Role Of The JNK Signal Pathway In Apoptosis Of PBMC In Chronic Hepatitis B Patients

Hepatitis B virus( HBV) often cause chronic infection,but the mechanism has not been completely clear.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 CHB patients has not been thoroughly expounded.In recent years,Studies have proposed that it was related with the activation-induced cell death(AICD).The apoptosis of activated mature lymphocytes(T or B)after activated again by stimulation(especially the TCR/ CD3 complex)is called AICD,Which is a physiological mechanisms of maintaining the stability of immunity and may be an important reason of peripheral immune tolerance.Peripheral blood mononuclear cells(PBMCs)are composed mainly by lymphocytes,and also include monocyte-macrophage 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 group.This phenomenon indicate that lymphocytes may be activated by HBV-specific antigen,and then apoptosis(AICD)happen.AICD may cause the quantity and the activity of lymphocytes to reduce,so that lead to cellular immunity response of CHB patients decrease,which may be an important factor of persistent infection of HBV,and an important mechanism of immune tolerance.So how to adjust AICD to reduce the apoptosis of lymphocyte and improve cellular immunity function of the CHB patients will be a possible way to break the state of immune tolerance and eliminates HBV in CHB patients.But the mechanism of AICD of PBMC in CHB patients has not been completely clear.The studies about the apoptosis pathway of AICD of PBMC in CHB patients mainly focused on the death receptor pathway which was mainly mediated by Fas/ FasL and the mitochondria pathway which was mainly regulated by Bcl-2 family,and proved that this two pathway might play an important role in the AICD of PBMC in CHB patients.c-Jun N-terminal kinases(JNKs)family,also referred to as stress-activated kinases(SAPKs), is a subfamily of the mitogen-activated protein kinases (MAPKs) superfamily.Its upstream dual specificity kinase MEK4/ 7 can phosphorylate the Thr183 and Tyr185 sites to activate JNK,and phosphorylation JNK(p-JNK) is its active form.A variety of apoptosis induced by extracellular stimuli (such as stress, Fas, TNFα) were mediated by JNK,and JNK signal pathway was involved in many apoptosis,and palyed an important role in the development of many diseases.The mechanism was that through the transcription-dependent or transcription-independent pathway, JNK could regulate the expression of downstream apoptosis-related target gene or the activity of target proteins,and mediated the apoptosis of the death receptor pathway and the mitochondrial pathway.C-Jun and Bim are important downstream molecules of the JNK-mediated apoptosis signal pathway.C- Jun is one of the nuclear transcription factor AP-1 proteins and the first discovered nuclear substrates of JNK, and phosphorylation c-Jun(p-c-jun) is its active form.Activated JNK(p-JNK) can phosphorylate the Ser63 and Ser73 sites to activate c-jun after translocation into the nucleus,thereby enhancing its transcription activity,and promote the transcription of downstream apoptosis-related target gene and the expression of apoptosis proteins(FasL,Bim,c-myc,et al),and mediate apoptosis of the death receptor pathway and the mitochondrial pathway.Bim is a member of BH3- only subfamily in Bcl-2 family and an important apoptosis regulatory protein. Activated JNK can transfer from the cytoplasm into the nucleus to increase the expression of Bim through activating the transcription factor c-jun;and can also directly phosphorylate Bim in the cytoplasm and mediate the apoptosis of mitochondrial pathway. In recent years, studies have shown that JNK mediated AICD of lymphocytes in a variety of diseases.A study found that application of JNK inhibitor SP600125 could fight the AICD of influenza epitope-specific human cytolytic T lymphocytes(CTL),and proved that JNK was involved in mediating AICD of the influenza epitope-specific human CTL.Another study found that JNK was involved in mediating AICD of the mouse spleen T cells.But whether the JNK signal pathway is also involved in mediating AICD of PBMC in CHB patients have not been reported.So we took the PBMC of CHB patients as the research object and simulated the process of AICD in vitro.We observed the expression of p-JNK,total-JNK and its downstream transcription factor p-c-jun and downstream protein Bim and the apoptosis rate of PBMC,and the change of the expression of this proteins and the apoptosis rate of PBMC after the application of JNK inhibitor SP600125,to research the role of JNK signal pathway in the process of AICD of PBMC in CHB patients.Objective:To study the role of JNK 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 criteriaWe 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+inhibitor group.All subjects were diagnosed according to the guide of prevention and treatment of chronic hepatitis B which was established in 2005 by Chinese Medical Association. HBVDNA greater than 1×103copies/ml and less than 1×107copies/ml,and ALT more than twice higher.All patients had not used antiviral drugs.We excluded HAV,HCV,HDV,HEV,HIV and acute infections.The patients who had used the drugs that can affect the immune function such as glucocorticoid and interferon nearly 3 months 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 years.2 PBMCs extraction and cells culturePBMCs 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+inhibitor group were pretreated with the JNK specific inhibitor SP600125(20μmol/l) for 2 hours before the use of anti-CD3 antibody.3 Detection of Cell apoptosisCultured 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 the apoptosis rate by flow cytometer.4 Detection of p-JNK,total-JNK,p-c-jun and Bim proteinProteins were extracted from remaining cells and preserved under -80℃.We collected enough samples and detected the expression of p-JNK, total- JNK,p-c-jun 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+inhibitor group(23.37%±5.52%),the discrepancy was statistically significant(P<0.05).The apoptotic rate of PBMC of CHB+inhibitor group(23.37%±5.52%) was higher than healthy control group(19.36%±5.90%),the discrepancy was statistically significant (P<0.05).2 The expression of p-JNK in PBMC of CHB group(1.09±0.16)was higher than healthy control group(0.83±0.12),the discrepancy was statistically significant(t=5.885,P<0.01),and also higher than CHB+inhibitor group(0.53±0.15),the discrepancy was statistically significant(t=11.502,P<0.01).The discrepancy of the expression of total-JNK in the three groups was not statistically significant.The expression of p-c-jun in PBMC of CHB group (1.21±0.15)was higher than healthy control group(0.96±0.13),the discrepancy was statistically significant(t=5.477,P<0.01),and also higher than CHB+ inhibitor group(0.62±0.14),the discrepancy was statistically significant(t= 12.899,P<0.01).The expression of Bim in PBMC of CHB group(0.96±0.14)was higher than healthy control group(0.58±0.12),the discrepancy was statistically significant(t=9.133,P<0.01), and also higher than CHB+inhibitor group(0.52±0.13),the discrepancy was statistically significant(t=10.086,P< 0.01).3 The apoptotic rate of PBMC had positive correlation with the expression of p-JNK in PBMC of CHB patients(r=0.823,P<0.01).Conclusion:1 There is the phenomenon of AICD of PBMC in CHB patients,and the apoptotic rate is higher than healthy persons.2 The JNK signal pathway is excessive activated in PBMC of CHB patients.3 The apoptotic rate of PBMC had positive correlation with the expression of p-JNK in PBMC of CHB patients.The expression of p-JNK and its downstream transcription factor p-c-jun and downstream protein Bim in PBMC of CHB patients and the apoptotic rate of PBMC were decreased after JNK was inhibited with the JNK specific inhibitor SP600125.This shows that JNK 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.