Results

In the view of the previously described beneficial effects of WGX-50 (Figure 1a) on Alzheimer's disease (AD)^1–6, pain relief⁷, drug-induced heart injury⁸ and skin health^9,10, we sought to investigate its impact on longevity of C. elegans because the underlying mechanism on longevity is uncertain. To investigate whether WGX-50 could prolong lifespan, we conducted a longevity test in C. elegans. Interestingly, WGX-50 significantly prolonged the lifespan of C. elegans and the best lifespan extension effect was observed at a concentration of 50 μM (Figure 1b). Interestingly, in accordance with our study, WGX-50 extended lifespan in w.t nematodes (unpublished data; dose range tested, 3.75 ~ 250 μM), in which the dose 15 M increased the median survival to 45% (from 22 to 32 days, Figure 1b) at 20 oC. To this end, we concluded that WGX-50 prolongs lifespan in C. elegans.

Figure 1: WGX-50 extends lifespan in N2 C. elegans. (a): WGX-50 was previously characterized as a novel drug candidate for AD, which is a natural product derived from Zanthoxylum bungeanum Maxim. (Image credit: Royalty-free photo from https://create.vista.com/, slightly changed by the authors). (b): WGX-50 extends lifespan in N2 nematodes at 20 °C (b, n > 280 worms per condition). **** p < 0.0001.
Four novel genes take no roles in WGX-50 mediated longevity owing to a combinatorial effect enabled by the drug

To decipher the mechanistic pathways behind the detected phenotypes, we performed RNA sequencing (RNA-seq) for adult worms treated with WGX-50 or vehicle. RNA sequencing strandedness allows researchers to determine which DNA strand (sense or antisense) a transcript came from. Compared to regular RNA sequencing methods, stranded RNA sequencing can find novel transcripts, distinguish transcripts from overlapping genes, find antisense sequences, and annotate genes.

Figure 2: Adult N2 C. elegans were treated with 50 μM WGX-50 or control (Ctrl) for 5 days at 25°C, collected worms were subjected to RNA-seq analysis. Volcano plot of differentially expressed genes (DEGs) in w.t C. elegans (n = 3). DEGs were selected based on predefined cutoff as p-adj < 0.05 and fold change (FC) ≥ 1.5 (dash-dotted line, vertical). Four down-regulated genes and some up-regulated genes (FC < 2) are in black. Genes with more than 4-fold changes are labeled with gene symbols. Colored dots indicate genes that had more than 2-fold changes varying at significance levels (red, p-adj < 0.05; gold, p-adj < 0.01; cyan, p-adj < 0.001). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001; ns or no indication, not significant.

Next, RNA interference (RNAi) was conducted to investigate the unexplored role of these genes in WGX-50 mediated longevity. RNAi is among the processes that use short RNAs as guides for sequence-specific silencing; these pathways are together referred to as RNA silencing pathways. Long double-stranded RNA causes sequence-specific mRNA destruction, which is known as RNAi. This phenomenon was first identified in C. elegans. As results show, WGX-50 medicated survival increase was not diminished by RNAi any of these genes.

Further measurement for these gene hits using RT-qPCR indicates that their expression levels were inconsistently changed upon WGX-50 in different treatment days (Figure 3), which is also incompatible with that in RNA-seq data. This may suggest their expression is dependent on the age of the worms, hence it differs for some hits. To address such inconsistency, we performed gene set enrichment analysis. As results show (Table 2), metabolisms in amino acids, vitamins, carbohydrates, and lipids were significantly enriched. In RNA-seq data, genes like ttr-1/6/17, drd-1, vit-1, far-1/2/6, lbp-3/6 whose expression had been significantly increased are predicted to be involved in lipid transport. Besides, T05E11.3 (enpl-1, human ortholog hsp90b1) including F44E5.4/.5, members of HSP family working as molecular chaperones, were increased in their transcripts (colored, un-texted, Figure 4). It was reported that T05E11.3 was activated even if at low levels in long lived worms¹¹. Collectively, our data herein demonstrated that WGX-50 possibly enables various longevity pathways, and thus, knocking down any of RNA-seq revealed hits doesn't attenuate the action of WGX50.

Figure 3: RNA-seq revealed gene hits didn't involve in WGX-50 longevity, which possibly owes to an activated paradigm consists of multiple pathways by WGX-50. Transcriptional levels of F44E5.4/.5, sri-40, F59B2.12, K09C6.9 were determined by RT-qPCR in WGX-50 (50 μM) treated C. elegans on day 3 or 5. Results are representative of two independent experiments (n = 2), showing in % relative mRNA levels normalized to gpd-1 (GAPDHase-l) for six biological replicates per condition (n = 6).* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001; ns or no indication, not significant.

Table 2: Metabolic pathways enriched by GSEA in WGX-50 treated C. elegans via RNA-seq.

Name	Metabolism type	Size	ES	NES	Nom p-val	FDR q-val	Core enrichment
Valine, leucine and isoleucine degradation	Amino acids	42	0.5117	1.7188	0.0025	0.0476	acdh-1, acdh-4, alh-12, acdh-9, ech-6, ech-7, Y44A6D.5, hach-1, hacd-1, B0250.5, alh-8, T09B4.8, T02G5.7, ard-1, alh-9
Glutathione metabolism	Amino acids	42	0.4965	1.7004	0.0025	0.045	gsto-1, gst-36, gst-5, gpx-5, gst-11, gst-6, gst-38, C02D5.4, spds-1, gpx-7, C44B7.7, Y7A9A.1, gpx-1, gpx-3, T25B9.9, gst-44, gst-10, gst-9, E01A2.1, gsto-2, idh-2
Cysteine and methionine metabolism	Amino acids	35	0.5464	1.761	0.0013	0.0438	C01G10.9, tatn-1, spds-1, cysl-4, cysl-1, Y44A6D.5, cth-1, ldh-1, cdo-1, sams-5, mpst-2, got-1.2, mpst-4, T09B4.8, ZC373.5, F42F12.4, cbs-2
Glycine, serine and threonine metabolism	Amino acids	25	0.5582	1.6836	0.0051	0.0496	Y51H7C.9, gldc-1, R102.4, gcsh-2, T25B9.1, cth-1, gcsh-1, F08F3.4, T09B4.8, cbs-2, agxt-1, alh-9, cbs-1, C31C9.2
Beta-alanine metabolism	Amino acids	20	0.5208	1.4857	0.0431	0.2041	upb-1, alh-12, ech-6, ech-7, spds-1, hach-1, alh-8, alh-9, alh-5, dhp-2
Folate biosynthesis	N-acyl-amino acids; vitamin B	11	0.6995	1.7402	0.0103	0.0454	ptps-1, F49H6.5, cat-4, mocs-1, moc-5, dhfr-1, qdpr-1
Pentose and glucuronate interconversions	Carbohydrates	22	0.5751	1.7081	0.0079	0.0462	ugt-55, ugt-62, ugt-48, ugt-46, ugt-6, Y71F9B.9, dhs-21, R04B5.5
Ascorbate and aldarate metabolism	Carbohydrates	20	0.6218	1.7942	0.0027	0.0381	ugt-55, ugt-62, ugt-48, alh-12, ugt-46, ugt-6
Retinol metabolism	Vitamin A	19	0.5719	1.655	0.0055	0.0605	ugt-55, ugt-62, ugt-48, ugt-46, ugt-6
Riboflavin metabolism	Vitamin B	13	0.6156	1.5851	0.0209	0.1043	pho-14, pho-1, pho-13, pho-4, pho-10
Metabolism of xenobiotics by cytochrome P450	Na.	31	0.624	1.971	0	0.0082	gsto-1, gst-36, gst-5, ugt-55, ugt-62, ugt-48, gst-11, ugt-46, gst-6, ugt-6, gst-38, C02D5.4
Steroid hormone biosynthesis	Lipids	22	0.6326	1.8647	0.0014	0.0208	stdh-2, dhs-25, ugt-55, ugt-62, ugt-48, ugt-46, ugt-6
Biosynthesis of unsaturated fatty acids	Lipids	18	0.557	1.5867	0.0307	0.1121	stdh-2, fat-7, acox-1.3, acox-1.5, fat-5, let-767, acox-1.4
Arachidonic acid metabolism	Lipids (PUFA)	18	0.5312	1.5002	0.0374	0.1936	C07E3.9, gpx-5, gst-11, gpx-7, gpx-1, gpx-3, daf-41, pges-2, ltah-1.2, gst-9
Ether lipid metabolism	Lipids	13	0.604	1.5421	0.0284	0.1422	T05C3.6, C07E3.9, T03G6.3, paf-1, paf-2

In RNA-seq data, HSP70 like F44E5.4/.5 genes were activated. Expectedly, the upstream hsf-1gene was tested to be transcriptionally elevated by WGX-50 in nematodes (Figure 5). Generally, hsf-1 responses to proteotoxic stress and thereby enables longevity in C. elegans via accumulating HSPs functioning as molecular chaperones¹². As quantified, HSPs forms such as hsp12.2 and hsp-90 were also transcriptionally increased (Figure 5). HSP90 has been reported to be required for nematodes' longevity¹³. It was confirmed that DAF-21, without influencing DAF-16D/F localization or transcriptional activity, particularly enhances daf-2 and heat-shock induced nuclear translocation of DAF-16A as well as the induction of DAF-16A-specific mRNAs. On the contrary, transcriptional levels of other tested HSPs were all decreased (Figure 5).

Figure 5: Heat shock protein genes were transcriptionally quantified by RT-qPCR for groups (50 μM of WGX-50 vs. Ctrl, 5 days). Results are representative of three independent experiments (n = 3), showing in % relative mRNA levels normalized to gpd-1 (GAPDHase-l) for biological replicates (n = 6).

As shown in Figure 6a and b, it was obviously that WGX-50 failed to activate the fluorescent reporter in hsp-16.2::GFP animals. Overall, our data indicate that WGX-50 activated HSPs longevity pathway despite down-regulation of other HSPs.

Figure 6: WGX-50 regulates HSPs longevity pathway. (a): 50 μM of WGX-50 didn't affect the protein levels of hsp-16.2 in transgenic CL2070 worms upon 5 days treatment at 25°C. Whole-body fluorescence for hsp-16.2::GFP (b) was visualized under an ECHO inverted fluorescence microscope. Signal intensities (c) was presented as integrated density of gray values ± SEM of 10 animals in each group (n = 10). Scale bar = 320 μm. Results were normally selected from two independent experiments. *** p < 0.001, **** p < 0.0001; ns or no indication, not significant.

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