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. 2022 Aug 22:13:931886.
doi: 10.3389/fphar.2022.931886. eCollection 2022.

Flavonol glycoside complanatoside A requires FOXO/DAF-16, NRF2/SKN-1, and HSF-1 to improve stress resistances and extend the life span of Caenorhabditis elegans

Lin Tan  1   2 , Zhuo-Ya Zheng  1 , Lv Huang  1 , Zhong Jin  3 , Su-Lian Li  4 , Gui-Sheng Wu  1   4   5 , Huai-Rong Luo  1   5   6
Affiliations

Flavonol glycoside complanatoside A requires FOXO/DAF-16, NRF2/SKN-1, and HSF-1 to improve stress resistances and extend the life span of Caenorhabditis elegans

Lin Tan et al. Front Pharmacol. .

Abstract

Aging is associated with the increased risk of most age-related diseases in humans. Complanatoside A (CA) is a flavonoid compound isolated from the herbal medicine Semen Astragali Complanati. CA was reported to have potential anti-inflammatory and anti-oxidative activities. In this study, we investigated whether CA could increase the stress resistance capability and life span of Caenorhabditis elegans. Our results showed that CA could extend the longevity of C. elegans in a dosage-dependent manner, while 50 μM of CA has the best effect and increased the life span of C. elegans by about 16.87%. CA also improved the physiological functions in aging worms, such as enhanced locomotor capacity, and reduced the accumulation of the aging pigment. CA could also reduce the accumulation of toxic proteins (α-synuclein and β-amyloid) and delay the onset of neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease, in models of C. elegans. Further investigation has revealed that CA requires DAF-16/FOXO, SKN-1, and HSF-1 to extend the life span of C. elegans. CA could increase the antioxidation and detoxification activities regulated by transcription factor SKN-1 and the heat resistance by activating HSF-1 that mediated the expression of the chaperone heat shock proteins. Our results suggest that CA is a potential antiaging agent worth further research for its pharmacological mechanism and development for pharmaceutical applications.

Keywords: Caenorhabditis elegans; DAF-16/FOXO; aging; complanatoside A; neurodegenerative diseases; oxidative stress; proteostasis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
CA extends the life span of C. elegans. (A) Chemical structure of CA; (B) survival curves of wild-type N2 worms raised at 20°C on NGM plates containing either no CA or 25, 50, 100, and 200 μM of CA; (C) survival curves of wild-type N2 worms treated from hatching and raised at 20°C on NGM plates containing either no CA or 50 μM of CA (p < 0.001, log-rank test). Statistical details and repeats of these experiments are summarized in Supplementary Table S1. (D) Body bending behavior of nematode N2 treated with 50 μM of CA for 5 and 10 days. The figure is the mean value of three independent experiments, and the SEM is represented by an error line. *** represents p < 0.001, calculated by two-tailed t-test. Statistical details and repeats of these experiments are summarized in Supplementary Table S3. (E) The fluorescence picture of lipofuscin in N2 fed with or without 50 μM of CA on the 10th day of adulthood. (F) Analysis of lipofuscin in N2 nematodes. The relative fluorescence intensity was calculated by ImageJ. The figure is the mean value of three independent experiments; SEM is represented by error line. *** represents p < 0.001, calculated by two-tailed t-test. Statistical details and repeats of these experiments are summarized in Supplementary Table S2.
FIGURE 2
FIGURE 2
CA could delay the progression of neurodegenerative diseases in models of C. elegans. The effect of CA on the paralysis rate of nematodes CL2006 (A-beta) (A) and CL4176 (A-beta) (B) strains; (C) the fluorescence picture of YFP-labeled α-synuclein in C. elegans NL5901 fed with or without 50 μM of CA on the sixth day of adulthood. Analysis of the accumulation of α-synuclein in PD model nematodes NL5901 (D) and accumulation of dopamine transporter in BZ555 (E). (F) The fluorescence picture of dopamine transporter conjugated with GFP in worms BZ555 fed with or without 50 μM of CA on the sixth day of adulthood, the positive control was used for levodopa. Statistical details and repeats of these experiments are summarized in Supplementary Table S8.
FIGURE 3
FIGURE 3
CA improves the oxidative stress resistance of C. elegans by activating SKN-1. (A) The survival percentage of wild-type worms cultured with 20 mM of paraquat and treated with 50 μM of CA (p < 0.001, log-rank test). Statistical details and repeats of these experiments are summarized in Supplementary Table S6. (B) ROS content detection of wild type N2 treated with 50 μM of CA. Paraquat (20 mM) and NAC (2 mM) are used as the negative and positive controls, respectively. Statistical details of these experiments are summarized in Supplementary Table S7. (C) Analysis of ROS in N2. (D) The fluorescence picture of SKN-1::GFP subcellular translocation in worms LD1 fed with or without 50 μM of CA on the L4 larvae or young adults. (E) Relative expression of downstream skn-1 genes in L4 wild-type worms (N2) treated with CA for 24 h. Statistical details and repeats of these experiments are summarized in Supplementary Table S10. (F) The fluorescence picture of SOD-3::GFP in worms CF1553 fed with or without 50 μM of CA on the sixth day of adulthood. The quantification of fluorescence intensity of SOD-3::GFP (G) and GST-4::GFP (H). (I) The fluorescence picture of GST-4::GFP in CL2166. (J) Survival curves of skn-1(zu67) raised at 20°C on the NGM plates containing CA in life span assays (p > 0.05). Statistical details of these experiments are summarized in Supplementary Table S4. CA significantly increased the expression of GST-4 and SOD-3; fluorescence intensity was calculated by Image J. The bar chart shows the mean value of three independently repeated experiments, and the error line represents SEM. *** represents p < 0.001, calculated by two-tailed t-test. Statistical details of these experiments are summarized in Supplementary Table S5.
FIGURE 4
FIGURE 4
CA improves the heat stress resistance of C. elegans by activating HSF-1. (A) The survival percentage of wild-type worms treated with 50 μM of CA at 35°C (p < 0.001, log-rank test). Statistical details and repeats of these experiments are summarized in Supplementary Table S6. (B) Relative expression of downstream hsf-1 genes in L4 wild-type worms (N2) treated with CA for 24 h. Statistical details and repeats of these experiments are summarized in Supplementary Table S10. The fluorescence picture of HSP-4::GFP (C), HSP-60::GFP (E), and HSP-6::GFP (G). The quantification of fluorescence intensity of heat shock protein HSP-4 (D), HSP-60 (F), and HSP-6 (H). (I) Survival curves of hsf-1(sy441)I raised at 20°C on NGM plates containing 50 μM of CA in life span assays (p > 0.05). Life span was analyzed using Kaplan–Meier analysis, and p values were calculated using log-rank test. Statistical details and repeats of these experiments are summarized in Supplementary Table S4. CA significantly increased the expressions of HSP-4, HSP-60, and HSP-6. Fluorescence intensity was calculated by ImageJ. The bar chart shows the mean value of three independently repeated experiments, and the error line represents SEM. *** represents p < 0.001. Statistical comparison analysis was calculated by two-tailed t-test. Statistical details and repeats of these experiments are summarized in Supplementary Table S5.
FIGURE 5
FIGURE 5
CA requires the transcription factor FOXO/DAF-16 to extend the longevity of C. elegans. (A) The survival curves of daf-16(mu86)I treated with CA in life span assays (p > 0.05). (B) CA induced DAF-16 nuclear localization. Worms of transgenic strain daf-16(zls356)IV were treated with CA at 20°C for 4 h. (C) Relative expression of downstream daf-16 genes in L4 wild-type worms (N2) treated with CA for 24 h. Statistical details and repeats of these experiments are summarized in Supplementary Table S10. The survival curves of daf-2(e1370)III (D), akt-1(ok525)V (E), and akt-2(ok393)X (F) treated with CA in life span assays (p > 0.05), Statistical details and repeats of these experiments are summarized in Supplementary Table S4.
FIGURE 6
FIGURE 6
The effect of CA on energy processing pathways. The survival curves of isp-1 (qm150) (A), clk-1(e2519)III (B), mev-1(kn1)III (C), and aak-2(ok524)X (D) treated with CA in life span assays (p > 0.05). Life span was analyzed using Kaplan–Meier analysis, and p values were calculated using log-rank test. Statistical details and repeats of these experiments are summarized in Supplementary Table S4.
FIGURE 7
FIGURE 7
The effect of CA on the reproductive signaling pathway. The survival curves of glp-1(e2141)III (A), daf-12 (rh274) (B), and nhr-80(tm1011)III (C) treated with CA in life span assays (p > 0.05). Life span was analyzed using Kaplan–Meier analysis, and p values were calculated using log-rank test. Statistical details and repeats of these experiments are summarized in Supplementary Table S4. (D) The effect of CA on the spawning rate of wild type N2. Statistical details and repeats of these experiments are summarized in Supplementary Table S9.
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