| BackgroundSepsis is defined as the host inflammatory response that occurs due to severe life-threatening infection. Sepsis is the most common cause of mortality in most intensive care units and the incidence of sepsis is increasing. Sepsis-induced immune cells apoptosis and following immunosuppression contribute to the morbidity and mortality of sepsis. Moreover,lymphocytes play a significant role in the immune protection.It has been demonstrated that sepsis induce plenty of lymphocytes apoptosis in immune organs and peripheral blood.Meanwhile, the apoptosis of those cells lead to following serious immunosuppression. According to the recent studies, extracellular histones play a key role in the pathogenesis and death of sepsis,and it is considered a potential target for the treatment of sepsis.Our previous studies have shown that massive release of extracellular histones leads to lymphocyte apoptosis through mitochondrial injury during sepsis.Grape seed proanthocyanindin extract (GSPE) is derived from grape seeds. Proanthocyanidins contain a number of phenolic hydroxyls. Polyphenolic compounds have a very important function of antioxidant, and they can clean off the free radicals, and reduce the membrane lipid peroxidation. Researches has demonstrated that GSPE has a wide range of biological effects, such as antioxidant, anti-inflammatory and mitochondrial protective effect in various experimental models.Because mitochondrial injury is a crucial way to histones-mediated lymphocytes apoptosis, we might have inferred that GSPE has an inhibiting effect on histones-mediated lymphocyte apoptosis.And we will make it clear about the effect and mechanism of GSPE on histone-mediated lymphocyte apoptosisby by the study.Objectives1.To investigate whether GSPE can suppress the lymphocyte apoptosis induced by histones;2.To explore whether GSPE inhibited histone-induced lymphocyte apoptosis through ROS-mediated pathway, MAPK p38 and mitochondrial pathway.Methods1.Extracting lymphocytes from human blood20ml blood was drawn from healthy volunteers and heparin anticoagulation, then divided into 5 tubes. Blood diluted average with PBS (1:1)was added upon the lymphocyte separation medium(4ml) and centrifuged of 2000rpm at 20℃ for 30min. Draw the white film of lymphocytes and wash with PBS resuspended with the 1640 medium of all lymphocytes in the same tube for counting.2.Drug treatments and groups of lymphocytesPeripheral blood was sponsored by healthy volunteers and cultured lymphocytes in 1640 culture medium after the step of separation.Lymphocytes were seeded in a 6-well plate at a concentration of 1×106 cells/well in 2ml),and divided those falling into 4 groups. Ⅰ control group;Ⅱ cultivated with histone (50ug/mL);Ⅲcultivated with GSPE (2ug/mL);Ⅳ GSPE+histone.Ⅲ and IVwere pretreated with GSPE for 2h, then IV incubated with 50μg/ml of histones for 2.5h.After incubation, the lymphocytes were collected for analysis.3. Cellular Assaysa) Cell viabilityCell viability was measured with the CCK8 assay. Human lymphocytes were seeded at a density of 1.25x104cells/well in 96-well plates, each plant hole is 200μl. After incubation with the indicated drugs, CCK8 cell viability assay measured after incubate 24 hours.b) Cytotoxicity AssayApoptotic or necrotic cell death was measured by flow cytometry, after drug treatment, cells were collected and re-suspended in Annexin-V and propidium iodide (PI) for 15min at room temperature. Following the samples were analyzed using a FACS Calibur flow cytometer.c) Measurements of Mitochondrial InjuryRhodamine-123(Rho-123), a cell-permeable, cationic, green-fluorescent dye that reversibly accumulates in mitochondria, which has been commonly used for monitoring membrane potential and apoptosis. Cells were labeled with Rhodamine 123 (1μM) at 37℃ for 30 min before the end of the drug incubation. After washing with PBS, the samples were analyzed by flow cytometry as described previouslyd) Detection of ROS productionIntracellular ROS production was determined by 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) staining followed by flow cytometry. Briefly, cells were loaded with 1μM DCFH-DA for 30 minutes, washed with PBS twice. Signals were detected through a 525 nm band-pass filter (FL1 channel) using a FACSCalibur flow cytometer.4. Determination of related protein expression in lymphocytes by Western Blota) Detection of mitochondrial membrane protein Bcl-2The prepared lymphocytes were centrifuged by 1200rpm in 10min and washed with lml PBS, then the supernatant was discarded. Cells were lysed in RIPA(1% SDS in 5 mM Tris-HCl, pH=7.4,1 mM EDTA,150 mM NaCl,1mM phenylmethanesulfonyl fluoride (PMSF),5μg/mL leupeptin, and 10μg/mL pepstatin) for 30min on ice. Samples were centrifuged at 14,000g for 10min at 4℃. GAPDH was included as a loading control to determine the amount of total protein in each group. Based on the volume, we take western blot to detect the expression of mitochondrial membrane protein Bcl-2.b) Detection of mitochondrial membrane protein p38The prepared lymphocytes were centrifuged by 1200rpm in 10min and washed with lml PBS, then the supernatant was discarded. Cells were lysed in RIPA(1% SDS in 5 mM Tris-HCl, pH=7.4,1 mM EDTA,150 mM NaCl,1 mM phenylmethanesulfonyl fluoride (PMSF),5μg/mL leupeptin, and 10μg/mL pepstatin) for 30min on ice. Samples were centrifuged at 14,000g for 10min at 4℃. GAPDH was included as a loading control to determine the amount of total protein in each group. Based on the volume, we take western blot to detect the expression of mitochondrial membrane protein p38.c) Detection of mitochondrial membrane protein caspase 3The prepared lymphocytes were cenrtifuged by 1200rpm in 10min and washed with 1ml PBS, then the supernatant was discarded. Cells were lysed in RIPA(1% SDS in 5 mM Tris-HCl, pH=7.4,1 mM EDTA,150 mM NaCl,1 mM phenylmethanesulfonyl fluoride (PMSF),5μg/mL leupeptin, and 10μg/mL pepstatin) for 30min on ice. Samples were centrifuged at 14,000g for 10min at 4℃. GAPDH was included as a loading control to determine the amount of total protein in each group. Based on the volume, we take western blot to detect the expression of mitochondrial membrane protein caspase 3.5.Statistical AnalysisData analysis was performed with SPSS 17.0 software. Results were expressed by means of ±EMs and analyzed with one-way ANOVA or 2-tailed Student’s t-test, as appropriate, followed by the relevant post hoc t test to determine P values. P values of less than 0.05 were considered statistically significant.Results1.Effects of GSPE on viability of human lymphocytesNo significant change in cell viability was observed, however, compared with control cell,viability of lymphocytes was significantly reduced with the treatment of histones (50μg/ml). (P<0.05).2.Effects of GSPE on lymptocyte apoptosis induced by histonesTo assess whether GSPE can protect lymphocytes from apoptosis induced by histones in vitro, lymphocytes were initially treated with GSPE for 2 hours and subsequently stimulated with histones for 2.5 hours. Apoptosis was analyzed using AnnexinV-FITC/PI staining and a FACSCalibur flow cytometer. The histones group showed a high proportion of apoptotic cells compared to the control group (16.58±7.17% vs 3.15±1.06%, P<0.01). The apoptotic cells were significantly reduced in the histones+GSPE group compared to the histones group (7.03±1.93% vs 16.58±7.17%, P<0.01). Limited apoptosis was detected in the GSPE group (2.74±1.98%).3.GSPE decreases intracellular ROS accumulation induced by histonesThere was a significant increase in ROS intensity between histones group and control group (2671.94±752.07 vs 1432.42±434.26, P<0.01). The ROS intensity was significantly reduced in the histones +GSPE group in contrast with the histones group (2101.13±773.22 vs 2671.94±752.07, P<0.01). GEPE group did not showed a significant decrease of ROS intensity compare to control group (1274.97±434.25 vs 1432.42±434.26, P>0.05).4.GSPE prevents mitochondrial from injury induced by histonesIt was observed that the proportion of healthy lymphocyte without mitochondrial injury decreased significantly in histones group in contrast with the control group (49.19±3.36 vs 68.85±4.07, P<0.01). The healthy lymphocyte was significantly increased in the histones+GSPE group in contrast with the histones group (66.83±3.3 vs 49.19±3.36, P<0.01). GEPE group did not show a significant decrease of healthy lymphocyte compare to control group (70.88±4.37 vs 68.85±4.07, P>0.05).5.GSPE resists the Bcl-2 down-regulation and sequential caspase 3 activation induced by histonesIt was observed that histones treatment resulted in Bcl-2 down-regulation, but GSPE treatment up-regulated Bcl-2 expression (P<0.05). Furthermore, the pretreatment of GSPE was able to counteract the down-regulation of Bcl-2 induced by histones treating (P<0.05). Consequently, cleaved caspase-3, the activating form of caspase-3 was up-regulated by histones treatment, and it was inhibited by GSPE pretreatment (P<0.05).6.GSPE inhibited p38 phosphorylation induced by histonesThe up-regulation of p38 phosphorylation was observed in lymphocyte treated with histones in contrast to control (P<0.05). Secondary, the effect of histones on p38 phosphorylation was compromised by the pretreatment of GSPE (P<0.05)Conclusion1. GSPE comprises an important protective part in apoptosis of lymphocytes induced by histone.2. GSPE inhibits histones-mediated apoptosis of lymphocytes may through ROS-mediated pathway, MAPK p38 and mitochondrial pathway. |
PDF Full Text Request