The Study Of Anti-aging Mechanism Of Zishen Yigan Fang In Vivo And Vitro

In recent years, with the serious problems of aging ratio in the total population, the prevalence of geriatrics, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), have been increasing continuously, resulting in greater and greater burden for the families even and community. Thus anti-aging and improving the life quality of the elder has attracted researchers more attentions. More and more anti-aging agents have been developed and discovered to be introduced for clinical researches, including single herb medicine and traditional Chinese medicinal formulations, however, few of them are successful.As a traditional folk formulation, Zishen Yigan Fang comprises some traditional Chinese medicinal herbs. The literature analysis indicates that it may have a strong anti-aging effect. So the aim of this study is to explore the effects of Zishen Yigan Fang on anti-aging and the underlying mechanisms in vivo and vitro.Objective To observe the effects of Zishen Yigan Fang on anti-aging and the underlying mechanisms in vivo and vitro. Methods1The animal experiments: ICR mice were introduced for this study and were divided into six groups: normal control group (ctrl), aging model group, vitamin E group, Zishen Yigan Fang at low, medium and high dosage (5,10,20g crude drugs·Kg-1·d-1) groups. Excepting the normal group, Others were subcutaneously injected D-Galactose to make aging model with150mg·Kg-1·d-1followed by the latter four groups were feed with the vitamin E, low, medium and high doses of Zishen Yigan Fang solution, respectively. After treatment for six weeks, the ability of learning and memory of every group were determined by mice s Escape Latency and the number of passing though the original platform in Morris water maze, then the blood was sampled by taking-out eyeball and the mice were killed by cervical dislocation. The spleen index and thymus index, SOD activity and level of NO in plasma, SOD, GSH-Px activity and MDA level of the liver as well as that of brain tissue were determined. Results1compared with the normal group, the Escape Latency of the model group extended (31.1±11.6s vs45.4±13.9s, P<0.05), and the number of passing though the original platform reduced (3±2.0vs1±1.0, P<0.01); the thymus and spleen index significantly reduced (0.1227±0.04vs0.0683±0.01and0.2312±0.02vs0.1942±0.02,P<0.01); SOD activity of plasma, liver and brain tissue declined (69.57±6.6U/ml vs60.43±4.7U/ml, P<0.05;29.88±2.2U/mgprot vs25.81±2.5U/mgprot, P<0.05, and45.62±5.0U/mgprot vs35.45±4.8U/mgprot, P<0.01, respectively); the MDA level of the liver and brain rise significantly (3.62±0.8nmol/mgprot vs6.76±1.6nmol/mgprot and6.15±0.4nmol/mgprot vs7.54±0.4nmol/mgprot, P<0.01); level of NO in plasma significantly increased (62.10±3.4μmol/L vs74.38±6.5μmol/L, P<0.01); the GSH-Px activity of the liver also obviously dropped from324.2±41.1U/mgprot to218.7±27.6U/mgprot (P<0.01). While compared with the model group, the Escape Latency of each group treated by Zishen Yigan Fang with low, medium and high dosage decreased to20.6±8.7s,28.5±6.6s and14.0±9.0s (P<0.01), and the number of passing though the original platform increased to3±1.6,4±2.0,5±1.7(P<0.01); the thymus and spleen index increased significantly and the data were0.0983±0.03,0.1165±0.03,0.1213±0.03(P<0.01);0.2476±0.03(P<0.05),0.2781±0.03(P<0.01),0.2319±0.02(P<0.01), respectively; SOD activity of plasma, liver and brain tissue obviously increased to83.42±7.0U/ml(P<0.01),81.55±8.0U/ml(P<0.01),67.01±9.0U/ml;33.35±3.0U/mgprot(P<0.01),31.66+2.7U/mgprot(P<0.01),29.12±3.5U/mgprot(P<0.05);54.83±4.6U/mgprot,53.32±3.8U/mgprot,48.48±5.9U/mgprot (P<0.01); the MDA level of the liver and brain dropped significantly to4.02±0.8nmol/mgprot,4.50±1.Onmol/mgprot,4.02±0.7nmol/mgprot(P<0.01);5.6410.7nmol/mgprot,5.76±0.5nmol/mgprot,4.87±0.7nmol/mgprot (P<0.01); levels of NO in plasma significantly declined to61.79±5.1μmol/L(P<0.01),66.99±11.2μmol/L,60.08±5.0pmol/L (P<0.01), respectively; and GSH-Px activity of liver also increased significantly to307.9±67.2U/mgprot,315.0±60.2U/mgprot,299.1±70.9U/mgprot (P<0.01). Methods2In vitro study:The protective effects of TSG and Zishen Yigan Fang solution on the oxi dative stress damage of PC12cells induced by H2O2were determined by MTT. The cells were divided into four groups:normal group, H2O2group, H2O2+TSG (0.1,1,5,10and50μmol/L) group, H2O2+Zishen Yigan Fang solution (0.16,0.4,0.8μmol/L) group. Firstly, to choose the best concentration of H2O2, the PC12cells were treated with different concentrations of H2O2, and the concentration of300μmol/L was selected at last. The cells were seeded into96-well plate, and treated firstly with TSG and Zishen Yigan Fang solution for24h, then H2O2was added to the96-well plate and treated for24h, too; after that, MTT was added and treated the cells for4h, then the OD values were determined to the calculate the survival rate of PC12cells. Results2After the cells were treated with H2O2, the cell survival rate reduced significantly from100±8.11%to85±5.02%(P<0.01). The survival rates of PC12cell pretreated with TSG and Zishen Yigan Fang solution were90.10±5.72%,90.78±9.23%,101.27±9.82%,108.8013.69%,109.63±13.72%;102.85±8.17%,106.27±9.19%,101.52±10.91%and the latter six groups ascended significantly (P<0.01). Conclusion The study of anti-aging and antioxidant of Zishen Yigan Fang in vivo and in vitro shows that Zishen Yigan Fang can delay ICR mice aging induced by D-galactose, and improve the ability of learning and memory and immune function of aging mice. The mechanism may be that Zishen Yigan Fang can clear away free radicals and improve the antioxidant system’s activity of the body to slow down the oxidative stress damage of the body induced by D-galactose, so that it can reach the aim of anti-aging.