A promising strategy for investigating the anti- aging effect of natural compounds: a case study of caffeoylquinic acids

Rong Li, Mingfang Tao, Ting Wu, Zhang Zhuo, Tingting Xu, Siyi Pan and Xiaoyun Xu *

Caffeoylquinic acids, as plant-derived polyphenols, exhibit multiple biological activities such as anti- oxidant, anti-inflammatory, and neuroprotective activities. However, only limited information about their effect on longevity is available. In the current study, molecular docking was employed to explore the interactions between six representative caffeoylquinic acids and the insulin-like growth factor-1 receptor (IGFR), which is an important target protein for longevity. The results indicated that all six compounds were embedded well in the active pocket of IGFR, and that 3,5-diCQA exhibited the strongest affinity to IGFR. Moreover, ASP1153, GLU1080, ASP1086, and ARG1003 were the key amino acid residues during the interaction of these 6 compounds with IGFR. Furthermore, the lifespan extension effect of caffeoylquinic acids was evaluated in a Caenorhabditis elegans (C. elegans) model. The results revealed that all the caffeoylquinic acids significantly extended the lifespan of wild-type worms, of which 3,5-diCQA was the most potent compound. Meanwhile, 3,5-diCQA enhanced the healthspan by increasing the body bending and pharyngeal pumping rates and reducing the intestinal lipofuscin level. Further studies demonstrated that 3,5-diCQA induced longevity effects by downregulating the insulin/insulin-like growth factor signaling (IIS) pathway. This study suggested that the combination of molecular docking and genetic analysis of specific worm mutants could be a promising strategy to reveal the anti-aging mecha- nisms of small molecule natural compounds.

1. Introduction
Aging is a progressive decline of physiological functions and recognized as a major risk for various chronic diseases.1 So far, there has been no therapy or drug able to cure aging- related diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), thus researchers have turned to alternative approaches. Nutritional intervention is considered as a key and effective strategy to promote healthy aging and improve life quality,2,3 and an increasing number of food- derived bioactive compounds have been reported to possess anti-aging activities.4–6
The insulin-like growth factor-1 receptor (IGFR) is a recep- tor tyrosine kinase, and it is engaged in the phosphoinositide 3-kinase (PI3K)/Akt kinase and RAS/MAP kinase signaling cascades, which play an important role in cell proliferation and apoptosis.7,8 Moreover, IGF signaling has been reported to negatively impact the lifespan by accelerating the aging process.9 The inactivation of the insulin/IGF-1 receptor, as a pivotal component of the IGF signaling pathway, has been found to prolong the lifespan of mice and even of humans.10–12 Therefore, the exploration of natural bioactive compounds inhibiting IGFR for promoting longevity has aroused great interest in recent years.
C. elegans has been widely used as an in vivo model to screen bioactive compounds with longevity effects and reveal the related mechanisms.13 In C. elegans, daf-2 encodes a recep- tor tyrosine kinase which shared more than 30% identical amino acids with the human IGF-1 receptor.14 The inhibition of DAF-2/IGFR, an initializer of the insulin/insulin-like growth factor 1 signaling pathway (IIS), reduces the IIS signaling and activates a critical transcription factor DAF-16/FOXO, resulting in lifespan extension in mammals and nematodes.15,16 Because of the high conservation of DAF-2/IGFR between nematodes and mammals, bioactive compounds with inhibi- tory effects on IGFR may have similar longevity promotion effects in C. elegans.
Caffeoylquinic acids are abundant plant-derived polyphenol compounds, and they are found in a wide range of materials such as coffee beans, tea, vegetables, and herbs.17 Caffeoylquinic acid has many isomers, and it is mainly com- posed of mono-caffeoylquinic acid and dicaffeoylquinic acid, according to the binding sites and number of caffeic acids on the quinic moiety. Notably, coffee consumption has been found to be closely associated with healthy aging. For example, it can improve cognitive function,18 and reduce the disease risk of cancers and diabetes.19,20 However, whether caffeoylqui- nic acids, the abundant components in coffee, have a direct effect on delaying the aging process remains to be further determined. Besides, one previous study has revealed that the longevity effect of Lonicera japonica extracts rich in caffeoylqui- nic acids is dependent on the daf-2 gene.21 Thus, it is plausible to assume that caffeoylquinic acids may have great potential in anti-aging effects, and that there might be a close relationship between caffeoylquinic acids and DAF-2/IGFR.
Molecular docking is an effective structure-based method to predict the biological activity of small molecule compounds by evaluating their interactions with potential targets.22 In the present study, we predicted the anti-aging effect of caffeoylqui- nic acid by molecular docking with the key target IGFR, veri- fied its longevity effects, and explored the underlying mecha- nisms in the C. elegans model. This study provides reference for screening other novel anti-aging natural compounds.

2. Materials and methods

2.1 Chemicals
Fluorodeoxyuridine (FudR 98%) was obtained from Sigma- Aldrich (St Louis, MO, USA). The standards ( purity > 98%) including 5-caffeoylquinic acid (5-CQA), 1,5-dicaffeoylquinic acid (1,5-diCQA), 1,4-dicaffeoylquinic acid (1,4-diCQA), 3,4- dicaffeoylquinic acid (3,4-diCQA), 3,5-dicaffeoylquinic acid (3,5-diCQA), and 4,5-dicaffeoylquinic acid (4,5-diCQA) were purchased from Biopurify Phytochemicals Ltd (Sichuan, China). The above six compounds were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions (100 mM), and then diluted to final concentrations of 25, 50, and 100 μM before use. The final concentration of DMSO in the control group was 0.1%.

2.2 Strains and maintenance conditions
C. elegans strains were purchased from Caenorhabditis Genetics Center (CGC) (University of Minnesota, USA), includ- ing N2, Bristol (wild-type), CB1370 [daf-2(e1370)III], TJ1052 [age-1(hx546)II], TJ356 [zIs356 IV (daf-16p::daf-16a/b::GFP + rol- 6 (su1006)], CF1038 [daf-16 (mu86)I], CF1553 [( pAD76) sod-3p:: GFP], and CL2070 [(dvls70)hsp-16.2::GFP]. Escherichia coli OP50 (E. coli OP50), RB759 [akt-1(ok525)V], and VC345[sgk-1(ok538)X] were kindly provided by Dr Luo (Southwest Medical University). All the strains were maintained on nematode growth medium (NGM) plates seeded with E. coli OP50 which grew overnight before use.

2.3 Molecular docking between IGFR and caffeoylquinic acids
Molecular docking was performed using the Schrödinger package (2018). The crystal structure of IGFR was obtained from RCSB Protein Data Bank (PDB ID: 5FXS).7 The structures of different caffeoylquinic acids were sketched and optimized using the ChemDraw software. The compounds with low energy conformation were selected and docked into the grid formed by the IGFR protein using a standard precision docking mode. The docking score reflected the affinity of the ligands and the protein.

2.4 Lifespan assay
Strains including N2, CB1370, TJ1052, CF1038, RB759, and VC345 were grown at 20 °C on nematode growth medium (NGM) plates seeded with live E. coli OP50. The lifespan assay was performed as previously described.23 Briefly, the synchro- nized L4-stage nematodes were transferred into a 96-well plate (10–15 worms per well) where the liquid S-complete medium was added with FudR (150 μM), the inactivated OP50 (65 °C for 30 min), and 0.1% DMSO (control) or caffeoylquinic acids (25, 50, and 100 μM, respectively). The survival rates were recorded every other day until all the worms died, and the entire assay was repeated 3 times.

2.5 Phenotype assay
Wildtype worms were treated as described in Section 2.4. Then, they were transferred to new NGM plates on the 3rd, 6th, and 9th day of adulthood. The rates of body bending and pharyngeal pumping within 20 s were measured by counting the number of continuous sinusoidal curves and the pharyn- geal contraction times of the worms, respectively.24 After a 10-day treatment, adult worms were anesthetized with 2% sodium azide and photographed using a Nikon fluorescence microscope (Nikon, Japan) with excitation and emission wave- lengths at 485 nm and 528 nm, respectively. Afterwards, the lipofuscin accumulation of each worm was analyzed using the ImageJ software. In the brood size assay, a single L4 larva cul- tured without adding FudR was placed onto a NGM plate seeded with E. coli OP50 to lay eggs, and the larva was trans- ferred daily to separate it from its offspring for a total of 6 days. Afterwards, the number of progeny produced during the whole reproduction period was counted.5 For body size measurement, photographs of worms were taken with an inverted microscope (Nikon, Japan) and the body length and width of each worm were measured with the ImageJ software. Each assay was performed in triplicate with at least 30 worms in each of the three independent groups.

2.6 Quantification of SOD-3::GFP and HSP-16.2::GFP expressions
SOD-3 and HSP-16.2 expressions were determined through the fluorescence intensities of the transgenic strains CF1553 and CL2070, respectively. The synchronized L1 larvae were incu- bated with 3,5-diCQA (50 μM) or 0.1% DMSO for 72 h, and then the worms were placed on glass slides and anesthetized with 2% sodium azide before photographing. The young adult nematodes of the CL2070 strain, were incubated at 37 °C for 2 h to induce the activity of HSP-16.2, and allowed to recover for 4 h at 20 °C. The fluorescence intensity of each worm was quantified by the ImageJ software. All the tests were repeated 3 times with more than 30 randomly selected nematodes per group.

2.7 Subcellular localization of DAF-16::GFP
The TJ356 strain (containing DAF-16-fused GFP protein) was utilized to detect intracellular DAF-16 localization. The syn- chronized L4 stage worms were incubated with 3,5-diCQA (50 μM) or 0.1% DMSO for 48 h, and then the worms were photographed using fluorescence microscopy (Nikon Eclipse 90i) to monitor the nuclear translocation of DAF-16::GFP. The nuclear translocation patterns of DAF-16::GFP were identified as “cytosolic”, “intermediate”, and “nuclear”. The DAF-16::GFP level was expressed as percentages of subcellular localization.

2.8 Gene expression assay by quantitative RT-PCR
The synchronized L4 larvae were incubated with or without 3,5-diCQA at 20 °C, as described in the lifespan assay. After a 3-day incubation, adult worms were collected for extracting total RNA according to the manufacturer’s protocol (Aidlab Biotechnologies Co., Ltd, Beijing, China). Afterwards, cDNA was prepared using a reverse transcription kit (Takara). The quantitative real-time PCR (qRT-PCR) was then performed (Mantel-Cox) test. The differences between two groups were analyzed by Student’s t test. p < 0.05 was considered signifi- cantly different. 3. Results 3.1 Molecular docking of IGFR with caffeoylquinic acids To investigate the interaction between caffeoylquinic acids and IGFR, we performed molecular docking of six typical caffeoyl- quinic acid compounds (Fig. 1) with the crystal structure of IGFR. The docking scores of these compounds with IGFR are listed in Table 1. As shown in Fig. 2A and B, all the com- pounds were embedded well in the IGFR active pocket mainly formed of the amino acid residues ASP1153, GLU1080, and ASP1086. In addition, the phenolic hydroxyl groups on 1,5- diCQA, 1,4-diCQA, and 3,4-diCQA formed intermolecular hydrogen bonds with GLU1004, ARG1092, and ILE1160 of IGFR, respectively. Compounds 5-CQA and 4,5-diCQA gener- ated hydrogen bond interactions with the side chains of LEU1005 and MET1082 residues, respectively (Fig. 2C). The compound 3,5-diCQA formed a hydrogen bond with the GLN1007 residue of IGFR, in addition, its carboxyl group gen- erated a strong electrostatic interaction with the side chain of ARG1003 (Fig. 2C). Among these compounds, 3,5-diCQA exhibited the strongest affinity to IGFR, which was even higher than that of 2-( pyrazol-4-ylamino)-pyrimidine,7 a medicine using SYBR Green PCR Master Mix (Takara) on a qTOWER 2.0 PCR system (Analytik JenaAG, Germany). The relative fold changes of the genes were calculated using the 2−ΔΔCT method after being normalized to the gene act-1. The primer sequences are presented in ESI Table 2.† 2.9 Statistical analysis Statistical analyses were performed using GraphPad Prism version 7.0 (GraphPad Software, CA, USA) and SPSS 19.0. The results of lifespan assays were analyzed using the log-rank Table 1 Molecular docking statistics of IGFR with different compounds Compounds Docking score 2-(Pyrazol-4-ylamino)-pyrimidine −7.225 3,5-diCQA −7.817 5-CQA −6.516 4,5-diCQA −6.436 1,5-diCQA −6.270 1,4-diCQA −6.219 3,4-diCQA −5.417 Fig. 1 Structures of six selected caffeoylquinic acids. (A) 3,5-diCQA, (B) 5-CQA, (C) 4,5-diCQA, (D) 1,5-diCQA, (E) 1,4-diCQA, (F) 3,4-diCQA. Fig. 2 Molecular docking of different caffeoylquinic acids and ligand with IGFR. (A) Docking of 7 selected compounds with IGFR: ligand (silver), 3,5- diCQA (dark blue), 5-CQA (light blue), 4,5-diCQA (green), 1,5-diCQA (light green), 1,4-diCQA (orange), 3,4-diCQA (red) and docking scores are pre- sented in Table 1. (B) The partially enlarged drawing of compounds docking with IGFR and bound amino acid residues. (C) The plan drawing of 3,5- diCQA, 5-CQA, and 4,5-diCQA docking with IGFR. inhibitor of IGFR. The results revealed the potential inhibitory effect of caffeoylquinic acids on IGFR. 3.2 Caffeoylquinic acids extended the lifespan of C. elegans In C. elegans, inactivation of DAF-2/IGFR was an effective strat- egy to prolong lifespan by lowering the insulin/IGF-1 signal- ing.25 Considering that caffeoylquinic acids had high binding ability to the inhibitory sites of IGFR, we investigated the long- evity extension effect of these compounds in C. elegans. In comparison with the control, all the caffeoylquinic acids at 50 μM significantly extended the lifespan of wild-type worms (Fig. 3A, Table 2). More importantly, among these compounds, 3,5-diCQA was found to exhibit an optimal effect on longevity extension with the mean lifespan increased by 16.64% ( p < 0.001, Table 2). Similar results were also observed at concen- trations of 25 μM and 100 μM (Fig. S1A and B, Table S1†). This was consistent with the molecular docking results that the docking score of 3,5-diCQA was the highest. Our data revealed Fig. 3 Effects of caffeoylquinic acid isomers on the lifespan of C. elegans and the role of daf-2 in 3,5-diCQA mediated lifespan extension. (A) Survival curves of wild type worms treated with 0.1% DMSO (control) or caffeoylquinic acid isomers at concentrations of 50 μM at 20 °C. (B) Survival curves of mutants daf-2(e1370) treated with 50 μM 3,5-diCQA. Statistical analysis was analyzed by the log-rank test using Prism 7.0 and detailed data are listed in Table 2. (C) The mRNA expression level of daf-2 was quantified by qRT-PCR, with act-1 as an internal control. (**p < 0.01; Student’s t test; mean ± SD, n = 3). Table 2 Effect of caffeoylquinic acids on the lifespan of C. elegans at 20 °C Strains Treatment (μM) Mean lifespan (d) (±SEM) Worm number Change (%) p value N2 Control 18.63 ± 0.60 112 — 3,5-diCQA (50) 21.73 ± 0.71 116 16.64 0.0004(***) 5-CQA (50) 21.31 ± 0.54 150 14.38 0.0040(**) 3,4-diCQA (50) 21.03 ± 0.67 123 12.88 0.0022(**) 1,5-diCQA (50) 20.51 ± 0.62 132 10.09 0.0092(**) 1,4-diCQA (50) 20.28 ± 0.58 134 8.86 0.0489(*) 4,5-diCQA (50) 20.24 ± 0.58 144 8.64 0.0218(*) daf-2(e1370)III Control 24.98 ± 0.99 120 3,5-diCQA (50) 25.08 ± 0.98 119 — 0.8422(ns) age-1(hx546)II Control 21.92 ± 0.56 117 3,5-diCQA (50) 21.13 ± 0.52 118 — 0.2739(ns) akt-1(ok525)V Control 19.98 ± 0.37 120 3,5-diCQA (50) 19.18 ± 0.33 123 — 0.0610(ns) sgk-1(ok538)X Control 19.32 ± 0.45 125 3,5-diCQA (50) 20.46 ± 0.38 123 — 0.2840(ns) daf-16(mu86)I Control 15.10 ± 0.30 125 3,5-diCQA (50) 14.39 ± 0.29 129 — 0.1246(ns) that 3,5-diCQA might be the most potent compound in life- span promotion. Since the lifespan-promotion effect of 3,5- diCQA was better at 50 μM than at 25 μM and 100 μM, we used 50 μM 3,5-diCQA for the subsequent experiments. 3.3 3,5-diCQA extended the lifespan of C. elegans by inhibiting DAF-2 It has been reported that daf-2 encodes orthologues of mam- malian insulin/IGF-1 receptors, and that this gene is a critical aging regulator in C. elegans.26 Considering the strong affinity of 3,5-diCQA to IGFR, the role of daf-2 in the lifespan extension mediated by 3,5-diCQA was further investigated. As shown in Fig. 3B and Table 2, daf-2 deletion led to elimination of the longevity-promotion effect of 3,5-diCQA ( p > 0.05), suggesting that daf-2 was essential for the action of 3,5-diCQA. Besides, the mRNA expression level of daf-2 was significantly reduced in worms treated with 3,5-diCQA (Fig. 3C, p < 0.01), which con- firmed the high binding ability of 3,5-diCQA to the inhibitory site of IGFR. 3.4 3,5-diCQA improved the healthspan of C. elegans Mutants with lower level of daf-2 have been reported to be more active and healthier than wild-type worms.26–28 To deter- mine whether 3,5-diCQA could promote healthspan, we inves- tigated several aging-associated phenotypes in wild-type worms. Although the rates of decline were similar between the 3,5-diCQA-treated worms and control worms at each stage examined, the 3,5-diCQA treatment group displayed a higher body bending rate than the control group (Fig. 4A). Similar results were also observed in the pharyngeal pumping assay (Fig. 4B). Furthermore, as shown in Fig. 4C and G, the intesti- nal lipofuscin level in the 3,5-diCQA treatment group was sig- nificantly decreased by 12.29%, compared with that in the control group ( p < 0.01). Meanwhile, no significant differences in progeny production and body size (body length and width) were observed between the 3,5-diCQA treatment group and control group (Fig. 4D–F). These results indicated that 3,5- diCQA could prolong healthspan in addition to extending lifespan. 3.5 3,5-diCQA extended the lifespan of C. elegans by regulating the IIS pathway Since the lifespan-extension effect of 3,5-diCQA was dependent on daf-2, a trigger of IIS signaling, we further performed a life- span assay of the mutants whose IIS pathway-related genes were deleted to reveal the underlying molecular mechanisms. The results showed that 3,5-diCQA treatment failed to prolong the lifespan of function-deficient mutants age-1(hx546)II, akt-1 (ok525)V, sgk-1(ok538)X, and daf-16 (mu86)I (Fig. 5A–D, Table 2), suggesting that 3,5-diCQA might extend lifespan by modulating the IIS pathway. 3.6 3,5-diCQA down-regulated IIS signaling and enhanced the expression of stress-related genes In C. elegans, DAF-2 was activated by binding insulin-like pep- tides, thus initiating a conserved cascade of phosphoinositide 3-kinase (AGE-1/PI3K)/Akt kinase, ultimately downregulating the activity of DAF-16, a FOXO transcription factor.29 To further determine the role of 3,5-diCQA in regulating the IIS pathway, we examined the expression levels of aging-associated genes (Fig. 6A). The results revealed that 3,5-diCQA signifi- cantly decreased the expression of age-1, a direct target gene of daf-2.In contrast, the expression of daf-16 was remarkably increased, indicating that 3,5-diCQA decreased the insulin/ IGF-1 signaling. In addition, the effect of 3,5-diCQA on the nuclear localization of DAF-16 was assessed using the trans- genic strain TJ356 (DAF-16::GFP). Results showed that the nuclear proportion of DAF-16 was increased from 17.75% to 45.21% ( p < 0.01), while the cytosolic proportion was reduced from 49.39% to 24.12% ( p < 0.05) in the transgenic strain TJ356 treated with 3,5-diCQA (Fig. 6B and C). Moreover, the stress-response genes downstream of the IIS pathway, sod-3 (superoxide dismutase) and hsp-16.2 (heat shock proteins) were obviously upregulated, whereas the expression of ctl-1 Fig. 4 Effects of 3,5-diCQA on the healthspan of N2 nematodes. (A) Body bending rates and (B) pharyngeal pumping rates were counted under an inverted microscope for 20 s on days 3, 6, and 9 of adulthood. (C) Relative fluorescence intensity of lipofuscin in worms treated with 50 μM 3,5- diCQA or 0.1% DMSO. (D) Total number of offspring. (E) Body length. (F) Body width. (G) Images of intestinal lipofuscin autofluorescence of worms on day 10 of adulthood. Data are expressed as means ± SD of three independent experiments. Statistical significance was calculated using Student’s t test. (***) p < 0.001. ns means no significance. (catalase) remained unchanged (Fig. 6A). Consistently, the expression levels of SOD-3::GFP and HSP-16.2::GFP were also enhanced by 10.58% and 31.47% in CF1553 and CL2070 worms exposed to 3,5-diCQA, respectively ( p < 0.01 and p < 0.001, Fig. 6D and E). Taken together, these data indicated that 3,5-diCQA diminished signaling of the IIS pathway. 4. Discussion In the present study, we found that six caffeoylquinic acids bound well to the inhibitory site of IGFR. Besides, both mono- caffeoylquinic acid and dicaffeoylquinic acids extended the lifespan of C. elegans, and 3,5-diCQA was the most potent compound. The 3,5-diCQA-induced lifespan extension was accompanied by aging process delay. The longevity promotion effect of 3,5-diCQA was dependent on the IIS signaling pathway. IGFR is a receptor tyrosine kinase, and it plays a key role in regulating lifespan.12,30 This study made the first attempt to employ IGFR as a key target to investigate the specific inter- actions between caffeoylquinic acids and IGFR by molecular docking. The molecular docking results indicated that six caffeoylquinic acids and one ligand inhibitor were embedded well in the active pocket, thus stabilizing the inactive confor- mation of IGFR. Besides, the caffeoylquinic acids with a substi- tuent group at C-5 had a higher docking score, implying that the esterification at C-5 might enhance the binding of the Fig. 5 The lifespan-extension effect of 3,5-diCQA was dependent on the insulin/insulin-like signaling pathway. Survival curves of mutants (A) age-1 (hx546), (B) akt-1(ok525), (C) sgk-1(ok538), (D) daf-16(mu86) treated with 50 μM 3,5-diCQA or 0.1% DMSO. The log-rank test was used for statistical analysis and detailed data are listed in Table 2. caffeoylquinic acids to IGFR. Among these compounds, 3,5- diCQA exhibited the highest affinity to IGFR, which might be attributed to its strong electrostatic interactions with ARG1003 and the intermolecular hydrogen bond formation by this com- pound and four amino acid residues (GLN1007, ASP1153, GLU1080, and ASP1086). The amino acid residues ASP1153, GLU1080, and ASP1086 also interacted with other active com- pounds to generate hydrogen bonds. Thus, ASP1153, GLU1080, ASP1086, and ARG1003 were the key amino acid resi- dues in the interaction between the abovementioned 6 com- pounds and IGFR. Taken together, our data suggested that caffeoylquinic acids might be potential inhibitors of IGFR. It has been reported that inhibiting IGFR is an effective strategy to prolong lifespan and alleviate aging-related dis- eases.9 Based on this, it could be hypothesized that the afore- mentioned caffeoylquinic acids might possess an anti-aging effect. To confirm our hypothesis, we examined the role of caffeoylquinic acids in extending the lifespan of C. elegans. The results showed that all six caffeoylquinic acids signifi- cantly prolonged the lifespan of C. elegans, of which 3,5-diCQA exhibited the optimal effect, which was substantially consist- ent with the molecular docking results of the six compounds with IGFR. Our findings also confirmed the feasibility of employing the molecular docking method to search for natural anti-aging compounds. Lifespan extension is not necessarily accompanied by health span promotion.31 Moreover, it seems more important to prolong the health span and the functional period of life than to extend only longevity. In the current study, 3,5-diCQA could not only extend the lifespan but also promote the health span of C. elegans. This was supported by our results that 3,5- diCQA remarkably delayed the aging progression, which was reflected by the increased body bending and pharyngeal pumping rates. Lipofuscin is an autofluorescent granule accu- mulated in the intestinal cells of C. elegans with aging.32 Supplementation with 3,5-diCQA considerably reduced the accumulation of lipofuscin, indicating the prolonged health- span. Previous studies have suggested that the extension of lifespan is closely related to the decrease in body size and progeny production in C. elegans.33,34 However, our data indi- cated that 3,5-diCQA had no effect on the body size and fecundity. All these data suggested that 3,5-diCQA could promote the health span of C. elegans without any observed impairments. Moreover, our findings provided a theoretical basis for explaining the relationship between drinking coffee and healthy aging. The gene daf-2 encodes a homologue of the insulin/IGF-1 receptor and regulates longevity, development, and metab- olism in C. elegans.35 Given that caffeoylquinic acids could bind well to the inhibitory site of IGFR, we further investigated the role of daf-2 in modulating longevity by deleting this gene. The results showed that 3,5-diCQA failed to extend the lifespan of daf-2 deletion mutants, indicating that the lifespan exten- sion effect of 3,5-diCQA was dependent on daf-2. This was in line with a previous report that daf-2 was essential for the life- span promotion effect of chlorogenic acid.36 In addition, the expression level of daf-2 was obviously decreased by 3,5-diCQA, suggesting that the functional deficiency of daf-2 might con- Fig. 6 3,5-diCQA extended the lifespan of C. elegans by downregulating the IIS signaling pathway. (A) The mRNA expression levels of age-1, daf-16, sod-3, ctl-1, and hsp-16.2 were quantified by qRT-PCR, with act-1 as an internal control. (B) Quantification of DAF-16::GFP localization. (C) Representative fluorescence photographs of transgenic strain TJ356 based on the localization of DAF-16: “cytosolic”, “intermediated” and “nuclear”. Fluorescence images and expressions of (D) SOD-3::GFP and (E) HSP-16.2::GFP in CF1553 and CL2070 strains. (*p < 0.05; **p < 0.01; ***p < 0.001; ns means no significance, Student’s t test; mean ± SD, n = 3). tribute to the lifespan extension effect of 3,5-diCQA. These results also demonstrated that the high binding ability of 3,5- diCQA to the inhibitory site of IGFR was closely associated with the inhibitory effect of this compound on daf-2. However, we were not able to determine the inhibitory effects of 3,5- diCQA on the protein DAF-2 due to the lack of available anti- bodies against DAF-2 in C. elegans. Therefore, it was too early to conclude that 3,5-diCQA downregulated DAF-2 transcrip- tionally and/or post-translationally based on the current results only. In C. elegans, insulin-like peptides regulate the insulin/ IGF-1 signaling pathway by binding to the IGFR orthologue DAF-2.37 Activation of DAF-2 initiates the sequential phos- phorylation cascade of several kinases including AGE-1/phos- phatidylinositol 3-kinase (PI3K), PDK-1, and AKT-1/2 kinases, ultimately antagonizing DAF-16, a human forkhead transcrip- tion factor orthologue.29,35,38 Considering that 3,5-diCQA could inhibit daf-2, the starting point of the IIS pathway, we further examined whether the lifespan extension effect of 3,5- diCQA was related to the IIS pathway. In the current study, the lifespan-extension effect of 3,5-diCQA was abolished in the function deficient mutants age-1(hx546)II, sgk-1(ok538)X, akt-1 (ok525)V, and daf-16 (mu86)I, which suggested that the longev- ity promotion effect of 3,5-diCQA was dependent on the IIS pathway. Our result was consistent with a previous report that Lonicera japonica extracts rich in caffeoylquinic acids pro- longed the lifespan of C. elegans by regulating the IIS pathway.21 A few previous studies have indicated that the downregula- tion of daf-2 results in reduced insulin/IGF-1 signaling, thus inducing the expression of downstream genes related to life- span extension and stress resistance enhancement.25,29,39 Considering the inhibitory effect of 3,5-diCQA on daf-2, we further examined whether 3,5-diCQA exerted its longevity effect by down-regulating the IIS pathway. The results showed that 3,5-diCQA decreased the expression of age-1, the direct downstream gene of daf-2. In addition, the expression level of daf-16, a transcriptional target of the IIS pathway, was obviously elevated. These results indicated that 3,5-diCQA downregulated the IIS pathway. Meanwhile, our data showed that 3,5-diCQA induced translocation of the DAF-16 protein from the cytoplasm to the nucleus, suggesting that the post- translational regulation of DAF-16 remarkably contributed to the lifespan extension effect of 3,5-diCQA. This was in line with the previous reports that DAF-16 activation was required for inhibiting the IIS pathway to extend the lifespan of C. elegans.35,40,41 When the IGF-1R level is decreased, FOXO proteins in mammals can induce stress-response genes.16,42,43 This was supported by our results that the transcript levels of sod-3 and hsp-16.2, downstream genes of DAF-16/FOXO, were increased in the 3,5-diCQA-treated worms. Furthermore, similar results were also observed in the GFP-fused transgenic strains CF1553 and CL2070. Collectively, these findings demonstrated that 3,5-diCQA lowered the signaling of the IIS pathway by inhibiting the activity of DAF-2/IGFR, resulting in the activation of DAF-16, thus inducing the expression of stress response genes. However, the stress resistance effect mediated by 3,5-diCQA remains to be further investigated in future. 5. Conclusion In conclusion, our findings reveal that caffeoylquinic acids may have great potential to serve as novel anti-aging com- pounds so as to promote healthy aging. Our data confirm that the combination of molecular docking and a worm mutant model is a promising strategy to predict and verify the anti- aging effects of natural compounds. It is worth noting that IGFR used in this study is only one of the target proteins to detect the possible anti-aging mechanisms of compounds such as caffeoylquinic acids through the above strategy. In future, more target proteins can be used to screen more natural compounds and explore their potential anti-aging mechanisms. Abbreviations C. elegans Caenorhabditis elegans IGFR Insulin-like growth factor-1 receptor IIS Insulin/insulin-like growth factor signaling DMSO Dimethyl sulfoxide E. coli OP50 Escherichia coli strain OP50 NGM Nematode growth medium 5- CQA 5-Caffeoylquinic acid 1,4-diCQA 1,4-Dicaffeoylquinic acid 3,4-diCQA 3,4-Dicaffeoylquinic acid 1,5-diCQA 1,5-Dicaffeoylquinic acid 3,5-diCQA 3,5-Dicaffeoylquinic acid 4,5-diCQA 4,5-Dicaffeoylquinic acid Author contributions Rong Li: conceptualization, methodology, software, validation, formal analysis, investigation, writing – original draft. Mingfang Tao: methodology, software, formal analysis. Ting Wu: supervision, writing – review & editing. Zhuo Zhang: soft- ware, writing – review & editing. Tingting Xu: software. Siyi Pan: supervision, writing – review & editing. 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