Effects of black garlic on the pacemaker potentials of interstitial cells of Cajal in murine small intestine in vitro and on gastrointestinal motility in vivo

ABSTRACT Black garlic (BG) is a newly explored food stuff obtained via fermentation of raw, healthy garlic, especially in Asian countries. Interstitial cells of Cajal (ICC) are the pacemaker cells of gastrointestinal (GI) motility. The purpose of this study was to investigate the effects of BG extract on the pacemaker potentials of the ICC in the small intestines of mice and the possibility of controlling GI motility. The antioxidant activity of BG extract was also investigated. The whole-cell electrophysiological method was used to measure pacemaker potentials of the ICC in vitro, whereas GI motility was measured using the intestinal transit rate (ITR) in vivo. BG extract depolarized the pacemaker potentials of the ICC. Y25130 and RS39604 5-HT receptor antagonists could not inhibit the effect of BG extract on the pacemaker potentials of the ICC, whereas the 5-HT receptor antagonist SB269970 could. Pre-treatment with external Na+ (5 mM) or Ca2+-free solution inhibited the BG extract-induced depolarization of the ICC. With SB203580, PD98059, or c-jun NH2-terminal kinase II inhibitor pre-treatment, BG extract did not induce pacemaker potential depolarization. Moreover, the ITR values were increased by BG extract. Elevation of the ITR due to BG extract was related with increased protein expression of the 5-HT7 receptors. In addition, BG extract showed antioxidant activity. Collectively, these results highlight the ability of BG extract to regulate GI motility and the possibility of using it to develop GI motility modulators in the future. Moreover, BG showed immense potential as an antioxidant.


Introduction
Garlic (Allium sativum L.) has been used as a spice and traditional medicine for eons. Several studies have shown that garlic has beneficial effects on human health, such as anti-inflammatory, anti-cancer, lipid lowering, maintenance of blood pressure, and blood glucose regulation (Kimura et al. 2017). However, unprocessed, raw garlic has a characteristic odor and spicy taste, which can limit its use because of gastrointestinal (GI) problems when consumed (Kodera et al. 2002). Black garlic (BG) is obtained after garlic has been fermented for a certain duration under high humidity and temperature conditions (Kimura et al. 2017). BG does not produce a strong off-flavor caused by the reduction of allicin, which converts it to an antioxidant during processing (Yuan et al. 2016). BG extract has demonstrated several bioactivities, including anti-oxidative, anti-allergic, anti-diabetes, anti-inflammation, anti-carcinogenic, and GI emptying effects (Jeong et al. 2016;Chen et al. 2018).
Interstitial cells of Cajal (ICC) are essential pacemaker cells that regulate the GI motility (Huizinga et al. 1995;Ward et al. 2000;Kim et al. 2005;Hwang et al. 2020. Thus, research on ICC plays a very important role in understanding GI motility regulation. However, little is reported on the effects of BG extract on ICC and GI motility. Thus, we investigated the effects of BG extract on the pacemaker potentials of ICC and GI motility. In addition, we assessed the antioxidant activity of BG extract.

Materials and methods
Preparation of the BG extract BG was purchased from Taewoo Food Co. (Daejeon, Korea). A total of 2 kg of BG was extracted in 70% ethyl alcohol (20 L) for 6 h at 80°C and filtered through a Whatman No. 4 filter paper. After concentrating the solvents using rotary evaporation at 50°C, the yield was approximately 19.8% on a dry weight (w/w) basis.

Preparation of ICC
Small intestines of mice were excised and after removing the mucous membrane, cut it into pieces. The cells were dispersed in a solution containing various enzymes, including collagenase, and were cultured in smooth muscle growth medium (Clonetics, San Diego, CA, U.S.A.) supplemented with a murine stem cell factor (Sigma-Aldrich, St. Louis, MO, U.S.A.) in a 95% O 2 incubator.

Intestinal transit rate (ITR)
Evans blue (5%, w/v) was administered to healthy ICR mice after administration of BG extract into the stomach. After 30 min of Evans blue administration, the ITR of the mice was checked.

GI motility dysfunction (GMD) model mice
We generated the GMD mouse models by using the acetic acid (AA, 0.6%, w/v, in saline)-induced peritoneal stimulation, as previously described (Lyu and Lee 2013). AA was injected intraperitoneally and the research process was carried out as described previously Wu et al. 2013).

Animals
Forty-nine mice (24 male and 25 female; 3-8-d-old) from ICR were used for the ICC experiments. In addition, 39 mice (male; 5-6-week-old) were used for the ITR experiments on heathy mice and mice with GI motility disease, whereas 10 mice (male; 5-6-week-old) were used for the protein expression experiments. The ICC and ITR experiments were completed within 12 h of culturing, respectively. We experimented according to The Institutional Animal Care and Use Committee at Pusan National University approved (approval no. PNU-2019-2462). Also, animals were handled according to the Guide for the Care and Use of Laboratory Animals.

Reactive oxygen species (ROS) scavenging activity
A 0.2-mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution was prepared by dissolving DPPH reagent in EtOH. The prepared solution and BG extract were mixed in a 1:1 ratio and incubated for 30 min in a dark room. Absorbance at 517 nm was measured and DPPH radical scavenging activity was calculated using the following formula: where A is the absorbance value of the sample solution and B is the absorbance value of the control solution.

Drugs
PD98059 and SB203580 were purchased from Tocris Bioscience (United Kingdom), whereas c-jun NH 2 -terminal kinase (JNK) II inhibitor was purchased from Calbiochem (San Diego, CA, U.S.A.). All other agents were purchased from Sigma-Aldrich.

Statistical analyses
Data are expressed as the mean ± standard error of the mean. Significant differences were evaluated using oneway analysis of variance (ANOVA) or the Student's t-test. P values < 0.05 were considered as significant.
Importance of extracellular Na + and Ca 2+ in BG extract-induced pacemaker potential depolarization of ICC Both external Na + and Ca 2+ play a key role in regulating GI motility (Ward et al. 2000). To investigate the importance of external Na + or Ca 2+ in the BG extractinduced responses, we used external Na + (5 mM) or Ca 2+ -free conditions. Pre-treatment with the external Na + or Ca 2+ -free solution suppressed the pacemaker potentials and inhibited BG extract-induced responses ( Figure 3A and 3B). The average degrees of depolarization were 1.4 ± 0.5 mV (n = 9; P < 0.0001) with Na + solution and 3.4 ± 0.6 mV (n = 13; P < 0.0001) with Ca 2 + -free solution ( Figure 3C). These results indicated that the BG extract-induced response was controlled by external Na + or Ca 2+ .
Importance of mitogen-activated protein kinase (MAPK) in BG extract-induced pacemaker potential depolarization of ICC It has been reported that MAPK regulates the proliferation and differentiation of the GI tract (Jeong et al. 2012). Therefore, we assessed whether MAPK signaling affects the efficacy of BG extract on pacemaker potentials by treatment with PD98059 (a p42/44 inhibitor), SB203580 (a p38 inhibitor), and JNK II inhibitor. With PD98059 (n = 11), SB203580 (n = 10), or JNK II (n = 10) inhibitor treatment, BG extract did not induce the pacemaker potential depolarization (Figure 4). These results indicated that the BG extract-induced response was dependent on MAPK signaling.

Regulation of BG extract-induced small intestinal 5-HT receptor expression
5-HT is mainly present in the GI tract, and an increase or decrease in the expression of 5-HT directly affects GI motility (Camilleri 2009). Moreover, 5-HT 3 , 4, and 7 receptors were found in ICC (Liu et al. 2011;Shahi et al. 2011). When BG extract was used, the expression of 5-HT 3 receptors decreased significantly, the expression of 5-HT 4 receptors did not change, and the expression of 5-HT 7 receptors increased significantly ( Figure 6A). The expression of 5-HT 3 receptors decreased by 69.3 ± 5.4% (n = 5; P < 0.01) whereas that of 5-HT 7 receptors increased by 228.9 ± 12.3% (n = 7; P < 0.0001) after BG extract treatment ( Figure 6B and 6D). However, the expression of 5-HT 4 receptors was unchanged (n = 6; Figure 6C). These results suggested that the ITR increase by BG extract was done by an increase in the expression of the 5-HT 7 receptors.

Discussion
Garlic has been used as a medicinal ingredient for a long time (Jeong et al. 2012). BG is a processed food, where fresh garlic is fermented at high humidity and high temperatures for 60-90 d ; Yang et al.     2019). Although BG has various activities, including influencing GI motility (Jeong et al. 2016;Chen et al. 2018), its effect on the regulation of ICC function has not been reported yet. The ingredients of BG extract may change for various reasons, such as the type of solvent used for extraction. However, in this experiment, instead of experimenting with extracts of several batches, the experiment was conducted with the extract of one batch. Although no analysis of the components of the BG extract was conducted, the results of previous studies showed that, as compared to regular garlic, BG contained large amounts of diallyl trisulfide and allyl methyl trisulfide and a small amount of epicatechin (Martínez-Casas et al. 2017). Furthermore, it has been reported that lactic acid is a major organic acid component of BG extract (Lu et al. 2017). Another study also showed that BG contains S-allyl-L-cysteine, Figure 6. Effects of BG extract on the protein expression of 5-HT 3 , 4 , and 7 receptors in mice. (A) 5-HT 7 receptor expression increased considerably, but 5-HT 4 receptors was unchanged. However, the expression of 5-HT 3 receptors decreased. (B-D) Band density is showed relative to CTRL. Mean ± SEs. **P < 0.01. ****P < 0.0001. BG: Black garlic. CTRL: Control. β-Actin was the loading control. Figure 7. ROS scavenging activity was measured using the DPPH reagent. BG extract was treated with 0.001, 0.01, 0.1, 1, and 10 mg/mL DPPH reagent, and 100% EtOH was used as a negative control (N.C). The results are from three independent experiments. Mean ± SEs. ****P < 0.0001. BG: Black garlic. DPPH: 1,1-diphenyl-2-picrylhydrazyl. S-allylmercaptocysteine, pyruvate, and amino acids (Kim et al. 2015). In this study, we checked the efficacy of BG extract but not the efficacy of the individual ingredients. We plan to conduct a study to reveal the effective ingredients of BG in the future. We found that BG extract modulated the ICC pacemaker potentials. BG extract depolarized the ICC pacemaker potentials (Figure 1). External Na + (5 mM) or Ca 2 + -free solution inhibited BG extract-induced pacemaker depolarization of ICC (Figure 3). BG extract increased the ITR. It also recovered the loperamide-induced decrease in ITR in vivo ( Figure 5A). Moreover, BG extract recovered the ITR in AA-induced GMD in mice ( Figure 5B). Therefore, it is thought that BG may control GI motility through the adjustment of the pacemaker potential of the ICC. ICC generate spontaneously active pacemaker potentials (Huizinga et al. 1995). 5-HT is secreted from enterochromaffin cells present mostly in the gut. Liu et al. (2011) showed that the ICC pacemaker activity was controlled through 5-HT 3 receptors, but Shahi et al. (2011) suggested that it was controlled through 5-HT 3 , 4 , and 7 receptors. In addition, Wouters et al. (2007) stated that 5-HT 2B receptors regulate the growth of ICC. However, in this study, the 5-HT 7 receptor antagonist SB269970 inhibited BG extract-induced responses, whereas the 5-HT 3 and 5-HT 4 receptor antagonists Y25130 and RS39604, respectively, did not. This shows that BG extract modulates pacemaker potentials due to the 5-HT 7 receptors ( Figure 2). In addition, BG extract-induced ITR increase was mediated by 5-HT 7 receptors ( Figure 6). Therefore, we hypothesize that 5-HT 7 receptors play a vital role in the regulation of GI motility by BG extract. 5-HT 7 receptors are present in lymphoid tissues, smooth muscle cells, ICC, and neurons within the gut (Tonini et al. 2005;Kim and Khan 2014). Various studies have demonstrated the relevance of 5-HT 7 receptors in GI motility regulation (Tonini et al. 2005). In the future, we will study the mechanisms and roles of 5-HT 7 receptors in GI motility and ICC in detail. In addition, MAPK signaling is also a major target for new treatments with GI motility disease (Ihara et al. 2011). In this study, MAPK inhibitors suppressed the effects of BG extract. It was observed that p38, p42/44, and JNK signaling is involved in the BG extract-mediated control of pacemaker potentials.
The human body has a variety of complex antioxidant defense mechanisms to counter the harmful effects of free radicals or other oxidants (Alam et al. 2013). Antioxidants are known to be very effective in preventing degenerative diseases and improving the quality of life (Alam et al. 2013). Compared to garlic, BG has approximately 10-fold stronger superoxide dismutase-like activity and antioxidant effects against hydrogen peroxide (Sato et al. 2006). In this study, we showed that the potent antioxidative effects of BG by using the DPPH radical scavenging assay (Figure 7).
Recently, natural herbal medicine has been attracting increasing attention as an alternative medicine with few side effects (Ekor 2014). Since many people have benefited from natural herbal medicine, we hope that research on the development of new treatments for GI diseases will be more active in the future.
Collectively, the results from the present study showed that BG extract depolarized the pacemaker potentials of the ICC via the 5-HT 7 receptors, extracellular Na + and Ca 2+ concentration regulation, and MAPK pathways ( Figure 8). Furthermore, BG extract increased the ITR in normal and GMD model mice. BG extractinduced ITR increase was mediated through the 5-HT 7 receptors. In addition, BG extract showed significant antioxidative effects. Therefore, BG might be a prokinetic agent that can cure or prevent GMD, and herbal medicine may become a very important strategy for the treatment of GI tract disorders.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This study has been worked in part with the support of a research grant of Kangwon National University in 2018 and also was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2021R1I1A3042479).