Abstract
Context: Fructus Aurantii, the unripe fruit of Citrus aurantium Linn (Rutaceae), is a Qi-regulating drug used in traditional Chinese medicine to improve gastrointestinal (GI) function. Vasoactive intestinal peptide (VIP) and 5-hydroxytryptamine (5-HT) regulate GI motility and fluid secretion.
Objective: We tested whether the Fructus Aurantii extract altered VIP and 5-HT expression levels in rats.
Materials and methods: Experimental rats were administered 0.3 g/ml Fructus Aurantii water decoction at 2.0 ml/100 g body weight per day for 10 days by gavage feeding, while control rats were gavage fed equal volumes of distilled water. Expression levels of 5-HT and VIP were measured by immunohistochemical staining and microscopic image analysis of the GI mucosa and myenteric nerve plexus.
Results: Average 5-HT staining intensity scores in the stomach antrum, duodenal mucosa and jejunal mucosa were significantly higher in the Fructus Aurantii treatment group than in the control group (antrum: 213% of control; duodenum: 193%; jejunum: 256%; p < 0.05 for all). In contrast, the average VIP density scores in the stomach antrum, duodenal mucosa and jejunal mucosa were significantly lower in the Fructus Aurantii group (antrum: 14% of control; duodenum: 15%; jejunum: 38%; p < 0.01 for all). Tissues from Fructus Aurantii-treated rats exhibited significantly greater numbers of 5-HT- and VIP-immunopositive cells in the gastric antrum, duodenum and jejunum mucosal layer but fewer VIP-expressing cells in the myenteric nerve plexus (p < 0.05 for both).
Conclusion: Fructus Aurantii can enhance gastrointestinal motility by altering 5-HT and VIP expression levels in the rat GI tract.
Introduction
Citrus aurantium L. belongs to the Rutaceae family of fruit trees that yield bitter “brigarade” oranges (Fructus Aurantii). In the lexicon of traditional Chinese medicine (TCM), the essential oils derived from Fructus Aurantii fruit downregulate “Qi,” which promotes digestion, removes stagnant air, and improves general gastrointestinal function (Wang et al., 2001). Gastrointestinal motility is regulated by a number of locally expressed peptide hormones and neuromodulators. Vasoactive intestinal peptide (VIP) and 5-hydroxytryptamine (5-HT, serotonin) levels in blood and tissue are correlated with gastrointestinal motility (Atkinson et al., Citation2006; Kamata et al., Citation1988). Pharmacological studies show that 95% of total body 5-HT is located in the gut, so it is reasonable to assume that most postprandial 5-HT in plasma is derived from the gut (Camilleri, Citation2009). Mammalian gastrointestinal motility is enhanced significantly by 5-HT, while motility is inhibited by VIP (Chen et al., Citation2001). Indeed, serum 5-HT is elevated in diarrhea and celiac disease and decreased during constipation (Camilleri, Citation2009), while VIP promotes intestinal fluid secretion (Palsson et al., Citation2004). To examine the physiological mechanisms underlying the effects of Fructus Aurantii on GI motility, we measured 5-HT and VIP expression in the gastrointestinal tract by immunohistochemistry in control rats and rats fed Fructus Aurantii extract.
Materials and methods
Preparation of Fructus Aurantii extract
Fruit sold as Fructus Aurantii was obtained from a market in Zhangshu City in 2008 and positively identified as the mature fruit of Citrus aurantium L. by Pro. Liu Qinghua, Jiangxi University of Traditional Chinese Medicine. Sections of Fructus Aurantii were soaked in water (1:5 v/v) for 30 min and then boiled for 2 h. The aqueous extract was filtered and stored at 4 °C until used.
Experimental animals
Specific pathogen-free male Sprague Dawley rats (200–250 g) (certificate no.: JZDW 2008-0217) were provided by Jiangxi University of Traditional Chinese Medicine Experimental Animal Center. All animal care and experimental procedures adhered to the guidelines of the National Institutes of Health and were approved by the National Institute of Mental Health Animal Care and Use Committee.
Main instruments
Biological tissue dehydration machine: Leica AS300s (Wetzlar, Germany); Tissue section embedding machine: Leica 1150H; Biological tissue slicer: Leica 2135 (Wetzlar, Germany) and Image analyzer: SCI-800 (China).
Figure 1. Gastrointestinal HE staining and negative contrast diagram: (A) gastric antrum HE staining; (B) gastric antrum negative contrast; (C) duodenum HE staining; (D) duodenum negative contrast; (E) jejunum HE staining; (F) jejunum negative contrast.
Figure 2. 5-HT immunoreactivity in the rat GI tract: (A) gastric antrum distilled water group; (B) gastric antrum Fructus Aurantii group; (C) duodenum distilled water group; (D) duodenum Fructus Aurantii group; (E) jejunum distilled water group; (F) jejunum Fructus Aurantii group.
Figure 3. VIP immunoreactivity in the rat GI tract: (A) gastric antrum distilled water group (B) gastric antrum Fructus Aurantii group; (C) duodenum distilled water group; (D) duodenum Fructus Aurantii group; (E) jejunum distilled water group; (F) jejunum Fructus Aurantii group.
Reagents
Xylene, formaldehyde, ethanol and other reagents were of analytical grade. Hematoxylin solution was made in house. Immunostaining kits for 5-HT and VIP were purchased from Shanghai Blue Gene Biotechnology Co., Ltd (Shanghai, China).
Table 1. Average concentration (%) of 5-HT-positive cells in the gastrointestinal mucosa (D n = 8).
Table 2. Average gray value of 5-HT positive cells in the gastrointestinal mucosa (D n = 8).
Table 3. Average concentration (%) of VIP-positive reactions in the gastrointestinal myenteric nerve plexus (D n = 8).
Table 4. Average gray value of VIP-positive reactions in the gastrointestinal myenteric nerve plexus (D n = 8).
Methods
Rats were divided into control and Fructus Aurantii groups with eight rats in each group. Rats in the Fructus Aurantii group were administered 0.3 g/ml Fructus Aurantii water decoction at 2.0 ml per 100 g body weight per day by gavage feeding, while the control rats were gavage fed the same volume of distilled water. After 7 days of daily treatment, rats were sacrificed and the abdominal cavity opened to excise the antrum, duodenum and jejunum. Tissues were placed in 10% neutral-buffered formalin fixation fluid for 24 h, embedded in paraffin, and then cut into 6 µm sections. Each sequence of three sections was sorted into three sets, one for HE staining and histological location check, one for 5-HT immunohistochemistry, and one for VIP immunohistochemistry. In one slice from each immunohistochemistry set, the primary antibody was replaced with normal rabbit serum, and in another, the primary antibody was replaced with PBS. The rest of the sections were stained using the SABC method. Briefly, paraffin-embedded sections were placed on polylysine-coated slides. The slides were placed in an oven at 60 °C for about 40 min to allow sections to adhere. Mounted sections were dewaxed in xylene, incubated in 3% aqueous H2O2 at room temperature for 5∼10 min to quench endogenous peroxidase activity, and rinsed three times in distilled water. Slides were placed in 0.01 M PBS buffer solution (pH 7.4) and heated to 100 °C in a microwave oven for 30 min to expose the antigens, allowed to cool, removed from the PBS, dripped with antigen repairing liquid for 10 min at room temperature, washed three times with distilled water, and blocked in normal serum sealing fluid at room temperature for 20 min. After gently removing excess fluid, slides were incubated in the primary antibody (1:1000 in PBS) at 4 °C overnight. Slides were then stained with a biotinylated goat anti-rabbit IgG at 20 °C ∼37 °C for 20 min, rinsed three times (2 min/wash) in 0.01 M PBS, dripped with SABC reagent at 20 °C∼37 °C for 20 min, and washed four times (5 min per wash) in 0.01 M PBS. A DAB staining kit was used to visualize the immunolabeling (5∼10 min in DAB). The hematoxylin sections were mildly restained, and all slices were then dehydrated and sealed.
Microscopic image analysis
Intramucosal 5-HT
The relative 5-HT content of each rat GI tract section was estimated by the average density and average gray value of 5-HT-positive cells in 10 randomly selected fields (40 µm2) from 10 randomly selected slides. The gastrointestinal mucosal layer was used as the reference area. The same method was used for estimation of myenteric nerve plexus VIP expression except that the gastrointestinal musculature was used as the reference area. The average 5-HT+ and VIP+ cell density and gray values were compared between control and extract-treated groups (Figures 1–3).
Results
The average 5-HT staining intensity (correlated with 5-HT+ cell number) was significantly higher in the gastric antrum, duodenum and jejunum mucosal layer of extract-treated rats compared to control rats (antrum: 213% of control; duodenum: 193%; jejunum: 256%; p < 0.05 for all) (Tables 1 and 2). In contrast, the average density of VIP+ cells was lower in the gastric antrum, duodenum and jejunum myenteric plexus of extract-treated rats compared to control rats (antrum: 14% of control; duodenum: 15%; jejunum: 38%; p < 0.01 for all). Thus, 5-HT content was increased over a broad area of the mucosal layer while VIP expression was downregulated in the myenteric plexus following treatment with Fructus Aurantii extract (Tables 3 and 4).
Discussion
The gastrointestinal tract is the largest and most complex mammalian endocrine organ. The GI epithelium and glands contain a multitude of distinct endocrine cells expressing different peptides and biogenic amines that regulate digestion, fluid transport/secretion and motility. Vasoactive intestinal peptide and serotonin are two important brain-gut signaling factors (Palsson et al., Citation2004). Serotonin plays a critical role in the regulation of gastrointestinal motility, secretion and sensation by activating 5-HT receptors widely expressed on intrinsic primary afferent neurons, smooth muscle cells, enterocytes and extrinsic afferent nerve fibers (Gershon & Tack, Citation2007). Endogenous 5-HT is synthesized by enterochromaffin cells, which utilize tryptophan hydroxylase-1 as the rate-limiting enzyme for biosynthesis (Gershon & Tack, Citation2007). Serotonin is usually stored in granules with other signaling molecules such as ATP. Local release of 5-HT activates Ach release from nearby cholinergic neurons and also stimulates nonadrenergic, noncholinergic intrinsic inhibitory neurons. In turn, Ach enhances longitudinal and circular smooth muscle excitability, leading to gastrointestinal contraction and higher gastrointestinal motility. The 5-HT selective reuptake transporter terminates the actions of 5-HT by removing it from the interstitial space (Wade et al., Citation1996).
The 28-amino acid vasoactive intestinal peptide is produced by many tissues, including gut, pancreas and the suprachiasmatic nucleus of the hypothalamus (Fahrenkrug & Emson, Citation1982; Said, Citation1986). In the intestinal wall, VIP is distributed mainly in the nerve plexus and smooth muscle layer, where it acts locally to regulate other neurotransmitter pathways. VIP relaxes smooth muscle and increases fluid secretion in the GI tract, thereby inhibiting GI motility (Sandgren et al., Citation2003). Previous pharmacological studies have shown that VIP is involved in sleep regulation; in particular, it stimulates rapid eye movement (REM) sleep. Several recent studies indicate that VIP can modulate immune function through G-protein-coupled receptors expressed by immune cells and is also an important component of a homeostatic neuroimmune control system (Delgado et al., Citation2004; Gonzalez-Rey & Delgado, 2005). The VIP pathway may also contribute to the development of obesity (Liu et al., 2005). Anti-inflammatory actions of VIP are mediated by two widely expressed VIP receptors, VPAC1 and VPAC2. Collectively, these results highlight the broad therapeutic potential of this peptide (Delgado et al., Citation2002, Citation2004; Martinez et al., Citation2002).
Immunohistochemistry demonstrated basal expression of 5-HT in the mucosal layer and VIP expression in the mucosal layer and myenteric plexus throughout the rat small intestine. Administration of Fructus Aurantii extract increased 5-HT throughout the mucosal layer and decreased VIP in the myenteric plexus, providing a possible explanation for increased GI motility following intake of the extract or raw fruit. The chemical components within the extract that mediate these effects on GI motility remain to be determined.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
References
- Atkinson W, Lockhart S, Whorwell PJ, et al. (2006). Altered 5-hydroxytryptamine signaling in patients with constipationand diarrhea-predominant irritable bowel syndrome. Gastroenterology 130:34–43
- Camilleri MD. (2009). Serotonin in the gastrointestinal tract. Curr Opin Endocrinol Diabetes Obes 16:53–9
- Chen JJ, Li Z, Pan H, et al. (2001). Maintenance of serotonin in the intestinal mucosa and ganglia of mice that lack the high-affinity serotonin transporter: Abnormal intestinal motility and the expression of cation transporters. J Neurosci 21:6348–61
- Delgado M, Jonakait GM, Ganea D. (2002). Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit chemokine production in activated microglia. Glia 39:148–61
- Delgado M, Pozo D, Ganea D. (2004). The significance of vasoactive intestinal peptide in immunomodulation. Pharmacol Rev 56:249–90
- Fahrenkrug J, Emson PC. (1982). Vasoactive intestinal polypeptide: Functional aspects. Br Med Bull 38:265–70
- Gershon MD, Tack J. (2007). The serotonin signaling system: From basic understanding to drug development for functional GI disorders. Gastroenterology 132:397–414
- Gonzalez-Rey E, Delgado M. (2005). Role of vasoactive intestinal peptide in inflammation and autoimmunity. Curr Opin Investig Drugs 6:1116–23
- Kamata K, Sakamoto A, Kasuya Y. (1988). Similarities between relaxations induced by vasoactive intestinal peptide and by stimulation of the non-adrenergic non-cholinergic neurons in the rat stomach. Pharmacol, 338:401–6
- Liu YJ, Guo YF, Zhang LS, et al. (2010). Biological pathway-based genome-wide association analysis identified the vasoactive intestinal peptide (VIP) pathway important for obesity. Obesity (Silver Spring) 18:2339–46
- Martinez C, Abad C, Delgado M, et al. (2002). Anti-inflammatory role in septic shock of pituitary adenylate cyclase-activating polypeptide receptor. Proc Natl Acad Sci USA 99:1053–8
- Palsson OS, Morteau O, Bozymski EM, et al. (2004). Elevated vasoactive intestinal peptide concentrations in patients with irritable bowel syndrome. Dig Dis Sci 49:1236–43
- Said SI. (1986). Vasoactive intestinal peptide. J Endocrinol Invest 9:191–200
- Sandgren K, Lin Z, Ekblad E. (2003). Differential effects of VIP and PACAP on survival of cultured adult rat myenteric neurons. Regul Pept 111:211–17
- Wade PR, Chen J, Jaffe B, et al. (1996). Localization and function of a 5-HT transporter in crypt epithelia of the gastrointestinal tract. J Neurosci 16:2352–64
- Wang CF, Yang DZ,Wei YQ, Xun QY. (2001). Study on the effect of Fructus aurantii immaturus on myoelectric activity of gastrointes tinal tract in rats. J Southeast Univ (Med Sci Ed) 20:153–4