| 1BackgroudAtherosclerosis is a slowly progressing disease in which endothelial dysfunction and damage play an initial role. Although the integrity and functional activity of the endothelial monolaver is essential forprotection against the initiation of atherosclerosis, many risk factors for atherosclerosis can lead to endothelial damage of the vessel.Endothelial dysfunction caused by endothelial cells apoptosis is recognized as an initial step in the atherosclerotic process. The repair of injured endothelium relies on the proliferation, migration, and remodeling of fullv differentiated endothelial cells. As mature endotheliaik cells are terminally differentiated cells with a low proliferative potential, endothelium repair needs the presence of other kinds of cells.Cells with the ability to mediate endothelial cell repair have been termed endothelial progenitor cells (EPCs).Bone marrow is the most defined source of circulating EPCs. EPCs derived from the bone marrow can be mobilized to the peripheral circulation upon a variety of stimuli including tissue ischemia through the release of growth factors such as vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1(SDF-1). EPCs are mobilized in response to vascular injury and migrate towards the region of injury where they adhere and facilitate repair of the endothelial cell layer.Almost all cardiovascular risk factors for atherosclerosis have been shown to exert detrimental effects on EPCs number and function. Not only EPCs count reflect cardiovascular risk and predict future events, but is also directly related to disease severity. As EPCs play an important role in the maintenance of vascular integrity, increased EPCs may be one of the mechanisms which could improve endothelial function and hence reduce atherosclerosis.According to traditional Chinese medicine(TCM) theory, the main pathogenesis mechanism of atherosclerosis is deficiency of qi and blood, with phlegm accumulating and stagnation. And recent findings suggest that formula functioning in enhancing qi and blood, promoting circulation and dissipating stasis could protect against atherosclerosis by improving the quantity and quality of EPCs. Danggui-Buxue decoction(DBD), a Chinese medicinal decoction contains Radix Astragali(RA, Huangqi) and Radix Angelicae Sinensis(RAS, Danggui) at a radio of5:1, functions in raising qi, nourishing blood, promoting circulation. Pharmacological results suggest that RA, RAS, as well as DBD all have effects on endothelial protection by lipid-regulation, anti-oxidization and anti-inflammation. Our former research findings also suggest that DBD could inhibit the progress of atherosclerosis by anti-inflammation through the p38mitogen-activited protein kinase and nuclear factor-κB signaling way.EPCs have the ability to counteract the process of inflammatory endothelium injury, and we hypothesis that the vascular protection of DBD may be closely related to its promotion of quantity and quality of EPCs.2AimEPCs, some kind of primitive cells found in bone marrows, can be differentiated towards endothelial cells. EPCs help in the regression of endothelial injury of atherosclerosis. There are encouraging data in our former research findings that DBD can regulate lipid, anti-oxidize, promote the growth of endothelial cell, so as to protect the vascular endothelial function.In this research, we may find the influence of DBD on EPCs in atherosclerotic progress, the molecular mechanism, and the possible signal transmitting way.This research will provide supplemental evidence for DBD’s effects on the prevention and therapy of atherosclerosis. It will also bring fresh ideas to the prevention and therapy of atherosclerosis by combining TCM with modern medicine.3Contents3.1To investigate the effects of DBD on the quantity and functional activities of EPCs from peripheral blood and bone marrow in atherosclerotic rabbits, the serum levels of VEGF and SDF-1. And to find the possible mechanism of DBD’ s influence on EPCs in vivo.3.2To observe the effects of DBD on the apoptosis and functional activities of EPCs from human peripheral blood induced by oxidized low-density lipoprotein (ox-LDL), the expression of Bcl-2mRNA and Bcl-2protein of EPCs. And to find the mechanism of DBD’s influence on EPCs induced by ox-LDL in vitro.3.3To observe the effects of drug-serum containing DBD on the functional activities of EPCs from human peripheral blood, the celluar excretion of nitric oxide (NO), the expression of endothelial nitric oxide synthase (eNOS) mRNA, the expression of Akt. And to find the possible signaling way of DBD’s influence on EPCs.4Methods4.1Influence of DBD on EPCs in the rabbit model of atherosclerosisRA and RAS at a radio of5:1were boiled in water over a moderate heat to reach a concentration at1g/mL. The model of atherosclerosis was established by using immune injury and fatty diet for4weeks in New Zealand rabbits (n=25). All modeled rabbits were randomized into5groups (each, n=5)according to the body weight. The control model group was intragastrically given distilled water, simvastatin group, aqueous suspension of simvastatin (1.7mg/kg), high-dose, mid-dose and low-dose groups were treated with DBD (6g/kg,3g/kg,1.5g/kg), respectively. All rabbits were treated once a day for two weeks. Then the mononuclear cells were isolated from rabbit peripheral blood and bone marrow by Ficoll density gradient centrifugation. Adherent cells cultured for7days were cultivated with Dil-ac-LDL and FITC-UEA-1together for EPCs identification. The number of colony forming units were calculated. The proliferation, migration, adhesion and tubule formation of EPCs were assayed using MTT method, transwell chambers, adhesion determination, and in vitro angiogenesis assay kit. The serum levels of VEGF and SDF-1were detected by using Elisa assay kit.4.2Influence and mechanism of DBD on EPCs induced by ox-LDL in vitroRA and RAS at a radio of5:1were boiled in water over a moderate heat to reach a concentration at0.48g/mL, then DBD was filter-sterilized using micro-pore filter. Peripheral blood mononuclear cells from healthy volunteers were isolated by Ficoll density gradient centrifugation and adherent cells were maintained for7days for EPCs identification.Samples of EPCs were randomized into eleven groups:A control group EPCs were incubated with M199medium containing0.2%bovine serum albuim for48h. B (5groups) EPCs were incubated with M199medium containing distilled water, simvastatin (0.1umol/L), DBD(1g/L,2g/L,4g/L) for24h, respectively, and with M199medium for another24h. C (5groups) EPCs were incubated with M199medium containing distilled water, simvastatin (0.1umol/L), DBD(1g/L,2g/L,4g/L) for24h, respectively, and with M199medium containing10mg/L ox-LDL for another24h. The proliferation, migration, adhesion and tubule formation of EPCs were assayed using MTT method, transwell chambers, adhesion determination, and in vitro angiogenesis assay kit. The cell apoptosis was detected by flow cytometry. The expression of Bcl-2mRNA were assayed by using realtime PCR, and the Bcl-2protein expression by Western-blot analysis.4.3Influence and mechanism of drug-serum containing DBD on EPCs in vitroNew Zealand rabbits were randomized into4groups (each, n=2) according to the body weight. The control group was intragastrically given distilled water. High-dose, mid-dose and low-dose groups were treated with DBD (30g/kg,15g/kg,3g/kg), respectively. All rabbits were treated twice a day for3days. After the last administration at the4th day morning, blood was obtained from rabbit artery2h later, and drug-serum was obtained by centrifugation and filter-sterilized.EPCs from human peripheral blood were cultured for7days and randomized into eight groups as follows:A control group EPCs were incubated with M199medium containing10%rabbit serum for24h. B EPCs of simvastatin group were incubated with M199medium containing10%rabbit serum and0.1umol/L simvastatin for24h. C(3groups) EPCs were incubated with M199medium containing10%rabbit drug-serum containing low-dosage, mid-dosage, high-dosage DBD for24h, respectively. D(3groups) EPCs were incubated with M199medium containing10%drug-serum containing low-dosage, mid-dosage, high-dosage DBD and wortamannin for24h, respectively. The secretion of NO was detected, and the proliferation, migration, adhesion and tubule formation of EPCs were assayed using MTT method, transwell chambers, adhesion determination, and in vitro angiogenesis assay kit. The expression of eNOS mRNA was assayed by using realtime PCR, and the Akt protein expression by Western-blot analysis.5Results5.1DBD could elevate the VEGF and SDF-1levels to improve the quantity and functional activities of EPCs in atherosclerotic processThe proliferation, migration, adhesion and tubule formation of EPCs from peripheral blood and bone marrow were all impaired in atherosclerotic rabbit. The number of EPCs colony forming units could reflect the cellular quantity, and there was no colony forming of EPCs from peripheral blood and few colonies of EPCs from spinal in model group. The quantity and functional activities of EPCs were all impaired in the pathological process of atherosclerosis. However, DBD and simvastatin could promote the colony formation and functional activities of EPCs. The levels of serum VEIF and SDF-1in DBD and simvastatin groups were much higher than that in model group. As VEGF and SDF-1play a vital role in functional regulation of EPCs, DBD could elevate the levels of circulating VEGF and SDF-1to regulate the quantity and functional activities of EPCs.5.2DBD could protect the apoptosis and activities of cultured BPCs induced by ox-LDL in vitroThe proliferation, migration, adhesion, tubule formation, and anti-apoptosis abilities of normal human EPCs, as well as EPCs induced by ox-LDL in vitro, were all improved in simvastatin and DBD (4g/L,2g/L, lg/L) treatment groups. Functional activities of EPCs induced by ox-LDL were impaired to some extent, and the cell apoptosis was obvious. DBD could protect the functional activities and help viability of EPCs induced by ox-LDL.DBD could improve the functional activities of EPCs by up-regulating the expression of Bcl-2mRNA and Bcl-2protein, as well as inhibit the low expression of Bcl-2mRNA and Bcl-2protein of EPCs induced by ox-LDL, which could be the possible mechanism of protective effects of DBD on EPCs.5.3DBD could regulate the functional activities of EPCs via PI3K/Akt pathwaySerum containing DBD and simvastatin could both improve the proliferation, migration, adhesion and tubule formation of EPCs, promote the cellular secretion of NO, up-regulate the expression of eNOS mRNA, the expression of total and phosphate Akt protein. However, the improvement of EPCs abilities by serum containing DBD were inhibited by PI3K/Akt pathway blocker, and the NO secretion, expression of eNOS mRNA, total and phosphate Akt protein were down-regulated by blocker. Above all suggested that PI3K/Akt pathway is one of the possible mechanisms of EPCs regulation by DBD.6Discussion and conclusionAtherosclerosis and atherosclerotic vascular disease are considered to be related with TCM pathogenesis mechanism " deficiency of qi and blood". Cardiovascular risk factors impair a patient’s "vital qi", resulting depletion of circulating EPCs, thus impair the quantity and functional activities of EPCs. EPCs represent the "promoters" of vascular endothelial repair. Following the most general principles of TCM,"vital qi" is some kind of protecting and curing functions of human itself. To some extent, the functional activities of EPCs are equal to the function of "vital qi" in TCM. Therefore, principles of TCM treatment emphasize on improvements of "qi" and circulatory function. DBD could enhance the"vital qi", promote the quantity and quality of EPCs, which could be a novel mechanism of its effects on endothelium protection.Our research findings suggested the quantity and functional activities of EPCs are impaired in the process of atherosclerosis. DBD could regulate the quantity and quality of EPCs impaired by atherosclerosis via the PI3K/Akt pathway, promote the mobilization of EPCs to the region of injury where they adhere and facilitate repair of the endothelial cell layer. Experiments in vitro with traditional water extracts of a formula are challenged by drug-serum pharmacology. Our data suggested that there is no difference in the regulation of EPCs between water extracts of DBD and drug-serum containing DBD.As characteristics of multi-target and multimechanism exist in TCM formula, it appears that the next logical step would be to characterize other mechanism whereby DBD alters number and function of EPCs. |
PDF Full Text Request