# Fructus Aurantii Extracts Ameliorate Intestinal Inflammation and Regulate the Intestinal Barrier through Fc Epsilon RI Signaling Pathway

Authors: Sicong Liu; Yongrui Bao; Shuai Wang; Tianjiao Li; Jiapeng Leng; Yi Zheng; Ying Meng; Ying Zheng; Bing Qi; Xi Luo; Xiansheng Meng
Journal: Journal of Food Biochemistry
 (2023)

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## Abstract

Fructus Aurantii (FA) is the dry and immature fruit ofCitrus aurantium L. and its rutaceous cultivars. FA has been widely used to treat digestive system diseases since ancient China, and it promotes gastrointestinal (GI) motility in functional dyspepsia (FD), but its potential therapeutic mechanisms remain unclear. We examined the effects of FA ethanol extracts in an iodoacetamide (IA)-induced FD rat model. Firstly, key FA therapy targets for FD were gathered using systematic pharmacology. Combined with systemic pharmacological analyses, plasma metabolomics based on UPLC-QTOF-MS were conducted. Then, MetaboAnalyst was used to jointly analyze systemic pharmacology targets and metabolomic metabolites to select key metabolic pathways. Finally, the key path is verified by experiments. FA exerted distinct therapeutic effects in anti-inflammation and promoting gastrointestinal motility in our IA-induced FD rat model. When compared with the model group, FA down-regulated the inflammatory factors interleukin 1β and tumor necrosis factor-α. At the same time, FA up-regulated tight junction proteins in the intestinal epithelial barrier. Through the integrated analysis of metabolomics and systemic pharmacology, we conducted experimental verification on Fc epsilon RI signaling pathway. When compared with the model group, FA down-regulatedphospho-mitogen activated protein kinase, phospho-extracellular signal regulated kinase1/2, myosin light chain kinase, and phospho-myosin regulatory light chain protein levels. Thus, FA ameliorated FD by regulating the Fc epsilon RI signaling pathway. Our integrated strategy identified underlying FA mechanisms toward FD treatment and provided a foundation for FA development as a clinical agent for FD.


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## Body

## 1. Introduction

Functional dyspepsia (FD) is defined as the presence of symptoms thought to originate in the gastroduodenal region in the absence of any organic, systemic, or metabolic disease that readily explains the complaints [1]. It is one of the most common functional gastrointestinal disorders, affecting up to 16%–26% of the population [2], which increases year on year and seriously affects the quality of life of sufferers. In recent years, with a deepened knowledge of its pathophysiology, FD pathogenetic research has shifted from the stomach to the duodenum and focused on mild duodenal inflammation and intestinal barrier damage [3]. Growing evidence has shown that low-grade duodenal inflammation in FD was mainly based on the histological detection of a slightly but significant increased number of mucosal immune cells, mainly eosinophils and mast cells [4]. In a previous study, it has demonstrated that the intestinal barrier damage in FD patients found a correlation between certain cell-to-cell adhesion proteins and low-grade inflammation [5]. Although the origin of this low-grade inflammation remains unidentified, it has been found that barrier dysfunction facilitates the passage of luminal antigens through the epithelium, thereby triggering an immune response. This, in turn, leads to low-grade inflammation, which contributes to the creation and persistence of gastrointestinal symptoms [6]. Currently, drug therapy is the major treatment for FD; while domperidone (Domp) and mosapride are effective treatments for the condition; their clinical use is restricted due to potential severe cardiovascular side effects [7]. Hence, safer and more efficacious FD treatments are required, and natural products may be an innovative source of such treatments. Presently, due to their benefits such as high compliance, remarkable curative effects, and fewer adverse effects, natural products are being used to treat and prevent FD.Fructus Aurantii (Chinese name Zhiqiao) is the dried unripe fruit ofCitrus aurantium L. that is collected from July. For a long time, the processed unripe fruits of bitter orange possesses homology of medicine and food characteristic, which is regarded to the health promotion effect in the digestive tract system. Since ancient times in China, Fructus Aurantii (FA) has been used for digestive system diseases. The “Supplementary Records of Famous Physicians” (the late Han Dynasty, approximately 220 A.D.) recorded that “FA eliminated flatulence and calmed the intestines and stomach.” FA also relieves gastrointestinal syndrome induced by FD and exerts significant curative effects for the condition [8, 9]. FA has diverse pharmacological activities, including antidiabetic, antidepression, anticancer, antikidney stone formation, and cardiovascular protection [10–12]. Clinical trials and pharmacological studies have confirmed its effects for treating GI dysfunction by accelerating GI transport and gastric emptying [9, 13]. FA reportedly elevates gastric emptying rates, intestinal propulsion, and motilin and gastrin serum levels in rats [14]. Also, the pharmacological components of FA, such as narirutin, naringin, and hesperetin, reportedly exert anti-inflammatory effects by inhibiting the lipopolysaccharide-mediated activation of NF-κB and MAPKs [15, 16]. Through 5-H4R/cAMP pathway activation, naringin, hesperidin, and hesperetin regulate intestinal epithelial tight junction (TJ) proteins and intestinal flora, thereby improving GI function [16, 17]. Although many studies have confirmed the therapeutic efficacy of FA for FD, its effective constituents and related anti-FD mechanisms are unclear, which complicates its clinical application to modern medicine. Hence, identification of efficacious FA constituents and their mechanisms of action are warranted.Metabolomics is an important drug research method, which can systematically detect the dynamic changes of small molecule metabolites in vivo [18, 19]. Given the complexity of multiple natural products constituents, it is difficult to screen for representative combinations of active constituents; the process is usually time-consuming, laborious, and leads to unsatisfactory results. Interestingly, metabolomics and natural products share similar system characteristics. Hence, metabolomics has been evaluated as the most common and effective method for the efficacy of natural products, comprehensively identifying and potentially explaining the molecular mechanisms underpinning different diseases and relevant natural products treatments [20]. In 2007, based on the interactive information from diseases, genes, targets, and drugs [21], systematic pharmacology [22] was proposed as an effective data mining tool. Currently, many systematic pharmacology studies have been performed and successfully predicted associations between herbal products and certain disease-related targets [23–25].In this study, metabolomics and systemic pharmacology were jointly applied to screen core signal pathways, and then experimental verification was conducted for the hub targets of the core pathway. Finally, a complete active herbal compound-target-pathway-biomarker network was constructed to explain the therapeutic efficacy of FA toward FD.


## 2. Materials and Methods

### 2.1. Materials and Reagents

FA (specimen number: 20210220, Quanjian, China) was identified asCitrus aurantium L. by Professor Zhang Hui, College of Pharmacy, Liaoning University of Traditional Chinese Medicine, Liaoning, China. Domp tablets (Lot. No. KJJOYG3) were supplied by Janssen, China. Standard reagents such as pentobarbital sodium (Merck, Germany), nobiletin, limonin, 3,4-dihydroxybenzaldehyde (purity ≥98%) were supplied by Weikeqi, China. Acetonitrile (LC-MS grade, Merck, Germany), methanol (LC-MS grade, Merck, Germany), and formic acid (LC-MS grade, Fisher, USA) were used. Enzyme-linked immunosorbent assays-tumor necrosis factor-α (TNF-α) (Lot: E-EL-R2856c, Elabscience, China) and interleukin-1β (IL-1β) (Lot: KE20005, Proteintech). PrimeScript™ RT reagent Kit (Lot. No. AT311, Transgen, China). Antibodies; Zonula occludens-1 (ZO-1), Occludin, Myosin light chain kinase (Mlck), and Myosin light chain2 (Mlc2) (Lot. No’s. 21773-1-AP, 27260-1-AP, 10906-1-AP, and 21642-1-AP, respectively Proteintech, UK), Extracellular signal regulated kinase (Erk), Mitogen activated protein kinase (Mek), phospho-Mek (P-Mek), and phospho-Mlc2 (P-Mlc2) (Lot. No’s. 4372S, 8727T, 9154S, and 3671T, respectively, Cell Signaling Technology, USA), P-Erk1/2 (Lot. No. 340767, Zenbio, China), and Iodoacetamide (IA) (Lot. No. 604E036, Solarbio, China).


### 2.2. FA Extract Preparation

The FA extraction method, comprising extraction solvents, methods, and times, was optimized in previous studies [26]. In our study, 100 g FA powder was added to 70% ethanol (1 : 10, w/v) boiled for 1 h and the process repeated three times. After decoction, the filtrates were merged and filtered by three layers of gauze. The filtrate was collected and concentrated to 0.27 g·mL−1 in the rotary evaporator. One ml of FA solution was diluted in 99 ml methanol and filtered using a 0.22 μm microporous membrane before qualitative analysis.


### 2.3. Qualitative Analysis of FA Active Ingredients

Qualitative analyses were performed using an Agilent 6550 precise mass quadrupole flight time instrument (Agilent Technology Co., Ltd.) using an Agilent pore shell SB-C18 column (100 × 2.1 mm, 2.7μm) at 40°C. The mobile phase consisted of 0.1% formic aqueous solution (A) and acetonitrile (B) over the following gradient: 5%–24% B at 0–9 min; 24%–43% B at 9–24 min; 43%–85% B at 24–33 min; 85%–100% B at 33–36 min. The flow rate was 0.4 ml/min and the injection volume was 1 μl. For MS detection, acquisition parameters were as follows: drying gas flow rate = 13 l/min; drying gas temperature = 250°C; nebulizer gas pressure = 45 psi; capillary voltage = 3500 V; fragmentor voltage = 125 V. The mass range was 50–1200 m/z. All samples were analyzed in negative and positive ion mode. Data were collected and processed using Agilent Mass Hunter software (version B.06.00).


### 2.4. Animals

In order to minimize animal suffering, we provided the best care. The experimental procedures followed the Guide for the Care and Use of Laboratory Animals (United States National Institutes of Health Publication No. 85–23, revised 1986). This study was reviewed and approved by the Institutional Animal Care and Use Committee of the Liaoning University of Traditional Chinese Medicine (approval No.: 2019YS(DW)-33-01). Male Sprague Dawley rats (10 days old, 24 ± 3 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd, China. Rats were kept at 25°C ± 2°C in a 12 h/12 h light/dark cycle, with ad libitum access to food and water. The experiment was carried out as describing in literature [27]. Rat pups were then intragastrically administered 0.1% IA plus 2% sucrose at 0.2 ml/100 g once a day for 6 days, whereas control rats received 2% sucrose (0.2 ml/100 g) only. At 6 weeks, IA-treated rats underwent alternate-day fasting for 2 weeks, after which rats were randomized into three groups (n = 8/group) as follows: FD group (gavaged with an equivalent normal saline volume), FA group (gavage with 2 ml FA extract (2.7 g/kg crude drug according to the optimal dose based on the previous pharmacodynamics study of the research group [23])), and the Domp group (gavaged with 2 ml Domp solution (at 0.14 g/kg in water) once a day for two weeks). From the first day of IA administration to the day before euthanasia, rat mental state, weight, and food intake data were monitored on a weekly basis. Rats were fasted for 12 h before the last administration. Then, they were anesthetized with 1% pentobarbital sodium (40 mg/kg), and blood and duodenal tissue were rapidly collected. After that, animals were euthanized immediately via pentobarbital sodium intraperitoneal injection (100 mg/kg) [28]. For histopathology, 1 cm long duodenal tissue samples were fixed in 4% paraformaldehyde, while remaining samples were immersed in liquid nitrogen and stored at −80°C until required (Supplementary Material S1).


### 2.5. Exploring Gastric Emptying and Intestinal Propulsion

After the last treatment, animals were fasted for 12 h. Rats had been fed with nutritious semisolid paste containing graphite powder (2 ml) by gavage. After 30 min, rats were humanely euthanized, and the abdominal cavity opened immediately. In the absence of obvious intra-abdominal abnormalities, pyloric orifice and gastric cardia ligation were performed, followed by whole stomach and small intestine removal. Thereafter, we weighed the whole stomach, then eliminated the stomach contents by immersing the stomach in 0.9% normal saline, and dried the stomach with sterile filter paper and weighed it. At the same time, we measured the distance the graphite powder moves from the pylorus and the length of the entire small intestine. Then, we evaluated gastrointestinal motility by gastric residual rate and intestinal propulsion rate (Supplementary MaterialS2).


### 2.6. Histopathological Examinations and Immunohistochemistry Analyses

We embedded 4% paraformaldehyde-fixed duodenal tissue in paraffin, sectioned tissue into 4μm slices, and hydrated and stained samples in hematoxylin and eosin (H & E) for microscopic analysis.Following euthanasia, rat duodenal tissues were quickly isolated and washed in normal saline after removing attached tissue. Then, 1 cm duodenal biopsy specimens were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, sliced into 4μm sections, stained in H & E and toluidine blue solution, and observed microscopically (Nikon Eclipse C1, Japan). Sections were incubated overnight at 4°C with primary antibodies for ZO-1 (1 : 1,000) and Occludin (1 : 400) and washed three times. After washing, a corresponding horseradish peroxidase (HRP)-labeled secondary antibody (1 : 300) was added to allow for a 50 min incubation period at room temperature. Then, after slightly drying, a 4′,6-diamidino-2-phenylindole solution was added to slides for 10 min at room temperature in the dark to stain nuclei. After washing, sections were dried slightly, mounted with antifluorescence quenching mounting agent, and observed under fluorescence microscopy. We captured 200x and 400x images which were quantitatively analyzed using Image J 8.0 (National Institutes of Health, MD, USA).


### 2.7. Inflammatory Cytokine ELISA

For ELISA, plasma inflammatory biomarkers were detected using IL-1β and TNF-α kits as per manufacturer’s instructions. Absorbance at 450 nm was measured using enzyme markers, standard curves plotted, and IL-1β and TNF-α levels determined.


### 2.8. Quantitative-Polymerase Chain Reaction (qPCR)

Total RNA was extracted using Trizol reagent according to manufacturer’s instructions. cDNA was synthesized using TransScript OneStep gDNA removal and cDNA Synthesis Super Mix kits according to manufacturer’s instructions. Subsequently, qPCR was conducted according to manufacturer’s protocols under the following conditions: denaturation for 30 s at 94°C, followed by 40 cycles of 5 s at 94°C and 30 s at 60°C on a Real-time Thermal Cycler 5100 using a SYBR-Green master mix. All samples were normalized against GAPDH expression. Relative gene expression was analyzed using the 2−ΔΔCt method. Primer sequences are shown in Table 1.Table 1
Primer sequences of the genes used for Q-PCR.
GenesForward primerReverse primerLYNGAA​GCC​TGG​ACA​ATG​GTG​GTTCCC​ATG​GCT​TCT​GTG​GTT​TGSYKTGC​CCA​TCC​TGT​GAC​TTG​GTCCT​CCC​GTT​GGC​TCA​TAG​GMEKCAG​TTG​GAA​GAT​ACC​CCA​TTC​CGAG​GTC​GGC​TAT​CCA​TTC​CATERKCTT​TCC​TCC​GGC​AAA​ATG​ACCCA​GGT​TGC​TGT​TCA​CCA​GAAGAPDHCAT​GTT​CGT​CAT​GGG​TGT​GAAGGC​ATG​GAC​TGT​GGT​CAT​GAG


### 2.9. Western Blotting

Total duodenum tissue proteins were extracted in RIPA lysis buffer (plus 1 mM PMSF and phosphatase inhibitors), and concentrations determined using a spectrophotometer (BioTeke ND5000, Beijing, China). Proteins were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, which were then blocked in 5% skimmed milk in Tris-buffered saline and 0.1% Tween 20 (TBST) for 1 h at 37°C, followed by overnight incubation with primary antibodies for ZO-1(1 : 2000), Occludin (1 : 2000), Mlck (1 : 2000), Mlc2 (1 : 2000), Erk (1 : 2000), Mek (1 : 1000), P-Mek (1 : 1000), P-Mlc2 (1 : 1000), P-Erk1/2 (1 : 500), andβ-actin (1 : 3000) at 4°C. Next, membranes were washed in TBST, incubated with an antirabbit lgG antibody conjugated to HRP at 37°C for 1 h and washed in TSBT. Protein signals in membranes were observed using an enhanced chemiluminescence kit following manufacturer’s instructions. Protein signal densities and immune response analyses were performed using the Tanon-5200 imaging system (Tanon Science and Technology Co. Ltd., Shanghai, China) and ImageJ software.


### 2.10. Metabolomics Profiling of Plasma Samples

Plasma samples were also generated for LC/MS analysis by protein precipitation with methanol; 600μl cold methanol was added to 100 μl plasma samples, vortexed for 60 s, and centrifuged at 12,000 rpm for 15 min at 4°C. Then, the supernatant was dried under nitrogen gas, and residues were redissolved in 30 μl 50% methanol aqueous solution. We also prepared internal quality control (QC) samples by mixing 10 μL of each test sample. All samples were maintained at 4°C during analyses. Mobile phase and gradient parameters were identical to aforementioned parameters (Section 2.3) and the injection volume was 3 μl.


### 2.11. Systematic Pharmacology Analysis

FA chemical constituents were imported into the TCMSP (https://lsp.nwu.edu.cn/index.php) database to filter active compounds through oral bioavailability (OB ≥ 30%) and drug-like properties (DL ≥ 0.18). Active compounds were imported into PharmMapper (https://www.lilabecust.cn/pharmmapper/index.html) and Swiss Target Prediction (https://www.swisstargetprediction.ch) databases to collect corresponding targets. FD-related target genes were obtained from DrugBank (https://www.drugbank.ca/), GeneCards (https://www.genecards.org/), OMIM (https://omim.org/), and DisGeNET (https://www.Disgenet.org/). Furthermore, we submitted the acquired target genes to STRING (https://STRING-db.org/) to recognize the functional protein association networks in accordance with calculation parameters and screening scores.


### 2.12. Joint-Pathway Analysis

Joint-pathway analysis of differentially expressed metabolites with target proteins was performed using MetaboAnalyst 5.0 (https://www.metaboanalyst.ca) to derive insights on biological mechanisms associated with FD treatment. Then, an interaction network of herb-activecompound-hub target-pathway-biomarkers of FA treating FD was established. This network was visualized in Cytoscape (3.7.1) to show FD mechanisms.


### 2.13. Statistical Analysis

Statistical analyses were performed in GraphPad Prism 8.0 (GraphPad, San Diego, CA, USA). Differences between two groups were assessed using one-way analysis of variance and presented as mean ± standard deviation.P<0.05 value was considered statistically significant.


## 2.1. Materials and Reagents

FA (specimen number: 20210220, Quanjian, China) was identified asCitrus aurantium L. by Professor Zhang Hui, College of Pharmacy, Liaoning University of Traditional Chinese Medicine, Liaoning, China. Domp tablets (Lot. No. KJJOYG3) were supplied by Janssen, China. Standard reagents such as pentobarbital sodium (Merck, Germany), nobiletin, limonin, 3,4-dihydroxybenzaldehyde (purity ≥98%) were supplied by Weikeqi, China. Acetonitrile (LC-MS grade, Merck, Germany), methanol (LC-MS grade, Merck, Germany), and formic acid (LC-MS grade, Fisher, USA) were used. Enzyme-linked immunosorbent assays-tumor necrosis factor-α (TNF-α) (Lot: E-EL-R2856c, Elabscience, China) and interleukin-1β (IL-1β) (Lot: KE20005, Proteintech). PrimeScript™ RT reagent Kit (Lot. No. AT311, Transgen, China). Antibodies; Zonula occludens-1 (ZO-1), Occludin, Myosin light chain kinase (Mlck), and Myosin light chain2 (Mlc2) (Lot. No’s. 21773-1-AP, 27260-1-AP, 10906-1-AP, and 21642-1-AP, respectively Proteintech, UK), Extracellular signal regulated kinase (Erk), Mitogen activated protein kinase (Mek), phospho-Mek (P-Mek), and phospho-Mlc2 (P-Mlc2) (Lot. No’s. 4372S, 8727T, 9154S, and 3671T, respectively, Cell Signaling Technology, USA), P-Erk1/2 (Lot. No. 340767, Zenbio, China), and Iodoacetamide (IA) (Lot. No. 604E036, Solarbio, China).


## 2.2. FA Extract Preparation

The FA extraction method, comprising extraction solvents, methods, and times, was optimized in previous studies [26]. In our study, 100 g FA powder was added to 70% ethanol (1 : 10, w/v) boiled for 1 h and the process repeated three times. After decoction, the filtrates were merged and filtered by three layers of gauze. The filtrate was collected and concentrated to 0.27 g·mL−1 in the rotary evaporator. One ml of FA solution was diluted in 99 ml methanol and filtered using a 0.22 μm microporous membrane before qualitative analysis.


## 2.3. Qualitative Analysis of FA Active Ingredients

Qualitative analyses were performed using an Agilent 6550 precise mass quadrupole flight time instrument (Agilent Technology Co., Ltd.) using an Agilent pore shell SB-C18 column (100 × 2.1 mm, 2.7μm) at 40°C. The mobile phase consisted of 0.1% formic aqueous solution (A) and acetonitrile (B) over the following gradient: 5%–24% B at 0–9 min; 24%–43% B at 9–24 min; 43%–85% B at 24–33 min; 85%–100% B at 33–36 min. The flow rate was 0.4 ml/min and the injection volume was 1 μl. For MS detection, acquisition parameters were as follows: drying gas flow rate = 13 l/min; drying gas temperature = 250°C; nebulizer gas pressure = 45 psi; capillary voltage = 3500 V; fragmentor voltage = 125 V. The mass range was 50–1200 m/z. All samples were analyzed in negative and positive ion mode. Data were collected and processed using Agilent Mass Hunter software (version B.06.00).


## 2.4. Animals

In order to minimize animal suffering, we provided the best care. The experimental procedures followed the Guide for the Care and Use of Laboratory Animals (United States National Institutes of Health Publication No. 85–23, revised 1986). This study was reviewed and approved by the Institutional Animal Care and Use Committee of the Liaoning University of Traditional Chinese Medicine (approval No.: 2019YS(DW)-33-01). Male Sprague Dawley rats (10 days old, 24 ± 3 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd, China. Rats were kept at 25°C ± 2°C in a 12 h/12 h light/dark cycle, with ad libitum access to food and water. The experiment was carried out as describing in literature [27]. Rat pups were then intragastrically administered 0.1% IA plus 2% sucrose at 0.2 ml/100 g once a day for 6 days, whereas control rats received 2% sucrose (0.2 ml/100 g) only. At 6 weeks, IA-treated rats underwent alternate-day fasting for 2 weeks, after which rats were randomized into three groups (n = 8/group) as follows: FD group (gavaged with an equivalent normal saline volume), FA group (gavage with 2 ml FA extract (2.7 g/kg crude drug according to the optimal dose based on the previous pharmacodynamics study of the research group [23])), and the Domp group (gavaged with 2 ml Domp solution (at 0.14 g/kg in water) once a day for two weeks). From the first day of IA administration to the day before euthanasia, rat mental state, weight, and food intake data were monitored on a weekly basis. Rats were fasted for 12 h before the last administration. Then, they were anesthetized with 1% pentobarbital sodium (40 mg/kg), and blood and duodenal tissue were rapidly collected. After that, animals were euthanized immediately via pentobarbital sodium intraperitoneal injection (100 mg/kg) [28]. For histopathology, 1 cm long duodenal tissue samples were fixed in 4% paraformaldehyde, while remaining samples were immersed in liquid nitrogen and stored at −80°C until required (Supplementary Material S1).


## 2.5. Exploring Gastric Emptying and Intestinal Propulsion

After the last treatment, animals were fasted for 12 h. Rats had been fed with nutritious semisolid paste containing graphite powder (2 ml) by gavage. After 30 min, rats were humanely euthanized, and the abdominal cavity opened immediately. In the absence of obvious intra-abdominal abnormalities, pyloric orifice and gastric cardia ligation were performed, followed by whole stomach and small intestine removal. Thereafter, we weighed the whole stomach, then eliminated the stomach contents by immersing the stomach in 0.9% normal saline, and dried the stomach with sterile filter paper and weighed it. At the same time, we measured the distance the graphite powder moves from the pylorus and the length of the entire small intestine. Then, we evaluated gastrointestinal motility by gastric residual rate and intestinal propulsion rate (Supplementary MaterialS2).


## 2.6. Histopathological Examinations and Immunohistochemistry Analyses

We embedded 4% paraformaldehyde-fixed duodenal tissue in paraffin, sectioned tissue into 4μm slices, and hydrated and stained samples in hematoxylin and eosin (H & E) for microscopic analysis.Following euthanasia, rat duodenal tissues were quickly isolated and washed in normal saline after removing attached tissue. Then, 1 cm duodenal biopsy specimens were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, sliced into 4μm sections, stained in H & E and toluidine blue solution, and observed microscopically (Nikon Eclipse C1, Japan). Sections were incubated overnight at 4°C with primary antibodies for ZO-1 (1 : 1,000) and Occludin (1 : 400) and washed three times. After washing, a corresponding horseradish peroxidase (HRP)-labeled secondary antibody (1 : 300) was added to allow for a 50 min incubation period at room temperature. Then, after slightly drying, a 4′,6-diamidino-2-phenylindole solution was added to slides for 10 min at room temperature in the dark to stain nuclei. After washing, sections were dried slightly, mounted with antifluorescence quenching mounting agent, and observed under fluorescence microscopy. We captured 200x and 400x images which were quantitatively analyzed using Image J 8.0 (National Institutes of Health, MD, USA).


## 2.7. Inflammatory Cytokine ELISA

For ELISA, plasma inflammatory biomarkers were detected using IL-1β and TNF-α kits as per manufacturer’s instructions. Absorbance at 450 nm was measured using enzyme markers, standard curves plotted, and IL-1β and TNF-α levels determined.


## 2.8. Quantitative-Polymerase Chain Reaction (qPCR)

Total RNA was extracted using Trizol reagent according to manufacturer’s instructions. cDNA was synthesized using TransScript OneStep gDNA removal and cDNA Synthesis Super Mix kits according to manufacturer’s instructions. Subsequently, qPCR was conducted according to manufacturer’s protocols under the following conditions: denaturation for 30 s at 94°C, followed by 40 cycles of 5 s at 94°C and 30 s at 60°C on a Real-time Thermal Cycler 5100 using a SYBR-Green master mix. All samples were normalized against GAPDH expression. Relative gene expression was analyzed using the 2−ΔΔCt method. Primer sequences are shown in Table 1.Table 1
Primer sequences of the genes used for Q-PCR.
GenesForward primerReverse primerLYNGAA​GCC​TGG​ACA​ATG​GTG​GTTCCC​ATG​GCT​TCT​GTG​GTT​TGSYKTGC​CCA​TCC​TGT​GAC​TTG​GTCCT​CCC​GTT​GGC​TCA​TAG​GMEKCAG​TTG​GAA​GAT​ACC​CCA​TTC​CGAG​GTC​GGC​TAT​CCA​TTC​CATERKCTT​TCC​TCC​GGC​AAA​ATG​ACCCA​GGT​TGC​TGT​TCA​CCA​GAAGAPDHCAT​GTT​CGT​CAT​GGG​TGT​GAAGGC​ATG​GAC​TGT​GGT​CAT​GAG


## 2.9. Western Blotting

Total duodenum tissue proteins were extracted in RIPA lysis buffer (plus 1 mM PMSF and phosphatase inhibitors), and concentrations determined using a spectrophotometer (BioTeke ND5000, Beijing, China). Proteins were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, which were then blocked in 5% skimmed milk in Tris-buffered saline and 0.1% Tween 20 (TBST) for 1 h at 37°C, followed by overnight incubation with primary antibodies for ZO-1(1 : 2000), Occludin (1 : 2000), Mlck (1 : 2000), Mlc2 (1 : 2000), Erk (1 : 2000), Mek (1 : 1000), P-Mek (1 : 1000), P-Mlc2 (1 : 1000), P-Erk1/2 (1 : 500), andβ-actin (1 : 3000) at 4°C. Next, membranes were washed in TBST, incubated with an antirabbit lgG antibody conjugated to HRP at 37°C for 1 h and washed in TSBT. Protein signals in membranes were observed using an enhanced chemiluminescence kit following manufacturer’s instructions. Protein signal densities and immune response analyses were performed using the Tanon-5200 imaging system (Tanon Science and Technology Co. Ltd., Shanghai, China) and ImageJ software.


## 2.10. Metabolomics Profiling of Plasma Samples

Plasma samples were also generated for LC/MS analysis by protein precipitation with methanol; 600μl cold methanol was added to 100 μl plasma samples, vortexed for 60 s, and centrifuged at 12,000 rpm for 15 min at 4°C. Then, the supernatant was dried under nitrogen gas, and residues were redissolved in 30 μl 50% methanol aqueous solution. We also prepared internal quality control (QC) samples by mixing 10 μL of each test sample. All samples were maintained at 4°C during analyses. Mobile phase and gradient parameters were identical to aforementioned parameters (Section 2.3) and the injection volume was 3 μl.


## 2.11. Systematic Pharmacology Analysis

FA chemical constituents were imported into the TCMSP (https://lsp.nwu.edu.cn/index.php) database to filter active compounds through oral bioavailability (OB ≥ 30%) and drug-like properties (DL ≥ 0.18). Active compounds were imported into PharmMapper (https://www.lilabecust.cn/pharmmapper/index.html) and Swiss Target Prediction (https://www.swisstargetprediction.ch) databases to collect corresponding targets. FD-related target genes were obtained from DrugBank (https://www.drugbank.ca/), GeneCards (https://www.genecards.org/), OMIM (https://omim.org/), and DisGeNET (https://www.Disgenet.org/). Furthermore, we submitted the acquired target genes to STRING (https://STRING-db.org/) to recognize the functional protein association networks in accordance with calculation parameters and screening scores.


## 2.12. Joint-Pathway Analysis

Joint-pathway analysis of differentially expressed metabolites with target proteins was performed using MetaboAnalyst 5.0 (https://www.metaboanalyst.ca) to derive insights on biological mechanisms associated with FD treatment. Then, an interaction network of herb-activecompound-hub target-pathway-biomarkers of FA treating FD was established. This network was visualized in Cytoscape (3.7.1) to show FD mechanisms.


## 2.13. Statistical Analysis

Statistical analyses were performed in GraphPad Prism 8.0 (GraphPad, San Diego, CA, USA). Differences between two groups were assessed using one-way analysis of variance and presented as mean ± standard deviation.P<0.05 value was considered statistically significant.


## 3. Results

### 3.1. LC-MS Analysis of FA and Compound Identification Using LC-MS Chromatograms

Chemical constituents in FA extracts were analyzed by UPLC-QTOF-MS. Based on retention time, m/z, fragments from secondary MS, reference to the literature, and reference comparisons, 38 compounds were obtained. Total ion chromatograms are shown (Figure1), while composition and other information are listed (Tables 2 and 3).Figure 1
Total ion chromatograph of FA sample in negative and positive ion mode.Table 2
Components of Fructus Aurantii in negative ion mode of MS.
No.Systematic nameMolecular formulaIon typePrecursor mass (m/z)Found at mass (m/z)Mass error (ppm)Rt (min)MS/MS fragmentations (m/z)1Citric acidC6H8O7M-H191.0197191.0205−4.050.943111.008523,4-Dihydroxybenzaldehyde∗C7H6O3M-H137.0244137.0245−0.63.142108.02123Chlorogenic acidC16H18O9M-H353.0878353.08741.153.797191.0564127.03944Caffeic acidC9H8O4M-H179.0350179.03443.254.389134.03705TroxerutinC33H42O19M + Cl777.2014777.2017−0.355.699579.1693458.1178271.06146Kaempferol-3-rutinoside∗C27H30O15M-H593.1512593.15080.666.756286.0479151.00217Eriocitrin∗C27H32O15M-H595.1668595.1672−0.66.914151.0039135.04548RutinC27H30O16M-H609.1461609.1462−0.157.075300.0207271.0243151.00209Limonin-7-O-beta-D-glucosideC32H42O14M-H649.2502649.2505−0.497.137606.2619444.2106228.147310NeoeriocitrinC27H32O15M-H595.1668595.1673−0.777.187460.1135151.0041135.045511Cynaroside∗C21H20O11M-H447.0923447.09271.317.651285.038712Narirutin∗C27H32O14M-H579.1719579.1726−1.168.48119.0501151.0042272.065313Naringin∗C27H32O14M-H579.1719579.1726−0.298.592460.1190272.0649151.0038119.050114Rhoifolin∗C27H30O14M-H577.1563577.15620.148.737459.1160271.0618269.0455151.0040119.050315Hesperidin∗C28H34O15M-H609.1825609.1826−0.179.376302.075116Nomilin glycosidesC34H46O15M-H693.2764693.2768−0.599.761566.267717HeterophyllinC27H32O14M-H579.1719579.1726−0.299.936272.0647151.004218Milinate-17-β-d-glucosideC34H48O16M-H711.2870711.28660.59.969652.2688608.278219Nominate-17-β-d-glucosideC34H48O16M-H711.2870711.28680.2210.081652.2658608.278320KaempferitrinC27H30O14M-H577.1563577.15620.1410.528269.0449151.003421Poncirin∗C28H34O14M-H593.1876593.18641.9911.424286.0805151.005322QuercetinC15H10O7M-H301.0354301.03472.2511.44107.0131121.0292151.003823Apigenin∗C15H10O5M-H269.0455269.0458−0.9413.645119.0506246′,7′-DihydroxybergamottinC21H24O6M-H371.1500371.14970.8414.109254.0579225.055725Hesperetin∗C16H14O6M-H301.0718301.07170.2114.317286.0393151.0039134.038426Diosmetin∗C16H12O6M-H299.0561299.05581.0414.333284.0323276′,7′-Dioxy bergamotC21H24O6M-H371.1500371.14980.5714.669225.055328Pine oleic acidC18H34O5M-H329.2333329.2334−0.1616.012171.1014139.1131Table 3
Components of Fructus Aurantii in positive ion mode of MS.
NoSystematic nameMolecular formulaIon typePrecursor mass (m/z)Found at mass (m/z)Mass error (ppm)Rt (min)MS/MS fragmentations (m/z)11,8,15,22,29-Pentaaza-cyclopentatriacontaneC30H55N5O5M + H566.4276566.42514.417.881548.4097453.3434435.32872Naringenin chalconeC15H12O5M + H273.0757273.07493.118.503153.0173147.0436119.04853Naringenin∗C15H12O5M + H273.0757273.07464.218.648153.0167119.0488147.0435111.00654HesperidinC28H34O15M + Na633.179633.17791.729.27548.4097453.3434435.3287311.09875Neohesperidin∗C28H34O16M + Na633.179633.17771.419.382487.1204331.0999185.04226MeranzinC15H16O4M + H261.1121261.11123.5816.387131.0489189.0544103.05427Limonin∗C26H30O8M + H471.2013471.19963.7118.177425.1991161.058483′,4′,7,8-TetramethoxylflavoneC19H18O6M + Na365.0996365.09784.8218.608335.04869Nobiletin∗C21H22O8M + H403.1387403.1374.3320.08373.0893327.0817358.0711345.1018387.0936211.0228103′,4′,3,5,6,7,8-HeptamethoxyflavoneC22H24O9M + H433.1493433.14754.1821.311403.1987385.096511Tangeretin∗C20H20O7M + H373.1282373.12664.2322.43343.0791300.0616271.0591211.0226∗Identified by comparing with the standards.


### 3.2. FA Attenuates IA-Induced FD Effects in Rats

To examine the FD-attenuating effects of FA in rats, 10 day old animals were given IA, and their mental state, food intake, and weight data were monitored on a weekly basis (Figure2(a)). When compared with the control group, weight gain in FD rats was significantly slow (P<0.01) and food intake was significantly reduced (P<0.05). After FA treatment, both weight gain (P<0.01) and food intake (P<0.05) in FD rats improved significantly. The weight gain and food intake of FA group were more than that of Domp group and close to that of control group (Figures 2(b) and 2(c)). FD rats exhibited markedly decreased GI motility, increased gastric residual rates (P<0.01), and decreased intestinal propulsion rates (P<0.01) when compared with controls. Also, when compared with the FD group, gastric residual rates (P<0.01) decreased and intestinal propulsion rates (P<0.01) increased in Domp and FA groups (Figure 2(k)). The effect of FA on promoting gastrointestinal motility in FD rats is similar to that of positive control drugs. A major pathological feature of FD is intestinal microinflammation. As expected, histopathological analyses showed the absence of apparent histopathological morphology in control animals, while in FD animals, duodenal mucosa exhibited defects and inflammatory cell infiltration. After FA and Domp administration, histopathology was improved to varying degrees (Figure 2(d)).Figure 2
FA alleviate IA-induced FD in rat. (a) Schematic diagram of the experimental process (created with Biorender.com). (b) Weight gain (n = 8). (c) Food intake (n = 8). (d) Representative images of duodenum pathologic damages with H & E staining, (scar bars: 100 μm, 200 μm). (e) Representative images of ZO-1 and occludin. (f) Detection of plasma IL-1β in rat of each group. (g) Detection of plasma TNF-α in rat of each group. (h) ZO-1 protein expressions (n = 3). (i) Occludin protein expressions (n = 3). (j) Immunofluorescence staining demonstrating the localization of ZO-1, occludin (scar bars:200 μm). (k) Gastrointestinal motility of rats in each group (n = 8). (l) The intensity of ZO-1 and occludin was determined by immunofluorescence (n = 3). Values are presented as the means ± SD. ∗P<0.05 and ∗∗P<0.01 were compared with the control group. #P<0.05 and ##P<0.01 were compared with the FD group.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)


### 3.3. FA Ameliorates Low‐Grade Duodenal Inflammation and Repairs Damaged Intestinal Barriers in FD Rats

Impaired duodenal integrity and low-grade inflammation are major pathological characteristics in FD [29]. When compared with controls, inflammatory cytokine IL-1β and TNF-α plasma levels significantly increased in the FD group (P<0.01). After FA administration, these plasma levels were significantly reduced (P<0.05) (Figures 2(f) and 2(g)). Intestinal barrier function is dependent on adjacent cell connections via junctional complexes at cell-cell contact sites and consists of tight junction, adherens junctions, and desmosomes [30]. TJ proteins include ZO-1 and occludin; ZO-1 connects to the transmembrane protein occludin and other cytoplasmic components, with key roles in TJ biology [31]. We observed that ZO-1 and occludin protein expression levels in the FD group had decreased (P<0.05) when compared with controls, but the level of FA groups is increased (P<0.05, P<0.01) (Figures 2(e), 2(g)–2(i), and 2(l)).


### 3.4. Systematic Pharmacology Analysis

Of the 38 compounds in FA extracts, 29 active substances were screened through OB and DL. By inputting these compounds into target databases, 1,018 FA targets were obtained. By gathering FD-related targets from disease databases, 1,207 FD targets were acquired (Figure3(a)). After interactive analysis of component and disease targets, 252 putative targets underlying anti-FD mechanisms mediated by FA extracts were obtained (Figure 3(b)). Thereafter, these intersected genes were imported in STRING database for constructing the protein-protein interaction (PPI) network and for obtaining PPI networks relations (Figure 3(c)). Then, we constructed a connection diagram of the potential target related pathways of FA potential material basis based on systematic pharmacological analysis (Figure 3(d)).Figure 3
Systematic pharmacology analysis. (a) Venn diagram of targets number in FD database. (b) Venn diagram of intersection targets of FD and FA. (c) Protein-protein interaction network of hub targets. (d) Connection diagram of FA-potential material basis-potentialtargets-related pathways of systematic pharmacology analysis. (e) Joint pathway analysis of biomarkers and target disturbed by FA. (f) Active compound-target-biomarker-pathway network (the red line circles the experimental validation pathway).
(a)(b)(c)(d)(e)(f)


### 3.5. Metabolomics Analysis

Metabolic plasma sample profiles, generated by UPLC-QTOF-MS, were used to evaluate metabolomic alterations in study rats. Our PCA score chart showed a distinct separation between FD and control groups. Importantly, QC samples were highly clustered, indicating good instrument-based stability (Figure4(a)). Additionally, 10 ions were extracted from basic QC sample peak intensity chromatograms for method validation. The RSDs of these 10 ions were in the 0.0981%–2.8325% region. To further identify different metabolites, we used supervised mode OPLS-DA (Figures 4(b) and 4(c)) to reduce the effects of intragroup differences on classification, thus maximizing separation between groups. To test the OPLS-DA model validity, a 200-permutation test was applied. All Q2 values on the left were <those on the right. The intersection value between the Q2 regression line and vertical axis was <0 (Figures 4(e) and 4(f)). This result (Q2 = 0.918) showed the model was valid and no over-fitting had occurred in OPLS-DA models. Therefore, all OPLS-DA scores plots (Figures 4(b) and 4(c)) showed that control and FA groups were separated from the FD group. The S-plots of OPLS-DA revealed a variety of metabolites (Figures 4(h) and 4(i)).Figure 4
Systems’ analysis of the metabolomic alterations in each group (n = 8). (a) PCA score plot in each group. (b) OPLS-DA score plot between control and FD (Y2 = 0.995 and Q2 = 0.851). (c) OPLS-DA score plot between FA and FD (Y2 = 0.987 and Q2 = 0.918). (d) Metabolic pathway analysis of different metabolites by metaboanalyst 5.0. (e) Validation diagram of the OPLS-DA model between control and FD. (f) Validation diagram of OPLS-DA model between FA and FD. (g) The heat map of metabolites in each group. Red means up-regulation, blue means down-regulation. (h) S-plot of OPLS-DA between control and FD. (i) S-plot of OPLS-DA between FA and FD.
(a)(b)(c)(d)(e)(f)(g)(h)(i)


### 3.6. Identification and Analysis of Endogenous Metabolites

Endogenous metabolites were identified according to VIP >1.0 andT-Test (P<0.05) values. In total, 54 different metabolites were identified based on secondary spectrum information, and the HMDB database was where 30 metabolites were up-regulated and 24 metabolites down-regulated. To further clarify the distribution of the 54 differential metabolites in different rat groups, unsupervised clustering was performed using hierarchical cluster heat map analysis. As shown (Figure 4(g)), model and control groups were separated into different clusters, while FA and control groups were gathered in the same cluster, suggesting FA had ameliorated the metabolic FD disorder. Metabolites were then introduced into the MetaboAnalyst program to investigate the metabolic effects of FA in FD rats. Metabolic pathways with an impact value >0.1 were related to the therapeutic effects of FA on FD. Our results showed that FA affected FD via different pathways, including linoleic acid, arachidonic acid, sphingolipid, tryptophan, pyrimidine, and purine metabolism, and also the pentose phosphate pathway, primary bile acid biosynthesis, glycerophospholipid metabolism, fatty acid biosynthesis, valine, leucine, and isoleucine degradation (Figure 4(d)) (Supplementary Material S3 and S5)


### 3.7. Joint Pathway Analysis of Targets and Metabolites

To explore key metabolic pathways, joint pathway analyses were performed on the 54 differential metabolites and 252 targets using MetaboAnalyst 5.0. As shown (Figure3(e)), the Fc epsilon RI signaling pathway was concurrently rich in network pharmacology targets and metabolomics differential metabolites. Based on the number of targets in network pharmacological pathways, Fc epsilon RI signaling was selected for further experimental verification. Combining systemic pharmacology and metabolomics, we established a complete active herbal compound-target-pathway-biomarker network to explain the therapeutic efficacy of FA toward FD (Figure 3(f)) (Supplementary Material S4).


### 3.8. Experimental Validation

Fc epsilon RI signaling pathway is an activation pathway for mast cells [32]. From our observations, mast cells aggregated and degranulation rates were increased in the FD group, while FA intervention attenuated these phenomena (Figure 5(d)). To further validate the influence of Fc epsilon RI signaling in FD rats, we experimentally verified the hub target and its upstream and downstream proteins (tyrosine-protein kinase (Lyn), spleen tyrosine kinase (Syk), Mek, P-Mek, Erk, P-Erk1/2, MLCK, Mlc2, and P-Mlc2) using qPCR and western blotting. The P-Mek, P-Erk, MLCK, and P-Mlc2 protein levels were significantly increased in model groups when compared with controls (Figure 5(a)). Western blot greyscale quantification P-Mek/Mek, P-Erk/Erk, P-Mlc2/Mlc2, and MLCK/β-actin analyses are shown (P<0.05, P<0.01, Figure 5(b)). P-Mek, P-Erk, MLCK, and P-Mlc2 expression decreased in the FA group when compared with the FD group (P<0.05), while ZO-1 and occludin expression increased in the FA group when compared with the FD group (P<0.05, P<0.01, Figures 2(e), 2(h) and 2(i)). The mRNA expression of Lyn, Syk, Erk, and Mek was significantly increased in model groups when compared with controls (P<0.01) and decreased in the FA group when compared with the FD group (P<0.01, Figure 5(c)).Figure 5
Effect of FA on the Fc epsilon RI signal pathway. (a) Representative images of P-Erk1/2, Erk, P-Mek, Mek, P-Mlc2, Mlc2, and Mlck. (b) P-Erk1/2, P-Mek, P-Mlc2, and MLCK protein expressions (n = 3). (c) Relative mRNA expression of Erk, Mek, Lyn, and Syk (n = 3). (d) Representative images for mast cell stained with toluidine blue solution (scar bars: 100 μm, 200 μm). Values are presented as the means ± SD. ∗P<0.05 and ∗∗P<0.01 were compared with the control group. #P<0.05 and ##P<0.01 were compared with the FD group.
(a)(b)(c)(d)


## 3.1. LC-MS Analysis of FA and Compound Identification Using LC-MS Chromatograms

Chemical constituents in FA extracts were analyzed by UPLC-QTOF-MS. Based on retention time, m/z, fragments from secondary MS, reference to the literature, and reference comparisons, 38 compounds were obtained. Total ion chromatograms are shown (Figure1), while composition and other information are listed (Tables 2 and 3).Figure 1
Total ion chromatograph of FA sample in negative and positive ion mode.Table 2
Components of Fructus Aurantii in negative ion mode of MS.
No.Systematic nameMolecular formulaIon typePrecursor mass (m/z)Found at mass (m/z)Mass error (ppm)Rt (min)MS/MS fragmentations (m/z)1Citric acidC6H8O7M-H191.0197191.0205−4.050.943111.008523,4-Dihydroxybenzaldehyde∗C7H6O3M-H137.0244137.0245−0.63.142108.02123Chlorogenic acidC16H18O9M-H353.0878353.08741.153.797191.0564127.03944Caffeic acidC9H8O4M-H179.0350179.03443.254.389134.03705TroxerutinC33H42O19M + Cl777.2014777.2017−0.355.699579.1693458.1178271.06146Kaempferol-3-rutinoside∗C27H30O15M-H593.1512593.15080.666.756286.0479151.00217Eriocitrin∗C27H32O15M-H595.1668595.1672−0.66.914151.0039135.04548RutinC27H30O16M-H609.1461609.1462−0.157.075300.0207271.0243151.00209Limonin-7-O-beta-D-glucosideC32H42O14M-H649.2502649.2505−0.497.137606.2619444.2106228.147310NeoeriocitrinC27H32O15M-H595.1668595.1673−0.777.187460.1135151.0041135.045511Cynaroside∗C21H20O11M-H447.0923447.09271.317.651285.038712Narirutin∗C27H32O14M-H579.1719579.1726−1.168.48119.0501151.0042272.065313Naringin∗C27H32O14M-H579.1719579.1726−0.298.592460.1190272.0649151.0038119.050114Rhoifolin∗C27H30O14M-H577.1563577.15620.148.737459.1160271.0618269.0455151.0040119.050315Hesperidin∗C28H34O15M-H609.1825609.1826−0.179.376302.075116Nomilin glycosidesC34H46O15M-H693.2764693.2768−0.599.761566.267717HeterophyllinC27H32O14M-H579.1719579.1726−0.299.936272.0647151.004218Milinate-17-β-d-glucosideC34H48O16M-H711.2870711.28660.59.969652.2688608.278219Nominate-17-β-d-glucosideC34H48O16M-H711.2870711.28680.2210.081652.2658608.278320KaempferitrinC27H30O14M-H577.1563577.15620.1410.528269.0449151.003421Poncirin∗C28H34O14M-H593.1876593.18641.9911.424286.0805151.005322QuercetinC15H10O7M-H301.0354301.03472.2511.44107.0131121.0292151.003823Apigenin∗C15H10O5M-H269.0455269.0458−0.9413.645119.0506246′,7′-DihydroxybergamottinC21H24O6M-H371.1500371.14970.8414.109254.0579225.055725Hesperetin∗C16H14O6M-H301.0718301.07170.2114.317286.0393151.0039134.038426Diosmetin∗C16H12O6M-H299.0561299.05581.0414.333284.0323276′,7′-Dioxy bergamotC21H24O6M-H371.1500371.14980.5714.669225.055328Pine oleic acidC18H34O5M-H329.2333329.2334−0.1616.012171.1014139.1131Table 3
Components of Fructus Aurantii in positive ion mode of MS.
NoSystematic nameMolecular formulaIon typePrecursor mass (m/z)Found at mass (m/z)Mass error (ppm)Rt (min)MS/MS fragmentations (m/z)11,8,15,22,29-Pentaaza-cyclopentatriacontaneC30H55N5O5M + H566.4276566.42514.417.881548.4097453.3434435.32872Naringenin chalconeC15H12O5M + H273.0757273.07493.118.503153.0173147.0436119.04853Naringenin∗C15H12O5M + H273.0757273.07464.218.648153.0167119.0488147.0435111.00654HesperidinC28H34O15M + Na633.179633.17791.729.27548.4097453.3434435.3287311.09875Neohesperidin∗C28H34O16M + Na633.179633.17771.419.382487.1204331.0999185.04226MeranzinC15H16O4M + H261.1121261.11123.5816.387131.0489189.0544103.05427Limonin∗C26H30O8M + H471.2013471.19963.7118.177425.1991161.058483′,4′,7,8-TetramethoxylflavoneC19H18O6M + Na365.0996365.09784.8218.608335.04869Nobiletin∗C21H22O8M + H403.1387403.1374.3320.08373.0893327.0817358.0711345.1018387.0936211.0228103′,4′,3,5,6,7,8-HeptamethoxyflavoneC22H24O9M + H433.1493433.14754.1821.311403.1987385.096511Tangeretin∗C20H20O7M + H373.1282373.12664.2322.43343.0791300.0616271.0591211.0226∗Identified by comparing with the standards.


## 3.2. FA Attenuates IA-Induced FD Effects in Rats

To examine the FD-attenuating effects of FA in rats, 10 day old animals were given IA, and their mental state, food intake, and weight data were monitored on a weekly basis (Figure2(a)). When compared with the control group, weight gain in FD rats was significantly slow (P<0.01) and food intake was significantly reduced (P<0.05). After FA treatment, both weight gain (P<0.01) and food intake (P<0.05) in FD rats improved significantly. The weight gain and food intake of FA group were more than that of Domp group and close to that of control group (Figures 2(b) and 2(c)). FD rats exhibited markedly decreased GI motility, increased gastric residual rates (P<0.01), and decreased intestinal propulsion rates (P<0.01) when compared with controls. Also, when compared with the FD group, gastric residual rates (P<0.01) decreased and intestinal propulsion rates (P<0.01) increased in Domp and FA groups (Figure 2(k)). The effect of FA on promoting gastrointestinal motility in FD rats is similar to that of positive control drugs. A major pathological feature of FD is intestinal microinflammation. As expected, histopathological analyses showed the absence of apparent histopathological morphology in control animals, while in FD animals, duodenal mucosa exhibited defects and inflammatory cell infiltration. After FA and Domp administration, histopathology was improved to varying degrees (Figure 2(d)).Figure 2
FA alleviate IA-induced FD in rat. (a) Schematic diagram of the experimental process (created with Biorender.com). (b) Weight gain (n = 8). (c) Food intake (n = 8). (d) Representative images of duodenum pathologic damages with H & E staining, (scar bars: 100 μm, 200 μm). (e) Representative images of ZO-1 and occludin. (f) Detection of plasma IL-1β in rat of each group. (g) Detection of plasma TNF-α in rat of each group. (h) ZO-1 protein expressions (n = 3). (i) Occludin protein expressions (n = 3). (j) Immunofluorescence staining demonstrating the localization of ZO-1, occludin (scar bars:200 μm). (k) Gastrointestinal motility of rats in each group (n = 8). (l) The intensity of ZO-1 and occludin was determined by immunofluorescence (n = 3). Values are presented as the means ± SD. ∗P<0.05 and ∗∗P<0.01 were compared with the control group. #P<0.05 and ##P<0.01 were compared with the FD group.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)


## 3.3. FA Ameliorates Low‐Grade Duodenal Inflammation and Repairs Damaged Intestinal Barriers in FD Rats

Impaired duodenal integrity and low-grade inflammation are major pathological characteristics in FD [29]. When compared with controls, inflammatory cytokine IL-1β and TNF-α plasma levels significantly increased in the FD group (P<0.01). After FA administration, these plasma levels were significantly reduced (P<0.05) (Figures 2(f) and 2(g)). Intestinal barrier function is dependent on adjacent cell connections via junctional complexes at cell-cell contact sites and consists of tight junction, adherens junctions, and desmosomes [30]. TJ proteins include ZO-1 and occludin; ZO-1 connects to the transmembrane protein occludin and other cytoplasmic components, with key roles in TJ biology [31]. We observed that ZO-1 and occludin protein expression levels in the FD group had decreased (P<0.05) when compared with controls, but the level of FA groups is increased (P<0.05, P<0.01) (Figures 2(e), 2(g)–2(i), and 2(l)).


## 3.4. Systematic Pharmacology Analysis

Of the 38 compounds in FA extracts, 29 active substances were screened through OB and DL. By inputting these compounds into target databases, 1,018 FA targets were obtained. By gathering FD-related targets from disease databases, 1,207 FD targets were acquired (Figure3(a)). After interactive analysis of component and disease targets, 252 putative targets underlying anti-FD mechanisms mediated by FA extracts were obtained (Figure 3(b)). Thereafter, these intersected genes were imported in STRING database for constructing the protein-protein interaction (PPI) network and for obtaining PPI networks relations (Figure 3(c)). Then, we constructed a connection diagram of the potential target related pathways of FA potential material basis based on systematic pharmacological analysis (Figure 3(d)).Figure 3
Systematic pharmacology analysis. (a) Venn diagram of targets number in FD database. (b) Venn diagram of intersection targets of FD and FA. (c) Protein-protein interaction network of hub targets. (d) Connection diagram of FA-potential material basis-potentialtargets-related pathways of systematic pharmacology analysis. (e) Joint pathway analysis of biomarkers and target disturbed by FA. (f) Active compound-target-biomarker-pathway network (the red line circles the experimental validation pathway).
(a)(b)(c)(d)(e)(f)


## 3.5. Metabolomics Analysis

Metabolic plasma sample profiles, generated by UPLC-QTOF-MS, were used to evaluate metabolomic alterations in study rats. Our PCA score chart showed a distinct separation between FD and control groups. Importantly, QC samples were highly clustered, indicating good instrument-based stability (Figure4(a)). Additionally, 10 ions were extracted from basic QC sample peak intensity chromatograms for method validation. The RSDs of these 10 ions were in the 0.0981%–2.8325% region. To further identify different metabolites, we used supervised mode OPLS-DA (Figures 4(b) and 4(c)) to reduce the effects of intragroup differences on classification, thus maximizing separation between groups. To test the OPLS-DA model validity, a 200-permutation test was applied. All Q2 values on the left were <those on the right. The intersection value between the Q2 regression line and vertical axis was <0 (Figures 4(e) and 4(f)). This result (Q2 = 0.918) showed the model was valid and no over-fitting had occurred in OPLS-DA models. Therefore, all OPLS-DA scores plots (Figures 4(b) and 4(c)) showed that control and FA groups were separated from the FD group. The S-plots of OPLS-DA revealed a variety of metabolites (Figures 4(h) and 4(i)).Figure 4
Systems’ analysis of the metabolomic alterations in each group (n = 8). (a) PCA score plot in each group. (b) OPLS-DA score plot between control and FD (Y2 = 0.995 and Q2 = 0.851). (c) OPLS-DA score plot between FA and FD (Y2 = 0.987 and Q2 = 0.918). (d) Metabolic pathway analysis of different metabolites by metaboanalyst 5.0. (e) Validation diagram of the OPLS-DA model between control and FD. (f) Validation diagram of OPLS-DA model between FA and FD. (g) The heat map of metabolites in each group. Red means up-regulation, blue means down-regulation. (h) S-plot of OPLS-DA between control and FD. (i) S-plot of OPLS-DA between FA and FD.
(a)(b)(c)(d)(e)(f)(g)(h)(i)


## 3.6. Identification and Analysis of Endogenous Metabolites

Endogenous metabolites were identified according to VIP >1.0 andT-Test (P<0.05) values. In total, 54 different metabolites were identified based on secondary spectrum information, and the HMDB database was where 30 metabolites were up-regulated and 24 metabolites down-regulated. To further clarify the distribution of the 54 differential metabolites in different rat groups, unsupervised clustering was performed using hierarchical cluster heat map analysis. As shown (Figure 4(g)), model and control groups were separated into different clusters, while FA and control groups were gathered in the same cluster, suggesting FA had ameliorated the metabolic FD disorder. Metabolites were then introduced into the MetaboAnalyst program to investigate the metabolic effects of FA in FD rats. Metabolic pathways with an impact value >0.1 were related to the therapeutic effects of FA on FD. Our results showed that FA affected FD via different pathways, including linoleic acid, arachidonic acid, sphingolipid, tryptophan, pyrimidine, and purine metabolism, and also the pentose phosphate pathway, primary bile acid biosynthesis, glycerophospholipid metabolism, fatty acid biosynthesis, valine, leucine, and isoleucine degradation (Figure 4(d)) (Supplementary Material S3 and S5)


## 3.7. Joint Pathway Analysis of Targets and Metabolites

To explore key metabolic pathways, joint pathway analyses were performed on the 54 differential metabolites and 252 targets using MetaboAnalyst 5.0. As shown (Figure3(e)), the Fc epsilon RI signaling pathway was concurrently rich in network pharmacology targets and metabolomics differential metabolites. Based on the number of targets in network pharmacological pathways, Fc epsilon RI signaling was selected for further experimental verification. Combining systemic pharmacology and metabolomics, we established a complete active herbal compound-target-pathway-biomarker network to explain the therapeutic efficacy of FA toward FD (Figure 3(f)) (Supplementary Material S4).


## 3.8. Experimental Validation

Fc epsilon RI signaling pathway is an activation pathway for mast cells [32]. From our observations, mast cells aggregated and degranulation rates were increased in the FD group, while FA intervention attenuated these phenomena (Figure 5(d)). To further validate the influence of Fc epsilon RI signaling in FD rats, we experimentally verified the hub target and its upstream and downstream proteins (tyrosine-protein kinase (Lyn), spleen tyrosine kinase (Syk), Mek, P-Mek, Erk, P-Erk1/2, MLCK, Mlc2, and P-Mlc2) using qPCR and western blotting. The P-Mek, P-Erk, MLCK, and P-Mlc2 protein levels were significantly increased in model groups when compared with controls (Figure 5(a)). Western blot greyscale quantification P-Mek/Mek, P-Erk/Erk, P-Mlc2/Mlc2, and MLCK/β-actin analyses are shown (P<0.05, P<0.01, Figure 5(b)). P-Mek, P-Erk, MLCK, and P-Mlc2 expression decreased in the FA group when compared with the FD group (P<0.05), while ZO-1 and occludin expression increased in the FA group when compared with the FD group (P<0.05, P<0.01, Figures 2(e), 2(h) and 2(i)). The mRNA expression of Lyn, Syk, Erk, and Mek was significantly increased in model groups when compared with controls (P<0.01) and decreased in the FA group when compared with the FD group (P<0.01, Figure 5(c)).Figure 5
Effect of FA on the Fc epsilon RI signal pathway. (a) Representative images of P-Erk1/2, Erk, P-Mek, Mek, P-Mlc2, Mlc2, and Mlck. (b) P-Erk1/2, P-Mek, P-Mlc2, and MLCK protein expressions (n = 3). (c) Relative mRNA expression of Erk, Mek, Lyn, and Syk (n = 3). (d) Representative images for mast cell stained with toluidine blue solution (scar bars: 100 μm, 200 μm). Values are presented as the means ± SD. ∗P<0.05 and ∗∗P<0.01 were compared with the control group. #P<0.05 and ##P<0.01 were compared with the FD group.
(a)(b)(c)(d)


## 4. Discussion

FD is a chronic GI disorder characterized by motility disturbances and visceral hypersensitivity and requires long-term effective pharmacological treatment. FA is the dried unripe fruit ofCitrus aurantium L. and its cultivated varieties, and it is a medicinal and edible fruit whose potential therapeutic effects reduce inflammation and promote GI motility [33, 34]. These key factors encouraged us to investigate the underlying FA mechanisms toward ameliorating FD. We confirmed these effects and demonstrated that anti-FD mechanisms were closely linked to mast cell-induced inflammatory responses. These findings facilitated the construction of a comprehensive network which improved our understanding of several interrelated FA mechanisms in improving FD in an IA-induced rat model.Rats in the IA-induced FD group had dull hair, slow weight gain, low food intake, impaired GI motility, and prolonged GI emptying time. After FA administration, the FD disease status improved to some extent. Also, inflammatory cell infiltration in the local mucosa of FD rat groups was observed in pathological sections, while no infiltration was observed in the FA group. Growing evidence now suggests a role for intestinal epithelial barrier disruption and inflammation in FD [35, 36]. Changes in TJ proteins disrupt the intestinal epithelial barrier, which allows microorganisms in the intestinal lumen to promote abnormal immune responses, with excessive bacterial antigen leakage from the mucosa gradually degrading TJs [37, 38]. TNF-α impairs intestinal barriers by inducing apoptosis in epithelial cells and altering TJ structure and function [37], while IL-1β increases TJ permeability in human intestinal epithelial cells [38]. In our study, TNF-α and IL-1β levels were elevated in the FD group, while the FA intervention down-regulated their secretion. Also, ZO-1 and occludin were down-regulated in the FD group, while this was reversed in the FA group. TJ defects may facilitate the passage of dietary antigens and bacterial products through the epithelial barrier, where they enter the lamina propria and possibly induce inflammatory changes [39]. Therefore, we confirmed the therapeutic potential of FA as it restored epithelial barrier structure and function to alleviate the effects of FD.To further explore mechanisms whereby FA generates anti-inflammation and repairing intestinal barrier properties, we performed an integrated study on each rat group by combining metabolomics and systemic pharmacology approaches. First of all, we identified the FA extract through UPLC-QTOF-MS/MS technology, screened the active components, collected the targets for the identified components through OB and DL, collected the disease targets according to the disease database of FD, and obtained the key targets of systemic pharmacology through interactive analysis. Then, we analyzed the differential metabolite through metabolomics technology, integrated the differential metabolite and key target, and obtained the core pathway. Biomarker is able to participate in the regulation of core pathway. Finally, we established an active herbal compound-target-pathway-biomarker network. Through the above analysis, we targeted Fc epsilon RI signaling pathway, which is a mast cell activation pathway, and downstream arachidonic acid metabolism as further experimental verification pathways.Mast cell infiltration and ultrastructural changes are reported in the duodena of FD patients, and such infiltration may be associated with chronic stress, intestinal flora, and acid hypersensitivity [40–42]. Thus, mast cell activation may be the pathological basis of mild intestinal inflammation in FD [31, 43]. Mast cell activation relies on several complex signal transduction steps; the main Src family kinase involved in these initial stages is Lyn [44]. When tyrosine residues in FceRI b and c chains are phosphorylated by Lyn, the phosphorylated immunoreceptor tyrosine-based activation motif of FceRI b and c chains provided the SH2 domain of Syk with high-affinity docking sites [45]. As the initial signaling molecule for mast cell activation, Syk regulates the phosphorylation of its downstream Mek, which phosphorylates both serine/threonine and tyrosine residues in Erk1/2, triggering its activation and subsequent cytosolic and nuclear activity. Erk1/2 is activated by MAPKs, which causes the nuclear translocation of AP-1, its transcription, and cytokine production, including IL-1β and TNF-α. Syk inhibition also leads to the downstream inhibition of MAPK, Mek, and Erk, suggesting a role in disease initiation and progression [46–48]. Mast cell degranulation releases TNF-α, IL-1β, and arachidonic acid metabolites. TNF-α and IL-1β bind to TNFR2 and IL-1R on intestinal epithelial cell surfaces to activate P38 and NF-κB, thereby inducing the transcription and enzyme activation of MLCK [49]. MLCK is a specific substrate enzyme of Mlc2 and specifically phosphorylates Ser18 and Thr19 residues in Mlc2. Then, p-Mlc2 activates the ATPase domain of the myosin heavy chain head, producing energy which causes cytoskeletal actin filaments to slide [50]. This causes the cell membrane endocytosis of TJ proteins and other transmembrane proteins (including occludin and ZO-1), which hinder TJ proteins in forming gaps, ultimately increasing epithelial permeability [51] and intestinal barrier disruption. Conversely, impaired barrier function may also lead to low-grade inflammation, as animal models showed that inflammation is weakened after increased intestinal permeability [52]. Arachidonic acid is also an important inflammatory mediator; it participates in and promotes inflammatory responses [53]. Many studies have reported that elevated levels of bodily arachidonic acid are closely associated with many diseases, including inflammatory bowel disease, arthritis, and stroke [54–56]. We showed that arachidonic acid levels increased in FD animals but decreased in the FA group, suggesting that FA blocked inflammation by regulating arachidonic acid metabolism (Figure 6).Figure 6
The molecular mechanisms of FA in treating FD (created with Biorender.com) FA reversed IA-induced FD in rats by regulating Fc epsilon RI signaling pathway to interfere with mast cell degranulation. FA treatment reduced mast cell activation and degranulation, decreased IL-1β and TNF-α secretion, and attenuated arachidonic acid pathway activation. In addition, it downregulated the expression of ZO-1 and occludin by reducing the phosphorylation of MLC2. It repaired the intestinal barrier, prevented the luminal antigen passing through the epithelial cells, and induced inflammatory reaction. ( direct action,  indirect action, and  pathogenic antigen).In conclusion, FA reversed IA-induced FD in rats by regulating Fc epsilon RI signaling pathway to interfere with mast cell degranulation. FA treatment lowered Lyn and Syk expression, down-regulated Mek and Erk phosphorylation, reduced mast cell activation and degranulation, decreased IL-1β and TNF-α secretion, and attenuated arachidonic acid pathway activation, thereby reducing the IL-1β and TNF-α induced downregulation of intestinal epithelial TJ proteins (ZO-1 and Occludin). Physiologically, these molecular events ultimately ameliorated IA-induced intestinal barrier function disruption and duodenal inflammation.


## 5. Conclusions

In the present study, an integrative metabolomics, and systematic pharmacology-based strategy was proposed to uncover the material basis and mechanisms of FA in treating FD. A total of 54 potential biomarkers and 252 targets related to 20 pathways were clarified, and 38 components of FA were identified. Then, we verified that FA can activate mast cells to treat FD through Fc epsilon RI signaling pathway. In short, we generated a new biochemical and molecular paradigm for FA actions against FD, these results confirm that FA has the potential to ameliorate FD. All our results also serve to explain the success of FA treatment of FD in humans in traditional Chinese medicine practice. This article only verified the molecular mechanism of FA in treating FD from Fc epsilon RI signaling pathway. Whether FA plays a role through other signaling pathways needs to be further studied. We hope it can be improved in the follow-up study.



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*Source: 8812517-2023-03-20.xml*