| Aging is a normal phenomenon in the biological world.It is a natural process and inevitable,but we can delay aging by some methods.Most people maintain a healthy lifestyle and anti-aging through dieting or exercising.In recent years,people also eat some natural products,extracts of natural products or drugs to acquire anti-aging effects.The traditional Chinese medicine,as a natural plant product,has been favored by researchers because they commonly have multi-functions and few side effects.Therefore,the new drug development of natural plants with anti-aging activity has been attracted extensive attention.According to the different processing technologies,ginseng can be divided into fresh ginseng,dried ginseng and red ginseng.Red ginseng is the dried root and rhizome obtained from fresh ginseng by steaming and processing.Panax ginseng C.A.Mey.,araliaceous is one of the most important traditional medicinal plant in China.Modern pharmacological studies had shown that ginseng have many biological functions of anti-aging,anti-depression,anti-senile dementia,anti-tumor and enhancing immunity,etc..It had been widely used as a food and drug in clinic.In our previous study,anti-aging experiments in Drosophila melanogaster(D.melanogaster)treated with ginseng,red ginseng and American ginseng had found that compared with ginseng and American ginseng,red ginseng could significantly extend the life span of D.melanogaster demonstrating red ginseng’s potential activity in antiaging,but the mechanism of the action was still unclear.Therefore,in this study,we further explored the anti-aging effects and its relative protein changes in both male and female D.melanogaster breeding with red ginseng powder(dried,crushed and sifted,RG)by observing the life-span and iTRAQ biology technique.The validation of the relative protein changes was detected by qRT-PCR and Western blotting.Futher more,the mechanism studies of RG on anti-aging effects in D.melanogaster had been explored.1.The anti-aging effects and relative protein changes in D.melanogaster fed with RGFirst,we analyzed the lifespan of D.melanogaster.After the female and male D.melanogaster fed with RG,the lifespan of both the female and male D.melanogaster were significantly extended,but the effective feeding dose were slightly different.RG with concentrations of 12.5 mg/mL,15.0 mg/mL and 17.5 mg/mL significantly extended the lifespan of female D.melanogaster.The 12.5 mg/mL and 15.0 mg/mL RG significantly extended the lifespan of male D.melanogaster.The lifespan of male and female D.melanogaster was reduced at a concentration of 20.0 mg/mL.The climbing ability evaluated as the autonomous activity ability in the aging male and female D.melanogaster treated with RG was determined.The results showed that female group treated with RG of 12.5 mg/ mL,15.0 mg/ mL and 17.5 mg/ mL had better climbing ability,male group treated with RG of 12.5 mg/ mL and 15.0 mg/ mL had better climbing ability.Secondly,iTRAQ analysis technique was used to identify the protein changes in D.melanogaster fed with RG.A total of 4313 proteins changes were founded in female and male drosophila.In female D.melanogaster,fifty-seven proteins had been found expressed significantly differently,including eleven overexpressed proteins and fortysix lower expressed proteins.In male D.melanogaster,ninety-four proteins had been found expressed significantly differently,including thirty-two overexpressed proteins and sixty-two lower expressed proteins.2.Validation of the differential proteins by qRT-PCR and Western blotting,and studies on the interaction of the differential proteins with ginsenosideThe differentially expressed proteins were verified by qRT-PCR and Western blotting.Autodock was used to map the possible docking locations of the interactions between the differential proteins and ginsenoside in male and female D.melanogaster.In the qRT-PCR experiment,the differentially expressed proteins’ genes of CG12895,Tim17 b,Dmel\CG12990,Dmel\CG6733,SLIRP1,GIP,s PLA2,Pebp1,Cyp6t3,spartin,smyd4-3 and Cyp6a18 in female D.melanogaster were consistent with the results of iTRAQ protein analysis.The differentially expressed proteins’ genes of CG9286,CG12374,CG10472,fk506-bp2,Akh,hgo,Su(P),ATPsyn Cf6,peng,Svil,CG31674,bocks,Lpin,BcDNA: RH44935,Reps,hppy,Chchd2,Dmel\CG6733 in male drosophila were consistent with the results of iTRAQ protein analysis.In the western blotting experiment,the results of female D.melanogaster showed that,the protein changes of Pebp1,spartin,Ent2,Tim17 b and TSG101 in the RG group were consistent with the protein changes in iTRAQ protein analysis compared with that of the control group.Results of Western blotting of male D.melanogaster showed that,the protein changes of hppy,Rheb,Ent2,Lpin and fk506-bp2 protein in the RG group were consistent with the changes of iTRAQ protein analysis compared with that of the control group.For the Autodock molecular docking software verification,Pebp1 showed a strong combination ability with ginsenoside of Re,Rf,Rb1,Rc,Rb2,Rb3 and Rd.Spartin showed a good combination ability with ginsenoside of Rb3 and Rc.Hppy showed a combination ability with ginsenoside of Rg1,Rf,Rb1,Rc,Rb2,Rb3 and Rd.Rheb showed a combination ability with ginsenoside of Rg1,Rc,Rb2 and Rb3.Lpin showed a combination ability with ginsenoside of Rg1,Rb1,Rc,Rb2,Rb3 and Rd.3.Mechanism studies on the anti-aging effects of RG on D.melanogasterIn order to explore the mechanism of the anti-aging effects of RG,western blotting technique was used to conform the effects of differentially proteins in the related pathways.It was found that the PI3K/AKT/FoxO pathway was possibly involved in prolonging the lifespan of female and male D.melanogaster fed with RG.In addition,ERK pathway was also involved in prolonging the lifespan of female D.melanogaster treated with RG.4E-BP was involved in prolonging the lifespan of male D.melanogaster treated with RG.In summary,the lifespan of female and male D.melanogaster could be prolonged fed with different concentrations of RG.Also,the protein changes were different between female and male D.melanogaster with the RG treatment.The mechanism studies showed that the PI3K/AKT/FoxO pathway,ERK pathway and 4E-BP were involved in regulating the anti-aging effects in D.melanogaster treated with RG.These results provided an experimental evidence for the development of RG as a promising anti-aging drug in clinic. |
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