The Role Of PI3K/PKB Singal Pathway In Apoptosis Of Pbmc In Chronic Hepatitis B Patients

Objective: Chronic hepatitis B is one of infective diseases to threaten human health which is mainly caused by continuing exist of hepatitis B virus. Hepatitis B virus damages hepatocytes by activating immune system. Repeating inflammations in the liver will lead to hepatic cirrhosis, even hepatic carcinoma if virus cannot be completely cleared.The mechanism resulted in chronic hepatitis is not clear to this day. Many studies have already confirmed that the pathogenesis of chronic viral hepatitis B (CHB) is closely related with the lower cellular immunity function of the patients, but the mechanism of the lower cellular immunity function of the patients has not been thoroughly expounded. In recent years, the relation of activation induced cell death ( AICD ) in T lymphocyte and Chronic Hepatitis B is paied close attention . AICD is a kind of apoptosis in reactivated T lymphocyte which means that reactivated mature lymphocyte (T or B) is reactived by activated singals ( especial TCR/CD3 complex ) and induce the expression of apoptotic effective factor such as Fas, FasL, TNF and its ligand. And transmit apoptotic signal pathway through the interaction of the ligand and receptor to induce the apoptosis in cell iteself. Peripheral blood mononuclear cells ( PBMCs ) are composed mainly by lymphocytes, and also include monocyte-macrophage cells and natural killer cells and other immune cells. PBMCs play an important role in body's immune response especially the cellular immune responses, and detection of apoptosis of PBMC can roughly reflect the apoptosis of peripheral blood lymphocytes. Studies have found that the ratio of AICD of peripheral blood lymphocyte and PBMC in chronic hepatitis B patients was higher than normal control. This phenomenon indicated that HBV might through induce PBMC apoptosis rise to reduce the quntity and the activity of immunocyte , so that led to immunity decreased in CHB patients, which might be an important mechanism for persistent infection of HBV and immune tolerance of chronic infection of HBV. so that resulted in persistent infection of HBV. So how to decrease the AICD of PBMC in CHB patients, and improve cellular immunity function of the CHB patients will be a possible way to break the state of immune tolerance and eliminate HBV in CHB patients. So the excess of AICD in lymphocyte result of HBV infection may be one of important mechanisms for chronic infection of HBV. The studies about the apoptosis pathway of AICD of PBMC in CHB patients mainly focused on the death receptor pathway which is mediated by Fas/ FasL etc and the mitochondria pathway which is mainly regulated by Bcl-2 family, and proved that this two pathway may play an important role in the AICD of PBMC in CHB patients.PI3K (phosphatidylinositoI3-kinase)/PKB (protein kinase B) singal pathway plays a vital important role in suppressing cell apoptosis. FOXO3a (FKHRL1) is a important downstream ttarget gene of the PI3K/PKB singal pathway, which is a member of Forkhead family of transcription factors. Dephosphorylated FOXO3a(FKHRL1) is in the nucleus, which could mediate the apoptosis by the death receptor pathway and the mitochondrial pathway through activate the downstream targe apoptosic gene (FasL,TRAIL,Bim ,ect). When FOXO3a(FKHRL1) is phosphorylated by PKB, it will lose its transcription function. When extracellular signals stimulate cell, they could active the PI3K on the cell membrane, which could phosphorylate PKB and activate PKB(p-PKB). The activated PKB could dephosphorylate and inactivated FOXO3a. The phosphorylated FOXO3a have low appetency with DNA , and could transfer from nucleus to cytoplasm. FOXO3a could combine with the chaperonin 14-3-3, which restrain FOXO3a return to nucleus. So that the transcription function of FOXO3a is suppressed, and suppress the transcription and translate of its downstream target gene Bim, and suppress the expression of proapoptotic protein Bim, and suppress the cell apoptosis. Reasearches have indicated that activating the PI3K/PKB singal pathway could supress the AICD in the T lymphocyte, while inhibiting the singal pathway could facilitate AICD in the T lymphocyte. Whether PI3K/PKB singal pathway mediate AICD of PBMC in CHB patients, research in this area has not been reported. So we take the PBMC of CHB patients as the research object and simulate the process of AICD in vitro. We observe the expression of p-PKB, PKB and its target transcription factor FOXO3a and proteine Bim in PI3K/PKB signal pathway and the apoptosis rate of PBMC, and the change of the expression of these proteins and the apoptosis rate of PBMC after the PI3K/PKB signal pathway is activated by the activator IGF-1.Research the role of PI3K/PKB signal pathway in the process of AICD of PBMC in CHB patients,and further explore the mechanism of AICD to provide a theoretical basis for the treatment of CHB.Methods:1 Inclusion criteria and exclusion criteria:We selected a total of forty CHB patients, including twenty-two males and eighteen females, aged from eighteen to sixty years, mean age thirty-five years, and they were randomly divided into two groups: twenty patients in CHB group and twenty patients in CHB+activator group. The HBV-DNA in patients in between 1×103copies/ml and 1×107copies/ml. The liver function is nuusual, the ALT is two times than the higher limit of the normal. All subjects were diagnosed according to the guide of prevention and treatment in chronic hepatitis B which was established in 2005 by Chinese Medical Association, and their hepatitis B markers HBsAg were positive, HBeAg or anti-HBe were positive and anti-HBc were positive. We have excluded HAV, HCV, HDV, HEV, and HIV. The patients who used the drugs that can affect the immune function such as glucocorticoid and interferon nearly 3 months were ruled out. And patients who once used antiviral drugs and recent obtained acute infections were ruled out. Twenty individuals obtained from healthy blood donors were included in the healthy control group, including twelve males and eight females, aged from eighteen to forty-five years, mean age thirty-five years. 2 PBMCs extraction and cells culture:PBMCs were extracted from fresh and heparinized blood by density gradient centrifugation under aseptic condition. PBMCs were stimulated by PHA(100μg/ml) for 16～18h , then removed PHA from nutrient medium and added exogenous IL-2(1000U/L). Cells were cultured at least 7 days and stimulated again by anti-CD3(500ug/ml) antibody for 48 hours. The members of CHB+activator group were pretreated with the IGF-1(100ng/ml) for 20 minutes before the use of anti CD3 antibody.3 Detection of Cell apoptosis:Cultured cells were fixed with 70% ethanol, and centrifuged at 4℃to remove the fixative. The cells were washed twice with cold PBS(phosphate buffered saline), and then added the PI dye about 1000μl containing RaseA. Placed at room temperature for 30 minutes, and then analyzed apoptosis rate by flow cytometer.4 Detection of p-PKB, total-PKB, p-FOXO3a and Bim protein:Proteins were extracted from remaining cells and preserved under -80℃. We collected enough samples and detected the expression of p-PKB, total- PKB, p-FOXO3a and Bim protein by Western blot.Result:1 The apoptotic rate of PBMC of CHB group (28.27%±4.61%) was higher than healthy control group (19.36%±5.90%), the discrepancy was statistically significant (P<0.05), and also higher than CHB+activator group (22.69%±3.47%), the discrepancy was statistically significant (P<0.05). The apoptotic rate of PBMC of CHB+ activator group (22.69%±3.47%) is higher than the healthy control group (19.36%±5.90%), the discrepancy was statistically significant (P<0.05).2 The expression of p-PKB in PBMC of CHB group (0.44±0.09) was lower than healthy normal control (0.55±0.10, t=3.649, P<0.01), and also lower than CHB+activator group(0.76±0.17, t=7.618, P<0.01). The discrepancy of the expression of total-PKB in the three groups was not statistically significant. The expression of p-FOXO3a in PBMC of CHB group (0.31±0.07) was lower than healthy normal control (0.43±0.05, t=6.131, P<0.01), and also lower than CHB+activator group(0.63±0.11, t=10.979, P<0.01). The expression of Bim in PBMC of CHB group (0.96±0.15) was higher than healthy normal control (0.58±0.12, t=9.110, P<0.01) , and also higher than CHB+activator group(0.53±0.10, t=10.997, P<0.01).3 The expression of p-PKB in PBMC of CHB patients had negative correlation with apoptotic rate(r=﹣0.839,P<0.01).Conclusion:1 There is the phenomenon of AICD in PBMC of CHB patients, and the apoptotic rate is higher than healthy persons. This may be an important reason for the lower cellular immunity function and the immune tolerance of the CHB patients.2 There is the phenomenon that the PI3K/PKB singal pathway is inhibited in the chronic hepatitis B patients. The rise of apoptotic rate in PBMC of CHB patients may associate with the decrease of PI3K/PKB singaling activity.3 PI3K/PKB signal pathway is involved in mediating AICD of PBMC in CHB patients, and is one of the mechanisms of increased PBMC apoptosis in CHB patients.