| The vascular endothelium,a selective barrier for liquid, proteins and cells, allows the high-efficient gas diffusion and transportation of electrolyte and inflammatory cells. The capillary wall, which composed of basal collagen, monolayer endothelial cells and intercellular tight junctions, is the base of material exchange and metabolism between extracellular fluid and blood. Under the state of inflammation, trauma and other pathological condition, the capillary permeability is increased because of the change of capillary blood flow, injury and apoptosis of endothelial cell and damage of tight junction protein. Therefore, capillary leakage is induced, and then multiple organs dysfunction appears. The mechanism of vascular endothelial barrier function regulation, as well as the reconstruction and treatment strategies for endothelial barrier under the pathological condition has become a research hotspot at present.Studies found that MMP9 could damage extracellular matrix and basement membrane of capillary and injure the barrier function. Our previous animal experiments also confirmed that MMP9 expression increased in aortic vascular endothelial layer of septic rat, which accompanied by the decrease of tight junction proteins(ZO-1 and claudin-5) and the increase of capillary permeability. The administration with rhubarb monomer by gavage in rats could protect the capillary endothelial cells of intestinal mucosa, reduce the capillary leakage of intestinal tissue, increase the number of functional capillary, expand the capillaries, reduce thrombosis in the intestinal mucosa, and ultimately improve the blood supply and oxygen supply in intestinal mucosa of septic rat. However, rhubarb is a complex with hundreds of components and various effective monomers. It is still unclear that which components can protect the micro vascular in sepsis or other severe infectious condition.In our previous research, 21 rhubarb monomers were extracted through the chromatography method. On the basis of the previous research, the monolayer model of Human Umbilical Vein Endothelial Cell(HUVEC) was established with the transwell chamber in present research. The effective monomers were selected through detecting the permeability of HUVEC monolayer and the proliferation of HUVEC, and the soluble cadherin, which impacted by the rhubarb monomers. The gene and protein expression of HUVEC VE-cadherin and tight junction(ZO-1, occludin, and claudin-5), the cell morphology and intensity of ligandin were detected through real-time quantitative PCR, Western blot and immunofluorescence respectively. Finally, the phosphorylation of Akt, the activation of Rac1 and the phosphorylation of MLC and the VE-cadherin protein expression intensity were analyzed to elucidate the signal transduction mechanism of rhubarb monomers on protecting the vascular endothelial barrier, which regulated Rac1 through PI3K/Akt signaling pathway, and then adjusted VE-cadherin expression and phosphorylation of MLC.PARTⅠEffects of rhubarb monomers on monolayer endothelial cells permeability and proliferationObjective: To select the effective rhubarb monomers and explain the mechanism through detecting the permeability of HUVEC monolayer, the proliferation of HUVEC and the soluble cadherin, which impacted by the rhubarb monomers.Methods: The HUVEC monolayer model was established by the Transwell chamber, which randomly assigned to control, MMP9, the 21 kinds of rhubarb monomers and Dexamethasone groups. The rhubarb monomers group was further divided into three subgroups according to the concentration of monomer, 1, 10, 50μmol/L. MMP9 was added to the cell culture media at the final concentration of 1mg/L in MMP9, rhubarb monomer and dexamethasone groups. In the meantime, the rhubarb monomer group was intervented with 21 kinds of rhubarb monomers at concentrations of 1, 10, 50 umol / L and the dexamethasone group was intervented with DEX at the concentrations of 10 umol / L. The control group was added the same amount of PBS as a negative control according to the dosing volume of MMP9. These groups were incubated for 24 hours in 5%CO2 cell incubation box at 37℃. The fluorescence of endocytosed fluorescein isothiocyanate(FITC)-dextran(FD40), which traversed the monolayer HUVEC, was detected to evaluated the permeability. The HUVEC proliferation and concentration of cadherin in cell culture supernatant were detected with MTT and ELISA respectively.Results:(1) The monolayer HUVEC permeability was increased by stimulating with MMP9 compared with the control group(P<0.05). However, the permeability injured by the MMP9 was improved by DEX and the rhubarb monomers, emodin, 3,8-dihydroxy-1-methyl-anthraquinone-2-carboxylic acid, 1-O-caffeoyl-2-(4-hydroxyl-Ocinnamoyl)-β-D-glucose, daucosterol linoleate, and rhein at 1μmol/L, as well as these monomers mixture(MM).(2) The HUVEC was prolifetated by the 1 μmol/L MM with 6.6% proliferation rate, but inhibited by the MM at concentration of 10、50μmol/L, the inhibition rate was 7.9% and 13% respectively.(3) The protein concentration of VE-cadherin in cell culture liquid of transwell upper and lower cabins was significantly increased after stimulation with MMP9, Which was decreased when the HUVEC was cultured with the five monomers or their mixture at 1μmol/L, or the DEX at 10μmol/L and MMP9.Conclusion: The rhubarb monomers, including Rhein, 3,8- dihydroxy 1-methylethyl anthraquinone 2 carboxylic acid, 1-O- coffee acyl- 2-(4- hydroxy-cinnamyl) beta D-glucose, daucosterol linoleic acid ester and emodin, can promote the HUVEC proliferation, decrease the shedding of VE-cadherin, and improve the permeability of monolayer HUVEC simulated by MMP9.PARTⅡ The Protect Effect of Rhubarb Monomer on the Vascular Endothelial Cells Nexin injuried by MMP9Objective: To investigate the effects of rhubarb monomers on the expression of HUVEC VE-cadherin and tight junction proteins, ZO-1, occluding and claudin-5 injuried by MMP9.Methods: The HUVEC monolayer model was established and randomly assigned to four groups: control, MMP9, MMP9 with rhubarb monomers mixture treated(MMP9+MM) and MMP9 with dexamethasone(MMP9+DEX) treated groups. MMP9 was added to the cell culture fluid at 1mg/L in the MMP9 group, MMP9+MM group and MMP9+DEX group, at the same time, the emodin, 3,8-dihydroxy-1-methylanthraquinone-2-carboxylic acid, 1-O-caffeoyl-2-(4-hydroxyl-O-cinnamoyl)-β-Dglucose, daucosterol linoleate, and rhein were added to the MMP9+MM group at 1μmol/L for each monomer and the DEX was added to the MMP9+DEX group at 10μmol/L. The control group was added the same amount of PBS as a negative control according to the dosing volume of MMP9. After cultured 24 h, the gene and protein expression of HUVEC VE-cadherin, the tight junction( ZO-1, occluding and claudin-5),and the cell morphology and intensity of ligandin were detected through real-time quantitative PCR, Western blot and immunofluorescence respectively.Results:(1) The gene expression of HUVEC VE-cadherin, and tight junction protein: stimulated by MMP9, the tight junction(ZO-1, occluding and claudin-5) was decreased in different degree compared with the control group(P<0.001), and the VE-cadherin decreased most significantly. After co-incubated with MM or DEX, the gene expression significantly increased than that in MMP9 group(P<0.001), and the MM was better than DEX(P<0.001).(2) The expression of HUVEC VE-cadherin, and tight junction protein: ZO-1, occludin, claudin-5 was decreased stimulated by MMP9 compared with the control, MMP9+MM and MMP9+DEX groups(P<0.001). And the protein expression was much higher in MMP9 and MM group compared with that in MMP9 and DEX group.(3) The morphology of cells was regular, the cell membrane was integrity, the connexin labeled with green fluorescent was continuous, and the structure was clear in control group. The fluorescent particles of connexin decreased and discontinuous significantly, and the morphology of the cells was obscured after stimulated by MMP9. The above situation was improved after treated with rhubarb monomers or DEX accompanied by MMP9 stimulation.Conclusion: The rhubarb monomers, including Rhein, 3,8- dihydroxy 1-methylethyl anthraquinone 2 carboxylic acid, 1-O- coffee acyl- 2-(4- hydroxy- cinnamyl) beta D-glucose, daucosterol linoleic acid ester and emodin, can increase the gene and protein expression of adhesion connection protein and tight junction protein and protect the morphology and syndeton of the endothelial cells.Part Ⅲ Signal Transduction Mechanism of Rhubarb Monomers Mixture on Improving Endothelial PermeabilityObjective: To detect the effect of rhubarb monomers on the PI3K/Akt-Rac1 signaling pathway and the phosphorylation of myosin, thus illuminate the signal transduction mechanism of rhubarb monomers on the protection of endothelial barrier.Methods: The HUVEC monolayer model was established and randomly assigned to six groups, control, MMP9, MMP9 with rhubarb monomers mixture treated(MMP9+MM) and MMP9 with dexamethasone(MMP9+DEX) treated groups, PI3 K inhibitor, wortmannin group and Rac1 inhibitor, nsc23766 group. The 1mg/L MMP9 was added into the cell culture fluid of MMP9, MM, DEX, wortmannin and NSC groups. At the same time, the emodin, 3,8-dihydroxy-1-methyl-anthraquinone-2-carboxylic acid, 1-O-caffeoyl-2-(4-hydroxyl-O-cinnamoyl)-β-D-glucose, daucosterol linoleate, and rhein were added to the MMP9+MM group at 1μmol/L for each monomer and the DEX was added to the DEX group at 10μmol/L. The wortmannin was added into wortmannin group at 1μmol/L, and the NSC23766 was added into the NSC group at 100μmol/L. After incubated for 24 h, the Rac1 activity was assaied using the Rac1 assay kit. The expression of AKT, p-AKT, VE-cadherin, MLC and p-MLC were detected using the Western blot method.Results:(1) The phosphorylation level of Akt stimulated by MMP9 was significantly higher than the control group(P<0.001). The expression of p-Akt was significantly decreased after intervention with MM, DEX and wortmannin(P<0.01), but it was increased by certain degree compared with the control group(P<0.01).(2) Compared with the normal group, the activity of Rac1 was significantly higher in MMP9 group(P<0.001). Compared with the MMP9 group, the activity of Rac1 was significantly decreased(P<0.01) and the protein expression of VE-cadherin was significantly increased(P<0.05) when intervented with MM, DEX or NSC.(3) The phosphorylation level of MLC stimulated by MMP9 was significantly higher than that in the control group(P<0.001). The expression of p-MLC was significantly decreased after the intervention with MM, DEX(P<0.01), but it was increased by certain degree than that in the control group(P<0.01).Conclusion: The rhubarb monomers, including Rhein, 3,8- dihydroxy 1-methylethyl anthraquinone 2 carboxylic acid, 1-O- coffee acyl- 2-(4- hydroxy- cinnamyl) beta D-glucose, daucosterol linoleic acid ester and emodin, can decrease the damage of the adherent junction protein by inhibiting the activation of Rac1 through the PI3K/Akt pathway. Meanwhile,it can reduce the phosphorylation of MLC and inhibite the contraction of endothelial cells. Ultimately, the permeability of monolayer endothelial cells was improved. |
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