| Object:The present study was performed to:(1) observe effects of histone deacetylase inhibitor on activation and permeability of microvascular endothelium cells in rats with 50%TBSA full-thickness scald injury; (2) study effects of HDACI on PMECs activation and permeability after TNF-a stimulation in vitro and their mechanisms, to provide some experimental evidences for the application of HDACI in early management of fatal scald shock.Methods:1.Animal experiments. Male Sprague-Dawley (SD) rats were randomly divided into four groups:①sham group, waterbath in 37℃.②scald group, waterbath in 80℃on rat back and both lower extremities 15s and abdomen 8s, total scald area for approximately 50%TBSA.③VPA group and④2M2P group, subcutaneous injection of either VPA or 2M2P(300mg/kg,100mg/ml) immediately postinjury. At 2,6 and 12h postinjury, survival rats were examined for pulmonary vascular permeability with Evans Blue staining method. Bloods were collected for tests of HCT and biochemistry indexes.Specimens of lung were harvested for determination of water content with oven drying method, assessment of VEGF, E-selectin and ICAM-1 contents with immunohistochemical analysis and evaluation of apoptosis rates of endothelium cells with TUNEL method.2. Vitro cell experiments. Pulmonary microvascular endothelium cells (PMECs) were harvested from male SD rats by tissue-sticking method and then cultured. Monolayer PMECs were established in upper room of 24-well Transwell plate to investigate permeability of PMEC after TNF-a stimulation and drug treatments. The experiment was devided into 4 groups:③control group;②TNF-a group;③VPA group and④2M2P group.②③④group were added with TNF-a at a final concentration of 500ng/ml.③④group were furtherly add with VPA or 2M2P (0.05mg/ml).All cells were incubated in 37℃,5%CO2 incubator for 6h and then added with bovine serum albumin(0.5mg/ml) in upper room for 45min. Supernatants were collected from both upper room and lower room of transwell plates for protein quantitation by BCA method and to calculate permeability coefficient. Furthermore cells were add to 24-well culture plate (106 cells per well) and treated as above mentioned. After 6h treatments supernatents were collected for determination of VEGF, sICAM-1, sE-selectin and HSP70 contents and cells were lysised to collect nuclear proteins for determination of NF-κB expression by ELISA method.Cell apoptosis rates were determined with flow cytometry.Results:1.Establishment of rat scald shock model Scald areas of the rat model were affirmed as approximately 50%TBSA by paper-sticking method.And deepnesses of scald injury were affirmed to be III degree by pathology of scald tissues.Diagnosis of shock was definited by symptoms, mean arterial pressure(MAP) and indexs of organ function.Mortality of 6h and 12h postinjury was 80% and 100%.2.Effect of VPA on permeability of rat scald shock model Compared to that of scald group, blood HCT, water contents of pulmonary tissues and permeability to EB were significantly lowered on VPA group and 2M2P group at 2h and 6h postinjury. Pathological changes of VPA group and 2M2P group were also allieviated compared to that of scald group.Such effects were dramatically stronger in VPA group than 2M2P group(p<0.05).3. Effects of VPA on endothelium activation and apoptosis of rat model Compared to that of sham group, VEGF, E-selectin, ICAM-1 positive staining rates and cell apoptosis rates of scald group were all dramatically raised both in 2h and 6h postinjury. And such cytokines positive rates and apoptosis rates of VPA group were significantly lowered compared to scald group.As for that of 2M2P group, cytokines positive rates were not statically different from scald group, while apoptosis rate lowered. 4.A PMEC cell line was established by tissue-sticking method and identified by morphology and immunofluorescence method.5.Effect of VPA on permeability of monolayer PMECs after TNF-a stimulation Compared to that of control group, permeability rate[×10-5 ml/(min*cm2)] of PMECs to BSA of TNF-a group was significantly raised (435.10±36.87 Vs.72.54±11.38, p<0.05).VPA and 2M2P treatment could dramatically depress this increase, which was much significant in VPA group (276.55±30.18 Vs.324.68±26.48,P<0.05) 6.Effect of VPA on PMEC cytokines expression Contents of VEGF,sE-selectin and sICAM-1 were much lowered in VPA group than that of TNF-a group(p<0.05),while there was no difference between 2M2P group and TNF-a group(P<0.05).7.Effect of VPA on NF-κB p65 expression of PMEC after TNF-a stimulation VPA notably inhibited the increase of NF-κB p65 expression in PMEC nuclear protein after TNF-αstimulation (872.31.55+133.48 Vs.1637.84.68±215.64 pg/ml, p<0.05), while 2M2P did not. 8.Effects of VPA on HSP70 expression and apoptosis rates of PMEC after TNF-αstimulation Contents of HSP70 were much increased in TNF-a group than that of control group (97.54±7.64 Vs.25.14±4.38, p<0.05), and apoptosis rates also increased(21.24±3.10% Vs.2.31±0.42%, p<0.05). With VPA and 2M2P treatments, contents of HSP70 were furtherly up-regulated (177.88±19.85Vs.126.35±14.89 pg/ml),while apoptosis rates were decreased (12.26±1.9%Vs.16.42±2.6%).The effect of VPA was much stronger than that of 2M2P (p<0.05).Conclusions: Histone deacetylase inhibitor could inhibit the activation of PMEC, lower cell apoptosis and thus ameliorate the increase of permeability of PMEC both in rat scald shock model and vitro cell model of TNF-a stimulation, through which lessened bloods volume losses caused by increase of blood vessel permeability. The mechanism of such effects was potentially relevant with inhibition of NF-κB signal transduction and enhancement of HSP70 expression. |
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