| Aging is an unavoidable"issue"in human destiny,which leads to functional regression,systemic disorders,and the occurrence of various aging-related diseases that seriously affect the quality of human health and lifespan.People are constantly searching for ways to slow down aging,and exploring the potential of human lifespan.Natural medicines have shown great potential in anti-aging and lifespan-extension,and taking medicine may be a relatively efficient and effective way of anti-aging and lifespan extension at present.A number of drugs with anti-aging and longevity effects have been identified,such as rapamycin,resveratrol,metformin,urolithin A,quercetin,etc.However,the targets,pharmacological mechanisms and side effects of many of these drugs are still unclear,and their safety limits the application prospects of the drugs.Therefore,there is an urgent need to develop safer and more effective new anti-aging drugs,and the exploration of their pharmacological mechanisms,which are particularly important for anti-aging research and future application to extend the healthy lifespan of human beings.The Chinese Pharmacopoeia not only contains evaluations of various aspects of herbs,such as species,source,and quality,but also records clinical dosages and therapeutic effects,making it an ideal data repository for mining and screening anti-aging drugs.The Pharmacopoeia records a variety of herbs with life-prolonging effects,such as ginseng,astragalus and rhodiola rosea.Among them,Rhodiola rosea is often used for the treatment of cardiovascular and neurological weaknesses,and studies have shown that these effects are mainly due to the rich content of various glycosides,flavonoids and coumarins in Rhodiola rosea.Salidroside is one of the main active ingredients in rhodiola rosea,and research has found that this substance has similar efficacy with rhodiola rosea,such as strong antioxidant properties,prevention of nerve damage,memory enhancement,enhancement of the body’s motor ability,and alleviation of the progression of aging-related diseases and so on.However,the effects of salidroside on lifespan,the identification of target proteins of salidroside,and their molecular mechanisms of lifespan regulatory of salidroside have rarely been reported.This severely limits the future clinical application of salidroside and needs to be addressed urgently.Therefore,in this study,we investigated the efficacy of salidroside on individual longevity and aging using silkworms,nematodes,and mice as experimental animal models,and identified the aging-related target proteins of salidroside to elucidate its molecular mechanisms in prolonging longevity and resisting aging.The following results were obtained:1.The effects of salidroside on lifespan and oxidation levelThe effects of salidroside on lifespan were explored by two administration methods:feeding and injecting in vitro.It was found that salidroside could significantly enhance the lifespan and maximum lifespan of male silkworms,but had no significant effect on female silkworms.By analyzing the duration of the three developmental stages（larval,pupal,and adult）of the silkworm,we found that the lifespan extension effect was mainly contributed to the adult stage of the silkworm.This suggests that salidroside are able to extend the lifespan of the adult stage of the silkworm without affecting its growth and development period,which is more meaningful for anti-aging drugs.Meanwhile,through the statistics of body weight,pupal weight and cocoon weight of individual silkworms,it was found that salidroside at effective lifespan-extending concentration did not have a negative effect on the above three physiological indexes of the silkworms,which suggests that salidroside may be able to prolong the lifespan of an individual without sacrificing the normal physiological energy.Meanwhile,the results of the study on nematodes,the classical animal model for longevity studies,replicated the effect of lifespan-extension on silkworms,suggesting that the efficacy of salidroside on longevity is conserved across species.According to previous studies,oxidative stress in individuals or cells can affect the homeostasis of the intrinsic systems,which is crucial for the onset and development of aging.Moreover,recent studies on salidroside have demonstrated their strong antioxidant capacity.Therefore,in order to investigate the lifespan-extending process of salidroside,whether it changes the level of oxidation and senescence on cells and individuals.Using hydrogen peroxide as a cellular senescence inducer,an oxidative senescence cell model was successfully constructed,and several concentration gradients of salidroside were added to cells under this stressful environment,it was found that salidroside was able to significantly reduce the level of reactive oxygen species produced by oxidative damage compared to the oxidative senescence damage group.Meanwhile,results in vitro demonstrated that the total antioxidant capacity of silkworm moth individuals was significantly increased on day 1 and 5 after injection of salidroside.These results of reduced individual oxidation levels were also observed in nematode studies.It has been shown that high temperatures cause oxidative stress in the body,so silkworm larvae at day 3 of 5th instar were placed at 37℃enviroment to observe the effects of salidroside on adversity ability of silkworms,and the results showed that salidroside significantly prolonged the survival of silkworms exposed to high temperatures compared to the control group.The same effect of salidroside was confirmed in nematode experiments.In a previous study,the expression levels of p53 and p21 genes were found to increase gradually with the onset of aging,and these two genes are closely related to aging.Hydrogen peroxide-induced senescence model treated with salidroside,RT-q PCR results showed that salidroside significantly slowed down the oxidation-induced increase in the expression levels of Bmp53 and Bmp21 in the senescence model.After that,we used salidroside to treat naturally aging mice,and found that salidroside was able to significantly improve the aging-induced loss of locomotor activity.The results of blood routine of mouse showed that the number of various leukocytes in the salidroside-treated mice tended to be rejuvenated,and the expression level of the aging related gene,p21WAF1,in the liver,kidney,and brain of the mice was significantly down-regulated.The above results indicated that salidroside significantly reduced the oxidation and aging levels of cells and individuals during the process of lifespan extension.2.Identification of target proteins of salidrosideWe found that salidroside extend individual lifespan and attenuate the level of oxidative senescence,but the specific molecular mechanisms of lifespan-extension of salidroside and its potential target molecules for lifespan extension are still unknown.Therefore,we explored the potential direct binding target proteins of salidroside using various molecular interaction assays such as drug affinity targeting method（DARTS）.By analyzing the mass spectrometry results of the differential bands in the DARTS experiment,we hypothesized that HSP90 protein（Heat Shock Protein 90）might be a potential target protein of salidroside.Afterwards,we verified that salidroside could directly bind to silkworm and human HSP90 proteins or exogenous overexpression of silkworm HSP90 protein by microscale thermophoresis（MST）and DARTS combined with Western blot,suggesting that the HSP90 protein is the target protein for direct binding by SA.In order to explore the binding pattern between the drug and the protein,the most probable binding sites of salidroside on the protein,predicted by molecular docking（Autodock Vina）,included two sites:（1）the N-terminal ATP binding site（-7.9 kcal/mol）,defined as Site 1,which was mainly in the protein’s ASN46,ASP88,and SER108,PHE129 and GLY132;（2）the ligand-binding site（-7.0 kcal/mol）,defined as Site 2,is the binding site of HSP90 protein to the ligand protein,which is mainly in the vicinity of the protein’s amino acid residues such as ASN303,LYS341 and ASN344.Because salidroside has a higher binding fraction to Site 1,the next step centered on the exploration of the interaction pattern between salidroside and Site 1.The four amino acid residues in Site 1that are critical in the binding of salidroside were mutated to alanine at the same time,and the vectors with single mutations of the four amino acids were also constructed separately,and which were overexpressed in the embryo cell of silkworm.The results of the DARTS experiments showed that the binding capacity of salidroside to the mutant HSP90 proteins was greatly weakened.This result suggests that these four amino acids are important for drug-protein interaction.3.The effect of salidroside mediated inhibition of HSP90 on lifespan and oxidative levelsHSP90,as a classical molecular chaperone,has important biological significance in the organism,and its N-terminal ATPase structural domain plays an important role in the conformation of HSP90 and its binding to downstream client proteins.Molecular docking simulations revealed that salidroside shared the ATP binding site on HSP90 and the amino acid residues on the site with ATP.This result suggesting that salidroside may affects ATPase activity at this site.Later,the addition of salidroside or geldanamycin,a known N-terminal inhibitor of HSP90,to the reaction between ATP and HSP90 protein had an inhibitory effect on the ATPase activity of HSP90 protein.The above results indicate that salidroside inhibit the ATPase activity of HSP90 by binding to the N-terminal ATP-binding domain of HSP90.Meanwhile,in the oxidation-induced senescence model,the addition of salidroside significantly up-regulated the expression levels of the downstream genes of HSP90─Bmhsp70 and Bmhsf-d,and such a trend of change was consistent with that produced by the inhibition of HSP90.Immunoprecipitation revealed that salidroside interfered with the binding between HSP90 protein and one of its client proteins,SMYD3protein.All of these results suggest that salidroside are able to bind HSP90 protein and inhibit its activity,which in turn has an effect on the binding of the client proteins and downstream regulation.By investigating the expression trend of Bmhsp90 in both male and female silkworm moths at the adult stage,it was found that the expression trend of Bmhsp90 at the adult stage was positively correlated with the onset of aging,which was also observed in nematodes.The expression trends of Bmp53 and Bmp21 genes,which are known to be associated with aging,were also investigated at the corresponding time points,and the results showed that the expression patterns of these three genes showed a similar trend in both sexes,which further confirmed the strong correlation between HSP90 and aging.Interfering with the expression of Bmhsp90 in male silkworm moths showed a significant extension of the Bmhsp90-RNAi group compared with the control,which was similar to the extension of adult lifespan in male moths injected with salidroside,and a weak interference with daf-21（a homologous gene of HSP90 in nematodes）in nematodes extended nematode lifespan as well.Weak interference with daf-21 in oxidation-related fluorescent nematode lines revealed that the fluorescence intensity,i.e.,oxidation level,was significantly reduced in the interference group compared to the control,which is similar to the antioxidant effect of salidroside in nematodes.Afterwards,in order to investigate whether SA mediates the effect of HSP90 on lifespan and oxidation.We continued to add salidroside while interfering with Bmhsp90 expression,and the lifespan statistics showed that lifespan was not further prolonged after the addition of salidroside,and at the same time,we found that there was no significant difference in the oxidation level within the salidroside group compared with control.Therefore,I believe that salidroside largely exerts antioxidant and lifespan-extending effects by relying on the inhibition of HSP90.In previous reports SMYD3 protein needs the binding and assistance of HSP90 protein to effectively enhance its methylation function,while the inhibition of SMYD3 protein can effectively delay cellular aging.After overexpression of SMYD3 in cells,it was found that the intracellular reactive oxygen species content was significantly elevated compared to the control group.The known inhibitors of SMYD3,BCI-121,and salidroside were added respectively,and they were effective in decreasing the rise of reactive oxygen species level caused by SMYD3 overexpression.This result suggests that SMYD3 has an important role in oxidation levels and lifespan,and that salidroside can effectively reduce oxidation levels in individuals and cells by interfering with the interactions between HSP90-SMYD3.In summary,we have demonstrated that salidroside have lifespan-extension effect in two species and reduce the levels of aging and oxidation in cell and individual level.At the same time,we have discovered a new target protein of salidroside,HSP90 protein,and we have a reason to propose the following molecular mechanism of lifespan-extension of salidroside:salidroside inhibits the functional activity of HSP90 and interferes with the interaction of HSP90 and its client protein,SMYD3,by directly binding to the N-terminal ATP-binding site of HSP90,which ultimately decrase the level of oxidative stress and aging,and extends the lifespan of the individual finally.This study not only provides theoretical reference for salidroside application in delaying aging and treating aging-related diseases,but also provides a new practical case for silkworm as an experimental animal for drug efficacy evaluation. |
PDF Full Text Request