Hesperidin depolarizes the pacemaker potentials through 5-HT4 receptor in murine small intestinal interstitial cells of Cajal

ABSTRACT Hesperidin, a citrus flavonoid, can exert numerous beneficial effects on human health. Interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal (GI) tract. In the present study, we investigated potential effects of hesperidin on pacemaker potential of ICC in murine small intestine and GI motility. A whole-cell patch-clamp configuration was used to record pacemaker potential in ICC, and GI motility was investigated in vivo by recording gastric emptying (GE) and intestinal transit rate (ITR). Hesperidin depolarized pacemaker potentials of ICC in a dose-dependent manner. Pre-treatment with methoctramine or 4-DAMP did not inhibit hesperidin-induced pacemaker potential depolarization. Neither a 5-HT3 receptor antagonist (Y25130) nor a 5-HT7 receptor antagonist (SB269970) reduced the effect of hesperidin on ICC pacemaker potential, whereas the 5-HT4 receptor antagonist RS39604 was found to inhibit this effect. In the presence of GDP–β–S, hesperidin-induced pacemaker potential depolarization was inhibited. Moreover, in the presence of U73122 and calphostin C, hesperidin did not depolarize pacemaker potentials. Furthermore, hesperidin accelerated GE and ITR in vivo. These results imply that hesperidin depolarized ICC pacemaker potential via 5-HT4 receptors, G protein, and PLC/PKC dependent pathways and that it increased GI motility. Therefore, hesperidin may be a promising novel drug to regulate GI motility.


Introduction
Polyphenols occur in various plants and are important for their defense systems; flavonoids are a group of polyphenols which are particularly common in edible plants that constitute a large part of human diet (Cho et al. 2018;Chung et al. 2018;Stevens et al. 2019). The predominant flavonoid in sweet fruits is hesperidin (Garg et al. 2001) which also occurs frequently in vegetables and beverages such as tea and red wine (Orallo et al. 2004;Bock et al. 2008). Hesperidin is known to exert various effects on humans including anti-inflammatory activity (Garg et al. 2001). Moreover, hesperidin has been reported to regulate gastrointestinal (GI) motility by reducing inflammatory reactions and stimulating calcium release (Xiong et al. 2016). GI motility is controlled by numerous different cell types in the GI tract, and among these, ICC plays a key role (Huizinga et al. 1995;Sanders 1996;Kim et al. 2005). ICC can generate electrical charges (Huizinga et al. 1995;Sanders 1996;Kim et al. 2005), and when ICC abundance and intercellular connectivity are reduced, GI motility also decreases (Der et al. 2000;Wei et al. 2014). Potential effects of hesperidin on GI motility have not yet been comprehensively investigated; therefore, in the current study, we assessed the effects of hesperidin on ICC in vitro and on GI motility in vivo.

Preparation of cell cultures
Animal experiments were conducted in compliance with the stipulations of the animal experiment ethics committee of Pusan National University (approval no. PNU-2018PNU- -1832. Small intestines of ICR mice were isolated, and the mucous membrane was excised. Small-intestinal muscles were equilibrated using Ca 2+ -free Hank's solution. Cells were enzymatically isolated using collagenase (Worthington Biochemical, Lakewood, NJ, USA) and were then cultured in smooth muscle growth medium ([SMGM]; Clonetics, San Diego, CA, USA) inside a CO 2 incubator and at 37°C.

Assessment of gastric emptying (GE)
Twenty minutes after administering phenol red solution, stomachs tissue was cut into several pieces which were placed in sodium hydroxide. Tissue preparations were then centrifuged with NaOH at 1050 × g for 10 min, and absorbance was measured using a spectrometer at 560 nm, according to previously published methods.

Intestinal transit rate (ITR) measurements
Mice were administered hesperidin, followed by oral administration of Evans Blue. Thirty minutes later, animals were euthanized, and ITR was measured according to the distance over which Evans Blue had been transported in the intestine (expressed as percentage of the length of the intestine).
Drugs 5-HT receptor antagonists were obtained from Tocris Bioscience (Bristol, United Kingdom). All other reagents including hesperidin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Statistical analyses
Results are shown as means ± standard error. We employed an ANOVA to test effects of hesperidin on the respective parameters using Prism 6.0 software (La Jolla, CA, USA). Statistical significance is reported at P < 0.05.

Discussion
We investigated effects of hesperidin on pacemaker potential in ICC and on GI motility. Hesperidin depolarized pacemaker potentials through 5-HT 4 receptors via G protein and PLC/PKC dependent pathways but not through muscarinic receptors, indicating that hesperidin can modulate ICC. Furthermore, hesperidin increased GE and ITR.
Hesperidin is commonly found in highly nutritious fruits such as oranges, tangelos, tangerines, grapefruits, and other citrus fruits (Suzuki et al. 2014). Hesperidin has been shown to exert numerous biological functions (Hou et al. 2012); therefore, hesperidin has been commonly used to treat various disorders including   neurological, psychiatric, and cardiovascular diseases (Li and Schluesener 2017). Furthermore, hesperidin is being used in combination with other drugs such as rikkunshito (Li and Schluesener 2017). Rikkunshito is a traditional herbal remedy to promote appetite and is commonly used in Japan to treat indigestion (Arai et al.  2012; Takiguchi et al. 2013). Hesperidin stimulates ghrelin secretion, thus synergistic effects of mixing hesperidin with rikkunshito produced promising good results. In addition, hesperidin is readily available and inexpensive, therefore it can be manufactured and commercialized for therapeutic purposes as well as in the food industry. In the present study, we found that hesperidin modulated pacemaker potentials in ICC. Therefore, we suggest that hesperidin may regulate GI motility via ICC pacemaker potential.
GI motility disorders are very common and occur throughout the GI tract, and the associated symptoms can substantially affect the quality of life (Pare et al. 2001;El-Serag and Talley 2003;Lacy and Weiser 2006;Lacy et al. 2018). Moreover, these disorders are of substantial economical concern (Sandler et al. 2002;Lacy et al. 2018). ICC are important for GI motility (Huizinga et al. 1995;Sanders 1996;Kim et al. 2005). ICC spontaneously generate active pacemaker potential, causing electrical and mechanical activity of smooth muscles (Huizinga et al. 1995;Sanders 1996;Kim et al. 2005). In ICC, a Ca 2+ -activated Cl − channel and a non-selective cation channel are required for pacemaking activity, and the ether-a-go-go-related K + channel is also one of the most important K + channels for maintaining and activating stable membrane voltage in ICC (Zhu et al. 2003;Kim et al. 2005;Zhu et al. 2009). Further studies on the involvement of ion channels in hesperidin-related effects are required.
Muscarinic receptors are important receptors for regulating GI motility (Hirst et al. 2002). In the present study, both methoctramine and 4-DAMP produced no effects on hesperidin reactions. Thus, muscarinic receptors were apparently not involved in hesperidin effects on ICC ( Figure 2). Furthermore, 5-HT plays a crucial role in regulating GI motility (Baker 2005). Previous studies suggested that 5-HT can control pacemaker activity through 5-HT 3 , 5-HT 4 , and 5-HT 7 receptors (Shahi et al. 2011). In the present study, RS39604 blocked hesperidin effects, whereas Y25130 and SB269970 showed no respective effect. Thus, 5-HT 4 receptors were apparently involved in hesperidin-induced effects on ICC (Figure 3). Furthermore, G-protein and PLC/PKC pathways are involved in hesperidin-induced effects in ICC (Figures 4  and 5). In addition, we found that hesperidin increased GE and ITR ( Figure 6).
Polyphenols occur naturally in various fruits and vegetables. However, potential effects of polyphenols on human health remain to be elucidated. Our results suggest that polyphenols such as hesperidin may be an option for prevention and treatment of GI disorders. Taken together, our results show that hesperidin depolarizes pacemaker potentials of ICC via 5-HT 4 receptors, G protein, and PLC/PKC dependent pathways, and increases GE and ITR.