4,4′-methylenediphenol was purchased from Chengdu Alfa Biotechnology Co. Ltd. (purity: 98%; 620–92-8). The worms were grown in nematode growth medium (NGM) containing sodium chloride, peptone, agar powder, cholesterol, anhydrous calcium chloride, anhydrous magnesium sulfate, and phosphate buffer. RNAi plates were prepared by adding ampicillin (96%; 7,177-48-2) and isopropyl-beta-D-thiogalactopyranoside (GC >8%; 367–93-1; Shanghai Macklin Biochemical Co., Ltd., China) to final concentrations of 100 μg/mL and 1 mM, respectively. To inhibit worm egg-laying, 5-fluoro-2′-deoxyuracil nucleoside (floxuridine, FUDR; 50–91–9; Shanghai Macklin Biochemical Co., Ltd., China) was added to the medium to a final concentration of 12 μM. The anesthetic levamisole hydrochloride (HPLC ≥98%; 16,595–80-5) was purchased from Sichuan Weikeqi-biotech Co. Ltd.; SuperKine Enhanced Antifluorescence Quencher (BMU104-CN) was supplied by Abbkine Scientific Co., Ltd.

The C. elegans lines used in this study were CL4176 {dvIs27 [myo-3p::A-Beta (1–42)::let-851 3′UTR) + rol-6(su1006)] X.}, CL2006 {dvIs2 [pCL12(unc-54/human Abeta peptide 1–42 minigene) + rol-6(su1006)]}, TJ356 {zIs356 [daf-16p::daf-16a/b::GFP + rol-6(su1006)]}, LD1 (ldIs7 [skn-1b/c::GFP + rol-6(su1006)]}, CF1553 {mu1s84[(pAD76) sod-3p::GFP) + rol-6(su1006)]}, CL2166 {dvIs19 [(pAF15) gst-4p::GFP::NLS] III}, and C. elegans N2 (wild-type strain). All worm strains were purchased from the C. elegans Genetics Center (CGC, University of Minnesota, Minneapolis, MN, United States). An uracil synthesis-deficient strain of Escherichia coli (OP50) was used as food. C. elegans CL4176 was grown and reproduced at 16°C; all other strains were grown and reproduced at 20°C.

4,4′-Methylenediphenol was weighed on an electronic precision balance, dissolved in deionized water [with DMSO (0.2%) as co-solvent] to a final concentration of 10 mM, filtered to remove bacteria, and stored at 4°C until needed. Different concentrations of the drug (0.125, 0.25, 0.5, 1, 2, and 4 mM) were generated by diluting the drug master mix with a solution containing inactivated OP50 bacteria (OD600 = 0.6, 30 min at 65°C). After mixing well, the mixture was added to the surface of the NGM, and allowed to air-dry before use. Worms in the Control group were given an equal volume of the bacterial solution. Subsequently, L1 stage-synchronized CL4176 worms were transferred to NGM plates with or without the drug (30–50 worms per plate). After incubation to the L3 stage at 16°C, the temperature was raised to 25°C, and incubation was continued for approximately another 24 h. The number of paralyzed worms was recorded every 2 h until all the worms were paralyzed. Paralysis was defined as rigid movement or only head movement following mechanical stimulation. The optimal effective concentration of the drug as determined by the paralysis assay was used the drug concentration in subsequent experiments.

L4 stage-synchronized CL4176 worms were transferred to plates with or without the drug (1 mM), which was recorded as day 0. FUDR (12 μM) was added to each plate to prevent egg-laying and larval hatching affecting the experimental counts. Three plates were run in parallel for each group (30–50 worms per plate). Thereafter, the number of surviving worms was recorded every 2 days, following which the surviving worms were transferred to fresh medium containing the same drug concentration, and culture was continued until all of the worms had died. Worms that did not respond to stimulation with a picker were considered dead. Worms that developed a bag-like phenotype and did not die within the area coated with the drug were excluded from the statistical analysis. The lifespan assay was repeated three times with no less than 60 worms each time.

L1 stage-synchronized N2 worms were cultured to the L4 stage at 20°C and then transferred to NGM plates with or without the drug (1 mM). After 10 days of incubation, worms from each group (≥10 worms per group) were transferred to PBS containing anesthetic and analyzed for lipofuscin autofluorescence under a fluorescence microscope (LSM900, Carl Zeiss, Germany). Fluorescence intensity values were determined using ImageJ software. The experiment was repeated independently three times.

N2 worms synchronized to the L4 stage were transferred to NGM plates (12 μM FUDR) with or without drug (1 mM), and the size of the worms was determined after continued incubation at 20°C for 2 days. Worms were collected with PBS buffer and anesthetized, and at least 20 worms in each group were imaged under an inverted biomicroscope (DMI1, Leica Microsystems (Shanghai) Co., Ltd.), and the pictures were saved. Worm body length (μm) was measured using Image J software. The experiments were repeated three times independently.

N2 worms synchronized at the L1 stage were transferred to NGM culture plates with or without the drug (1 mM) (~50 worms per plate) and incubated at 20°C to the L4 stage. Then, 5 mM paraquat solution (methyl viologen dichloride hydrate; 1910-42-5; Shanghai Aladdin Biochemical Technology Co., Ltd) was added to the NGM plates for 4 h, after which the worms were collected and washed three times with PBS. Subsequently, 50 μL of 10 μM DCFH-DA solution (Reactive Oxygen Species Detection Kit; S0033S; Beyotime Biotech. Inc.) was added, and the worms were incubated at 37°C for 30 min. After washing three times with PBS, 30 μL of worm-containing fluid was placed on a slide, coverslipped, and observed under a fluorescence microscope (LSM900, Carl Zeiss), and adjust the parameters, save the picture, and measure the fluorescence intensity value using Image J software. At least 10 worms in each group.

L1 stage-synchronized CL2006 worms were transferred to NGM plates with OP50 bacterial solution only, incubated to the L4 stage at 20°C, and then transferred to NGM plates with or without the drug (1 mM). After 2 days of incubation, the worms were collected in M9 buffer, washed three times, and fixed in 1 mL of 4% paraformaldehyde (pH 7.4; BL539A; Labgic Technology Co., Ltd) at 4°C for 24 h. After discarding the fixative, the worms were incubated in 1 mL of permeabilizing solution [5% β-hydrophobic ethanol [60–24-2; Shanghai Macklin Biochemical Co., Ltd], 1% Triton X-100 [9002-93-1; Meilunbio Biotechnology Co., Ltd], and 125 mM Tris [pH 7.4; 77–86-1; Beijing Solarbio Science & Technology Co., Ltd]] at 37°C for 24 h and rinsed three times with TBST solution. Then, 100 μL of the fluorescent dye thioflavin S (0.125%) (1326-12-1; Shanghai Yuanye Bio-Technology Co., Ltd) was added, followed by incubation at room temperature for 2 min. After discarding the staining solution, the worms were cleared twice with 50% ethanol, and Aβ protein deposition in the head of worms (i.e., the part before the pharyngeal pump) was observed under a fluorescence microscope (LSM900, Carl Zeiss). For imaging, 30 μL of worm-containing fluid was aspirated and placed on a slide, coverslipped, and the parameters adjusted. At least 10 worms were assessed in each group.

For all gene expression experiments involving C. elegans GFP reporter strains, L1-stage worms were incubated on NGM plates with or without the drug (1 mM) for 72 h, transferred to slides, anesthetized, and fixed in 2% levamisole. Images were captured under a fluorescence microscope (LSM900, Carl Zeiss) at 10/40× magnification and analyzed using ImageJ software. As the positive controls for the TJ356 strain, worms were cultured on plates containing blank medium for 72 h, and the temperature was then increased to 37°C for 30 min for induction. SKN-1::GFP fluorescence intensity was measured in the intestinal region of LD1 worms under the pharynx. The nuclear localization of DAF-16 was expressed as the number of fluorescence puncta per TJ356 worm (Kang et al., 2022). Finally, the whole-body fluorescence intensity associated with SOD-3::GFP and GST-4::GFP expression was evaluated in CF1553 and CL2166 worms, respectively. The experiments were independently replicated three times, with a minimum of 10 worms per group per strain.

After synchronization, the resulting eggs of CL4176 worms were transferred to culture plates coated with R13H8.1 E. coli containing daf-16 dsRNA to knock down daf-16 and incubated at 16°C. Eggs incubated on culture plates coated with the HT115 E. coli strain expressing the L4440 empty vector served as controls. After passing to the third generation, the worms were synchronized, and the resulting eggs were placed in M9 buffer and incubated at 16°C. RNAi efficiency was subsequently assessed by qPCR. Worms successfully subjected to RNAi were then transferred to NGM plates coated with a solution of either RNAi-expressing bacteria or the empty vector-expressing strain and with or without the drug (1 mM), and were cultured to the L3 stage at 16°C. Subsequently, the plates were transferred to 25°C for heat shock, which led to the expression of high levels of Aβ protein in the muscle cells of the body wall of the worms. This resulted in continuous Aβ peptide aggregation, leading to muscle paralysis in the worms. After 24 h, the number of paralyzed worms in each group was counted every 2 h until the proportion of paralyzed worms in the blank control group accounted for more than 80% of the total number of worms. Three plates were run in parallel, with a least 90 worms per group. The experiment was independently repeated three times.

The data were statistically analyzed and plotted using GraphPad Prism 8.0 and the results are expressed as means ± standard deviation. Differences between and among groups were analyzed by the t-test or one-way ANOVA, respectively. Survival was analyzed using the log-rank test. p-values <0.05 were considered significant (*p < 0.05, **p < 0.01, ***p < 0.001). The experiments were independently repeated 3 times.

Under normal conditions, the body of CL4176 transgenic worms is curved (i.e., C-shaped), and the worms can move normally in culture medium. However, when the culture temperature is increased, CL4176 worms express large quantities of the Aβ_1–42 peptide, which exerts toxic effects on the muscle cells of the animals through aggregation (Sola et al., 2015; Fu et al., 2023). To investigate the potential effect of 4,4′-methylenediphenol on the progression of Aβ toxicity-induced paralysis, we administered different concentrations (0.125, 0.25, 0.5, 1, 2, and 4 mM) of the drug to CL4176 worms and undertook a paralysis assay. The data showed that the time to paralysis was longer in all the 4,4′-methylenediphenol treatment groups than in the Control group (Figure 1A). Except for the group administered the lowest dose (0.125 mM), the time to paralysis for 50% (PT50) of the nematodes was greater in all dosing groups than in the Control group (Table 1). Although the PT50s of the 2 and 4 mM administration groups were longer than that of the 1 mM group, the difference was minimal, and the 1 mM concentration exerted a more significant effect in prolonging the overall paralysis rate of worms (Figure 1B). Based on the principle of selecting the lowest concentration among those with comparable efficacy, 1 mM was deemed to be the optimal effective concentration for this drug and was used for subsequent experiments. In conclusion, the above results indicated that 4,4′-methylenediphenol delayed the onset of paralysis in worms and had a protective effect against β-amyloid-induced toxicity.

The lifespan of worms is closely related to senescence, which plays an important role in the development of AD (Santoro et al., 2020). Here, we investigated the effect of 4,4′-methylenediphenol (1 mM) on the lifespan of AD model worms. The maximum lifespan of worms in the Control and 4,4′-methylenediphenol administration group was 22 and 26 days, respectively. Compared with the Control group, the median and mean lifespan of worms in the drug treatment group were significantly prolonged (p < 0.05 and p < 0.01, respectively), with an average lifespan extension of 20.053%. Survival curves showed a rightward shift after 4,4′-methylenediphenol treatment, indicative of a significantly prolonged lifespan in AD model worms (Table 2; Figure 2A). In addition, lipofuscin accumulates in the intestinal tract of worms as their lifespan increases, so lipofuscin levels are strongly associated with worm aging (Höhn et al., 2010). Lipofuscin fluorescence intensity was stronger in the Control group than in the 4,4′-methylenediphenol administration group, and worms in the former group accumulated significantly higher levels (p < 0.001) of lipofuscin in their bodies (Figures 2B–D). The above findings indicated that 4,4′-methylenediphenol can reduce lipofuscin levels in worms, and further suggested that the drug can delay aging in the animals.

The effect of a drug on helminth senescence can be indirectly reflected by the determination of the effect of the drug on the reproductive function of the animals (Hyun et al., 2021). The results showed that the difference in the number of offspring between the Control group and the 1 mM 4,4′-methylenediphenol administration group was not significant (p > 0.05), indicating that the drug did not affect the reproductive capacity of the worms (Figure 2E). In addition, measurements of worm body length showed no statistically significant (p > 0.05) differences in worm body length between groups on day 2 after drug administration, indicating that the drug had no effect on worm development (Figures 2F,G). Under physiological conditions, the ability of worms to pump their pharynx is indicative of their ability to feed, which, in turn, reflects the extent of senescence (Gao et al., 2015). We found that there was no effect on the twitching ability of the worms on day 4 post-drug administration (p > 0.05); in contrast, on days 6 and 8 after treatment, the number of twitching events in the worms of the 4,4′-methylenediphenol administration group was significantly higher (p < 0.001) than that of the Control group. The motility of worms also has a large correlation with lifespan (Cogliati et al., 2020). Importantly, locomotor ability is the most basic index reflecting the function of the nervous system and is widely used in the toxicological assessment of drugs and poisons (Wang et al., 2021). Our data showed that, compared with the Control group, the motility of the worms in the treatment group was enhanced on days 4, 6, and 8 post-administration (p < 0.001). In conclusion, our findings implied that 4,4′-methylenediphenol enhanced motility in AD model worms, including pharyngeal pump beating and locomotion, and also delayed the aging of the animals (Figures 2H,I).

Stress resistance refers to the ability of an organism to resist a stressful environment and there is a strong positive correlation between increased longevity and enhanced stress resistance (Tan L. et al., 2022). To investigate the effect of 4,4′-methylenediphenol on stress resistance in the nematodes, we first explored the changes in the ability of N2 worms to resist heat stress (37°C) following 4,4′-methylenediphenol administration. The results showed that the overall survival time of worms in the 4,4′-methylenediphenol (1 mM) administration group was prolonged compared with that in the Control group (p < 0.001), even after 7 h of administration (p < 0.01), indicating that treatment with 1 mM 4,4′-methylenediphenol augmented resistance to heat stress in the animals (Figures 3A,B). Next, to test the ability of the drug to protect against oxidative damage, we exposed N2 worms to juglone (300 μM), a strong oxidant that led to their deaths, and measured the survival rate of the worms. The results showed that compared with the Control group, the survival curve of worms in the 4,4′-methylenediphenol administration group was shifted to the right, and the survival rate was significantly increased (p < 0.001). After 7 h of treatment, the survival rates of worms in the Control and drug administration groups were 0 and 14.634%, respectively (p < 0.001), showing that 4,4′-methylenediphenol can improve the ability of worms to resist externally induced oxidative stress (Figures 3C,D).

When animals are subjected to a variety of harmful stimuli in vivo, ROS production is accelerated, leading to an imbalance between the oxidative and antioxidant systems, which then triggers a series of molecular-mechanical reactions. In AD model animals, it has been shown that these events contribute to Aβ accumulation, which, in turn, enhances ROS production and promotes neuronal damage and death, leading to the occurrence of AD. Accordingly, the determination of ROS levels is crucial in the assessment of the effects of AD-targeting drugs (Song et al., 2022). Here, we used paraquat (5 mM) to induce an increase in ROS contents in AD model worms and found that the average ROS fluorescence intensity in the drug administration group was significantly reduced compared with that in the Control group (p < 0.01), indicating that 4,4′-methylenediphenol inhibits the production of free radicals in worms to a certain extent, thereby reducing Aβ-induced toxicity (Figures 3E–G).

Transgenic C. elegans of the CL2006 strain become progressively paralyzed. As the worms age, Aβ gradually accumulates and forms plaques that become toxic, leading to paralysis. Here, we explored the effect of 4,4′-methylenediphenol on the formation of Aβ protein deposits using an in vivo Aβ aggregation assay (Fay et al., 1998). Thioflavin S is a homogeneous chemical mixture that can be used for the staining and observation of β-amyloid plaques in AD (DanQing et al., 2021). The results showed that there was no Aβ protein deposition in wild-type N2 worms. In CL2006 worms, however, 4,4′-methylenediphenol (1 mM) administration significantly reduced Aβ protein aggregation in the head of the worms compared with that seen with the Control treatment (p < 0.001). These findings revealed that 4,4′-methylenediphenol can reduce Aβ protein deposition in worms, thus minimizing Aβ-induced toxic effects (Figures 4A–D).

We performed a broad-spectrum TM analysis to investigate the effect of 4,4′-methylenediphenol treatment on the metabolite profile of the AD-like C. elegans CL4176 strain. First, Pearson’s correlation analysis was performed on the QC samples, and it was found that the correlation coefficient (r) of the QC samples was close to 1, which indicated that the assay process was stable and the data quality was high (Figure 5A). Secondly, the coefficient of variation (CV) distribution plots of all the samples also showed that the percentage of substances with CVs lower than 0.3 in the QC samples was higher than 75%, indicating that the experimental data were stable (Figure 5B). Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA), which combines orthogonal signal correction and PLS-DA and decomposes X-matrix information into two categories, namely, Y-related and irrelevant, and filters the discrepant variables by removing irrelevant differences (Wang et al., 2022). As shown in Figure 5C, OPLS-DA demonstrated that there was significant separation between the two groups. Next, we screened for differential metabolites using VIP >1 and p-value <0.05 as the screening criteria. As shown in the volcano plot in Figures 5D, a total of 669 differentially abundant metabolites were identified, 627 of which were downregulated and 42 upregulated. To facilitate the observation of the relative changes in metabolite content, we generated a heatmap of differential metabolite hierarchical clustering. The first-level classification of the differential metabolites in the clustering heatmap indicated that they were related to amino acids and their metabolites, organic acids and their derivatives, nucleotides and their metabolites, coenzymes and vitamins, alkaloids, flavonoids, and terpenoids (Figure 5E). Finally, we performed KEGG pathway enrichment analysis on the metabolites identified as showing differential abundance (Figure 5F). We found that the top 20 p-value-ranked pathways included ABC transporter protein; ubiquinone and other terpene quinone biosynthesis; fatty acid metabolism; unsaturated fatty acid biosynthesis; glycine, serine, and threonine metabolism; and phenylalanine, tyrosine, and tryptophan biosynthesis, among other pathways.

The insulin signaling pathway plays a key role in the regulation of lifespan. DAF-16 (FOXO transcription factor homolog) is a major regulator of the insulin signaling pathway and its overexpression can delay aging and extend lifespan in worms (Wang et al., 2016). skn-1, gst-4, and sod-3 are target genes of DAF-16, and all are associated with longevity and protection against oxidative stress (Liu et al., 2023; Wang et al., 2023). In this study, we first determined the nuclear localization of DAF-16 and the expression of SKN-1. The results showed that treatment with 4,4′-methylenediphenol accelerated the nuclear translocation of DAF-16 in strain TJ356 (p < 0.001) (Figures 6A–D), and upregulated SKN-1 expression in LD1 worms (Figures 6E–G). This suggested that the 4,4′-methylenediphenol-mediated mitigation of Aβ toxicity and enhancement of antioxidant properties was, at least partly, due to the activation of DAF-16. Next, we examined the expression of SOD-3 and GST-4. The results showed that 4,4′-methylenediphenol treatment enhanced the fluorescence intensity of SOD-3::GFP in the CF1553 strain (Figures 6H–J), and the fluorescence of the CL2166 strain expressing GST-4::GFP was also significantly increased after 4,4′-methylenediphenol treatment (Figures 6K–M). These results showed that 4,4′-methylenediphenol enhanced the expression of sod-3 and gst-4 in the worms (p < 0.001), thereby delaying paralysis onset and prolonging the lifespan of the worms.

DAF-16 acts as a major regulator of the insulin/insulin-like growth factor 1 signaling (IIS) pathway and plays an important role in mediating stress resistance, development, reproduction, metabolism, and longevity (Yang et al., 2016; Zullo et al., 2019). Because we found that 4,4′-methylenediphenol activates the nuclear translocation of DAF-16 in TJ356 worms, we next investigated whether the effect of 4,4′-methylenediphenol on paralysis onset in AD model worms was mediated through DAF-16 by knocking down DAF-16 using RNAi. To this end, we performed RNA interference by feeding to knock down DAF-16 in AD worms to verify whether 4,4′-methylenediphenol could continue to delay the paralytic process of the worms. The results showed that when DAF-16 was knocked down in the worms, there was no significant difference in the ability of the 1 mM administration group to delay the paralysis of the worms compared with the Control group (p > 0.05). In contrast, the ability to delay Aβ-induced paralysis was significantly enhanced in the 1 mM-administered group in the control group of worms in which DAF-16 was not knocked down (Figures 7A,B). This indicated that DAF-16 plays a crucial role in mediating the neuroprotective effect of 4,4′-methylenediphenol in AD model nematodes.