Inhibition Of Sodium Butyrate On The Increased Permeability Of Human Umbilical Vein Endothelial Cells Induced By Hypoxia And Its Mechanism

ObjectiveThe increase of vascular endothelial permeability is the pathological basis of many diseases.Hypoxia is a common pathological process,and it is an important pathogenic factor of many diseases.Studies have shown that hypoxia can change connection between endothelial cells and damage vascular endothelial integrity,leading to cell contraction,increased intercellular space,destruction of intercellular connection complex structure,and increased permeability of vascular endothelial cells.How to protect the integrity of vascular endothelial cells and inhibit the increased permeability of vascular endothelial cells induced by hypoxia is still lack of effective measures. Sodium butyrate sodium butyrate(NAB), as a common short chain fatty acid, produced indirectly by digestion of the intestinal microflora in fibrous food. Previous studies showed that sodium butyrate could play an important role in the prevention and treatment of atherosclerosis and vascular remodeling by inhibiting hypoxia and inflammatory factors. In addition, sodium butyrate also plays a role in the maintenance of vascular cell barrier,it can increase the tight junction protein, maintaining normal vascular endothelial permeability.Sodium butyrate has received extensive attention in the application of vascular diseases.However,the effect of sodium butyrate on the endothelial permeability of endothelial cells induced by hypoxia and the mechanism of its action is not clear.The aim of this study was to study the effect of sodium butyrate on the permeability of human umbilical vein endothelial cells induced by hypoxia,and to explore the mechanism of it.MethodsHuman umbelical vein endothelial cells(HUVECs)were divided into normoxic group(21%O2)、hypoxia group(1%O2)and hypoxia plus sodium butyrate group(n=3). Three gas incubator were used to contain the cells of two latter groups,and the normoxic group was cultured in CO2 incubator.1.The permeability of HUVECs was tested by Transwell system; immunofluorescence assay was used to detect the transcellular permeability of HUVECs by cy5-BSA.2.Western blot was performed to detect protein expression of p120 and β-catenin;the fluorescence intensity of protein p120 were observed by immunofluorescence assay.3. Western blot was performed to detect protein expression of p120 treated by CHX and MG-132.4. Western blot was performed to detect protein expression of p120 treated by Carboxy-PTIO and si RNA(e NOS).5. Western blot was performed to detect protein expression of e NOS; Level of NO in cell supernatant and cell protein were detected by Nitric Oxide Assay Kit; the location of p120 and e NOS were detected by immunofluorescence assay.Results1. Effects of sodium butyrate on the permeability of human umbilical vein endothelial cells induced by hypoxia:the permeability of HUVECs was significantly increased in 12 hour hypoxia group than the normoxic group(P<0.05)(normoxic group10%,hypoxia group16%);After addition of 1mmol/L Na B,the increased HUVECs permeability of hypoxia group was inhibited significantly(P <0.05).The fluorescence intensity of cy5-BSA in hypoxia group and hypoxia plus sodium butyrate(1mmol/L) group was not significantly different from that of the normoxic group.2. Effects of sodium butyrate on the p120 expression of human umbilical vein endothelial cells induced by hypoxia: The protein level of p120 instead of β-catenin was reduced in hypoxia group than the normoxic group(P<0.05),but increased with Na B(1mmol/L) treatment in hypoxia group(P<0.05).Consistently,immunofluorescence data showed that p120 fluorescence intensity was reduced by hypoxia,but Na B(1mmol/L) reversed this situation.3. Changes of p120 protein stability induced by hypoxia:the p120 expression of HUVECs was significantly increased in 12 hour hypoxia plus MG-132(25μmol/L)group than the hypoxia group(P<0.05);The half-life of p120 protein in HUVECs in the normoxic group was 16 h, The half-life of p120 protein in HUVECs in the hypoxia group was 11 h, The half-life of p120 protein in HUVECs in the hypoxia plus sodium butyrate(1mmol/L) group was 25 h.4. Effect of NO on the expression of hypoxia p120 protein: the p120 expression of HUVECs was significantly increased in 12 hour hypoxia plus Carboxy-PTIO(0.3mmol/L)group than the hypoxia group(P<0.05)5. Effect of e NOS on the expression of hypoxia p120 protein: After transfection with si RNA(e NOS), the expression of p120 protein in HUVECs was significantly lower than that of the control group(P < 0.05)6. Effects of sodium butyrate on the expression of e NOS and the secretion of NO: the e NOS expression of HUVECs in hypoxia plus sodium butyrate group was not significantly different from that of the hypoxia group(P>0.05);There was no significant difference in the NO level of cell culture supernatant and cell protein between normoxic group, hypoxia group, hypoxia plus sodium butyrate group( P > 0.05).7. Effect of sodium butyrate on e NOS/p120 co-location: Compared with hypoxia group, the 12 hour fluorescence co-localization of e NOS/p120 in HUVECs was decreased in hypoxia plus sodium butyrate(1mmol/L) group.Conclusion1.hypoxia increased the permeability of HUVECs permeability, but Sodium butyrate inhibited the increased permeability of HUVECs induced by hypoxia.2.hypoxia reduced the p120 expression of HUVECs,but sodium butyrate increased p120 protein expression under hypoxia,suggesting a protective effect of sodium butyrate on permeability is achieved by increasing the expression of p120 protein under hypoxia.3. Sodium butyrate can increase the stability of p120 protein and increase the expression of p120 protein in HUVECs induced by hypoxia;4. The decrease of e NOS protein level promotes the down-regulation of p120 expression under hypoxia;the decrease of NO levels inhibited the down-regulation of p120 protein induced by hypoxia.5. Sodium butyrate had no significant effect on the expression of e NOS and NO levels under hypoxia, suggesting that other NO related mechanisms mediate the inhibition of sodium butyrate on the decreased expression of p120 in HUVECs induced by hypoxia.