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 prepare and purify the polyclonal antibodies against human myofibrillogenesis regulator 1 (hMR-1), then to characterize the purity, titer, specificity and the availability.METHODS: Two polypeptides named peptide 1 and 2 were synthesized based on the bioinformatics analysis of the sequence of hMR-1 by using software TMHMM and DNAStar, then coupled with keyhole limpet hemocyanin (KLH) for immunization. These peptides for immunization were mixed and injected into New Zealand rabbits to prepare antibodies specifically against hMR-1. ELISA assay was used to detect the titers of the antibodies. After purification by immunoaffinity chromatography, antibodies were identified by Western blotting and immunocytofluorescent assays. Applications of the antibodies on neonatal rat cardiomyocytes were also employed.RESULTS: (1)The titers of antibodies were 1:10~5. In WB assay, a specific 17kD band was detected, corresponding to the predicted molecular weight of hMR-1; the positive fluorescent signals were distinct. (2)On the neonatal rat cardiomyocytes model, we observed a peri-nucleus location. The fluorescent signal of hMR-1 overexpression group was much stronger than that in vector control and normal control groups.CONCLUSION: All these results indicate that the antibodies obtained from poly peptides mixture immunization have either human original or rat original antigens. The antibody is available for using in Western blotting or immunofluorescent assays.