Free and Easy Wanderer Ameliorates Intestinal Bloating‐Dependent Avoidance Behavior of Caenorhabditis elegans Through Gut‐Germline‐Neural Signaling

The following bacterial strains were used: E. c. and P. a. Bacterial cultures were grown in Luria‐Bertani (LB) broth at 37°C.

C. elegans hermaphrodites were maintained on E. c at 20°C, except HH142 fer‐1 (b232) and CB4037 glp‐1(e2141) strains that were maintained at 15°C. Bristol Wild‐type N2 was used as the wild‐type control. HH142 fer‐1(b232), CB4037 glp‐1(e2141), CF1038 daf‐16(mu86), and CF1139 daf‐16(mu86) I; muIs61 strains were obtained from the Caenorhabditis elegans Genetics Center (University of Minnesota, Minneapolis).

The traditional Chinese herb formula FAEW was purchased from Guangdong Zhengyuan Molecular Chinese Medicine Co. A highly concentrated FAEW stock solution was prepared by dissolving molecular weight FAEW in distilled water and then filtering through a 0.22 μm membrane. Long‐term storage was at −4°C. A gradient diluted FAEW plate was prepared by mixing FAEW stock solution into the medium. Under the condition that the growth of E. c was not affected, high and low doses were selected to carry out the experiment. Fluoxetine hydrochloride was purchased from Shanghai Eon Chemical Technology Co Ltd. (Shanghai, China). It was filtered through a 0.22 μm filter membrane and added to the plate according to the experimental method described above.

The bacterial lawns were generated by isolating individual colonies of P. a, which were then cultured in 2 μL of LB medium and incubated at 37°C for 12 h on a shaker. Subsequently, a 20 μL aliquot of the culture was applied to the center of a 3.5‐cm modified nematode growth medium (NGM) plate and incubated at 37°C for an additional 12 h. For each experimental condition, 20 synchronized hermaphroditic animals were utilized. The transfer of these animals into the bacterial lawn was followed by counting the number of animals present both on and off the lawn at specified time points during each experiment. The experiments were predominantly conducted at 25°C. The calculation of percent occupancy was determined by the formula (N_on‐lawn/N_total) × 100.

RNAi experiments were conducted according to established protocols detailed in prior studies [12]. In brief, E. c strains carrying the relevant vectors were cultured in LB broth supplemented with ampicillin (100 μg/mL) at 37°C overnight. The cultured bacteria were then plated onto NGM plates containing 100 μg/mL ampicillin and 3 mM isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) for RNAi induction (referred to as RNAi plates). Following an overnight incubation at 37°C, RNAi‐expressing bacterial colonies were generated. Subsequently, synchronized animals at the L4 developmental stage were transferred onto the RNAi plates and cultured for 24 h at 25°C. It is important to note that all RNAi clones were obtained from the Ahringer RNAi library.

Wild‐type N2 animals grown on HK E. c for 24 h were used for the pharyngeal pumping assay with animals grown on live E. c as controls. The number of contractions of the terminal bulb was counted over 1 min. A contraction was defined as the backward movement of the grinder in the terminal bulb of the pharynx. The pumping rates for 20 worms were recorded for each condition.

Wild‐type N2 animals grown on HK E. c for 24 h were used for the defecation assay, with animals grown on live E. c as controls. The defecation cycle length was scored by assessing the time between expulsions. Five cycles each were measured for six different animals per condition.

The C. elegans killing assay was conducted using wild‐type P. a lawn, which was incubated at 37°C for 12 h. 50 μL of an overnight culture of P. a grown at 37°C was evenly spread on the entire surface of 3.5‐cm‐diameter SK plates. The plates were then incubated at 37°C for 12 h before seeding with synchronized young gravid adult hermaphroditic animals. The killing assays were conducted at 25°C, with live animals transferred daily to fresh plates. Animals were assessed at specified time points and considered dead if unresponsive to touch.

The animals were transferred onto new plates containing E. c. These plates were supplemented with streptomycin (100 mg/mL), kanamycin (50 mg/mL), and nystatin (10 mg/mL) to prevent contamination. A 350 μL drop of the HK bacteria was plated on a 6 cm plate and incubated at 25°C. Survival assessments were conducted at specified times, with live animals transferred to fresh plates as needed. Each experiment was replicated three times, with 50 worms in each group and triplicate sets.

The animals were then washed off the plates with M9 buffer three to four times and frozen in TRIzol (Accurate Biotechnology (Hunan) Co. Ltd. Chang Sha, China). Subsequently, 10 μg of total RNA was reverse transcribed with random primers using the Evo M‐MLV Reverse Transcription Kit (Accurate Biotechnology (Hunan) Co. Ltd. Chang Sha, China). Quantitative RT‐PCR was performed using SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology (Hunan) Co. Ltd. Chang Sha, China) on a BIO‐RAD CFX96 real‐time PCR machine in a 96‐well plate format, utilizing 5 μL in each reaction. The relative fold‐changes of the transcripts were calculated using the comparative CT (2^−ΔΔCT) method and normalized to pan‐actin values. Data analysis was conducted using the CFX96 Touch Real‐Time PCR Detection System (BIO‐RED, US). Three technical replicates of samples were utilized, and the experiment was repeated three times for robustness and reliability. The primers used to measure the indicated genes were listed in Table S1.

The worms were collected and washed with M9 three times. The adult worms were paralyzed with 5 mM Levamisole hydrochloride and then mounted to a 2% agar pad. The anterior and posterior intestine regions were taken on a Positive fluorescence microscope (AXIO SCOPE.A1, Zeiss, Germany).

A precisely measured dried sample of FAEW weighing 1 g was placed into a centrifuge tube. Subsequently, 10 mL of water was added to the tube, and the resulting solution was filtered. The analysis was performed using a Waters ACQUITY I‐Class Plus UPLC system from Waters, USA, featuring a TurboIonSpray source (AB SCIEX, USA) and a quadrupole time‐of‐flight mass spectrometer SCIEX X500R (AB SCIEX, USA). Both ESI positive‐ and negative‐ion scanning modes were employed in the study. The acquired UPLC–Q‐TOF/MS data were processed and interpreted using SCIEX OS software. Target compounds were identified utilizing the secondary database of the Traditional Chinese Medicine (TCM) MS/MS Library.

Synchronized young adult animals were exposed to either P. a or E. c for 24 h at 25°C. Subsequently, worms were washed with M9 and resuspended in a fixing solution (KCl, Tris–HCl (pH 7.4), NaCl, Na₂EGTA, EDTA, spermidine HCl, PIPES (pH 7.4), Triton X‐100, methanol, formaldehyde) before being snap‐frozen in liquid nitrogen. The worms were fixed on ice for 4 h and briefly washed in T buffer (Tris–HCl (pH 7.4), EDTA, Triton X‐100) before a 15 min incubation in T buffer supplemented with β‐mercaptoethanol at 37°C. Following this, the worms were washed with borate buffer and then incubated in borate buffer containing DTT for 15 min, followed by H₂O₂ incubation for an additional 30 min. Subsequently, the worms were blocked in PBST containing 1% BSA for 30 min. They were then incubated overnight with anti‐H4K8ac antibody (1:100; ab15823, Abcam) and Alexa Fluor 594 secondary antibody (1:300; ab150080, Abcam). The worms were mounted on a microscope slide and observed using a stereofluorescence microscope (AXIO SCOPE.A1, Zeiss, Germany). The fluorescence intensity was quantified using Image J.

Statistical analyses were performed with Prism 8 (GraphPad). A two‐tailed Student t‐test for independent samples was used to analyze the data. For comparing the means of more than two groups, a one‐way ANOVA with post hoc analysis was performed. The Kaplan‐Meier method was used to calculate the survival fractions, and statistical significance between survival curves was determined using the log‐rank test. All the experiments were repeated at least three times, and error bars represent the standard deviation. The data were judged to be statistically significant when p < 0.05, and the exact p‐values are listed in Table S2. "ns" indicates non‐significant. "n" represents the number of animals for each experiment.

The Chinese medicinal herb formula FAEW originates from "Taiping Huimin Heji Ju Fang," representing a widely employed formula. This prescription comprises Chaihu (Bupleurum chinensie DC.), Fulin (Poria cocos Schw. Wolf), Baizhu (Atractylodes Macrocephala), Baishao (Paeoniae Radix Alba), Danggui (Radix Angelicae Sinensis), Bohe (Mentha haplocalyx Briq.), Gancao (Radix Glycyrrhizae), and Shengjiang (Zingiber officinale Roscoe). Clinically in China, FAEW is frequently employed in the treatment of neuropsychiatric disorders, gastrointestinal diseases and various other ailments [13]. To elucidate the primary compounds responsible for the pharmacological effects of FAEW, we initially used UPLC‐Q‐TOF/MS for the comprehensive analysis and identification of its components. As depicted in Figure 1A,B, a total of five major components of FAEW were successfully identified. Noteworthy constituents include chlorogenic acid, paeoniflorin, ferulic acid, glycyrrhizin, and glycyrrhetinic acid (Figure 1C,D).

In the defense against pathogens, C. elegans typically employs an avoidance response to enhance survival. Previous research by Alejandro Aballay's team revealed a connection between the avoidance behavior of C. elegans and the colonization of its gut by P. a. As P. a accumulates in the intestine, the intestinal lumen of C. elegans swells, leading to a significant upregulation of various immune genes such as clec‐60 and cpr‐2 [14]. Additionally, neuropeptide receptors, including flp‐21 and npr‐1, are activated, triggering avoidance behavior. The onset of avoidance behavior in C. elegans is closely tied to immune function, with animals exhibiting lower immunity displaying earlier avoidance behavior.

To assess the impact of FAEW on C. elegans' avoidance behavior, we used high (40 mg/mL) and low (0.4 mg/mL) dosages of FAEW to treat worms. Additionally, 0.4 μM fluoxetine (FXT) was used as a positive control. As shown in Figure 2A, a significant delay was observed in avoidance behavior upon the treatment of FAEW, suggesting that FAEW effectively postpones the onset of avoidance behavior in C. elegans. To investigate whether the immune system is involved in the modulation of avoidance behavior by FAEW, we conducted a survival assay to assess the impact of FAEW on immunity. Contrary to our expectations, FAEW did not enhance the innate immunity of C. elegans (Figure S1A). The relative mRNA levels of three PMK‐1/p38‐dependent immunity genes and the clec‐60 gene were elevated after P. a infection; however, only F08G5.6 and clec‐60 levels were relatively reduced after FAEW administration (Figure S1B–E). This finding suggests that multiple signaling pathways are involved in the innate immunity of C. elegans, and FAEW did not affect the innate immune response towards P. a infection.

Studies suggest that in the absence of defecation, motor program defects, or pathogenic microbial infections, C. elegans exhibited intestinal lumen distension and early avoidance. However, their pharyngeal pumping rate and defecation cycle remained unaltered compared to the normal diet group [15, 16, 17, 18]. To explore whether FAEW's impact on avoidance behavior is linked to gut lumen distension, we examined avoidance behavior and gut lumen distension in the presence of FAEW by feeding animals with HK E. c. The results showed that FAEW significantly prolonged C. elegans' aversive behavior towards P. a after HK E. c feeding (Figure 2B) and improved intestinal lumen distension (Figure 2C). Nevertheless, the pharyngeal pumping rate and defecation cycle did not change (Figure 2D,E). This study suggests that FAEW improved intestinal lumen distension, consequently delaying avoidance behavior in C. elegans.

C. elegans' ability to sense pathogens is predominantly mediated by its nervous system, which perceives and responds to molecular inputs in the environment, akin to higher animals. The gene tph‐1 plays a crucial role in the biosynthesis of 5‐hydroxytryptamine [19, 20]. Furthermore, it has been reported that the chemosensation of phenazine by pathogenic P. a activates DAF‐7/TGF‐β in ASJ neurons, impacting pathogen avoidance behavior [21]. Additionally, Intestinal infections and bloating, dependent on P. a virulence, regulate both pathogen avoidance and aversive learning through modulation of the DAF‐7/TGF‐β pathway and the NPR‐1/GPCR pathway, influencing chemotactic behavior and aversion learning [22]. Although the studies differ in some respects, they concur that the nervous system of C. elegans detects pathogen‐specific molecular signals, ultimately enabling the avoidance of pathogenic bacteria.

To explore the impact of FAEW on neuroendocrine signaling related to intestinal infections and bloating, we examined the gene expression of key components in the serotonin biosynthesis pathway (tph‐1) [23], the DAF‐7/TGF‐β pathway (daf‐7), and the NPR‐1/GPCR pathway (npr‐1, flp‐18, and flp‐21). As shown in Figure 3A–E, compared to the control group, the levels of tph‐1, daf‐7, npr‐1, flp‐18, and flp‐21 in HK E. c‐fed animals significantly increased, while they were significantly decreased upon the treatment of FAEW. Additionally, the fluorescence of flp‐21p::GFP (NY2087) transgenic animals increased significantly after HK E. c feeding, and the effect diminished upon FAEW treatment, (Figure 3F,G). Taken together, these findings suggest that FAEW's effect in enhancing avoidance behavior of C. elegans is associated with a reduction in the expression of genes involved in multiple neuroendocrine signaling pathways at the molecular level.

To delve into the molecular mechanisms by which FAEW inhibits the avoidance behavior of C. elegans, we conducted RNA sequencing. The results showed that FAEW intervention resulted in the altered expression of multiple genes (Figure S2A–C). The Venn diagrams illustrating the differential genes in each group are shown in Figure 4A. Bioinformatics was performed to analyze the interactions between DEGs using the Ouyi Cloud platform, and KEGG pathway enrichment analysis indicated that DEGs were mainly associated with the involvement of the Longevity regulating pathway—worm (Figure 4B,C). Specific KEGG pathway maps are shown in Figure S2D,E. We are concerned that the C. elegansftt‐2 and par‐5 genes act together on the 14‐3‐3 protein during germline ablation, and it has been documented that DAF‐16 is able to accumulate in the nucleus when 14‐3‐3 protein expression is reduced, which correlates with an increase in its transcription factor activity [24]. These results suggest that FAEW may affect C. elegans longevity and is complexly intertwined with avoidance behaviors. This conclusion is supported by the GSEA results (Figure 4D). The relative expression of specific genes in the longevity regulation pathway was shown in Figure 4E.

The research team had previously identified elevated levels of acetylated H4K8ac in C. elegans following P. a infection, a phenomenon associated with the regulation of avoidance behavior. Increased H4K8ac levels were observed irrespective of luminal distension induced by P. a infection for 24 h or normal feeding in constipated aex‐5 and eat‐2 mutants, indicating that H4K8ac elevation results from intestinal distension, suggesting gut‐germline‐neural signaling mediated pathogen avoidance behavior [25]. To understand the impact of FAEW on the H4K8ac levels in the germline, immunohistochemistry was employed. As shown in Figure 5A,B, HK E. c feeding induced H4K8ac expression with intensive red fluorescence, while both the positive control and FAEW can downregulate H4K8ac with weakened fluorescence.

Previous Co‐IP experiments have proved that the 14–3‐3 chaperone protein PAR‐5 can bind to H4K8ac and affect avoidance behavior. Silencing of the par‐5 gene leads to reduced H4K8ac and delayed avoidance of P. a. In our study, we found that FAEW treatment decreased the expression of par‐5 mRNA levels induced by HK E. c (Figure 5C). To further prove the effect, we observed the pathogen avoidance behavior of FAEW upon the treatment of par‐5 RNAi. As shown in Figure 5D, FAEW had no effect on the delayed avoidance behavior conferred by gene silencing of par‐5. Similarly, FAEW did not affect H4K8ac levels in par‐5 RNAi animals (Figure 5E,F). Together with the findings mentioned above (Figure 2B), it is inferred that FAEW delayed the adverse response of C. elegans to P. a following HK E. c feeding, which is correlated with enhanced intestinal lumen expansion, resulting in subsequent changes in par‐5‐mediated histone acetylation H4K8ac in the germline.

Furthermore, the glp‐1 mutants, incapable of germline development due to the meiosis process, do not exhibit expression of H4K8ac. In this study, avoidance experiments using the glp‐1 mutants (Figure 5D) showed that the effects of FAEW did not significantly impact the delayed avoidance caused by glp‐1. Thus, avoidance behavior resulting from intestinal lumen distension correlates with H4K8ac levels in the germline, and the potential mechanism underlying the inhibition of C. elegans avoidance behavior by FAEW may be linked to the amelioration of H4K8ac down‐regulation induced by intestinal lumen distension.

It has been established that both the par‐5 gene and the glp‐1 signaling pathway in C. elegans are associated with increased lifespan [26, 27]. To confirm the effect of FAEW on aging and the potential signaling pathway suggested in the RNA sequencing data, firstly, we observed the effect of FAEW on lifespan, as shown in Figure 6A; FAEW significantly extended the lifespan of wild‐type animals. Secondly, a qRT‐PCR experiment was performed to validate the RNA sequencing data by taking some aging‐related genes; the results show the consistency between the two‐experiment methods of qRT‐PCR and RNA sequencing Figure 6B–D.

Numerous signaling pathways, including the insulin/IGF‐1 signaling pathway, mTOR signaling, AMPK pathway, JNK pathway, and germline signaling, have been identified as playing a role in the processes of aging and longevity [28, 29, 30, 31, 32]. DAF‐16/FOXO, serving as a pivotal transcription factor, possesses the capability to integrate diverse signals emanating from these pathways [33]. This integration allows for the modulation of aging and longevity by shuttling between the cytoplasm and nucleus. To confirm whether the effect of FAEW on avoidance behavior is through activating DAF‐16, we selected daf‐16 mutants and the rescued animals to test the avoidance behavior of FAEW. As shown in Figure 6E,F, daf‐16 mutants exhibit earlier avoidance behavior than wild‐type, while the delayed avoidance behavior' effect of FAEW was affected in daf‐16 mutants, showing no difference compared with daf‐16 mutants but restored in daf‐16 rescued animals. Taken together, these results indicate that FAEW regulates avoidance behavior of C. elegans through activating DAF‐16.