N. nucifera leaves were procured from Yongyi TCM Co., Ltd. (Hebei, China) and deposited in Southwest Medical University with a voucher specimen (No. SWMU-VS202211). To obtain the ethanolic extract of N. nucifera leaves, approximately 100 g of the leaves was meticulously chopped and then pulverized to achieve a particle size ranging from 40 to 60 mesh. The resulting powder was subjected to extraction thrice using 95% ethanol (500 mL each time) for a total duration of 2.5 h at 65°C. The resultant liquids from these extractions were amalgamated, sifted, and then concentrated in vacuum using a rotary evaporator. This crude concentrate was later redissolved in dimethylsulfoxide (DMSO) for further analysis. All subsequent experimental solutions were prepared by diluting this stock after ensuring that the final DMSO concentration was below 0.1%.

The chemical composition analysis of NLE involved an ultrahigh-performance liquid chromatography system equipped with diode array detection (Shimadzu, Kyoto, Japan) and coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-DAD-Q/TOF-MS/MS). Separation of NLE component was performed on an Agilent Zorbax EcLipse Plus C18 column (1.8 μm, 100 mm × 2.1 mm) at a flow rate of 0.3 mL/min, with the column maintained at 40°C. The mobile phase comprising eluent A (0.1% formic acid in water, v/v) and eluent B (0.1% formic acid in acetonitrile, v/v) was obtained in accordance with a previously established methodology (Pei et al., 2021), where the electrospray ionization (ESI) parameters were set in accordance with prior specifications (Pei et al., 2021). Subsequent data analysis was performed using Peak View^® 1.4 software (AB SCIEX, Foster City, CA, United States).

To assess the impacts of NLE on Aβ-induced pathology, antioxidant capacity, and autophagy activation in worms, multiple C. elegans strains were used in the current study, namely Bristol N2, CL802 [rol-6(su1006)] II, CL4176 dvIs27 [Pmyo-3:human A-beta 1–42; let-851 3′UTR; rol-6(su1006)], CL2006 dvIs2 [Punc-54:human A-beta 3–42; pRF4 rol-6(su1006)], CL2331 dvIs37 [myo-3p:GFP::A-beta (3–42) + rol-6(su1006)], CL2355 pCL45 [Psnb-1:human A-beta 1–42:3′UTR (long); Pmtl-2:GFP], CL2122 dvIs15 [(pPD30.38) unc-54(vector) + (pCL26) mtl-2:GFP], SJ4100 zcIs13[hsp-6:GFP], SJ4005 zcIs4 [hsp-4:GFP] V, CL2166 dvIs19 [(pAF15)gst-4p:GFP::NLS] III, CF1553 muIs84 [(pAD76) sod-3p:GFP + rol-6(su1006)], DA2123 adIs2122 [lgg-1p:GFP:lgg-1 + rol-6(su1006)], and BC12921 sIs10729 [rCes T12G3.1:GFP + pCeh361]. All these strains were procured from the Caenorhabditis Genetics Center (CGC, University of Minnesota, Minneapolis, MN, United States). The worms were then cultured at 20°C, except for CL4176, CL2331, and CL2355, which were grown at 15°C on nematode growth medium (NGM) agar plates supplemented with Escherichia coli (either OP50 or transformed HT115). To achieve synchronized L1 larvae, the embryos were isolated via a bleaching solution and subsequently incubated in M9 buffer. Post incubation, the L4 larvae were placed on NGM plates infused with 5 mg/L 5-fluoro-2′-deoxyuridin (FUDR, sourced from Sigma) to prevent offspring hatching.

Paralysis assessments were performed with the CL4176 and CL2006 worms based on methodologies reported in a previous study (Long et al., 2023b). Initially, synchronized L1 larvae of the CL4176 worms were cultured on NGM plates at 15°C for 36 h. Then, they were transferred to a 25°C setting to trigger accelerated Aβ_(1–42) protein expression culminating in paralysis. On the contrary, the CL2006 larvae were raised at 20°C until they attained the late L4 stage. Thereafter, they were relocated to fresh NGM plates containing 5 mg/L FUDR to prevent egg hatching. For paralysis detection, a platinum wire was utilized to gently tap the worm heads. Worms with mobile heads but immobile bodies were designated as “paralyzed.” Each experiment necessitated a minimum of 50 worms per group, and the paralysis assessments were performed at least three times. For the rolling movement assay of the CL4176 worms, the nematodes were subjected to treatment with or without NLE for a duration of 24 h. Subsequently, the numbers of rolls made by the nematodes were counted over a period of 20 s. Each experiment required 15 worms per group, and the assay was conducted at least three times.

To assess the impact of NLE on Aβ aggregation in worms, the CL2331 strain that constitutively expresses the Aβ_(3–42)-GFP fusion protein in its body wall muscle was utilized. This continuous expression of Aβ_(3–42)-GFP enables real-time visualization of Aβ aggregation in vivo. Initially, the synchronized L1 larvae were treated with NLE at 15°C until they progressed to the late L4 stage. Thereafter, they were shifted to a 25°C environment and exposed to NLE for an additional 48 h. Upon treatment completion, these worms were harvested using M9 buffer and positioned onto slides equipped with 2% agarose foundation. Fluorescent images of the nematodes were then captured using a positive fluorescence microscope (Leica DM6B, Leica Microsystems GmbH, Germany), and the images were magnified to count the Aβ_(3–42) aggregates within the worms. Each experiment involved a minimum of 20 worms per group, and the aggregation assessments were performed at least three times.

The food-sensing behavior assay was performed to assess the effects of Aβ on the functions of neurons in C. elegans. Initially, the synchronized L1 larvae of the CL2355 worms that express the human Aβ_(1–42) protein throughout their neurons and control worms (CL2122) were maintained at 15°C for 36 h. Thereafter, the temperature was increased to 25°C, and the worms were further incubated for 36 h. Subsequently, the worms were repositioned centrally on NGM agar plates that either contained or lacked E. coli OP50. After 2 min, their locomotion rates were noted in 20-s intervals. A specific formula was applied to compute the slowing rate as (N _{without food}–N _{with food})/N _{without food}, where N indicates the total body bends in the presence or absence of bacteria. Each experiment involved 20 worms per group, and the behavior examinations were conducted at least thrice.

For the thermal tolerance assays, synchronized L1 larvae of the N2 worms were shifted to fresh NGM plates with or without NLE until they reached the late L4 stage. Then, the worms were moved to new NGM plates containing FUDR to prevent offspring hatching and were maintained at 20°C for 5 days. After this treatment, the environmental temperature was increased to 35°C, and the non-responsive worms (assessed through platinum wire touch) were recorded hourly. For the oxidative stress resistance experiments, the synchronized L1 larvae were initially placed on fresh NGM plates with NLE and allowed to grow at 20°C until they reached the late L4 stage. They were then transferred to new NGM plates containing 50 mM H₂O₂ to induce acute oxidative stress. Hourly assessments were then conducted, with the worms unresponsive to platinum wire touch being classified as deceased. At least 50 worms per group were required in each experiment, and each of these tests was replicated thrice. The survival patterns were analyzed using the Kaplan–Meier approach, while the statistical relevance was evaluated using the log-rank test by employing GraphPad Prism 9.0 toolkit.

To quantify the ROS levels in the worms, NLE-treated CL4176 or CL802 (control worms) were gathered and exposed to 100 μM dihydroethidium (DHE) staining in a 500 μL solution for 1 h at 20°C. After immobilization with 10 mM sodium azide, the worms were mounted on glass slides using 2% agarose pads. Fluorescent images of the worms were then captured using a positive fluorescence microscope, and ImageJ software was employed to measure the red fluorescence intensity to serve as an indicator of ROS level. This quantification involved measuring the intensities of the segmented regions of interest in the worms and normalizing them with respect to the body area to calculate the average pixel intensity. Each experiment was performed in triplicate, and 15 worms were assessed in each replication. To evaluate the SOD, GSH-Px, and CAT activities in the worms, NLE-treated CL4176 worms were collected, ground in phosphate-buffered saline (PBS), and centrifuged to collect the supernatant. The supernatant was then used to assay the SOD, GSH-Px, and CAT activities with commercial test kits from Beyotime Biotechnology in China as per manufacturer instructions. Each experiment was performed in triplicate to ensure the robustness and reliability of the results.

The fluorescence intensity quantification assay was conducted to assess the impacts of NLE on the expressions of various proteins, including hsp-4::GFP, hsp-6::GFP, sod-3p::GFP, and gst-4p::GFP in the transgenic worms SJ4005, SJ4100, CF1553, and CL2166, respectively. In brief, the synchronized L1 worms were transferred to NGM plates containing 200 μg/mL NLE and incubated at 20°C for 2 days. Subsequently, the treated worms were collected in M9 buffer and placed on agarose pad slides with 10 mM sodium azide. Representative images of the worms were captured using a positive fluorescence microscope, and the fluorescence intensity was quantified using ImageJ software by measuring the intensities of the segmented regions of interest in the worms and normalizing them to the body areas to calculate the average pixel intensities. Each experiment involved 15 worms per group, and the assay was repeated for at least three independent experiments.

The impacts of NLE on autophagy initiation were evaluated in the BC12921 and DA2123 strains by monitoring the green fluorescence protein (GFP) expressions associated with p62/SQST-1-GFP and quantifying the GFP-tagged LGG-1/Atg8 puncta in the worms in accordance with established protocols (He et al., 2023). In brief, BC12921 and DA2123 worms were cultured on NGM plates with or without NLE or rapamycin (Rap, used as a positive control) at 20°C for 48 h. After treatment, the worms were collected in M9 buffer and placed on glass slides with 2% agarose pads to capture representative images using a fluorescence microscope. The fluorescence intensity representing p62 expression in the BC12921 strain was determined by measuring the intensities within the segmented regions of interest in the worms and normalizing them with respect to the body area to calculate the average pixel intensity. GFP::LGG-1/Atg8 puncta quantification in the DA2123 strain was conducted by direct counting. Each group in every experiment consisted of at least 20 worms, and the experiments were replicated thrice independently. To ascertain the protein levels of p62/SQST-1-GFP and ratios of phosphatidylethanolamine (PE) conjugated GFP::LGG-1 (PE-GFP::LGG-1) to non-lipidated GFP::LGG-1 in C. elegans, the BC12921 and DA2123 worms were subjected to 48 h of treatment with NLE. Subsequently, the worms were collected using M9 buffer and employed in a Western blotting assay following established protocols from previous studies (Chen Y.-L. et al., 2019).

The RNAi assays were performed by exposing the worms to bacteria expressing RNAs targeting specific genes, consistent with established methodologies (Grishok et al., 2000). In brief, RNAi bacteria of bec-1 and vps-34 genes as well as control bacteria HT115 with the empty L4440 vector were cultured overnight at 37°C in Luria–Bertani liquid supplemented with ampicillin (100 mg/L). Subsequently, the bacteria were collected and spread onto NGM plates containing 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce double-stranded RNA synthesis. The synchronized L1 larvae were then placed on the IPTG-treated NGM plates infused with either NLE or DMSO. The RNAi clones targeting bec-1 and vps-34 were obtained from Ahringer’s RNAi library, and the interference effects of these target genes in C. elegans have been elucidated in a previous study (Zhu et al., 2023).

Statistical evaluations were performed on data collated from at least three separate experiments. For evaluating the paralysis frequency and stress resilience, the Kaplan–Meier survival curves were employed. The significances in these contexts were determined by the log-rank test. The other data sets are presented in terms of mean values and their standard deviations (SDs). The p-values were analyzed by the two-tailed t-test. All statistical analyses were conducted using GraphPad Prism version 9.0, where p-values less than 0.05 were considered statistically significant.

In this study, we characterized the bioactive constituents of NLE using UHPLC-DAD-Q/TOF-MS/MS to ensure sample quality. Representative base peak chromatograms (BPCs) of the NLE in the positive and negative ion modes for this analysis are depicted in Figure 1A. A comprehensive list of the components identified in NLE, including their chemical names, molecular formulas, masses, and retention times, is presented in Table 1. The primary constituents encompass a diverse array of alkaloids, flavonoids, phenols, and other substances, such as nuciferine, leonticine, dauriciline, sinomenine, chrysoeriol, and kaempferol. These findings are aligned with the components identified in a previous study (Pei et al., 2021).

To evaluate the potential of NLE in mitigating Aβ-induced toxicity, the transgenic C. elegans strain CL4176 was employed. This strain features temperature-sensitive expression of the human Aβ_(1–42) protein in its body wall muscle cells under the control of the muscle-specific unc-54 promoter, resulting in the formation of toxic aggregates and progressive paralysis (Wu et al., 2006). Following treatments with various concentrations of NLE from 25 μg/mL to 500 μg/mL, the paralysis rates of these worms were assessed. The findings depicted in Figures 1B,C reveal that a concentration of 200 μg/mL of NLE was most effective in reducing paralysis. Consequently, this concentration was chosen for subsequent anti-AD studies. Additionally, the NLE treatment showed decreased paralysis rates in the CL4176 worms at multiple time points (33 h, 36 h, and 39 h) after shifting the temperature to 25°C (Figure 1D). To further substantiate these findings, another transgenic strain CL2006 characterized by continuous Aβ_(1–42) expression was utilized. The paralysis assay revealed that NLE supplementation decelerated the progression of paralysis in the CL2006 worms (Figure 1E). Recognizing the potential negative impact of Aβ accumulation on muscle function, the physical effects of NLE on the CL4176 worms were assessed by quantifying the number of rolls. The CL4176 strain featuring a rolling coinjected marker results in rolling of the transgenic nematodes instead of sinusoidal movements. The rolling movement assay of CL4176 demonstrated that NLE treatment enhanced mobility compared to untreated worms (Figure 1F), as evidenced by the increased number of rolls in an interval of 20 s, indicating its potential to alleviate Aβ-induced motor deficits in C. elegans. Furthermore, the impact of NLE on neurotoxicity induced by the Aβ protein was investigated. In the transgenic strain CL2355 expressing the human Aβ_(1–42) protein throughout the neurons (DanQing et al., 2021), it was observed that the worms faced challenges in food searching relative to the control strain CL2122. However, NLE administration significantly improved their food-searching capabilities, as indicated by the recovered slowing rate (Figure 1G). Overall, these results demonstrate that NLE can counteract Aβ-induced toxicity in C. elegans.

Previous studies have emphasized that the early accumulation of Aβ aggregates in the brain are precursors to AD and that these often manifest years before the emergence of distinct clinical symptoms (Hampel et al., 2021). This study investigates the impacts of NLE on Aβ accumulation in C. elegans. Specifically, the CL2331 strain was utilized as it expresses the human Aβ_(3–42) protein combined with GFP in a temperature-sensitive manner in its muscle cells. Fluorescence analysis showed that there was a significant reduction in Aβ aggregates in the NLE-treated CL2331 worms compared to the untreated worms (Figures 2A,B), suggesting the effectiveness of NLE in obstructing in vivo Aβ aggregation. Abnormal Aβ aggregates in AD have been linked to mitochondrial dysfunction, subsequently leading to oxidative stress and neuronal cell death (Eckert et al., 2003). To explore NLE’s protective properties, the DHE staining method was employed to quantify the ROS levels stemming from the Aβ aggregates. The data illustrated in Figures 2C,D indicate increased ROS generation in the CL4176 worms compared to the wild-type strain CL802, probably owing to Aβ aggregation. However, NLE treatment noticeably reduced the ROS levels in both worm strains. Additionally, the activities of key antioxidant enzymes, including SOD, GSH-Px, and CAT, were diminished in the CL4176 worms than the CL802 worms but were restored upon NLE treatment (Figures 2E–G). These findings collectively underscore the dual capability of NLE to inhibit Aβ aggregate formation and mitigate oxidative stress induced by these aggregates in C. elegans.

As organisms age, their ability to withstand external stressors diminishes, exacerbating the aging process (Long et al., 2022). To investigate whether NLE enhances stress resistance in C. elegans, experiments involving oxidative and heat stresses were conducted. In the oxidative stress assay, N2 worms underwent a pretreatment phase with NLE for 2 days at 20°C, followed by exposure to fresh NGM plates containing 50 mM of H₂O₂ to induce acute oxidative stress. Remarkably, the application of NLE resulted in a substantial extension of the lifespans of the worms, as evidenced in Figure 3A. Additionally, NLE treatment incited elevated expressions of sod-3p::GFP and gst-4p::GFP fusion proteins in transgenic strains CF1553 and CL2166, respectively (Figures 3C,D). These proteins are well-recognized markers associated with oxidative stress resistance in C. elegans. In the heat stress assay, N2 worms were pretreated with NLE for 5 days at 20°C before being exposed to a temperature of 35°C. As illustrated in Figure 3B, NLE treatment significantly increased the survival rates of the worms subjected to thermal stress. To determine the underlying mechanisms, an examination was conducted on whether NLE influenced the expression of heat shock proteins (HSPs) that are known for their close association with heat stress resistance (Jacob et al., 2017). The findings revealed that NLE treatment substantially upregulated the expressions of HSP-4:GFP and HSP-6:GFP fusion proteins in transgenic strains SJ4005 and SJ4100, respectively (Figures 3E,F). Collectively, the results provide compelling evidence that NLE treatment fortifies the resistance of C. elegans to both oxidative and thermal stressors, indicating its potential as an enhancer of stress resilience in this model organism.

The process of autophagy has garnered considerable attention in contemporary research, with studies underscoring its paramount significance in counteracting the adverse effects of aging and related diseases (Hughes et al., 2020). In this study, the potential of NLE to modulate autophagy was investigated using transgenic C. elegans models BC12921 and DA2123. The BC12921 model manifests a p62/SQST-1-GFP fusion protein, which diminishes during elevated autophagy activity (He et al., 2023). Fluorescence evaluations revealed a marked dip in GFP brightness (indicative of p62 protein amounts) in the NLE-administered BC12921 worms as opposed to the control group (Figures 4A,B). Moreover, Western blot analyses demonstrated that NLE decreased the protein levels of p62/SQST-1-GFP in BC12921 worms (Figure 4E), indicating degradation induced by NLE treatment. On the other hand, the DA2123 strain emits an LGG-1-GFP fusion protein that adheres to the autophagosome membrane, forming distinct structures upon triggering autophagy. The results in Figures 4C,D show that untreated DA2123 worms exude a spread-out fluorescence in various tissues, while the NLE treatment induces emergence of LGG-1⁺ structures specifically within their seam cells. Additionally, NLE elevated the ratio of phosphatidylethanolamine (PE) conjugated GFP::LGG-1 (PE-GFP::LGG-1) to non-lipidated GFP::LGG-1 in the DA2123 worms (Figure 4F). Collectively, these data demonstrate that NLE induces autophagy in C. elegans.

Considering the autophagy-inducing attributes of NLE in the C. elegans model organism, we examined whether the activation of autophagy could serve as a plausible mechanism to counteract the toxic effects induced by the Aβ protein. To achieve this, RNAi was employed as a highly effective gene silencing method in C. elegans to selectively inhibit two crucial genes integral to the autophagy process. Following the suppression of these genes, the extent of toxicity induced by Aβ in the AD models of C. elegans was evaluated. The results presented in Figures 5A,B indicate substantial alleviations in the paralysis rates in CL4176 worms when exposed to the control E. coli strain HT115 after NLE intervention. However, a marked contrast was observed when the worms consumed RNAi bacteria, leading to inhibition of bec-1 and vps-34 genes; under such conditions, the earlier reductions in the paralysis rates were conspicuously compromised. Simultaneously, the capability of NLE to mitigate the accumulation of Aβ aggregates was equivalently invalidated under the aforementioned conditions (Figures 5C,D). Together, the aforementioned findings provide compelling evidence suggesting a profound connection between the neuroprotective effects conferred by NLE and activation of the autophagy pathway in counterbalancing the toxic impacts mediated by Aβ in C. elegans.