基于秀丽隐杆线虫的中药抗衰老成分研究进展

刘 静，石京山

(遵义医科大学 基础药理教育部重点实验室暨特色民族药国际合作联合实验室，贵州 遵义 563099)

1 Introduction

Aging is characterized by progressive,degenerative changes in many organ systems.Age-related degenerative diseases are major contributor to human disability and death,seriously affect the quality of life of the rapidly growing elderly population,therefore,age-related degeneration treatments are more urgent than ever before[1].Aging is inevitable for all living creatures,but advances in science and technology can delay aging.Model organisms currently used for anti-aging research includeSaccharomycescerevisiae,Caenorhabditiselegans(C.elegans),fruit flies,zebrafish,Senescence-accelerated mouse prone 8 (SAMP8) and rhesus monkey[2-7].In the last few years,nematodes have proven to be the most commonly used model organism in anti-aging studies[8].Specifically,the terrestrial nematodeC.eleganshas become an outstanding model organism for studying aging.C.eleganshas been widely used to study the mechanisms of aging processes and aging-related diseases such as Alzheimer’s disease,Parkinson’s disease,and Huntington’s disease[9-11].In this review,we provide an introduction toC.elegansand its applications for anti-aging studies of traditional Chinese medicine,with the aim of encouraging scientists to adoptC.elegansas an experimental model for screening on various traditional Chinese medicine ingredients.

1.1CaenorhabditiselegansC.elegans,a kind of multicellular and lower eukaryotic systems,lives in the soil and feeds on bacteria.The body ofC.elegansis transparent,and the adult worm is about 1 mm in length.They are divided into hermaphrodites and males.In the natural environment,the majority are hermaphrodites,male nematodes only account for 0.05%[12].C.elegansincludes the embryonic phase,four (L1～L4) larval stages,and the adult stage.They take approximately 6 days from egg to adult at 15 ℃,only take 3～4 days at 20 ℃,and less than 3 days at 25 ℃.What’s more,20 ℃,at which temperature the nematodes have the fastest reproduction speed.The offspring number ofC.elegansare 200-350,the life cycle are about 2～3 weeks,and they are extremely sensitive to the environment[13-14].In the 1960s,the molecular geneticist Sydney Brenner applied firstly theC.elegansin the developmental biology and neurobiology field,which laid the groundwork ofC.elegansas a model organism for the anti-aging study[15].ThenC.eleganshas become the excellent model organism for studying aging because of several advantages.Firstly,The lifetime ofC.elegansare shorter than other animals such as SAMP8 mice and zebrafish.Their offspring can breed faster and culture easily under laboratory conditions,and they can be observed under the stereomicroscope[16-17].As an in-vivo system,nematodes exist the advantage of identifying complex phenotypic compounds in drug screening[18-19].They can be stored and recovered at -80 ℃ as well as cells,an advantage that model organisms such as fruit fly and mice do not possess.Secondly,C.elegansare the first multicellular eukaryotes to complete genome-wide sequencing,the genetic backgrounds are clear,and homologous to human genes are 60%～80%[20-21].Above all,the cycle of aging aboutC.eleganshave been greatly shorten,and that will facilitate anti-aging research.

1.2 Traditional Chinese medicine The resources of traditional Chinese medicine are vast,and some of them play a role in anti-aging research.One of the main goals of anti-aging research is to identify Chinese medicine ingredients,which can prolong life,improve age-related diseases and human health.Based on recent progress of anti-aging experimental studies,we roughly classify the anti-aging components of traditional Chinese medicines divided into polysaccharides,flavonoids,phenols and so on.

2 Anti-aging ingredients in traditional Chinese medicine

2.1 Polysaccharides Plant polysaccharides extracted from Chinese herbs are the most common ingredient in existing resources and drug in China.Studies have found that many types of polysaccharides could scavenge free radicals,increase antioxidant enzyme activity,inhibit lipid peroxidation,play a role both in protecting cell membrane and delaying aging[22-23].Polysaccharides fromDendrobiumofficinaleKimura et Migo,DendrobiumchrysotoxumLindl andDendrobiummoniliforme(L.)Sw.could increase the average lifespan by nearly 20%～35% inC.elegans.Meanwhile,the above three polysaccharide components could improve the ability of nematodes to resist acute heat stress,and enhance nematode locomotor activity[24].In addition,a study reported that polysaccharides extracted fromBletillastriatacould also increase the locomotion behavior,improve the ability of resisting heat stress,and extend nematodes lifetime[25].Compared with the negative control group,4 g/L polysaccharides fromSophoramoorcroftianaseeds could increase lifespan to 27.3 days (15.9 days in the control group) inC.elegans,extend the average lifespan of worms under oxidative stress and thermal stress.Enhance the offspring number ofC.elegansand extend spawning cycle to the eighth.However,it could not obviously influence the motility ofC.elegansincluding head wiggle frequency,body bending frequency and swallowing frequency[26].CoptischinensisFranch polysaccharide (CCP) at the dose of 100 mg/L had the effect on extending the lifetime and delaying the paralysis rate ofC.elegans.Thioflavine S staining showed that the deposition of Aβ protein in the brain of CL4176 nematodes was significantly reduced by treating with 100 mg/L CCP,and real-time fluorescence quantitative PCR showed the transcription levels of HSP16.2 (Y46H3A.D) and HSP16.41 (Y46H3A.E) were higher than the control group[27].What’s more, Reishi polysaccharide were able to show a life-prolonging effect ofC.elegansvia activating TIR-1 receptor and mitogen-activated protein kinase (MAPK) signaling pathway,and promoting the expression ofdaf-16[28].Astragaluspolysaccharides(astragalan) at the dose of 1 g/L and 2.5 g/L could prolong the lifespan of wild-type nematodes,daf-2 andage-1 mutant nematodes[29].In summary,polysaccharides extracted from traditional Chinese medicines will be a huge resource to search anti-aging drugs based on these studies ofC.elegans.

2.2 Flavonoids Flavonoids are a class of polyphenolic compounds that exist widely on traditional Chinese medicines,natural medicines,fruits,and vegetables[30-31].They exhibit broad pharmacological activities includes antioxidant,anti-tumor,anti-arrhythmia and anti-hypertension[32].In addition,flavonoids have been widely reported that they could improve the health and longevity ofC.elegans.Studies have shown that 2.5 g/L total flavonoids of Boju,quercetin,and kaempferol were able to increase lifespan in the model organismC.elegans[33-34].50 mg/LEpimediumflavonoids could prolong the average lifespan and maximum lifespan ofC.elegans,delay nematodes entered the stage of death,reduce their age-dependent mortality and does not influence the reproductive ability of nematodes[35].In addition,45 μmol/L icariin was the best longevity for wild typeC.elegans,and the average lifespan were extended 20.67% at 25 ℃.20 μmol/L icariside II could prolong the maximum lifespan ofC.elegans,and the mechanism of prolonging lifespan was related to Insulin/insulin-like signaling pathway (IIS pathway).Icariin and icariside II were capable of extending the average lifespan ofC.elegansunder heat stress(35 ℃) and oxidative stress(10 mmol/L or 15 mmol/L H2O2).Simultaneously,drug administration group could enhance muscle function ofC.elegansand alleviate Aβ toxicity[36].Not only the wild-type nematodes，but also some mutant nematodes were used to anti-aging researches.For example,three mutant strainsklo-1(ok2925),klo-2(ok1862),andklo-2(ok1830) were treated with 200 μmol/L quercetin-3-O-glucoside(Q3Glc) could significantly increase lifespan of nematodes,included median lifespan and maximum lifespan[37].The mechanisms of life extension were related to the regulation of signaling pathway or antioxidant genes such as quercetin extended lifespan of nematodes related to the regulation ofage-1,daf-2,unc-43,andsek-1[38].50 mg/LHerbaScutellariaeBarbataeflavonoids (HF) increased activities of superoxide dismutases (SOD) and catalases (CAT) enzymes by 15.7% and 39.3%,respectively.30 mg/L HF could up-regulate the expression of antioxidant genesdaf-16 andsir-2.1[39].

2.3 Phenols Phenol,a wide range of compounds,was found in plant species and was able to antioxidant activities[40].Studies have shown that phenols could significantly increase the survival rate of nematodes induced by juglone and reduce intracellular reactive oxygen species (ROS) concentration inC.elegans[41].In recent years,many studies have confirmed that the phenols in traditional Chinese medicines had an effect on anti-aging and could prolong the life time ofC.elegans.Incubation with the best concentration of the polydatin (1 mmol/L) increased the survival by 30.7%,and it also slowed down the life expectancy of nematodes caused by high sugar and oxidative stress.The polydatin administration group could extend nematodes lifetime by 62.1% under the 500 μmol/L Cu2+stress[42-43].Resveratrol (RSV) is a class of polyphenolic compounds.100 μmol/L RSV,act as an antioxidant,could shift the survival curve of wild-typeC.elegansrightward and significantly extend its lifespan.Used the mutant nematodes in the relevant life tests,the results showed that RSV shortened the lifespan of mutantsclk-1 andisp-1 with altering mitochondrial function,and extended life of the five SODs deletion mutant nematodes.However,the longevity of RSV was independent from insulin/insulin-like signaling pathway and mitochondria[44-45].The optimum concentration 25 mg/L of mulberry leaf polyphenol (MLP) extended nematodes life cycles.Lifespan of wild typeC.eleganswere prolonged by 25% at 20 ℃ and prolonged by 22.46% at 25 ℃ after administration of MLP.MLP could improve the anti-oxidative ability,usedmev-1 (kn1) mutant nematodes to verify its anti-oxidation effect,which further proved that MLP could significantly improve the survival rate of the mutant nematodes.MLP prolonged the lifespan of nematodes accompanied with reducing egg production and the lipid content of nematodes.25 mg/L MLP has no effect on extending lifespan indaf-12,daf-16,pha-4,andnhr-80 mutant nematodes,which upregulateddaf-12 (fard-1),daf-16 (lipil-4,sod- 3),pha-4 (unc-51),nhr-80 (fat-6) genes expression.In summary,above all indicated that MLP could extend the lifespan ofC.elegansand adjust lipid metabolism related to germline signaling pathway[46].

2.4 The other Chinese medicine ingredients 25 mg/L,37.5 mg/L,50 mg/L ethyl acetate extract fromPolygonummultiflora(EPM) could shift the life curve of nematodes to the right,which revealed that EPM could prolong the life of nematodes.EPM at dose of 37.5 mg/L was the best concentration,it could extend the average lifespan by 34.3% compared with the blank group[47].The unsupervised mode principal component analysis of H-NMR spectra showed that AS-IV could improve the aging of nematodes[48].1 mg/mlPhyllanthusemblicaL.extracts prolonged normal life,the life time under the condition of oxidative stress and heat stress,natural life among them was the first choice of anti-aging research.The extracts had no effect on the mobility and the muscle contraction function of the nematodes while the lifespan extension of theC.elegans[49].The supplementation with three different extract concentrations ( 0.05 g/L,0.1 g/L,0.5 g/L) of sea buckthorn seed extracts (SBSE) results in more prolonged average life span,longest lifespan and median longevity extensions inCaenorhabditiselegans. This increase in life span caused by SBSE were significant under 35℃ and the strong oxidative environment constructed by Juglans mandshurica,that is the ability of resisting thermal or oxidative stress were increased.The lipofuscin level ofC.eleganswere obvious reduction after SBSE treatment.Activity of SOD,CAT,and glutathione peroxidases(GSH-Px) inC.eleganswere increased clearly,and the expression levels ofage-1,daf-2,andsod-3 were decreased.Thesir-2.1 gene expression of the insulin/insulin-like signaling pathway to regulate the expression levels of nematode senescence-related genes,reduce lipofuscin accumulation,increase anti-oxidation and anti-heat stress ability,thus delayed the aging of nematodes[13].The following summarizes the anti-aging ingredients of traditional Chinese medicine based onC.elegansin table 1.

表1 Summary of anti-aging traditional Chinese medicine compounds inC.elegans

CategoryDrugOptimum concentrationExtensionPotential mechanism ReferencePolysaccharidesDendrobium officinale Kimura et Migo80 mg/L30.54%Increases heat stress resistance [24]Dendrobiumchrysotoxum Lindl80 mg/L21.3%Increases heat stress resistance [24]Dendrobiummoniliforme(L.)Sw.80 mg/L33.21%Increases heat stress resistance [24]Reishi polysaccharide fraction 3 100 mg/L20%～30%DAF-16 dependent [28]Panax notoginseng polysaccharides0.1 g/L5.2%～21%Antioxidant effects[50]Polysaccharides from Sophora- moorcroftiana seeds4 g/L71.70%Increases stress resistance [26]Coptis chinensis Franch polysaccharide100 mg/L--Inhibits Aβ deposition and reduces Aβ-in-duced toxicity[27]Astragalanpolysaccharide 1.0,2.5 g/L30%DAF-16/FOXO dependent [29]Bletilla striata polysaccharides50 mg/L10%～20%Insulin/IGF-1 signaling pathway [25]FlavonoidsEpimedium flavonoids50 mg/L--Increases heat stress resistance [35]Icariin45 μmol/L20.67%Insulin/IGF-1 signaling pathway [36]Icariside II20 μmol/L～20%Insulin/IGF-1 signaling pathway [36]Kaempferol100 μmol/L5%～6%Independent daf-16[34]Flavonoids of Boju2.5 g/L--Antioxidant effects[33]Herba Scutellariae Barbatae flavonoids50 mg/L--Increases stress resistance Antioxidant effects [39]Quercetin-3-o-glucoside25 μmol/L23%DAF-16/FOXO dependent [37]Quercetin200 μmol/L10%Antioxidant effects[37][54]Naringin5 μmol/L7.7%～15.4%AMPK signaling pathway [52]Luteolin100 μmol/L6.3%～6.7%AMPK signaling pathway [52]Chrysin10 μmol/L6.7%～30.7%AMPK signaling pathway [52] PhenolsMulberry leaf polyphenols 25 mg/L25%(20℃)22.46%(25℃)Germline signaling pathway[46]Resveratrol100 μmol/L30.08%Independent insulin/IGF-1 signaling pathway[44]Polydatin1 mmol/L30.7%insulin/IGF-1 signaling pathway[43]Echinacoside300 μmol/L15.82%DAF-16 dependent[53]The other ingredientsLignans from Arctium lappa100 μmol/L25%DAF16-mediated signaling pathway[51]Ethyl acetate extract from Folygonal multiflora37.5 mg/L34.3%Increases stress resistance [47]Sea buckthorn seed extracts0.05 g/L13.7%Antioxidant effects insulin/IGF-1 signaling pathway[13]

3 The prospect of anti-aging composition of traditional Chinese medicine based on C.elegans.

Many traditional Chinese medicines are able to anti-aging.At present,anti-aging research of Chinese medicine based onC.elegansare pay close attention to polysaccharides,flavonoids,polyphenols,etc.However,the other traditional Chinese medicines still exist great anti-aging potential such as acids,alkali and so on.Researchers can take advantage ofC.elegansto fully develop the anti-aging effect of Chinese herbal ingredients.Some studies have proven that the nematodeC.eleganscould be used for in-depth mechanism studies andC.elegansresearches are gradually maturing with transgenic nematodes and mutant nematodes are used to further anti-aging mechanism study.Based on a series of advantages and experiences of nematode research,the beneficial combination ofC.elegansand traditional Chinese medicine anti-aging ingredients will bring a great gospel to human anti-aging researches.However,C.elegansare far from the human genetic background,related anti-aging researches need to be further validated on other animal models.