Carpesii fructus extract exhibits neuroprotective effects in cellular and Caenorhabditis elegans models of Parkinson's disease

SCH772984 was purchased from Topscience Co., Ltd.; N‐acetyl‐L‐cystine (NAC, A7520), Hoechst 33342 (B2261), PI (P4170), and dihydroethidium (DHE, 309800) were purchased from Sigma Chemical Co. Dulbecco's modified Eagle's medium (DMEM), Trypsin–EDTA, and penicillin–streptomycin were purchased from Thermo Fisher Scientific Inc. Fetal bovine serum (FBS, #164210) was obtained from Procell Life Science & Technology Co., Ltd. YFP‐CL1 (11950), EGFP‐α‐synuclein‐WT (40822), EGFP‐α‐synuclein‐A53T (40823), and pHM6‐α‐synuclein‐A53T (40825) plasmids were purchased from Addgene (Addgene Inc.). EGFP‐N1 (6085/1/1), EGFP‐α‐synuclein‐A30P (PPL01823↗ ‐2b), and EGFP‐α‐synuclein‐E46K (PPL01823↗ ‐2c) plasmids were purchased from Public Protein/Plasmid Library (PPL). The commercially available antibodies used in this study include Bax (14,796S), Bcl‐2 (3498S), ERK (4695S), JNK (4668S), α‐actinin (3134S), and p‐JNK (4668S) from Cell Signaling Technology Inc. (CST); p38 MAPK (sc‐7972), p‐p38 MAPK (sc‐166,182), Caspase‐3 (sc‐390,394), and β‐actin (sc‐47,778) from Santa Cruz Biotechnology Inc; GAPDH (60004‐1‐Ig), Caspase‐9 (10380‐1‐AP), and PARP1 (13371‐I‐AP) from Proteintech Group, Inc; p‐ERK (ap0974) from ABclonal Biotechnology Co., Ltd.; and GFP (MO49‐3) from MBL International. The specificity of all antibodies was verified by the suppliers.

Dried Carpesii fructus was purchased from Tong Ren Tang Company and authenticated by Professor Can Tang. A voucher specimen (No. CF‐2021‐001) has been deposited at Southwest Medical University for future reference. Carpesii fructus extract (CFE) was prepared using a previously established method.27 Briefly, the dried fruit was ground into a fine powder and extracted with 75% ethanol using an ultrasonication‐assisted extraction technique. The extract was then filtered, concentrated under reduced pressure, and lyophilized to obtain a powdered form. The extract was stored at −20°C until further use.

The constituents of the CFE were identified using a Shimadzu UHPLC system. This system encompassed an LC‐3 AD solvent dispenser, SIL30ACXR autosampler, CTO‐30 AC column oven, DGU‐20A3 degasser, and CBM‐20A controller. To separate the samples, an Agilent UHPLC Zorbax EcLipse Plus C₁₈ column (100 mm × 2.1 mm, 1.8 μm) with a flow rate of 0.3 mL/min was utilized. The column temperature remained constant at 40°C throughout the analysis. The mobile phase consisted of a solution of water with 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The elution gradient was programmed as follows: From 0 to 15 min, there was a gradual increase from 10% to 95% of solvent B; from 15.01 to 19 min, there was a linear increase from 95% to 10% of solvent B. An AB SCIEX triple TOF X500R instrument equipped with a Duo Spray ion source was employed for mass spectrometry analysis. Negative electrospray ionization (ESI) mode was used, with the following parameters: ion spray voltage set to −4500 V, curtain gas maintained at 35 psi, ion source temperature set at 550°C, declustering potential (DP) set at −100 V, nebulizer gas (GS1) at 55 psi, and heater gas (GS2) at 55 psi. The mass scan range for the MS scan was set from 50 to 1600 Da. For data processing and interpretation, PeakView 1.4 software was employed in the analysis.

PC‐12 cells, a commonly used model for studying neuronal function, were obtained from the American Type Culture Collection (ATCC). Stable GFP‐LC3 U87 cells were provided by Dr. Xiaoming Zhu at Macau University of Science and Technology. Cells were cultured in high‐glucose DMEM or α‐MEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (PS). The cells were maintained in a humidified incubator at 37°C with 5% CO₂.

To evaluate the effect of CFE on ROS levels in PC‐12 cells, a fluorescent probe, dihydroethidium (DHE), was used. After treatment, PC‐12 cells were incubated with DHE (10 μM) for 30 min at 37°C. The cells were then washed with phosphate‐buffered saline (PBS) to remove excess DHE. The intracellular distribution and intensity of DHE fluorescence, reflecting ROS levels, were visualized and analyzed using a Nikon ECLIPSE 80i fluorescence microscope. Representative images were captured from multiple fields, ensuring a sufficient number of cells were analyzed. The fluorescence intensity of DHE in individual cells was measured, and the mean fluorescence intensity (MFI) was calculated. Additionally, the ROS levels were measured by a BD FACSVerse™ flow cytometer (BD Bioscience). Briefly, following the incubation period, the cells were washed with PBS to remove excess DHE. The fluorescence intensity of DHE was measured by the flow cytometer. A sufficient number of events (10,000 cells) were acquired for each sample.

Cell viability was determined using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay.28 After the treatment period, the culture medium was removed, and the cells were incubated with MTT solution (0.5 mg/mL) for 4 h at 37°C. The formazan crystals formed were solubilized by adding dimethyl sulfoxide (DMSO) and gently shaking the plates for 10 min. The absorbance was measured at a wavelength of 570 nm using a microplate reader.

Cell apoptosis was evaluated using flow cytometry analysis with Annexin V‐FITC/PI cell apoptosis kit (4A biotech).29 After the treatment period, PC‐12 cells were harvested and washed with cold PBS. The cells were then resuspended in Annexin V binding buffer and stained with Annexin V‐FITC and PI according to the manufacturer's instructions. The stained cells were analyzed using a flow cytometer, and data were acquired for at least 10,000 events per sample.

For plasmid transfection, PC‐12 cells were seeded in 6‐well culture plates at a density of 2 × 10⁵ cells per well and allowed to adhere overnight. The cells were then transfected with the desired plasmids expressing the target gene or control plasmids using a transfection reagent from Vazyme Biotech Co., Ltd. for DNA transfection. The transfection complex was prepared by diluting the plasmid DNA in serum‐free Opti‐MEM and mixing it with the transfection reagent. The mixture was added to the cells, and the transfection process was allowed to proceed for the recommended duration. Control cells were transfected with an empty vector or a non‐targeting plasmid.

Mitochondrial membrane potential (MMP) was assessed using JC‐1 kit (MCE),30 a fluorescent probe that exhibits a potential‐dependent dual emission. After the treatment period, PC‐12 cells were washed with pre‐warmed PBS and incubated with JC‐1 staining solution (5 μg/mL) in serum‐free DMEM at 37°C for 30 min in the dark. Subsequently, the cells were washed again with PBS to remove excess JC‐1 staining solution. To visualize the MMP changes, the stained cells were examined using a fluorescence microscope. In healthy cells with intact MMP, JC‐1 forms aggregates, emitting red fluorescence. Conversely, in cells with disrupted MMP, JC‐1 remains in the monomeric form, emitting green fluorescence. The fluorescence images were captured, and representative images were selected for further analysis using the ImageJ software (NIH). Additionally, MMP was detected by a flow cytometer. In brief, PC‐12 cells were harvested and resuspended in PBS. The fluorescence intensity of JC‐1 aggregates (red) and JC‐1 monomers (green) was measured. The ratio of green to red fluorescence was calculated as an indicator of MMP.

Cell death was evaluated using Hoechst 33342/propidium iodide (PI) staining, a widely used method for distinguishing between live and dead cells.31 After the treatment period, PC‐12 cells were washed with PBS and incubated with Hoechst 33342 staining solution (10 μg/mL) and PI staining solution (5 μg/mL) for 15 minutes at room temperature in the dark. The stained cells were examined using a fluorescence microscope. Live cells displayed normal nuclear morphology with uniform Hoechst staining, while dead cells exhibited condensed and fragmented nuclei. Representative images were captured, and the percentage of cell death was determined based on the fluorescence intensity of cells stained with PI to Hoechst using the ImageJ software.

After the treatment period, PC‐12 cells were washed with ice‐cold PBS and lysed using an appropriate lysis buffer supplemented with protease and phosphatase inhibitors. The lysates were collected and centrifuged to obtain the protein‐containing supernatant. Protein concentrations in the supernatant were determined using the Bradford reagent. Equal amounts of protein samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE). The proteins were separated and electrophoresed under specific conditions. Following electrophoresis, the proteins were transferred from the gel onto a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. The membrane was then blocked with 5% non‐fat milk to prevent nonspecific binding of antibodies. The blocked membrane was incubated overnight at 4°C with primary antibodies specific to the target proteins of interest. After washing, the membrane was incubated with secondary antibodies at room temperature for 1 hour. The immunoreactive protein bands on the membrane were visualized using UltraSignal Hypersensitive ECL Chemiluminescent Substrate (4A Biotech). The chemiluminescent signals were captured using a ChemiDoc MP Imaging System (Bio‐Rad). The band intensities were quantified using the ImageJ analysis software.

C. elegans strains, including N2 and transgenic strains, were obtained from the Caenorhabditis Genetics Center (CGC) or other reliable sources. The worms were maintained on nematode growth medium (NGM) agar plates seeded with Escherichia coli (E. coli) OP50 as a food source. To obtain a synchronized population of worms, adult hermaphrodites were allowed to lay eggs on NGM plates for 4 h. Subsequently, the adult worms were removed, and the plates containing eggs were incubated at 20°C until the worms reached the desired developmental stage.

After the treatment period, NL5901 worms were anesthetized using sodium azide. The worms were mounted on agarose pads, and the fluorescence of GFP‐tagged α‐synuclein was visualized using a fluorescence microscope. Representative images were captured from multiple fields, ensuring a sufficient number of worms were analyzed. For quantitative analysis, the fluorescence intensity of GFP in individual worms was measured using the ImageJ software, and the MFI was calculated.

Motor function was assessed by measuring the locomotion behavior of NL5901 worms.32 The synchronized L4 stage nematodes were cultured until the 5th or 10th day post‐synchronization. To perform the assay, a 35 mm petri dish was prepared by adding 1 mL of M9 buffer solution. An appropriate number of nematodes were carefully picked using a nematode picker and transferred to the petri dish containing the buffer solution. The nematodes were allowed to settle for 2 min to acclimate to the new environment. After the acclimation period, the nematodes were observed under a stereomicroscope. Within a 20 s interval, the number of body swings or thrashes made by each nematode was recorded.

After the treatment period, BZ555 worms were immobilized using sodium azide. The worms were mounted on agarose pads, and the fluorescence of GFP‐labeled dopaminergic neurons was visualized using a fluorescence microscope. Representative images were captured from multiple fields, and the fluorescence intensity of GFP in individual worms was measured using the ImageJ software.

The food‐sensing ability of BZ555 worms was evaluated by observing their chemotaxis behavior toward food sources.32 Using a nematode picking needle, nematodes were randomly selected and transferred onto NGM plates. The nematodes were allowed to acclimate to the sterile environment for 2 min. Subsequently, the nematodes were observed under a microscope, and the number of movements exhibited within a 20‐second period was recorded. To determine the reduction rate of nematode food perception, the following formula was employed with reference to the previous method.12

The pharyngeal pumping behavior of N2 worms was assessed by counting the number of contractions made by the pharyngeal muscles within a 20‐second period.33 The worms were transferred to agarose pads, and their pharyngeal pumping was observed using a stereomicroscope. The contractions were visually counted by an observer blinded to the treatment groups, and the pumping rate was calculated.

The reproductive fitness of N2 worms was assessed by measuring brood size.33 The worms were individually transferred to new NGM plates and allowed to lay eggs per 24 h until the loss of reproductive ability. The number of offspring (brood size) produced by each worm was counted.

The lifespan of N2 worms was assessed by monitoring the survival of individual worms over time.27 The worms were transferred to fresh NGM plates at regular intervals (e.g., every 1 day) to prevent overcrowding and maintain optimal conditions. The number of live and dead worms was recorded daily until all the worms in a particular group had expired. For quantitative analysis, the survival curves were generated by plotting the percentage of surviving worms against time. The median lifespan, defined as the time at which 50% of the worms had expired, was determined for each treatment group and control group.

Lipofuscin, a fluorescent pigment associated with aging and cellular damage, was determined in N2 worms.33 The worms were immobilized using sodium azide and mounted on agarose pads. The fluorescence of lipofuscin was visualized using a fluorescence microscope. Representative images were captured from multiple fields, and the intensity of lipofuscin fluorescence was measured and quantified for individual worms.

The stress resistance of N2 worms was assessed by subjecting the worms to various stressors known to induce physiological stress, such as heat stress, oxidative stress, or pathogenic bacteria.33 For heat stress, the worms were transferred to pre‐warmed NGM plates and incubated at 35°C. For oxidative stress, the worms were exposed to 30 mM H₂O₂. For pathogenic bacteria, the worms were exposed to pseudomonas aeruginosa (PA14). The survival and health of the worms under these stress conditions were recorded. The survival rate was calculated as the percentage of worms that survived the stress exposure compared to the initial population.

The ROS levels in worms were determined using the DHE reagent. In brief, after treatment, the worms were incubated with the DHE solution, and representative images were captured by a fluorescence microscope. The fluorescence intensity was measured and quantified for individual worms using the ImageJ software.

To investigate the functional role of specific genes in C. elegans, worms were subjected to RNAi by feeding them with bacteria expressing double‐stranded RNA (dsRNA) targeting the respective genes.21 The synchronized NL5901 and BZ555 worms were transferred to NGM plates seeded with mpk‐1 RNAi bacteria and allowed to grow until the desired stage or age for subsequent experiments. Control worms were cultured on NGM plates seeded with HT115 bacteria.

Data were expressed as the mean ± standard deviation (SD) and were derived from at least three independent experiments. Analyses were conducted using GraphPad Prism 8.0 software. The normality of the data was ascertained using the Shapiro–Wilk test, while the Brown–Forsythe test was used to check for the homogeneity of variances. The Kaplan–Meier method was employed to evaluate the lifespan, paralysis, survival rate, and stress resistance parameters. The significance of differences was determined using the log‐rank test to compute p‐values. For comparisons among multiple groups, a one‐way analysis of variance (ANOVA) followed by post hoc Tukey's test was used, and a two‐way ANOVA was applied when appropriate. A p‐value of less than 0.05 was considered statistically significant.

Oxidative stress has emerged as a critical pathological feature of PD, implicating the imbalance between reactive oxygen species (ROS) and antioxidant defense mechanisms in the neurodegenerative process, shedding light on potential therapeutic strategies to mitigate disease progression.34 In this study, we employed H₂O₂‐induced PC‐12 cells and investigated the neuroprotective effect of CFE, which is the ethanol extract of Carpesii fructus (Figure 1A). First, the constituents in CFE were identified using UHPLC‐DAD‐Q/TOF‐MS/MS. Figure S1 and Table S1 show that 17 main constituents were characterized based on the literature35 and the Dictionary of Natural Products (DNP) database, accessible at https://dnp.chemnetbase.com↗. After examining the cytotoxicity of CFE and H₂O₂ in PC‐12 cells (Figure 1B and S2), we investigated the effect of CFE at concentrations of 50–100 μg/mL on the inhibition of H₂O₂‐induced cell death. The results in Figure S3 and Figure 1C–E demonstrate that CFE dose‐dependently improved cell morphology, increased cell viability, and reduced cell death in H₂O₂‐induced PC‐12 cells. Furthermore, we evaluated mitochondrial damage by measuring the mitochondrial membrane potential (MMP) using JC‐1 staining and ROS production using DHE staining. Figures 1D,F, and S2 show that CFE significantly restored MMP, as evidenced by the decreased ratio of JC‐1 monomers (green fluorescence) to JC‐1 aggregates (red fluorescence). Moreover, Figures 1D,G, and S4 illustrate that CFE reduced ROS production, as evidenced by the DHE intensity (red fluorescence). Additionally, we examined the inhibitory effect of CFE on H₂O₂‐induced cell apoptosis, and the results in Figure 1D,H showed that CFE remarkably decreased the apoptosis rate of H₂O₂‐induced PC‐12 cells. Collectively, the above data suggest that CFE protects PC‐12 cells against H₂O₂‐induced cytotoxicity, possibly by inhibiting mitochondria‐mediated apoptosis.

The 6‐OHDA‐induced model of PD has provided valuable insights into the pathogenesis of dopaminergic neuronal degeneration, offering a versatile tool to investigate molecular mechanisms and explore neuroprotective interventions.22 In this study, we investigated the neuroprotective effect of CFE in 6‐OHDA‐induced PC‐12 cells after examining the cytotoxicity of 6‐OHDA in PC‐12 cells (Figure S5). The results in Figures 2A,D,E, and S6 show that CFE significantly increased cell viability, reduced cell death, and improved cell morphology in 6‐OHDA‐induced PC‐12 cells. Then, we examined mitochondrial damage by measuring the mitochondrial membrane potential (MMP) using JC‐1 staining and reactive oxygen species (ROS) production using DHE staining. Figures 2D,F, and S7 show that CFE significantly decreased the ratio of JC‐1 monomers to JC‐1 aggregates, suggesting a restoration of MMP in 6‐OHDA‐induced PC‐12 cells. Additionally, Figure 2B–D,G shows that CFE remarkably attenuated the DHE intensity, indicating a reduction in ROS production. Furthermore, CFE inhibited 6‐OHDA‐induced apoptosis in PC‐12 cells, as evidenced by the decreased apoptosis rate (Figure 2D,H) and the expression of apoptosis‐related proteins (Figure 2I–K). Thus, CFE protects PC‐12 cells against 6‐OHDA‐induced cytotoxicity.

α‐synuclein, a protein abundantly expressed in neurons, has emerged as a key player in the pathogenesis of PD, with its abnormal aggregation and deposition contributing to the formation of Lewy bodies and the subsequent neurodegenerative cascade.12 In this study, we employed and transiently transfected plasmids, including EGFP‐N1, EGFP‐α‐synuclein‐WT, EGFP‐α‐synuclein‐A53T, EGFP‐α‐synuclein‐A30P, and EGFP‐α‐synuclein‐E46K into PC‐12 cells. Then, we determined the expression of α‐synuclein using fluorescence microscopy, flow cytometry, and Western blotting methods. Figure S8 and Figure 3A–E demonstrate that CFE had no effect on the transfection efficiency but significantly decreased the intensity of GFP. This decrease reflects the expression of EGFP‐α‐synuclein‐WT, EGFP‐α‐synuclein‐A53T, EGFP‐α‐synuclein‐A30P, and EGFP‐α‐synuclein‐E46K, respectively. Flow cytometry analysis results revealed that CFE reduced the percentage of cells with GFP signal in transfected PC‐12 cells, suggesting CFE reduced α‐synuclein expression (Figure S8 and Figure 3F–I). Additionally, we employed a GFP antibody to determine the protein expression of GFP, and Figure 3J–Q demonstrated a significant reduction in GFP expression in these transfected PC‐12 cells treated with CFE at indicated concentrations. Furthermore, we determined the inhibitory effect of CFE on ROS production in CFE‐treated pHM6‐α‐synuclein‐A53T‐overexpressing PC‐12 cells, and Figure 3R,S displays that CFE remarkably reduced the intensity of DHE in PC‐12 cells, suggesting a reduction in ROS production. Collectively, CFE reduces α‐synuclein expression and its induced ROS production.

In the context of neurodegeneration in PD, aberrant MAPK signaling has been implicated as a key mediator of neuronal death in response to diverse insults such as 6‐OHDA.36 In this study, we employed Western blotting to investigate the mechanism of action of CFE. Figure 4A–D shows that CFE dose‐dependently upregulated ERK but downregulated JNK and p38 signaling pathways. In 6‐OHDA‐induced PC‐12 cells, the ratio of p‐JNK/JNK and p‐p38/p38 was significantly increased, where CFE treatment successfully reversed these increasing trends (Figure 4E–G). The above data suggest that CFE regulates the MAPK signaling pathways. Furthermore, we employed SCH772984, a specific inhibitor of ERK, to cotreat with CFE, and we found that CFE counteracted the effect of SCH772984 on the improvement of cell viability and the inhibition of cell death (Figure 4H–J). Collectively, CFE exhibits a neuroprotective effect in 6‐OHDA‐induced PC‐12 cells via regulating the MAPK signaling pathway.

To investigate the neuroprotective effect of CFE in vivo, we employed NL5901 strain‐expressing α‐synuclein in the body wall muscle. Figure 5A,B shows that CFE significantly reduced the expression of α‐synuclein, as evidenced by reduced GFP intensity. Additionally, we examined the motility of NL5901 worms, and Figure 5C shows that CFE significantly increased the body bends at days 5 and 10, suggesting an improvement in motility. Furthermore, we measured the ROS production in NL5901 worms, and Figure 5D,E shows that CFE decreased the DHE intensity in worms, indicating a reduction in ROS production. Collectively, CFE alleviates α‐synuclein pathology in the NL5901 strain.

The BZ555 strain, a genetically modified model organism, has emerged as a valuable tool in the study of dopaminergic neurons in PD research. In this study, we examined the viability of dopaminergic neurons in 6‐OHDA‐induced BZ555 worms with or without CFE. Figure 6A,B demonstrates a progressive loss of dopaminergic neurons, reflected by the reduced GFP signal in the anterior cephalic neurons (CEPs). However, the treatment with CFE or L‐Dopa successfully restored the expression of GFP, suggesting that CFE and L‐Dopa rescue the dopaminergic neurons in 6‐OHDA‐induced BZ555 worms. Then, we investigated the improvement effect of CFE on the food‐sensing ability, and Figure 6C shows that CFE and L‐Dopa significantly increased the slowing rate of 6‐OHDA‐induced BZ555 worms. Additionally, we detected the ROS production in BZ555 worms using DHE staining. Figure 6D,E demonstrates an obvious increase in fluorescence intensity in 6‐OHDA‐induced BZ555 worms, whereas the CFE or L‐Dopa treatment significantly inhibited the DHE intensity. Collectively, CFE inhibits the degeneration of dopaminergic neurons in BZ555 worms.

Aging represents a key determinant in the pathogenesis of PD.37 The multifaceted role of aging involves complex interplays of proteostasis decline, mitochondrial dysfunction, neuroinflammation, and cellular senescence, culminating in the selective vulnerability of dopaminergic neurons. In this study, we investigated the effect of CFE on extending aging and improvement of aging‐related phenotypes in N2 worms. Figure 7A shows that CFE significantly prolonged the lifespan of N2 worms, as evidenced by the increased survival rate. The accumulation of lipofuscin is recognized as a marker of aging.33 With aging, the intensity of blue fluorescence reflecting the content of lipofuscin increases. However, CFE treatment distinctly reduced the intensity of blue fluorescence (Figure 7B,C). In addition, the motility decreases with aging, and CFE treatment improved motility, as evidenced by the increased body bends within 20 s (Figure 7D). Studies have shown that decreased pharyngeal pumping rate and diet restriction are correlated with the extension of aging in nematodes.38 Here, we found that the pharyngeal pumping rate of worms increased at Day 5 and Day 10 after CFE treatment, suggesting that the anti‐aging effect of CFE is independent of diet restriction (Figure 7E). Furthermore, the reproductive ability of worms upon CFE treatment was evaluated. As shown in Figure 7F, G, CFE significantly increased the body length and brood size of N2 worms, suggesting that CFE may have non‐reproductive toxicity and promote the growth and development of worms. Collectively, CFE prolongs lifespan and promotes healthy aging in C. elegans.

C. elegans serves as an exceptional model to study stress resistance during aging.33 In this study, we examined the effect of CFE on the resistance of worms to various stresses, including heat stress, oxidative stress, and pathogenic bacteria. Figure 8A shows that CFE significantly increased the survival rate of N2 worms when the incubation temperature was shifted from 25°C to 35°C. Under the exposure of 30 mM H₂O₂, the treatment of CFE and NAC remarkably increased the survival rate of N2 worms (Figure 8B). Concurrently, the DHE staining images displayed that CFE and NAC reduced the intensity of fluorescence in N2 worms (Figure 8C,D). Additionally, we examined the expression of sod‐3 and gst‐4, two important antioxidative genes. Figure 8E–G shows that CFE significantly increased the intensity of GFP, reflecting the expression of sod‐3 in CF1553 strain and gst‐4 in CL2166 strain, respectively. Therefore, CFE exhibits an antioxidative effect in worms. Furthermore, CFE treatment could increase the survival rate of N2 worms treated with PA14, suggesting that CFE resists pathogenic bacteria (Figure 8H). Taken together, CFE increases the stress resistance of C. elegans.

As a member of the MAPK family, mpk‐1 plays a crucial role in transducing extracellular signals to regulate diverse cellular processes, including development, stress response, and longevity.39 Its functional homology to mammalian ERK makes mpk‐1 an essential and well‐studied component in understanding signaling pathways and cellular behaviors in C. elegans. In this study, we fed worms with mpk‐1 RNAi bacteria to knock down the expression of mpk‐1 in NL5901 and BZ555 worms. As shown in Figure 9A,B, the feeding of mpk‐1 RNAi bacteria significantly reversed the inhibitory effect on the expression of α‐synuclein, as evidenced by the increased intensity of GFP. Additionally, the treatment of CFE and L‐Dopa remarkably restored the viability of dopaminergic neurons in 6‐OHDA‐induced BZ555 worms, whereas the feeding of mpk‐1 RNAi bacteria attenuated the beneficial effect of CFE on dopaminergic neurons (Figure 9C,D). Collectively, CFE inhibits α‐synuclein expression and the degeneration of dopaminergic neurons via activating mpk‐1.

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.