| BackgroundTraditional Chinese medical theory holds that the kidney is the origin of congenital constitution. The essence theory with the "Kidney stores the essence" as a representative is an important basic theory of Chinese medicine. The kidney essence, mainly mediating growth and reproduction, is the fundamental of human growth and development. The acquired essence of kidney, is the subtle substance of sustaining life, which is closely related to growth. The kidney essence could produce marrow, which would fill in brain. "Supplementing the kidney essence to strengthen the brain" is a classical and effective method used to treat for aging related diseases in Chinese medicine area. In recent years, with the increase of the incidence of Central nervous system diseases, the clinical applications of "Supplementing the kidney essence to strengthen the brain" are more and more. However, the material basis of the kidney essence is still unknown.According the source and effect of erythropoietin (EPO), we assume that EPO may be a biological material basis for the kidney essence, and the neuroprotective effect of EPO may be the biological mechanism for the theory that kidney well versed in brain. In this study, we co-cultured three kinds of rat brain cells to mimic cerebral neurovascular unit (CNVU) model in vitro and applied serum pharmacology to observing the proliferation effect of EPO on the NVU in vitro model. Additionally, BushengYijing Recipe was used to promote EPO production and the growth of brain cells, so that verifying the hypothesis.ObjectiveTo investigate the protective effects of rHuEPO and the traditional Chinese Medicine Bushenyijingprescription on CNVU in vitro model.Methods1. Effects of rHuEPO on CNVU cell growth and proliferation in vitroWe studied the effect of rHuEPO on pre-established model of CNVU which is a co-culturing model composed of three kinds of rat brain cells.The growth curve and growth cycle of astrocytes and brain microvascular endothelial cells were determined using MTT colorimetric method and flow cytometry, respectively. The axonal growth of neuron were observed by inverted microscopy with micrometer. The expression of GAP-43 was detected by Western blot.2 Quality control of Youguiyin, Rehmannnia radix praeparata, Polygoni multiflori radixpraeparata decoction and preparation of medicated serum:Quality control of Youguiyin, Rehmannnia radix praeparata, Polygoni multiflori radix praeparata①Youguiyin:chromatographic conditions for loganin determination:the mobile phase was acetonitrile-water (15:85) and UV detection wavelength was set at 240 nm; chromatographic conditions for cinnamic aldehyde determination:the mobile phase was acetonitrile-water (35:65), with UV detection wavelength 290 nm; and chromatographic conditions forVerbascoside determination:the mobile phase was acetonitrile-0.1% acetic acid (15:85) and the UV detection wavelength was 334 nm.②Rehmannnia radix praeparata:chromatographic conditions for Verbascoside determination:the mobile phase was acetonitrile-0.1% acetic acid (15:85) and the UV detection wavelength was 334 nm.③Polygoni multiflori radix praeparata: chromatographic conditions for stilbene glucoside determination:the mobile phase was acetonitrile-water (20:80) and the UV detection wavelength was 320 nm.3 The effect of Bushenyijing recipes on cell proliferation and secretion of EPO in CNVU modelCNVU cells were divided into four groups, control serum group, youguiyin medicated serum group (YMS), Rehmannnia radix praeparata medicated serum (RMS) and Polygoni multiflori radix praeparata medicated serum (PMS) groups. ELISA was used to detect the expression of EPO in supernatants and the expression of cell EPOR was detected by Western blot. MTT colorimetric method was used to determine the activity of astrocytes and brain microvascular endothelial cells. BrdU was used to detect the ratio of cell proliferation.4 Neuroprotective effects of Bushenyijing recipe on the oxygen and glucose deprive /reoxygenation injured NVU modelCo-cultured Cellsin CNVU model were divided into control group, oxygen and glucose deprive/reoxygenation(OGD/R) group, OGD/R+control serum group, OGD/R+YMS group, OGD/R +RMS group, and OGD/R+ PMS group. After suffering hypoxia for 6h and reoxygenation for 12h, the cells were determined with activity by MTT colorimetric method. The expression of EPO and EPOR was analyzed by Western blot.Results1 rHuEPO promoted brain cell growth and proliferation in CNVU modelCompared to the control group, rHuEPO 12.5U/mL group and 25 U/mL group significantly promoted neuron axonal growth and the expression of GAP-43(P<0.05), markedly promotedthe proliferation of astrocytes and brain microvascular endothelial cells (P<0.05), and also increased the proportion of the cells in S and G2/M phases (P <0.05), point out rHuEPO promoted neuron axonal growth and proliferation of astrocytes and brain microvascular endothelial cells in CNVU model.2 Bushenyijing recipes promoted cell proliferation and increased secretion of EPO in CNVU modelCompared to 5%control serum,5%YMS,5% RMS, and 5%PMS increased Brdu+ astrocytes (P<0.05), Compared to 10%control serum,10%YMS,10%RMS, and 10%PMS increased Brdu+ brain microvascular endothelial cells (P<0.05), point out Bushenyijing recipespromotedthe proliferation of astrocytes and brain microvascular endothelial cells. Compared to control serum, YMS, RMS, and PMSpromoted the secretion of EPO and the expression of EPOR in neuron, astrocytes and brain microvascular endothelial cellsto some extent, but without significance(P>0.05).3 Neuroprotective effects of Bushenyijing recipeson the oxygen and glucose deprive /reoxygenation injured NVU CellsThe cell activity and the expression of EPO/EPOR in the H/R injured CNVU model were lower than that the cells in normal control group (P<0.05). After the treatment of YMS, RMS, and PMS, astrocytes and brain microvascular endothelial cells activity was improved obviously (P<0.05). Compared with the cells in normal control, control serum promoted the expression ratios of EPO/EPOR in neurons, astrocytes, and brain microvascular endothelial cells significantly in the OGD/R injured CNVU model(P<0.05). Compared to the control serum, YMS and RMS promoted the expression ratios of EPO/EPOR in neurons, astrocytes, and brain microvascular endothelial cells significantly in the OGD/R injured CNVU model(P<0.05). However, PMS did not induce significant changes in the level of EPO/EPORO(P>0.05). The result point out Youguiyin,Re hmannia,andPolygoni multiflori radix praeparata decreased the cell apoptosis and protected NVU cells from OGD/R injury, probably via the EPO/EPOR pathway.Conclusions1 rHuEPO promoted the neuron axonal growth and increased the proliferation of astrocytes and brain microvascular endothelial cells in CNVU model2 The Bushenyijing recipes promoted the cell proliferation in CNVU model.3 Youguiyin, Rehmannia,andPolygoni multiflori radix praeparata decreased the cell apoptosis and protected NVU cells from OGD/R injury, probably via the EPO/EPOR pathway.4 rHuEPO and the traditional Chinese Medicine Bushenyijing recipes both promoted cell growth and proliferation in CNVUmodel, with the increasedexpression of EPO/EPOR, which proved the similarity of the EPO and the kidney essence,verifyingthe hypothesis that "EPO may be a biological material basis for the kidney essence, and neuroprotective of EPO may be the biological mechanism forthe theory that kidney well versed in brain."... |
PDF Full Text Request