| BackgroundTonic herbs were used to cure the brain disease based on the Chinese medicine theory of Shen-Jing Tong Yu brain. But what is Shen-Jing and how it performed the role of Neuroprotection is still unclear. The present study showed that erythropoietin(EPO) was similary to Shen-Jing in function. In present study, we utilized the aging model to simulate kidney essence insufficiency syndrome, and then Tonic herbs were used to invigorate kidney for improving essence, and observed the change of brain EPO/EPOR expression in the different condition. The study was proved by the sourch of National Natural science Foundation Item(81473549).ObjectiveThe present study aimed at verificating the similarity between EPO and Shen-Jing using aging model and probing the underlyling mechanism of neuroprotection of tonic herbs.Methods1. Establishment of aging rat and the change of EPO expressionEstablishment of naturel aging rat and the change of EPO expression in related tissues Male Sprague-Dawley rats, aged either 2 or 24 months, 2 months rats weighting 200±20g, 24 months weighting 500±50g, were divided into 2 groups, including young group and naturel aging group(n=13). Then the rat weight and smoothness of fur were observed and active movement was measured by open field test. Then the assay kit detected the level of MDA and SOD activation. At last, EPO expressions in the brain, kidney, bone marrow, serum were measured by Western Blot or enzyme-linked immuno sorbent assay(ELISA).Establishment of D-gal-induced aging rat model and the change of EPO expression in brain Male Sprague-Dawley rats, aged 2 months, weighting 200±20g, were randomly divided into 4 groups, including control-treated group,50mg/kg D-gal-treated group, 150mg/kg D-gal-treated group and 250mg/kg D-gal-treated group(n=13). 50mg/kg D-gal-treated group, 150mg/kg D-gal-treated group and 250mg/kg D-gal-treated group individually received subcutaneous injected of D-gal(Amresco, USA) at dose of 50mg/kg, 150mg/kg and 250mg/kg daily for 8 weeks. The control-treated group was injected subcutaneously with the same volume of vehicle(0.9% saline) daily for 8 weeks. Then the rat weight and smoothness of fur were observed. And we assessed cognitive impairment using Morris water maze(MWM) and examined the amount of β-galactosidase, lipid peroxidation product malondialdehyde(MDA) level and superoxide dismutase(SOD) activity in the hippocampus by assay kits. The levels of EPO, and EPOR in the hippocampus were detected by western blot.2. Antiaging effect of tonic herbs and the effect of notic herbs on the EPO expressionAntiaging effect of tonic herbs in natural aging model and the effect of notic herbs on the EPO expression Male Sprague-Dawley rats, including 2 months old(n=13) and 24 months old(n=52), 24 months rats were divided into 5 groups, including young group, naturel aging group, You gui yin(YGY)-treated group, radix rehmanniae praeparata(REP)-treated group and Radix Polygoni Multiflori Preparata(RPMP)-treated(n=13). Then the rat weight and smoothness of fur were observed and active movement was measured by open field test. After anesthetizd with chloral hydrate, the level of MDA and SOD activation in serum were detected. The expression of EPO/EPOR in the brain, kidney, bone marrow were measured by Western Blot and the expression of EPO in serum was measured by enzyme-linked immuno sorbent assay(ELISA).Antiaging effect of tonic herbs and the effect of notic herbs on the EPO expression in D-gal-induced aging rat model aging model Male Sprague-Dawley rats, aged 2 months, weighting 200±20g, were randomly divided into 3 groups, including control-treated group,150mg/kg D-gal-treated group and radix rehmanniae praeparata(REP)-treated group(n=13).D-gal-treated group was received subcutaneous injection of D-gal at dose of 150mg·kg-1 daily for 8 weeks. The REP-treated group group was given WERRP oral gavage at a dose of 4g·kg-1 daily starting from the 5th week. Morris water maze(MWM) test was used to assess the spatial learning and memory. SOD, MDA and β-Galactosidase in brain were examined by Assay Kits. The expression of EPO/EPOR in the brain was measured by Western Blot to observed the effect of notic herbs on the EPO expression.3. The neuroprotection of tonic herbs and the study of molecule mechanismThe neuroprotection of tonic herbs The SOD activity and MDA level in the brain were measured by Assay Kits to reflect the anti-oxidation of tonic herbs. The Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL) and the western blot for Caspase 3 were performed to detecte the anti-apoptosis of tonic herbs. Last the the number of Endothelial progenitor cells in the born marrow was measured through flow cytometer labeled by the antibody of VEGFR2 and CD133 and the VIII factor expressionwas detected through immunofluorescence, and The expression of VEGFR2 in the brain was measured by western blot to reflect the angiogenesis of tonic herbs.The study of molecule mechanism The HIF-2α, P-AKT and P-STAT 5 expression in hippocampuse were detected by WB to study the molecule mechanism of neuroprotection.Result1. Establishment of aging rat and the change of EPO expressionEstablishment of naturel aging rat and the change of EPO expression in related tissues Compared with the young rat group, the fur of naturel aging rat was rough and the colour was grey yellow. Compared with the young group, a significant decrease in locomotor activity, in the SOD activity(P<0.05), a significant increased in the amount of β-galactosidase(P<0.05) in the brain, in the MDA level(P<0.01) in the serum were observed in the natured aging rat, which indicated that the aging rat model was established successfully. Then the results of western blot showed the expression of EPO/EPOR in the brain, born marrow and kidney as well as the secretion of EPO in the serum and supernate of born marrow were all significantly decressed(P<0.05).Establishment of D-gal-induced aging rat model and the change of EPO expression in brain Compared with the control group, the fur of D-gal-induced aging rat was rough and the colour was grey yellow. D-gal-treated group showed significant longer latency to platform and less times of cross the platform(P<0.05) in MWM. After treated with D-gal, SOD was drastically decreased and MDA and β-Galactosidase were remarkably increased in brains compared with vehicle(0.9% saline)-treated rats(P<0.01). In addition, the expression of EPO and EPOR was significantly decreased in the brains of D-gal-treated rats compared with vehicle-treated rats.2. Antiaging effect of tonic herbs and the change of EPO, EPOR expressionAntiaging effect of tonic herbs and the change of EPO, EPOR expression in natural aging model Compared with naturel aging model, Youguiyin(YGY)-treated group, radix rehmanniae praeparata(REP)-treated group and Radix Polygoni Multiflori Preparata(RPMP)-treated group the locomotor activity had no siggificant difference in the locomotor activity(P>0.05). While compared with naturel aging model, a significant decress in the amount of galactosidase, in the MDA level(P<0.05) and a significant increase in the SOD activity(P<0.05) in the serum were observed in the YGY-treated group, REP-treated group and RPMP-treated group. Compared with natural aging group, YGY-treated group, REP-treated group and RPMP-treated group had a significant increase on the EPO/EPOR expression in the brain. While in the kidney, EPO expression was significanted decreased(P<0.05) compared with natural aging group, and EPOR expression has no signifacant change(P>0.05). In the bone marrow, the western blot result showed EPOR expression has no signifacant change(P>0.05) while EPO in supernatant of born marrow had a signifiant increase in the group of YGY-treated and REP-treated(P<0.05). In the serum, the ELISA result showed the EPO expression was significantly increased in the group of YGY-treated and REP-treated(P<0.05) while the RPMP-treated group had on significant increase compared with naturel aging model(P>0.05).Antiaging effect of tonic herbs the change of brain EPO, EPOR expression in D-gal-induced aging rat model aging model. WERRP-treated rats showed significant improvement of spatial learning and memory, decreased of MDA level(P<0.05), and increased of SOD activity(P<0.05) compared with D-gal-treated rat which indicated WERRP had the role of antiaging. The western blot and immunofluorescence results showed enhancement of EPO/EPOR in the brain of the REP-treated group compared with D-gal-treated rat(P<0.05).3. The neuroprotection of tonic herbs and the study of molecule mechanismThe neuroprotection of tonic herbs Compared with natural aging rats, brain SOD activity had a significant increase and brain MDA level had a significant decrease(P<0.05) in the YGY-treated group, REP-treated group and RPMP-treated group which indicated the tonic herbs had the effect of antioxidation. The TUNEL result showed that the positive cells in YGY-treated group, REP-treated group and RPMP-treated group had a significant decrease(P<0.05) compared with natural aging model. On the other hand, the western blot showed that the caspase 3 expression was significantly decreased in the brain of YGY-treated group, REP-treated group and RPMP-treated group compared with natural aging model. Compared with natural aging model, the number of Endothelial progenitor cells in the bone marrow was significantly increased(P<0.05), Endothelial cell in the brain was significantly increased(P<0.05) as well as the VEGFR2 expression in YGY-treated group, REP-treated group and RPMP-treated group.The molecule mechanism of neuroprotection of tonic herbs Compared with natural aging model, the expression of HIF-2α and p-STAT were singificantly increased in the group of tonic herbs(P<0.05).ConclusionsEPO was similary to Shen-Jing in the source and function and we proposed that EPO may be one of basis material for the "kidney essence". On the same time, Youguiyin, radix rehmanniae praeparata and Radix Polygoni Multiflori Preparata increased the expression of EPO/EPOR in the brain and delayed brain senescence which was similary to the function of EPO. |
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