Para-Hydroxybenzyl Alcohol Delays the Progression of Neurodegenerative Diseases in Models of Caenorhabditis elegans through Activating Multiple Cellular Protective Pathways

Abstract

The traditional Chinese medicine Gastrodia elata (commonly called "Tianma" in Chinese) has been widely used in the treatment of rheumatism, epilepsy, paralysis, headache, and dizziness. Phenolic compounds, such as gastrodin, para-hydroxybenzyl alcohol (HBA), p-hydroxybenzaldehyde, and vanillin are the main bioactive components isolated from Gastrodia elata. These compounds not only are structurally related but also share similar pharmacological activities, such as antioxidative and anti-inflammatory activities, and effects on the treatment of aging-related diseases. Here, we investigated the effect of para-hydroxybenzyl alcohol (HBA) on neurodegenerative diseases and aging in models of Caenorhabditis elegans (C. elegans). Our results showed that HBA effectively delayed the progression of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease in models of C. elegans. In addition, HBA could increase the average lifespan of N2 worms by more than 25% and significantly improve the age-related physiological functions of worms. Moreover, HBA improved the survival rate of worms under stresses of oxidation, heat, and pathogenic bacteria. Further mechanistic investigation revealed that HBA could activate FOXO/DAF-16 and SKN-1 to regulate antioxidative and xenobiotic metabolism pathway. HBA could also activate HSF-1 to regulate proteostasis maintenance pathway, mitochondrial unfolded stress response, endoplasmic stress response and autophagy pathways. The above results suggest that HBA activated multiple cellular protective pathways to increase stress resistance and protect against aging and aging-related diseases. Overall, our study indicates that HBA is a potential candidate for future development of antiaging pharmaceutical application.

Figure 1

HBA delays the progression of Alzheimer's disease (AD) in models of C. elegans. (a) The chemical structure of HBA. (b) Paralysis analysis of C. elegans CL4176 dvIs27 [myo-3p::A-Beta (1-42)::let-851 3′UTR) + rol-6(su1006)] X under ddH₂O (control) and HBA (50 μM, 100 μM, 200 μM, and 400 μM). (c) Paralysis analysis of strain CL2006 dvIs2 [pCL12(unc-54/human Abeta peptide 1-42 minigene) + rol-6(su1006)] under ddH₂O (control) and HBA (50 μM, 100 μM, 200 μM, and 400 μM). (d) Thioflavin S straining of Aβ_1-42 in wild-type N2 and transgenic strain CL2006 (dvIs2) fed with or without 200 μM HBA. Scale bar, 25 μm. (e) The quantification of Aβ protein aggregation spots indicated by thioflavin S staining in the head of worms CL2006 (p < 0.001). (f) Gene expression of Aβ_1-42 in C. elegans CL4176 fed with or without 200 μM of HBA (mean ± SD, n = 3). (g) The chemotactic index (CI) of strain CL2355 and the transgenic control strain CL2122 fed with vehicle or 200 μM of HBA. Data were obtained from three repeated experiments with 80 worms in each group. Prism 6.0 was used for statistical analysis, and a t-test or log-rank test was used to express statistical significance in p value (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001). Statistical details and repeats of these assays are summarized in Tables S1, S2, S5, and S11 (supplementary information).

Figure 2

HBA delays the progression of Parkinson's disease (PD) and Huntington's disease (HD) in models of C. elegans. (a) The fluorescence picture of α-synuclein conjugated with yellow fluorescent protein in C. elegans NL5901 pkIs2386 [unc-54p::alpha-synuclein::YFP + unc-119(+)], fed with or without 200 μM of HBA on the 3rd and 7th day of adulthood. (b) The quantification of α-synuclein protein aggregation in strain NL5901 (p < 0.001). (c) The fluorescence picture of dopaminergic neurons in C. elegans strain BZ555 fed with or without 200 μM of HBA after being treated with 6-hydroxydopamine. (d) The quantification of the fluorescence intensity of dopaminergic neurons in the C. elegans strain BZ555 egIs1 [dat-1p::GFP] (p < 0.001). (e) The fluorescence picture of poly-Q conjugated with yellow fluorescent protein in strain AM141 rmIs133 [unc-54p::Q40::YFP] fed with or without 200 μM of HBA. (f) The quantification of the aggregation of poly-Q protein in the strain AM141. Prism 6.0 was used for statistical analysis, and the values were expressed as mean ± SEM. The t-test or log-rank test were used to express statistical significance in p value (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001). Statistical details and repeats of these assays are summarized in Table S2 (supplementary information).

Figure 3

HBA reduces the accumulation of ROS in C. elegans. The body bending behavior (body bending times every 20 seconds) on the 3rd and 7th day of adulthood of wild-type (a) N2 and (b) CL2006 fed with 200 μM of HBA (p < 0.001). (c) The pictures of lipofuscin deposition on the 7th day of adulthood of wild-type N2 and CL2006 fed with 200 μM of HBA. The quantification of lipofuscin on the 7th day of adulthood of wild-type (d) N2 and (e) CL2006 by using ImageJ. Values are expressed as mean ± SEM. (p < 0.001). The mRNA levels of genes in wild-type (f) N2 and (i) null mutant daf-16 (mu86) fed with HBA (mean ± SD, n = 3). Lifespan analysis of (g) N2, (h) null mutant daf-2(e1370), (j) null mutant daf-16 (mu86), (k) null mutant akt-1(ok525) V, and (l) null mutant akt-2(ok393), after being fed with HBA. The (m) fluorescent picture and (n) quantification of CF1553 sod-3::GFP expressing SOD-3 conjugated with GFP fed with HBA. Prism 6.0 was used for statistical analysis, and the values were expressed as mean ± SEM. Use the t-test or log-rank test to express statistical significance in p value (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001). Statistical details and repeats of these assays are summarized in Tables S3, S4, S9, S10, and S11 (supplementary information).

Figure 4

HBA enhances the antioxidant capacity of C. elegans. The lifespan expectancy of C. elegans wild-type (a) N2 and (b) strain CL2006 were fed with or without 200 μM HBA for 7 days and then exposed to 20 mM of paraquat. (c) The lifespan expectancy of null mutant skn-1(zu67) fed with or without 200 μM HBA (p > 0.05). The expression of skn-1 and its target genes in wild-type (f) N2 and (i) mutant skn-1(zu67). The CM-H₂DCFDA staining picture of wild-type (d) N2 and (g) CL2006 was fed with or without HBA (200 μM) or NAC (5 mM). The quantification of ROS levels indicated in pictures (e) and (h) using ImageJ (p < 0.001). (h) The green fluorescence pictures of C. elegans transgenic strain LD1 (Is007) [Pskn-1::skn -1b/c::GFP; pRF4 rol-6 (su1006)]) (top) and CL2166 (dvIs19)[(pAF15)gst-4p::GFP] (bottom) fed with or without 200 μM HBA. (j) The quantification of the fluorescence intensity of SKN-1::GFP (I) and GST-4:: GFP. (k) The superoxide dismutase (SOD) activity of strain CL4176 fed with 200 μM HBA (p < 0.001). Prism 6.0 was used for statistical analysis, and the values were expressed as mean ± SEM. The t-test or log-rank test were used to calculate the statistical significance in p value (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001). Statistical details and repeats of these assays are summarized in Table S6, S7, S8, and S9 (supplementary information).

Figure 5

HBA enhances the stress resistance of C. elegans. The survival span of (a) wild-type N2 and (b) transgenic CL2006 under ambient temperature of 35°C after being treated with or without 200 μM HBA for 7 days. (c) The lifespan expectancy of null mutant hsf-1(sy441) fed with or without 200 μM HBA (p > 0.05). (f) The fluorescent pictures and the fluorescence quantification of C. elegans strain (from top to bottom) (g) SJ4005 zcIs4 [hsp-4::GFP] V, (h) SJ4058 zcIs9 [hsp-60::GFP + lin-15(+)], (i) SJ4100 zcIs13 [hsp-6p::GFP + lin-15(+)], (j) BC12921 sIs10729 [rCes T12G3.1::GFP + pCeh361] fed with or without 200 μM HBA. The survival span of the wild-type (d) N2 and (e) CL2006 fed with pathogenic bacteria Pseudomonas aeruginosa (PA14) after being treated with or without 200 μM HBA for 7 days. The results showed that HBA could significantly prolong the lifespan of N2 and CL2006 by 15.21% (p < 0.001) and 20.37% (p < 0.001), respectively. (k) The mRNA levels of genes in wild-type N2 fed with 200 μM HBA (mean ± SD, n = 3). The mRNA levels of genes in wild-type (l) N2 and (m) CL4176 fed with 200 μM HBA (mean ± SD, n = 3). Prism 6.0 was used for statistical analysis, and the values were expressed as mean ± SEM. Use t-test or log-rank test to express statistical significance in p value (^∗p < 0.01, ^∗∗p < 0.05, and ^∗∗∗p < 0.001). Statistical details and repeats of these assays are summarized in Table S6, S9, S10, and S11 (supplementary information).

Figure 6

HBA requires FOXO/DAF-16 to extend the healthy lifespan of C. elegans. The lifespan analysis of eat-2 null mutant eat-2(ad1116) II (a) (p < 0.05), null mutant aak-2(ok524) (b) (p > 0.05), rsks-1(ok1255) (c) (p > 0.05), sir-2.1(ok434) (d) (p > 0.05), isp-1(qm150) (e) (p > 0.05), and clk-1(e2519) (f) (p > 0.05) fed with 200 μM HBA. Prism 6.0 was used for statistical analysis, and the values were expressed as mean ± SEM. Use t-test or log-rank test to express statistical significance in p value (^∗p < 0.05). Statistical details and repeats of these assays are summarized in Table S10 (supplementary information).