Application of Caenorhabditis elegans in screening active ingredients of traditional Chinese medicine

Ling Zhao,Mengyue Zhang,Yingying Wu,Rutian Jin,Lingxuan Li,Ying Long,Xin Wu

School of life science and biopharmaceutics,Shenyang Pharmaceutical University,Shenyang 110016,China

Abstract People continue to recognize the important role of Chinese medicine in the treatment of diseases,and there is an urgent need to clarify the mechanism of Chinese medicine.Because of the multi-target and multi-component characteristics of traditional Chinese medicine,it is difficult to elaborate from a single perspective.As a high-quality animal model,Caenorhabditis elegans with the characteristics of small size and fast reproduction,has been widely used in traditional Chinese medicine activity experiments.In this article,Caenorhabditis elegans,as model organisms used to screen the active components of traditional Chinese medicine were reviewed.

Keywords: active ingredients in traditional Chinese medicine;Caenorhabditis elegans;model organisms

1 Introduction

As a precious treasure of nature,Chinese medicine has shown infinite vitality after thousands of years of development.In recent years,with the continuous implementation of Healthy China Strategy,people have further recognized the important role of traditional Chinese medicine,and the modernization process of traditional Chinese medicine has been continuously promoted.Compared with synthetic drugs,Chinese medicine has attracted more attention in the process of new drug development because of its diverse active ingredients and less adverse reactions.After many times of extraction and separation the screening method is developed,and the active monomer components of traditional Chinese medicine are obtained.With the wide application of computer technology,such as molecular docking and highthroughput screening,the characteristics of the multi-component,multi-target,and synergistic effect of traditional Chinese medicine,are gradually applied to identify specific disease targets,and achieve comprehensive and objective screening [1].

The evaluation of the efficacy activity,mechanism elucidation,and toxicity evaluation of the active ingredients of traditional Chinese medicine obtained after preliminary screening is important for the further experiment.Caenorhabditiselegans,a model organism for organ development and apoptosis mechanism research,has gradually been developed and applied by researchers,and now nematodes have become the classic model animals in basic research [2].

Caenorhabditis elegansis a non-parasitic nematode that feeds on Escherichia coli (E.coli)in the laboratory.The adult is transparent,about 1 mm long,and has a reproductive cycle of three d and,a lifespan about two to three week,The basic anatomical structure includes the mouth,pharynx,intestine,gonads,and collagen stratum corneum [3],Nematodes are simple in anatomy,with behavioral characteristics such as movement,foraging,egg laying,defecation,and sensitive sensory organs.It has a very unique gender system: male or hermaphroditic [4].Hermaphrodites have both sperm and ovaries,which makes self reproduction possible,Each adult can produce about 300 eggs.Starting from one cell of the fertilized egg and developing through cell division and differentiation,nematodes have rich behavioral properties and can sense temperature,smell and taste.Caenorhabditis eleganshas a small genome of about 100 million base pairs,including 6 chromosomes and 1 mitochondrial genome [3].It is the first multicellular organism in the world to complete the whole gene sequencing.Its genes are highly homologous to human genes,and highly conserved.With the continuous development of genetic modification technology and the use of different technical means such as gene editing and another nematode genetic modification,the construction of a series of different genotypes of nematode strains,is considered a good animal model.

Compared with other animal models,Caenorhabditis eleganshas been widely used in the screening of compounds in the treatment of neurological diseases,apoptosis,body aging,intestinal flora,pathological toxicology,drug screening,and other aspects due to its stable genetic properties and low test costs.

2 Application of Caenorhabditis elegans in different disease models

2.1 Application in anti-aging models

Aging is the process of gradual accumulation of various molecular and cellular damage in the body over time,and it is a normal and irreversible physiological phenomenon of the human body.With the aging of the body,various functions decline,the ability to resist external environmental damage and restore homeostasis decreases,increasing disease and accelerating death [5].By identifying physiological indicators of nematodes,such as lifespan,reproductive ability,exercise ability,stress experiments,body swing frequency,pharyngeal pump frequency,and antioxidant damage ability,the active ingredients of anti-aging Chinese medicine can be screened and evaluated for activity.

There is evidence that chlorogenic acid can regulate glycolipid metabolism in animal models,and N2 wild-typeCaenorhabditis eleganshas a long life after chlorogenic acid treatment [6].Given the extract of Artemisia annua leaves,the body bending and pharynx blood pumping rate ofCaenorhabditis elegansincreased,the accumulation of intracellular reactive oxygen species (ROS) decreased,and the healthy life ofCaenorhabditis eleganswas prolonged.It was found that caffeic acid in the extract played a major role.In addition,ASTE further studies showed that ASTE induced the activity of transcription factor DAF-16/FOXO by activating the insulin/insulin-like growth factor signaling pathway [7].Dendrobium officinale polysaccharide extracted from Dendrobium officinale was given to N2 wild-type Caenorhabditis nematode,and the results showed that Dendrobium officinale leaf polysaccharides can effectively scavenge free radicals,improve the body’s antioxidant stress ability,and improve the average life and maximum life of nematodes [8].Li et al.feed N2 wild-typeCaenorhabditis eleganswith different concentrations of red deer antler aqueous extracts and found that 1 mg/mL of red deer antler water extract could significantly extend the life span of wildtype N2Caenorhabditis elegans(P＜0.05),which could activate the mitochondrial signaling pathway,upregulate the expression of relevant antioxidant genes,reduce the level of reactive oxygen radicals in nematodes to enhance their antioxidant stress capacity,reduce oxidative damage,and delay the aging rate [9].Li et al.identified the phytochemical composition of Hedyotis diffusa Willd [10].They found that after feedingCaenorhabditis elegans,it extended the life of nematodes by stimulating growth and development,reducing the deposition of pigments in the elderly,increasing the activity of superoxide dismutase and glutathione peroxidase dismutase,and reducing reactive oxygen species levels without impairing fertility.Fig.1 and Table 1 showed a brief introduction about the retrieved anti-aging Chinese medicine ingredients and the mechanisms involved.

Table 1 Application of Caenorhabditis elegans in anti-aging models

Fig.1 Introduction to the anti-aging mechanism [15]

2.2 Application in neurological disease models

Neurodegenerative lesions refer to cognitive impairment due to loss of neuronal structure and function,including neuronal death and glial cell balance [16].Alzheimer’s disease(AD),Parkinson’s disease,and Huntington’s disease are the main types of neurodegenerative diseases.However,currently,there is no effective treatment.

2.2.1 Application in the Alzheimer’s model

The two main neuropathological changes in the body of AD patients are age spots and nerve fiber tangles [17].Aβ1-42,one of the 26 forms of Aβprotein produced by amyloid precursor protein throughγ-secretase andβ-secretase-mediated lysis of peptide--amyloid in the cerebrospinal fluid,is considered to be the main component of age spots because of greater hydrophobicity,and Tau protein leads to nerve fiber entanglement due to a variety of factors such as phosphorylation,oxidative stress,neuroinflammation,and other effects.An important cause of AD is neurological disorders caused by abnormal acetylcholinergic neurotransmitters due to the loss of basal forebrain cholinergic neurons.Researchers transferred the expression of human Aβ1-42 into the muscle cells of nematodes to nematodes,and the Aβ1-42 expressed in the nematodes accumulated,forming deposition plaques,damaged cells,and finally causing nematodes to appear paralyzing phenotypic AD-like pathological features.Zhou et al.applied Caestrophyllum Elegans CL4176 based on the starch cascade hypothesis to find that the six-flavored Rehmannia extract could significantly delay the paralyzing phenotype caused by the overexpression and accumulation of Aβ1-42 in nematodes to form deposition spots [18].Further research has shown that Dihuang extract can prolong the life after oxidative damage,reduce the level of reactive oxygen species,and alleviate Aβtoxicity.Natural products isolated from garlic and synthetic derivatives were found to delay the paralysis of Caenorhabditis elegans CL4176 and prolong life of nematodes.Further studies have shown that the compound relieves paralytic manifestations by inhibiting the expression of genes that regulate inflammation and stress,and combats the Aβtoxicity of Caenorhabditis elegans [19].Cui et al.isolated a natural product from salvia Sinensis,and found that it alleviated AD-like features associated with Aβ1-42 toxicity in muscle cells as well as neuronal cells.Further studies showed that CTS alleviated oxidative stress,reduced levels of abnormally aggregated proteins,reduced reactive oxygen species levels in nematodes,and enhanced superoxide dismutase activity [20].Hyacinthosis [21] and Deer antler extract [22] exhibited an anti-toxic neuroprotective effect on Aβ-induced transgenic ADCaenorhabditis elegansby reducing the accumulation of toxic Aβand enhancing the ability of nematodes to resist oxidative stress,which could delay the paralysis phenotypic paralysis rate of nematodes and prolong the life of nematodes,and might become an effective treatment method to alleviate Alzheimer’s symptoms.

2.2.2 Application in Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disorder.At present,it is considered a chronic disease of dysregulation of dopamine and acetylcholine imbalance caused by impaired dopamine nerve function in the striatal system of the substantia nigra.The main pathological feature is the accumulation of synuclein (α-syn) in the body ofα[23].Parkinson’s syndrome model construction was induced by nematodes overexpressingαsynuclein in different neurons or by treatment with the neurotoxic drug MPTP (1-methyl phenyl-1,2,3,6-tetrahydropyridine).

Piperine component 7-ethoxy-4-methyl coumarin.is an agonist of the Dopamine D2 receptor,and the accumulation ofα-synuclein inCaenorhabditis elegansNL5901 overexpressedα-syn gene was significantly improved by feeding 7-ethoxy-4-methyl coumarin [24] the constituent and the underlying mechanism remain unclear.This study aimed to investigate the effects of 7-ethoxy-4-methylcoumarin EMC.The flavonoid boxy line extracted from passionflower reduced the aggregation toxicity ofCaenorhabditis elegansOW13α-nucleoprotein,enhanced the activity of ubiquitin-like proteasome and superoxide dismutase in transgenic Caenorhabditis nematode models,and reduced reactive oxygen species levels [25].It also reduces BZ555 Caenorhabditis nematode dopamine neuronal degeneration and extends the lifespan of nematodes.Ferulic acid significantly inhibits the accumulation of NL5901 wormsα-nucleoprotein and improves movement disorders [26].At the same time,it reduces the degradation of dopaminergic (DA) neurons inCaenorhabditis elegansBZ555,improves food perception behavior,and reduces levels of reactive oxygen species (ROS).Wu et al.found that Schisandra extract alleviated the ethanol concentration and free radical content ofCaenorhabditis eleganstissues,alleviated oxidative stress,and extended its lifespan,which can be used to treat ethanol-induced neurotoxicity and protectCaenorhabditis elegansethanolinduced neurotoxicity [27].Further research found that Schizandra extract inhibited persistent dopamine excitation and played a protective role in dopaminergic neurons.The main alkaloid in fenugreek,has anti-inflammatory,antioxidant,and anti-pathogenic effects in the neurodegenerative disease of theCaenorhabditis elegansmodel.The active ingredient from Gastrodia produces protective effects on dopamine neurons and reduces the accumulation ofα-synuclein inCaenorhabditis elegansthrough the insulin-like pathways DAF-2 and DAF-16 [28].Hydroxyl-substituted flavonols isolated from the fruit of Sichuan pepper (DC)and citrus kernel extract showed anti-Parkinson’s effects by reducingα-synuclein levels and lipid buildup [29]accompanied with age-associated neurological disorders such as Parkinson’s disease (PD).

2.2.3 Application in Huntington’s disease

Huntington’s disease (HD) is a single-gene genetic disorder in neurodegenerative diseases that is autosomal dominant.Abnormal amplification of CAG repeats in the exon region of the HTT gene located on chromosome 4 results in variablelengthN-terminal polyglutamine (poly Q) chains,producing huntingtin protein toxicity and leading to neurodegeneration [30].

To simulate the neurotoxicity of poly Q protein,researchers have genetically manipulated fragments of different lengths of poly Q protein to specifically transfer and express the protein to specific parts of Nematoda neuronal cells,muscle cells,and intestinal cells [31].Cordeiro et al.have found that the flavonoid rutin has a neuroprotective effect in a model of Huntington’s C.Elegans,and further research has found that treatment maintains the function of multimodal sensory (ASH) neurons and reduces the degeneration of their sensory termination [32].The acidic polysaccharide EbPS-A1,isolated in Epimedium by Xiang et al.inhibits polyglutamine(poly Q)-mediated behavioral dysfunction in nematode models by reducing reactive oxygen species levels,and enhancing antioxidant enzyme activity [33].Xiao et al.found that salidroside with antioxidant activity reduces neuronal death and behavioral dysfunction mediated by poly Q expressed in multimodal sensory (ASH) neurons through the oxidative stress pathway [34].Guarana extract has a protective effect in HD and AD models ofCaenorhabditis elegans,and GHE reduces poly Q protein aggregation in muscles and can combat protein misfolding and accumulation [35].

2.3 Application in anti-cancer research

The receptor tyrosine kinase RAS signaling pathway in mammalian cells controls many physiological functions of the cell.This pathway has a high mutation rate and is associated with a variety of cancers after over activation,which accelerates the cell proliferation and differentiation cycle and leads to unlimited tumor proliferation.The nematodes are highly homologous to mammalian genes,and the presence of the RAS homologous gene Let-60 in nematodes can be achieved by detecting tumors expressing Let-60 in nematodes.

After extracting different parts of red peony [36]and crow bile [37],MTZ124 extract ofCaenorhabditis elegans,with acute toxicity and antitumor activity,was used to preliminarily screen out the tumor active ingredients that inhibited the ras mutation at different extraction sites of red peony and crow bile.Escudero et al.found that betain,which occurs naturally in foods such as beetroot and prickly pear,could prevent and treat cancer [38],The therapeutic effect of betaine was evaluated using an animal model,Caenorhabditis elegansJK1466,and the results showed that the tumor volume was reduced and the life of nematodes was extended through DAF-16 transcription factors and insulin signaling pathways.Liu et al.applied the proven antioxidant bitter water rose to inhibit overactivation of the Ras/MAPK pathway by modulating oxidative stress-related proteins (DAF-16),glutathione S-transferase 4 (GST-4),and superoxide dismutase (SOD) [39].Through the nematode nl046gf model,a screening system was established,the Chinese medicine Huang Lian water extract with good phenotypic activity inhibition of nematode was screened,and the mechanism of pantethine antitumor activity was preliminarily explored by Ras point mutant cells,laying a foundation for the clinical application of Huang Lian anti-tumor research [40].

2.4 Application in antibacterial research

Antibacterial drugs have been widely used due to their good efficacy.“Antibacterial drug abuse” has caused adverse consequences,such as the occurrence of multiple drug resistance.In 2006,through the establishment of an enterococcus faecalis infection model of nematodes,researchers first used nematodes for antibacterial drug screening [41].They realized that it was possible to establish a pathogenic bacteria screening model through nematodes to screen new anti-infective drugs.

At present,many screening models of antibacterial drugs have been established,such as Candida albicans and extensively drug-resistant Acinetobacter baumannii (XDRAB),Pseudomonas aeruginosa,Staphylococcus aureus,etc.Scientific researchers use nematode models to find active ingredients with strong antibacterial activity from traditional Chinese medicine,providing support for the development of new antibacterial drugs.Luo et al.found that baicalein in Scutellaria baicalensis Georgi exerted a dose-dependent inhibition on the virulence phenotype regulated by quorum sensing QS in Pseudomonas aeruginosa,regulateed the expression of QS gene,and reduced the pathogenicity of Pseudomonas aeruginosa inCaenorhabditis elegans[42].Zhang et al.explored the mechanism of Xue Bi Jing in treating Pseudomonas aeruginosa infection and found that Xue Bi Jing exhibited no obvious antibacterial activity by inhibiting the formation of pseudomonas biofilm and reducing the expression of virulence genes and quorum sensing (QS) related genes [43].Jiang et al.found that the soluble ethyl acetate extract of Salvia miltiorrhiza had anti-infective activity against Staphylococcus aureus in the infection model ofCaenorhabditis elegans[44].Further research found that Tanshinone IIB could reduce RNAIII and PSM-α.The expression ofβlactamase showed strong anti-infective activityinvitroandin vivo.Shu et al.found that thymol,a phenolic compound from thyme,could inhibit the formation and increase of candida Albicans colony in the small intestine ofCaenorhabditis elegansby the model ofCaenorhabditis elegans[45].Among the p38 MAPK signal pathway genes ofCaenorhabditis elegans,thymol treatment could restore the expression imbalance of pmk-1 and sek-1 genes caused by Albicans infection,as well as the phosphorylation level of PMK-1.Duan used glp-4 in the early stage through the optimization research group [46].The XDRAB infection model established by sek-1 double gene mutation nematode was used to find the effective components with antibacterial effects from 73 Chinese herbal medicines with heatclearing and detoxification,and the peony bark with antibacterial activity was obtained,further verifying the antibacterial activity of its active component paeonolin vivoandin vitro.Exploration of the antibacterial activity of 43 kinds of traditional Chinese medicines against nematodes,showed that 8 different kinds had antibacterial effects [47].

2.5 Others

In addition,the nematode model is also used as sugar and lipid metabolism models.Yang et al.used the type 2 diabetes model ofCaenorhabditis elegansdaf-2 mutant to screen the prescription of traditional Chinese medicine [48].Juan et al.found that balsam pear saponin BMS had an inhibitory effect on nematode fat deposition,which might depend on the fatty acid synthesis pathway mediated by fatty acid desaturase to prolong the life span of nematodes.Except as an animal model.Caenorhabditis eleganshas also been used to evaluate the biological toxic effects of traditional Chinese medicine [49].Miao et al.measured the toxic effects of different concentrations of aqueous extracts of Coptis Chinensis with the indicators of nematode mortality,maximum life span and median lethal time,ontogeny,number of eggs laid,and movement behavior.Yinghui Lu et al.used model organisms to determine the toxic effects of different proportions of Coptis and Cornus officinalis extracts [50].

3 Expectation

Traditional Chinese medicine emphasizes the balance of yin and yang in treating diseases.The imbalance of yin and yang in the human body leads to the occurrence of diseases.Traditional Chinese medicine is used to replenish qi and blood,strengthen the body,coordinate the functions of viscera and meridians,and restore the body to a normal state.However,traditional Chinese medicine has been questioned in its application due to the complexity of its mechanism and target.In the modernization process of traditional Chinese medicine,different components of traditional Chinese medicine need to be deeply explored to provide support for the research and development of traditional Chinese medicine.The model ofCaenorhabditis elegansprovides new research ideas for drug screening and the discovery of new drugs.Compared with mammalian models,nematodes can obtain better time and economic benefits because of their small size,rapid reproduction,short life cycle,and transparent body.Compared with single-celled organisms,nematodes,as a whole animal,can be used to preliminarily screen the active ingredients of traditional Chinese medicine for a more comprehensive effect.

Since the discovery ofCaenorhabditis elegans,it has been widely used as an animal modelin vivoafter nearly half a century of development.However,there are relatively few studies on the application of nematode models to the screening of active substances in traditional Chinese medicine.Caenorhabditis eleganshas unique advantages in application,but it can not completely simulate the physiological state of the human body due to its significant difference from mammalian species,such as lack of immune function,heart,and other organs.Therefore,it is difficult to study the mechanism of some human diseases.However,based on the progress made in the application ofCaenorhabditis elegansas a research model,nematodes will become an important tool for drug discovery.The nematode model,combined with high-throughput drug screening platform,proves to be a potential strategy for drug target identification and drug discovery.

4 Acknowledgments

This work was financially supported by Scientific Research Fund Project of Education Department of Liaoning Province (LJKZ0960) and Science Foundation of Shenyang Pharmaceutical University (GGJJ2021105).