酸地塞米松组(DXM,4 mg/kg)。观察HPE对急性缺氧大鼠肝、肾组织病理学变化的影响,并测定其中氧化应激指标和相关炎症因子的变化。【结果】与模型组比较,给予HPE干预后的大鼠肝、肾组织中丙二醛(MDA)和过氧化氢(H_(2)O_(2))含量下降(P<0.01,P<0.05),谷胱甘肽(GSH)含量增加(P<0.01,P<0.05),总超氧化物歧化酶(T-SOD)和过氧化氢酶(CAT)活力有所提高(P<0.01,P<0.05)。同时,大鼠肝、肾组织中炎症因子白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)和血管内皮生长因子(VEGF)的释放均被抑制(P<0.01,P<0.05)。【结论】HPE对急性缺氧大鼠肝、肾损伤具有一定程度的改善作用。

加味涤痰汤调控miR-149对慢性间歇低氧大鼠肝损伤的影响

目的探讨加味涤痰汤调控mi R-149对慢性间歇低氧大鼠肝损伤的影响及作用机制。方法将18只雄性SD大鼠随机分为对照组、模型组和加味涤痰汤组,每组6只。模型组和加味涤痰汤组于低氧箱中造模,造模期间加味涤痰汤组予加味涤痰汤灌胃,连续12周。HE染色观察肝组织病理变化,透射电镜观察肝组织超微结构,TUNEL染色观察肝细胞凋亡情况,检测肝组织丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、超氧化物歧化酶(SOD)、丙二醛(MDA)含量,RT-qPCR和Western blot检测肝组织miR-149、转录激活因子(ATF)6、葡萄糖调节蛋白-78(GRP78)、C/EBP同源蛋白(CHOP)mRNA和蛋白表达。分析肝组织ATF6 mRNA与miR-149 mRNA表达相关性。结果与对照组比较,模型组大鼠肝组织少量炎性细胞浸润,肝细胞排列不规则、不同程度水肿,内质网扩张,核糖体脱落,线粒体膜破裂,肝细胞凋亡率升高(P<0.05),肝组织ALT、AST、MDA含量升高(P<0.05),SOD含量降低(P<0.05),miR-149 mRNA表达降低(P<0.05),ATF6、GRP78、CHOP mRNA和蛋白表达升高(P<0.05);与模型组比较,加味涤痰汤组大鼠肝组织未见明显水肿及炎性细胞浸润,肝细胞排列规则,内质网稍扩张,核糖体分布较均匀,线粒体膜相对完整,肝细胞凋亡率降低(P<0.05),肝组织ALT、AST、MDA含量降低(P<0.05),SOD含量升高(P<0.05),miR-149m RNA表达升高(P<0.05),ATF6、GRP78、CHOPmRNA和蛋白表达降低(P<0.05)。ATF6 mRNA与miR-149 mRNA水平呈显著负相关(r=-0.766)。结论加味涤痰汤可能通过调控miR-149/ATF6通路抑制慢性间歇低氧大鼠肝脏氧化应激反应,改善肝损伤。

组蛋白去乙酰化酶抑制剂SAHA对慢性间歇性低氧小鼠肝损伤的影响及其机制

目的 探索组蛋白去乙酰化酶抑制剂SAHA对凋亡蛋白Caspase-9/Caspase-3的表达和慢性间歇性低氧小鼠肝损伤的影响及其机制。方法 将雄性C57小鼠随机分为正常组(control组)、慢性间歇性低氧组(CIH组)和SAHA干预组(CIH+SAHA组),将CIH+SAHA组与CIH组小鼠放入低氧仓内进行间歇性低氧处理,每天8 h,持续4周,从第3周起,每日造模前给予实验小鼠腹腔注射SAHA 50 mg/(kg·d),给药持续2周。实验第4周后,测量小鼠体质量,然后处死小鼠,HE染色观察肝组织形态学变化,测定血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)水平,Western blot检测小鼠肝组织中裂解型胱天蛋白酶3(c-Caspase-3)和Caspase-9的蛋白水平,TUNEL试剂盒检测小鼠肝组织细胞凋亡情况。结果 与control组相比,间歇性低氧4周后,CIH组小鼠肝指数显著升高且肝组织损伤明显,肝组织细胞凋亡相关蛋白Caspase-9和c-Caspase-3的表达水平显著增高,肝组织细胞凋亡比例增加(P<0.05);与CIH组相比,CIH+SAHA组肝指数、ALT、AST水平以及Caspase-9和c-Caspase-3蛋白表达水平、肝组织细胞凋亡指标降低(P<0.05)。结论 SAHA可能通过抑制凋亡相关蛋白Caspase-9/Caspase-3,进而改善慢性间歇性低氧引起的小鼠肝损伤。

非酒精性脂肪性肝病患者血清HIF-1α、HMGB1和脂联素水平变化及其与颈动脉粥样硬化的关系研究

目的探讨非酒精性脂肪性肝病(NAFLD)患者血清低氧诱导因子-1α(HIF-1α)、高迁移率族蛋白1(HMGB1)和脂联素(APN)水平变化及其与颈动脉粥样硬化(CAS)的关系。方法2018年5月~2023年3月我院诊治的NAFLD患者158例(其中合并CAS者71例),使用Fibrotouch弹性成像仪诊断脂肪肝,使用超声诊断仪检测颈动脉斑块形成。采用ELISA法检测血清HIF-1α、HMGB1和APN水平,应用二元Logistic回归分析NAFLD合并CAS的影响因素,应用受试者工作特征曲线下面积(AUC)评估血清指标预测NAFLD患者合并CAS的效能。结果合并CAS组收缩压为(137.1±10.3)mmHg,显著高于未合并CAS组【(132.9±8.2)mmHg,P<0.05】;合并CAS组血清TC、LDL-C、HIF-1α和HMGB1水平分别为(6.5±2.3)mmol/L、(3.7±0.6)mmol/L、(25.7±6.5)pg/L和(9.4±2.3)ng/ml,显著高于未合并CAS组【分别(5.1±1.7)mmol/L、(2.8±0.3)mmol/L、(17.2±4.1)pg/L和(6.1±1.5)ng/ml,P<0.05】,而血清APN为(7.5±3.0)mg/L,显著低于未合并CAS组【(12.8±4.6)mg/L,P<0.05】;多因素Logistic回归分析显示,TC(OR=1.411,95%CI:1.133~1.757)、LDL-C(OR=1.419,95%CI:1.128~1.785)、HIF-1α(OR=1.504,95%CI:1.182~1.914)、HMGB1(OR=1.520,95%CI:1.206~1.916)和APN(OR=1.530,95%CI:1.226~1.909)均是影响NAFLD患者合并CAS的独立危险因素(P<0.05);ROC曲线分析显示,血清HIF-1α、HMGB1和APN水平联合预测NAFLD患者合并CAS的AUC为0.863,其敏感度为94.5%,特异度为75.0%,优于各指标单独预测(P<0.05)。结论NAFLD患者血清HIF-1α和HMGB1水平升高而血清APN水平降低是发生CAS的危险因素,应及时发现和给予必要的干预。

基于肝脏转录组测序对不同海拔喜马拉雅旱獭的低氧适应性研究

为探究低、中、高3个不同海拔梯度下喜马拉雅旱獭(Marmota himalayana)基因表达是否存在差异,筛选该物种与高原低氧及强紫外辐射环境适应的相关候选基因,采用Illumina HiSeq 2500高通量测序平台对喜马拉雅旱獭肝脏组织进行无参转录组学分析,通过对质控后测序数据与参考基因组的比较分析,筛选差异表达基因后进行GO功能注释和KEGG富集分析。结果显示:乐都样品(低海拔组,LD)、玉树样品(高海拔组,YS)喜马拉雅旱獭肝脏组织中差异表达基因差异较大,刚察样品(中海拔组,GC)与其他两组间均具有较小的基因表达差异模式。上调表达基因在19个GO Terms和6个KEGG通路上有显著富集,氧化磷酸化相关基因(HiF-1α、MPKA3)、矿物质吸收及脂质代谢相关基因(PPARs)在高海拔组中呈现上调表达,推测这些基因可能直接或间接在喜马拉雅旱獭适应高海拔低氧等极端环境中,通过调整细胞代谢、提高氧化还原能力以及增强免疫反应来维持自身的生理功能和生存能力,为后续深入研究高原野生动物基因多样性以及喜马拉雅旱獭低氧适应性提供基因组学数据。

肝癌组织中缺氧诱导因子-1α的表达及其与血管内皮生长因子的相关性分析

目的探讨肝癌组织中缺氧诱导因子-1α(HIF-1α)的表达及其与血管内皮生长因子(VEGF)的相关性。方法选取2020年1月至2021年12月江西省肿瘤医院收治的94例肝癌患者作为研究对象。比较患者手术前后视觉模拟评分法(VAS)评分和血清HIF-1α、VEGF水平,比较术后1 d和术后7 d不同肝功能Child-Pugh分级、巴塞罗那分期(BCLC)患者的HIF-1α水平,分析HIF-1α水平与肝癌患者各因素的相关性。结果术前及术后1、7 d,患者VAS评分及血清HIF-1α、VEGF水平比较差异有统计学意义(P<0.05);术后1、7 d,患者VAS评分及血清HIF-1α、VEGF水平均高于术前,且术后7 d低于术后1 d,差异有统计学意义(P<0.05)。术后7 d,肝功能Child-Pugh不同分级和BCLC不同分型的患者HIF-1α水平均低于术后1 d,差异有统计学意义(P<0.05)。Pearson相关分析结果显示,患者HIF-1α水平与VAS评分(r=0.485,P<0.001)、VEGF水平(r=0.476,P<0.001)、肝功能ChildPugh分级(r=0.284,P=0.006)、BCLC分期(r=0.250,P=0.016)呈正相关。结论血清HIF-1α与VEGF水平在肝癌患者不同疼痛程度上呈正相关,检查HIF-1α与VEGF水平对肝癌患者具有一定临床意义。

慢性间歇性低氧激活NLRP1炎性小体引起小鼠肝损伤

目的 探讨慢性间歇性低氧(CIH)是否能激活含pyrin结构域核苷酸结合寡聚结构域样受体家族蛋白1(NLRP1)炎性小体引起肝损伤。方法 将C57BL/6雄性小鼠随机分为对照组、 CIH组。CIH组小鼠放入CIH仓进行造模(每天8 h,连续4周)。造模后,采用HE染色观察肝组织细胞形态、试剂盒检测小鼠血清中丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)水平,二氢乙啶(DHE)标记检测肝组织活性氧(ROS)的水平,免疫组织化学染色法检测小鼠肝组织NLRP1、含胱天蛋白酶激活和募集结构域凋亡相关斑点样蛋白(ASC)和胱天蛋白酶1(caspase-1)的表达和定位,Western blot法检测小鼠肝组织中NLRP1、 ASC、 caspase-1、白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)的蛋白表达,ELISA检测小鼠血清IL-1β、 TNF-α水平。结果与对照组相比,CIH组肝细胞病变明显,细胞出现破裂、坏死、炎症细胞聚集,ALT、 AST、 ROS、 IL-1β和TNF-α水平显著升高;NLRP1、 ASC、caspase-1、IL-1β和TNF-α的蛋白表达升高。结论 CIH通过激活NLRP1炎性小体引起肝损伤。

肝细胞癌中HIF-1α依赖性的ATP2C1过表达指示不良预后

缺氧诱导因子(hypoxia-inducible factor, HIF)与肝细胞癌的发生发展相关。HIF-1α在包括肝细胞癌在内的多种癌症类型的发生发展中发挥着重要作用,但其在肝细胞癌中的靶基因尚未完全确定。为找到HIF-1α在肝癌中新的致癌靶点,通过整合HIF-1α敲除的RNA-seq数据,HIF-1α的ChIP-Seq数据,HIF-1α在肝癌中的共表达基因,以及肝癌相关的GEO(Gene Expression Omnibus)数据集,寻找HIF-1α的潜在靶基因。通过分析TCGA(The Cancer Genome Atlas)肝癌数据库、GEO和HPA(Human Protein Atlas)数据集,研究HIF-1α与ATP2C1的相关性,ATP2C1在肝癌中的表达及预后。通过建立物理和化学(氯化钴)缺氧模型验证ATP2C1与低氧及HIF-1α的关系。通过GO(Gene Ontology),KEGG(Kyoto Encyclopedia of Genes and Genomes)和GSEA(Gene Set Enrichment Analysis)分析探索ATP2C1的生物学功能。通过设计体外实验证实ATP2C1对HCC的作用。利用STRING和BioGRID两个蛋白互作在线数据库获得ATP2C1的互作蛋白,并研究其在肝癌中的表达及相关性。通过整合及筛选数据,ATP2C1被鉴定为一个HIF-1α的潜在靶基因。ATP2C1与HIF-1α高度相关,在肝细胞癌中高表达,且伴随有不良预后。富集分析与体外实验的结果表明ATP2C1参与调控HCC细胞的增殖迁移。蛋白互作数据表明ATP2C1与TMEM165存在互作关系,生存分析表明TMEM1651高表达的肝癌患者预后较差。相关性分析的结果显示ATP2C1与肝癌中TMEM165和MMP2的表达高度相关,表明ATP2C1可能与TMEM165和MMP2存在互作关系,并参与了肝癌的进展过程。结果表明,ATP2C1是HIF-1α的靶基因和肝细胞癌的生物标志物,其敲低抑制了HCC的增殖和迁移。

黄芪甲苷基于Nrf2/HO-1通路对模拟急进高原大鼠肝脏氧化应激损伤的保护性作用研究

目的:探讨缺氧不同时间对肝脏氧化应激水平的影响及肝肾功能发生的变化,研究黄芪甲苷基于Nrf2/HO-1通路对模拟急进高原大鼠肝脏的保护性作用。方法:建立模拟高原急性缺氧动物模型,56只SD大鼠随机分为对照组(Control),缺氧组(H),黄芪甲苷组(AS-IV+H),H组和AS-IV+H组进一步分为12 h,24 h和48 h亚组,每组8只,共7组。AS-IV+H组给予黄芪甲苷灌胃(80 mg/kg),1次/d,连续7d。将实验组大鼠放入低压氧舱(模拟海拔5000 m),到达预定时间后,分别检测大鼠血清肝肾功,肝组织中超氧化物歧化酶(SOD),丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-PX)的含量;蛋白印记法和荧光定量PCR分别检测肝脏组织中Nrf2、HO-1蛋白表达及其基因表达水平;肝组织的病理学表现通过HE染色观察。结果:与常氧环境的对照组比较,不同缺氧时间段的H组和AS-IV+H组肝肾功指标均高于对照组,且随缺氧时间的增加,呈现增高的趋势(P<0.05或P<0.01)。组织匀浆中抗氧化酶除MDA高于对照组外,其余抗氧化酶超氧SOD、GSH-PX水平均低于对照组(P<0.05或P<0.01)。肝组织中Nrf2、HO-1蛋白及mRNA表达随缺氧时间的延长而升高,在缺氧24h达到高峰,缺氧48h表达较缺氧24h有所下降,但在各时间段均高于对照组(P<0.05或P<0.01)。与同时间缺氧组比较,黄芪甲苷组肝肾功,SOD,GSH-PX均低于同时间缺氧,但MDA高于同时间缺氧组(P<0.05或P<0.01)。肝组织中Nrf2、HO-1蛋白及mRNA表达均高于同时间缺氧组(P<0.05或P<0.01)。HE染色显示缺氧组肝细胞水肿,黄芪甲苷组水肿减轻。结论:急进高原缺氧环境导致肝肾功能出现异常,肝脏组织发生相关的病理学改变,黄芪甲苷干预能提高高原低氧环境下肝脏对抗氧化应激损伤的能力,保护低氧环境下的肝肾功能,提高机体在高原低氧环境下的适应性能力。