Effects of Chaihu-Shugan-San on Small Intestinal Interstitial Cells of Cajal in Mice

Abstract

Chaihu-Shugan-San (CSS) has been widely used as an alternative treatment for gastrointestinal (GI) diseases in East Asia. Interstitial cells of Cajal (ICCs) are pacemakers in the GI tract. In the present study, we examined the action of CSS on pacemaker potentials in cultured ICCs from the mouse small intestine in vitro and on GI motility in vivo. We used the electrophysiological methods to measure the pacemaker potentials in ICCs. GI motility was investigated by measuring intestinal transit rates (ITR). CSS inhibited the pacemaker potentials in a dose-dependent manner. The capsazepine did not block the effect of CSS. However, the effects of CSS were blocked by glibenclamide. In addition, N^G-nitro-L-arginine methyl ester (L-NAME) also blocked the CSS-induced effects. Pretreatment with SQ-22536 or with KT-5720 did not suppress the effects of CSS; however, pretreatment with ODQ or KT-5823 did. Furthermore, CSS significantly suppressed murine ITR enhancement by neostigmine in vivo. These results suggest that CSS exerts inhibitory effects on the pacemaker potentials of ICCs via nitric oxide (NO)/cGMP and ATP-sensitive K⁺ channel dependent and transient receptor potential vanilloid 1 (TRPV1) channel independent pathways. Accordingly, CSS could provide the basis for the development of new treatments for GI motility dysfunction.

INTRODUCTION

Traditional medicines are widely prescribed in many countries and improves the healing power of the body and helps the body recover to its natural state.¹⁾ Many recent studies have indicated that herbal preparations and their extracts have favorable effects on the treatment of various diseases.²⁾ Among various diseases, an estimated 51% of patients have gastrointestinal (GI) disorders and 10% of alternative medicines are being used for digestive symptoms.³⁾ These GI conditions have been reduced the quality of human life and increased the cost of maintaining health.⁴⁾ However, these various available drugs treatment for these diseases are chemical drugs and these chemical drugs have a large number of adverse effects.⁴⁾ Traditional medicine may be an alternative medicine with a naturalistic approach and be known to have fewer side effects on GI diseases. Therefore, currently, many people are interested in treating GI tract diseases by natural medicine.

Chaihu-Shugan-San (CSS; 柴胡疏肝散) is a traditional medicine consisting of seven herbs, Chuanxiong Rhizoma, Citri Reticulatae Pericarpium Viride, Bupleuri Radix, Cyperi Rhizoma, Aurantii Fructus Immaturus, Paeoniae Radix Alba and Glycyrrhizae Radix.⁵⁾ CSS has been widely used as an herbal prescription to treat various symptoms. CSS is commonly efficient in treating neurologic impairment and depression.^6–8) Also, CSS controls the inflammatory reaction in the liver by controlling the insulin response and regulates the phospholipids to liver damage caused by chronic stress.^9,10) In addition, CSS has an effect on nonalcoholic fatty liver disease with insulin resistance in rats.¹¹⁾ Furthermore, CSS has been used for centuries to improve some GI disorders that are similar to functional dyspepsia, gastric ulcers, diarrhea, and gastritis.^5,12,13)

Interstitial cells of Cajal (ICCs) are pacemaker cells in the GI tract and generate pacemaker potentials.^14,15) The damage and decrease in numbers of these are associated with many GI diseases.^16–18) Therefore, this study is of considerable importance to the study of GI function. Many neurotransmitters and hormones affect these ICCs and thus regulate GI motility.^17,18) To date, however, whether CSS has effects on ICCs and GI motility has not been clarified. Therefore, in this study, we examined the efficacy of CSS on the pacemaker potentials of ICCs in small intestine in vitro and the function of GI motion by measuring intestinal transit rates (ITRs) in vivo in mice.

MATERIALS AND METHODS

Chemical Profiling of CSS by Ultra Performance Liquid Chromatography (UPLC)

UPLC was conducted using a Waters ACQUITY™ ultra performance LC system (Milford, MA, U.S.A.). Waters ACQUITY™ photodiode array detector (PDA; Milford) and HPLC Column were used for Waters ACQUITY™ BEH C₁₈ column (1.7 µm, 2.1 × 100; Milford). The software was used to operate the system Empower (Milford). In addition, samples were extracted using an ultrasonic cleaner 8210R-DTH (Branson Company; San Jose, CA, U.S.A.). The reagents used for this experiment were methanol (Junsei, Tokyo, Japan), acetonitrile (JT-BAKER; Radnor, PA, U.S.A.), water (tertiary distilled water), and dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, U.S.A.). CSS (Chuanxiong Rhizoma 0.43 g, Citri Reticulatae Pericarpium Viride 0.48 g, Bupleuri Radix 0.26 g, Cyperi Rhizoma 0.4 g, Aurantii Fructus Immaturus 0.39 g, Paeoniae Radix Alba 0.34 g, Glycyrrhizae Radix 0.17 g; Lot number: 137H035) was donated from HANKOOKSHINYAK Pharmaceutical Co. LTD. (Nonsan, Republic of Korea). The dried extract powder of CSS was used in this present study. The components and indications of CSS are described in details in Table 1. The standard preparations of this experiment were obtained from Cayman Chemical (Ann Arbor, MI, U.S.A.) and Sigma-Aldrich. The right amount preparations of saikosaponin A (Sigma-Aldrich; Lot number: PHL89526-10 mg), poncirin (Sigma-Aldrich; Lot number: PHL82628-10 mg), naringin (Cayman Chemical; Lot number: 17923), glycyrrhizinic acid (Sigma-Aldrich; Lot number: PHL89217-20 mg), liquiritigenin (Sigma-Aldrich; Lot number: 78825), albiflorin (Sigma-Aldrich; Lot number: SMB00082-10 mg), paeoniflorin (Sigma-Aldrich; Lot number: P0038-25 mg) and hesperidin (Cayman Chemical; Lot number: 18646) were measured accurately and the standard compounds were melted by DMSO and methanol. After that, they were made from a standard undiluted solution which contain 1 µg per mL. In succession, the right amounts of standard undiluted solution were diluted with methanol to give 1, 5, or 10 ng per mL standard solutions. A standard curve determination coefficient (R₂) value of all standard materials was more than 0.999.

Table 1. Contents of Eight Marker Compounds of CSS by UPLC

Compound	Content (ppm)
Saiko saponin A	11.65 ± 0.39
Poncirin	0.57 ± 0.06
Paeoniflorin	294.01 ± 8.07
Naringin	33.81 ± 0.85
Glycyrrhizinic acid	99.15 ± 0.91
Liquiritigenin	1.96 ± 0.08
Albiflorin	172.55 ± 6.28
Hesperidin	109.13 ± 5.51

Values are expressed as the means ± standard deviation (S.D.) of three independent experiments. CSS was analyzed for its saikosaponin A, poncirin, paeoniflorin, naringin, glycyrrhizinic acid, liquiritigenin, albiflorin, and hesperidin content using UPLC. CSS: Chaihu-Shugan-San. UPLC: Ultra Performance Liquid Chromatography.

Quantitation of CSS Extract

The test liquid for quantitative analysis was prepared by homogeneously mixing the CSS with 30% MeOH. In succession, that sample was added on the 30% methanol to be contained 500 mg per mL. Finally, that sample was extracted in a microwave for one hour. This test liquid was filtered through a 0.2 µm pore size filter and then selected as the test liquid. During PDA, the following wavelengths were used (nm): saikosaponin A (203), poncirin and naringin (313), glycyrrhizinic acid (254), liquiritigenin (276), albiflorin and paeoniflorin (230), hesperidin (280). The mobile phase used for analysis was a mixture of acetonitrile and water containing 0.1% formic acids (FA; Sigma-Aldrich). The injection volume was 2 µL and the flow rate was 0.4 mL/min. Qualitative analyses were based on retention time and quantities were determined by the peak area method (Tables 2, 3).

Table 2. Mobile Phase Condition of UPLC

Time (minutes)	0.1% FA/water (%)	0.1% FA/acetonitrile (%)	Flow rate (mL/min)
0	98	2	0.40
1.0	98	2	0.40
2.0	90	10	0.40
3.0	85	15	0.40
4.0	70	30	0.40
6.0	60	40	0.40
8.0	50	50	0.40
9.0	20	80	0.40
10.0	10	90	0.40
12.0	0	100	0.40
14.0	98	2	0.40
16.0	98	2	0.40

FA: Formic acids. UPLC: Ultra Performance Liquid Chromatography.

Table 3. Retention Time of Reference Standards

Reference standard	Reference standard retention time (min)
Saikosaponin A	10.155
Poncirin	8.024
Paeoniflorin	5.432
Naringin	6.476
Glycyrrhizinic acid	9.611
Liquiritigenin	5.916
Albiflorin	5.193
Hesperidin	6.638

UPLC: Ultra Performance Liquid Chromatography.

Preparation of Cells and Cell Cultures

Animal experiments have complied with the rules of the animal experiment ethics committee of Pusan National University (No. PNU-2018-2110). Small intestine of ICR mice (3–7 d) were taken out and removed the mucous membrane. Small intestinal muscle was equilibrated with Ca²⁺-free Hank’s solution. Cells were isolated by enzyme such as collagenase (Worthington Biochemical, Lakewood, NJ, U.S.A.), bovine serum albumin (BSA; Sigma-Aldrich) and trypsin inhibitor (Sigma-Aldrich) and then cultured at 37°C in a 95% O₂/5% CO₂ incubator in smooth muscle growth medium (SMGM) (Clonetics, San Diego, CA, U.S.A.) supplemented with 2% antibiotics/antimycotics (Gibco, Grand Island, NY, U.S.A.) and murine stem cell factor (5 ng/mL; Sigma-Aldrich). All other methods were carried out in a generally well-known manner.^{14,15,17–19)}

Patch Clamp Experiments

Na⁺-Tyrode solution was used in bath and the pipette solution was KCl 140, MgCl₂ 5, K₂ATP 2.7, NaGTP 0.1, creatine phosphate disodium 2.5, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) 5, and ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) 0.1. Whole-cell patch clamp configuration techniques were conducted in a general method and results were analyzed using pClamp and Origin software (version 6.0, Microcal, U.S.A.).

In Vivo Intestine Motility Measurements

Male ICR mice (Samtako Bio Korea Co., Ltd., Osan, Republic of Korea) weighing 23–30 g were used to investigate the in vivo effects of CSS on the GI tract. After CSS administration, Evans Blue (5% (w/v), in distilled water (DW)) was administered through the mouth. After 30 min, animals were sacrificed and ITR was measured Evans Blue as the length past in intestine. ITR was measured the length of the entire length as a percentage of the length that it had passed.

Western Blot Analysis

The total proteins of homogenates were determined using with RIPA buffer containing protease inhibitor (Roche, Indianapolis, IN, U.S.A.) and phosphatase inhibitor cocktail (Calbiochem, Darmstadt, Germany). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis by using 6% polyacrylamide gels and transferred to a polyvinylidene difluoride (PVDF) membrane. After 1 h treatment with blocking buffer in Tris-buffered saline (TBS) containing 5% non-fat milk at room temperature, PVDF membranes were analyzed by anti-c-kit (eBioscience, San Diego, CA, U.S.A.), and anti β-actin (Santa Cruz Biotechnology, Dallas, TX, U.S.A.) antibodies, respectively. An enhanced chemiluminescence reagent kit (Advansta, Menlo Park, CA, U.S.A.) was used for detection. All other procedures were generally carried out in a way.¹⁷⁾

Drugs

All drugs were purchased from Sigma-Aldrich. Chemicals were dissolved in Na⁺-Tyrode solution to their final concentrations immediately before use.

Statistical Analysis

Results are expressed as the means ± standard error of the means (S.E.M.s). For multiple comparison analysis, we used one-way ANOVA with Bonferroni’s post hoc comparison. For statistical analyses, we used Prism 6.0 (GraphPad Software Inc., La Jolla, CA, U.S.A.) and Origin version 8.0 (OriginLab Corporation, Northampton, MA, U.S.A.). p Values of <0.05 were considered statistically significant. The n values reported in the text refer to the number of cells used in patch-clamp experiments.

RESULTS

Analysis of CSS

CSS was analyzed for its saikosaponin A, poncirin, paeoniflorin, naringin, glycyrrhizinic acid, liquiritigenin, albiflorin, and hesperidin content using UPLC. The concentrations of the eight compounds were calculated from a calibration curve of standards (Table 1, Fig. 1). The method validation confirmed its stability and reliability and resulted in consecutive separation of the eight major compounds in CSS.

Fig. 1. Ultra Performance Liquid Chromatography (UPLC) Chromatogram of the Eight Major Compounds Identified in Chaihu-Shugan-San (CSS)

(A) UPLC chromatogram of the commercial standard compounds. (B) UPLC chromatogram of the eight major compounds in CSS. The chromatograms were obtained at 203 nm (saikosaponin A), 230 nm (albiflorin and paeoniflorin), 254 nm (glycyrrhizinic acid), 276 nm (liquiritigenin), 280 nm (hesperidin) and 313 nm (poncirin and naringin).

CSS Decreases the Amplitude of Pacemaker Potentials in Cultured ICCs

ICCs had a mean resting membrane potential of −57.3 ± 0.8 mV and produced electrical pacemaker potentials (n = 42). The frequency of this pacemaker potential was 18.2 ± 1.8 cycles/min with an amplitude of 23.7 ± 0.8 mV (n = 42; Fig. 2). The addition of CSS (5–100 mg/mL) decreased the amplitude of the pacemaker potentials (Figs. 2A–2E), but the resting membrane potentials hyperpolarized only in CSS 50 and 100 mg/mL (Figs. 2D, 2E). The amplitudes were 21.1 ± 0.6 mV at 5 mg/mL CSS (n = 7), 11.2 ± 0.7 mV at 10 mg/mL CSS (n = 6), 2.6 ± 0.5 mV at 30 mg/mL (n = 5), 2.5 ± 0.6 mV at 50 mg/mL (n = 7) and 1.4 ± 0.4 mV at 100 mg/mL (n = 5) (Fig. 2F). The resting membrane potentials were −56.8 ± 1.1 mV at 5 mg/mL, −57.2 ± 0.7 mV at 10 mg/mL, −58.1 ± 0.8 mV at 30 mg/mL, −64.1 ± 0.6 mV at 50 mg/mL, and −68.8 ± 1.7 mV at 100 mg/mL (Fig. 2G). CSS is mainly composed of saikosaponin A, poncirin, naringin, glycyrrhizinic acid, liquiritigenin, albiflorin, paeoniflorin and hesperidin. Therefore, we examined the effects of these components on the pacemaker potentials of ICCs. Saikosaponin A depolarized pacemaker potential in a concentration-dependent manner (Fig. 3A). In the presence of saikosaponin A, the mean amplitudes were 1.1 ± 1.0 mV at 1 µM, 2.3 ± 1.1 mV at 3 µM, 5.6 ± 1.9 mV at 5 µM, 13.1 ± 1.8 mV at 10 µM and 26.2 ± 1.5 mV at 30 µM (EC₅₀ = 10.8 ± 2.1 µM; Fig. 3H, n = 5). Also, glycyrrhizinic acid (1–30 µM) inhibited pacemaker potentials and decreased their amplitudes in a concentration-dependent manner (EC₅₀ = 9.9 ± 1.4 µM).¹⁹⁾ Liquiritigenin decreased pacemaker potential amplitudes in a concentration-dependent manner (Fig. 3D). In the presence of liquiritigenin, the mean amplitudes were 24.1 ± 0.8 mV at 1 µM, 24.7 ± 1.1 mV at 10 µM, 23.5 ± 1.7 mV at 50 µM, 22.1 ± 2.5 mV at 100 µM, 12.2 ± 2.1 mV at 300 µM and 3.5 ± 1.3 mV at 500 µM (IC₅₀ = 303.4 ± 3.3 µM; Fig. 3I, n = 5). In addition, albiflorin also decreased pacemaker potential amplitudes in a concentration-dependent manner (Fig. 3E). In the presence of albiflorin, the mean amplitudes were 24.6 ± 0.1 mV at 1 µM, 25.1 ± 1.1 mV at 10 µM, 18.5 ± 2.7 mV at 50 µM, 1.5 ± 1.4 mV at 100 µM, and 1.1 ± 1.0 mV at 500 µM (IC₅₀ = 58.3 ± 1.5 µM; Fig. 3J, n = 5). Hesperidine depolarized pacemaker potential in a concentration-dependent manner (Fig. 3G). In the presence of hesperidine, the mean amplitudes were 2.5 ± 1.0 mV at 1 µM, 5.5 ± 1.3 mV at 5 µM, 13.9 ± 1.5 mV at 10 µM and 24.7 ± 2.3 mV at 30 µM (EC₅₀ = 9.8 ± 1.3 µM; Fig. 3K, n = 6). However, poncirin, naringin and paeoniflorin had no effects on pacemaker potential (Figs. 3B, 3C, 3F). These results showed that CSS decreased the amplitude of ICC pacemaker potentials in a dose-dependent manner mainly through liquiritigenin and albiflorin.

Fig. 2. The Effects of CSS on the Pacemaker Potentials of Cultured ICCs from Mouse Small Intestine

(A–E) Pacemaker potentials of interstitial cells of Cajal (ICCs) exposed to CSS (5–100 mg/mL). CSS decreased the amplitude of pacemaker potentials in a concentration-dependent manner. (F, G) Bar graph of the decrease of amplitude and the change of resting membrane potentials with CSS. Results are expressed as the mean ± standard error of the mean (S.E.M.). ** p < 0.01. CSS, Chaihu-Shugan-San. CTRL, control. RMP, resting membrane potentials.

Fig. 3. Effects of Saikosaponin A, Poncirin, Naringin, Liquiritigenin, Albiflorin, Paeoniflorin and Hesperidin, the Major Components of CSS, on the Pacemaker Potentials of Cultured ICCs from Mouse Small Intestine

(A) Saikosaponin A depolarized pacemaker potential. (B and C) Poncirin and naringin had no effects on pacemaker potential. (D and E) Liquiritigenin and albiflorin decreased pacemaker potential amplitudes. (F) Paeoniflorin had no effects on pacemaker potential. (G) Hesperidine depolarized pacemaker potential. (H) Concentration–response curve with various saikosaponin A concentrations represents mean ± S.E. (I) Concentration–response curve with various liquiritigenin concentrations represents mean ± S.E. (J) Concentration–response curve with various albiflorin concentrations represents mean ± S.E. (K) Concentration–response curve with various hesperidine concentrations represents mean ± S.E. CSS, Chaihu-Shugan-San. EC₅₀, Half maximal effective concentration. IC₅₀, Half maximal inhibitory concentration.

No Involvement of Transient Receptor Potential Vanilloid 1 (TRPV1) Receptor on CSS-Induced Effects on Pacemaker Potentials in Cultured ICCs

To check the involvement of various ion channels on CSS-induced effects on pacemaker potentials in cultured ICCs, we examined the relevance of TRPV1 channel.²⁰⁾ The inhibitory response of CSS (30 mg/mL) was not suppressed by the pretreatment of TRPV1 antagonist capsazepine (10 µM) (Fig. 4A). The amplitudes before and after the treatment of capsazepine with co-treatment of CSS were 23.7 ± 0.8 mV and 2.4 ± 0.5 mV (n = 6; Figs. 4A, 4B). The above results indicate that the TRPV1 channel is not involved in the response of the CSS.

Fig. 4. Effect of CSS in the Presence of Transient Receptor Potential of the Vanilloid Type 1 Receptor Antagonist, Capsazepine, on the Pacemaker Potentials of Cultured ICCs from Mouse Small Intestine

(A) CSS (30 mg/mL) that shows inhibitory efficacy by capsaicin (10 µM). (B) Bar graph of the decrease of amplitude with CSS and capsazepine. Results are expressed as the means ± S.E.M. ** p < 0.01. CSS, Chaihu-Shugan-San. CTRL, control.

Involvement of ATP Sensitive K⁺ Channels on CSS-Induced Effects on Pacemaker Potentials in Cultured ICCs

Various types of K⁺ channel blockers were used to check the involvement of K⁺ channels in CSS-induced responses. In the presence of Ca²⁺-activated K⁺ channel blocker tetraethylammonium chloride (TEA; 10 mM), CSS inhibited pacemaker potential (Fig. 5A). In addition, CSS also inhibited pacemaker potential when co treated with transient voltage-dependent K⁺ channel blocker 4-aminopyridine or Ca²⁺-activated K⁺ channel blocker apamin (Figs. 5B, 5C). In a previous study, we found that ATP sensitive K⁺ channels may be involved in the regulation of pacemaker potentials in cultured ICCs.^21,22) Therefore, we examined the effects of CSS on pacemaker potentials in the presence of an ATP sensitive K⁺ channel blocker glibenclamide. CSS had no effects on pacemaker potentials after glibenclamide pretreatment (Fig. 5D) and CSS (30 mg/mL) reduced the amplitude of the pacemaker potentials, while the addition of glibenclamide (10 µM) reversed these effects (Fig. 5E). A summary of the effects of CSS and K⁺ channel blockers on pacemaker potentials are provided in Fig. 5F. The results suggest that the inhibitory effects of CSS on pacemaker potentials in cultured ICCs are mediated by ATP sensitive K⁺ channels.

Fig. 5. The Effects of Various Types of K⁺ Channel Blockers, TEA, 4-Aminopyridine, Apamin, Glibenclamide and CSS on the Pacemaker Potentials of Cultured ICCs from Mouse Small Intestine

(A) TEA (Ca²⁺-activated K⁺ channel blocker, 10 mM), (B) 4-aminopyridine (transient voltage-dependent K⁺ channel blocker, 5 mM), or (C) apamin (Ca²⁺-activated K⁺ channel blocker, 1 mM) did not affect the CSS-induced effects. (D) Glibenclamide (an ATP sensitive K⁺ channel blocker) alone did not affect pacemaker potentials, and then, CSS did not inhibit pacemaker potentials. (E) The decreased amplitude of pacemaker potentials caused by CSS was prevented by glibenclamide (10 µM). (F) Bar graph of the amplitude with CSS, glibenclamide, TEA, 4-aminopyridine and apamin. Results are expressed as the means ± S.E.M. ** p < 0.01. CSS, Chaihu-Shugan-San. CTRL, control. TEA, tetraethylammonium chloride. 4-ami., 4-aminopyridine. Gliben., glibenclamide.

The Involvement of Nitric Oxide on CSS-Induced Effects on Pacemaker Potentials in Cultured ICCs

The effects of N^G-nitro-L-arginine methyl ester (L-NAME) were examined to investigate the possible regulation of pacemaker potentials by CSS. L-NAME (10 µM) was pretreated for 10 min before application of CSS (30 mg/mL). The inhibitory effects of CSS were blocked by pretreatment of L-NAME (Fig. 6A). In the presence of L-NAME, the amplitude by CSS was 23.7 ± 0.5 mV (n = 5; Fig. 6B). These results suggest that L-NAME had effects on pacemaker potentials.

Fig. 6. The Effects of CSS in the Presence of Nitric Oxide (NO) Synthase Inhibitor, L-NAME, on the Pacemaker Potentials of Cultured ICCs from the Mouse Small Intestine

(A) Pretreatment with L-NAME (10 µM) did not have any effect on normal pacemaker potentials and the inhibitory effect of CSS was not blocked, even after pretreatment with L-NAME. (B) Bar graph of the amplitude with CSS and L-NAME. Results are expressed as the mean ± S.E.M. CSS, Chaihu-Shugan-San. CTRL, control.

Involvement of Guanylate Cyclase and Protein Kinase G (PKG) on CSS-Induced Effects on Pacemaker Potentials in Cultured ICCs

Adenylate cyclase inhibitor (SQ-22536) and guanylate cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one; ODQ) were used to determine the relevance of CSS-induced response to cyclic nucleotide dependent pathway. In the presence of SQ-22536 (100 µM), CSS still inhibited pacemaker potentials (Fig. 7A). However, ODQ (100 µM) suppressed CSS-induced pacemaker potentials inhibition (Fig. 7B). In the presence of SQ-22536, the mean amplitude of CSS-induced pacemaker potentials was 2.4 ± 0.5 mV (n = 5; Fig. 7E) and the ODQ corresponding value was 23.5 ± 1.3 mV (n = 6; Fig. 7E). In addition, in the presence of KT-5720 (a protein kinase A (PKA) inhibitor; 1 µM), CSS inhibited pacemaker potentials (Fig. 7C); however, preincubation with KT-5823 (a protein kinase G (PKG) inhibitor; 1 µM) suppressed CSS-induced pacemaker potentials inhibition (Fig. 7D). These results suggest that cGMP and PKG may play a vital role in CSS-induced responses.

Fig. 7. The Effects of CSS on the Relevance of Cyclic Nucleotide Dependent Pathway on the Pacemaker Potentials of Cultured ICCs from the Mouse Small Intestine

(A) SQ-22536 (an adenylate cyclase inhibitor, 100 µM) had no effect on pacemaker potentials inhibition by CSS. (B) ODQ (a guanylate cyclase inhibitor, 100 µM) blocked pacemaker potentials inhibition by CSS. (C) KT-5720 (a protein kinase A inhibitor, 1 µM) had no effect on pacemaker potential inhibition by CSS. (D) KT-5823 (a protein kinase G inhibitor, 1 µM) blocked these inhibitory effects of CSS. (E) Bar graph of the amplitude with CSS, SQ-22536, ODQ, KT-5720, and KT-5823. Results are expressed as the mean ± S.E.M. ** p < 0.01. CSS, Chaihu-Shugan-San. CTRL, control. SQ., SQ-22536.

CSS Inhibited Neostigmine-Induced Intestinal Hyperactivity

Next, we looked at the effect of CSS on ITRs in vivo. Previous researches have suggested that neostigmine increases intestinal motility.^23,24) In neostigmine-administered mice, application of 0.1 g/kg or 1 g/kg CSS reduced the increase in ITR caused by neostigmine (Fig. 8).

Fig. 8. The Effect of CSS on Intestinal Motility in Vivo

Neostigmine increases intestinal motility. CSS at 0.1 g/kg or 1 g/kg inhibited ITR increased by neostigmine. Results are expressed as the mean ± S.E.M. ** p < 0.01. CSS, Chaihu-Shugan-San. CTRL, control.

Administration of CSS Treatment Decreased Protein Expression of c-Kit

c-Kit is a transmembrane protein that represents the quantity and density of these ICCs.²⁵⁾ After treatment with neostigmine and CSS, the protein expression levels of c-Kit were checked by Western blot method. Western blot analysis results showed that the degree of expression levels of this were considerably higher after neostigmine treatment (Fig. 9A). The increased c-Kit expression level in neostigmine treatment mice significantly decreased by 30.7% after treating the mice with CSS (p < 0.05) (Fig. 9B).

Fig. 9. CSS Treatment Decreased Protein Expression of the Quantity and Density Biomarker of ICCs, c-Kit

(A) The protein expression of c-Kit was determined by using Western blot analyses. (B) The protein c-kit expression levels are presented as band densities relative to CTRL. Results are expressed as the mean ± S.E.M. * p < 0.05. ** p < 0.01. CSS, Chaihu-Shugan-San. CTRL, control.

DISCUSSION

In this study, we looked the efficacy of CSS on pacemaker potentials and related mechanisms in cultured ICCs from the mouse small intestine Our observations suggest that CSS inhibits the pacemaker potentials of ICCs via NO/cGMP and ATP-sensitive K⁺ channel dependent and TRPV1 independent pathways. These findings show that CSS offers a basis for the basis for the development of new treatments for GI motility dysfunction.

In China, an ancient traditional Chinese medicine (TCM) formula called CSS is efficiently applied to the treatment of various diseases; it is used for its neurologic impairment and depression treatment, anti-inflammatory with insulin signaling improvement, liver toxicity recovery effects, and nonalcoholic fatty liver regeneration effects, as well as to treat GI disorders with functional dyspepsia, gastric ulcers, diarrhea, and gastritis.^6–11,13) Chronic stress can affect neuroendocrine and behavior and cause depression; however, administration of CSS under these conditions has been shown to regulate hypothalamic-pituitary-adrenocortical systems and exert anti-depressive effects in rats.²⁶⁾ Additionally, CSS was traditionally used to treat chronic fatty liver diseases.⁷⁾ Many studies have demonstrated that CSS protects against insulin resistance and lipid peroxidation.^11,27) Some studies have also shown that it could cure a variety of GI diseases.^5,12,13) Moreover, CSS exerts prokinetic properties on the GI tract.⁵⁾ Additionally, CSS markedly accelerated gastric emptying (GE) and intestinal transit (IT) and significantly promoted ileum peristalsis in rats.⁵⁾ However, the effects of CSS on ICCs have not been previously investigated.

ICCs act as gut pacemaker cells and coordinate peristaltic movements.^14,15,17,18) In addition, hormones and neurotransmitters can modulate ICCs activity to influence gut motility.^28,29) The results of the present study showed CSS decreased the amplitude of pacemaker potentials in a concentration-dependent manner in ICCs (Fig. 2) mainly through liquiritigenin and albiflorin (Fig. 3), and that these effects were mediated not in TRPV1 channels (Fig. 4), but in ATP-sensitive K⁺ channels (Fig. 5). Moreover, NO/cGMP pathways were involved in the effects of CSS (Figs. 6, 7). The TRPV1 channel is also called the capsaicin receptor, which stimulates the TRPV1 channel and generates various reactions. Immunohistochemical studies have shown that there are many TRPV1 channels in the GI tract.^30,31) Therefore, this TRPV1 channel has become the most important pharmacological targets in GI studies, and capsaicin has been used to activate this TRPV1 channel in the GI tract.²⁰⁾ In this study, the inhibitory effect of GSS on pacemaker potentials was similar to that of capsaicin²⁰⁾ (Fig. 2). Therefore, we investigated the involvement of TRPV1 channels on CSS-induced effects on pacemaker potentials in cultured ICCs. However, similar to the capsaicin-induced effects,²⁰⁾ TRPV1 channels were not involved in CSS-induced effects on pacemaker potentials in ICCs (Fig. 4).

The K⁺ channel in smooth muscle has the function of regulating cell membrane potential and cell excitability. ATP-sensitive K⁺ channels stabilize the resting membrane potential and causes hyperpolarization of the cell membrane and reduces electrical excitability.³²⁾ ATP-sensitive K⁺ channels have also been reported in GI smooth muscle cells, and act as targets of neurotransmitters and peptides.^33,34) In this study, CSS acted as neurotransmitters and peptides in ICCs and regulated the pacemaker potentials by modulating the ATP-sensitive K⁺ channels (Fig. 5). Therefore, we think that CSS acts on ATP-sensitive K⁺ channels of the intestinal ICCs. GI motility patterns are highly integrated and require coordination with smooth muscle cells, neurons, endocrines and immune cells.²⁵⁾ Therefore, further investigations are needed to investigate the effects of CSS on other cells in the GI tract.

In this study, CSS exerts inhibitory effects on the pacemaker potentials of ICCs via NO/cGMP and ATP-sensitive K⁺ channel dependent pathways. Based on the findings described in this, we propose the following model of the effects of CSS in ICCs (Fig. 10). CSS is composed of saikosaponin A, poncirin, naringin, glycyrrhizinic acid, liquiritigenin, albiflorin, paeoniflorin and hesperidin. These eight components showed generally the reduction of NO. Saikosaponin A or poncirin significantly inhibited the expression of inducible nitric-oxide synthase (iNOS) and finally resulted in the reduction of NO.^35–38) Naringin could regulate the glutamate-nitric oxide cGMP pathway and enhanced the glutamine synthetase (GS) activity in hyperammonemic rats with neurotoxicity.³⁹⁾ Also, in rats with high blood pressure caused by glycyrrhizic acid, the renal protein expression of endothelial nitric oxide synthase (eNOS) and iNOS was increased and however, the cGMP production was not changed.⁴⁰⁾ Liquiritigenin or Albiflorin exerted anti-inflammatory effects, which results from the inhibition of nuclear factor (NF)-kappaB activation in macrophages, thereby decreasing production of iNOS.⁴¹⁾ Paeoniflorin, isolated from the root of Paeonia lactiflora pall, protected RAW264.7 macrophages from lipopolysaccharide (LPS)-induced cytotoxicity and genotoxicity with the decrease of NO and preserved endothelial cells from hypoxic damage by enhancing the production of NO.^42,43) Also it was involved in the cardioprotective effects by eNOS pathway.⁴⁴⁾ In pentylenetetrazole (PTZ)-kindled mice, hesperidin showed the neuroprotective effect through the NO–cGMP pathway.⁴⁵⁾ Also, hesperidin exerted antidepressant-like effects with the possible role of L-arginine–NO–cGMP pathway.^46,47) However, the NO–cGMP pathway effects of these eight components in GI tract have not been known. In addition, these eight components did not have any the electrophysiological effects on ATP sensitive K⁺ channels. Therefore, the effects of these eight components on GI tract and ATP sensitive K⁺ channel require experiments

Fig. 10. Hypothetical Schematic Signaling Pathway of CSS-Induced ICCs Hyperpolarization

CSS-induced membrane hyperpolarization seems to be mediated by a NO/cGMP dependent manner through ATP-sensitive K⁺ channels, not TRPV1 channels.

Taken together, our data suggest that CSS inhibits the amplitude of the pacemaker potentials of ICCs in a NO/cGMP dependent manner through ATP-sensitive K⁺ channels, not TRPV1 channels. In addition, CSS also suppressed ITR increases caused by neostigmine in vivo. Further findings are needed to identify CSS as a basis for the development of new treatments for GI motility dysfunction.

Acknowledgments

This research was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea Government (MSIP) (No. NRF-2017R1A2B2003764).

Conflict of Interest

The authors declare no conflict of interest.

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