上调SERCA2a基因对慢性心力衰竭大鼠的作用及机制

目的研究上调心肌肌浆网Ca^(2+)-ATP酶(SERCA)2a基因对慢性心力衰竭大鼠的作用及机制。方法选取SPF级SD健康大鼠40只,随机选其中10只作为正常组,其余30只建立慢性心力衰竭模型,建模成功后将30只慢性心力衰竭模型大鼠随机分为模型组、下调组和上调组各10只,构建SERCA2a基因慢病毒载体,行慢病毒滴度测定,将10μl SERCA2a慢病毒悬液在无菌环境下注射至上调组大鼠心脏中,将10μl LV-SERCA2a(抑制载体)慢病毒悬液(病毒滴度为108TU/ml)在无菌环境下注射至下调组大鼠心脏中,正常组、模型组大鼠不做任何处理。观察各组大鼠心肌组织病理变化,检测心功能、血流动力学、心肌细胞凋亡率及心肌细胞中Ca^(2+)浓度、心肌组织中受磷蛋白(PLB)、内质网分子伴侣葡萄糖调节蛋白(GRP)78、凋亡蛋白CCAAT/增强子结合蛋白同源蛋白-磷酸化的c-Jun氨基末端激酶-半胱氨酸天冬氨酸蛋白酶(CHOP-JNK-Caspase)-12通路蛋白表达量。结果与模型组相比,下调组大鼠左心室舒张末内径(LVEDD)、左心室舒张末压(LVEDP)、心肌细胞凋亡率、心肌细胞中Ca^(2+)浓度、心肌组织中GRP78、CHOP、p-JNK、Caspase-12蛋白表达量升高,LVEDD、左室射血分数(LVEF)、左心室短链缩短率(FS)、左心室收缩末压(LVESP)、左心室内压上升速率(+dp/dt)、左心室内压下降速率(-dp/dt)、PLB蛋白表达量降低,上调组大鼠LVEDD、LVEDP、心肌细胞凋亡率、心肌细胞中Ca^(2+)浓度、心肌组织中GRP78、CHOP、p-JNK、Caspase-12蛋白表达量降低,LVEDD、LVEF、FS、LVESP、+dp/dt、-dp/dt、PLB蛋白表达量升高(P<0.05);与下调组相比,上调组大鼠LVEDD、LVEDP、心肌细胞凋亡率、心肌细胞中Ca^(2+)浓度、心肌组织中GRP78、CHOP、p-JNK、Caspase-12蛋白表达量降低,LVEDD、LVEF、FS、LVESP、+dp/dt、-dp/dt、PLB蛋白表达量升高(P<0.05)。结论上调SERCA2a可有效改善慢性心力衰竭大鼠心功能,其机制可能和抑制心肌内质网应激相关的心肌凋亡有关。

Panax quinquefolium saponin attenuates cardiomyocyte apoptosis induced by thapsigargin through inhibition of endoplasmic reticulum stress

Background Endoplasmic reticulum （ER） stress-related apoptosis is involved in the pathophysiology of many cardiovascular diseases, and Panax quinquefolium saponin （PQS） is able to inhibit excessive ER stress-related apoptosis of cardiomyocytes following hypoxia/reoxygenation and myocardial infarction. However, the pathway by which PQS inhibits the ER stress-related apoptosis is not well understood. To further investigate the protective effect of PQS against ER stress-related apoptosis, primary cultured eardiomyocytes were stimulated with thapsigargin （TG）, which is widely used to model cellular ER stress, and it could induce apoptotic cell death in sufficient concentration. Methods Primary cultured cardiomyocytes from neonatal rats were exposed to TG （1 μmol/L） treatment for 24 h, following PQS pre-treatment （160 μg/mL） for 24 h or pre-treatment with small interfering RNA directed against protein kinase-like endoplasmic reticulum kinase （Si-PERK） for 6 h. The viability and apoptosis rate of cardiomyocytes were detected by cell counting kit-8 and flow cytometry respectively. ER stress-related protein expression, such as glucose-regulated protein 78 （GRP78）, calreticulin, PERK, eukaryotic translation initiation factor 2α （elF2c0, activating transcription factor 4 （ATF4）, and C/EBP homologous protein （CHOP） were assayed by western blotting. Results Both PQS pre-treatment and PERK knockdown remarkably inhibited the cardiomyocyte apoptosis induced by TG, increased cell viability, decreased phosphorylation of both PERK and eIF2α, and decreased protein levels of both ATF4 and CHOP. There was no statistically significant difference between PQS pre-treatment and PERK knockdown in the cardioprotective effect. Conclusions Our data indicate that the PERK-eIF2α-ATF4-CHOP pathway of ER stress is involved in the apoptosis induced by TG, and PQS might prevent TG-induced cardiomyocyte apoptosis through a mechanism involving the suppression of this pathway. These findings provide novel data regarding the molecular mechanisms by which PQS inhibits cardiomyocyte apoptosis.

Effect of Huanglian Jiedu Decoction on cardiac endoplasmic reticulum stress in spontaneously hypertensive rats

Huanglian Jiedu Decoction(HLJDD)is a quintessential prescription renowned for its heat-clearing and detoxifying properties.It is primarily prescribed to counteract the syndrome characterized by the excessive heat of the Sanjiao fire.Notably,the hyperactivity of liver fire is frequently linked with hypertension,where wind fire and wind toxicity emerge as pivotal pathogenic factors.This study aimed to investigate the impact of HLJDD on the endoplasmic reticulum in spontaneously hypertensive rats(SHR),further delving into the interplay between endoplasmic reticulum stress(ERS)and myocardial remodeling and damage.Fifty SHR rats were stratified randomly into five cohorts:model,low-dose HLJDD,medium-dose HLJDD,high-dose HLJDD,and captopril groups.For comparison,a set of Wistar-Kyoto(WKY)rats served as the baseline control group,with each group comprising 10 rats.While the model and control groups received equivalent volumes of normal saline via gavage,the other groups were administered the respective drug dosages through the same route daily for a span of 6 weeks.Upon the experiment’s conclusion,metrics such as the heart mass index(HWI)and left ventricular mass index(LVWI)were assessed.Cardiac tissue anomalies were identified using H&E staining,while ERS-related protein and mRNA expression levels were ascertained via Western blotting analysis and qPCR.Moreover,TUNEL staining was employed to detect cardiomyocyte apoptosis.The findings indicated that increasing HLJDD concentrations corresponded with escalated HWI and LVWI in rat hearts(P<0.05).There was a marked enhancement in myocardial structural integrity,accompanied by a notable reduction in collagen fibers.The mRNA and protein expressions of myocardial inositol-dependent enzyme 1α(IRE1α),X-box binding protein 1(XBP1),glycoregulatory protein 78(GRP78),and CCAAT enhancer binding protein homologous protein(CHOP)in the medium and high-dose groups saw significant declines(P<0.05).These effects mirrored those observed in the captopril group.The study underscored HLJDD’s efficacy in mitigating myocardial tissue damage in SHR.This therapeutic effect was potentially attributed to the downregulation of IRE1α,XBP1,GRP78,and CHOP,curbing excessive ERS,diminishing cardiomyocyte apoptosis,and thereby conferring cardioprotection.

Salusins protect myocardium against ischemic injury by alleviating endoplasmic reticulum stress

Salusins are regulatory peptides that affect cardiovascular function. We previously reported that salusin-a and -β protected cultured cardiomyocytes from serum deprivation-induced cell death through upregulating glucose-regulated protein 78 （GRP78）, an endoplasmic reticulum （ER） resident protein whose overexpression acts as a marker and suppressor of ER stress. The present study examined whether salusin-α and -β inhibit ER stress in ischemic myocardium. In a rat model of myocardial infarction created by ligating the left anterior descending coronary artery （LAD）, salusin-α or -β was intravenously injected at 5 or 15 nmol kg-1 15 min prior to 2 h of LAD occlusion. The high dose of salusin-α and -β3 significantly improved heart function and hemodynamics in LAD-occluded rats, but had no effects in sham-operated rats. The arrhythmias caused by LAD oc- clusion were markedly attenuated by salusin-α and -β. The apoptotic rate in ischemic myocardium was reduced from 31.5%±3.7% to 19.8%±2.2% and 12.3%±2.2%, and the infarct size was reduced from 53.4%±4.0% of the risk area to 26.5%±9.7% and 23.7%±8.9% by 15 nmol kg-1 salusin-α and -β, respectively. Furthermore, salusin-α and -β prevented the ac- tivation of GRP78 and ER stress-specific apoptotic effectors caspase-12 and CHOP （C/EBP homologous protein）, and attenu- ated the reduction of an ER stress-associated antiapoptotic protein Bcl-2 in ischemic cardiac tissue. The salusins also inhibited the ER stress induced by tunicamycin in cultured rat H9c2 cardiomyocytes. These results indicate that salusins protect myo- cardium against ischemic injury by inhibiting ER stress and ER stress-associated apoptosis.