丁酸钠通过调节SIRT1/AMPK信号通路对慢性肾衰竭大鼠肾功能的影响

摘要 目的：探讨丁酸钠（NaB）通过调节沉默信息调节因子1（SIRT1）/单磷酸腺苷活化蛋白激酶（AMPK）信号通路对慢性肾衰竭（CRF）大鼠肾功能的影响。方法：选用SD大鼠72只,随机分为6组（12只/组）：control组、Model组、NaB低剂量组（100 mg/kg）、NaB中剂量组（200 mg/kg）、NaB高剂量组（400 mg/kg）、抑制剂组（400 mg/kg NaB+2 mg/kg SIRT1/AMPK通路抑制剂EX527）;采用饲喂0.5%腺嘌呤饲料以建立CRF大鼠模型,建模成功后,灌胃和腹腔注射相应药物。使用试剂盒检测大鼠24 h尿蛋白（24 h U-pro）、血清肌酐（SCr）、尿素氮（BUN）水平;采用苏木精-伊红（HE）及Masson染色法观察肾脏病理改变,并计算胶原容积分数（CVF）;采用茜素红染色法与主动脉钙含量测定评估主动脉钙化情况;采用酶联免疫吸附法（ELISA）检测各组大鼠肾脏组织白细胞介素（IL）-6、IL-β、肿瘤坏死因子（TNF）-α水平;采用过氧化氢酶（CAT）、丙二醛（MDA）、活性氧（ROS）检测试剂盒检测大鼠肾脏组织中CAT、MDA、ROS水平;采用蛋白免疫印迹法（WB）检测各组大鼠SIRT1/AMPK信号通路及骨形态发生蛋白2（BMP2）、Runt相关转录因子2（Runx2）蛋白的表达。结果：与control组比较,Model组大鼠肾脏组织损伤严重、胶原纤维沉积显著、主动脉钙化严重,CAT活性、SIRT1、p-AMPK/AMPK表达水平显著降低（P<0.05）,CVF、主动脉钙含量和SCr、BUN、24 h U-pro、IL-6、IL-β、TNF-α、MDA、ROS水平及BMP2、Runx2蛋白表达水平显著升高（P<0.05）;与Model组比较,NaB低、中、高剂量组大鼠肾脏组织损伤减轻、胶原纤维沉积面积明显减少、主动脉钙化程度减轻,CVF、主动脉钙含量和SCr、BUN、24 h U-pro、IL-6、IL-β、TNF-α、MDA、ROS水平及BMP2、Runx2蛋白表达水平显著降低（P<0.05）,CAT活性、SIRT1、p-AMPK/AMPK表达水平显著升高（P<0.05）;SIRT1/AMPK通路抑制剂EX527可降低高剂量NaB对CRF大鼠主动脉钙化和肾功能的改善作用（P<0.05）。结论：NaB可能通过激活SIRT1/AMPK信号通路,减轻肾脏组织炎症、氧化应激损伤、主动脉钙化和肾纤维化,从而起到改善CRF大鼠肾功能的作用。     

槲皮素调节AMPK/SIRT1/NF-κB通路对乙型肝炎大鼠肝组织损伤的影响

摘要 目的：探究槲皮素调节AMPK/SIRT1/NF-κB通路对乙型肝炎（HB）大鼠肝组织损伤的影响。方法：采用随机数字表法将65只Wistar大鼠分为Ctrl组、HB组、槲皮素低剂量组（槲皮素L组,50 mg/kg）、槲皮素高剂量组（槲皮素H组,200 mg/kg）及槲皮素H+AMPK抑制剂组（200 mg/kg槲皮素+10 mg/kg Compound C）,每组各13只,采用尾静脉注射携带1.3拷贝HBV基因组的重组8型腺相关病毒（rAAV8-1.3HBV）法建立HB大鼠模型（Ctrl组除外）;酶联免疫吸附实验（Elisa）检测血清中HB表面抗原（HBsAg）、HB e抗原（HBeAg）、肝功能指标[谷丙转氨酶（ALT）、谷草转氨酶（AST）、总胆红素（TBIL）]水平及肝组织中炎性因子[白介素-1β（IL-1β）、肿瘤坏死因子α（TNF-α）]水平;实时荧光定量PCR（qRT-PCR）检测血清中HBV-DNA水平;苏木素-伊红（HE）染色、曼森氏（Masson）染色观察肝组织病理改变;免疫印迹法（WB）检测肝组织中AMPK/SIRT1/NF-κB通路蛋白表达水平。结果：与Ctrl组比较,HB组血清中HBsAg、HBeAg、HBV-DNA、ALT、AST、TBIL水平均升高（P<0.05）;肝组织可见肝静脉扩张、充血,肝细胞排列紊乱、水肿、坏死,同时发生明显纤维化;肝组织中AMPK磷酸化、SIRT1蛋白水平均降低,IL-1β、TNF-α水平及核NF-κB蛋白水平均升高;经槲皮素L、槲皮素H干预后上述情况均得到改善,且槲皮素H干预改善更明显（P<0.05）;而增加AMPK抑制剂干预后,槲皮素H干预的改善作用被削弱（P<0.05）。结论：槲皮素能够减轻HB大鼠肝组织损伤,保护其肝功能,其机制可能与调节AMPK/SIRT1/NF-κB通路有关。     

白细胞介素-1通过JNK信号通路调控Schlafen2基因的表达

目的：在多效生长因子（Ptn）基因稳定沉默的小鼠胚胎成纤维细胞Ptn-siRNA B/MEF241中,研究白细胞介素-1（IL-1）调控Schlafen2（Slfn2）基因表达的机制。方法：应用Northernblot检测Ptn沉默细胞Ptn-siRNAB/MEF241处于不同生长密度时Slfn2基因的表达变化,以确定Ptn沉默细胞中Slfn2基因的表达是否受到某种分泌性细胞因子的调控;用不同浓度的IL-1α中和抗体及IL-1受体拮抗剂处理Ptn沉默细胞,通过Northern blot检测细胞内Slfn2表达的抑制情况;用不同浓度的IL-1α中和抗体及IL-1受体拮抗剂处理Ptn沉默细胞不同时间,通过Western blot检测细胞中JNK磷酸化水平;Northern blot检测SP600125（JNK/MAPK通路抑制剂）对Ptn沉默细胞中Slfn2基因表达的影响。结果：Ptn沉默细胞中Slfn2基因的表达水平同细胞密度相关;用中和抗体和受体拮抗剂阻断IL-1通路,Slfn2表达受到显著抑制;IL-1受到抑制会影响JNK通路的活化;阻断JNK通路,Slfn2的表达受到显著抑制。结论：IL-1可以通过JNK通路诱导Slfn2的表达。     

白细胞介素-1信号转导通路上的两个新分子

白细胞介素 1β(interleukin 1β,IL 1β)为重要的多效性细胞因子,参与炎症反应、神经内分泌调控、大脑学习和记忆等功能,探究其受体后信号转导通路的分子组成,以及分子间相互作用,是揭示其生物学作用的重要步骤。目前,除2 0 0 0年瑞士学者发现并命名的新分子Tollip (Toll inte     

α-硫辛酸通过激活AMPK/mTOR通路改善2型糖尿病大鼠肝脏病变

目的:探讨α-硫辛酸是否通过激活腺苷酸活化蛋白激酶(AMPK)/雷帕霉素靶蛋白(mTOR)通路改善2型糖尿病(T2DM)大鼠肝损伤。方法:应用高糖高脂饮食联合链脲佐菌素27.5 mg/(kg·d)腹腔注射法制备T2DM大鼠模型。将32只成模大鼠随机分为4组：T2DM组、α-硫辛酸组、Compound C(AMPK抑制剂)组和α-硫辛酸+Compound C组,每组各8只。另8只健康Sprague-Dawlay(SD)大鼠作为正常对照组。α-硫辛酸注射液100 mg/(kg·d)腹腔注射,Compound C 20 mg/(kg·d)腹腔注射,药物干预共持续8周。检测相关生化指标;计算肝脏质量指数;进行光镜、电镜观察;采用Western blot方法检测大鼠肝脏中AMPK、p-AMPK、mTOR、p-mTOR蛋白的表达水平。结果:与正常对照组相比,T2DM组大鼠肝脏质量指数、胰岛素抵抗指数、空腹血糖、谷丙转氨酶、谷草转氨酶、γ–谷氨酰转肽酶、甘油三酯水平均升高(P均<0.05);肝组织结构损伤,脂肪变明显;肝脏组织中p-AMPK表达显著减少(P<0.05),p-mTOR表达显...     

白细胞介素-10、白细胞介素-6对慢性肺源性心脏病预后评价的临床意义

目的:检测慢性肺源性心脏病患者血清白细胞介素-10和白细胞介素-6的水平,探讨细胞因子对慢性肺源性心脏病患者预后评价的临床意义。方法:将我院住院治疗的慢性肺源性心脏病患者40例,按照心功能情况分为肺源性心脏病组21例(对照组)和肺源性心脏病合并心力衰竭组19例(观察组),进行血清白介素-6、白介素-10定量检测。结果:观察组血清白介素-6水平较对照组明显升高,而白细胞介素-10却明显低于对照组(P值均<0.05)。结论:检测白细胞介素-6、白细胞介素-10的水平,可作为监测肺源性心脏病心功能恶化的预测指标。     

白细胞介素-6与肝纤维化的关系

肝纤维化是肝纤维结缔组织异常增殖的一个重要病理变化,也是形成肝硬化的必要条件。白细胞介素6(interleukin 6,IL-6)是一种具有多效生物活性的炎性细胞因子,其在肝纤维化形成过程中起着关键性的作用。大量研究表明IL-6与肝纤维化的发生发展密切相关。本文就IL-6与病毒、药物、毒物、肥胖、酒精、胆汁淤积性及自身免疫等原因所致肝纤维化的相关性做简要阐述,以期能为肝纤维化的诊断及其预后提供一种新的非侵入性标志物。     

中枢白细胞介素-1在应激反应中的作用

白细胞介素-1（interleukin-1,IL-1）系统分子广泛分布于中枢神经系统。中枢IL-1具有极其丰富的生物学功能,作为经典炎性细胞因子,在多种生理、病理生理过程中起重要作用。近几年来,中枢IL-1的应激介质作用备受关注。本文综述了中枢IL-1在应激反应中作用的最新进展,包括应激对中枢IL-1系统的影响,中枢IL-1对应激反应的启动和介导作用;参与中枢IL-1-应激介导作用的神经环路和细胞信号转导通路,以及中枢IL-1对应激时脑高级功能和行为反应的影响。     

白细胞介素-1的分子生物学

本文先简述了IL-1的来源及生物功能,接着对不同生物的IL-1α、IL-1β前体蛋白进行了同源性比较和分析,然后就人类IL-1β分子前结构与功能的关系及空间结构进行了探讨。     

白细胞介素-1家族细胞因子研究进展

随着老龄化时代的到来,各种自身免疫性疾病和肿瘤性疾病更加频繁发生,给人类的生命健康和生活质量带来了影响,其中白细胞介素-1(interleukin-1, IL-1)家族成员在这些疾病中起着重要作用。目前,对IL-1家族的研究发展迅速,已鉴定了11种细胞因子和10种受体,其生物学功能、与疾病的关系及应用越来越受到各国科学家的青睐。现对IL-1家族细胞因子、受体、信号通路和生物学功能作一概述,并展望IL-1家族在药物研发和临床治疗方面的应用前景。     

Vasostatin 1 activates eNOS in endothelial cells through a proteoglycan‐dependent mechanism

Accumulating evidences point to a significant role for the chromogranin A (CgA)‐derived peptide vasostatin 1 (VS‐1) in the protective modulation of the cardiovascular activity, because of its ability to counteract the adrenergic signal. We have recently shown that VS‐1 induces a PI3K‐dependent‐nitric oxide (NO) release by endothelial cells, contributing to explain the mechanism of its cardio‐suppressive and vasodilator properties. However, the cellular processes upstream the eNOS activation exerted by this peptide are still unknown, as typical high‐affinity receptors have not been identified. Here we hypothesize that in endothelial cells VS‐1 acts, on the basis of its cationic and amphipathic properties, as a cell penetrating peptide, binding to heparan sulfate proteoglycans (HSPGs) and activating eNOS phosphorylation (Ser1179) through a PI3K‐dependent, endocytosis‐coupled mechanism. In bovine aortic endothelial cells (BAE‐1 cells) endocytotic vesicles trafficking was quantified by confocal microscopy with a water‐soluble membrane dye; caveolin 1 (Cav1) shift from plasma membrane was studied by immunofluorescence staining; VS‐1‐dependent eNOS phosphorylation was assessed by immunofluorescence and immunoblot analysis. Our experiments demonstrate that VS‐1 induces a marked increase in the caveolae‐dependent endocytosis, (115 ± 23% endocytotic spots/cell/field in VS‐1‐treated cells with respect to control cells), that is significantly reduced by both heparinase III (HEP, 17 ± 15% above control) and Wortmannin (Wm, 7 ± 22% above control). Heparinase, Wortmannin, and methyl‐β‐cyclodextrin (MβCD) abolish the VS‐1‐dependent eNOS phosphorylation (P^Ser1179eNOS). These results suggest a novel signal transduction pathway for endogenous cationic and amphipathic peptides in endothelial cells: HSPGs interaction and caveolae endocytosis, coupled with a PI3K‐dependent eNOS phosphorylation. J. Cell. Biochem. 110: 70–79, 2010. © 2010 Wiley‐Liss, Inc.     

Calorie restriction in overweight males ameliorates obesity-related metabolic alterations and cellular adaptations through anti-aging effects,possibly including AMPK and SIRT1 activation

Background

Calorie restriction (CR) is accepted as an experimental anti-aging paradigm. Several important signal transduction pathways including AMPK and SIRT1 are implicated in the regulation of physiological processes of CR. However, the mechanisms responsible for adaptations remain unclear in humans.

Scope of review

Four overweight male participants were enrolled and treated with 25% CR of their baseline energy requirements for 7 weeks. Characteristics, including body weight (BW), body mass index (BMI), %fat, visceral fat area (VFA), mean blood pressure (MBP) and VO₂ max, as well as metabolic parameters, such as insulin, lipid profiles and inflammatory makers and the expression of phosphorylated AMPK and SIRT1 in peripheral blood mononuclear cells (PBMNCs), were determined at baseline and then after 7 weeks. In addition, we assessed the effects of the serum collected from the participants on AMPK and SIRT1 activation and mitochondrial biogenesis in cultured human skeletal muscle cells.

Major conclusions

After CR, BW, BMI, %fat, VFA and MBP all significantly decreased, while VO₂ max increased, compared to those at baseline. The levels of fasting insulin, free fatty acid, and inflammatory makers, such as interleukin-6 and visfatin, were significantly reduced, whereas the expression of phosphorylated AMPK and SIRT1 was significantly increased in PBMNCs collected after CR, compared to those at baseline. The skeletal muscle cells that were cultured in serum collected after CR showed an increase in AMPK and SIRT1 activity as well as mitochondrial biogenesis.

General significance

CR is beneficial for obesity-related metabolic alterations and induces cellular adaptations against aging, possibly through AMPK and SIRT1 activation via circulating factors.     

金雀异黄酮通过减少卵巢切除大鼠心肌小凹蛋白-1的表达而增加一氧化氮的生成

观察金雀异黄酮(genistein)替代治疗对卵巢切除大鼠心肌中一氧化氮(nitric oxide,NO)和内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)的影响.成年雌性Sprague-Dawley大鼠经双侧卵巢切除术,假手术组作为对照,术后三周将行卵巢切除术的大鼠随机分为低剂量genistein(0.5 mg/kg·d1)、高剂量genistein(5.0 mg/kg·d-1)、17-β雌二醇(0.1 mg/kg·d-1)和模型组(100μl/d芝麻油),各组均皮下注射给药并给予不含大豆的饲料喂养6周,测定大鼠尾动脉血压、心率,麻醉后放血处死大鼠称量子宫重量;放免法检测血浆中总雌二醇,亚硝酸还原酶法检测心肌匀浆中NO,Western blot检测心肌中eNOS的表达以及eNOS的调节蛋白小凹蛋白-1(caveolin-1)和钙调素(calmodulin)的表达情况.结果显示各组间大鼠血压无显著性差异,同17-β雌二醇一样,genistein能呈剂量依赖性地增加心肌组织中eNOS表达量和NO生成,同时genistein能明显降低内源性eNOS活性抑制物caveolin-1的表达,而不影响eNOS活性正性调节蛋白钙调素的表达.与溶媒对照组比较,0.5 mg/kg·d-1的genistein不增加子宫重量,5.0 mg/kg·d-1的genistein增加子宫重量3倍,但较17-β雌二醇(增加6倍)的作用小(P＜0.01).上述结果提示,植物雌激素genistein剂量依赖性地上调心肌组织eNOS的活性并增加NO的生成,减少抑制eNOS活性的小凹蛋白-1表达.     

Metformin overcomes high glucose-induced insulin resistance of podocytes by pleiotropic effects on SIRT1 and AMPK

Podocyte insulin sensitivity is critical for glomerular function, and the loss of appropriate insulin signaling leads to alterations and disorders featuring diabetic nephropathy. Energy-sensing pathways, such as AMP-dependent protein kinase (AMPK) and protein deacetylase SIRT1, have been shown to play an important role in insulin resistance. The absence of a stimulating effect of insulin on glucose uptake into podocytes after exposure to hyperglycemic conditions has been demonstrated to be related to a decreased level and activity of SIRT1 protein, leading to reduced AMPK phosphorylation.The present work was undertaken to investigate metformin's ability to restore the insulin responsiveness of podocytes by regulating SIRT1 and AMPK activities.Primary rat podocytes cultured with standard or high glucose concentrations for 5 days were transfected with siRNAs targeting SIRT1, AMPKα1, or AMPKα2. SIRT1 activity was measured by a fluorometric method. Insulin-stimulated changes in glucose uptake were used to detect insulin resistance. Podocyte permeability was measured by a transmembrane albumin flux assay to examine podocytes functioning.Our results demonstrated that metformin activated SIRT1 and AMPK, prevented hyperglycemia-induced reduction of SIRT1 protein levels, ameliorated glucose uptake into podocytes, and decreased glomerular filtration barrier permeability. Furthermore, metformin activated AMPK in a SIRT1-independent manner, as the increase in AMPK phosphorylation after metformin treatment was not affected by SIRT1 downregulation. Therefore, the potentiating effect of metformin on insulin-resistant podocytes seemed to be dependent on AMPK, as well as SIRT1 activity, establishing multilateral effects of metformin action.     

Berberine modulates deacetylation of PPARγ to promote adipose tissue remodeling and thermogenesis via AMPK/SIRT1 pathway

Pharmacological stimulation of adipose tissue remodeling and thermogenesis to increase energy expenditure is expected to be a viable therapeutic strategy for obesity. Berberine has been reported to have pharmacological activity in adipose tissue to anti-obesity, while the mechanism remains unclear. Here, we observed that berberine significantly reduced the body weight and insulin resistance of high-fat diet mice by promoting the distribution of brown adipose tissue and thermogenesis. We have further demonstrated that berberine activated energy metabolic sensing pathway AMPK/SIRT1 axis to increase the level of PPARγ deacetylation, which leads to promoting adipose tissue remodeling and increasing the expression of the thermogenic protein UCP-1. These findings suggest that berberine that enhances the AMPK/SIRT1 pathway can act as a selective PPARγ activator to promote adipose tissue remodeling and thermogenesis. This study proposes a new mechanism for the regulation of berberine in adipose tissue and offers a great prospect for berberine in obesity treatment     

Atractylenolide III ameliorates Non-Alcoholic Fatty Liver Disease by activating Hepatic Adiponectin Receptor 1-Mediated AMPK Pathway

Background: Nonalcoholic fatty liver disease (NAFLD) is the most frequent cause of chronic liver diseases worldwide. At present, there are no effective pharmacological therapies for NAFLD except lifestyle intervention-mediated weight loss. Atractylenolide III (ATL III), the major bioactive component found in Atractylode smacrocephala Koidz, has been shown to exert anti-oxidant, anti-tumor, anti-allergic response, anti-bacterial effects and cognitive protection. Here we investigate the therapeutic potential and underlying mechanisms of ATL III for the treatment of NAFLD.Methods: Male C57BL/6J mice were fed a high-fat diet (HFD) and treated with ATL III. Lipid accumulation was analyzed by Oil Red O staining in liver tissues and free fatty acids (FFAs)-treated hepatocytes. AMP-activated protein (AMPK) and sirtuin 1(SIRT1) signaling pathways were inhibited by Compound C and EX527 in vitro, respectively. Small-interfering RNA (siRNA) was used to knockdown adiponectin receptor 1 (AdipoR1) expression in HepG2 cells.Results: ATL III treatment ameliorated liver injury and hepatic lipid accumulation in the HFD-induced NAFLD mouse model as demonstrated by that ATL III administration significantly reduced serum levels of alanine aminotransferase, glutamic oxaloacetic transaminase, triglycerides, total cholesterol and low-density lipoprotein. Furthermore, treatment with ATL III alleviated hepatic oxidative stress, inflammation and fibrosis in the HFD feeding model. To study the underlying mechanisms, we performed Computer Aided Design assay and found that open-formed AdipoR1 and adiponectin receptor 2 were the potential receptors targeted by ATL III. Interestingly, HFD feeding or FFAs treatment only reduced hepatic AdipoR1 expression, while such reduction was abolished by ATL III administration. In addition, in vitro treatment with ATL III activated the AdipoR1 downstream AMPK /SIRT1 signaling pathway and reduced lipid deposition in HepG2 cells, which was diminished by silencing AdipoR1. Finally, inhibition of AMPK or SIRT1, the AdipoR1 downstream signaling, abolished the protective effects of ATL III on lipid deposition and oxidative stress in FFAs-treated HepG2 cells.Conclusion: Our findings suggest that ATL III is a therapeutic drug for the treatment of NAFLD and such protective effect is mediated by activating hepatic AdipoR1-mediated AMPK/SIRT1 signaling pathway.     

The N-terminal portion of autoinhibitory element modulates human endothelial nitric-oxide synthase activity through coordinated controls of phosphorylation at Thr495 and Ser1177

NO production catalysed by eNOS (endothelial nitric-oxide synthase) plays an important role in the cardiovascular system. A variety of agonists activate eNOS through the Ser¹¹⁷⁷ phosphorylation concomitant with Thr⁴⁹⁵ dephosphorylation, resulting in increased ·NO production with a basal level of calcium. To date, the underlying mechanism remains unclear. We have previously demonstrated that perturbation of the AIE (autoinhibitory element) in the FMN-binding subdomain can also lead to eNOS activation with a basal level of calcium, implying that the AIE might regulate eNOS activation through modulating phosphorylation at Thr⁴⁹⁵ and Ser¹¹⁷⁷. Here we generated stable clones in HEK-293 (human embryonic kidney 293) cells with a series of deletion mutants in both the AIE (Δ594–604, Δ605–612 and Δ626–634) and the C-terminal tail (Δ14; deletion of 1164–1177). The expression of Δ594–604 and Δ605–612 mutants in non-stimulated HEK-293 cells substantially increased nitrate/nitrite release into the culture medium; the other two mutants, Δ626–634 and Δ1164–1177, displayed no significant difference when compared with WTeNOS (wild-type eNOS). Intriguingly, mutant Δ594–604 showed close correlation between Ser¹¹⁷⁷ phosphorylation and Thr⁴⁹⁵ dephosphorylation, and NO production. Our results have indicated that N-terminal portion of AIE (residues 594–604) regulates eNOS activity through coordinated phosphorylation on Ser¹¹⁷⁷ and Thr⁴⁹⁵.     

MARCH5 restores endothelial cell function against ischaemic/hypoxia injury via Akt/eNOS pathway

MARCH5 is a critical regulator of mitochondrial dynamics, apoptosis and mitophagy. However, its role in cardiovascular system remains poorly understood. This study aimed to investigate the role of MARCH5 in endothelial cell (ECs) injury and the involvement of the Akt/eNOS signalling pathway in this process. Rat models of myocardial infarction (MI) and human cardiac microvascular endothelial cells (HCMECs) exposed to hypoxia (1% O₂) were used in this study. MARCH5 expression was significantly reduced in ECs of MI hearts and ECs exposed to hypoxia. Hypoxia inhibited the proliferation, migration and tube formation of ECs, and these effects were aggravated by knockdown of MARCH5 but antagonized by overexpressed MARCH5. Overexpression of MARCH5 increased nitric oxide (NO) content, p-eNOS and p-Akt, while MARCH5 knockdown exerted the opposite effects. The protective effects mediated by MARCH5 overexpression on ECs could be inhibited by eNOS inhibitor L-NAME and Akt inhibitor LY294002. In conclusion, these results indicated that MARCH5 acts as a protective factor in ischaemia/hypoxia-induced ECs injury partially through Akt/eNOS pathway.