ABSTRACT
OBJECTIVE@#To investigate the effects and underlying mechanisms of Panax quinquefolium saponin (PQS) on energy deficiency in hypoxia-reperfusion (H/R) induced cardiomyocytes.@*METHODS@#The H/R injury involved hypoxia for 3 h and then reperfusion for 2 h. Cardiomyocytes recruited from neonatal rat ventricular myocytes (NRVMs) were randomly divided into control, H/R, H/R+compound C (C.C), H/R+PQS, and H/R+C. C+PQS groups. BrdU assay, lactase dehydrogenase (LDH) leakage and early apoptosis rate were evaluated to assess cell damages. Contents of high energy phosphate compounds were conducted to detect the energy production. Protein expression levels of adenosine monophosphate-activated protein kinase a (AMPKα), glucose transporter 4 (GLUT4), phosphate fructose kinase 2 (PFK2), fatty acid translocase/cluster of differentiation 36 (FAT/CD36), and acetyl CoA carboxylase 2 (ACC2) in the regulatory pathways were measured by Western blotting. Immunofluorescence staining of GLUT4 and FAT/CD36 was used to observe the mobilization of metabolic transporters.@*RESULTS@#PQS (50 mg/L) pretreatment significantly alleviated H/R-induced inhibition of NRVMs viability, up-regulation of LDH leakage, acceleration of early apoptosis, and reduction of energy production (P<0.05). Compared with the H/R group, up-regulated expression of AMPKα, GLUT4, PFK2, FAT/CD36 and ACC2 were observed, and more GLUT4 and FAT/CD36 expressions were detected on the membrane in the H/R+PQS group (P<0.05). These effects of PQS on H/R-induced NRVMs were eliminated in the H/R+C.C+PQS group (P<0.05).@*CONCLUSION@#PQS has prominent advantages in protecting NRVMs from H/R-induced cell damages and energy metabolic disorders, by activation of AMPKα-mediated GLUT4-PFK2 and FAT/CD36-ACC2 pathways.
ABSTRACT
AIM:To study the effect of Panax quinquefolium saponin (PQS) on cardiomyocyte apoptosis in-duced by thapsigargin ( TG) .METHODS:Primary cultured cardiomyocytes from neonatal SD rats were divided into con-trol group, TG group, PQS (40 mg/L, 80 mg/L and 160 mg/L) +TG group, si-PERK+TG group, and mock+TG group.The cells were treated with 1 μmol/L TG for 24 h to induce apoptosis.The PERK gene in the cardiomyocytes was knocked down by RNAi.The cell viability was detected by CCK-8 assay.Apoptosis was analyzed by flow cytometry.Wes-tern blotting was used to determine the expression of ERS molecules GRP78, CRT, ATF4 and CHOP, anti-apoptosis pro-tein Bcl-2 and pro-apoptosis protein Bax.RESULTS: Compared with control group, TG significantly and the apoptosis, reduced the cell viability (P<0.05), increased the phosphorylation of PERK and eIF2α, increased the expression of GRP78, CRT, ATF4, CHOP and pro-apoptosis protein Bax, and decreased the expression of anti-apoptosis protein Bcl-2 (P<0.05).Compared with TG group, PQS treatment (160 mg/L) significantly reduced the apoptosis and increased the cell viability (P<0.05).All the 3 different concentrations of PQS significantly increased the expression of anti-apoptosis protein Bcl-2 and reduced the expression of pro-apoptosis protein Bax (P<0.05) in a dose-dependent manner.PQS pre-treatment and knockdown of PERK both reduced the protein levels of GRP78, CRT, PERK, p-PERK, eIF2α, p-eIF2α, ATF4, CHOP and pro-apoptosis protein Bax, and increased the expression of anti-apoptosis protein Bcl-2 (P<0.05). CONCLUSION:PQS at concentration of 160 mg/L attenuated cardiomyocyte apoptosis induced by TG.PQS had the simi-lar effect as PERK knockdown on cardiomyocyte apoptosis.The mechanism may be associated with inhibiting PERK-eIF2α-ATF4-CHOP pathway of ERS-related apoptosis.
ABSTRACT
AIM:To investigate whether mitochondrial membrane potential (ΔΨm ) and the mitochondrial ap-optotic pathway are involved in the protective mechanism of Panax quinquefolium saponin ( PQS) against cardiomyocyte ap-optosis after ischemia/reperfusion ( I/R) injury in rat myocardium .METHODS: Ninety healthy male SD rats were ran-domly divided into sham group , I/R group, PQS (200 mg· kg -1 · d-1 ) +I/R group, cyclosporine A ( CsA) group, CsA (10 mg· kg-1 ) +I/R group and PQS +CsA +I/R group.The model of myocardial I/R injury in vivo was established by ligating the left anterior descending artery ( LAD) for 30 min followed by 120 min of reperfusion in the rats .The serum ac-tivity of lactate dehydrogenase ( LDH ) was measured by automatic chemistry analyzer .The myocardial infarct size was measured by Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining.Cardiomyocyte apoptosis was detected by in situ TDT-mediated dUTP nick end labeling (TUNEL).The protein levels of Bcl-2, Bax, cleaved caspase-3 and cytosol-ic cytochrome C were determined by Western blotting .ΔΨm was measured by laser scanning confocal microscopy and fluo-rescence microplate reader .RESULTS:Compared with I/R group, the serum content of LDH ,the infarction size in PQS+I/R group, CsA+I/R group and PQS +CsA+I/R group and the myocardial apoptotic index were decreased .Compared with I/R group, the fluorescence intensity of mitochondria after JC-1 staining was enhanced in PQS +I/R group, CsA+I/R group and PQS+CsA+I/R group, and the relative fluorescence units (RFU) ofΔΨm were improved in those 3 groups. In PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group, the protein expression of Bcl-2 was increased, and that of Bax was decreased compared with I/R group.Moreover, in those 3 groups, the protein levels of cleaved-caspase-3 and cytosolic cytochrome C were decreased compared to I /R group, respectively.CONCLUSION:PQS attenuates myocardial injury and cardiomyocyte apoptosis during I /R, and the protective mechanisms of PQS were associated with the modulation ofΔΨm and the inhibition of mitochondrial apoptosis pathway .