| Objectives: (1) To investigate the association of serum fetuin-A level with the severity of liver damage, the disease progrosis and tumor necrosis factor alpha (TNF-α) level in chronic severe hepatitis. (2) To observe the effect of fetuin-A on the production of TNF-αand IL-6 by LPS-stimulated human PBMCs in vitro. (3) To develop a simple, convenient, efficient and stable mouse model of fulminant hepatic failure (FHF)mouse model, and observe the dynamic changes of fetuin-A expression in the model and evaluate the protection effect of fetuin-A. The possible mechanisms were further explored in vivo.Methods: (1) Three studied groups were observed, including healthy group, chronic hepatitis group and chronic severe hepatitis group; the chronic severe hepatitis group was divided into two subgroups based on the patients recovered or unrecovered. Serum fetuin-A, TNF-α, prothrombin activity (PTA), serum albumin and total bilirubin (T-Bil) were examined. (2) Human peripheral blood mononuclear cells (PBMC) were cultured in the presence of fetuin-A, spermine and LPS, then the supernatant was collected to assay the level of TNF-αand IL-6 in order to investigate the effect of fetuin-A. (3) Factorial experiment was used to establish the standard mouse model of fulminant hepatic failure which was assessed by three indexes including the mouse's death rate within 24 hours, liver function and liver histopathology. (4) The dynamic changes of fetuin-A expression in FHF model were investigated by semi-quantitative RT-PCR (mRNA), Western blot (protein) and immunohistochemical staining. (5) The effect of fetuin-A on FHF model was evaluated through observing the serum level of TNF-αand IL-6, the death rate within 24 hours, liver function, apoptotic index (AI) and liver histopathology.Results: (1) Serum fetuin-A level was significantly lower in chronic severe hepatitis patients (114.02±89.11μg/ml) than that in the people of healthy group (317.75±107.83μg/ml) (P＜0.01) and chronic hepatitis group ( 253.14±206.45μg/ml ) (P＜0.01) respectively. Among the chronic severe hepatitis patients, those recovered patients had higher serum fetuin-A level (203.05±112.94μg/ml) than the unrecovered patients (82.53±47.90μg/ml)(P＜0.01). In chronic severe hepatitis group, serum fetuin-A level was correlated with PTA (Rho=0.493, P＜0.01) and serum albumin level positively (Rho=0.420, P＜0.01), but negative correlation was shown between serum fetuin-A level and TNF—α(Rho=—0.378, P＜0.01) or T-Bil (Rho=—0.463, P＜0.01) respectively. (2) The production of TNF-α(24.89±7.71 pg/ml) and IL-6 (112.91±37.80 pg/ml) by LPS-stimulated human PBMC was significantly reduced by a given doseof fetuin-A and spermine (P＜0.01). (3) A mouse model of FHF was developed successfully and two administration regimens were choosen and verified. They are D-Galactosamine 600 mg/kg combined with LPS 0.5 mg/kg, or D-Galactosamine 800 mg/kg combined with LPS 0.04mg/kg. (4) The reduction of fetuin-A mRNA was earlier than the protein. Immunohistochemical staining showed that the expression of fetuin-A was mainly in cytokinesis, partly in cell membrane. (5) Fetuin-A could effectively decrease the death rate within 24hs (from 83% to 25%, P＜0.05) and serum ALT level (from 291370.76±29241.68 nkat/L to 31585.15±4086.35 nkat/L, P＜0.05); and inhibit the production of TNF-α(from 515.48±46.31 to 233.87±48.03, P＜0.01 ) and IL-6 (from 1093.93±153.15 to 546.73±56.42, P＜0.01); and reduce the degree of inflammation, necrosis and apoptosis in liver of our FHF model.Conclusions (1) Serum fetuin-A level is correlated with the severity of liver damage, serum TNF—αlevel in patients with chronic severe hepatitis and prognosis of them. (2) The production of TNF-αand IL-6 by LPS-stimulated human PBMC could be reduced by a given dose of fetuin-A plus spermine. (3) A standard, convenient and effective mouse model of FHF was established. Fetuin-A expression was decreased along with the damage of liver in FHF model. Fetuin-A could effectively reduce the fulminant hepatic failure induced by D-Galn and LPS. One of the possible way is by inhibiting production of TNF-αand IL-6. |
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