Expression And Clinical Significance Of GSK3β In Liver Failure

Objective: Liver failure, a serious liver disease caused by a variety of factors, is a common clinical syndrome and has a extremely low survival rate. Over the years, scholars from all over the world explore the pathogenesis of liver failure continuously, which has not been fully elucidated. Glycogen synthase kinase 3β is the subtype of Glycogen synthase kinase 3. Studies showed that GSK3 is an important positive regulator in inflammatory process, and GSK3β proteins are reported to produce an effect on NF-κb activity. Our study aims to explore impact of GSK3β, p-GSK3β in the evolution of the liver failure by investigating the GSK3β, p-GSK3β levels in serum and liver tissues among the different research objects.Methods: This experiment included the following two parts.1 Animal experimentTo induce ALF, the mice(except for the control) were injected intraperitoneally with D-Gal N. The levels of alanine transaminase(ALT), aspartate transaminase(AST), total bilirubin(TB) and prothrombintime(PT) in serum were measured, the liver pathology was observed by hematoxylin-eosin(HE) staining, the GSK3β m RNA expressions were detected in liver tissues by real-time polymerase chain reaction(RT-PCR), the distributioins of GSK3β, p-GSK3β in liver tissues were investigated by immune- histochemical staining and the expressions of GSK3β, p-GSK3β in liver tissues were analyzed by Western blot.2 Clinical studyThe blood serum of outpatients or inpatients in the Third Hospital of Hebei Medical University from January 2015 to Octobor 2015 was enrolled in our study. Furthermore, we also recruited the sample of healthy people from the medical examination center of the same hospital. Then a total 67 subjects were enrolled in our study and divide into 3 groups including 22 chronic hepatitis B patients, 25 HBV related acute on chronic liver failure patients and 20 healthy controls. At the same period, 39 liver tissues were obtained from 12 HBV-related ACLF patients, 23 CHB patients and 4 liver donors by liver transplantations and liver biopsy. Liver function parameters, coagulation function parameters, serum virology and serum HBV DNA loads were determined. Then enzyme-linked immunosorbent assays(ELISA) was performed to value the serum levels of p-GSK3β and TNFα. In addition, the GSK3β m RNA expressions in liver tissues were analyzed by RT-PCR. Further, immunohistochemical staining and Western blot were performed to illustrate the distributions and amounts of liver GSK3β, p-GSK3β in different groups. In the end, we also explored the relationship between the p-GSK3β level and the inflammation of liver failure.Results: 1 Animal experiment 1.1 The ALF models in mice were built successfully. Normal mice were in good spirits and with good appetite, as well as in normal response to external stimuli and pain. ALF mice were in low spirites and with poor appetite, and response to external stimuli diminishedly.1.2 The levels of serum ALT, AST, TB and PT in ALF mice were obviously higher than healthy control(P <0.01). 1.3 Normal hepatocytes were observed to be arranged radially from the central veins by HE staining in healthy control group. Histopathological examinations of liver biopsies in the ALF model showed that the structure of liver tissues was destroyed and large necrosis area and significant inflammatory cellular infiltration were present. 1.4 The higher expressions of GSK3β m RNA in ALF liver tissues were observed comparaed with healthy control(P <0.01). 1.5 The expressions of GSK3β, p-GSK3β were showed mainly in hepatocyte cytoplasm by the immunohistochemical staining. The GSK3β expressions of liver tissues in all enrolled groups detected by western blot were no differences(P >0.05), and the lower expression of p-GSK3β in ALF mice were observed comparaed with healthy control(P <0.01). 2 Clinical study2.1 The demographic data of the subjects were comparable for age and sex. The ACLF patients displayed obviously higher levels of ALT, TB, DB and INR, but a lower Alb levels(P<0.01). There were no significant differences in AST level and HBV DNA loads between ACLF patients and CHB patients(P >0.05). 2.2 The levels of serum TNFα in CHB patients were higher than healthy control. The levels of serum TNFα in ACLF patients displayed obviously higher than the CHB patients and healthy control(P<0.01).The levels of serum p-GSK3β in CHB patients were higher than healthy control, and the levels of serum p-GSK3β in ACLF patients displayed obviously higher comparaed with the CHB patients and healthy control(P<0.01). 2.3 The expressions of liver GSK3β m RNA in ACLF patients were higher than healthy control and CHB patients(P<0.01), but there was no statistical significance between CHB patients and healty control patients(P >0.05). 2.4 The expressions of GSK3β, p-GSK3β were showed mainly in hepatocyte cytoplasm by the immunohistochemical staining. The GSK3β expressions of liver tissues in all enrolled groups detected by western blot were no differences(P >0.05), and the lower expressions of p-GSK3β in CHB patients were observed comparaed with healthy control(P <0.01). Moreover, the expressions of p-GSK3β in ACLF patients were much lower than the CHB patients and healthy control(P<0.01). 2.5 The expressions of serum p-GSK3β in ACLF patients were negatively correlated with the levels of serum TNFα.Conclusions:1 GSK3β hosphorylation/dephosphorylation was triggered in liver failure.2 Activity of GSK3β was enhanced in ACLF patients along with higher TNFα levels, reminding GSK3β ight have a role in the progression of ACLF by participating synthetic section and/or secretion of TNFα.