To evaluate the inhibitory effect of butyric acid (BA) as a histone deacetylase (HDAC) inhibitor on lipopolysaccharide (LPS)-induced pulmonary fibrosis and its mechanism.

Thirty C57/BL6 mice were randomly divided into three groups according to the random number method, namely control group (physiological saline was given intraperitoneally and by gavage), LPS challenge group (LPS-induced murine model of pulmonary fibrosis was reproduced with intraperitoneal injection of 10 mg/kg LPS), and BA preconditioning + LPS challenge group (10 mg/kg BA was given followed by intraperitoneal injection of 10 mg/kg LPS), with 10 mice in each group. Mice were sacrificed painlessly, and lung tissue samples were harvested at 2 weeks and 4 weeks respectively (five samples every group each time). HDAC activity was evaluated with fluorescence analysis kit. Protein expression of acetylated-histone H3 (Ace-H3), acetylated-histone H4 (Ace-H4) and thymocyte differentiation antigen 1 (Thy-1) were determined by Western Blot. The mRNA expression of Thy-1 was assessed by real-time reverse transcription- polymerase chain reaction (real-time RT-PCR). The degree of lung inflammation and fibrosis were microscopic detected after hematoxylin-eosin (HE) staining and Masson collagen staining. The deposition of lung collagen was detected by hydroxyproline content measurement kit.

Compared to control group, the degree of lung inflammation and fibrosis was aggravated after LPS challenge, as manifested by increased hydroxyproline content (μg/mg, 2 weeks: 8.384±0.632 vs. 4.388±0.334, 4 weeks: 8.308±0.244 vs. 4.370±0.342, both P < 0.01), increased HDAC activity (μmol/L, 2 weeks: 7.243±0.384 vs. 3.628±0.641, 4 weeks: 6.479±0.202 vs. 3.238±0.524, both P < 0.01), increased deacetylation degree of histone H3 and H4 [relative expression of Ace-H3 (gray value): 0.516±0.115 vs. 1.005±0.359 at 2 weeks, 0.633±0.143 vs. 1.092±0.193 at 4 weeks, both P < 0.05; relative expression of Ace-H4 (gray value): 0.402±0.164 vs. 0.759±0.187 at 2 weeks, P > 0.05; 0.426±0.098 vs. 0.858±0.177 at 4 weeks, P < 0.01], and lowered Thy-1 mRNA and protein expression [Thy-1 mRNA (2^-ΔΔCt): 0.606±0.066 vs. 1.005±0.109 at 2 weeks, P < 0.01; 0.824±0.101 vs. 1.210±0.400 at 4 weeks, P > 0.05; relative expression of Thy-1 protein (gray value): 0.725±0.284 vs. 1.249±0.297 at 2 weeks, 0.589±0.139 vs. 1.372±0.343 at 4 weeks, both P < 0.05]. Compared with LPS group, BA precondition could inhibit above processes, as manifested by decreased hydroxyproline content (μg/mg: 5.943±0.726 vs. 8.384±0.632 at 2 weeks, 4.938±0.209 vs. 8.308±0.244 at 4 weeks, both P < 0.01), decreased HDAC activity (μmol/L: 4.386±0.117 vs. 7.243±0.384 at 2 weeks, 4.863±0.096 vs. 6.479±0.202 at 4 weeks, both P < 0.01), increased Thy-1 mRNA expression at 2 weeks (2^-ΔΔCt: 0.884±0.216 vs. 0.606±0.066, P < 0.05), increased acetylation degree of histone H4 and Thy-1 protein expression at 4 weeks [relative expression of Ace-H4 (gray value): 0.715±0.145 vs. 0.426±0.098, P < 0.05; relative protein expression of Thy-1 (gray value): 0.939±0.098 vs. 0.589±0.139, P < 0.01].

LPS-induced pulmonary fibrosis was related with activation of HDAC, deacetylation of histone H3 and H4 and Thy-1 gene silencing. HDAC inhibitor BA could inhibit LPS-induced pulmonary fibrosis and Thy-1 gene silencing through inhibiting activation of HDAC and deacetylation of histone H4.

Pulmonary fibrosis; Lipopolysaccharide; Histone deacetylase; Butyric acid; Epigenetic regulation