The Pharmacological Effects Of Histone Deacetylase Inhibitor Valproic Acid On Mouse Lymphocytes And Its Action Mechanism

Aim:The aim of this study is to analyze the effects of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, on the proliferation, activation, apoptosis and syntheses of cytokines of murine activated lymphocytes. The pharmacological effects of VPA on apophasis-related proteins, MAPKs signal pathway, STAT3 signal molecules and chromosome related proteins were further investigate in order to elucidate the cellular and molecular mechanism of immunomodulatory effects of VPA in lymphocytes.Methods:Lymphocytes were isolated from lymph nodes of BALB/c mouse. After staining with CFSE, mouse lymphocytes were stimulated with polyclonal activators ConA. The proliferation rate of mouse lymphocytes was analyzed with flow cytometry. The expression levels of CD69 on T lymphocytes stimulated with ConA were evaluated with flow cytometry after staining with fluorescent monoclonal antibody. Cell-cycle distribution of the lymphocytes was analyzed by propidium iodide staining. Nuclear morphological changes were observed by Hoechst33342 staining combined with laser scanning confocal microscopy. The lymphocytes were stimulated with phorbol ester (PDB) and inomycin in the presence or absence of different concentrations of VPA, and the syntheses of IL-2, IFN-y and IL-6 of different subsets of mouse T lymphocytes were analyzed using flow cytometry. Furthermore, using Western blot analysis, we investigated the effects of difference concentrations of VPA on the protein expression of pro-apoptotic Bax, anti-apoptotic Bcl-2, the phosphorylation levels of H2A.X, STAT3, as well as the phosphorylation levels of p38, ERK and JNK which belong to MAPKs signal pathway. In addition, the acetylation level of histone H3 was detected.Results:Our results showed that VPA inhibited the proliferation of Con A-activated lymphocytes in a dose-dependent manner. Low-dose VPA (≤1.1 mmol/L) enhanced CD69 expression on the activated lymphocytes whereas at high doses (≥3.3 mmol/L) it decreased CD69 expression. Furthermore, VPA reduced activation-induced apoptotic cell death at low doses, but at high doses it promoted apoptotic cell death of activated lymphocytes dramatically. Most of the nuclei of the lymphocytes were uniform and blue at low doses of VPA, whereas more nuclei showed chromatin condensation, nuclear fragmentation and pyknosis after being treated with high concentration of VPA. Additionally, VPA inhibited the aggregated colony sizes of Con A-activated lymphocytes in a dose-dependent manner. Intracellular cytokine staining results showed that after stimulated with PDB and ionomycin, VPA obviously reduced the expression rate of IL-6+, IFN-γ+ and IL-6+IFN-γ+ in CD3+ T cells in a dose-dependent manner (P<0.01). Similarly, VPA dose-dependently diminished the percentage of CD4+ and CD8+ T cell subsets that expressed IL-2+ and IFN-γ+ (P<0.01). There was similar inhibitory effect on the IL-2+IFN-γ+ double positive CD4+ and CD8+ T cells (P<0.01). Moreover, the inhibitory effect of VPA on the expression of IFN-y in CD8+ T cells was stronger than in CD4+ T cells. It was found that the Bax/Bcl-2 ratio and phosphorylation of histone H2A.X were decreased at low doses of VPA but increased at high doses. The phosphorylation of STAT3 was also differential regulated by different doses of VPA. VPA of 5 mmol/L induced the phosphorylation of p38 but not JNK and ERK1/2. In addition, VPA induced a dose-dependent increase in the acetylation of histone H3.Conclusion:VPA inhibited the proliferation or cytokine synthesis of Con A or PDB+Ion-activated lymphocytes in a dose-dependent manner. Furthermore the effect of VPA on the expression of CD69 (activation marker) and the apoptotic cell death of lymphocytes in response to Con A stimulation was biphasic. CD69 surface expression on Con A-stimulated lymphocytes was further increased while the Con A-induced apoptosis was alleviated under treatment with low doses of VPA. However VPA inhibit the expression of CD69 on Con A-stimulated lymphocytes and the proportions of apoptotic cells were markedly increased at high doses. At the early phases, the modulation of Bax/Bcl-2 balance may play a role in regulating apoptosis induced by VPA; at the later phase, regulation of STAT3 and p38 may be associated with the modulation of apoptosis. Additionally, its effect on yH2A.X levels was biphasic, which may be relevant to the biphasic effect of VPA on apoptosis.