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. 2024 May 22:16:1414956.
doi: 10.3389/fnagi.2024.1414956. eCollection 2024.

P-hydroxybenzaldehyde protects Caenorhabditis elegans from oxidative stress and β-amyloid toxicity

Xingzhi Yu  1 , Jie Tao  1 , Tian Xiao  1 , Xiaohua Duan  1
Affiliations

P-hydroxybenzaldehyde protects Caenorhabditis elegans from oxidative stress and β-amyloid toxicity

Xingzhi Yu et al. Front Aging Neurosci. .

Abstract

Introduction: Gastrodia elata is the dried tuber of the orchid Gastrodia elata Bl. It is considered a food consisting of a source of precious medicinal herbs, whose chemical composition is relatively rich. Gastrodia elata and its extracted fractions have been shown to have neuroprotective effects. P-hydroxybenzaldehyde (p-HBA), as one of the main active components of Gastrodia elata, has anti-inflammatory, antioxidative stress, and cerebral protective effects, which has potential for the treatment of Alzheimer's disease (AD). The aim of this study was to verify the role of p-HBA in AD treatment and to investigate its mechanism of action in depth based using the Caenorhabditis elegans (C. elegans) model.

Methods: In this study, we used paralysis, lifespan, behavioral and antistress experiments to investigate the effects of p-HBA on AD and aging. Furthermore, we performed reactive oxygen species (ROS) assay, thioflavin S staining, RNA-seq analysis, qPCR validation, PCR Array, and GFP reporter gene worm experiment to determine the anti-AD effects of p-HBA, as well as in-depth studies on its mechanisms.

Results: p-HBA was able to delay paralysis, improve mobility and resistance to stress, and delay aging in the AD nematode model. Further mechanistic studies showed that ROS and lipofuscin levels, Aβ aggregation, and toxicity were reduced after p-HBA treatment, suggesting that p-HBA ameliorated Aβ-induced toxicity by enhancing antioxidant and anti-aging activity and inhibiting Aβ aggregation. p-HBA had a therapeutic effect on AD by improving stress resistance, as indicated by the down-regulation of NLP-29 and UCR-11 expression and up-regulation of PQN-75 and LYS-3 expression. In addition, the gene microarray showed that p-HBA treatment played a positive role in genes related to AD, anti-aging, ribosomal protein pathway, and glucose metabolism, which were collectively involved in the anti-AD mechanism of p-HBA. Finally, we also found that p-HBA promoted nuclear localization of DAF-16 and increased the expression of SKN-1, SOD-3, and GST-4, which contributed significantly to inhibition of Aβ toxicity and enhancement of antioxidative stress.

Conclusion: Our work suggests that p-HBA has some antioxidant and anti-aging activities. It may be a viable candidate for the treatment and prevention of Alzheimer's disease.

Keywords: Alzheimer’s disease; Aβ protein; Caenorhabditis elegans; neuroprotection; oxidative stress; p-hydroxybenzaldehyde.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1
Effect of p-HBA on the paralyzed phenotype of AD transgenic CL4176 worms. Synchronized CL4176 worms were treated with different concentrations of p-HBA, respectively, and an equal volume of OP50 bacterial solution without drug was used as a blank control (Control group), which was incubated at 16°C for 48 h and then transferred to 25°C to induce the paralytic phenotype. p-HBA treatment had a positive effect on delaying worm paralysis. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 2
Figure 2
Effects of p-HBA on senescence, motility and growth and development of worms. (A) Effect of p-HBA on lifespan of AD transgenic CL4176 worms: CL4176 worms were treated with 0 mM (blank control) and 0.5 mM p-HBA. Treatment prolonged the median and mean lifespan of the worms, and the time to half-survival was also significantly prolonged (p < 0.01). The experiment was repeated three times independently. Lipofuscin levels were examined in worms: (B) Analysis of lipofuscin fluorescence intensity (p < 0.001). (C) Representative fluorescence images of the control group. (D) Representative images of 0.5 mM treatment group. p-HBA on AD worm motility study: (E) Frequency of pharyngeal pump beating of worms within 30 s on days 4, 6, and 8. (F) Treatment and observation times were the same as in the pharyngeal pump beating experiment and the frequency of sinusoidal movements of worms within 30 s on days 4, 6, and 8. p-HBA effects on worm growth and development: (G) Analysis of the total number of eggs laid in the control and 0.5 mM groups (p > 0.05). (H) Worm body length was analyzed using Image J software measurements (p > 0.05). (I) Representative images of worm body length in the control group. (J) Representative images of worm body length in the 0.5 mM treatment group. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3
Figure 3
Effect of p-HBA on stress resistance and in vivo ROS levels in N2 worms. 0.5 mM p-HBA treatment improves the resistance of worms: (A) Survival curves of worms under high temperature stress at 37°C. (B) Survival curves of worms in Juglone (300 μM) constructs for oxidative stress damage environment were analyzed. The experiment was independently repeated three times. Moreover, p-HBA reduced ROS levels in N2 worms. Paraquat (5 mM) was used to induce ROS production in worms, and pictures were taken under a fluorescence microscope after staining with DCFH – DA solution. (C) Representative pictures of N2 worms without p-HBA treatment. (D) Pictures of 0.5 mM p-HBA-treated N2 worms. (E) Fluorescence intensity was measured and analyzed using Image J software. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4
Figure 4
Determination of the number of Aβ protein deposits by thioflavin S staining in transgenic worms CL2006. In CL2006 worms, Aβ protein stained with thioflavin S produces distinct fluorescent patches near the pharynx, with white arrows indicating Aβ deposition sites. (A) Wild-type N2 worms served as a negative control with no Aβ protein deposition. (B) Representative images of CL2006 worms not treated with p-HBA, with more deposits. (C) Representative images of worms under 0.5 mM p-HBA treatment with significantly less deposits. (D) Number of Aβ protein deposits in each group ***p < 0.001.
Figure 5
Figure 5
We performed RNA-seq analysis and qPCR validation of the AD worm CL4176. Worms of CL4176 synchronized to L1 stage were transferred to NGM plates with or without drug (0.5 mM), incubated at 16°C for 48 h, then warmed up to 25°C and continued to be incubated for about 30 h. Worms were collected in EP tubes with M9 buffer and washed 2–3 times with sterile water. Sequencing analysis was performed after snap-freezing with liquid nitrogen. (A) Differential genes between groups, and the number of up- and down-regulations. (B) Differential pathways analyzed by GO enrichment. (C) The 15 pathways identified by KEGG enrichment analysis. (D) Based on the results of RNA-seq analysis, qPCR analysis was performed to validate four genes related to stress response. The expression trend of these genes was the same as that of RNA-seq analysis, and the mechanism of p-HBA anti-AD may be related to its anti-stress effect. **p < 0.01, ***p < 0.001 Subsequently, PCR Array was used for analytical validation of AD-related genes and pathways. Sample preparation was consistent with RNA-seq analysis experiments. Then RNA was extracted and reverse transcribed into cDNA, which was operated according to the instructions of the Gene Chip kit, and the data were exported for analysis. (E) Volcano plots reflecting differential genes and expression trends between groups. A total of 10 differentially expressed genes were screened with |log2Fold Change| > =1 and p < 0.05 as the condition, including two genes with down-regulated expression and eight genes with up-regulated expression, which were related to AD, aging/anti-aging, ribosomal and glucose metabolism pathways, respectively.
Figure 6
Figure 6
Effect of p-HBA on the expression of DAF-16::GFP, SKN-1::GFP, SOD-3::GFP and GST-4::GFP in several reporter-gene worms. TJ356 worms: fluorescence images were obtained under a laser confocal microscope, and green fluorescent spots indicated the nucleus localization of DAF-16 in TJ356 worms (with white arrows to indicate the loci), treated at 37°C for 30 min as a positive control. (A) Control group. (B) Positive control group. (C) 0.5 mM p-HBA group. (D) Number of fluorescence points in each group (p < 0.001). LD1 worms: (E) Control group. (F) 0.5 mM p-HBA group. (G) Relative fluorescence intensity measurements of SKN-1::GFP in LD1 worms (p < 0.01). CF1553 worms: (H) Control group. (I) 0.5 mM p-HBA group. (J) Fluorescence intensity expression analysis of SOD-3::GFP in worms (p < 0.001). CL2166 worms: (K) Control group. (L) 0.5 mM p-HBA group. (M) Relative fluorescence intensity of GST-4::GFP expression in CL2166 worms (p < 0.001). Experiments were independently repeated 3 times.
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Grants and funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. The present study was supported by the Xingdian Talent Support Program – Special for Young Talent (grant no. XDYC-QNRC-2022-0284), the Highlevel Talents Projects of Yunnan University of Chinese Medicine – Fifth Level Talents, the National Administration of Traditional Chinese Medicine High-level Key Discipline Construction Project “Minority medicine (Dai Medicine)” (No. Zyyzdxk-2023193), the Open Project of Yunnan Key Laboratory of Dai and Yi Medicines (No. 202210SS2209).