| Backgroud and AimsAcute lung injury (ALI) is most frequent acute disease and associated with high casualty clinically. The available therapy for ALI is not satisfactory. It is believed that the pro-inflammatory cytokines play a pivotal role in the pathogenesis and development of ALI. And the relevant studies have shown that the inhibition of pro-inflammatory cytokines production could alleviate the pulmonary inflammation in ALI. Interestingly, histone deacetylase inhibitors (HDACi), developed as promising anti-tumor drugs, exhibit their anti-inflammatory properties due to their effects on reduction of inflammatory cytokines. This study was undertaken to investigate the protective effect of butyrate on ALI and the relevant mechanism.Methods1.Citrated blood was obtained from consenting healthy donors and peripheral blood mononuclear cells (PBMCs) were isolated. PBMCs were cultured with lipopolysacchride (LPS) or butyrate, and 24 h later the supernatants were collected. The concentrations of pro-inflammatory cytokins tumor necrosis factor (TNF) -α, interleukin (IL) -1βand IL-8 in collected supernatants were measured respectively by enzyme-linked immunosorbent assay (ELISA). The effect of butyrate inhibiting the por-inflammatory cytokin production by LPS-stimulated PBMCs was confirmed by statistical analysis.2.The animal model of ALI was established by intratracheal instillation of LPS in BALB/c mice. After the exposure of LPS, the bronchoalveolar lavage fluid (BALF) and lung tissues were harvested at specific time point. The concentrations of TNF-αand IL-1βin BALF were measured by ELISA. Pulmonary histological changes were evaluated by hematoxylin-eosin stain and lung wet/dry weight ratios were observed. The concentrations of nitric oxide (NO) and myeloperoxidase (MPO) activity in lung tissue homogenates were measured by ELISA. Whether the characterization of this murine model of LPS-induced ALI met the demand of the further study in vivo was concluded from the data above.3.Before 1 h of LPS instillation, the mice received butyrate or saline orally. After the exposure of LPS, the bronchoalveolar lavage fluid (BALF) and lung tissues were harvested at specific time point. The concentrations of TNF-αand IL-1βin BALF were measured by ELISA. Pulmonary histological changes were evaluated by hematoxylin-eosin stain and lung wet/dry weight ratios were observed. The concentrations of nitric oxide (NO) and myeloperoxidase (MPO) activity in lung tissue homogenates were measured by ELISA. Expression of nuclear factor (NF) -κB p65 in cytoplasm and nucleus was determined by Western blot analysis respectively. Based on these data, we could evaluate the protective effect of butyrate on LPS-induced ALI and investigate the mechanism. Results1.Compared with control and butyrate groups, the concentrations of TNF-α,IL-1βand IL-8 in supernatants were significantly increased with the stimulation of LPS. And the concentrations of TNF-α, IL-1βand IL-8 were 223.5±13.5 pg/ml, 106.2±10.6 pg/ml and 89.1±6.9 pg/ml respectively. Pretreatment of butyrate efficiently reduced the production of TNF-α,IL-1βand IL-8, and the concentrations of TNF-α, IL-1βand IL-8 dropped to 130.9±10.4 pg/ml, 57.7±8.6 pg/ml and 48.6±5.5 pg/ml respectively.2.After intratracheal instillation of LPS 3 h, the concentrations of TNF-αand IL-1βin BALF, measured by ELISA, peaked to 3678±16 pg/ml and 638±23pg/ml respectively. The peak concentrations of TNF-αand IL-1βin control group were 119±9 pg/ml and 46±6 pg/ml respectively. Compared with control group, the concentrations of TNF-αand IL-1βin LPS group significantly increased. The peak activity of MPO and the peak concentrations of NO in lung homogenates reached to 44.2±2.3 uIU/ml and 92.8±2.4 umol/L respectively after LPS instillation. Compared with control group, the activity of MPO and the concentrations of NO in LPS group markedly increased. After intratracheal instillation of LPS 24 h, the lung wet/dry ratios peaked to 5.32±0.38. In control group, the the lung wet/dry ratio was 4.22±0.23. Compared with control group, the lung wet/dry ratios in LPS group significantly increased. Lung tissues from the control group showed a normal structure and no histopathological changes under a light microscope. In LPS group, the lungs stained with hematoxylin-eosin indicated widespread alveolar wall thickness caused by edema, severe hemorrhage in the alveolus, alveolus collapse and obvious inflammatory cells infiltration.3.The concentrations of TNF-αand IL-1βin BALF were significantly increased at 1 h and peaked at 3 h after LPS administration. Butyrate pretreatment efficiently reduced the production of TNF-αand IL-1βat different time point. After LPS administration, the MPO activity in lung tissues was significantly and continuously increased compared with the control and butyrate groups from 1 to 24 h. In addition, the concentrations of NO were significantly increased at 1 hour and peaked at 3 h after LPS administration. However, in LPS + butyrate group, butyrate pretreatment markedly decreased the MPO activity and NO concentrations at different time point. Compared with the control and butyrate groups, the lung wet/dry weight ratios were significantly and continuously increased from 1 to 24 h after LPS administration. The increase of the lung wet/dry weight ratios was significantly reduced by butyrate administration at different time point. Lung tissues from the control and butyrate groups showed a normal structure and no histopathological changes under a light microscope. In LPS group, the lungs stained with hematoxylin-eosin indicated widespread alveolar wall thickness caused by edema, severe hemorrhage in the alveolus, alveolus collapse and obvious inflammatory cells infiltration. In LPS + butyrate group, the histopathological changes of lung were minor compared with those in LPS group, especially in inflammatory cells infiltration. After LPS administration, the expression of NF-κB p65 in nucleus markedly increased and peaked at 3 h. The expression of NF-κB p65 in nucleus induced by LPS was significantly suppressed at 1, 3 and 6 hours by butyrate pretreatment. On the contrary, the expression of NF-κB p65 in cytoplasm was significantly reduced by LPS administration, and these changes were inhibited by butyrate pretreatment. No changes of expression of NF-κB p65 in cytoplasm and nucleus, same as control group, were observed with administration of butyrate alone at each time point.Conclusions1.In vitro, the butyrate significantly inhibited the production of TNF-α, IL-1βand IL-8 by LPS-induced PBMCs. The results indicated that the butyrate exerted its anti-inflammatory effect in vitro primarily by suppressing the production of pro-inflammatory cytokines.2.In the murine model of LPS-induced ALI, the increased concentrations of TNF-αand IL-1βin BALF, the enhanced activity of MPO and the increased concentrations of NO in lung homogenates, the increased lung wet/dry ratios, and pulmonary histopahtological changes suggested that the model was potent to perform further study in vivo.3.Pretreatment with butyrate led to significant attenuation of LPS induced evident lung histopathological changes, alveolar hemorrhage, and neutrophils infiltration with evidence of reduced MPO activity. The lung wet/dry weight ratios were reduced by butyrate administration. Butyrate also repressed the production of TNF-α, IL-1βand NO. These results indicated that butyrate attenuated LPS-induced ALI in mice. Furthermore, the expression of NF-κB p65 in nucleus was markedly suppressed by butyrate pretreatment, which suggested that the anti-inflammatory effects of butyrate in ALI may primarily rely on inhibition of the nuclear translocation of NF-κB, and consequently inhibiting the production of cytokines regulated by NF-κB.In this study, we investigated the anti-inflammatory effects of butyrate in vitro and in vivo, and illustrated elementarily the mechanism. Thus, butyrate might be useful in situations where current anti-inflammatory therapies are unsatisfactory.
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