The Effect And Mechanism Of Histones On Apoptosis Of Lymphocytes In Sepsis

BackgroundSepsis is defined as systemic inflammation induced by bacterial, fungal or viral infection, which is a common cause of shock and organ failure. Sepsis with rapid progress, high mortality, has become a major cause of death in Intensive Care Unit. The complex mechanism of occurrence and development increase the difficulty in clinical treatment of sepsis.A plenty of studies have shown:it presents during sepsis that large numbers of immune and non-immune cells have happened to apoptosis, leading to immune suppression and organ damage. There’s significant difference among all immune and non-immune cells with different susceptibility to apoptotic stimuli. So the intestinal epithelial cells and immune cells are most likely to occur to apoptosis due to active metabolite. Subsequent effect of apoptosis on immune system is the key to sepsis death. Apoptosis results that decrease of immune cells for apoptosis and immunosuppressive factors working on vicinity secreted by apoptotic immune cells lead to dominant role of negative T cell, so that innate immunity and acquired immunity are affected. Apoptosis found in peripheral blood of patients with sepsis was more than non-septic patients significantly, which mediated immune suppression promoting deterioration in sepsis. As the key of death in sepsis, it can get better treatment to prevent or inhibit apoptosis of immune system in sepsis.Histones, as the basic structure of eukaryotic chromosomes, including H1, H2A, H2B, H3, H4of five types, constitute the core of the nucleosome. In addition to participating in chromosome structure, histones also have such physiological function as constitution of heterochromatin formation, gene imprinting, X chromosome inactivation, transcriptional control and so on. In healthy people, or patients, extracellular histones may exist, which are released from dying cells. The mechanism is still not clear that histones released to extracellular. It can cause high concentration of histones in circulation, if nucleosomes released from the apoptotic or necrotic cells in death process are beyond the body’s normal ability to clear or impair. Neutrophil extracellular traps(NETs) released from neutrophil granulocyte are compounds of DNA, histones and cell particles with scavenging pathogenic microorganisms, in which histones play a key role. NETs, large released during sepsis, induce extensive damage of immune and tissue cells accompanied with antimicrobial.It has been proved that the extracellular histones is a major cause of death in septic. Immunosuppression induced by apoptosis of immunocytes is crucial to death of sepsis. It’s unknown that whether histones can induce apoptosis of immunocytes.However, histones can results inflow of calcium ions and injury of organelles. Therefore, we hypothesize that histones-induced apoptosis of immunocytes through receptor of p38and injury of mitochondria to active Caspase3. This study has explained the mechanism of histones-mediated apoptosis of immunocytes, enrich the pathogenesis of sepsis and provide new theories for the treatment of sepsis.Objective1. To investigate histones release into the blood in sepsis;2. To explore the apoptosis of lymphocytes induced by histones, and the strength of it; 3. To explore whether histones induce apoptosis of lymphocytes through MAPK p38and mitochondrial pathway;4. To investigate whether histone antibody can reduce sepsis-induced apoptosis of lymphocytes.Methods1. Detection of the expression of histone H4in peripheral blood of mice with septic model.a) Groups of animals18male C57mice (8-12weeks old) selected were randomly assigned to three groups:normal control group, sham operation group (sham group), sepsis group (CLP group). Among them, mice in the control group were received no treatment. In mice of sham group we only open the abdominal cavity, and then find the cecum out and into the abdominal cavity, close the abdominal cavity. Mice in sepsis group were operated with cecal ligation and puncture (Cecal Ligation and Puncture, CLP), Observed at room temperature of25℃for6hours. Taking blood about1mL, of which0.3mL taken for obtaining leukocytes,0.7mL of for blood plasma. The experiment was repeated three times.b) Establishing septic modelMice were fixed, skin prepared under anesthesia. We incide the skin and abdominal peritoneum on white line, find and ligate1/3from the end of cecum, then piece with a7-gauge needle, close peritoneal cavity at last. The mice were placed at room temperature of25℃for observation.c) Detection of extracellular histone H4After centrifugation from0.7mL whole blood specimens for plasma, we diluted it for5-folds and denatured, centrifuged, kept in-20℃for preservation. We measured the concentration of total protein with BCA technology. Based on the same volume, we take western blot to detect the expression of histone H4in each group.d) Detection of apoptosis in lymphocytes of miceLymphocytes were separated with density gradient centrifugation, and then were stained with Annexin V-FITC/PI. Apoptosis of lymphocytes were detected by flow cytometry.2. The effect and strength of apoptosis of lymphocytes induced by histonesa) Groups of lymphocytesWe drew blood from healthy volunteers and cultured lymphocytes in1640culture medium after the step of separation.(1).Histone-induced apoptosis of lymphocytes on concentration-dependence:Lymphocytes were divided into four major groups, including12tubes. Each tube with lymphocytes of2.5×105were cultivated with histone Oug/mL (control group),50ug/mL,100ug/mL,200ug/mL respectively at37℃for2h, taking the same volume of1640culture medium as control.(2) Histone-induced apoptosis of lymphocytes on time-dependence: Lymphocytes were divided into three major groups, including nine tubes. Each tube with lymphocytes of2.5x105were cultured with histones100ug/ml at37℃in0h (control group),2h,3h, taking the same volume of1640culture medium as control.b) Extracting lymphocytes from human blood20mL blood was drawn from healthy volunteers and divided into five tubes, blood diluted with PBS was added upon the lymphocyte separation medium and centrifuged of1200g at25℃for30min. Draw the white film of lymphocytes and wash with PBS resuspended with the1640medium of all lymphocytes in one same tube for counting.c) Detection of apoptosis in lymphocytesThe prepared lymphocytes were centifuged by250g in10min, and washed with lml PBS. After supernatant was discarded, lymphocytes were stained with AnnexinV-FITC/PI apoptosis detection kit, then detected with resuspeding of PBS by flow cytometry.d) Groups of animals20male C57mice (8-12weeks old) were selected.14for survival study were randomly assigned to two groups:normal control group and histone group. Histones of75mg/kg were injected into the tail vein, while an equal volume of PBS was injected in the control group, survival rate was observed in mice within2hours.6mice for detection of apoptosis in leukocytes were randomly assigned to two groups: histone group and control group, histones of50mg/kg was injected into tail vein, while an equal volume of PBS was injected in the control group. Mice were observed for6h and got blood of0.1mL for detection of lymphocytes.e) Detection of apoptosis in lymphocytes of miceLymphocytes were separated with density gradient centrifugation, and then were stained with AnnexinV-FITC/PI. Apoptosis of lymphocytes were detected by flow cytometry.3. Histones induced apoptosis of lymphocytes through MAPK p38mitochondrial pathwaya) Groups of lymphocytesWe drew blood from healthy volunteers and cultured lymphocytes in1640culture medium after the step of separation,(1)test of mitochondrial membrane: Lymphocytes were divided into four major groups, including12tubes. Each tube with lymphocytes of2.5×105were cultivated with histone Oug/mL (control group),50ug/mL,100ug/mL,200ug/mL respectively at37℃for2h, taking the same volume of1640culture medium as control.(2)test of apoptotic protein:Lymphocytes were divided into three major groups, including12tubes. Each tube with lymphocytes of10×105/ml were cultivated with histone Oug/mL (control group), 50ug/mL,100ug/mL respectively at37℃for2h, taking the same volume of1640culture medium as control.b) Extracting lymphocytes from human bloodThe details were the same as before.c) Detection of membrane injury of mitochondrionThe prepared lymphocytes were centrifuged and supernatant was discarded.Lymphocytes were stained with Rhodaminel230.5ug/ml at37℃for flow cytometry to detect damage of mitochondrial membrane.d) Detection of mitochondrial membrane protein Bcl-2The prepared lymphocytes were centifuged by250g in10min and washed with lml PBS, then the supernatant was discarded. Cell lysates were added to extract protein, which were denatured and kept at-20℃for preservation. We took GAPDH protein as a reference for immunoblotting (western blot) 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.d) Detection of the phosphorylation of p-38The prepared lymphocytes were centifuged by1000rpm in10min and washed with lml PBS, then the supernatant was discarded. Cell lysates were added to extract protein, which were denatured and kept at-20℃for preservation. We took GAPDH protein as a reference for immunoblotting (western blot) to determine the amount of total protein in each group. Based on the volume, we take western blot to detect the phosphorylation of Caspase3.e) Detection of the activation of Caspase3The prepared lymphocytes were centifuged by1000rpm in10min and washed with1ml PBS, then the supernatant was discarded. Cell lysates were added to extract protein, which were denatured and kept at-20℃for preservation. We took GAPDH protein as a reference for immunoblotting (western blot) to determine the amount of total protein in each group. Based on the volume, we take western blot to detect the activation of Caspase3.4. Histone H4antibody reduce sepsis-induced apoptosis in lymphocytea) Groups of lymphocytesWe drew blood from healthy volunteers and cultured lymphocytes in1640culture medium after the step of separation. Lymphocytes were divided into six groups of three tubes, normal control group, sham control group, sepsis group, IgG antibody group, group of histone H4antibody lOug and group of histone H4antibody20ug. Lymphocytes in sepsis group and different antibody groups were cultured with40uL plasma of CLP model mice, each antibody group were added to the same volume IgG antibody25ug/mL, histone H4antibody lOug/mL, histone H4antibody25ug/mL respectively, incubated for2h at37℃, taking the same volume of1640culture medium as control.b) Extracting lymphocytes from human bloodThe details were the same as before.c) Detection of apoptosis in lymphocytesThe prepared lymphocytes were centifuged by250g in10min, and washed with lml PBS. After supernatant was discarded, lymphocytes were stained with AnnexinV-FITC apoptosis detection kit, then detected with resuspeding of PBS by flow cytometry.5. Statistical methodsGraphs were drawn and statistical analyses were performed using SPSS19.0statistics. Results are expressed as mean±standard error and compared by one-way analysis of variance (ANOVA) followed by Dunnett’s t test and Bonferroni’s t test. A p value less than0.05was considered statistically significant. The survival study was analyzed using Kaplan-Meier method and compared by log-rank test.Results1. Level of histone H4and apoptosis of lymphocytes in septic miceAnalysis of western blot showed that histone H4in peripheral blood of mice was expressed in CLP group rather than the sham group and control group. Apoptosis of lymphocytes in CLP mice was significantly increased as13.23±1.50%higher than the normal control group, which is different from the percentage increased3.23±1.39%in the sham group compared with the control group(F=101.895. p=0.000).2. The intensity of apoptosis in lymphocytes induced by histones: concentration-dependent and time-dependentThe apoptosis of lymphocytes in different groups of histones were significantly different (F=35.921, p=0.000). Apoptotic rate of histone Oug/mL (control group),50ug/mL,100ug/mL and200ug/mL groups were15.64±2.44%,77.98±2.90%,93.61±2.86%,94.30±3.31%.There was no significant difference of apoptosis in lymphocytes (p>0.05) among groups at different time induced by histones100ug/mL. Apoptotic rates of lymphocytes were14.93±2.87%,93.76±3.43%,97.55±2.73%in Oh,2h and3h. Early apoptotic rate of lymphocytes was89.54±2.02%in2hours. However, late apoptotic rate of lymphocytes was91.80±1.54%in3hours.Apoptotic rate of lymphocytes in histone group increased significantly compared with the control group (F=70.41, p=0.001). Survival time of histone group decreased significantly compared with the control group (x2=27.56, p=0.000).3. Effects of histone on MAPK p38and mitochondrial pathway of apoptosis in lymphocytesThe percentage of mitochondrial injury was significantly different among different histone groups (p<0.05). The percentages of mitochondrial injury in lymphocytes of histone Oug/mL (control group),25ug/mL,50ug/mL,100ug/mL and200ug/mL were65.27±3.83%,42.15±11.13%,34.19±12.38%,18.49±2.02%,6.30±1.15%.Analysis of western blot showed that expression of Bcl-2in group of50ug/mL,100ug/mL decreased significantly compared with the control group (F=73.465, p=0.000). In the meantime, the phosphorylation of p38in groups of50ug/mL and100ug/mL decreased significantly compared with the control group (F=239.503, p=0.000). Activated caspase3in groups of50ug/mL and100ug/mL was significantly different from the control group (F=105.470, p=0.000).4. The effects of histone H4antibody on apoptosis of lymphocytesApoptosis of lymphocytes in group of histone H4antibody was significantly different (F=21.140,*p=0.000) form IgG group and CLP group. The apoptotic rates of lymphocytes in sham group, CLP group, IgG group and histone H4antibody lOug/mL,25ug/mL group were44.20±9.61%,73.18±2.44%,68.45±1.56%,55.68±2.60%,35.29±1.34%respectively.Conclusion1. Extracellular histones play an important role in apoptosis of lymphocytes in sepsis.2. Histones induce apoptosis of lymphocytes through phosphorylation of MAPK p38, injury of mitochondrion and activation of Caspase3.