4-Hydroxybenzyl alcohol, 4-hydroxybenzaldehyde and 4,4-dihydroxydiphenylmethane were purchased from Chengdu Alfa Biotechnology Co., Ltd.; trolox and 2,4,6-tripyridine-s-triazine (TPTZ) were purchased from Macklin Biochemical Co., Ltd.; 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was purchased from Shanghai Yi En Chemical Technology Co., Ltd.; and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

The C. elegans strains employed in the present study were: Wild-type N2 (Bristol), the transgenic strains CL2006 {dvls2 [pCL12(unc-54/human Aβ1-42) + rol-6 (su1006)]}, CL4176 {dvIs27 [myo-3p::Aβ (1-42)::let-851 3'UTR] + rol-6(su1006) X}. Escherichia coli OP50 was obtained from the Caenorhabditis Genetics Center (University of Minnesota). CL4176 is a temperature-sensitive transgenic model in which the Serine/threonine-protein kinase 1 system is inactivated when the temperature increases from 16 to 25˚C, which leads to overexpression of Aβ in muscles and a paralysis phenotype. All C. elegans were cultured on solid nematode growth medium (NGM; consisting of 300 ml deionized water including 0.82 g peptone, Oxoid Limited; Thermo Fisher Scientific; 1.2 g NaCl, Tianjin Fengchuan Chemical Reagent Co., Ltd.; 5.15 g agar, Beijing Solarbio Science & Technology Co., Ltd.; autoclaved and supplemented 1 mM MgSO₄, 1 mM CaCl₂, Tianjin Fengchuan Chemical Reagent Co., Ltd.; 12.9 mM cholesterol solution, Macklin, Inc.) containing E. coli OP50. CL4176 was maintained at 16˚C, while N2 and CL2006 were maintained at 20˚C.

Gastrodia elata Bl. was purchased from Yunnan Huide Pharmaceutical Co., Ltd. The samples were collected from artificially cultivated Gastrodia elata in Xiaocaoba (Yiliang County, Zhaotong, Yunnan, China), and analyzed by Associate Professor Zili Yin, Yunnan University of Chinese Medicine (Kunming, China). Drug extraction was performed as previously described (19). The specified amount of Gastrodia elata Bl. was extracted three times with 95% ethanol under heating reflux at 85˚C. The organic solvent was evaporated under reduced pressure and the remaining extract was dissolved in deionized water and extracted with ethyl acetate three times. Samples were concentrated in a rotary evaporator at 60˚C under reduced pressure, and dried. The obtained EEGE was analyzed by HPLC (Agilent 1290 InfinityIIHPLC system; Agilent Technologies, Inc.). The gradient elution process was performed as follows on a ZORBAX SB-C18 column (4.6x250 mm; 5 µm; Agilent Technologies, Inc.). Mobile phase A was acetonitrile, and mobile phase B was water (0-25 min, from 13% A to 58% A; 25-26 min, from 58% A to 100% A). The detection wavelength was 221 nm. EEGE and standards were dissolved in methanol, the flow rate was 1.0 ml/min, and the column was at room temperature, with a sample quantity of 10 µl.

Total phenolic content of EEGE was measured by the Folin-Ciocalteu method (20). Briefly, gallic acid of known concentration was used as a standard and Folin-Ciocalteu reagent was allowed to react with different concentrations of standard and sample for 5 min, Na₂CO₃ was then added, mixed thoroughly, and incubated at room temperature in the dark for 2 h. Absorbance at 760 nm was then measured. The total flavonoid content was determined with reference to Navarro-Hortal et al (21). Rutin was used as a standard. The samples were reacted with NaNO₂ for 6 min and then incubated with AlCl₃ for 5 min (at room temperature). Finally, 4 ml NaOH was added, thoroughly mixed and reacted at room temperature for 5 min. Absorbance was determined at 510 nm. The results for total phenolic and flavonoid content are expressed as mg gallic acid equivalent/g dry extract and mg rutin equivalent/g dry extract, respectively. The total antioxidant capacity of EEGE was evaluated by three methods including ferric reducing antioxidant power (FRAP), DPPH and ABTS. In the FRAP method (22), when iron and TPTZ are complexed in sodium acetate solution, the color changes and the absorbance is measured at 593 nm, which can be used to evaluate the ability of samples to reduce Fe³⁺ to Fe²⁺. DPPH detection was performed according to Qadir et al (23). At a wavelength of 517 nm, the stronger the reducing power of the compound in the sample, the faster the color elimination of DPPH. Finally, oxidation of ABTS by K₂S₂O₈ results in green ABTS⁺ free radicals (24). At a wavelength of 734 nm, the stronger the anti-oxidation ability of the sample, the faster the color elimination of ABTS⁺ radicals. Absorbance was measured using a Varioskan Flash instrument (Thermo Fisher Scientific, Inc.). Each experiment was repeated three times. The results are expressed as mM trolox equivalent/g dry extract.

CL4176 worms synchronized to the L1 stage were transferred to 35 mm culture plates with or without EEGE (0.125, 0.25, 0.5, 1 and 2 mg/ml) and cultured at 16˚C for 36 h. The temperature was then raised to 25˚C for transgene induction. After culture at 25˚C for 24 h the paralyzed C. elegans were observed every 2 h. Worms were considered paralyzed when they did not move and did not respond to platinum wire stimulation.

L4 stage CL4176 nematodes were transferred to NGM plates with or without EEGE. Three replica plates and no less than 70 nematodes for each group were prepared. The nematodes were incubated at 16˚C. To prevent the influence of egg and larval development on the nematode counts, oviposition was inhibited by adding 12 mM fluorouracil to the NGM medium. The number of nematodes surviving on each culture plate was counted every 2 days until all nematodes died. C. elegans death was determined by the absence of movement and swallowing, and no reaction after being touched by a platinum wire. Nematodes that burrowed into the agar or climbed the wall of the plate and died of desiccation were excluded from the statistics. The experiment was repeated independently three times.

L4 stage N2 nematodes were transferred to NGM plates with or without EEGE. Three replica plates and no less than 70 nematodes for each group were prepared. The nematodes were incubated at 20˚C for 48 h. The temperature was then changed to 35˚C, and the number of nematodes surviving in each culture plate was counted every hour until all nematodes died. C. elegans death was determined by the absence of movement and swallowing, and no reaction after being touched by a platinum wire. The experiment was repeated independently three times.

The effect of EEGE on juglone-induced oxidative stress was evaluated using wild-type N2 worms (10). N2 nematodes were cultured to the L1 stage after synchronization, and transferred to NGM plates with or without EEGE. A total of three replica plates for each group were prepared. The nematodes developed to the L4 stage at 20˚C and were then transferred to NGM plates supplemented with juglone (300 µM) (Shanghai Yuanye Biotechnology Co., Ltd.). The survival of the nematodes was observed every hour until all had died. Worms that were rigid and unresponsive to light and slight vibrations were recorded as dead.

The reproductive and locomotor abilities of C. elegans are physiological markers related to senescence (25). After synchronization, N2 nematodes were cultured at 20˚C to the L4 stage and transferred to NGM medium with or without EEGE (0.5 or 1 mg/ml), with three parallel plates in each group and at least 15 nematodes in each plate. After culture for 48 h, nematodes were transferred to blank NGM medium to observe the number of sinusoidal movements within 20 sec. A reproduction assay was performed according to Meng et al (26); two N2 nematodes at the L4 stage were selected from each group and fed separately at 20˚C (three replica plates were prepared for each group). This was recorded as the first day of the reproduction assay. They were transferred to new plates every 24 h until the reproductive capacity of the nematodes was lost. The egg-laying boards were incubated at 20˚C for 48 h and the number of offspring was counted (in this experiment, the number of nematode offspring indirectly reflected the number of eggs laid).

Cytoplasmic ROS were detected as reported (27) after nematodes were incubated for 4 h with a fluorescent probe, 2,7-dichlorofluorescein diacetate at 25 µM. The probe becomes fluorescent after combining with reactive oxygen species in the cytoplasm. N2 nematodes were synchronized and cultured to the L1 stage, then transferred to an NGM culture plate with or without EEGE, and developed to the L4 stage at 20˚C. Then, 5 mM paraquat (Aladdin) was added to the NGM plate and the nematodes incubated for 4 h. The nematodes were then rinsed with M9 buffer solution, placed on a slide and covered with a cap. The nematodes were observed using a positive fluorescence microscope (Axio Scope A1; Carl Zeiss AG). The fluorescence intensity was quantified using ImageJ v1.8.0. software (National Institutes of Health).

Transgenic C. elegans strain CL2006 synchronized to the L4 stage (early adult stage) was inoculated on NGM plates with or without EEGE. N2 worms were used as a negative control for Aβ deposition (28). After incubation at 20˚C for 48 h, worms were collected with M9 buffer and fixed in 4% paraformaldehyde/PBS (pH 7.4) (cat. no. BL539A; Biosharp Life Sciences) at 4˚C for 24 h. The worms were then incubated in 5% β-mercaptoethanol (cat. no. M828395; Macklin, Inc.), 1% Triton X-100 (cat. no. MB2486; meilunbio) and 125 mM Tris (pH 7.4) (cat. no. T8060; Beijing Solarbio Science & Technology Co., Ltd.) at 37˚C for 24 h. The worms were then stained with 0.125% thioflavin S (cat. no. S19293; Shanghai Yuanye Biotechnology Co., Ltd.) in 50% ethanol at room temperature for 2 min, and then rinsed in 50% ethanol 2-3 times. The worms were then placed on a glass slide for observation under a laser scanning confocal microscope (LSM900; Carl Zeiss AG). The amount of thioflavin S deposition in the prepharyngeal region of each nematode was scored to quantify amyloid deposits.

With reference to Song et al (6), CL4176 nematodes were synchronized at 16˚C for 48 h, cultured at 25˚C for 40 h then rinsed twice with M9 buffer to remove E. coli. The nematodes were then homogenized and protein abundance was measured by the bicinchoninic acid assay (Beyotime Institute of Biotechnology). The levels of MDA, SOD and CAT were determined using Total Superoxide Dismutase Assay (Beyotime Institute of Biotechnology), Catalase Assay (Beyotime Institute of Biotechnology) and Malondialdehyde Detection (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. ROS accumulation was determined with reference to Wang et al (29). Briefly, 50 µl of nematode supernatant was added to each well of a 96-well plate and 50 µl of 100 µM DCFH-DA solution (a fluorescent probe) (Beyotime Institute of Biotechnology) was added to give a final DCFH-DA concentration of 50 µM, The solutions were thoroughly mixed by shaking for 30 sec. Fluorescence detection was performed using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Detection was conducted once every 10 min, and ROS changes within 100 min were counted.

Gene expression in transgenic CL4176 nematodes treated with EEGE (1 mg/ml) and controls was analyzed by RNA-seq. Extraction and purification of total RNA, library construction and sequencing were performed at Beijing Fruit Shell Biotechnology Co., Ltd. using the Illumina Novaseq 6000 system (Illumina, Inc.). The quality of the data sets was evaluated using an Agilent bioanalyzer 2100 (Agilent Technologies, Inc.). Transcript levels were estimated using fragments per kilobase of transcript per million mapped reads values to allow different genes or samples to be compared. Settings: Two-fold change in expression levels and a false discovery rate with a P-value <0.05 were used to screen the RNA-seq data for differentially expressed genes (DEGs). All analyses were performed at Beijing Fruit Shell Biotechnology Co., Ltd. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DEGs was carried out using the DAVID (https://david.ncifcrf.gov/) online platform. The R package ggplot2 (version 3.3.2) (30) was used to generate volcano, GO and KEGG bubble maps to visualize the distribution of DEGs.

To verify the RNA-seq results, synchronized L1 stage CL4176 nematodes were transferred onto NGM plates with or without EEGE (1 mg/ml) (~500 nematodes per plate) and cultured at 15˚C for 36 h. The temperature was then raised to 25˚C, and the culture continued for ~40 h. The nematodes were then collected with M9 buffer into an EP tube, and washed three times. Total RNA was extracted from nematodes using an RNA extraction kit (Tiangen Biotech Co., Ltd.) according to the manufacturer's instructions. RNA concentration and purity were measured using an ultra-micro spectrophotometer (SMA6000; Merinton Instrument, Ltd.). cDNA (Promega Beijing Biotech Co., Ltd.) was generated by RT-PCR in a PCR instrument (Veriti™ 96-Well Fast Thermal Cycler; Applied Biosystems; Thermo Fisher Scientific, Inc.), and then the target mRNA was quantified (GoTaq^® qPCR and RT-qPCR Systems, Promega Beijing Biotech Co., Ltd.) using a real-time PCR instrument (C1000 Touch PCR; Bio-Rad Laboratories, Inc.). RT-qPCR was performed with the following cycling conditions: 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec, 55˚C for 30 sec, 72˚C for 30 sec and then maintained at 4˚C. Relative gene expression was calculated by the 2^-∆∆Cq method, using β-actin as a housekeeping gene (31). The analysis was performed in triplicate for each group. Primer 3 Plus (https://www.primer3plus.com/) was used to design the primers and they were synthesized by TsingKe Biological Technology. The primers are listed in Table SI.

Using GraphPad prism 8.0.0 (GraphPad Software, Inc.) statistical software for analysis and processing, if the data conformd to the normal distribution and the variance was uniform (P>0.05), ANOVA followed by Bonferroni's multiple comparison test was used. For lifespan and paralysis assays, Kaplan Meier survival was utilized and P-values were calculated using the log-rank test. P<0.05 was considered to indicate a statistically significant difference, and all values were expressed as means ± standard deviation. All experiments were repeated three times.

As revealed by HPLC analysis, three main compounds were detected in EEGE, namely: 4-Hydroxybenzyl alcohol, 4-hydroxybenzaldehyde, 4,4-Dihydroxydiphenylmethane (Fig. 1A and B). All three compounds have benzene rings in their structures (Fig. 1C). The total phenols, total flavonoids, total antioxidant capacity (DPPH, FRAP and ABTS) and relative contents of main compounds of EEGE are presented in Table I. These results indicate that EEGE has a strong antioxidant capacity in vitro.

The present study first studied the effect of different concentrations of EEGE (0.125, 0.25, 0.5, 1 and 2 mg/ml) on the Aβ-induced toxicity of the transgenic C. elegans strain CL4176. None of the concentrations were lethally toxic to CL4176 nematodes (Fig. 2A). Compared with the control group, 0.125 mg/ml EEGE had no significant effect on CL4176 nematodes (P>0.05). EEGE (0.25, 0.5, 1 and 2 mg/ml) could prolong the paralysis time of nematodes. The drug effect was dose-dependent within the concentration range of 1 mg/ml, and the efficacy was weakened when the drug concentration reached 2 mg/ml. Therefore, 1 mg/ml was the optimal concentration for EEGE. These results suggested that the potential of EEGE to protect CL4176 from Aβ-induced toxicity.

Senescence plays an important role in the development of AD. Survival analysis showed that EEGE treatment significantly shifted the survival curve of CL4176 to the right (Fig. 2B). Compared with the control group, 0.5 and 1 mg/ml EEGE increased the maximum lifespan of CL4176 nematodes by 10.0% (P<0.01), 20.0% (P<0.01), respectively. These data indicated that EEGE could delay the senescence of CL4176 nematodes.

Compared with the control group, 0.5 and 1 mg/ml EEGE could delay the survival rate of N2 nematodes under heat stress induced by high temperature (P<0.01; Fig. 2C), although the mortality time of C. elegans could not be prolonged, the overall survival rate of C. elegans was increased. The results showed that EEGE could improve the heat stress resistance of C. elegans.

The present study further revealed that EEGE could also protect wild-type N2 nematodes from oxidative stress produced by juglone, and after 6 h of juglone stress, all nematodes in the control group died, which was not significantly different compared with the control group with 0.5 mg/ml EEGE (P>0.05), and the maximum survival rate of the worms in the 1 mg/ml EEGE group was significantly improved by 16.7% (P<0.01; Fig. 2D). The results showed that the worms subjected to EEGE intervention exhibited significant protection against oxidative stress induced by juglone (300 µM).

As the locomotor behavior of C. elegans decreased with age, the present study investigated whether EEGE affected the locomotor ability of C. elegans. Compared with the control group, EEGE intervention increased the activity of N2 wild-type nematodes (Fig. 2E), and the effect of 1 mg/ml EEGE was the most significant (P<0.01). Nematodes begin to lay eggs when they enter the adult stage. As the nematodes gradually aged, their egg-laying rate gradually decreased and reached the peak of growth on the 3rd day of adult life. Compared with the blank group, the egg laying rate of the EEGE group was not significantly different on Days 1 to 3 (P>0.05; Fig. 2F), and the egg laying rate was significantly increased on Days 4 to 6. 1 mg/ml EEGE could significantly increase the total oviposition of nematodes (P<0.05), while 0.5 mg/ml EEGE had no statistical significance (P>0.05). The results showed that EEGE could increase the number of nematode progeny and improve the reproductive ability of nematode.

Paraquat can induce the increase of free radicals in C. elegans, and EEGE has strong antioxidant capacity in vitro (27). The present study further determined the effect of EEGE on ROS in C. elegans. The higher the amount of green fluorescence in nematodes, the more ROS accumulation. Compared with the control group, the green fluorescence in EEGE group was decreased (Fig. 3A), and the fluorescence intensity of ROS per unit area was significantly decreased (P<0.01; Fig. 3B). The results showed that EEGE could inhibit the generation of free radicals and improve the antioxidant capacity of C. elegans.

The present study observed the formation of amyloid fibrils using the thioflavin-S fluorescence method to study the effect of EEGE on the aggregation of Aβ. The transgenic C. elegans strain CL2006 used in the present study expressed Aβ protein fragments that are associated with the development of AD. C. elegans demonstrated that the expression and aggregation of Aβ in muscle led to progressive paralysis. CL2006 were stained with triterpenes for Aβ at the end of EEGE treatment (showing a green fluorescent spot). Fluorescence imaged of the heads of CL2006 nematodes demonstrated that, compared with untreated worms (negative control), Aβ deposition in C. elegans treated with EEGE was significantly decreased. Wild N2 strain has no Aβ deposition in the whole animal (Fig. 3C). EEGE (0.5 and 1 mg/ml) significantly reduced the number of Aβ oligomers (P<0.01; Fig. 3D), and these results indicated that EEGE directly inhibited the aggregation and deposition of Aβ in transgenic nematode muscle cells, thereby delaying nematode paralysis.

Oxidative stress has been shown to play an important role in Aβ-induced toxicity (32). The present study investigated the effects of EEGE on Aβ-induced SOD, CAT, MDA and ROS. As shown in Fig. 4, compared with the control group, EEGE was able to increase the CAT and SOD activities in nematodes (P<0.01; Fig. 4A and B), reduce the MDA level in nematodes (P<0.01; Fig. 4C), and inhibit the rising trend of ROS in vivo (P<0.01; Fig. 4D), indicating that after intervention with EEGE, the expression of antioxidant enzymes such as CAT and SOD in nematodes was increased, the accumulation of lipid peroxides was reduced, and the generation of free radicals was reduced to improve the antioxidant capacity of the body, thereby reversing the symptoms of AD to exert neuroprotective function.

A total of 763 DEGs were identified by RNA-Seq analysis, including 145 upregulated genes and 618 downregulated genes (Fig. 5A). The present study demonstrated the biological process in GO analysis. GO analysis showed that the regulatory mechanisms of EEGE involved 25 biological processes such as ‘innate immune response’, ‘transmembrane transport’ and ‘lipid metabolic process’ (Fig. 5B). In addition, nine pathways were identified by KEGG enrichment analysis, which were related to, for example, ‘metabolic pathways’, ‘lysosome’ and ‘longevity regulating pathway-worm’ (Fig. 5C). The results demonstrated that the treatment of diseases with complex pathogenesis by a single target is limited, highlighting the advantages of multi-component and multi-target treatment of diseases by traditional Chinese medicine.

To verify the results of RNA-Seq, qPCR analyses were performed. A total of 11 genes involved in nematode longevity, oxidative stress, immunity, aging and regulation of Aβ protein were selected for verification. These 11 genes and their functions were considered to be closely related to AD pathogenesis (Table II). Compared with the control group, the expression levels of gst-4, gst-25, hsp-12.3, hsp-12.6, ugt-37 and ugt-63 genes were upregulated (Fig. 6A) and the expression levels of fat-7, ins-7, ins-23, rgba-1 and dod-22 genes were downregulated (P<0.05) (Fig. 6B). These 11 genes each showed the same tendency as observed in the RNA-Seq experiments, and the present study inferred that the EEGE regulation mechanism was likely related to the insulin pathway based on the function and characteristics of the genes.