Male SPF grade ICR mice (18–22 g) were procured from Speforward (Beijing) Biotechnology Co., Ltd. (License No. SCXK (Beijing) 2024-0001). Mice were housed under standard laboratory conditions (18–22 °C, 50–60% relative humidity) with a 12 h light/dark cycle and had ad libitum access to food and water. Animals were acclimatized to the laboratory environment for one week prior to experimentation. All animal procedures were performed in accordance with the guidelines of the Animal Ethics Committee of Dali University (Ethics No. 2024-P2-110).

The whole plant of M. delavayi was collected in Dali City, Yunnan Province, China, and authenticated morphologically by Dr. Yong-Zeng Zhang at the College of Pharmacy, Dali University, Yunnan Province, China. A voucher specimen was deposited at the Yunnan Key Laboratory of Screening and Research on Anti-pathogenic Plant Resources from Western Yunnan, Dali University (plant sample preservation number: 20180627-4). Two kilograms of plant samples were milled and extracted three times with 95% ethanol at room temperature (24 h, 48 h, and 48 h, respectively). The resulting extracts were combined, filtered, and concentrated under reduced pressure using a rotary evaporator, yielding 445 g of freeze-dried powder. Prior to administration, the powders were suspended in a 0.5% CMC-Na solution. CMC-Na (Batch No: IS9000) was purchased from Solarbio Biotechnology Co., Ltd., Beijing, China. Domperidone was purchased from Shanghai Yuan-Ye Co., Ltd. (Batch No: S26612) and used as a positive control drug.

L-arg (Batch No: 150905) and superoxide anion (O₂⁻) detection kit (Batch No: BC1290) were purchased from Solarbio Biotechnology Co., Ltd., China. Evans Blue dye (Batch No. 991107) was purchased from the Shanghai Chemical Reagents Procurement and Supply Station. Detection kits of motilin (MTL, Batch No: H182-1-2) and gastrin (GAS, Batch No: H239-1-2) were sourced from Nanjing Jiancheng Bioengineering Co., Ltd., China. Detection kits of vasoactive intestinal peptide (VIP, Batch No: E-EL-M1234) and acetylcholinesterase (AChE, Batch No: E-EL-M26) were purchased from Elabscience Biotechnology Co., Ltd., China. Detection kits of soluble guanylate cyclase (sGC, Batch No: ml925127V), protein kinase G (PKG, Batch No: ml037829V), inducible nitric oxide synthase (iNOS, Batch No: ml057773V), and neuronal nitric oxide synthase (nNOS, Batch No: ml0930900) were sourced from Shanghai Enzyme-linked Biotechnology Co., Ltd., China. The primary antibodies against sGC (Batch No: 19011-1-AP), PKG (Batch No: 21646-1-AP), nNOS (Batch No: 29231-1-AP), iNOS (Batch No: 22226-1-AP), and $\beta$-actin (Batch No: 20536-1-AP), as well as the horseradish peroxidase (HRP)-linked secondary antibody (Batch No: RGAR001, 1:6000), were purchased from Proteintech Group, Inc., China.

After acclimation, sixty mice were randomly assigned to six groups (n = 10 per group): control, model, domperidone (0.1 g/kg, positive control), and M. delavayi extract (1.0, 0.5, and 0.25 g/kg). All treatments were administered once daily by gavage for 7 days at an administration volume of 20 mL/kg. From days 4–7, all mice except those in the control group received L-arg (3 g/kg, 1 h before drug treatment) intragastrically to induce FD. The dosage and administration period were determined based on preliminary experiments and previously published studies.

A blue-colored semi-solid nutritional paste (300 mL) was prepared by dissolving 10 g sodium carboxymethyl cellulose in 250 mL distilled water, followed by sequential addition of 12 g sucrose, 16 g milk powder, and 10 g starch to form a suspension under continuous stirring, with the final addition of 0.1 g Evans Blue dye as a visual tracer. The paste was stored at 4 °C and warmed to room temperature prior to oral administration. After seven days of administration, mice were for fasted 12 h with ad libitum access to water. The next morning, each mouse was administered 0.8 mL of the prepared paste through gavage. Twenty minutes post-gavage, the blood of each mouse was collected. Following collection, mice were euthanized by cervical dislocation, and their abdomen was excised. After ligation of both the cardia and pylorus, the entire stomach was removed and rinsed with saline to eliminate blood contaminants. The total weight of stomach was weighed, then the stomach was opened along the greater curvature, and the gastric contents were washed with saline. The emptied stomach was wiped dry with filter paper before measurement of the net weight of the stomach. Gastric emptying rate (%) was calculated as: [Weight of administered paste – (total weight of stomach – net weight of stomach)] / Weight of administered paste $\times$ 100%. Concurrently with stomach isolation, the entire small intestine was excised from the distal pylorus to the ileocecal junction. Both the total length of the small intestine and the propulsion distance of the paste were measured to calculate intestinal propulsion rate (%) as: Propulsion distance of the paste / Total length of the small intestine $\times$ 100%.

Blood samples were collected from the retro-orbital plexus under brief isoflurane (3% for induction and 1.5–2% for maintenance, administered via inhalation using a calibrated vaporizer with oxygen flow) anesthesia and allowed to stay at room temperature for 2 h. Then, serum was prepared by centrifugation (3000 rpm for 10 min) at 4 °C, stored at –80 °C, and used to analyze the levels of gastrointestinal hormone. The gastric antrum specimen was isolated from the stomach and fixed in 4% paraformaldehyde for histopathological examination. The ileum tissue was obtained from the small intestine and homogenised on ice for subsequent analysis, including Enzyme-Linked Immunosorbent Assay (ELISA) and WB.

Following fixation, gastric specimens were processed through standard H&E staining protocols, including paraffin embedding, sectioning, xylene dewaxing, graded ethanol rehydration, H&E staining, ethanol dehydration, xylene clearing, and neutral balsam mounting. Histopathological changes were observed under a light microscope.

ELISA kits were employed to quantify the levels of gastrointestinal hormones and the contents of the key NO-sGC-PKG signaling pathway components in mice. After the preparation of serum, the contents of MTL, GAS, VIP, and AChE were measured according to the kit protocols. The samples of ileum tissues were homogenized and centrifuged, and the resulting supernatant was used to measure the contents of sGC, PKG, nNOS, iNOS and O₂⁻ with the kit instructions.

Ileal tissues were lysed in high-efficiency RIPA lysis buffer (Solarbio, Beijing, China; Batch No. 20220211) supplemented with a 50$\times$ protease–phosphatase inhibitor cocktail (Beyotime Biotechnology, Shanghai, China; Batch No. 020221210218). Protein concentrations were determined using a BCA protein assay kit (Biosharp Biotechnology Co., Ltd., China; Batch No. 21362908). Equal amounts of protein were resolved by SDS–PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Germany; Batch No. R1AB03348) using a Mini Trans-Blot transfer system (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% non-fat dry milk (Solarbio, Beijing, China; Batch No. 107B053) for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies against sGC (1:4000), PKG (1:3000), nNOS (1:4000), iNOS (1:1000), and $\beta$-actin (1:6000) (Proteintech Group, Wuhan, China). After three washes with TBST, membranes were incubated with HRP-conjugated secondary antibodies (goat anti-rabbit IgG, Proteintech) for 2 h at room temperature. Protein bands were visualized using an ultrasensitive ECL chemiluminescence detection kit (Beyotime Biotechnology, Shanghai, China; Batch No. 090921220310) and imaged with a ChemiDoc XRS+ system (Bio-Rad, USA). Relative protein levels were normalized to $\beta$-actin.

A total of 49 compounds isolated from the 95% ethanol extract of M. delavayi (as reported by Tang [22]) were subjected to network pharmacology analysis. Potential molecular targets of the identified compounds were predicted using the SwissTargetPrediction database (http://www.swisstargetprediction.ch/), and targets with a probability $>$0.15 were retained for further analysis. FD-related genes were retrieved from the GeneCards database and subsequently refined through additional screening via OMIM and DisGeNET databases. The overlapping targets between M. delavayi compounds and FD-related genes were identified through a Venn diagram using the Jvenn online platform. These targets were regarded as potential candidates for M. delavayi in treating FD and imported into the STRING database (https://string-db.org/) to construct a protein-protein interaction (PPI) network, with parameters set to Homo sapiens species and a minimum interaction confidence score of 0.7. The PPI results were visualized and topologically analyzed using Cytoscape 3.9.1 software. The key nodes were selected based on the median centrality (betweenness), proximity to centrality (closeness), and degree of freedom (degree). Subsequently, the analysis of Gene Ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed using the DAVID functional annotation tool. Hierarchical clustering heatmaps and bubble plots were generated to visualize the enriched pathways, with statistical significance set at p 0.05.

Data were presented as mean $\pm$ standard deviation (SD). Statistical analysis was performed using GraphPad Prism 10.0 (GraphPad Software, San Diego, CA, USA), with one-way analysis of variance (ANOVA) followed by post hoc tests for multiple group comparisons. Tukey’s test was used for uniform variance, while the Dunnett’s T3 test was applied for non-uniform variance. p 0.05 was considered statistically significant.

Compared to the control group, L-arg administration significantly reduced gastric emptying rate and intestinal propulsion rate, both of which are key indicators of abnormal gastrointestinal motility observed in the model group (p 0.01) (Fig. 1A,B). As expected, both the positive drug domperidone and M. delavayi treatment markedly restored gastric emptying rate and small intestinal propulsion rate compared to the model group (p 0.01) (Fig. 1A,B), except for the 0.25 g/kg M. delavayi, which had no obvious effect on gastric emptying rate. These findings indicate that M. delavayi alleviates FD symptoms by promoting gastrointestinal peristalsis.

Fig. 1.

Effect of M. delavayi on gastrointestinal motility and gastric tissue damage in FD mice. (A) Gastric emptying rate (n = 10). (B) Small intestinal propulsion rate (n = 10). ^#⁢# p 0.01 vs. the control, **p 0.01 vs. the model. (C) H&E staining images of gastric antrum tissue (scale bar = 100 µm). Green arrows indicate desquamation of epithelial cells, red arrow indicates focal necrosis of gastric glandular cells, and black arrows indicate infiltration of inflammatory cells. Data are presented as mean $\pm$ SD. M. delavayi, Megacarpaea delavayi; FD, functional dyspepsia; H&E, Hematoxylin and Eosin.

In the control group, H&E staining of the gastric antrum tissue exhibited uniformly stained cells and orderly arranged muscle fibers in the muscularis layer. The lamina propria contained abundant gastric glands with intact morphology. There were no obvious pathological alterations or inflammatory cell infiltration observed (Fig. 1C). In contrast, the model group displayed gastric tissue abnormalities, including desquamation of epithelial cells (green arrows) and focal necrosis of gastric glandular cells (red arrows, pyknotic nuclei) in the lamina propria, and marked infiltration of inflammatory cells (black arrows) in the submucosal layer (Fig. 1C). After domperidone and M. delavayi treatment, gastric antrum tissue exhibited varying degrees of improvement in structure, with relatively preserved epithelial integrity and reduced inflammatory infiltration (Fig. 1C).

Gastrointestinal hormones play important roles in regulating gastric motility and peristaltic rhythm. Compared to the control group, serum levels of motilin MTL and GAS were significantly reduced in the model group (p 0.01) (Fig. 2A,B), while levels of VIP and AChE were markedly elevated (p 0.01) (Fig. 2C,D). After drug intervention, compared with the model group, M. delavayi at 0.25, 0.5, and 1 g/kg, as well as domperidone, significantly increased MTL and GAS levels (p 0.01) (Fig. 2A,B), except for the 0.5 g/kg M. delavayi, which showed no significant effect on GAS. All the treatments also exhibited marked suppression of AChE activity (p 0.01) (Fig. 2D), leading to elevated ACh levels. Notably, VIP levels remained unchanged in all M. delavayi treatment groups (p $>$ 0.05); only a significant decrease in VIP levels was observed in the domperidone group (p 0.05) (Fig. 2C). These results demonstrate that M. delavayi ameliorates impaired gastrointestinal motility in FD mice by restoring physiological levels of excitatory gastrointestinal hormones (MTL, GAS, and ACh).

Fig. 2.

Effect of M. delavayi on serum levels of gastrointestinal hormones and AChE in FD mice (n = 6). (A) MTL level. (B) GAS level. (C) VIP level. (D) AChE level. ^#⁢# p 0.01 vs. the control, **p 0.01, *p 0.05 vs. the model. MTL, motilin; GAS, gastrin; VIP, vasoactive intestinal peptide; AChE, acetylcholinesterase. Data are presented as mean $\pm$ SD.

Based on previous phytochemical investigations, 49 active compounds were identified from M. delavayi. Among these, 35 compounds (Supplementary Table 1) were predicted to have corresponding protein targets. After target prediction using the SwissTargetPrediction database and removal of duplicate targets, 337 potential protein targets were obtained. Meanwhile, 2093 FD-associated genes were retrieved from three databases. Comparative analysis of compound-related targets and disease-associated genes yielded 102 overlapping targets, which were visualized in a Venn diagram (Fig. 3A).

Fig. 3.

Network pharmacology prediction. (A) Venn diagram of overlapping target genes between FD and M. delavayi. (B) PPI network visualization of the total of 102 overlapping target genes. (C) PPI network visualization of the top 16 targets and their corresponding compounds in M. delavayi. (D) Hierarchical network plot for GO enrichment analysis (top 10 items). (E) Sankey diagram (left) and bubble plot (right) for KEGG enrichment analysis (top 20 items). BP, biological process; MF, molecular function; CC, cellular component; KEGG, Kyoto Encyclopedia of Genes and Genomes.

The PPI network of the 102 overlapping targets was analyzed using the STRING database and constructed using Cytoscape software. The network consisted of 102 nodes and 847 edges, with an average node degree of 6.48 (Fig. 3B). These targets were further filtered and explored through PPI network analysis, leading to the identification of 16 core targets. Among these, six are associated with inflammation, including IL6, TNF, NOS1, EGFR, SRC, and STAT3 (Fig. 3C).

GO enrichment analysis was performed across the categories of biological process (BP), molecular function (MF), and cellular component (CC), using a significance threshold of p 0.05 with false discovery rate correction (q 0.05) (Fig. 3D). The top 10 enriched GO entries were visualized using a hierarchical network plot (Fig. 3D). Notable BP mainly involved positive regulation of the ERK1/2 cascade, response to oxidative stress, and positive regulation of the NO biosynthetic process. The CC mainly included the plasma membrane, neuronal cell body, and postsynaptic membrane. The MF primarily involved protein kinase binding, enzyme binding, and neurotransmitter receptor activity.

KEGG pathway enrichment analysis identified 120 enriched entries, and the top 20 significantly enriched signaling pathways were visualized using a bubble plot (Fig. 3E). The results demonstrated that the nitrogen metabolism, calcium signaling pathway, cholinergic synapse, and reactive oxygen species (ROS) were the main enriched pathways.

Integration of the core targets with GO and KEGG enrichment analysis revealed that NO and its associated inflammatory signaling pathways play a pivotal role in the action of M. delavayi, requiring further validation.

Firstly, the ELISA assay was used to quantify the levels of key components in the NO-sGC-PKG signaling pathway, which is closely associated with neurotransmitter receptor modulation, inflammatory responses, and oxidative stress regulation. Compared to the normal group, L-arg intervention in FD mice significantly elevated O₂⁻ contents (p 0.01, Fig. 4A) and suppressed levels of sGC and PKG (p 0.01, Fig. 4B,C) in intestinal tissues, while differentially modulated the contents of NOS isoforms by inhibiting nNOS expression (p 0.01, Fig. 4D) but enhancing iNOS content (p 0.01, Fig. 4E). Similar results were observed by WB analysis (Fig. 4F).Administration of M. delavayi at all doses ameliorated L-arg-induced abnormal activities of the NO-sGC-PKG signaling pathway in a dose-dependent manner. Specifically, all dose of M. delavayi (1, 0.5, and 0.25 g/kg) obviously decreased O₂⁻ contents (p 0.01, Fig. 4A) and elevated the levels of sGC and nNOS (p 0.01, Fig. 4B,D). Only the 1 and 0.5 g/kg M. delavayi markedly increased PKG level (p 0.01 or p 0.05, Fig. 4C). The 0.5 and 0.25 g/kg M. delavayi did not significantly alter iNOS levels (p $>$ 0.05, Fig. 4E), whereas the 1.0 g/kg dose unexpectedly induced a pronounced elevation of iNOS (p 0.01, Fig. 4E), suggesting a dose-dependent but complex regulatory effect on iNOS synthesis.

Fig. 4.

Effect of M. delavayi on the NO-sGC-PKG signaling pathway in FD mice. (A–E) The levels of O₂⁻, sGC, PKG, nNOS, and iNOS were measured by ELISA (n = 8). (F) Western Blot image. (G–J) The relative protein levels of sGC, PKG, nNOS, and iNOS were measured by Western Blot (n = 4). ^#⁢# p 0.01 vs. the control, **p 0.01, *p 0.05 vs. the model. O₂⁻, superoxide anion; sGC, soluble guanylate cyclase; PKG, protein kinase G; nNOS, neuronal nitric oxide synthase; iNOS, inducible nitric oxide synthase. Data are presented as mean $\pm$ SD.

Similar results were observed by WB analysis. Compared to the model group, 1 and 0.5 g/kg M. delavayi significantly increased the protein expression levels of sGC, PKG, and nNOS (p 0.01 or p 0.05, Fig. 4G–I). Meanwhile, 0.5 and 0.25 g/kg M. delavayi had no significant effect on iNOS expression (p $>$ 0.05, Fig. 4J), whereas 1 g/kg M. delavayi markedly upregulated iNOS protein levels (p 0.01, Fig. 4J).