| ObjectiveThis paper was designed to study characters of the kidney deficiency rats induced by high dose of hydrocortisone and the injury of PC 12 cells induced by D-Gal, and the therapeutic effects of Youguiyin, classic traditional Chinese medicine (TCM) in treating the syndrome. Detected the level of EPO and EPOR in the kidney deficiency rats and injuryed of PC 12 cells by treated with Youguiyin decoctum and discusses the protective effects of Youguiyin may mediate EPO/EPOR pathway.Methods1. Detection of main components content in Youguiyin decoctum:using high performance liquid chromatograph, Loganin, Cinnamaldehyde and Catalpol in YOU GUI YIN were detected. The determination condition of Loganin:the mobile phase was acetonitrile-water(15:85) and UV detection wavelength was set at 240 nm, the flow rate was 1.0 mL · min-1 and the column temperature was 30℃. The determination condition of Cinnamaldehyde:the mobile phase was acetonitrile-water(35:65) and UV detection wavelength was set at 290 nm, the flow rate was 1.0 mL·min-1 and the column temperature was 30℃. The determination condition of Catalpol:the mobile phase was acetonitrile-water(2:98) and UV detection wavelength was set at 210 nm, the flow rate was 1.0 mL·min-1 and the column temperature was 30℃.2. Protective effects of Youguiyin on Kidney Deficiency Syndrome(KDS) Rats:60 Sprague-Dawley (SD) rats were randomly caged in groups of 2, normal control group (n=12), treatment group (n=50, respectively). After one-week habituation, normal control group were administrated intraperitoneally normal saline and the rest of rats were injected intraperitoneally hydrocortisone at a dose of 10 mg/kg body weight once daily for 15 days. Then normal control group were raised normally and treatment rats were caged in groups of 4 (n=12, respectively) including model group were given equal volume of normal saline administration by gavage, three groups were received YGY decoction via oral administration at graded dose of low-dose(5.86 g.kg-1), a medium-dose(11.7 g.kg-1)and high-dose(23.4g.kg-1), respectively. All of rats were administrated once daily for the following 15 days. After the last treatment, fasting but water ad libitum 4 hours, all rats were weighed, anesthetized, and sacrificed. Whole blood was collected from the suborbital vein under chloral hydrate anesthesia. The brain, thymus, spleen, kidney, testis of five rats in each group were removed, weighted.3. Protective effects of Youguiyin on PC12 cells induced by D-Gal:At the cellular level, PC 12 cells were cultured, we studyed the effects of Youguiyin decoctum on D-Gal injury PC 12 cells model. After intervention of Youguiyin, MTT colorimetric method was used to determine cell activity, FCM(Flow Cytometry) was used to determine cell apoptosis. Additionally, the levels of apoptotic related proteins Bcl-2, Bax and EPO、EPOR were measured by Western blotting analysis.Results1. Detection of main components content in Youguiyin decoctum:Loganin, Cinnamaldehyde and Catalpol content were 0.341 mg·g-1,0.039 mg·g-1,0.026 mg·g-1.2. Protective effects of Youguiyin on Kidney Deficiency Syndrome (KDS) Rats: Signs of KDS rats induced by hydrocortisone were sleepy, unactived,humid and loss of appetite. The biochemical changes in 4 groups which were treated by hydrocortisone were significantly different from the normal control group (P<0.05). While Youguiyin treatment can ameliorated KDS rats towards normal level, such as weight back, increasing in activity, the content of ACTH, T, T3 and T4 were increased。 Youguiyin elevate organ index of KDS rats, the rats of thymus index, renal index and testis index in model group markedly reduced than normal control group (P<0.05). While, compared with the model group, the thymus index, adrenal glands index and testis index were increased in the Youguiyin treatment groups. Administration of Youguiyin (23.4 g·kg-1), the thymus index, renal index and testis index were dramaticlly increased compared with the model group (P<0.05). Treatment Youguiyin (11.7 g·kg-1), renal index and testis index were dramaticlly higher than model group (P<0.05), but with no significance increase in the thymus index (P>0.05). Then, the testis index of Youguiyin (5.86 g·kg-1) treatment group had notably increased than the rats of model group (P<0.05), but there is no significant difference in the thymus index and renal index (P>0.05). The rats in model group had significantly lower WBC, RBC, HGB, PLT and lymphocytes proportion decreased significantly than normal control group (P<0.05), while, treatment with Youguiyin (23.4 g·kg-1) resulted significantly increased WBC and lymphocytes proportion compared with KDS rats in model group(P<0.05), rats were treated with Youguiyin(11.7 g·kg-1) also had notably increased RBC, HGB and lymphocytes proportion (P<0.05), but 5.86 g·kg-1 with no significance changing in all cells except RBC (P<0.05), and no significant difference in the others cells (P>0.05). The expressions of EPO, EPOR and Bcl-2/Bax ratio in model group were significantly lower than that in the normal control group (P<0.05). While the expressions of EPO, EPOR and Bcl-2/Bax ratios were obviously increased by treatment with Youguiyin, which indicated that the Youguiyin can protect KDS rats by affecting the expression of EPO, EPOR and Bcl-2/Bax ratios in brain tissues.3. Protective effects of Youguiyin on PC12 cells induced by D-Gal:D-Gal injury PC12 cells model, cell activity and apoptosis ratio were lower than control group (P<0.05). After intervention of Youguiyin and EPO, cell activity improved obviously (P<0.05) and cell apoptosis ratio were lower than model group (P<0.05) determined by FCM(Flow Cytometry). Additionally, the levels of apoptotic related proteins Bcl-2/Bax ratio and EPO、EPOR were lower than control group (P<0.05). After intervented by Youguiyin and EPO, proteins Bcl-2/Bax ratio and EPO、EPOR were lower than control group (P<0.05). Especially 0.1mg·mL-1 and 0.2mg·mL-1 have no significance changing in the level of Bcl-2/Bax ratio and EPO、EPOR measured by Western blotting analysis.Conclusion1. Success to establish testing method of main components of Youguiyin decoctum and ensure the reliability and repeatability of this research.2. Success to duplicate the Kidney Deficiency Syndrome (KDS) Rats:and applied to Youguiyin decoctum in the related study.3. Youguiyin decoctum improve KYDS rats level of EPO and EPOR protein in brain tissue, and the ratio of Bcl-2/Bax were increased, which has proved that the decoctum of Youguiyin may be mediate the pathway of EPO/EPOR to protect the KDS rats.4. Discusses the protective effects of Youguiyin and exogenous EPO in PC12 cells injuryed by D-Gal, it proved that Youguiyin and exogenous EPO can promote the content pf endogenous EPO, and decrease the cell apotosis and increase the level of Bcl-2 protein which can protect the PC 12 from D-Gal, point out Youguiyin may through the EPO/EPOR pathway to protect cells. |
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