The chemical structure of Berberine hydrochloride (BH) was showed in Figure 1A . To test whether Berberine hydrochloride was able to increase pathogenic resistance, C. elegans were exposed to the human opportunistic pathogen Pseudomonas aeruginosa (PA14). Wild-type worms were treated with varying concentrations of Berberine hydrochloride (0 μM, 5 μM, 10 μM, 20 μM). The results indicated that 10 μM Berberine hydrochloride significantly improved survival to P. aeruginosa ( Figure 1B ; Supplementary Table S1 ). These findings suggested that Berberine hydrochloride enhances innate immunity in C. elegans. To determine whether this effect was due to inhibition of bacterial growth, a bacterial growth assay was conducted, showing that 10 μM Berberine hydrochloride did not suppress the proliferation of P. aeruginosa PA14 ( Figure 1C ). Moreover, our findings revealed that treatment with 10 μM Berberine hydrochloride has no effects on pathogen avoidance behavior in C. elegans ( Figure 1D ). Bacterial clearance is an essential aspect of host defense against pathogen infection (2, 19). To investigate whether Berberine hydrochloride affects bacterial accumulation, we examined its impact on Pseudomonas aeruginosa fusing with green fluorescent protein (GFP). Compared to control group, Berberine hydrochloride-treated worms showed a reduced accumulation of P. aeruginosa/GFP ( Figure 1E ). Furthermore, we assessed the effect of Berberine hydrochloride on the expression of the antimicrobial peptide gene irg-1 (20), which was induced in exposure of P. aeruginosa. Our results revealed that Berberine hydrochloride increased the expression of irg-1p::GFP ( Figure 1F ). These findings indicate that Berberine hydrochloride may enhance pathogen resistance by lowering bacterial load in the C. elegans intestine.

To determine whether Berberine hydrochloride enhances resistance to other pathogens, we exposed worms to the Gram-negative bacterium Salmonella enterica and the Gram-positive bacterium Listeria monocytogenes. Supplementation with 10 μM Berberine hydrochloride significantly improved host survival ( Figures 2A,B ; Supplementary Table S1 ). To assess whether this effect was due to inhibition of bacterial growth, we conducted bacterial growth assays. The results demonstrated that 10 μM Berberine hydrochloride did not inhibit the growth of S. enterica or L. monocytogenes ( Figures 2C, D ). Since bacterial clearance is a critical aspect in host defense (2, 19). We next investigated whether Berberine hydrochloride affected bacterial accumulation. Compared to control animals, those treated with Berberine hydrochloride exhibited reduced accumulation of S. enterica and L. monocytogenes tagged with green fluorescent protein (GFP) ( Figures 2E, F ). These findings suggest that Berberine hydrochloride enhances resistance in both Gram positive and negative pathogens.

To elucidate the molecular mechanisms underlying Berberine hydrochloride protective effects against pathogen infection, we examined several signaling pathways associated with innate immunity in C. elegans, including the p38 MAPK/PMK-1 pathway (13), DAF-2/DAF-16 insulin-like pathway (15), ERK MAPK/MPK-1 pathway (21). We found that Berberine hydrochloride failed to enhance resistance to P. aeruginosa PA14 infection in pmk-1(km25) mutants, compared to WT (N2) worms ( Figures 3A, B ; Supplementary Table S1 ). However, it significantly increased survival rates in daf-2(e1370) and mpk-1(n2521) mutants following P. aeruginosa PA14 infection ( Figures 3C, D ; Supplementary Table S1 ). We further investigated conserved signaling pathways, CRH-1/CREB, AAK-2/AMPK, and JNK-1/JNK, results revealed that Berberine hydrochloride enhanced survival in crh-1(tz2), aak-2(ok524), and jnk-1(gk7) mutants after infection, similar to its effects in wild-type worms ( Supplementary Figure S1A, D ). We then tested the core components of the p38 MAPK pathway, including the MAPK kinase NSY-1 and the MAPK kinase SEK-1. Berberine hydrochloride failed to confer resistance to P. aeruginosa PA14 infection in nsy-1(ag3) and sek-1(ag1) mutants ( Figures 3E, F ; Supplementary Table S1 ). These findings indicate that Berberine hydrochloride may enhance innate immunity in C. elegans primarily through the p38 MAPK pathway.

To determine whether Berberine hydrochloride activates the p38 MAPK pathway, we assessed the phosphorylation levels of PMK-1, a key marker of its activation (8, 22, 23). Our results showed that Berberine hydrochloride significantly increased the levels of phosphorylated PMK-1 protein in C. elegans ( Figure 4A ). We next examined the expression of PMK-1 downstream targeted genes, K08D8.5, lys-2, and F35E12.5 (2, 24). Quantitative real-time PCR analysis revealed that these genes were up-regulated in Berberine hydrochloride-treated worms compared to control ( Figure 4B ). However, this up-regulation was abolished in pmk-1 RNAi-treated worms ( Figure 4B ). Additionally, we evaluated the expression of K08D8.5 and T24B8.5 using transgenic worms expressing K08D8.5p::GFP and T24B8.5p::GFP. Higher GFP fluorescence levels were observed in Berberine hydrochloride-treated worms but were not detected in pmk-1 RNAi worms ( Figures 4C, D ). These findings collectively demonstrate that Berberine hydrochloride may activate the p38 MAPK pathway in C. elegans after exposure to pathogens.

To evaluate the tissue-specific role of PMK-1 in response to P. aeruginosa infection following Berberine hydrochloride treatment, we conducted tissue-specific knockdown experiments using TU3401 (25, 26), NR350 (27), NR222 (27), and VP303 strains (28). Knockdown of pmk-1 in neurons, muscle, or hypodermis after Berberine hydrochloride treatment enhanced host survival during P. aeruginosa PA14 infection ( Figures 5A–C ; Supplementary Table S1 ). However, RNAi-mediated knockdown of pmk-1 in VP303 completely abolished the protective effect of Berberine hydrochloride ( Figure 5D ; Supplementary Table S1 ). These results indicated that Berberine hydrochloride mediated-pathogen resistance depended on the intestinal activation of PMK-1. To further confirm whether Berberine hydrochloride relies on intestinal PMK-1 to regulate innate immunity, we utilized AY102 transgenic worms, which express pmk-1 under the intestinal vha-6 promoter in a pmk-1(km25) mutant background. Expression of pmk-1 in the intestine fully restored the survival rate of pmk-1(km25) mutants ( Figure 5E ; Supplementary Table S1 ). These results demonstrate that Berberine hydrochloride-induced innate immunity requires the intestinal activity of PMK-1.

To investigate whether Berberine hydrochloride protects mice against P. aeruginosa infection, mice were treated with 10 mg/kg body weight of Berberine hydrochloride (29), while mice were infection with P. aeruginosa PA14 (1.0 × 10⁶ CFUs/mouse). Berberine hydrochloride-treated mice exhibited enhanced resistance to P. aeruginosa PA14 infection compared to control mice ( Figure 6A ). Since, the p38 MAPK pathway, conserved from worms to mammals (8), was assessed to determine if it mediates the Berberine hydrochloride-induced immune response. Mice were treated with 10 mg/kg Berberine hydrochloride (29) or 20 μg/kg of the p38 inhibitor SB202190 (8, 30), followed by P. aeruginosa PA14 infection (1.0 × 10⁶ CFUs/mouse). SB202190-treated mice showed increased susceptibility to infection compared to controls, and the inhibitor blocked the enhanced resistance induced by Berberine hydrochloride ( Figure 6B ). Bacterial loads in the lungs were quantified by colony-forming units (CFUs). Berberine hydrochloride-treated mice displayed significantly fewer CFUs of P. aeruginosa compared to controls ( Figure 6C ), whereas SB202190-treated mice had increased bacterial loads. However, the bacterial reduction conferred by Berberine hydrochloride was not observed when SB202190 was administered ( Figure 6C ). Additionally, Berberine hydrochloride significantly increased active PMK-1 levels in mice ( Figures 6D, E ). Collectively, these results suggest that Berberine hydrochloride enhances innate immunity in mice via the p38 MAPK pathway.

Berberine hydrochloride was obtained from Sigma Chemical Co. (St. Louis, MO) and dissolved in Dimethyl sulfoxide (DMSO) as a stock solution at a 100 mM concentration and stored in aliquots at -20°C. The experimental groups with different molar concentrations (5, 10, and 20 μM) were obtained by adding 5 μL, 10 μL, and 20 μL of 100 mM Berberine hydrochloride to 100 mL of Nematode Growth Medium (NGM). During this procedure, the concentration of DMSO in the medium ends up being much less than 0.02%. This will ensure the accuracy of the experimental results during the experiment.

N2 Bristol wild-type, AU3 nsy-1(ag3), AU1 sek-1(ag1), KU25 pmk-1(km25), CB1370 daf-2(e1370), SD184 mpk-1(n2521), YT17 crh-1(tz2), CL691 skn-1(zu67), VC8 jnk-1(gk7), RB754 aak-2(ok524), AU133 (irg-1::GFP), AU78 (T24B8.5p::GFP), SAL144 (K08D8.5p::GFP), AY102 [pmk-1(km25);acEx102], NR222 [rde-1(ne219);kzIs9], NR350 [rde-1(ne219);kzIs20], VP303 [rde-1(ne219) V; kbIs7], TU3401 [sid-1(pk3321)V;uIs69V] were obtained from the Caenorhabditis Genetics Center (CGC at the University of Minnesota, USA).

The E. coli strain HT115(DE3) expressing dsRNA and the bacteria strain HT115 (DE3) containing the empty vector L4440 as a control were each added to LB liquid medium containing 100 μg/mL ampicillin, overnight culture at 37 ℃. The amplified bacterial solution was added to the NGM culture plate containing IPTG, dried and placed in a constant temperature incubator at 25 ℃ for 12 h. Then the synchroized larvae growing to L1 stage were added to the culture plate. These nematodes were cultured at 20 ℃ until L4 stage, and then verified by gene knock-down experiment, or used for later RNAi nematode pathogen infection experiment.

The strains of E.coli used for RNAi were obtained from the Ahringer library (41). Unc-22 RNAi was included as a positive control in all experiments to account for RNAi efficiency.

Escherichia coli OP50, Listeria monocytogenes, Salmonella enterica SL1344, and Pseudomonas aeruginosa PA14 were grown overnight in LB broth at 37°C, and took 100 μL of bacterial solution and spread to NGM plates. Then the NGM plate coated with the bacterial solution is placed in a biosafety cabinet to let the bacterial solution dry. The culture plate was incubated at 37 ℃ for 12 h to activate the virulence factors of Listeria monocytogenes, Salmonella enterica SL1344, and Pseudomonas aeruginosa PA14. All infection assay were performed on NGM agar plates or NGM plates supplemented with or without Berberine hydrochloride (0, 5, 10, 20 μM). The number of living worms were counted at 12 h intervals. Immobile adult worms unresponsive to touch were scored as dead (19, 42). Animals that climbed down from plates or showed unnatural deaths were censored. Unnatural death mainly refers to the death of nematodes caused by external factors or experimental operations. For example, improper use of worm pickers can cause mechanical damage to the worm bodies. Uneven temperature can cause nematodes to drill into the NGM plate or lead to their loss. All experiments were performed three times independently.

Liquid bacterial growth was performed in 96-well microtiter plates containing the different bacterial strains as previously described (31, 43). The absorbance (OD 600 nm) was measured every 5 h for an 36 h incubation period with regular shaking at 37°C, 180 rpm. All experiments were performed three times independently.

Nematodes were synchronized and treated for 1 day with or without 10 μM Berberine hydrochloride starting at L4 larvae stage. The images were obtained by using Zeiss Axioskop 2 plus fluorescence microscope. Fluorescence intensity was quantified by using the ImageJ software (NIH). All experiments were performed three times independently.

Nematodes were synchronized and treated for 1 day with or without 10 μM Berberine hydrochloride starting at L4 larvae stage. Total RNA was extracted from worms with TRIzolReagent (Invitrogen) as previously described (8). Using pmp-3 for an internal controlas previously described (8). All experiments were performed three times independently. The following primers were used in this study:

pmp-3 primers:

K08D8.5 primers:

lys-2 primers:

F35E12.5 primers:

Synchronized worms were cultivated on E.coli OP50 at 20°C until L4 larvae stage. Then, worms were transferred to NGM agar plates (supplemented with or without 10 μM Berberine hydrochloride) containing P. aeruginosa/GFP, S. enterica/GFP, or L. monocytogenes/GFP for 48h at 25°C (2, 23). The images were obtained by using a Zeiss Axioskop 2 plus fluorescence microscope. Fluorescence intensity was quantified by using the ImageJ software (NIH). All experiments were performed three times independently.

Worms or lung tissue were homogenized in liquid nitrogen. Then, the homogenate was lysed on ice for 30 minutes in lysis buffer. 1. The lysates of total protein were loaded (60μg per well) and separated on a 10% SDS polyacrylamide gel. The preparation method refers to the instruction manual of the SDS-PAGE gel preparation kit. 2. Electrotransfer was performed after electrophoresis. A PVDF membrane with a diameter of 0.22 μm was soaked in methanol until the membrane became translucent. Then, the PVDF membrane was soaked in the electrotransfer solution. A membrane sandwich was made by placing the gel on it and covering it with the PVDF membrane. Electrotransfer was carried out at 300 mA for 90 min. 3. After being blocked with 5% skim milk powder for 2 h, the PVDF membrane was washed three times with TBST. 4. Phosphorylated PMK-1 protein was detected by using anti-active p38 polyclonal antibody from rabbit (Abcam, ab4822, 1:1000 dilution), and anti-beta actin antibodies (Abcam, ab227387,1:1000 dilution). Band intensities were measured using ImageJ software. All experiments were performed three times independently.

C57BL/6mice were inoculated with P. aeruginosa-laden agarose beads, as previously described (8, 44). The average 50 µl agar-beads suspension contained 1.0 × 10⁶ CFUs/mouse. The agar-beads suspension was ready for inoculation in the lungs of mice by an intratracheal injection. Animals received daily doses of 10 mg/kg body weight Berberine hydrochloride and SB202190 (20 μg/kg/d) through intraperitoneal injection for 7 d. Each group had 10 animals.

ARRIVE guidelines Compliance: All methods are reported in accordance with ARRIVE guidelines.

Data were presented as mean ± SEM. Graphs were generated with GraphPad Prism 7.0 software (GraphPad, San Diego, CA, USA). Statistical analyses for all data were carried out using Student’s t-test (unpaired, two-tailed) or ANOVA after testing for equal distribution of the data and equal variances within the data set. Survival data were analyzed by using the log-rank (Mantel-Cox) test. All experiments were performed three times independently. P< 0.05 was considered significant.