RESUMEN
Abstract Osteoarthritis (OA) encompasses degeneration of articular cartilage, subchondral bone erosions and sclerosis. Chondrocyte apoptosis and an oxygen-deprived microenvironment are essential factors in OA pathogenesis. PAR-4 (Prostate apoptosis response-4) is a pro-apoptotic protein implicated in many pathologies as well as in chondrocyte cell death mechanism. Vitamin D supplementation has been identified as a therapeutic tool for a variety of inflammatory pathologies. In the present manuscript, we investigated whether first, PAR-4 expression is influenced by chondrocytes in a model of OA, in vitro, and second, whether vitamin D modulates PAR-4 expression in the same model. To test our hypothesis, we used the primary culture of murine chondrocytes isolated from the femoral and tibial condyles of wistar rats. The expression of the pro-inflammatory effect interleukin IL-1β was evaluated in the presence and absence of vitamin D. Western blot and immunofluorescence analysis confirmed protein expression. In the normoxia condition, the chondrocytes expressed PAR-4 in the cell nucleus, and in the hypoxic condition, PAR-4 was expressed in the cell cytoplasm. We disclosed that the treatment with Vitamin D decreased PAR-4 (p= 0.0137) and caspase-3 (p= 0.0007) expression. Thus, the results suggested that PAR-4 and caspase-3 proteins could be potential targets for OA.However, we believe that research is needed to identify the mechanisms implicated in the regulation of PAR-4 in OA.
RESUMEN
Objective To investigate the effects of Par-4 gene silence on hydrogen peroxide-induced apoptosis in alveolar epithelial cells. Method The alveolar epithelial cells A549 were cultured and exposed to hydrogen peroxide. The siRNA sequences targeted Par-4 gene was chemically synthesized and transfected to A549 cells with or without the exposure of hydrogen peroxide. The cells were divided into normal control groups, hydrogen peroxide-treated group(The cells were treated with 0. 1 mmol/L hydrogen peroxide), hydrogen peroxide and Par-4-siRNA-treated group(The cells were treated with 0. 1 mmol/L hydrogen peroxide after transfection of Par-4-siRNA), Non-specific DNA sequence transfection control group. The apoptosis of A549 cells was quantified by flow cytometry. The expression of Smac protein was detected by Western blot.Electrophoretic mobility shift assay was applied for evaluating the change of E2F1 DNA binding activity. Relative activity of Caspase-3 was detected by clolorimetric assay. Results The percent of apoptotic cells in hydrogen peroxide and Par-4-siRNA-treated group was (29.7 ± 2.3) %, which was significantly lower than that of hydrogen peroxide-treated group [(54.2 ± 4.1)%, q= 8.91, P ＜ 0.01)]. Par-4 siRNA could significantly suppress the increase of Smac protein, E2F1 DNA binding activity and caspase-3 activity induced by hydrogen peroxide in A549 cells. Conclusions Par-4 gene silence induced by siRNA might inhibit the apoptosis of alveolar epithelial cells, which might be resulted from suppression of the up-regulation of Smac gene expression, E2F1 DNA binding activity and caspase-3 activity.
RESUMEN
Objective To observe expressions of mRNA for Par-4 and WT1 in bone marrow cells from acute leukemia patients and non-leukemia patients, and to approach the correlation between CR rate and Par-4, WT1 expression level. Methods To detect Par-4 and WT1 mRNA expression level in bone marrow cells from 78 acute leukemia patients and 23 non-leukemia patients by means of Real-time Fluorescent Quantitation RT-PCR. Results FQ-RT-PCR result showed that Par-4 mRNA was expressed in bone marrow cells from 78 acute leukemia patients and 23 non-leukemia patients. Compared with control groups, the expression levels of Par-4 mRNA were significantly suppressed (9.35×10-4±8.4×10-5, P ＜0.05). Compared with initial treatment groups and relapse groups, the expression levels of Par-4 mRNA in remission groups were significantly up-regulated (1.26×10-3±1.1×10-4) but were still significantly lower than that in control groups (3.25×10-3±2.9×10-4). There was no significance difference between initial treatment groups and relapse groups. No apparent association was found between Par-4 expression level and CR rate (P ＞0.05). WT1 gene was overexpressed in bone marrow cells from acute leukemia patients(2.98× 10-3±2.1×10-4), but the expression levels of WT1 mRNA were significantly lower in bone marrow cells from control groups (7.25×10-5±6.7×10-6,P ＜0.05). Compared with initial treatment groups and relapse groups, the expression levels of WT1 mRNA in remission groups were significantly down-regulated (6.86×10-4±5.2× 10-5) but were still significantly higher than that in control groups. There was no significant difference between initial treatment groups and relapse groups.There was significant difference between different WT1 expression levels and CR rates (P ＜0.05). Conclusion The result of FQ-RT-PCR testing confirmed that Par-4 mRNA expression is lower, while WT1 is higher in acute leukemia. Par-4 and WT1 gene present mutually exclusive expression patterns. There was no apparent association between Par-4 expression level and CR rate.
RESUMEN
Objective To explore the changes of Par-4 and WT1 genes expression during human leukemic K562 cells apoptosis induced by arsenic trioxide (As2O3). Methods After the K562 cells were treated with arsenic trioxide (2-10 μmol/L) for 24-72 hours, cell survival rate was evaluated by MTT assay and apoptosis was analyzed using flow cytometry. The expressions of mRNA and protein for par-4 and WT1 were tested by Real-time quantitative reverse transcriptase polymerase chain reaction (RQ-RT-PCR) method and Western blotting in K562 cells with various concentrations of arsenic trioxide at different time points. Results After the K562 cells were treated with As2O3 of different concentrations, arsenic trioxide could efficiently inhibit the growth of K562 cells and induce apoptosis with the increase of As2O3 concentration. The same time, the mRNA and protein expressions of Par-4 were up-regulated, while the mRNA and protein expression of WT1 were down-regulated. Conclusion Arsenic trioxide could inhibit the growth of K562 cells and induce apoptosis. Par-4 and WT1 genes could participate in the apoptosis of K562 cells induced by arsenic trioxide.
RESUMEN
Objective To construct an eukaryotic expression vector of human Par-4 gene with green fluorescent protein gene which iS named pIRES2-EGFP/Par-4 and transfect jt into K562 cell line. Methods Using pDNR/Par-4 plasmid as a template.the full length Par-4 cDNA was amplified by PCR and subsequently cloned into T-A vector.Then subcloned into pIRES2-EGFP vector.After identified by digestion of restriefive endonucleases.pIRES2-EGFP/Par-4 was further confirmed by sequencing.Then it was transfected into K562 cells with Superfect reagents.The total proteins were isolated and Par-4 was detected by Western blotting. Results The exact sequences of pIRES2-EGFP/Par-4 vector were confirmed by digestion of restrictive endonucleases and sequencing.After transfection,the expressions of green fluorescent protein were present.The protein expression of Par-4 has been detected in transfected ceils hv Western blotting.Conclusion The vector pIRES2-EGFP/Par-4 has been constructed and could Successfully express Par-4 gene in K562 cells.
RESUMEN
Objective To study the effect of procyanidins (PC) on mRNA and protein expression of par-4 and bcl-2 genes in PC12 cells induced by A?_ 25-35 . Methods Cell survival rate was evaluated by MTT assay and apoptosis was analyzed by Hoechst 33258-PI fluorescence staining. The expressions of mRNA and protein for par-4 and bcl-2 were tested by RT-PCR and Western blotting. Results Pretreatment with different concentrations of PC (5、10、20, and 30 mg/L) for 1 h increased the survival rate of PC12 cell in a dose-dependent manner. PC prevented the PC12 cells nuclei from shrinkage, condensation, and cleavage induced by A?_ 25-35 . PC decreased the expression of par-4 mRNA and protein, and increased the expression of bcl-2 mRNA and protein. Conclusion PC can protect PC12 cells from apoptosis induced by A?_ 25-35 in a dose dependent manner. The mechanism of protection is likely related to decreasing the par-4 gene expression and increasing the bcl-2 gene expression.
RESUMEN
AIM To study the mechanism of the protective effects of sodium ferulate (SF) on apoptosis of PC12 cells induced by Hydrogen peroxide(H 2O 2). METHODS Based on the model of apoptosis of PC12 cells induced by H 2O 2, cell viability was tested by MTT assay. The expressions of mRNA and protein were tested by RT-PCR and Western blot. RESULTS Pretreatment with different concentrations of SF (25～250 ?mol?L -1) for 1h increased the survival rate of PC12 cells, decreased the expressions of par-4, caspase-3 mRNA and Par-4 protein, while there was no obvious change of the activity fregment of caspase-3 P20 and caspase-3 activity. CONCLUSION SF can prevent the PC12 cells against H 2O 2 neurotoxicity in a concentration-dependent fashion. The mechanism of protection is likely related to decreasing the Par-4 level, and the relation with Caspase-3 needs further study.