| Background and ObjectiveAcute on chronic hepatitis B liver failure (ACHBLF) is a serious disease with high mortality. It was commonly accepted that pro-inflammatory cytokines play an important role in the pathogenesis of ACHBLF. Peroxisome proliferator-activated receptor gamma (PPAR-y) has been demonstrated to be involved in anti-inflammatory reactions, but its role in acute-on-chronic hepatitis B liver failure (ACHBLF) is unclear. Therefore, DNA methylation patterns and expression level of PPAR-y gene were detected and the possible role of PPAR-y in ACHBLF was analyzed.Patients and MethodsIn our study,161 patients were enrolled including 81 patients with ACHBLF,50 patients with chronic hepatitis B (CHB), and 30 healthy controls. Level of mRNA in peripheral blood mononuclear cells (PBMCs) was detected by real-time polymerase chain reaction (RT-PCR). The methylation status of PPAR-y was detected by methylation-specific PCR (MSP). Levels of plasma TNF-a and IL-6 were detected by enzyme linked immunosorbent assay (ELISA). All data were analyzed using SPSS 17.0 software. Comparison of methylation status among different groups was analyzed by chi-square test or Fisher’s exact test. Changes in mRNA concentration were measured by Student t test or Mann-Whitney U test. Spearman test was applied for correlation analysis. P value< 0.05 was considered statistically significant.Results1. The PPAR-y mRNA level was significantly elevated in ACHBLF patients compared with CHB patients (Z=-4.003, P<0.001). However, PPAR-y mRNA level was significantly lower in ACHBLF patients than that in healthy controls (Z=-4.632, P<0.001). Meanwhile, expression of PPAR-y was significantly lower in CHB patients than healthy controls (Z=-6.037, P<0.001).2. Linear correlation analysis was conducted between PPAR-y mRNA level and clinical parameters of patients with ACHBLF, including serum alanine aminotransferase (ALT), total bilirubin (TBIL), international normalized ratio (INR), creatinine (Cr) and hepatitis B virus-DNA (HBV-DNA) load. The results showed that expression of PPAR-γ was negatively correlated with TBIL (r=-0.280, P=0.011) and INR (r=-0.230, P=0.039). No significant correlation was found between PPAR-γ mRNA level and ALT, Cr or HBV-DNA load in patients with ACHBLF3. The methylation frequency of PPAR-γ promoter in ACHBLF patients was significantly decreased compared with CHB patients (CpG-1, X2=8.918, P=0.003; CpG-2, x2=9.268, P=0.002). Still, methylation frequency in ACHBLF patients was higher than healthy controls (CpG-1, X2=6.691, P=0.009; CpG-2, x2=5.050, P=0.025). Also, methylation frequency of both CpG islands in CHB patients was prominently higher than healthy controls (CpG-1, x2= 21.333, P<0.001; CpG-2, x2= 19.448, P<0.001).4. The mRNA level of PPAR-y in subjects with or without methylation was compared. The results showed that relative expression of PPAR-y was significantly decreased with either CpG-1 (Z=-6.613, P<0.001) or CpG-2 (Z=-7.712, P<0.001) island methylation, and the suppression seemed to be superposable (both MU vs. one MU, Z=-4.085, P<0.001).5. The plasma levels of TNF-α and IL-6 were assessed by ELISA in the subjects. Levels of both cytokines was significantly increased in ACHBLF patients compared with CHB patients (TNF-α, t=6.649, P<0.001; IL-6, t=6.784, P<0.001) and healthy controls (TNF-α, t=5.582, P0.001; IL-6, t=5.961, P<0.001). In patients with ACHBLF, plasma cytokines levels in methylated group were significantly higher than unmethylated group (TNF-α, t=2.312, P=0.023; IL-6, t=2.639, P-0.012).6. The three-month mortality was 54.32%(44/81), and the mean survival time is 53.47 (SE 4.057,95%CI 45.39-61.54). The PPAR-γ mRNA level of survivors was significantly higher than non-survivor (Z=-3.489, P<0.001). Prognosis of methylated group was significantly poorer compared with unmethylated group (x2=11.140, P<0.001). We noted that PPAR-γ expression was significantly increased with time extending in survivors, while remained low level in non-survivors. Prognostic performance was evaluated and the receiver operating characteristic (ROC) curve for PPAR-γ methylation, PPAR-γ mRNA and model for end-stage liver diseases (MELD) score was illustrated. The areas under the ROC curve (AUC) were 0.726 (SE 0.059,95%CI 0.611-0.841),0.657 (SE 0.062,95%CI 0.535-0.778) and 0.725 (SE 0.056,95% CI 0.615-0.836) respectively.ConclusionsIn conclusion, we found decreased methylation frequency and elevated PPAR-γ expression level of PPAR-γ promoter in ACHBLF patients compared with CHB patients. Promoter methylation and expression level of PPAR-γ was closely associated with severity and prognosis of ACHBLF. Also, PPAR-γ may have a potential role in improving prognosis of patients with ACHBLF by down-regulating TNF-α and IL-6. Promoter methylation and expression level of PPAR-γ were of great value in predicting short-term prognosis of ACHBLF. |
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