Exploring the Impact of Onobrychis cornuta and Veratrum lobelianum Extracts on C. elegans: Implications for MAPK Modulation, Germline Development, and Antitumor Properties

Abstract

In an era of increasing interest in the potential health benefits of medicinal foods, the need to assess their safety and potential toxicity remains a critical concern. While these natural remedies have garnered substantial attention for their therapeutic potential, a comprehensive understanding of their effects on living organisms is essential. We examined 316 herbal extracts to determine their potential nematocidal attributes in Caenorhabditis elegans. Approximately 16% of these extracts exhibited the capacity to induce diminished survival rates and larval arrest, establishing a correlation between larval arrest and overall worm viability. Certain extracts led to an unexpected increase in male nematodes, accompanied by a discernible reduction in DAPI-stained bivalent structures and perturbed meiotic advancement, thereby disrupting the conventional developmental processes. Notably, Onobrychis cornuta and Veratrum lobelianum extracts activated a DNA damage checkpoint response via the ATM/ATR and CHK-1 pathways, thus hindering germline development. Our LC-MS analysis revealed jervine in V. lobelianum and nine antitumor compounds in O. cornuta. Interestingly, linoleic acid replicated phenotypes induced by O. cornuta exposure, including an increased level of pCHK-1 foci, apoptosis, and the MAPK pathway. Mutants in the MAPK pathway mitigated the decline in worm survival, underscoring its importance in promoting worm viability. This study reveals complex interactions between herbal extracts and C. elegans processes, shedding light on potential antitumor effects and mechanisms. The findings provide insights into the complex landscape of herbal medicine's impact on a model organism, offering implications for broader applications.

Keywords: DNA repair; O. cornuta; V. lobelianum; germline; linoleic acid; meiosis.

Figure 1

Herb extracts have an inhibitory effect on worm survival and development but do not affect bacterial growth. (A) Experimental workflow depicting the process of extracting compounds from herbs. The extracted compounds were then subjected to a phenotypic analysis, using the C. elegans model system. A 1 kg sample of the air-dried whole plant of each plant was milled to a coarse powder and extracted with methanol (3 × 6 L). The pooled methanol extract was concentrated in vacuo to afford a tarry residue. The methanol extract was dissolved in 90% (aqueous) methanol and extracted with n-hexane. The residual hydroalcoholic phase was freed of the solvent in vacuo, suspended in water, and then sequentially extracted with dichloromethane and n-butanol (n-BuOH) to afford a gross separation into hexane-, dichloromethane-, butanol-, and water-soluble fractions. Three solvents, i.e., hexane, butanol, and water, were designated -H, -B, and -A, respectively. The herbal extracts used in the screening process are listed in Supplementary Table S1. (B) The impact of plant extracts on C. elegans survival and development overview. A total of 316 (168 + 97 + 51) extracts were tested, revealing significant nematocidal effects. Survival (%), adult (%), and HIM (%) of C. elegans were monitored after a 24 h treatment of herb extract and monitored for 48 h. (C) Dose-dependent effects of herb extracts on survival, adult formation, and HIM phenotype. Investigation into the dose-dependent relationship between herbal extract concentrations and C. elegans phenotypes. (Top, middle) Survival rates and adult formation were inversely correlated with increasing herbal extract doses. Dose–response trends were consistent across extracts from different solvents (-A, -B, and -H). (Bottom) Induction of the HIM phenotype in response to herb extracts. V.l. and O.c. extracts were treated at a final concentration of 0.03, 0.3, and 3 µg/mL. The p-values were determined by the two-tailed Mann–Whitney test. Statistical significance between +DMSO and the samples is indicated by asterisks, using the two-tailed t-test. (D) Evaluation of herb extracts’ impact on bacterial growth. Analysis over 24 h revealed no significant bacterial growth defect, suggesting that the nematocidal effects were not primarily attributed to compromised bacterial growth. V.I and O.c were added to a final concentration of 0.03 μg/mL. Asterisks indicate statistically significant.

Figure 2

Defective germline development induced by herbal extracts. (A) Nuclei arrangement during germline development in control and herb extract-exposed C. elegans hermaphrodites. Exposure to V. lobelianum (+V.l.) and O. cornuta (+O.c.) extracts resulted in increased gaps indicated by arrows between nuclei in the premeiotic tip–transition zone (PMT-TZ) and pachytene stages. The distances between adjacent nuclei were significantly greater in herb extract-treated worms than in control worms. Asterisks indicate statistical significance according to the two-tailed Mann–Whitney test. Bar = 2 µm. (B) Aberrant transition from mitosis to meiosis and chromatin bridge formation. Herbal extract exposure led to aggregates of crescent-shaped nuclei (top row, indicated by arrows) and chromatin bridges in mitotic gut cells (bottom left). Additionally, herb extract-exposed worms exhibited reduced DAPI-stained bodies during diakinesis (bottom right), indicating a faulty DNA recombination process. Asterisks indicate statistical significance according to the two-tailed Mann–Whitney test. Bar = 2 µm. (C) Decreased length of PMT, TZ, and pachytene stages. V. lobelianum and O. cornuta extracts led to reduced lengths of the PMT, TZ, and pachytene stages compared to the control. Asterisks indicate statistical significance according to the two-tailed Mann–Whitney test. Right, the overall shape of the germline upon herb extract exposure. Bar = 10 µm. (D) Impaired fertility due to defective germline development. Exposure to herbal extracts resulted in a significant reduction in the number of progenies produced by hermaphrodite worms over a span of four days. V.I. and O.c. were added to a final concentration of 0.03 μg/mL. Asterisks indicate statistical significance according to the two-tailed Mann–Whitney test. All experiments were performed on C. elegans hermaphrodites. Data are presented as the mean ± SEM.

Figure 3

Exposure to herbal extracts of V. lobelianum and O. cornuta activates the DNA damage response pathway in C. elegans, resulting in increased levels of key DNA damage checkpoint components and phosphorylated CHK-1. (A) ATM and ATR kinases collaboratively regulate downstream CHK-1 processes in the intricate DNA damage response pathway, playing essential roles in DNA damage repair. (B) Exposure to herbal extracts leads to the increased expression of ATM-1 and ATL-1, validating the activation of the DNA damage response pathway. Gene expression levels between live and autoclaved OP50 were comparable, indicating that the nematocidal effects attributed to the herbal extracts may not be influenced by bacterial metabolism. Asterisks indicate statistical significance according to the two-tailed Mann–Whitney test. (C) Elevated pCHK-1 foci observed in germlines underscore the active DNA damage response following exposure to herbal extracts, confirming checkpoint activation. Bar = 2 µm. (D) Quantification of the number of pCHK-1 foci presented in (C). Asterisks indicate statistical significance according to the two-tailed Mann–Whitney test. (E) The pachytene stage revealed an increased level of apoptosis, suggesting that unrepaired DNA damage triggers checkpoint activation and subsequent apoptosis in the germline. V.I. and O.c. were added to a final concentration of 0.03 μg/mL. Asterisks indicate statistical significance according to the two-tailed Mann–Whitney test. Bar = 20 µm.

Figure 4

Spectrum of phytochemicals identified in both V. lobelianum and O. cornuta. Ten compounds were found in both herb extracts. Gray marking and the numbers indicate the predicted fragmentation of compounds provided by Analyst 1.6, an in silico analysis tool. The x-axis in an LC–MS graph represents the mass-to-charge ratio (m/z), which indicates the size and charge of ions. The y-axis represents the intensity in counts per second (CPS), showing how many ions of a particular m/z are detected.

Figure 5

mRNA expression profiles and effects of herbal extracts on DNA damage checkpoint activation, apoptosis, and hedgehog pathway components. (A) mRNA expression profile of hedgehog signaling components in C. elegans treated with V. lobelianum extract. wrt-1 and hog-1 were significantly downregulated. (B) Upregulation of the hedgehog signaling pathway when jervine was used alone. (C) Jervine extract did not increase the number of pCHK-1 foci (1.4 vs. 0.6 in control and +jervine, p < 0.0001). Bar = 2 µm. (D) V. lobelianum extract did not induce apoptosis (1.4 vs. 1.0 in control and +jervine, p = 0.1077). Bar = 20 µm. (E) O. cornuta extract led to the upregulation of the hedgehog pathway components wrt-1, hog-1, ptc-3, and qua-1, distinct from V.l. extract. V.I. and O.c. were added to a final concentration of 0.03 μg/mL. The concentration of Jervine was 20 μM. Asterisks indicate statistical significance according to the two-tailed Mann–Whitney test.

Figure 6

MAPK pathway activation and its role in response to O. Cornuta treatment. (A) Induction of the MAPK pathway components mek-2 and sos-1 upon O. cornuta treatment. mek-2 expression increased by 2.53-fold (p = 0.0006), and sos-1 expression increased by 6.86-fold (p = 0.0002) compared to the control. Additionally, mpk-1 and let-60 expression was induced by 1.74-fold (p = 0.0003) and 1.43-fold (p = 0.0003), respectively. Asterisks indicate statistical significance according to the two-tailed Mann–Whitney test. (B) Activation of the MAPK pathway by individual compounds in O. cornuta. For instance, sugiol treatment increased mek-2 expression by 1.7-fold (p = 0.0002) and sos-1 expression by 3.65-fold (p = 0.0002). Thymol treatment resulted in a 2.54-fold increase in mek-2 expression (p = 0.0009) and a 4.0-fold increase in sos-1 expression (p = 0.0009). Intriguingly, linoleic acid induced all four MAPK components, while other compounds induced one or two genes. Various O. cornuta compounds (sugiol, thymol, linoleic acid, luteolin, and DHT) altered MAPK pathway expression. DHT, dihydrotanshinone. (C) Linoleic acid-induced DNA damage checkpoint activation. Increased pCHK-1 foci during the pachytene stage (p < 0.0001). Arrows indicate pCHK-1 foci. Asterisks indicate statistical significance against the control group. Bar = 2 µm. (D) Linoleic acid-triggered apoptosis in germline nuclei similar to O. cornuta treatment (p < 0.0001). Other O. cornuta compounds did not show a simultaneous increase in both pCHK-1 foci and apoptosis. Asterisks indicate statistical significance against the control group. (E) The role of the MAPK pathway in the O. cornuta response validated by the mpk-1(ga111) mutant strain. Reduced survivability was reversed in the mutant (p = 0.4256 on day 5, p = 0.4208 on day 10 by the two-tailed t-test). Relative survivability is presented. Asterisks indicate statistical significance against the control group. (F) Suppressed apoptosis in the mpk-1 (ga111) mutant, supporting MAPK-dependent DNA damage pathway activation and survivability (p = 0.0173 in mpk-1 +O.c. and wild type +O.c.). Thymol (2 mM), dihydrotanshinone I (5 μM), luteolin (20 μM), linoleic acid (0.3 μM), and sugiol (10 μM) were employed in a qPCR study. O.c. extracts were introduced at a final concentration of 0.03 μg/mL Please refer to the Section 4 for details. Arrows indicate nuclei undergoing apoptosis. Asterisks indicate statistical significance against the control group. Bar = 20 µm.

Figure 7

O. Cornuta extract induces the upregulation of the MAPK kinase pathway, resulting in germline abnormalities and influencing worm survival. Our observation highlights that not only does O. cornuta itself lead to the upregulation of the MAPK pathway, but the compounds within the O. cornuta extract also play a significant role in this process. This collective activation of the MAPK pathway underscores its pivotal involvement in influencing germline abnormalities and impacting worm survival. Our observation highlights that not only does O. cornuta itself lead to the upregulation of the MAPK pathway, but the compounds within the O. cornuta extract also play a significant role in this process. This collective activation of the MAPK pathway underscores its pivotal involvement in influencing germline abnormalities and impacting worm survival. A photograph capturing the exquisite bloom of Onobrychis cornuta (L.) Desv. Photographed by Ori Fragman-Sapir (https://flora.org.il/en/plants/ (accessed on 13 December 2023) [46]).