Attenuation Of Reactive Nitrogen Species-induced Cardiac Injury By Lipoic Acid

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 Methods1. Attenuation of SNP-induced injury in cardiomyoblasts by lipoic acid1.1Cell culture and treatmentRat 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 morphologyAfter 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 apoptosisNuclear 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 nitrotyrosineThe 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 activationThe 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 experimentsCells 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 acid2.1Animal treatmentTo 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 examinationCardiac 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 studyAnimals 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 proteinsThe expressions of Bcl-2and Bax proteins in the myocardium were evaluated byWestern blot.2.5Examination of iNOS, eNOS, NO and nitrotyrosineThe 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 myocardiumThe expressions of I-Bα and p-I-Bα were evaluated by Western blot.2.7Examination of PI3K/Akt activationThe 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 experimentsFor 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.Results1.1Dose-response of SNP on the viability in H9c2cellsWhen 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 challengeLA 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 expressionThe 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 LAThe 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 acid2.1LA attenuated cardiac dysfunction in mice following LPS challengeLPS 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 miceLA 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 myocardiumLPS 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 stimulationA 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 LPSstimulationLPS 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 stimulationResults 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 dysfunctionThe results of echocardiographic showed that WM abolished LA-inducedprotection in cardiac dysfunction following LPS challenge.Conclusions1. 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.