Effects And Mechanism Of Traditional Chinese Medicine,Gengnianchun On Anti-aging

Background:Female aging is a complex physiological phenomenon,through menopause transition,ovarian functions decline,during which period women show vasomotor disorders and mental nerve symptoms,endure perimenopausal syndromes,such as hot flashes,sweating,insomnia,depression.In the postmenopausal women,the aging rate of the body accelerates,and the tissues and organs were degenerative.Female aging is often associated with age-related diseases such as hypertension,diabetes,Alzheimer's disease(AD),osteoporosis,and cataracts,which harm the health and life quality of women.As the global population lives longer,female aging and age-related diseases have become widespread medical problems.Many factors contribute to aging,especially free radical theory focus on that free radical accumulation damage cytomembrane,nucleic acid,protein and other enzymes,and subsequently disrupting cellular and organic functions and resulting in aging.In addition,free radicals are one of the important causes of ovarian aging.In recent years,research on anti-aging drugs has begun to receive more and more attention.It is more important to extend a healthy lifespan than to simply prolong life.Nowadays,it is believed that extending the lifespan,while improving the anti-stress ability,delaying the development of age-related diseases,is namely,extending a healthy lifespan.Extending healthy life is the key to achieving a healthy aging and improving the quality of life for the elderly.In anti-aging area of female,extending the life of women,delaying the menopausal transition and beginning of ageing,preventing and treating age-related diseases is the key to extend the healthy life of women.However,drugs that can effectively delay aging are still lacking.Traditional Chinese medicine(TCM)has profound theoretical foundation and practical experience in delaying aging,among which,"toning kidney" is a dominant way to delay aging.In the early 1990s,Jin Yu,a professor from Obstetrics and Gynecology of Fudan University,developed the traditional Chinese medicine,Gengnianchun(GNC).GNC has been used for nearly thirty years.According to clinical and experimental data,GNC has therapeutic effects on menopausal symptoms.Furthermore,animal experiments showed that GNC attenuated age-related diseases,such as skin aging,memory declining,osteoporosis,etc.However,it is not clear whether GNC has the effect of prolonging healthy life and its mechanism to improve women's perimenopause syndromes and delay senility,which need further investigation.Caenorhabditis elegans is an international animal model in aging research because of its short lifespan and clear genetic background,which is very suitable for the evaluation and mechanism research of anti-aging drugs.In this study,C.elegans was used as experimental animals,and the effect and mechanism of traditional Chinese medicine GNC,which was clinically significant in alleviating the symptoms of perimenopausal syndrome,were studied.Objective:In this study,we used C.elegans as experimental animals to investigate the effect of GNC on extending longevity,increasing oxidative stress resistance,protecting against A? toxicity and its related mechanisms.We aimed to evaluate the function and mechanism of GNC to delay healthy lifespan and provide theoretical support for the clinical value of traditional Chinese medicine GNC in anti-aging and improving the symptoms of menopause in women.Methods:1.Drug concentrations and animal models:Wild C.elegans nematodes N2 were treated with different gradient concentrations of GNC,in order to choose the appropriate concentrations of GNC.In this study,the following nematodes were selected:C.elegans Bristol N2(wild-type);CB1370(daf-2 gene mutation in insulin/IGF-1 signaling pathway);TJ1052(age-1 gene mutation in insulin/IGF-1 signaling pathway);CF1038(FOXO homologous gene--daf-16 gene mutation);DA465(eat-2 gene mutation,dietary restrictions model);RB1206(rsks-1 gene mutation in mTOR/S6K signaling pathway);CF1903(glp-1 gene mutation in reproductive signaling pathway);AD model(A? transgenic nematodes)CL4176,etc.2.The effect of GNC on lifespan and its mechanism:Lifespan assays,oxidative and heat stress resistance assays,chemotaxis assays,reproduction assays were performed in wild type C.elegans N2 with different treating method.Gene expression levels were analyzed using RNA-seq.Bioinformatics and real time PCR were used to analyze and verify gene differential expression.Lifespan assays were conducted with different mutant strains.3.The effect of GNC on anti-oxidative stress and its mechanism:Cr6+-induced oxidative stress resistance assays were performed.ROS levels were also tested.Stress-induced genes were analyzed using real time PCR method.Oxidative stress resistance assays were conducted with different mutant strains.4,The effect of GNC on A? toxicity and its mechanism in AD model nematodes:Temperature induced paralysis tests were conducted using AD model nematodes.The A? species in the transgenic C.elegans strains were identified by Western blotting.Intracellular ROS levels were measured using H2DCF-DA.qRT-PCR was used to test genes expression.To test whether daf-16 is required for the protective effect of GNC against A? toxicity,we performed the experiments by using RNAi knock down of daf-16 expression and examined the translocation of daf-16.Results:1.GNC experimental concentrations:The wild-type nematodes N2 were cultivated in liquid medium containing 0.00394 mg/mL~7.88 mg/mL GNC for 48 h,and the survival rate was 100%.2.The effect of GNC on lifespan:0.394 mg/mL~7.88 mg/mL doses of GNC significantly increased the mean lifespan of adult worms compared with the control and the effect was in a dose-dependent manner;3.94 mg/mL provided the maximum lifespan increase(31.3%,P<0.0001);Under H2O2-induced oxidative stress,GNC pretreatment provided significant protection,with 23.33%versus 9.33%(control)of the worms surviving(P=0.023);Under paraquat-induced oxidative stress,the survival rate of worms pretreated with GNC was 70.00%versus 28.89%(P=0.011);Under Cr6+ and acute thermal stress at 35?,the overall survival increased significantly with GNC pretreatment compared with the control(P<0.0001);In GNC-treated worms,the chemotaxis behaviour was significantly improved(P=0.0289);There was no significant difference in total progeny production between the GNC and control groups.3.The mechanism of GNC increasing lifespan:The RNA-seq results showed that GNC changed the 1444 genes expression,of which 1120 genes were up-regulated and 324 were down-regulated.Through bioinformatics analysis and literature review,we found ten genes--vang-1,R04A9.7,cyd-1,fat-7,skr-7,skr-8,skr-9,kgb-2,skr-5,and gsto-2 may be associated with the longevity effect of GNC.Real time PCR results showed that vang-1,R04A9.7,cyd-1,fat-7,skr-7 genes were down-regulated,while kgb-2,skr-5,gsto-2 genes were up-regulated,with statistical significance;These genes each showed the same tendency as them observed in the RNA-seq experiments.Compared with control,eat-2 mutant strain DA465 nematodes(model of dietary restriction),rsks-1(S6 kinase homologous gene)mutant strain RB1206 nematodes,glp-1 mutant strain CF1903 nematodes(reproductive signal)treated with GNC,the mutant nematodes mean life expectancy extended by 29.3%,19.2%and 16.4%,respectively,with statistical significance(P<0.01).However,GNC could not prolong the mean lifespan of daf-2,age-1 daf-16 mutant strains CB1370,TJ1052,CF1038 nernatodes in insulin/IGF-1 signaling(IIs)pathway(P>0.05),suggesting that GNC extends lifeapan of C.elegans mainly via IIs pathway.4.The effect and mechanism of GNC increasing anti-oxidative stress ability:GNC increased the mean lifespan of wild-type C.elegans N2 under Cr(Vl)-induced oxidative stress in a dose-dependent manner.GNC significantly reduced total ROS levels(P<0.001)compared with the vehicle control.GNC up-regulated the expression levels of sod-3,mtl-1 hsp-12.6 and hsp-16.2(P<0.01).Under oxidative stress conditions,compared with control,daf-2 mutant strain CB1370 nematodes,age-1 mutant strain TJ1052 nematodes treated with GNC,the mean survival time extended by 28.1%and 21.4%,respectively(P<0.0001);eat-2 mutant strain DA465 nematodes,rsks-1 mutant strain RB1206 nematodes,glp-1 mutant strain CF1903 nematodes treated with GNC,the mutant nematodes mean survival time extended by 31.5%,38.8%,24.2%(P<0.0001).However,GNC did not enhance stress resistance in daf-16 mutant strain CF1038,indicating that FOXO/DAF-16 function is essential for the observed GNC-mediated increase in oxidative stress resistance.5.The effect of GNC on A? toxicity and its mechanism in AD model nematodes:GNC delayed A?-paralysis in a dose-dependent manner.GNC resulted in a 49%decrease in the expression of the A? transgene(P=0.0044),and a significant 35%reduction in A? levels(P?O.0383).GNC reduced total ROS levels by 35.6%(P= 0.024)compared with the vehicle control.GNC up-regulated the expression levels of daf-16(1.8-fold),sod-3(2.3-fold)and hsp-16.2(2.7-fold)(P<0.01).GNC enhanced the translocation of daf-16 from the cytoplasm to nuclei in juglone-oxidative stress.When daf-16 gene of CL4176 was inhibited by dsRNA,the mRNA expression level was reduced to 38%;GNC could not delay the paralysis time of the worms,suggesting that FOXO/DAF-16 is required for the protective effects of GNC on A? toxicity.Conclusions:1.GNC extends the lifespan of C.elegans in a dose-dependent manner.GNC increases the chemotaxis ability and has no effect on the fecundity of C.elegans.2.GNC plays an anti-aging role via the insulin/IGF-1 signalling pathway,not through germline signalling pathway,DR or mTOR/S6K signalling pathway.3.GNC enhances oxidative stress resistance in C.elegans by modulating FOXO/DAF-16.4.In Alzheimer's Disease(AD)model nematodes,GNC delayed the paralysis and ameliorated P-amyloid peptide toxicity through FOXO/DAF-16.Above all,traditional Chinese medicine GNC which has curative effect on women's menopause syndrome could prolong the healthy lifespan,its antioxidant effect may be helpful to delay aging of ovary and other organs;GNC may be a potential anti-aging agent in women.