Inhibitory Effects of Jakyakgamcho-Tang ( Glycyrrhiza uralensis and Paeonia lactiﬂora ) on the Pacemaker Potential of the Interstitial Cells of Cajal in the Murine Small Intestine

: Jakyakgamcho-tang (JYGCT) has been used to treat various diseases. The interstitial cells of Cajal (ICC) regulate gastrointestinal (GI) motility as pacemaker cells. Here, we examined the effects of JYGCT on the pacemaker potential of the ICC in the small intestine. We observed that JYGCT inhibited the pacemaker potential in a dose-dependent manner. Glibenclamide did not affect the pacemaker potential and on these conditions, JYGCT also had no effect on the pacemaker potential. Pretreatment with capsazepine or SB452533 blocked the JYGCT-induced effects. In the presence of SQ-22536, JYGCT did not inhibit the pacemaker potential. Additionally, JYGCT inhibited spontaneous [Ca2+]i oscillations and JYGCT-induced ITR increase was associated with TMEM16A, motilin and substance P activation. Moreover, JYGCT was effective in alleviating the symptoms of irritable bowel syndrome. Our results suggest that JYGCT inhibited the pacemaker potential of the ICC via KATP, the TRPV1 or the cyclic AMP pathway, and intracellular Ca2+ regulation, indicating that JYGCT can affect ICC and thus have the function of regulating GI motility. Therefore, JYGCT may be used as a GI motility disorder regulator or disease prevention agent.


Introduction
Jakyakgamcho-tang (JYGCT; Glycyrrhiza uralensis and Paeonia lactiflora) is a traditional herbal medicine and has been used to treat various disease conditions, including muscle pain and acute abdominal pain [1][2][3][4][5][6][7][8][9][10]. JYGCT shows excellent efficacy in enhancing spleen function and relieving liver function and is used clinically in the treatment of various indications, such as muscle and abdominal pain, and bronchial asthma [2][3][4]. JYGCT has the function of decreasing the paclitaxel-induced painful neuropathy, such as allodynia and hyperalgesia [6] and reduces muscle cramps without any side effects [7]. It shows an anti-inflammatory effect and an antispasmodic effect on skeletal muscle [1,8]. It also reduces the serum uric acid levels and increases the sympathetic activities in hyperuricemic vegetarians [9]. In addition, JYGCT also affects the gastrointestinal (GI) tract. It is effective in relieving abdominal pain in acute GI inflammation [11], and it inhibits the transient receptor potential (TRPV) 1 channel and suppresses serotonin production, thereby alleviating the visceral hypersensitivity [12].
GI motility is a highly regulated process and an essential factor determining the quality of life [13]. The interstitial cells of Cajal (ICC) are essential in GI motility and are considered the pacemakers of the GI tract [14][15][16]. Injury of ICC leads to various diseases related to GI motility [17]. Therefore, research on these cells is important to understand the regulation of GI motility. However, the question remains as to whether JYGCT exerts regulatory effects on ICC or GI motility and whether the underlying mechanisms require investigation. Therefore, we investigated the regulatory effect of JYGCT on ICC physiological signaling pathways and GI motility in mice.

Preparation of Cell Cultures
The Animal Experiment Ethics Committee of Pusan National University (no. PNU-2020-2831) guidelines were followed. Institute of Cancer Research (ICR) mice were sacrificed by cervical dislocation and then the small intestines were isolated and the mucous membranes were removed. Cells were isolated using collagenase and cultured in smooth muscle growth medium (SMGM; Clonetics, San Diego, CA, USA) in a CO 2 incubator at 37 • C.

Patch Clamp Experiments
We used the patch clamp technique to record ICC pacemaker potential. A Na+-Tyrode bath and KCl (140 mM) pipette solution were used. Electrophysiological techniques were conducted and results were analyzed using pClamp and Origin software (version 6.0, Microcal, Northampton, MA, USA).

Intracellular Free Calcium Ion Concentration [Ca2+]i
The cultured ICC were loaded with fura-2 AM and measured [Ca2+]i with a PTI Delta scan illuminator (Photon Technology International Inc., Birmingham, NJ, USA).

ITR Measurement
After intragastric administration of JYGCT to ICR mice, Evans blue (5%, w/v; 0.1 mL/kg) was intragastrically administered 30 min later. ITR was measured 30 min after the Evans blue administration.

GI Motility Dysfunction (GMD) Model Mice
We made the GMD mouse models by using the acetic acid. AA was injected intraperitoneally and allowed to rest for 30 min. ITR was measured 30 min after the Evans blue administration.

Gut Hormone Levels
After JYGCT (0.5 g/kg) was fed once a day for 5 days, MTL, substance P (SP), somatostatin (SS), and vasoactive intestinal polypeptide (VIP) hormone levels were detected using commercial kits.

Irritable Bowel Syndrome (IBS) Experiments
C57/BL6 mice (6 weeks) induced colitis by 0.1 mL zymosan suspension (30 mg/mL) which was administered trans-anally via a feeding needle into the colons of mice. Zymosan, PBS or JYGCT were administered daily for 3 consecutive days. After 3 days of administration, the experiment was conducted on the 4th day. The zymosan-injected mice were divided into three groups (n = 9 per group) and treated with PBS (control), zymosan or JYGCT (500 mg/kg).

Macroscopic Scoring
The weight of the large intestine was measured after removing the stool, and the length of the large intestine was measured from the cecum to the anus. The individual scores were graded: stool score (0, normal; 1, loose/moist; 2, amorphous/sticky; and 3, diarrhea). The stool status was measured by three researchers using the blind method.

Drugs
The drugs used in the experiments were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Statistical Analysis
Results are represented as the mean ± standard error (SE). The results were analyzed using Prism 6.0 (La Jolla, CA, USA) and Origin version 8.0 (OriginLab Corporation, Northampton, MA, USA). One-way analysis of variance (ANOVA) or Student's t-test for unpaired data were used to compare control and experimental groups. A p-value < 0.05 was considered statistically significant.

JYGCT Inhibits the Pacemaker Potential of the ICC in the Murine Small Intestine
We performed whole-cell patch clamp experiments using the cultured ICC to examine the pacemaker potential of these cells. We noted that, under these conditions, the ICC spontaneously generated a pacemaker potential with a mean frequency of 11.3 ± 0.9 cycles/min and a mean amplitude of 24.8 ± 1.3 mV. When JYGCT (1-10 mg/mL) was applied, the pacemaker potential decreased ( Figure 1A). The mean frequency was 9.3 ± 0.5 cycles/min (p < 0.01) at 1 mg/mL, 5.6 ± 0.5 cycles/min (p < 0.0001) at 5 mg/mL, and 1.5 ± 0.4 cycles/min (p < 0.0001) at 10 mg/mL ( Figure 1E); the mean amplitude was 23.9 ± 1.0 mV, 2.4 ± 0.5 mV (p < 0.0001), and 2.1 ± 0.8 mV (p < 0.0001) ( Figure 1F). Thus, these results suggest that JYGCT inhibits the pacemaker potential of the ICC.

ATP-Sensitive K+ Channels Are Involved in the JYGCT-Induced Pacemaker Potential Inhibition in the ICC
To investigate the involvement of the K+ channels, we used several K+ channel blockers. In the presence of TEA, JYGCT inhibited the pacemaker potential in the ICC (Figure 2A). In addition, JYGCT inhibited the pacemaker potential when co-treated with 4-aminopyridine or apamin ( Figure 2B,C). Moreover, glibenclamide (an ATP-sensitive K+ (KATP) channel blocker) did not affect the pacemaker potential and, under these conditions, JYGCT also showed no effect on the pacemaker potential ( Figure 2D); further, glibenclamide reversed the effects of JYGCT ( Figure 2E). In the presence of 5 mg/mL JYGCT, the mean frequency and amplitude were 5.6 ± 0.6 cycles/min and 2.6 ± 0.4 mV for TEA, 5.3 ± 0.5 cycles/min and 2.6 ± 0.4 mV for 4-aminopyridine, 5.5 ± 0.6 cycles/min and 2.5 ± 0.4 mV for apamin, and 12.1 ± 0.9 cycles/min (p < 0.0001) and 22.7 ± 1.0 mV (p < 0.0001) for glibenclamide ( Figure 2F,G). These results suggest that JYGCT activates the KATP channels in the ICC.

ATP-Sensitive K+ Channels Are Involved in the JYGCT-Induced Pacemaker Potentia Inhibition in the ICC
To investigate the involvement of the K+ channels, we used several K+ channel blo ers. In the presence of TEA, JYGCT inhibited the pacemaker potential in the ICC (Fig  2A). In addition, JYGCT inhibited the pacemaker potential when co-treated with 4-a nopyridine or apamin ( Figure 2B,C). Moreover, glibenclamide (an ATP-sensitive (KATP) channel blocker) did not affect the pacemaker potential and, under these con tions, JYGCT also showed no effect on the pacemaker potential ( Figure 2D); furth glibenclamide reversed the effects of JYGCT ( Figure 2E). In the presence of 5 mg/ JYGCT, the mean frequency and amplitude were 5.6 ± 0.6 cycles/min and 2.6 ± 0.4 mV TEA, 5.3 ± 0.5 cycles/min and 2.6 ± 0.4 mV for 4-aminopyridine, 5.5 ± 0.6 cycles/min a 2.5 ± 0.4 mV for apamin, and 12.1 ± 0.9 cycles/min (p < 0.0001) and 22.7 ± 1.0 mV (p < 0.00 for glibenclamide ( Figure 2F,G). These results suggest that JYGCT activates the KA channels in the ICC.

Transient Receptor Potential Vanilloid 1 (TRPV1) Channels Are Involved in the JYGCT-Induced Pacemaker Potential Inhibition in the ICC
Many studies show that TRPV1 is involved in the physiological functions of the tract [19][20][21]; therefore, we investigated the relevance of the TRPV1 channel. We incuba cells with capsazepine or SB452533, a TRPV1 receptor antagonist, for 5 min. Pretreatm with capsazepine or SB452533 blocked the JYGCT-induced effects ( Figure 3A,B). The quency of the pacemaker potential with JYGCT and capsazepine or SB452533 was 11 0.9 cycles/min (p < 0.0001) or 11.9 ± 1.0 cycles/min (p < 0.0001), with a corresponding a plitude of 22.7 ± 1.1 mV (p < 0.0001) or 22.8 ± 1.7 mV (p < 0.0001) ( Figure 3C,D). Thus, th results suggest that the TRPV1 channels regulate the JYGCT-induced responses.

Transient Receptor Potential Vanilloid 1 (TRPV1) Channels Are Involved in the JYGCT-Induced Pacemaker Potential Inhibition in the ICC
Many studies show that TRPV1 is involved in the physiological functions of the GI tract [19][20][21]; therefore, we investigated the relevance of the TRPV1 channel. We incubated cells with capsazepine or SB452533, a TRPV1 receptor antagonist, for 5 min. Pretreatment with capsazepine or SB452533 blocked the JYGCT-induced effects ( Figure 3A,B). The frequency of the pacemaker potential with JYGCT and capsazepine or SB452533 was 11.4 ± 0.9 cycles/min (p < 0.0001) or 11.9 ± 1.0 cycles/min (p < 0.0001), with a corresponding amplitude of 22.7 ± 1.1 mV (p < 0.0001) or 22.8 ± 1.7 mV (p < 0.0001) ( Figure 3C,D). Thus, these results suggest that the TRPV1 channels regulate the JYGCT-induced responses.

Effects of JYGCT on Macroscopic Score of Zymosan-Induced Colon Changes
We checked the colon length, weight, and stool status by JYGCT in the large intestine administered with zymosan. Administration of zymosan reduced the length of the colon compared to normal mice, and thus it can be seen that colitis was induced. However, when the mice administered with JYGCT were compared with mice administered with zymosan, the length of the colon was increased (Naïve: 9.45 ± 0.8 cm, CTRL: 7.55 ± 0.6 cm (p < 0.05), JYGCT: 9.36 ± 0.7 cm (p < 0.05), Figure 6A). In addition, although the colon weight increased compared to normal, JYGCT decreased the colon weight (Naïve: 0.25 ± 0.01 g, CTRL: 0.30 ± 0.02 g (p < 0.05), JYGCT: 0.23 ± 0.01 g (p < 0.05), Figure 6B). The feces of normal mice are dark brown and have the form of hard lumps, but in mice administered with zymosan, it can be seen that diarrhea is induced in light brown or yellow color with water-like mucus. The stool score was increased by comparing the zymosan-administered mice with the normal mice, but decreased by JYGCT (Naïve: 0.09 ± 0.03, CTRL: 2.46 ± 0.11 g (p < 0.0001), JYGCT: 0.54 ± 0.11 (p < 0.0001), Figure 6C). Thus, these results indicate that JYGCT might inhibit zymosan-induced colitis and diarrhea. Appl. Sci. 2022, 12, x FOR PEER REVIEW 8 of 11

Discussion
In this study, we investigated the effect of JYGCT on the pacemaker potential in the ICC obtained from the murine small intestine. JYGCT inhibited the pacemaker potential via KATP, the TRPV1, the cAMP pathway, and intracellular Ca2+ regulation, indicating that JYGCT can affect the ICC and may be a novel prokinetic agent in the GI tract.
JYGCT is a traditional medicine for various purposes [10]. It has been applied to treat various disease conditions; it has been used in the prevention of muscle cramps and the inhibition of skeletal muscle contractions [7], uric acid reduction, autonomic function regulation [9], and intestinal smooth muscle relaxation [25]. JYGCT has the function of decreasing the neuropathy [3] and shows an anti-inflammatory effect [1]. It also has antispasmodic effect on skeletal muscle [8], reduces the serum uric acid levels, and increases the sympathetic activities [9]. However, although JYGCT has been used as an herbal remedy to regulate the GI tract [11,12], its regulation of GI motility has not yet been reported.
In this study, we demonstrated that JYGCT modulated the pacemaker potential in the ICC. Therefore, it can be suggested that JYGCT may regulate GI motility via its effects on the ICC pacemaker potential. In a forthcoming study, we plan to investigate the effects of JYGCT's components, Jakyak and Gamcho, on the pacemaker potential in the ICC.
Ion channels are important in the physiological control of GI motility [26]. Among the various ion channels, KATP channels are enriched at various locations in the GI tissues [27,28]. KATP channels in the ICC are composed of SUR2B and Kir 6.2 in the small intestine and SUR2B and Kir 6.1 in the colon [29]. Previous studies have shown that KATP channels exist in cultured murine small intestinal ICC and reduce GI motility by causing relaxation of the smooth muscles; this makes them potential candidates in the treatment of GI motor diseases [30]. In addition, the TRPV1 channels are expressed throughout the alimentary canal where they play important roles in GI motility through the ICC [18]. Various studies show that the TRPV1 channels are involved in the treatment of gastric esophageal reflux disease and GI pain hypersensitivity in the GI tract [18,31]. In this study, JYGCT inhibited the pacemaker potential in the ICC via KATP (Figure 2) and the TRPV1 (Figure 3). Therefore, we believe that KATP and the TRPV1 channels may get involved in

Discussion
In this study, we investigated the effect of JYGCT on the pacemaker potential in the ICC obtained from the murine small intestine. JYGCT inhibited the pacemaker potential via KATP, the TRPV1, the cAMP pathway, and intracellular Ca2+ regulation, indicating that JYGCT can affect the ICC and may be a novel prokinetic agent in the GI tract.
JYGCT is a traditional medicine for various purposes [10]. It has been applied to treat various disease conditions; it has been used in the prevention of muscle cramps and the inhibition of skeletal muscle contractions [7], uric acid reduction, autonomic function regulation [9], and intestinal smooth muscle relaxation [25]. JYGCT has the function of decreasing the neuropathy [3] and shows an anti-inflammatory effect [1]. It also has antispasmodic effect on skeletal muscle [8], reduces the serum uric acid levels, and increases the sympathetic activities [9]. However, although JYGCT has been used as an herbal remedy to regulate the GI tract [11,12], its regulation of GI motility has not yet been reported. In this study, we demonstrated that JYGCT modulated the pacemaker potential in the ICC. Therefore, it can be suggested that JYGCT may regulate GI motility via its effects on the ICC pacemaker potential. In a forthcoming study, we plan to investigate the effects of JYGCT's components, Jakyak and Gamcho, on the pacemaker potential in the ICC.
Ion channels are important in the physiological control of GI motility [26]. Among the various ion channels, KATP channels are enriched at various locations in the GI tissues [27,28]. KATP channels in the ICC are composed of SUR2B and Kir 6.2 in the small intestine and SUR2B and Kir 6.1 in the colon [29]. Previous studies have shown that KATP channels exist in cultured murine small intestinal ICC and reduce GI motility by causing relaxation of the smooth muscles; this makes them potential candidates in the treatment of GI motor diseases [30]. In addition, the TRPV1 channels are expressed throughout the alimentary canal where they play important roles in GI motility through the ICC [18]. Various studies show that the TRPV1 channels are involved in the treatment of gastric esophageal reflux disease and GI pain hypersensitivity in the GI tract [18,31]. In this study, JYGCT inhibited the pacemaker potential in the ICC via KATP (Figure 2) and the TRPV1 (Figure 3). Therefore, we believe that KATP and the TRPV1 channels may get involved in the JYGCT-induced inhibition of pacemaker potential. Next, we intend to study the specific processes and molecular control strategies of KATP and the TRPV1 channels in the ICC. The intracellular regulation of Ca2+ flux has a role in the pacemaker activity in the ICC [24,32]. Ca2+ flux regulation is important for generating the pacemaker activity in the ICC [33]. In the present study, spontaneous [Ca2+]i responses were inhibited by JYGCT ( Figure 5). Therefore, we concluded that JYGCT inhibited the pacemaker potential and probably regulates the pacemaker activity through intracellular Ca2+ modulations. In addition, three cell surface proteins, TRPM7, c-Kit, and TMEM16A, are used for identifying the ICCs activation [15,[22][23][24]. In this study, JYGCT increased the ITR and the expression of TMEM16A was only increased by JYGCT, but the expression of c-Kit and TRPM7 was not ( Figure 5A,B). In addition, we looked at changes in GI hormones. GI hormonal changes can alter GI motility [34,35]. The roles of GI hormones are diverse and extensive, and although not fully understood yet, they are known to have a significant impact on the physiological functions of the GI tract [36]. In particular, it is known that hormones are responsible for regulating the process of food being digested and sent down through the GI tract [37]. These hormones are believed to regulate the functions of the GI tract as well as the bile and pancreas [38]. GI hormones are thought to have many unexplained functions and are attracting attention as a new research field in the future. Currently, studies on how these GI hormones act when certain GI diseases occur are being actively conducted, and, in particular, the use of hormones as therapeutic agents for GI motility diseases in food digestion is being attempted. In the present study, changes in four representative hormones MTL, SP, SS, and VIP were investigated. By JYGCT, MTL and SP hormone levels increased, but SS and VIP hormone levels did not ( Figure 5C). Therefore, it is thought that JYGCT-induced ITR increase is induced by the increase of TMEM16A protein and MTL and SP hormones. In this experiment, ICR mice that are good for observing the GI tract were used for the study of ICC because they are large in size, which are generally used for GI-related experiments. However, in the IBS-related study, C57/BL6 mice, which are mainly used for observing biochemical changes given relatively gentle and specific conditions, were used as black mice. We do not think that there is any particular difference, but the experiment was conducted using commonly used animal species.
Abnormalities and reductions in ICC are often found in GI diseases [17]. A recent study reports that JYGCT protects the ICC in the sphincter of Oddi (SO) and regulates the movement of SO [39]. In addition, the most important IBS mechanisms are GI dysmotility, and, therefore, ICC may be involved in the pathogenesis of IBS [40,41]. In this study, JYGCT restored colon length, weight, and stool status to normal in zymosan-induced IBS-like symptoms ( Figure 6). However, research on the association between GI motor disease and the ICC is still underway. Therefore, traditional medicine, which has fewer side effects due to its naturalistic approach, is a very attractive alternative [42,43]. Currently, there is a keen interest in treating GI tract diseases using natural medicine; accordingly, natural substances have the potential to reveal the mechanisms of action of pacemaker potential generation in the ICC and assist in the development of new drugs.

Conclusions
The present study shows that JYGCT inhibits the pacemaker potential of the ICC via KATP, the TRPV1 channels, the cAMP pathway, and intracellular Ca2+ regulation, increases ITR and alleviates IBS, and indicates that herbal medicine can be used for GI motor control. This study is on the GI tract efficacy of JYGCT in mice, showing the potential for use in GI diseases in mice. However, studies on the efficacy of JYGCT in the human GI tract have not yet been conducted. It is thought that further studies on the efficacy of JYGCT in humans are needed in the future.