Hypomethylation of thymosin β4 promoter is associated with glucocorticoid therapy in patients with acute-on-chronic hepatitis B-induced liver failure

Abstract

Background: We aimed to determine whether the methylation status of thymosin β4 (Tβ4) promoter reflects the severity of acute-on-chronic hepatitis B liver failure (ACHBLF) and whether glucocorticoids affect this status.

Methods: Fifty-six patients with ACHBLF, 45 with chronic hepatitis B (CHB) and 32 healthy controls (HCs), were retrospectively enrolled. Methylation-specific PCR and real-time PCR were used to detect Tβ4 methylation frequency and mRNA level. The expression of Tβ4 was measured before and after glucocorticoid treatment in patients with ACHBLF. Clinical and laboratory parameters were obtained.

Results: Tβ4 mRNA expression of patients with ACHBLF was lower than in patients with CHB or HCs, but the methylation frequency was higher. Tβ4 promoter methylation frequency was correlated with serum total bilirubin, prothrombin activity and model for end-stage liver disease score. Moreover, Tβ4 promoter methylation frequency decreased and demethylation occurred during glucocorticoid therapy. After glucocorticoid therapy, Tβ4 mRNA expression and liver function were better in patients with low levels of methylation than in those with higher levels. After 90 d, the survival of patients with low levels of methylation was significantly higher than those with high levels.

Conclusions: Patients with ACHBLF who have low levels of Tβ4 methylation may show a more favorable response to glucocorticoid treatment.

Keywords: acute-on-chronic hepatitis B liver failure; glucocorticoid therapy; methylation; thymosin β4 promoter.

Figure 1.

Flowchart depicting the selection process of the participants.

Figure 2.

PBMC expression and the plasma concentration of thymosin β4 (Tβ4) in participants with ACHBLF or CHB and HCs. (A) The frequency of methylation of the Tβ4 promoter in peripheral blood mononuclear cells (PBMCs) in participants with acute-on-chronic hepatitis B liver failure (ACHBLF) or chronic hepatitis B (CHB) and healthy controls (HCs). (B) Tβ4 mRNA expression in the PBMCs of each group of participants. (C) Plasma Tβ4 concentration in participants in each group. (D) Representative Tβ4 methylation obtained using methylation-specific PCR. A 50 bp DNA ladder marker is shown. M, methylated sequence; NC, negative control; PC, positive control; U, unmethylated sequence.

Figure 3.

Effect of glucocorticoid treatment on liver function and the thymosin β4 (Tβ4) promoter methylation status and mRNA expression in peripheral blood mononuclear cells (PBMCs) of participants with acute-on-chronic hepatitis B liver failure (ACHBLF). (A–C) Liver function of the participants after 0, 7 and 28 d of glucocorticoid therapy. (D and E) Tβ4 mRNA expression and methylation frequency in PBMCs at each time point. (F) Plasma Tβ4 concentration of patients at each time point. *p<0.05.

Figure 4.

(A) Kaplan–Meier survival curves for the high and low methylation groups. The log-rank test was used to compare these. (B) 90-d survival of the two groups, compared using the χ² test. *p<0.05.

Figure 5.

ROC curve for Tβ4 methylation and the MELD score for predicting the 3-mo mortality of patients with ACHBLF.