Induction of Klotho Gene Expression by Natural Product Compounds and the Anti-aging Analysis
|School||East China Normal University|
|Keywords||klotho gene D-galactose induced aging-model mice anti-aging effect (+)-marmesin|
Klotho gene encodes a membrane protein. Null mutation of klotho gene resulted in premature aging, and overexpression of the gene led to the extension of life span and suppression of aging in mice. Therefore, klotho gene has been considered as an aging-suppressing gene. Identification of compounds that can activate the expression of klotho gene could be used for the development of anti-aging agents.A stable cell line （HEK293/kl） was established by transfecting a reporter gene vector containing klotho promoter and luciferase gene to HEK293 cells. A model for cellular aging was generated by treating HEK293/kl cells with 0.006% H2O2 for 6h Luciferase activity was measured to identify compounds that can regulate klotho promoter activity. A number of plan extracts, including Notopterygium incisum Ting., Semen Raphani and Epimdium biolba, EC50 of which is 128.90μg/ml, 3.57 mg/ml and 1.62 mg/ml respectively, were found to be able to activate the promoter of klotho gene.The extract of Notopterygium incisum Ting, increased luciferase activity in the HEK293/kl cells. We also examined the klotho mRNA level in primary cultured kidney cells from mice. Cells were treated with H2O2 and then with the extract from Notopterygium incisum Ting.. Our results showed that the extract enhanced the expression of klotho gene.Aging animal model has been established by injection of D-galactose into ICR mice. After 4 weeks of treatment, the D-galactose-induced aging mice treated with the extract of Notopterygium incisum Ting.. After 4 weeks of administration, the molecular marker of aging and expression of klotho gene were measured.In the aging models, the serum SOD activity and serum NO concentration were significantly decreased. Serum cholesterin and liver LPO were also increased. We found that the expression of klotho in the kidney was significantly down-regulated. After Notopterygium incisum Ting, treatment, the activity of serum SOD significantly increased; LPO in liver and serum phosphorus reduced; the concentration of NO in the blood increased, and the expression of klotho in the kidney were significantly increased. These results suggested that the extract of Notopterygium incisum Ting, could enhance the expression of klotho, protect cardio-vascular system, and increase the antioxidation and anti-aging activity.Furthermore, we have isolated, separated and purified the active compounds based their bioactivity. An active compound was separated and purified by filtering, extracting and column chromatography from the Notopterygium incisum Ting. extracts. The structure of the compound was determined as（+）-marmesin by 1H-NMR, EI-MS and optical rotation.