The Protective Effects Of HDACIs And Their Mechanism On Scald-induced Gut Barrier Dysfunction In Rats

Objective: Recent researches have found that hypoxia-inducible factor1(HIF-1) was activated after severe burn injury, result in injuring the function ofintestinal barrier, but histone deacetylase inhibitors(HDACIs) protects someorgans against burn injury which induced by trauma, shock and burn, and reducedthe intestinal origin of systemic inflammation and remote organ dysfunction,however the definite mechanism was indistinct, the researches assumed thatHDACIs Protects the Intestinal Barrier Structure because of increasing HIF-1acetylation levels, reducing expression of HIF-1in burned Rat. The purpose ofthis study:(1) the SD rats50%TBSA Ⅲ degree burn model was used to studywhether it could reduce the expression of HIF-1, then improve intestinal barrierdysfunction or not by injecting histone deacetylase inhibitor intraperitoneally;(2)Researching the mechanism of HDACIs influencing the expression of HIF-1byculturing intestinal epithelial cellsMethod:1Animal experimentGroups:64male SD rats, weight240g-260g, adaptability feeding a week,randomly divided into four groups①Sham Control(S group)②scald+normalsaline(SN group)③scald with SAHA administration(SS group)④scald withVPA administration(SV group); For the different observation time, each groupwere randomly separated into two time:3h,6h after scald burns;Rat burn model: After hocusing by injecting pentobarbital sodium (50mg/kg) intraperitoneally, barbering the back, abdomen, scald group were subjected toscald injury by means of immersing the back of the trunk for15s and theabdomen for8s in100°C water, resulting in third degree50%TBSA burns, thenseverally treated with0.25ml normal saline, SAHA(7.5mg/kg, dissolved in0.25ml normal saline), VPA(300mg/kg, dissolved in0.25ml normal saline), Sham Control group were disposed in37°C water, then treated with0.25ml normalsaline.3h or6h after scald, taking blood specimen from the abdominal aorta andintestinal tissue specimens from the terminal ileum.Measurements and methods:①②the activity of DAO and ZO-1detectedby laser confocal microscopy were measured to evaluate the intestinal mucosabarrier function;③Z O-1, HIF-1α was detected by Western blot;④Intestinalmucosal blood flow (IMBF) was detected by laser Doppler;2Cell experimentsCell culture and groups: Taking frozen Caco-2cells, culturing cells afterreviving. Cells were randomly separated into four groups,①normal controlgroup (NC group)②hypoxiagroup (H group)③hypoxia with SAHAadministration group (5uM)(HS group)④h ypoxia with valproate administrationgroup (2mM)(HV group).Methods: When cells achieved confluence to95%or more, wererespectively stimulated with DMSO, Cocl2(1mM)+DMSO, Cocl2(1mM)+SAHA(5uM), Cocl2(1mM)+VPA (2mM)+DMSO, Taking the specimens from thesupernatant and lysed cells After24hours of stimulationMeasurements and methods:①measuring the activity of supernatant DAO;②H IF-1α was detected by Western blot;③detecting acetylation levels of HIF-1αby immunoprecipitationResults:Animal experiments:1DAO: compared with Sham Control Group, The DAO activity ofscald+NS group was significantly increased,(P<0.05), scald+VPA group,scald+SAHA group were markedly decreased compared with scald+NS group,(P<0.05), scald+SAHA group was significantly lower than scald+VPA group,(P<0.05).2ZO-1:(1) The expression of ZO-1was lower in scald+NS group than inSham Control group by immunoblotting,(P<0.05); scald+VPA group,scald+SAHA group was significantly higher than scald+NS group(P<0.05);scald+SAHA group was higher than scald+VPA group(P<0.05);(2) Frozen sections for immunofluorescence studies of the intestinal epithelial ZO-1wereobserved, Sham Control group’ ZO-1showed a stronger and continuouser thanscald+NS group, scald+VPA group and scald+SAHA group was higher thanscald+NS group.3HIF-1α: compared with Sham Control group, The expression of HIF-1αwas higher in scald+NS by immunoblotting,(P<0.05); scald+VPA group,scald+SAHA group was significantly lower than scald+NS group(P<0.05); thegroup of scald+SAHA was lower than the scald+VPA group(P<0.05).4Intestinal mucosal blood flow: compared with Sham Control Group, theIMBF of scald+NS group was decreased,(P<0.05), scald+VPA group,scald+SAHA group were increased compared with scald+NS group,(P<0.05),scald+SAHA group was significantly higher than scald+VPA group,(P <0.05).Cell experiments1DAOCompared with normal control group, the DAO activity of hypoxia groupwas significantly elevated,(P<0.05), hypoxia+VPA group, hypoxia+SAHA groupwere sharp fell compared with hypoxia group,(P<0.05), hypoxia+SAHA groupwas significantly lower than hypoxia+VPA group,(P<0.05).2HIF-1α: compared with normal control group, hypoxia group’s theexpression of HIF-1α was higher by immunoblotting,(P<0.05); hypoxia+VPAgroup, hypoxia+SAHA group was much lower than hypoxia group (P<0.05);hypoxia+SAHA group was lower than the hypoxia+VPA group(P<0.05).3The acetylation levels of HIF-1α: hypoxia group was not obvious bycoimmunoprecipitation, but hypoxia+VPA group, hypoxia+SAHA group wasmuch higher than hypoxia group (P <0.05).Conclusion:1Intestinal barrier function was protected by lessening the degradation ofZO-1and reducing the damage of intestinal mucosa permeability because ofHDACIs. The protective of SAHA was significant better than VPA.2HDACIs can increase acetylation levels of HIF-1α, reduce the expressionof HIF-1α, protect the intestinal barrier, which may be one of the important mechanism of HDACIs protection intestinal mucosal barrier...