Effect of TongXie-YaoFang on Cl<sup><b>−</b></sup> and <svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-4.3443pt" id="M1" height="18.596pt" version="1.1" viewBox="-0.0657574 -14.2517 51.9931 18.596" width="51.9931pt"><g transform="matrix(.017,0,0,-0.017,0,0)"><path id="g121-70" d="M729 650H468V622C546 615 554 610 554 527V365H213V527C213 610 221 616 297 622V650H38V622C120 616 128 611 128 527V123C128 39 120 34 40 28V0H303V28C221 34 213 40 213 123V322H554V123C554 39 546 34 460 28V0H728V28C647 34 639 39 639 123V527C639 610 647 615 729 622V650Z"/></g><g transform="matrix(.017,0,0,-0.017,12.937,0)"><path id="g121-65" d="M614 175C564 76 510 21 408 21C256 21 146 149 146 336C146 488 235 629 402 629C510 629 570 586 597 480L626 488C620 541 614 582 606 638C578 643 510 665 429 665C206 665 44 527 44 316C44 157 153 -15 402 -15C474 -15 558 5 586 11C604 45 629 119 643 165L614 175Z"/></g><g transform="matrix(.017,0,0,-0.017,24.349,0)"><path id="g121-77" d="M381 665C170 665 44 498 44 318C44 125 188 -15 369 -15C552 -15 703 117 703 333C703 534 550 665 381 665ZM359 629C491 629 601 517 601 306C601 114 502 21 390 21C249 21 146 158 146 346S248 629 359 629Z"/></g><g transform="matrix(.012,0,0,-0.012,37.167,4.134)"><path id="g50-52" d="M290 377C321 398 342 415 358 430C378 450 389 473 389 502C389 578 329 635 238 635H237C184 635 137 610 109 578L64 515L88 493C112 529 154 573 208 573S303 542 303 482C303 409 233 370 141 341L149 308C165 313 190 319 215 319C272 319 341 283 341 193C342 98 292 43 222 43C163 43 122 72 96 94C88 101 79 100 70 94C61 87 47 73 46 60C44 47 48 37 62 23C76 10 118 -12 165 -12C238 -12 430 62 430 223C430 297 379 359 290 375V377Z"/></g><g transform="matrix(.012,0,0,-0.012,43.745,-7.578)"><path id="g54-33" d="M556 236V289H56V236H556Z"/></g></svg> Transport in Diarrhea-Predominant Irritable Bowel Syndrome Rats

Lu, Xiaofang; Zhang, Shengsheng; Yang, Cheng; Wang, Zhengfang; Zhao, Luqing; Wu, Zhenyu; Xie, Jing

doi:https://doi.org/10.1155/2016/7954982

Evidence-Based Complementary and Alternative Medicine

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Abstract Introduction Materials and Methods Results Discussion Acknowledgments References Copyright Related Articles

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Complementary and Alternative Therapies for Functional Gastrointestinal Diseases 2016

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Research Article | Open Access

Volume 2016 | Article ID 7954982 | https://doi.org/10.1155/2016/7954982

Effect of TongXie-YaoFang on Cl⁻ and Transport in Diarrhea-Predominant Irritable Bowel Syndrome Rats

Xiaofang Lu,¹Shengsheng Zhang,¹Cheng Yang,¹Zhengfang Wang,¹Luqing Zhao,¹Zhenyu Wu,¹and Jing Xie¹

Academic Editor: Jieyun Yin

Received15 Feb 2016

Revised05 May 2016

Accepted10 May 2016

Published14 Jun 2016

Abstract

TongXie-YaoFang (TXYF) can effectively alleviate the symptoms of diarrhea-predominant irritable bowel syndrome (D-IBS) patients. However, the curative mechanism has not been fully clarified. The study was designed to investigate the effect of TXYF on the colonic ion transport induced by serotonin (5-HT) in D-IBS rats. A method of multiple stress (neonatal maternal separation (NMS) combined with restraint stress (RS)) was used to induce the D-IBS model. The model rats were randomly divided into two groups: NMS + RS group and TXYF-formula group, and the normal control (no handling) rats were classified as NH group. In the NMS + RS group, the change of short-circuit current () induced by 5-HT was lower than that in the NH and TXYF-formula groups. After removing of the extracellular Cl⁻ or or basolateral Na⁺ or blocking the cystic fibrosis transmembrane conductance regulator (CFTR), Na⁺-K⁺-2Cl⁻ cotransporter (NKCC), cotransporter, exchanger, K⁺ channel, or Na⁺/K⁺-ATPase, respectively, there was no difference in 5-HT-induced among the three groups. These data suggest that TXYF can regulate 5-HT-induced Cl⁻ and secretion, possibly mediated by the combined action of CFTR, NKCC, Na⁺- cotransporter, exchanger, K⁺ channel, and Na⁺/K⁺-ATPase.

1. Introduction

Diarrhea-predominant irritable bowel syndrome (D-IBS) is a chronic functional gastrointestinal disease, which is characterized by abdominal pain/discomfort and diarrhea without the demonstrable pathological evidence according to Rome III criteria [1]. As one of the most frequent subtypes of IBS patients, the D-IBS patients have lower quality of life than the other subtypes [2, 3]. However, as a multifactorial disease, the pathogenesis of D-IBS is complex and has not been fully understood as yet. Hence, the therapeutic effect has not been satisfactory for patients, although multiple medications have been developed as the optional therapies including loperamide, bile acid sequestrants, antispasmodics, tricyclic antidepressants, and rifaximin [4–9]. Currently, a series of randomized controlled trials have shown that Traditional Chinese Medicine (TCM) such as the representative formula TongXie-YaoFang (TXYF) can significantly alleviate the symptoms and improve the quality of life in D-IBS patients [10, 11]. However, the mechanism of the therapeutic effect has not been known, which limits the application of TXYF on a larger scale.

Serotonin (5-HT) is an important monoamine neurotransmitter in the brain-gut axis, which makes a great contribution to the onset and development of D-IBS. The hyperactivity of 5-HT has been found in D-IBS patients, which is recognized to be related with the disturbed visceral sensitivity, epithelium secretion, and smooth muscle contraction [12–14]. In the physiological studies, it has been shown that 5-HT as a potent secretagogue produces a significant effect on the intestinal ion transport including Cl⁻ or secretion via multiple ion channels, cotransporters, and exchangers such as the cystic fibrosis transmembrane conductance regulator (CFTR), Na⁺-K⁺-2Cl⁻ cotransporter (NKCC), Na⁺- cotransporter, Cl⁻/ exchanger, or K⁺ channel, all of which are related to the creation of osmotic pressure for dragging fluid into the enteric cavity [15, 16]. At present, much work has confirmed the changes of 5-HT-induced intestinal ion transport under stress [17] or in diarrheal diseases [18]. However, in D-IBS as a disease which is closely related to chronic stress and could be manifested by the symptom of diarrhea [19], it still remains unclear whether the altered ion transport induced by 5-HT is present. Further whether TXYF-formula can work on the ion transport also needs to be explored. Hence, the aims of this study were to investigate the effect of TXYF on 5-HT-induced ion transport in D-IBS rats and meanwhile explore the related mechanism.

2. Materials and Methods

2.1. Animals

Sprague-Dawley male rats (postnatal day 1) were obtained from Vital River Laboratories Animal Technology Co., Ltd. (Beijing, China), and kept in Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine. All the rats were housed under a 12-hour light/dark cycle (lights on at 8:00 a.m.) with a standard temperature (21–23°C) and humidity (50% ± 5%) and had access to food and water ad libitum. In this study, female rats were excluded to eliminate the possible effects of estrogen or the other female hormones on the secretory and sensory responses in the intestine [20]. All manipulations were performed between 8:00 a.m. and 11:00 a.m. every day to minimize the influence of circadian rhythms. On postnatal day 22, all the litters were weaned and kept in the individual cages with 3-4 pups per cage.

The study was performed according to the institutional ethical guidelines and conformed to the requirements of the Institutional Animal Care and Use Committee of Beijing University of Chinese Medicine. All animal care and experimental procedures were approved by the Animal Ethics Committee of Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine.

2.2. Neonatal Maternal Separation (NMS) Combined with Restraint Stress (RS)

On postnatal days 2–21, the neonatal maternal separation was conducted as previously described [21, 22]: the NMS + RS litters were removed from their home cages and separated from their maternal rats for 3 hours each day. During the period of separation, the litters stayed alone in the compartments, where the temperature was maintained at 23 ± 0.5°C in a thermally regulated facility. The litters were returned to their home cages immediately after separation. The normal control litters remained in their home cages with their maternal rats all the time.

On postnatal days 50–59, the restraint stress was performed as follows [23, 24]: the NMS + RS rats were placed in a transparent plastic restraint cylinder (4 × 4 × 18 cm³) for 3 hours, in which they could move forward and backward but could not turn around.

2.3. TXYF Composition and Administration

TXYF-formula was composed of the following traditional Chinese herbal medicines as recorded by the “Formulas of Chinese Medicine” [25]: Baizhu (Rhizoma Atractylodis Macrocephalae) 93.75 g, Baishao (Radix Paeoniae Alba) 62.5 g, Chenpi (Pericarpium Citri Reticulatae) 46.875 g, and Fangfeng (Radix Saposhnikoviae) 31.25 g, which were composed in 6 : 4 : 3 : 2 proportion. All raw materials in the formula were examined according to the quality control criteria in the Chinese Pharmacopoeia [26]. It was manufactured by the Preparation Room for TCM of the Beijing Hospital of Traditional Chinese Medicine.

The NMS + RS rats were divided into two groups (NMS + RS group and TXYF-formula group), and the normal control (no handling) rats were classified as NH group. From postnatal day 60, the rats in the TXYF-formula group were given intragastric administration with TXYF-formula (4.92 g/100 g/d). And the NH group and NMS + RS group were given distilled water. The delivery volumes in each group were always 2 mL/100 g/d, serving twice a day, for 14 consecutive days.

2.4. Reagents and Apparatuses

The preparation of Krebs-Henseleit solution (K-HS, in mmol/L) was as follows: NaCl 117, KCl 4.7, MgCl₂ 1.2, NaHCO₃ 24.8, KH₂PO₄ 1.2, CaCl₂ 2.56, and glucose 11.1; K-HS without Cl⁻: NaCl, KCl, MgCl₂, and CaCl₂ were replaced by sodium gluconate, potassium gluconate, magnesium gluconate, and calcium gluconate, respectively; K-HS without : NaHCO₃ was replaced by NaCl and the solution was buffered with 10 mmol/L HEPES-free acid, bubbled with 100% O₂; K-HS without Cl⁻ and : sodium gluconate took the place of NaCl and NaHCO₃ and potassium gluconate, magnesium gluconate, and calcium gluconate took the place of KCl, MgCl₂, and CaCl₂, respectively, with being buffered with 10 mmol/L HEPES and gassed with 100% O₂; K-HS without Na⁺: N-methyl-D-glucamine chloride (NMDGCl) and choline HCO₃ took the place of NaCl and NaHCO₃, respectively. If needed, the osmolality was adjusted to 290 mOsmol/L with D-mannitol.

The following were used: 5-HT, batch number: 1001156278; indomethacin, batch number: 1001087688; N-phenylanthranilic acid (DPC), batch number: 101078880; CFTR(inh)-172, batch number: 6311; glibenclamide, batch number: 1001068037; 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS), batch number: 1001339605; bumetanide, batch number: 101016760; amiloride, batch number: 101093389; 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), batch number: 1001208418; BaCl₂, batch number: 1398; ouabain, batch number: 101354363. All reagents were purchased from Sigma-AldrichCo.

A multichannel voltage-current clamp (VCC MC6) was purchased from Physiologic Instruments Corporation; the bridge amplifier (ML228) and the recording and analysis system for the physiological data (Power Lab) were purchased from AD Instruments Corporation.

2.5. Tissue Preparation

After treatment, all the rats in the three groups were killed with the method of cervical dislocation. The distal colon (6-7 cm from the anus) was removed quickly, cut longitudinally along the mesenteric border, and flushed with ice-cold K-HS. The tissue with the mucosal side down was fixed onto a petri dish with silica gel in the bottom. The petri dish was filled with ice-cold K-HS containing 95% oxygen and 5% carbon dioxide. The serosal, muscular, and submucous layers were carefully separated from the mucosal layer with fine tweezers under a microscope. Then the mucosal layer was cut into small sheets with areas of more than 0.5 cm² for further measurement. Two sheets could be obtained from one segment of the distal colon.

2.6. Ussing Chamber Experiment

The mucosal preparations from the distal colon were mounted in the Ussing chamber to measure the short-circuit current (). K-HS (5 mL) was injected into the two adjacent compartments of Ussing chamber, respectively, with circulating 95% oxygen and 5% carbon dioxide; the pH was maintained at 7.35–7.45 and the temperature was at 37°C. The tissues were left to incubate for 60 min to stabilize the electrical parameters. The voltage across the tissues was clamped to zero and the basal was measured. After adding the drugs or blockers to the basolateral or apical side for 30 min, 5-HT (10 μmol L⁻¹) was added to the basolateral side. were recorded continuously by a chart recorder. The changes in (Δ) were calculated to evaluate the ion transport by the difference value before and after 5-HT stimulation and were normalized as current per unit area of mucosa (μA/cm²). To measure the transmembrane resistance, electrical stimulation of 1 mV was applied and the resistance was calculated according to Ohm’s Law. Indomethacin (10 μmol L⁻¹) was routinely added to the basolateral side of the tissue to block the influence of endogenous prostaglandins in the experiment [27].

2.7. Statistical Analysis

The statistical analyses were performed by using SPSS 17.0 software (SPSS, Chicago, IL, USA) and each value was expressed as means ± SEM. All the original data in the study was distributed normally and conformed to homogeneity of variance. The differences among the three groups were analyzed using one-way analysis of variance (ANOVA) followed by least-significant difference (LSD) test to compare the differences between two groups. was considered statistically significant.

3. Results

3.1. Effect of TXYF on the Basic Electrophysiological Properties and 5-HT-Induced Δ

There were no significant differences in the basal , potential difference (PD), and transmembrane resistance (TR) among the three groups (, Figures 1(a)–1(c)). After addition of 5-HT (10 μmol L⁻¹) in the basolateral side, Δ in the NMS + RS group was reduced more than that in the NH group; after treatment with TXYF, Δ in the TXYF-formula group was significantly increased compared with the NMS + RS group (, Figure 1(d)).

(a)

(b)

(c)

(d)

3.2. Effects of TXYF on 5-HT-Induced Δ after Removal of Cl⁻, , or Na⁺ from K-HS

When the Cl⁻ and were removed from K-HS, respectively, there was no significant difference in 5-HT-induced Δ among the three groups; in the Cl⁻- and -free K-HS, no significant difference was found among the three groups as well (, Figure 2(a)). In addition, after removing the apical Na⁺ from K-HS, 5-HT-induced Δ was lower in the NMS + RS group compared with the NH group (, Figure 2(b)) and TXYF-formula group (, Figure 2(b)). In contrast, when the Na⁺-free K-HS was applied to the basolateral side of the colonic mucosa, no significant difference was found in 5-HT-induced Δ among the three groups (, Figure 2(c)).

(a)

(b)

(c)

3.3. Effects of TXYF on the 5-HT-Induced after Application of the Channel Blockers

Following the apical application of a nonselective Cl⁻ channel blocker, namely, DPC (1 mmol L⁻¹), or glibenclamide (1 mmol L⁻¹) or CFTR(inh)-172 (100 μmol L⁻¹), known as the cystic fibrosis transmembrane conductance regulator (CFTR) blockers, Δ induced by 5-HT had no significant differences among the three groups (, Figure 3(a)). In contrast, after a Ca²⁺-dependent Cl⁻ channel (CaCC) blocker, namely, DIDS (500 μmol L⁻¹), was added to the apical side, 5-HT-induced Δ remained lower in the NMS + RS group than that in the TXYF-formula group and NH group (, Figure 3(a)). Similarly, with the apical application of amiloride (100 μmol L⁻¹), an epithelial Na⁺ channel (ENaC) blocker, Δ induced by 5-HT was higher in NH and TXYF-formula groups than that in NMS + RS group (, Figure 3(b)). However, after basolateral application of the K⁺ channel inhibitor, namely, BaCl₂ (5 mmol L⁻¹), there was no statistical difference in 5-HT-induced Δ among the three groups (, Figure 3(c)).

(a)

(b)

(c)

3.4. Effects of TXYF on the 5-HT-Induced Δ after Application of the Cotransporter or Exchanger Inhibitors

In the presence of bumetanide (100 μmol L⁻¹) in the basolateral side, an inhibitor of Na⁺-K⁺-2Cl⁻ cotransporter (NKCC), there was no significant difference among the three groups in Δ induced by 5-HT ( > 0.05, Figure 4). After the basolateral addition of SITS (100 μmol L⁻¹), an inhibitor of Na⁺- cotransporter or Cl⁻/ exchanger, Δ induced by 5-HT had no significant difference among the three groups ( > 0.05, Figure 4). Similarly, after the basolateral side was treated with ouabain (1 mmol L⁻¹), a Na⁺/K⁺-ATPase inhibitor, Δ was statistically insignificant among the three groups ( > 0.05, Figure 4).

4. Discussion

In the present study, it was found that TXYF could regulate the secretory maladjustment of Cl⁻ and in D-IBS rats; when the colonic mucosa was pretreated with the inhibitors of CFTR, NKCC, Na⁺- cotransporter, Cl⁻/ exchanger, K⁺ channel, or Na⁺/K⁺-ATPase, no difference between TXYF and NMS + RS groups was found, indicating that TXYF could regulate the anion transport via these elements as CFTR, NKCC, Na⁺- cotransporter, Cl⁻/ exchanger, K⁺ channel, and Na⁺/K⁺-ATPase.

TXYF as a popular formula in TCM was first recorded in Dan Brook Heart Law, which is a comprehensive medical book edited in Yuan Dynasty. Four Chinese herbs are included in the prescription, which are Baizhu (three Taels), Baishao (two Taels), Chenpi (one and a half Taels), and Fangfeng (one Tael). TXYF is a representative formula for antidiarrhea and analgesia and recognized to be an alternative therapy for D-IBS. A clinical study performed by Pan et al. [10] showed that TXYF could significantly relieve the abdominal pain/discomfort and improved the stool property in D-IBS patients. A recent animal experiment also demonstrated that TXYF could increase the pain threshold and decrease the number of fecal pellets in IBS rats [28]. However, the curative mechanism has not been clear although much work has been done.

At present, although the pathogenesis of D-IBS has not been understood clearly, the dysfunction of the brain-gut axis has been recognized as one of the most important pathophysiologies, which is mainly regulated by a variety of brain-gut peptides including 5-HT [29]. In D-IBS patients, increased platelet-depleted plasma 5-HT concentration under fasting and fed conditions was found [13]. Besides, in an animal experiment, the 5-HT level in the distal colon, spinal cord, and hypothalamus rose in D-IBS model rats as well [30], which demonstrated that the upregulated level of 5-HT contributed to the onset of D-IBS. In addition to regulating intestinal motility and sensitivity, 5-HT as a potent intestinal secretagogue plays an important role in maintaining the balance of intestinal electrolyte and fluid, which is related to the occurrence of diarrhea [31]. In our study, when exogenous 5-HT was added to the basolateral side of colonic mucosa, increased in all the three groups. Besides, in NMS + RS group, a decline in Δ was elicited compared with the NH group. The result could be supported by another in vitro study conducted by Goldhill et al. [32], in which it was found that the wrap restraint stress rats displayed a reduced response to 5-HT. Further, after treatment with the TXYF-formula, the mucosal response to 5-HT was restored to the similar level to that observed in NH group, which showed that TXYF-formula was able to regulate the alteration of colonic secretion induced by 5-HT in D-IBS rats.

However, interestingly, the secretion induced by 5-HT in the colonic mucosa of the NMS + RS group was lower than that in the NH group and TXYF-formula group, which seemed contradictory to the hypersecretion of diarrhea. Consider the possible reason that may be attributed to the differences between in vivo and in vitro tests and related to increase of 5-HT level or desensitization of 5-HT receptors in the D-IBS rats. As in our previous study, the increased 5-HT level and decreased 5-HT₄ receptor expression in D-IBS rats have been demonstrated [33], but the specific mechanism needed further discussion.

It is well known that, under normal conditions, Cl⁻ transport is the key link in maintaining the balance of the intestinal electrolyte, which is secreted into the enteric cavity and generates the osmotic driving force for water [34]. The general process of Cl⁻ secretion has been elaborated by Frizzell and Hanrahan [35]: the Cl⁻ enters across the basolateral membrane mediated by NKCC and is secreted into the enteric cavity from the apical side via cAMP- and Ca²⁺-dependent Cl⁻ channels. In the present study, we found that TXYF-formula was able to regulate the secretion of Cl⁻ induced by 5-HT. The supporting evidence was displayed as follows: (1) in the normal K-HS, 5-HT-induced Δ in the NMS + RS group differed significantly from that in the TXYF-formula group, but when the extracellular Cl⁻ was removed, the difference disappeared; (2) after the apical application of CFTR blockers or basolateral application of NKCC inhibitor in the tissue, Δ had no statistical difference between the NMS + RS group and TXYF-formula group. Besides, these results also showed that CFTR and NKCC participated in the effect of TXYF on the colonic Cl⁻ transport. However, when the CaCC was blocked, the statistical difference between the NMS + RS group and TXYF-formula group still remained, which implied that TXYF-formula regulated 5-HT-induced Cl⁻ secretion via cAMP-dependent Cl⁻ channels instead of CaCC. The inference was supported by a physiological experiment, in which it was shown that CaCC had no effect on the 5-HT-induced Cl⁻ secretion in the rat colon [15].

In a recent experiment, it has been shown that secretion induced by 5-HT was present in the rat colon [16]. In our study, in addition to the regulation of Cl⁻ secretion, we found that TXYF also altered secretion in the colonic mucosa. When the extracellular was removed or Na⁺- cotransporter or Cl⁻/ exchanger was blocked, 5-HT-induced Δ did not differ between the NMS + RS group and TXYF-formula group. Besides, the same result was obtained when the extracellular Cl⁻ and were both removed. Thus, it was demonstrated that TXYF-formula could alter the colonic secretion by regulating the transport of both Cl⁻ and .

Besides, we further explored more possible regulatory mechanisms of TXYF on the anion secretion and found that, in addition to the specific channels, transporters, and exchangers, the transport of K⁺ and basolateral Na⁺ were involved in the secretion of Cl⁻ and , which was demonstrated by the following evidence. (1) The previous study has reported that the basolateral K⁺ conductance can maintain the electrical driving force for the anion secretion [35]. In our study, in the presence of the nonselective K⁺ channel blocker (BaCl₂), the 5-HT-induced Δ was similar between the NMS + RS group and TXYF-formula group, which suggested that the effect of TXYF on anion secretion might be related with the basolateral K⁺ transport. However, because at least two types of K⁺ channels had been found, the precise regulatory subtypes needed further studies. (2) In addition to the K⁺ transport, it was also found that the basolateral Na⁺ participated in the process, which was associated with the generation of electrochemical driving force for anion secretion [36]. As shown in our study, after removal of the basolateral Na⁺ or in the presence of the Na⁺-K⁺-ATPase inhibitor (ouabain), 5-HT-induced Δ did not differ between the TXYF-formula and NMS + RS groups, indicating that the regulation of TXYF on anion secretion was partly dependent on the basolateral Na⁺ transport.

Moreover, in our study, we also investigated the effect of TXYF on Na⁺ absorption. After removal of the apical Na⁺ or application of the epithelial Na⁺ channel blocker (amiloride), Δ still differed between the NMS + RS group and TXYF-formula group. Therefore, it can be seen that TXYF-formula had little effect on the Na⁺ absorption. That was probably because, in the physiological condition, the Na⁺ absorption played a tiny role in the 5-HT-induced secretory response in the rat colon, as reported by Ning et al. [15].

In conclusion, the present study has demonstrated that TXYF was able to regulate 5-HT-induced Cl⁻ and secretion in D-IBS-like rats, possibly mediated by the transport elements as CFTR, NKCC, Na⁺- cotransporter, Cl⁻/ exchanger, K⁺ channel, and Na⁺/K⁺-ATPase, at least in part.

Competing Interests

The authors declare that there are no competing interests regarding the publication of this paper.

Acknowledgments

This study was supported by grants from (1) the National Natural Science Foundation of China (no. 81473644); (2) the Clinical Medicine Development Project of Beijing Municipal Administration of Hospitals (no. ZYLX201411); (3) the Project of Health Technical Personnel of the Beijing Health System (no. 2011-2-13). The authors thank all members of the three projects above for their partnership and support.

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Copyright © 2016 Xiaofang Lu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Evidence-Based Complementary and Alternative Medicine

Complementary and Alternative Therapies for Functional Gastrointestinal Diseases 2016

Effect of TongXie-YaoFang on Cl− and Transport in Diarrhea-Predominant Irritable Bowel Syndrome Rats

Abstract

1. Introduction

2. Materials and Methods

2.1. Animals

2.2. Neonatal Maternal Separation (NMS) Combined with Restraint Stress (RS)

2.3. TXYF Composition and Administration

2.4. Reagents and Apparatuses

2.5. Tissue Preparation

2.6. Ussing Chamber Experiment

2.7. Statistical Analysis

3. Results

3.1. Effect of TXYF on the Basic Electrophysiological Properties and 5-HT-Induced Δ

3.2. Effects of TXYF on 5-HT-Induced Δ after Removal of Cl−, , or Na+ from K-HS

3.3. Effects of TXYF on the 5-HT-Induced after Application of the Channel Blockers

3.4. Effects of TXYF on the 5-HT-Induced Δ after Application of the Cotransporter or Exchanger Inhibitors

4. Discussion

Competing Interests

Acknowledgments

References

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Effect of TongXie-YaoFang on Cl⁻ and Transport in Diarrhea-Predominant Irritable Bowel Syndrome Rats

3.2. Effects of TXYF on 5-HT-Induced Δ after Removal of Cl⁻, , or Na⁺ from K-HS