| Objective: Capillary only consists of monolayer endothelial cells and basement membrance, is the central point of exchange information and material. The tight junction of endothelial cells is one of the main factors that decide the microvascular permeability. Many pathological processes, accompanied by endothelial cells damage, vascular permeability, lead to intravascular fluids and cells through the vessel wall. Hypoxia is one of the common and important injury factors. Mesentery microvascular is abundant and sensitive to hypoxia. Therefore, there is great of significance to explore effective protection measures to viscera injury iduced by acute hypoxia.Tongxinluo(TXL) is a compound traditional Chinese medicine. Although the existing research shows that TXL can improve endothelial cell function, but its molecular mechanism is unclear. Studies have found that micro RNA-155(mi R-155) is closely related to many cardiovascular diseases. But the function of mi R-155 in vascular endothelial cells rarely was reported. This study focus on TXL effect on mesentery microvascular permeability induced by acute hypoxia and the expression of mi R-155, and investigate the molecular mechanism of TXL regulation of mi R-155 and tight junction proteins expression in endothelial cells.Methods: Experimental mice were randomly assigned to C57BL/6Jacute hypoxia group, C57BL/6J acute hypoxia + TXL group and mi R-155-/-acute hypoxia group. TXL group was intragastrically administered TXLevery day, C57BL/6J acute hypoxia group and mi R-155-/- acute hypoxiagroup intragastrically administered an equal amount of saline. After threedays, we established the model of acute hypoxia, and observed mesenterymicrovascular permeability by Leica DMIRB, and recorded the survival timeand respiratory rate. The level of mi R-155 m RNA in human cardiacmicrovascular endothelial cells(HCMEC) was detected by real-time PCR.The expression of tight junction proteins in HCMEC was detected byWestern blot and immunofluorescence staining. The expression of p47, p22and HO-1in HCMEC and human vascular smooth muscle cells(HVSMC)were detected by Western blot. SPSS 13.0 was used for statistical analysis.The dates are expressed as mean ± S.D., and intergroup comparison involvedone-way ANOVA. Probabilities of 0.05 or less were considered to bestatistically significant.Results:1 TXL improved the survival time and respiratory rate of acute hypoxia miceTXL extended the survival time and respiratory rate of acute hypoxia mice group of C57BL/6J mice. The survival time of mi R-155-/- mice were also improved. The results showed TXL increased the survival time of acute hypoxia mice may be associated with down-regulation of mi R-155.2 TXL inhibited the mesentery microvascular permeability induced by acute hypoxiaAfter tail vein injected of fluorescence labeled BSA(FITC-BSA), Leica DMIRB recorded the mesentery microvascular permeability of acute hypoxia mice. The results showed the mesentery microvascular permeability of TXL group was obviously less than that of the control group; mi R-155-/- group also decreased the permeability compared with the wild-type mice. These results suggested that TXL reduced mesentery microvascular permeability induced by acute hypoxia may through down-regulation of mi R-155.3 TXL inhibited the expression of mi R-155 induced by TNF-αTo verify above speculation, we cultivated human cardiac microvascular endothelial cells(HCMEC) and treated with TNF-α, and observed the effection of different concentrations TXL to the expression of mi R-155. The results showed that TXL could significantly inhibit the expression of mi R-155 induced by TNF-α.4 mi R-155 affected the expression tight junction proteins in HCMECIn order to further clarify the relationship between mi R-155 and the tight junction proteins, Western blot and immunofluorescence staining was used. The results showed that overexpression of mi R-155 could decrease the expression of tight junction proteins on the cell membrane, including claudin1, beta-catenin and ve-cadherin. On the contrary, knock down the expression of mi R-155 could increase the expression of tight junction proteins on the cell membrane, which indicated that mi R-155 participated in regulating micro vascular permeability by regulating the expression of tight junction proteins.5 TXL could reverse the effect of mi R-155 on the expression of HCMEC tight junction proteinsTo further study the molecular mechanism of TXL regulating the expression of tight junction proteins by mi R-155, Western blot and immunofluorescence staining was used to detect the expression and localization of tight junction proteins in HCMEC. The results showed that overexpression of mi R-155 before incubation with TXL increased the expression of tight junction proteins on the cell membrane. On the contrary, knock down the expression of mi R-155 decreased the expression and distribution of tight junction proteins on the cell membrane. These results demonstrated that TXL could completely reverse the expression of tight junction proteins on the cell membrane induced by mi R-155.6 mi R-155 participated in the oxidative stress process of HCMECTo clear the molecular mechanism of mi R-155 regulating tight junction proteins, we observed the effect of mi R-155 on the oxidative stress in HCMEC. Western blot showed that overexpression of mi R-155 inhibited the expression of p47 and p22, and promoted the expression of HO-1; While knock down the expression of mi R-155 got the opposite results.7 TXL reversed the effect of mi R-155 on the expression of HO-1 in HCMECTo further prove the effect of TXL on oxidative stress, we detected the expression of p47, p22, and HO-1 in HCMEC overexpression with mi R-155 before incubation with TXL. The results showed that TXL incubation increased the expression of p47 and p22, and decreased the expression of HO-1. On the contrary, knock down the expression of mi R-155 decreased the expression of p47 and p22, suggesting TXL regulated the expression of p47, p22, and HO-1 in HCMEC by mi R-155.8 TXL had no effect on the expression of HO-1 in HVSMCTo determine whether TXL has the above effect on vascular smooth muscle cells, human vascular smooth muscle cells(HVSMC) was used. Western blot showed that overexpression of mi R-155 promoted the expression of p47, p22, and HO-1 in HVSMC. TXL incubation had no effect on the expression of p47, p22, and HO-1, prompting TXL reversed the above several kinds of protein expression in HCMEC by mi R-155 with endothelial specificity.Conclusions:1 TXL improved the survival time and respiratory rate of acute hypoxia mice, and inhibited the mesentery microvascular permeability which were associated with down-regulation of mi R-1552 TXL could reverse the effect of mi R-155 on the expression of HCMEC tight junction proteins3 TXL could reverse the function of mi R-155 which were related to the regulation of HO-1... |
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