| ObjectiveThis study will discusses the mechanism of Qiongyugao acting on the spleen of D-galactose-induced aging rats by regulating the PI3K/Akt signal pathway. In the previous experiments we had been screened target genes or proteins which are associated with aging on the aging rats spleen. In this experiment we will use the fluorescence quantitative PCR, immunohistochemistry (IHC) and western blot (WB) method, based on these candidate target genes and proteins of the signal pathway.analysis its protein expression, explore in depth of the mechanism of Qiongyugao anti-aging in aging rats through this experiment, to provide certain theoretical and experimental basis for the further research and development of Qiongyugao.MethodsForty two Sprague-Dawley rats were randomly divided into three groups (Control, Model:D-galactose (D-gal)-administration, and QYG: D-gal-administration+QYG-treatment groups). After continuous treatments of D-gal (250 mg/kg) and QYG (14 g/kg) daily for 62 days, the Morris water maze test was employed to measure spatial learning and memory, HE staining was used to observe pathological changes in spleen, the fluorescence quantitative PCR and western blot (WB) were used to detected the expression of HSPBP1 and TIMM8B mRNA and proteins. Immunohistochemical was used to detected the expression of differential proteins of P-Akt, P-mTOR, Caspase 3, Nrf2 and P53.Results1. Using the Morris circular water maze test for the evaluation of Subacute aging rat model.Compared with blank group, The model group have obvious rised in 3,4, 5, and 6 days (K0.05), and the incubation period is on the decline; Compared with model group, The Qiongyugao group have obvious decreased in 3,4,5, and 6 days (/K0.05), and the incubation period is on the decline;there is no difference between the Qiongyugao group and blank group.2. Using the fluorescence quantitative PCR to detect the expression of HSPBP1 and TIMM8B mRNA in the spleen tissue.Compared with blank group, the expression of HSPBP1 mRNA in the model group rised obviously(P＜0.01), and the expression of TIMM8B mRNA decreased obviously (P＜0.05);Compared with model group, the expression of HSPBP1 mRNA in the Qiongyugao group decreased obviously (P＜0.01), and the expression of TIMM8B mRNA rised obviously (P<0.05); there is no obvious difference between the Qiongyugao group and blank group of the expression of HSPBP1 mRNA and TIMM8B mRNA.3. Using western blot (WB), detect the proteins expression of HSPBP1 and TIMM8B in the spleen tissue.Compared with blank group, the proteins expression of HSPBP1 in the model group rised obviously(K0.01), and the proteins expression of TIMM8B decreased obviously (P＜0.05);Compared with model group, the proteins expression of HSPBP1 in the Qiongyugao group decreased obviously (P＜0.01), and the proteins expression of TIMM8B rised obviously(P＜<0.05);there is no obvious difference between the Qiongyugao group and blank group of the proteins expression of HSPBP1, the proteins expression of TIMM8B have obvious difference(P＜0.001).4. Using the method of immunohistochemical, detect the proteins expression of P-Akt, P-mTOR, Caspase 3, Nrf2 and P53 of the spleen tissue protein.Compared with blank group, the proteins expression of P-akt and mTOR in the model group decreased obviously (P＜0.01), the proteins expression of caspase-3^ P53 and Nrf2 rised obviously; Compared with model group, the proteins expression of P-akt^ mTOR and Nrf2 in the Qiongyugao group rised obviously (P＜0.01), the proteins expression of caspase-3 and P53 decreased obviously (P＜0.01).Conclus i on1.The subacute aging rat model is successful, the Qiongyugao has the ability to improved the learning and memory in the rat model of aging.2. The Qiongyugao can downregulated the expression of HSPBP1 mRNA and protein, can upregulated the expression of TIMM8B mRNA and protein.3. The Qiongyugao can upregulated the proteins expression of P-Akt, P-mTOR and Nrf2, can downregulated the protein expression of Caspase 3 and P53, play a role of resistance to apoptosis.This experimental study show that The Qiongyugao can regulate the PI3K/Akt signal pathways play a role of anti-aging on the spleen of D-galactose-induced aging rats. |
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