| Ventricular remodeling (VR) after acute myocardial infarction (AMI) is a complicated pathological process, including thinning of the ventricular wall, dilatation of the ventricle and myocardial hypertrophy. The underlying mechanism of VR is not clear so far, though most researchers suppose that it involves apoptosis, hypertrophy of cardiomyocytes and fibrosis of the myocardial matrix. Endoplasmic reticulum stress (ERS) triggered by myocardial ischemia induces cardiomyocyte apoptosis, which presented as the up-regulation of ERS-induced apoptosis pathway glucose regulating protein78(GRP78), calreticulin (CRT), and C/EBP homologous protein (CHOP). Our previous study showed that CHOP-mediated ERS-related apoptosis was involved in rat myocardial hypertrophy induced by abdominal aortic constriction and in rat myocardial ischemia/reperfusion injury. However, whether the CHOP-mediated ERS-related apoptosis contributes to the VR after AMI remains to be studied.Panax quinquefolium saponin (PQS) has been identified beneficial for VR after AMI, myocardial ischemia/reperfusion (I/R) injury, and hypoxia-reoxygenation (H/R) injury of cardiomyocytes in vitro. We found that PQS alleviates H/R injury and I/R injury by inhibiting excessive ERS in cardiomyocyte. Whether the effect of PQS on VR after AMI involves CHOP-mediated ERS-related apoptosis remains to be studied.We established the AMI model by ligating the left anterior descending coronary artery of rats to determine the effects of PQS on LV remodeling after AMI, and explored its possible mechanism involving CHOP-mediated ERS-related apoptosis. The cardioprotective effects of PQS were studied on thapsigargin-treated cardiomyocytes from neonatal Sprague-Dawley (SD) rats to induce ERS-induced apoptosis. Recombinant adenovirus or RNAi were used to exogenous express or knockdown protein kinase R-like ER kinase (PERK), which aimed to explore the signal pathway of PQS inhibiting ERS related apoptosis in cardiomyocytes. Part Ⅰ The improvement effect of PQS on VR after AMIObjective:To study the effect of PQS on VR after AMI.Methods:SD rats subjected to AMI were randomly treated with either driking water, PQS (50mg/kg/d,100mg/kg/d or200mg/kg/d) or taurine, an ERS inhibitor (300mg/kg/d) for4weeks. Left ventricle (LV) fractional shortening (FS), ejection fraction (EF) and structure were then evaluated using echocardiography. The hemodynamic parameters were obtained by carotid artery intubation, and the plasma BNP level was detected by BIOSITE Triage equipment. Myocardial infarct size was measured by Evans blue and2,3,5-triphenyhetrazolium chloride (TTC) staining. The hydroxyproline content in myocardium was determined using the colorimetric method.Results:Compared with the AMI group, PQS treatment (50mg/kg/d,100mg/kg/d, and200mg/kg/d) significantly decreased LVESD and LVEDD and increased FS, EF, the mean arterial pressure, left ventricular systolic pressure, the maximal rates of rise and decline of left ventricular pressure (P<0.05) and significantly increased the left ventricular end-diastolic pressure and plasma BNP levels (P<0.05). A similar trend was observed between rats treated with taurine and rats treated with PQS200mg/kg/d. Conclusion:PQS improved the VR after AMI by attenuating cardiac structural changes, functional damage and fibrosis.Part Ⅱ The inhibitory effects of PQS on ERS related apoptosis after AMIObjective:To study the effect of PQS on ERS-related apoptosis after AMI.Methods:Twenty-four hours after the surgery, the AMI rats were randomly divided into3groups as follows:AMI group, PQS200mg/kg/d group and taurine (300mg/kg/d) group. Another15rats underwent the same procedure except for the ligation of the coronary artery as the sham group. Cardiomyocyte apoptosis was detected using Terminal Deoxynucleotidyl Transferase Mediated dUTP Biotin Nick End Labeling (TUNEL). In addition, expression of ERS molecules in the non-infarcted myocardium was detected using Western blotting.Results:Compared with the AMI group, PQS treatment (200mg/kg/d) significantly decreased the apoptosis index, the expression of GRP78, calreticulin (CRT), CHOP and Bax protein, as well as increased Bcl-2protein expression in non-infarcted myocardium. PQS200mg/kg/d treatment mimicked the results achieved from the taurine-treated rats. CHOP expression positively correlated with the apoptosis index of cardiomyocytes in the non-infarcted myocardium (r=0.797, P<0.01).Conclusion:PQS treatment significantly attenuated apoptosis in the non-infarcted myocardium, which might be attributed to inhibiting CHOP-mediated ERS-related apoptosis.Part Ⅲ The effect of PQS on decreasing cardiomyocytes apoptosis induced by thapsigarginObjective:To study the effect of PQS on cardiomyocytes apoptosis induced by thapsigargin.Methods:Primary cultured cardiomyocytes from neonatal SD rats were divided into6groups as follows:Control group, TG group (cells were treated with1μM TG for24h), PQS40μg/ml+TG group (cells were pretreated with40μg/ml PQS for24h and then treated with1μM TG for24h), PQS80μg/ml+TG group, PQS160μg/ml+TG group, taurine+TG group. Cell viability was detected by CCK-8. Apoptosis was detected by flow cytometry. Western blotting was used to detecte the expression of ERS molecules GRP78, PERK, CHOP and apoptosis related protein Bcl-2and Bax.Results:Compared with TG group, PQS treatment (160μg/ml) significantly reduced the apoptosis and increased the cell viability (P<0.05). All the three different concentration of PQS significantly reduced the expression of GRP78, PERK, CHOP and Bax, and increased the expression of Bcl-2(P<0.05), in a dose dependent manner.Conclusion:PQS160μg/ml attenuated cardiomyocyte apoptosis induced by thapsigargin through inhibiting excessive ERS. Part Ⅳ The inhibitory effect of PQS on apoptosis through the PERK-eIF2a-ATF4-CHOP pathwayObjective:To explore the mechanism by which PQS inhibiting apoptosis induced by thapsigargin.Methods:Primary cultured cardiomyocytes from neonatal SD rats were divided into6groups as follows:control group, TG group, PQS+TG group, Si-PERK+TG group, Random double-stranded RNA-transfected+TG group, PERK overexpression group (Ad-PERK), PQS+PERK overexpression group; Cell viability was detected by CCK-8. Apoptosis was detected by flow cytometry. RT-PCR and Western blotting was used to detecte the expression of ERS molecules including GRP78, PERK, eIF2a, and CHOP.Results:Compared with TG group, both PERK knockdown and PQS pretreatment significantly reduced the rate of cardiomyocyte apoptosis, increased the cell viability, decreased the transcription and translation of ERS molecules GRP78, ATF4and CHOP (P<0.05) and decreased the phosphorylation of PERK and eIF2a (P<0.05). Compared with TG group, overexpression of PERK significantly increased the apoptosis rate, decreased the cell viability, and increased the transcription and translation of ERS molecules GRP78, ATF4and CHOP (P<0.05) and decreased the phosphorylation of PERK and eIF2a (P<0.05); PQS pretreatment significantly decreased the changes induced by PERK overexpression (P<0.05).Conclusion:PQS pretreatment mimicked the effects of PERK knockdown, which would be related to the PERK--eIF2a-ATF4-CHOP apoptotic pathway of ERS. |
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