α-硫辛酸对活性氮介导心肌损伤的保护作用及其机制研究
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摘要
背景近年来，在心血管疾病的发病机制中活性氮及其产生的硝化应激已逐渐成为国内外学者的研究热点。所谓硝化应激(nitrative stress)，是由过量NO或由NO衍生的活性氮族（reactive nitrogen species, RNS）与活性氧族共同联合发生的生物化学反应，其特征是细胞内蛋白质中的酪氨酸硝化成3-硝基酪氨酸(3-nitrotyrosine，NT)，从而引起蛋白质结构和功能改变，进一步对细胞产生多种毒性作用，导致细胞损伤或凋亡的病理状态。硝化应激的标志物主要为活性氮，活性氮指的是NO（nitric oxide）与由NO与ROS衍生的自由基或非自由基活性产物，如NO2、HNO2、ONOO-、NO2C1等。在心血管方面，活性氮介导的硝化应激参与了多种疾病的病理生理过程。如心肌缺血-再灌注损伤、内毒素血症心功能异常、动脉粥样硬化、高血压等。
α-硫辛酸亦称硫辛酸（lipoic acid, LA），二氢硫辛酸(dihydrolipoic acid，DHLA)为其还原形式。大量研究表明，LA及DHLA具有很强的抗氧化性， LA对于糖尿病和糖尿病并发症、各种脑和神经退行性疾病、肝脏系统疾病、衰老等均有良好的预防和治疗作用。尤为重要的是，LA在心血管疾病中所起的作用日益受到人们的关注。在体内和体外实验模型中，LA均被证实对心血管系统有重要的保护作用。例如减轻缺血再灌注损伤、调节脂质代谢、降低血压等。但LA对活性氮和硝化应激介导的心肌损伤是否有保护作用，还知之甚少。
目的本研究拟通过以下两部分实验，分别以硝普钠作为外源性NO的供体，以脂多糖作为内源性NO诱导物，研究内外源性活性氮对心肌的损伤作用，进一步探索LA对其是否具有保护作用，并重点关注其保护机制是否通过PI3K/Akt依赖的信号通路实现，为LA在心血管疾病中的应用提供理论依据。
内容与方法
1.α-硫辛酸对硝普钠所致心肌细胞损伤的保护作用及机制
1.1心肌细胞培养和分组
配置含10%胎牛血清的低糖DMEM培养基和0.25%的胰蛋白酶消化液，H9c2大鼠心肌细胞于37℃，含5％CO2的细胞培养箱中进行细胞培养和传代。分为4组处理：(1)对照组(Con)：不加任何药物；(2) α-硫辛酸组(LA)：在培养基中加入终浓度为500μM的LA；(3)硝普钠组(SNP)：在培养基中加入1mM的SNP；(4) α-硫辛酸+硝普钠组(LA+SNP)：在培养基中加入终浓度为500μM的LA，12h后加入终浓度为1mM的SNP。另组实验中在LA处理之前，加入API(1.5M)作用30min。
1.2细胞形态及细胞增殖率测定
首先用MTT比色实验测定不同浓度SNP对细胞存活的影响，选择合适的SNP浓度作为损伤模型。通过相差倒置显微镜（200×）观察细胞生长状态以及形态改变，MTT比色实验测定细胞增殖活力。
1.3细胞凋亡测定
分别用荧光核染料Hoechst33342进行细胞凋亡染色、JC－1行线粒体膜电位测定、Western blot检测Bax、Bcl-2表达反映细胞凋亡水平。
1.4NO及蛋白酪氨酸硝基化测定
通过硝酸还原酶法检测NO含量，Western blot检测蛋白酪氨酸硝基化水平。
1.5PI3K/Akt信号通路活性测定
通过Western blot检测p-Akt、p-GSK-3β表达水平。另外，在LA处理之前，加入Akt抑制剂API，观察各组心肌细胞p-Akt、p-GSK-3β的表达水平。
1.6抑制PI3K/Akt信号通路对细胞形态、细胞增殖及细胞凋亡的影响
在LA处理之前，加入Akt抑制剂API，分别用相差倒置显微镜、MTT比色实验、Hoechst33342、JC－1进行细胞形态、细胞增殖及细胞凋亡检测。
2.α-硫辛酸对脂多糖所致心肌损伤的保护作用及机制
2.1动物分组及处理
8-10周龄C57BL/6J，分成以下四组，8-15只/组；(1)对照组(Con)：等体积无菌生理盐水腹腔注射作为对照；(2) α-硫辛酸组(LA)：腹腔注射LA（100mg/kg）两次，间隔12小时；(3)脂多糖组(LPS)：单独腹腔注射LPS（10mg/kg）诱导内毒素血症模型；(4) α-硫辛酸+脂多糖组(LA+LPS)：先腹腔注射LA（100mg/kg）两次，间隔12小时，第二次注射后1小时，腹腔注射LPS（10mg/kg）。
2.2小鼠心脏功能测定
LPS腹腔注射后6小时，用Vevo770超声心动图测定小鼠心功能，于左室乳头肌水平短轴切面测量各项指标，测3-5个心动周期，取均值。
2.3小鼠生存率测定
上述8-10周龄C57BL/6J小鼠，分成两组，分别为脂多糖组(LPS)：单独腹腔注射LPS（20mg/kg）及α-硫辛酸+脂多糖组(LA+LPS)：先腹腔注射LA（100mg/kg）两次，间隔12小时，第二次注射后1小时，腹腔注射LPS（20mg/kg）。每隔2小时观察小鼠死亡情况，记录小鼠5天生存率，并描绘生存曲线。
2.4小鼠心肌组织凋亡蛋白测定
Western blot法检测小鼠心肌组织Bax、Bcl-2表达。
2.5小鼠心肌组织iNOS、eNOS、NO含量及蛋白酪氨酸硝基化测定
通过硝酸还原酶法检测NO含量，Western blot检测iNOS、eNOS、蛋白酪氨酸硝基化水平。
2.6小鼠心肌组织IκBα磷酸化测定
通过Western blot检测小鼠心肌组织p-IκBα、IκBα表达水平。
2.7PI3K/Akt信号通路活性测定
通过Western blot检测小鼠心肌组织p-Akt、p-GSK-3β表达水平。另外，在LA处理之前，加入PI3K抑制剂WM，观察各组心肌组织p-Akt、p-GSK-3β表达水平。
2.8抑制PI3K/Akt信号通路对小鼠心功能的影响
在LA处理之前，加入PI3K抑制剂WM，用超声心动图观察抑制PI3K/Akt信号通路对小鼠心功能的影响。
结果
1.α-硫辛酸对硝普钠所致心肌细胞损伤的保护作用及机制
1.1不同浓度SNP对细胞存活的影响
用1mM、1.5mM、3mM的SNP处理后，与对照组相比，其细胞增殖抑制率分别为43.56%,79.46%和96.41%,且抑制率随SNP剂量的增加而逐渐增加，差异具有统计学意义(P <0.01)。
1.2LA显著减轻SNP引起的H9c2细胞增殖抑制和形态学损伤
MTT法检测细胞增殖活性示：在1mM SNP作用后24h，SNP组与对照组相比，其细胞增殖抑制率达42.78%(P<0.01)，而与SNP组比较，LA预处理使细胞存活率增加42.66%(P<0.01)。同时，LA预处理显著减轻了SNP诱导的细胞形态变化。
1.3LA显著减轻SNP引起的H9c2细胞凋亡
(1)SNP作用后48h时间点，SNP组细胞出现固缩变形，Hoechst33342染色出现致密浓染，其凋亡率与Con组比较，增加了34.39倍(P<0.01)，而与SNP组比较，LA预处理使细胞凋亡率减少了70.27%(P<0.01)。(2)在SNP作用后24h，SNP组的细胞线粒体膜电位（ΔΨm）较Con组下降了12.67%(P<0.01)，而与SNP组比较，LA预处理使ΔΨm显著增加10.54%(P<0.01)。(3)凋亡相关蛋白Bax/Bcl-2比值在LPS组明显增高82.01%(P <0.01)，而LA+LPS组与LPS组比较，Bax/Bcl-2比值降低28.60%(P <0.01)。
1.4LA对SNP诱导的NO生成及蛋白酪氨酸硝基化的影响
采用硝酸还原酶法检测心肌细胞NO显示：在SNP作用后24h，SNP组与对照组相比，其心肌细胞NO生成量增高约2.96倍(P<0.01)，与SNP组比较，LA预处理使心肌细胞NO生成量减少，但无明显统计学差异。通过Western blot可观察到蛋白酪氨酸硝基化显著增多。而与SNP组比较，LA预处理使细胞蛋白酪氨酸硝基化程度明显减轻64.21%(P<0.01)。
1.5PI3K/Akt信号通路参与LA对SNP诱导H9c2细胞损伤的保护作用
用Western blot法检测PI3K/Akt信号通路途径发现：SNP作用后1h时间点，SNP组p-Akt和p-GSK-3β的水平较对照组相比明显降低49.03%和51.93%(P<0.01)，而LA+SNP组与SNP组比较，p-Akt和p-GSK-3β水平明显增加79.85%和86.56%(P<0.01)。API预处理使LA+SNP组相对增加的p-Akt及p-GSK-3β水平明显减低。API+SNP和API+LA+SNP组之间p-Akt和p-GSK-3β水平无明显差异。
1.6抑制PI3K/Akt信号通路去除了LA对SNP所致细胞形态、细胞增殖抑制及细胞凋亡的保护作用
API预处理抑制PI3K/Akt信号通路活性，去除了LA减轻SNP致H9c2细胞形态学损伤及对细胞增殖抑制和细胞凋亡的保护作用。
2.α-硫辛酸对脂多糖所致心肌损伤的保护作用及机制
2.1LA显著减轻LPS诱导的小鼠心功能不全
注射LPS后小鼠心功能明显下降，与生理盐水对照组小鼠相比LPS组小鼠EF%、FS%、SV和CO分别下降63.01%、68.82%、68.53%和76.14%（P<0.01）；LA预处理后与LPS组小鼠相比EF%、FS%、SV和CO分别提高55.64%、63.33%、70.02%和76.21%（P<0.01或P<0.05）。
2.2LA显著提高小鼠生存率
当5日观察期结束时，LPS组的15只小鼠存活1只，生存率为6.67%，而LA+LPS组的15只小鼠存活6只。生存率为40%、结果显示，与LPS单独处理组比较，LA预处理使小鼠生存率明显提高(P<0.01)。
2.3LA显著抑制LPS引起的小鼠心肌细胞凋亡
与对照组相比，心肌组织Bax/Bcl-2比值在LPS组明显增高1.41倍(P <0.01)，而LA+LPS组与LPS组比较，Bax/Bcl-2比值明显降低32.21%(P <0.01)。
2.4LA显著减轻LPS所致的iNOS/eNOS比例失衡,减少LPS诱导的NO生成量及小鼠心肌蛋白酪氨酸硝基化
通过Western blot可观察到LPS组小鼠心肌组织中iNOS表达量显著增高，eNOS表达水平明显下降。但LA+LPS组与LPS比较，iNOS表达量减少32.51%（P<0.01），而eNOS表达水平增高81.11%。LPS组与对照组相比，其心肌细胞NO生成量增高约2.24倍(P<0.01)，与LPS组比较，LA预处理使心肌细胞NO生成量明显减少48.40%。同时，LA预处理使细胞蛋白酪氨酸硝基化程度较LPS组明显减轻43.19%(P<0.01)。
2.5LA显著抑制LPS诱导的心肌组织IκBα磷酸化
LPS刺激后，小鼠心肌组织p-IκBα较对照组明显升高3.81倍（P<0.01），但与LPS组小鼠相比，LA预处理使鼠p-IκBα升高水平减少了49.11%（P<0.01）；相反，LPS作用后，小鼠心肌组织的IκBα水平较对照组下降88.12%（P<0.01），但LA+LPS组小鼠IκBα水平与LPS小鼠相比，升高了2.82倍（P<0.01）。LA显著减轻了LPS诱导的小鼠心肌组织IκBα磷酸化水平。
2.6LA维持了LPS小鼠心肌PI3K/Akt信号通路活性
在LPS处理后1小时小鼠心肌p-Akt、p-GSK-3β水平较其对照组分别下降49.01%和47.22%（P<0.01），但与LPS组小鼠比较，LA预处理组小鼠（LA+LPS组）的p-Akt、p-GSK-3β水平的下降程度分别减轻了68.63%和58.24%（P<0.01）
2.7抑制PI3K/Akt信号通路去除了LA对LPS所致小鼠心功能不全的保护作用
WM预处理抑制PI3K/Akt信号通路活性，去除了LA对LPS所致小鼠心功能不全的保护作用。
结论
1. LA可减轻外源性活性氮供体SNP所诱导的心肌细胞损伤和凋亡，LA对心肌细胞的保护作用至少部分是通过维持PI3K/Akt通路的激活实现。
2. LA可减轻LPS所致内源性活性氮诱导的小鼠心功能不全，改善生存率，LA对小鼠心功能的保护作用至少部分是通过维持PI3K/Akt通路的激活实现。
Background Most recently, it is well known that the reactive nitrogen species-provoked nitrative stress could play a causative role in the pathophysiology ofcardiovascular abnormalities. Nitrative stress is a biological process derived from thebiochemical interaction of nitric oxide or nitric oxide-derived secondary productsreactive nitrogen species with reactive oxygen species，leading to the concomitantformation of3-nitrotyrosine, which will cause cell injury and apoptosis by increasingmitochondrial permeability, changing structure and functions of important proteins,and disrupting the calcium transportation system. Many recent studies demonstratedthat nitrative stress played a key role in the pathophysiology of several majorcardiovascular diseases such as myocardial ischemical reperfusion injury，endotoxin-induced cardiac dysfunction, atherosclerosis，and hypertension．
Alpha-lipoic acid (LA) and its reduced form DHLA have been long noted toact as powerful antioxidants. There is an avalanche rise in the number of publicationsconfirming beneficial effects of lipoic acid in therapy of many diseases, includingdiabetes and diabetic complications, degenerative processes in neurons, diseases ofliver and aging. Of particular interest to this study, LA has been shown an effectiveprotection in cardiac diseases such as attenuating cardiac ischemia/reperfusion,regulating lipometabolism and lowering blood pressure. However, whether LA exertscardioprotective effects against nitrative stress induced cardiac injury has not beeninvestigated.
Objective In the present study, we separately evaluated the cardiac toxic effects of SNP-induced exogenous NO and LPS-induced endogenous NO as well as protectiveeffect of LA. Furthermore, we attempted to elucidate whether the protective action ofLA was mediated through a PI3K/Akt-dependent mechanism.
Materials and Methods
1. Attenuation of SNP-induced injury in cardiomyoblasts by lipoic acid
1.1Cell culture and treatment
Rat cardiomyoblast H9c2cells were maintained in DMEM supplemented with10%FBS. Cells were assigned to four groups when they reached75-80%confluence:(1) untreated control group (Con);(2) LA group: cells were treated with LA (500M)for indicated times;(3) SNP group: cells were stimulated with SNP (1mM) forindicated hours;(4) LA+SNP group: cells were pretreated with LA (500M) for12hr and followed by stimulation with SNP (1mM) for indicated hours. For PI3K/Aktinhibition experiments, cells were treated with API (1.5M)30min prior to LAadministration.
1.2Examination of cell viability and morphology
After stimulation with SNP (1mM) for24hr, cell viability was determined byMTT assay. In another set of experiments, cell morphology was examined usingphase-contrast light microscope with a magnification of200.
1.3Examination of cardiomyoblast apoptosis
Nuclear condensation was examined by Hoechst33342staining. Mitochondrialtransmembrane potential was evaluated by JC-1. The expressions of Bcl-2and Baxproteins were evaluated by Western blot.
1.4Examination of NO and nitrotyrosine
The concentration of NO was measured as nitrite (the final stable state of NO)accumulation in the culture medium using a NO assay kit according to the manufacture’s instruction. The expressions of nitrotyrosine were detected withWestern blot.
1.5Examination of PI3K/Akt activation
The expressions of p-Akt and p-GSK-3β were evaluated by Western blot. ForPI3K/Akt inhibition experiments, cells were treated with API (1.5M)30min priorto LA administration and then expressions of p-Akt and p-GSK-3β were evaluated byWestern blot.
1.6PI3K/Akt inhibition experiments
Cells were treated with API (1.5M)30min prior to LA administration and thencell viability, cell morphology and cell apoptosis were evaluated as mentioned above.
2. Attenuation of LPS-induced cardiac injury in mice by lipoic acid
2.1Animal treatment
To induce endotoxemia, male C57BL/6J mice were treated with LPSintraperitoneally at a dosage of10mg/kg body weight according to our previousstudies. To evaluate the roles of LA on LPS-induced cardiac injury, mice wereinjected with LA (100mg/kg body weight, intraperitoneally)13hr and1hr prior toLPS administration. In PI3K/Akt inhibition experiments, mice were pretreated with aselective PI3K inhibitor WM (1mg/kg body weight)1hr before the last injection ofLA.
2.2Echocardiographic examination
Cardiac function was examined by using the Vevo770High-Resolution In VivoImagine System equipped with a35-MHz transducer6hr after LPS challenge.Parameters of cardiac function were measured digitally on the M-mode tracings. Datafrom three to five consecutive selected cardiac cycles were analyzed and averaged.
2.3Survival study
Animals were injected intraperitoneally with LPS at a concentration of20mg/kg body weight. To evaluate the roles of LA on LPS-induced mortality, mice wereinjected intraperitoneally with LA (100mg/kg body weight)13hr and1hr prior toLPS administration. The animal death was carefully monitored and recorded onceevery2hr for up to120hr.
2.4Examination of cardiac apoptotic proteins
The expressions of Bcl-2and Bax proteins in the myocardium were evaluated byWestern blot.
2.5Examination of iNOS, eNOS, NO and nitrotyrosine
The expressions of iNOS, eNOS and nitrotyrosine in the myocardium wereevaluated by Western blot. The concentration of NO was measured as mentionedabove.
2.6Examination of I-Bα phosphorylation in myocardium
The expressions of I-Bα and p-I-Bα were evaluated by Western blot.
2.7Examination of PI3K/Akt activation
The expressions of p-Akt and p-GSK-3β were evaluated by Western blot. ForPI3k/Akt inhibition experiments, mice were pretreated with a selective PI3K inhibitorWM (1mg/kg body weight)1hr before the last injection of LA. and then expressionsof p-Akt and p-GSK-3β were evaluated by Western blot.
2.8PI3K/Akt inhibition experiments
For PI3k/Akt inhibition experiments, mice were pretreated with a selective PI3Kinhibitor WM (1mg/kg body weight)1hr before the last injection of LA. and thencardiac function was evaluated as mentioned above.
Results
1.1Dose-response of SNP on the viability in H9c2cells
When the concentrations of SNP increased to1,1.5and3mM, cell viability was decreased by43.56,79.46and96.41%, respectively, in comparison with the untreatedcontrols (P<0.01).
1.2LA improves cell survival following SNP challenge
LA pretreatment increased viability by42.66%in SNP-challenged cellscompared with the cells challenged with SNP alone (P<0.01). The protective effectsof LA on cell survival were confirmed by the observations in cell morphology.
1.3LA reduces SNP-induced apoptosis
(1) SNP increased the percentage of condensed nuclei by34.39-fold comparedwith controls (P<0.01). LA pretreatment significantly decreased the SNP-inducednuclear condensation by70.27%compared with the cells stimulated with SNP alone(P<0.01)(2) SNP decreased m by12.67%compared with controls (P<0.01).However, the SNP-induced loss of m was significantly attenuated by LApretreatment. LA increased m by10.54%in SNP-treated cells compared with thecells treated with SNP alone (P<0.01)(3) SNP increased Bax/Bcl-2by82.01%compared with controls (P<0.01). LA pretreatment significantly decreased theSNP-induced increase in the level of Bax/Bcl-2by28.60%compared with the cellsstimulated with SNP alone (P<0.01).
1.4Effects of LA on SNP-induced NO production and nitrotyrosine expression
The NO content was significantly increased by2.96-fold in SNP-challenged cellswhen compared with controls (P<0.01). LA pretreatment showed no significanteffects on SNP-induced NO generation (P>0.05). SNP challenge significantlyupregulated the expression of nitrotyrosine compared with controls (P<0.01).However, pretreatment with LA markedly decreased the levels of nitrotyrosine by64.21%in SNP-challenged cells compared with the cells challenged with SNP alone(P<0.01).
1.5LA rescues the SNP-provoked decreases in the levels of p-Akt and p-Gsk-3
Result shows a significant decrease in p-Akt level in SNP-challenged cellscompared with controls (P<0.01). LA pretreatment increased the p-Akt level by79.85%in SNP-challenged cells compared with the cells challenged with SNP alone(P<0.01). Similar with the observations in Akt phosphorylation, SNP alsosignificantly decreased p-Gsk-3level compared with untreated controls (P<0.01).However, the SNP-induced decrease in Gsk-3phosphorylation was attenuated by86.56%by LA pretreatment. API administration prevented the LA-induced increasesin p-Akt and p-Gsk-3levels following SNP challenge. No significant difference ofp-Akt and p-Gsk-3levels was detected between LA+SNP group and SNP group inthe presence of API (P>0.05).
1.6Blockade of Akt activation abrogates the cytoprotective effects of LA
The results of MTT assay, cell morphology and apoptosis examination showedthat API abolished LA-induced protection in cell viability and apoptosis followingSNP challenge.
2. Attenuation of LPS-induced cardiac injury in mice by lipoic acid
2.1LA attenuated cardiac dysfunction in mice following LPS challenge
LPS treatment significantly decreased ejection fraction (EF%) by63.01%,fractional shortening (FS%) by68.82%, stroke volume (SV) by68.53%and cardiacoutput (CO) by76.14%, respectively, when compared with control mice (P<0.01).LA pretreatment increased EF%by55.64%, FS%by63.33%, SV by70.02%and COby76.21%in LPS-challenged mice, respectively, compared with the LPS-treatedmice that did not receive LA (P<0.01or P<0.05).
2.2LA improved survival in LPS-treated mice
LA pretreatment significantly improved survival in LPS-treated mice, incomparison with those treated with LPS only (P<0.01). At72hr after LPS challenge,the survival rate in LPS-treated mice was40%in the presence of LA whereas was only6.67%in the absence of LA (P<0.01).
2.3LA reduces LPS-induced apoptosis in the myocardium
LPS increased Bax/Bcl-2by1.41-fold compared with controls (P<0.01). LApretreatment significantly decreased the LPS-induced increase in the level ofBax/Bcl-2by32.21%compared with the mice stimulated with LPS alone (P<0.01).
2.4LA attenuated the increases in iNOS, NO and nitrotyrosine production anddecrease in eNOS in myocardium following LPS stimulation
A significant increase of iNOS expression was observed in LPS-treated micecompared with controls (P<0.01). LA pretreatment attenuated the LPS-induced iNOSexpression by32.51%compared with LPS-challenged mice that did not receive LA(P<0.01). On the other hand, LPS significantly decreased eNOS level by56.01%compared with controls. However, LA preadministration significantly increasedeNOS level by81.11%in LPS-challenged mice, when compared withLPS-challenged mice that did not receive LA (P<0.01). The NO and nitrotyrosinecontent were significantly increased by2.24and1.77-fold in LPS-challenged micewhen compared with controls (P<0.01). However, pretreatment with LA markedlydecreased the levels of NO and nitrotyrosine by48.40%and43.19%inLPS-challenged mice compared with the mice challenged with LPS alone (P<0.01).
2.5LA suppressed I-Bα phosphorylation in myocardium following LPSstimulation
LPS stimulation significantly increased phosphorylation level of I-Bα by3.81-fold in myocardium compared with controls (P<0.01). However, LApretreatment significantly suppressed the LPS-induced increase in I-Bαphosphorylation by49.11%, in comparison with LPS-challenged mice that did notreceive LA (P<0.01). Conversely, LPS significantly decreased total I-Bα level by88.12%compared with controls. LA increased total I-Bα level by2.82-fold inLPS-challenged mice, when compared with LPS-challenged mice that did not receive LA (P<0.01).
2.6LA prevented the decreases in the phosphorylation levels of Akt and Gsk-3in myocardium following LPS stimulation
Results showed a significant decrease in p-Akt level by49.01%in themyocardium of LPS-challenged mice compared with controls (P<0.01). LApretreatment increased the p-Akt level by68.63%in LPS-challenged mice comparedwith the mice challenged with LPS alone (P<0.01). The LPS-induced decrease inGsk-3phosphorylation was also attenuated by58.24%by LA pretreatment (P<0.01).WM abrogated the LA-induced preserved activation of the PI3K/Akt signalingfollowing LPS challenge.
2.7PI3K inhibition abrogated the protective effect of LA on LPS-inducedcardiac dysfunction
The results of echocardiographic showed that WM abolished LA-inducedprotection in cardiac dysfunction following LPS challenge.
Conclusions
1. LA attenuated SNP-induced injury and apoptosis in cardiomyoblasts. Themechanisms may involve the preserved activation of PI3K/Akt signaling pathway.
2. LA attenuated cardiac dysfunction and improved survival in mice following LPSchallenge. The mechanisms may involve the preserved activation of PI3K/Aktsignaling pathway.
α-硫辛酸亦称硫辛酸（lipoic acid, LA），二氢硫辛酸(dihydrolipoic acid，DHLA)为其还原形式。大量研究表明，LA及DHLA具有很强的抗氧化性， LA对于糖尿病和糖尿病并发症、各种脑和神经退行性疾病、肝脏系统疾病、衰老等均有良好的预防和治疗作用。尤为重要的是，LA在心血管疾病中所起的作用日益受到人们的关注。在体内和体外实验模型中，LA均被证实对心血管系统有重要的保护作用。例如减轻缺血再灌注损伤、调节脂质代谢、降低血压等。但LA对活性氮和硝化应激介导的心肌损伤是否有保护作用，还知之甚少。
目的本研究拟通过以下两部分实验，分别以硝普钠作为外源性NO的供体，以脂多糖作为内源性NO诱导物，研究内外源性活性氮对心肌的损伤作用，进一步探索LA对其是否具有保护作用，并重点关注其保护机制是否通过PI3K/Akt依赖的信号通路实现，为LA在心血管疾病中的应用提供理论依据。
内容与方法
1.α-硫辛酸对硝普钠所致心肌细胞损伤的保护作用及机制
1.1心肌细胞培养和分组
配置含10%胎牛血清的低糖DMEM培养基和0.25%的胰蛋白酶消化液，H9c2大鼠心肌细胞于37℃，含5％CO2的细胞培养箱中进行细胞培养和传代。分为4组处理：(1)对照组(Con)：不加任何药物；(2) α-硫辛酸组(LA)：在培养基中加入终浓度为500μM的LA；(3)硝普钠组(SNP)：在培养基中加入1mM的SNP；(4) α-硫辛酸+硝普钠组(LA+SNP)：在培养基中加入终浓度为500μM的LA，12h后加入终浓度为1mM的SNP。另组实验中在LA处理之前，加入API(1.5M)作用30min。
1.2细胞形态及细胞增殖率测定
首先用MTT比色实验测定不同浓度SNP对细胞存活的影响，选择合适的SNP浓度作为损伤模型。通过相差倒置显微镜（200×）观察细胞生长状态以及形态改变，MTT比色实验测定细胞增殖活力。
1.3细胞凋亡测定
分别用荧光核染料Hoechst33342进行细胞凋亡染色、JC－1行线粒体膜电位测定、Western blot检测Bax、Bcl-2表达反映细胞凋亡水平。
1.4NO及蛋白酪氨酸硝基化测定
通过硝酸还原酶法检测NO含量，Western blot检测蛋白酪氨酸硝基化水平。
1.5PI3K/Akt信号通路活性测定
通过Western blot检测p-Akt、p-GSK-3β表达水平。另外，在LA处理之前，加入Akt抑制剂API，观察各组心肌细胞p-Akt、p-GSK-3β的表达水平。
1.6抑制PI3K/Akt信号通路对细胞形态、细胞增殖及细胞凋亡的影响
在LA处理之前，加入Akt抑制剂API，分别用相差倒置显微镜、MTT比色实验、Hoechst33342、JC－1进行细胞形态、细胞增殖及细胞凋亡检测。
2.α-硫辛酸对脂多糖所致心肌损伤的保护作用及机制
2.1动物分组及处理
8-10周龄C57BL/6J，分成以下四组，8-15只/组；(1)对照组(Con)：等体积无菌生理盐水腹腔注射作为对照；(2) α-硫辛酸组(LA)：腹腔注射LA（100mg/kg）两次，间隔12小时；(3)脂多糖组(LPS)：单独腹腔注射LPS（10mg/kg）诱导内毒素血症模型；(4) α-硫辛酸+脂多糖组(LA+LPS)：先腹腔注射LA（100mg/kg）两次，间隔12小时，第二次注射后1小时，腹腔注射LPS（10mg/kg）。
2.2小鼠心脏功能测定
LPS腹腔注射后6小时，用Vevo770超声心动图测定小鼠心功能，于左室乳头肌水平短轴切面测量各项指标，测3-5个心动周期，取均值。
2.3小鼠生存率测定
上述8-10周龄C57BL/6J小鼠，分成两组，分别为脂多糖组(LPS)：单独腹腔注射LPS（20mg/kg）及α-硫辛酸+脂多糖组(LA+LPS)：先腹腔注射LA（100mg/kg）两次，间隔12小时，第二次注射后1小时，腹腔注射LPS（20mg/kg）。每隔2小时观察小鼠死亡情况，记录小鼠5天生存率，并描绘生存曲线。
2.4小鼠心肌组织凋亡蛋白测定
Western blot法检测小鼠心肌组织Bax、Bcl-2表达。
2.5小鼠心肌组织iNOS、eNOS、NO含量及蛋白酪氨酸硝基化测定
通过硝酸还原酶法检测NO含量，Western blot检测iNOS、eNOS、蛋白酪氨酸硝基化水平。
2.6小鼠心肌组织IκBα磷酸化测定
通过Western blot检测小鼠心肌组织p-IκBα、IκBα表达水平。
2.7PI3K/Akt信号通路活性测定
通过Western blot检测小鼠心肌组织p-Akt、p-GSK-3β表达水平。另外，在LA处理之前，加入PI3K抑制剂WM，观察各组心肌组织p-Akt、p-GSK-3β表达水平。
2.8抑制PI3K/Akt信号通路对小鼠心功能的影响
在LA处理之前，加入PI3K抑制剂WM，用超声心动图观察抑制PI3K/Akt信号通路对小鼠心功能的影响。
结果
1.α-硫辛酸对硝普钠所致心肌细胞损伤的保护作用及机制
1.1不同浓度SNP对细胞存活的影响
用1mM、1.5mM、3mM的SNP处理后，与对照组相比，其细胞增殖抑制率分别为43.56%,79.46%和96.41%,且抑制率随SNP剂量的增加而逐渐增加，差异具有统计学意义(P <0.01)。
1.2LA显著减轻SNP引起的H9c2细胞增殖抑制和形态学损伤
MTT法检测细胞增殖活性示：在1mM SNP作用后24h，SNP组与对照组相比，其细胞增殖抑制率达42.78%(P<0.01)，而与SNP组比较，LA预处理使细胞存活率增加42.66%(P<0.01)。同时，LA预处理显著减轻了SNP诱导的细胞形态变化。
1.3LA显著减轻SNP引起的H9c2细胞凋亡
(1)SNP作用后48h时间点，SNP组细胞出现固缩变形，Hoechst33342染色出现致密浓染，其凋亡率与Con组比较，增加了34.39倍(P<0.01)，而与SNP组比较，LA预处理使细胞凋亡率减少了70.27%(P<0.01)。(2)在SNP作用后24h，SNP组的细胞线粒体膜电位（ΔΨm）较Con组下降了12.67%(P<0.01)，而与SNP组比较，LA预处理使ΔΨm显著增加10.54%(P<0.01)。(3)凋亡相关蛋白Bax/Bcl-2比值在LPS组明显增高82.01%(P <0.01)，而LA+LPS组与LPS组比较，Bax/Bcl-2比值降低28.60%(P <0.01)。
1.4LA对SNP诱导的NO生成及蛋白酪氨酸硝基化的影响
采用硝酸还原酶法检测心肌细胞NO显示：在SNP作用后24h，SNP组与对照组相比，其心肌细胞NO生成量增高约2.96倍(P<0.01)，与SNP组比较，LA预处理使心肌细胞NO生成量减少，但无明显统计学差异。通过Western blot可观察到蛋白酪氨酸硝基化显著增多。而与SNP组比较，LA预处理使细胞蛋白酪氨酸硝基化程度明显减轻64.21%(P<0.01)。
1.5PI3K/Akt信号通路参与LA对SNP诱导H9c2细胞损伤的保护作用
用Western blot法检测PI3K/Akt信号通路途径发现：SNP作用后1h时间点，SNP组p-Akt和p-GSK-3β的水平较对照组相比明显降低49.03%和51.93%(P<0.01)，而LA+SNP组与SNP组比较，p-Akt和p-GSK-3β水平明显增加79.85%和86.56%(P<0.01)。API预处理使LA+SNP组相对增加的p-Akt及p-GSK-3β水平明显减低。API+SNP和API+LA+SNP组之间p-Akt和p-GSK-3β水平无明显差异。
1.6抑制PI3K/Akt信号通路去除了LA对SNP所致细胞形态、细胞增殖抑制及细胞凋亡的保护作用
API预处理抑制PI3K/Akt信号通路活性，去除了LA减轻SNP致H9c2细胞形态学损伤及对细胞增殖抑制和细胞凋亡的保护作用。
2.α-硫辛酸对脂多糖所致心肌损伤的保护作用及机制
2.1LA显著减轻LPS诱导的小鼠心功能不全
注射LPS后小鼠心功能明显下降，与生理盐水对照组小鼠相比LPS组小鼠EF%、FS%、SV和CO分别下降63.01%、68.82%、68.53%和76.14%（P<0.01）；LA预处理后与LPS组小鼠相比EF%、FS%、SV和CO分别提高55.64%、63.33%、70.02%和76.21%（P<0.01或P<0.05）。
2.2LA显著提高小鼠生存率
当5日观察期结束时，LPS组的15只小鼠存活1只，生存率为6.67%，而LA+LPS组的15只小鼠存活6只。生存率为40%、结果显示，与LPS单独处理组比较，LA预处理使小鼠生存率明显提高(P<0.01)。
2.3LA显著抑制LPS引起的小鼠心肌细胞凋亡
与对照组相比，心肌组织Bax/Bcl-2比值在LPS组明显增高1.41倍(P <0.01)，而LA+LPS组与LPS组比较，Bax/Bcl-2比值明显降低32.21%(P <0.01)。
2.4LA显著减轻LPS所致的iNOS/eNOS比例失衡,减少LPS诱导的NO生成量及小鼠心肌蛋白酪氨酸硝基化
通过Western blot可观察到LPS组小鼠心肌组织中iNOS表达量显著增高，eNOS表达水平明显下降。但LA+LPS组与LPS比较，iNOS表达量减少32.51%（P<0.01），而eNOS表达水平增高81.11%。LPS组与对照组相比，其心肌细胞NO生成量增高约2.24倍(P<0.01)，与LPS组比较，LA预处理使心肌细胞NO生成量明显减少48.40%。同时，LA预处理使细胞蛋白酪氨酸硝基化程度较LPS组明显减轻43.19%(P<0.01)。
2.5LA显著抑制LPS诱导的心肌组织IκBα磷酸化
LPS刺激后，小鼠心肌组织p-IκBα较对照组明显升高3.81倍（P<0.01），但与LPS组小鼠相比，LA预处理使鼠p-IκBα升高水平减少了49.11%（P<0.01）；相反，LPS作用后，小鼠心肌组织的IκBα水平较对照组下降88.12%（P<0.01），但LA+LPS组小鼠IκBα水平与LPS小鼠相比，升高了2.82倍（P<0.01）。LA显著减轻了LPS诱导的小鼠心肌组织IκBα磷酸化水平。
2.6LA维持了LPS小鼠心肌PI3K/Akt信号通路活性
在LPS处理后1小时小鼠心肌p-Akt、p-GSK-3β水平较其对照组分别下降49.01%和47.22%（P<0.01），但与LPS组小鼠比较，LA预处理组小鼠（LA+LPS组）的p-Akt、p-GSK-3β水平的下降程度分别减轻了68.63%和58.24%（P<0.01）
2.7抑制PI3K/Akt信号通路去除了LA对LPS所致小鼠心功能不全的保护作用
WM预处理抑制PI3K/Akt信号通路活性，去除了LA对LPS所致小鼠心功能不全的保护作用。
结论
1. LA可减轻外源性活性氮供体SNP所诱导的心肌细胞损伤和凋亡，LA对心肌细胞的保护作用至少部分是通过维持PI3K/Akt通路的激活实现。
2. LA可减轻LPS所致内源性活性氮诱导的小鼠心功能不全，改善生存率，LA对小鼠心功能的保护作用至少部分是通过维持PI3K/Akt通路的激活实现。
Background Most recently, it is well known that the reactive nitrogen species-provoked nitrative stress could play a causative role in the pathophysiology ofcardiovascular abnormalities. Nitrative stress is a biological process derived from thebiochemical interaction of nitric oxide or nitric oxide-derived secondary productsreactive nitrogen species with reactive oxygen species，leading to the concomitantformation of3-nitrotyrosine, which will cause cell injury and apoptosis by increasingmitochondrial permeability, changing structure and functions of important proteins,and disrupting the calcium transportation system. Many recent studies demonstratedthat nitrative stress played a key role in the pathophysiology of several majorcardiovascular diseases such as myocardial ischemical reperfusion injury，endotoxin-induced cardiac dysfunction, atherosclerosis，and hypertension．
Alpha-lipoic acid (LA) and its reduced form DHLA have been long noted toact as powerful antioxidants. There is an avalanche rise in the number of publicationsconfirming beneficial effects of lipoic acid in therapy of many diseases, includingdiabetes and diabetic complications, degenerative processes in neurons, diseases ofliver and aging. Of particular interest to this study, LA has been shown an effectiveprotection in cardiac diseases such as attenuating cardiac ischemia/reperfusion,regulating lipometabolism and lowering blood pressure. However, whether LA exertscardioprotective effects against nitrative stress induced cardiac injury has not beeninvestigated.
Objective In the present study, we separately evaluated the cardiac toxic effects of SNP-induced exogenous NO and LPS-induced endogenous NO as well as protectiveeffect of LA. Furthermore, we attempted to elucidate whether the protective action ofLA was mediated through a PI3K/Akt-dependent mechanism.
Materials and Methods
1. Attenuation of SNP-induced injury in cardiomyoblasts by lipoic acid
1.1Cell culture and treatment
Rat cardiomyoblast H9c2cells were maintained in DMEM supplemented with10%FBS. Cells were assigned to four groups when they reached75-80%confluence:(1) untreated control group (Con);(2) LA group: cells were treated with LA (500M)for indicated times;(3) SNP group: cells were stimulated with SNP (1mM) forindicated hours;(4) LA+SNP group: cells were pretreated with LA (500M) for12hr and followed by stimulation with SNP (1mM) for indicated hours. For PI3K/Aktinhibition experiments, cells were treated with API (1.5M)30min prior to LAadministration.
1.2Examination of cell viability and morphology
After stimulation with SNP (1mM) for24hr, cell viability was determined byMTT assay. In another set of experiments, cell morphology was examined usingphase-contrast light microscope with a magnification of200.
1.3Examination of cardiomyoblast apoptosis
Nuclear condensation was examined by Hoechst33342staining. Mitochondrialtransmembrane potential was evaluated by JC-1. The expressions of Bcl-2and Baxproteins were evaluated by Western blot.
1.4Examination of NO and nitrotyrosine
The concentration of NO was measured as nitrite (the final stable state of NO)accumulation in the culture medium using a NO assay kit according to the manufacture’s instruction. The expressions of nitrotyrosine were detected withWestern blot.
1.5Examination of PI3K/Akt activation
The expressions of p-Akt and p-GSK-3β were evaluated by Western blot. ForPI3K/Akt inhibition experiments, cells were treated with API (1.5M)30min priorto LA administration and then expressions of p-Akt and p-GSK-3β were evaluated byWestern blot.
1.6PI3K/Akt inhibition experiments
Cells were treated with API (1.5M)30min prior to LA administration and thencell viability, cell morphology and cell apoptosis were evaluated as mentioned above.
2. Attenuation of LPS-induced cardiac injury in mice by lipoic acid
2.1Animal treatment
To induce endotoxemia, male C57BL/6J mice were treated with LPSintraperitoneally at a dosage of10mg/kg body weight according to our previousstudies. To evaluate the roles of LA on LPS-induced cardiac injury, mice wereinjected with LA (100mg/kg body weight, intraperitoneally)13hr and1hr prior toLPS administration. In PI3K/Akt inhibition experiments, mice were pretreated with aselective PI3K inhibitor WM (1mg/kg body weight)1hr before the last injection ofLA.
2.2Echocardiographic examination
Cardiac function was examined by using the Vevo770High-Resolution In VivoImagine System equipped with a35-MHz transducer6hr after LPS challenge.Parameters of cardiac function were measured digitally on the M-mode tracings. Datafrom three to five consecutive selected cardiac cycles were analyzed and averaged.
2.3Survival study
Animals were injected intraperitoneally with LPS at a concentration of20mg/kg body weight. To evaluate the roles of LA on LPS-induced mortality, mice wereinjected intraperitoneally with LA (100mg/kg body weight)13hr and1hr prior toLPS administration. The animal death was carefully monitored and recorded onceevery2hr for up to120hr.
2.4Examination of cardiac apoptotic proteins
The expressions of Bcl-2and Bax proteins in the myocardium were evaluated byWestern blot.
2.5Examination of iNOS, eNOS, NO and nitrotyrosine
The expressions of iNOS, eNOS and nitrotyrosine in the myocardium wereevaluated by Western blot. The concentration of NO was measured as mentionedabove.
2.6Examination of I-Bα phosphorylation in myocardium
The expressions of I-Bα and p-I-Bα were evaluated by Western blot.
2.7Examination of PI3K/Akt activation
The expressions of p-Akt and p-GSK-3β were evaluated by Western blot. ForPI3k/Akt inhibition experiments, mice were pretreated with a selective PI3K inhibitorWM (1mg/kg body weight)1hr before the last injection of LA. and then expressionsof p-Akt and p-GSK-3β were evaluated by Western blot.
2.8PI3K/Akt inhibition experiments
For PI3k/Akt inhibition experiments, mice were pretreated with a selective PI3Kinhibitor WM (1mg/kg body weight)1hr before the last injection of LA. and thencardiac function was evaluated as mentioned above.
Results
1.1Dose-response of SNP on the viability in H9c2cells
When the concentrations of SNP increased to1,1.5and3mM, cell viability was decreased by43.56,79.46and96.41%, respectively, in comparison with the untreatedcontrols (P<0.01).
1.2LA improves cell survival following SNP challenge
LA pretreatment increased viability by42.66%in SNP-challenged cellscompared with the cells challenged with SNP alone (P<0.01). The protective effectsof LA on cell survival were confirmed by the observations in cell morphology.
1.3LA reduces SNP-induced apoptosis
(1) SNP increased the percentage of condensed nuclei by34.39-fold comparedwith controls (P<0.01). LA pretreatment significantly decreased the SNP-inducednuclear condensation by70.27%compared with the cells stimulated with SNP alone(P<0.01)(2) SNP decreased m by12.67%compared with controls (P<0.01).However, the SNP-induced loss of m was significantly attenuated by LApretreatment. LA increased m by10.54%in SNP-treated cells compared with thecells treated with SNP alone (P<0.01)(3) SNP increased Bax/Bcl-2by82.01%compared with controls (P<0.01). LA pretreatment significantly decreased theSNP-induced increase in the level of Bax/Bcl-2by28.60%compared with the cellsstimulated with SNP alone (P<0.01).
1.4Effects of LA on SNP-induced NO production and nitrotyrosine expression
The NO content was significantly increased by2.96-fold in SNP-challenged cellswhen compared with controls (P<0.01). LA pretreatment showed no significanteffects on SNP-induced NO generation (P>0.05). SNP challenge significantlyupregulated the expression of nitrotyrosine compared with controls (P<0.01).However, pretreatment with LA markedly decreased the levels of nitrotyrosine by64.21%in SNP-challenged cells compared with the cells challenged with SNP alone(P<0.01).
1.5LA rescues the SNP-provoked decreases in the levels of p-Akt and p-Gsk-3
Result shows a significant decrease in p-Akt level in SNP-challenged cellscompared with controls (P<0.01). LA pretreatment increased the p-Akt level by79.85%in SNP-challenged cells compared with the cells challenged with SNP alone(P<0.01). Similar with the observations in Akt phosphorylation, SNP alsosignificantly decreased p-Gsk-3level compared with untreated controls (P<0.01).However, the SNP-induced decrease in Gsk-3phosphorylation was attenuated by86.56%by LA pretreatment. API administration prevented the LA-induced increasesin p-Akt and p-Gsk-3levels following SNP challenge. No significant difference ofp-Akt and p-Gsk-3levels was detected between LA+SNP group and SNP group inthe presence of API (P>0.05).
1.6Blockade of Akt activation abrogates the cytoprotective effects of LA
The results of MTT assay, cell morphology and apoptosis examination showedthat API abolished LA-induced protection in cell viability and apoptosis followingSNP challenge.
2. Attenuation of LPS-induced cardiac injury in mice by lipoic acid
2.1LA attenuated cardiac dysfunction in mice following LPS challenge
LPS treatment significantly decreased ejection fraction (EF%) by63.01%,fractional shortening (FS%) by68.82%, stroke volume (SV) by68.53%and cardiacoutput (CO) by76.14%, respectively, when compared with control mice (P<0.01).LA pretreatment increased EF%by55.64%, FS%by63.33%, SV by70.02%and COby76.21%in LPS-challenged mice, respectively, compared with the LPS-treatedmice that did not receive LA (P<0.01or P<0.05).
2.2LA improved survival in LPS-treated mice
LA pretreatment significantly improved survival in LPS-treated mice, incomparison with those treated with LPS only (P<0.01). At72hr after LPS challenge,the survival rate in LPS-treated mice was40%in the presence of LA whereas was only6.67%in the absence of LA (P<0.01).
2.3LA reduces LPS-induced apoptosis in the myocardium
LPS increased Bax/Bcl-2by1.41-fold compared with controls (P<0.01). LApretreatment significantly decreased the LPS-induced increase in the level ofBax/Bcl-2by32.21%compared with the mice stimulated with LPS alone (P<0.01).
2.4LA attenuated the increases in iNOS, NO and nitrotyrosine production anddecrease in eNOS in myocardium following LPS stimulation
A significant increase of iNOS expression was observed in LPS-treated micecompared with controls (P<0.01). LA pretreatment attenuated the LPS-induced iNOSexpression by32.51%compared with LPS-challenged mice that did not receive LA(P<0.01). On the other hand, LPS significantly decreased eNOS level by56.01%compared with controls. However, LA preadministration significantly increasedeNOS level by81.11%in LPS-challenged mice, when compared withLPS-challenged mice that did not receive LA (P<0.01). The NO and nitrotyrosinecontent were significantly increased by2.24and1.77-fold in LPS-challenged micewhen compared with controls (P<0.01). However, pretreatment with LA markedlydecreased the levels of NO and nitrotyrosine by48.40%and43.19%inLPS-challenged mice compared with the mice challenged with LPS alone (P<0.01).
2.5LA suppressed I-Bα phosphorylation in myocardium following LPSstimulation
LPS stimulation significantly increased phosphorylation level of I-Bα by3.81-fold in myocardium compared with controls (P<0.01). However, LApretreatment significantly suppressed the LPS-induced increase in I-Bαphosphorylation by49.11%, in comparison with LPS-challenged mice that did notreceive LA (P<0.01). Conversely, LPS significantly decreased total I-Bα level by88.12%compared with controls. LA increased total I-Bα level by2.82-fold inLPS-challenged mice, when compared with LPS-challenged mice that did not receive LA (P<0.01).
2.6LA prevented the decreases in the phosphorylation levels of Akt and Gsk-3in myocardium following LPS stimulation
Results showed a significant decrease in p-Akt level by49.01%in themyocardium of LPS-challenged mice compared with controls (P<0.01). LApretreatment increased the p-Akt level by68.63%in LPS-challenged mice comparedwith the mice challenged with LPS alone (P<0.01). The LPS-induced decrease inGsk-3phosphorylation was also attenuated by58.24%by LA pretreatment (P<0.01).WM abrogated the LA-induced preserved activation of the PI3K/Akt signalingfollowing LPS challenge.
2.7PI3K inhibition abrogated the protective effect of LA on LPS-inducedcardiac dysfunction
The results of echocardiographic showed that WM abolished LA-inducedprotection in cardiac dysfunction following LPS challenge.
Conclusions
1. LA attenuated SNP-induced injury and apoptosis in cardiomyoblasts. Themechanisms may involve the preserved activation of PI3K/Akt signaling pathway.
2. LA attenuated cardiac dysfunction and improved survival in mice following LPSchallenge. The mechanisms may involve the preserved activation of PI3K/Aktsignaling pathway.
引文
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