Zhonghua wei zhong bing ji jiu yi xue · 2017
[Protective effects of valproic acid on gut barrier function after major burn injury and its mechanism].
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Luo Hongmin, Hu Sen, Bian Huining, Zheng Shaoyi, Xiong Bing, Huang Zhifeng, Liu Zuan, Sun Chuanwei, Ma Lianghua, Li Hanhua, Yu Wen, Du Minghua, Chen Huade, Lai Wen
Аннотация
OBJECTIVE: To investigate the potential protective effects of valproic acid (VPA) on gut barrier function after major burn injury in rats and its mechanism.
METHODS: Forty male Sprague-Dawley (SD) rats were divided into sham + normal saline (NS), sham + VPA, scald + NS, and scald + VPA groups, with 10 rats in each group. Rat with 55% total body surface area (TBSA) third-degree severe-burns model was reproduced by immersing into 80 °C water, and the rats in sham groups were given sham-burns by immersing into 37 °C water. The rats after severe-burns were immediately treated with 0.25 mL of 300 mg/kg VPA or NS by subcutaneous injection. Rats were sacrificed at 2 hours and 6 hours after injury, and abdominal aortic blood and ileal tissue were harvested. The levels of vascular endothelial growth factor (VEGF) were determined by enzyme-linked immunosorbent assay (ELISA). The intestinal permeability was evaluated by fluorescein isothiocyanate-dextran (FITC-dextran) determination. The histomorphological changes in gut barrier were evaluated by Chiu grading system. Levels of acetylated lysine at the ninth position of histone 3 protein (Ac-H3K9), hypoxia-inducible factor 1α (HIF-1α), zona occludens 1 (ZO-1) and myosin light chain kinase (MLCK) were determined by immunofluorescence staining and Western Blot.
RESULTS: Compared with sham + NS group, rats in scald + NS group showed intestinal mucosal damage 2 hours after burn injury, as well as increased mucosal permeability, protein expression levels of HIF-1α, VEGF, MLCK, and lowered levels of AC-H3K9 and ZO-1. These changes were much more prominent at 6 hours after injury. VPA treatment significantly attenuated the burn-induced intestinal damage. Compared with scald + NS group, the protective effects in scald + VPA group was not evident at 2 hours after injury; however, intestinal damage was much less severe at 6 hours after injury (Chiu score: 2.03±0.27 vs. 3.12±0.15), intestinal permeability was significantly decreased [FITC-dextran (μg/L): 709±76 vs. 1 138±75], histone acetylation was enhanced [Ac-H3K9 (gray value): 1.55±0.12 vs. 0.48±0.12], ZO-1 degradation was significantly inhibited (gray value: 0.69±0.12 vs. 0.43±0.16), the protein expression levels of VEGF and MLCK were significantly down-regulated [VEGF (ng/mg): 51.7±3.7 vs. 71.2±4.3, MLCK (gray value): 1.98±0.20 vs. 2.80±0.24], while the HIF-1α protein expression levels were significantly reduced at both 2 hours and 6 hours after injury (gray value: 2.50±0.39 vs. 3.88±0.42 at 2 hours, 1.83±0.42 vs. 4.42±0.41 at 6 hours, all P < 0.05).
CONCLUSIONS: Severe burn injury can induce histone deacetylation, ZO-1 degradation and intestinal barrier dysfunction. VPA can improve the levels of histone acetylation and ZO-1, and protect intestinal epithelial barrier function. These may probably be mediated through inhibiting HIF-1α and its downstream gene VEGF and MLCK.
METHODS: Forty male Sprague-Dawley (SD) rats were divided into sham + normal saline (NS), sham + VPA, scald + NS, and scald + VPA groups, with 10 rats in each group. Rat with 55% total body surface area (TBSA) third-degree severe-burns model was reproduced by immersing into 80 °C water, and the rats in sham groups were given sham-burns by immersing into 37 °C water. The rats after severe-burns were immediately treated with 0.25 mL of 300 mg/kg VPA or NS by subcutaneous injection. Rats were sacrificed at 2 hours and 6 hours after injury, and abdominal aortic blood and ileal tissue were harvested. The levels of vascular endothelial growth factor (VEGF) were determined by enzyme-linked immunosorbent assay (ELISA). The intestinal permeability was evaluated by fluorescein isothiocyanate-dextran (FITC-dextran) determination. The histomorphological changes in gut barrier were evaluated by Chiu grading system. Levels of acetylated lysine at the ninth position of histone 3 protein (Ac-H3K9), hypoxia-inducible factor 1α (HIF-1α), zona occludens 1 (ZO-1) and myosin light chain kinase (MLCK) were determined by immunofluorescence staining and Western Blot.
RESULTS: Compared with sham + NS group, rats in scald + NS group showed intestinal mucosal damage 2 hours after burn injury, as well as increased mucosal permeability, protein expression levels of HIF-1α, VEGF, MLCK, and lowered levels of AC-H3K9 and ZO-1. These changes were much more prominent at 6 hours after injury. VPA treatment significantly attenuated the burn-induced intestinal damage. Compared with scald + NS group, the protective effects in scald + VPA group was not evident at 2 hours after injury; however, intestinal damage was much less severe at 6 hours after injury (Chiu score: 2.03±0.27 vs. 3.12±0.15), intestinal permeability was significantly decreased [FITC-dextran (μg/L): 709±76 vs. 1 138±75], histone acetylation was enhanced [Ac-H3K9 (gray value): 1.55±0.12 vs. 0.48±0.12], ZO-1 degradation was significantly inhibited (gray value: 0.69±0.12 vs. 0.43±0.16), the protein expression levels of VEGF and MLCK were significantly down-regulated [VEGF (ng/mg): 51.7±3.7 vs. 71.2±4.3, MLCK (gray value): 1.98±0.20 vs. 2.80±0.24], while the HIF-1α protein expression levels were significantly reduced at both 2 hours and 6 hours after injury (gray value: 2.50±0.39 vs. 3.88±0.42 at 2 hours, 1.83±0.42 vs. 4.42±0.41 at 6 hours, all P < 0.05).
CONCLUSIONS: Severe burn injury can induce histone deacetylation, ZO-1 degradation and intestinal barrier dysfunction. VPA can improve the levels of histone acetylation and ZO-1, and protect intestinal epithelial barrier function. These may probably be mediated through inhibiting HIF-1α and its downstream gene VEGF and MLCK.