| Objective: To investigate the anti-aging effect of Cordyceps militaris Protein Extract(CMPE) and its related mechanism.Methods: CMPE was prepared by ammonium sulfate precipitation.DPPH,superoxide anion radical and hydroxyl radical scavenging activity were used to evaluate the antioxidant capacity of CMPE in vitro.Establishment of aging model of mouse embryonic fibroblasts (NIH/3T3)induced by D-Gal (D-Galactose,800 m M),and the proliferation capacity of CMPE for NIH/3T3 cells was detected by MTT method.The aging cells were stained by-galactosidase staining,and the Superoxide dismutase (SOD) activity,Malondialdehyde (MDA) level,Catalase (CAT) activity and reactive oxygen species (ROS) activity in NIH/3T3 cells were determined by spectrophotometry.The m RNA expression levels of Nrf2,GCLM,NQO1,GCLC,P53,P16,P21,CDK4 and Cyclin D1 in NIH/3T3 cells were detected by RT-PCR,The D-Gal induced mouse senescence model was used to divide the ICR mice into four groups according to the random grouping method.MOD group (double distilled water was given to the stomach by subcutaneous injection of 400 mg·kg-1 D-Gal);PC group (800 mg·kg-1 piracetam gavage,400mg·kg-1 D-Gal subcutaneous injection);In the CMPE group (800mg·kg-1 CMPE gavage,400mg·kg-1 D-Gal subcutaneous injection),the mice were subjected to dark avoiding,platform jumping and Morris water maze behavior experiments after 10 w.The levels of SOD,CAT,MDA,Glutathione peroxidase (GSH-Px) and 8-hydroxy-2deoxygu-anosine (8-OHd G) in brain tissue of mice were analyzed by spectrophotometry.Serum levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) in aging mice were detected by ELISA;HE staining method was used to observe the pathology of hippocampal area of brain tissue in mice,and double immunofluorescence labeling method was used to observe the situation of new proliferating nerve cells in dentate gyrus in mice.The expression levels of Nrf2,GCLM,NQO1,GCLC,P53,P16,P21,CDK4 and Cyclin D1 m RNA in brain tissues of mice were detected by RT-PCR.Results: the maximum scavenging rate of DPPH,superoxide anion radical and hydroxyl radical by CMPE reached 76.16%,75.14% and 66.80%,respectively.In MOD group,the survival rate of NIH/3T3 cells was 67.1% (p < 0.01),and that of CMPE (100 ug/m L) pretreated survival rate of NIH/3T3 cells was 97.23% (p < 0.01)..Compared with MOD group,CMPE can significantly improve SOD (p < 0.05) and CAT (p < 0.05) activities in NIH/3T3 cells,and reduce MDA (p <0.01) and ROS(p < 0.05)levels.Compared with MOD group,m RNA expression levels of GCLC,NQO1 and GCLM in CMPE group were significantly increased (p < 0.05),mRNA expression levels of P53 and P21 in CMPE group were significantly decreased (p < 0.05),and m RNA expression levels of cyclin D1 and CDK4 in CMPE group were significantly increased (p < 0.05).The behavioral results of the aging model mice showed that the incubation period of the diving platform was significantly prolonged (p < 0.05),and the number of errors was significantly reduced (p < 0.05).In the dark avoidance experiment,the incubation period of dark avoidance was significantly extended (p < 0.01),and the number of errors was significantly reduced (p < 0.05).Morris water maze experiment,compared with MOD group,CMPE group mice positioning navigation latency significantly shortened (p < 0.01);Compared with MOD group,CMPE can significantly increase the levels of SOD (p < 0.01),GSH-Px (p < 0.05)and CAT (p < 0.01)in brain tissue,and reduce the content of MDA (p < 0.05) in brain tissue.Compared with MOD group,the morphology and number of neurons in CMPE group were significantly improved.Compared with MOD group,CMPE significantly increased the number of Brdu/Neu N double-labeled positive cells (p < 0.01).Compared with MOD group,the m RNA expression levels of Nrf2,NQO1,GCLC and GCLM in brain tissue of CMPE group were significantly increased (p < 0.05),while the m RNA expression levels of P53 and P21 in brain tissue of CMPE group were significantly decreased (p < 0.05).In CMPE group,the expression levels of cyclin D1,CDK4 and m RNA were significantly increased (p < 0.01).Conclusion: CMPE has significant antioxidant activity in vitro.CMPE has anti-aging effects,which may be achieved by down-regulating the expression of genes related to the P53/P21 signaling pathway and activating the expression of key genes related to the antioxidant signaling pathway Nrf2-ARE. |
PDF Full Text Request