# Rhubarb Tannins Extract Inhibits the Expression of Aquaporins 2 and 3 in Magnesium Sulphate-Induced Diarrhoea Model

Authors: Chunfang Liu; Yanfang Zheng; Wen Xu; Hui Wang; Na Lin
Journal: BioMed Research International
 (2014)

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## Abstract

Tannins, a group of major active components of Chinese rhubarb and widely distributed in nature, have a significant antidiarrhoeal activity. Aquaporins (AQPs) 2 and 3 play important roles in regulating water transfer during diarrhoea. The present study aims to determine the effect of the total tannins extract of rhubarb on aquaporins (AQPs) 2 and 3 in diarrhoea mice and HT-29 cells both induced by magnesium sulphate (MgSO4). Our results showed that rhubarb tannins extract (RTE) significantly decreased the faecal water content in colon and evaluation index of defecation of diarrhoea mice. Interestingly, RTE could markedly reduce the mRNA and protein expression levels of AQPs 2 and 3 in apical and lateral mucosal epithelial cells in the colons of diarrhoea mice and HT-29 cells both induced by MgSO4 in a dose-dependent manner. Furthermore, RTE suppressed the production of cyclic monophosphate- (cAMP-) dependent protein kinase A catalytic subunits α (PKA C-α) and phosphorylated cAMP response element-binding protein (p-CREB, Ser133) in MgSO4-induced HT-29 cells. Our data showed for the first time that RTE inhibit AQPs 2 and 3 expression in vivo and in vitro via downregulating PKA/p-CREB signal pathway, which accounts for the antidiarrhoeal effect of RTE.


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## Body

## 1. Introduction

Diarrhoea is the frequent passage of unformed, loose, or watery stools, usually three or more times in 24 hours [1, 2]. In the pathogenesis of diarrhea, transepithelial hypersecretion of fluid in the gastrointestinal (GI) tract and defects in water absorption in the colon are both important factors. The regulation of transepithelial fluid transport in the GI tract is based on ion transport and also on water transport by aquaporin- (AQP-) type water channels [3]. In particular, a change in expression of AQPs in the colon appears to be correlated with the severity of diarrhoea in animals and patients [4]. AQPs constitute a family of small integral membrane proteins that are selectively permeable to water and driven by osmotic gradients [5–8]. It has been reported that a defect or an increase in the expression and/or function of AQPs 2, 3, 4, and 8 underlies many kinds of diarrhoea [9–15]. Of these, extensive research has been conducted on AQPs 2 and 3, which are considered to play an important role in regulating water transfer in the colon during diarrhoea [9, 16, 17]. Noteworthily, increasing knowledge about the structure and function of AQPs led to new approaches using AQPs as drug targets [18, 19]. Thus, AQPs 2 and 3 are likely to prove central to the pathophysiology of diarrhoea and, ultimately, they could be a target for therapy in diarrhoea.Diarrhoea is a common cause of death in developing countries and the second most common cause of infant deaths worldwide. About four billion cases occur each year and 3-4 million individuals die as a result annually [20]. In many cases of diarrhoea, the treatments utilized are oral rehydration therapy and pharmacological intervention such as antibiotics. Although oral rehydration salt has greatly improved clinical outcome in cholera and other diarrheas, there remains significant mortality from infectious diarrheas, with recurrent major outbreaks. Antibiotics used as antidiarrheal drugs sometimes provoke adverse effects and microorganisms tend to develop resistance toward them [21]. In this regard, use of traditional medicines to combat the consequences of diarrhea has been emphasized by World Health Organization in its Diarrhoea Control Programme [22, 23]. It is therefore important to identify and evaluate available natural drugs as alternatives to current antidiarrheal drugs, which are not always free from adverse effects.Rhubarb, an old and well-known traditional Chinese herbal medicine, has been used as a folk remedy for gastrointestinal disease including diarrhea and constipation for over two thousand years in China. Today, it is already officially listed in Chinese, European, and Japanese Pharmacopoeia [24–26]. Rhubarb has many pharmacological activities including purgation [27], antidiarrhoeal effect [28], anti-inflammation [28], and immunoregulation [29]. On the other hand, rhubarb contains lots of compounds, mainly including anthraquinones (anthraquinone derivatives), tannins, polysaccharides, and other substances. Among these, tannins, widely distributed in nature and present in almost all plant foods and some beverages, are the major active compounds of rhubarb; moreover, they are often the active compounds of the other medicinal plants in which they occur [30, 31]. It is demonstrated that the antidiarrhoeal activity of many medicinal plants which are rich in tannins is related to the existence of tannins [2, 32]. In particular, the purgative activity of rhubarb extract could be significantly increased by reducing the content of tannins [33], and some processed products from crude rhubarb with decrease of purgative anthraquinones and relative increase of tannins even exhibited an antidiarrhoeal activity [34]. The first direct convincing evidence of antidiarrhoeal activity of rhubarb tannins is that successive administration of the total tannins extract of rhubarb produces the antidiarrhoeal effect on magnesium sulphate- (MgSO4-) and castor oil-induced diarrhoea mice by protein-precipitating reaction to the GI mucosa [35]. Besides, previous studies have demonstrated that tannins reduce significantly intraluminal fluid accumulation by the antisecretory activity and promoting reabsorption of water and decrease intestinal motility [36–38]. However, the effect of tannins on AQPs 2 and 3 remains unknown, which play important roles in regulating water transfer in the colon during diarrhoea. Therefore, we here investigated the effect of the total tannins extract of rhubarb on intestinal fluid accumulation and the expression of AQPs 2 and 3 in MgSO4-induced diarrhoea mouse model, and the results were further confirmed in HT-29 cells. In addition, the mechanism of the changes induced by rhubarb tannins in the expression of AQPs 2 and 3 was examined.


## 2. Materials and Methods

The study was approved by Research Ethics Committee of China Academy of Chinese Medical Sciences, in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All animals were treated in accordance with the guidelines and regulations for the use and care of animals of the Center for Laboratory Animal Care, China Academy of Chinese Medical Sciences.


### 2.1. Preparation of RTE

The dried root and rhizoma ofRheum palmatum, produced in Yushu county of Qinghai province of China, were purchased from the Yushu Pharmaceuticals Company and were identified by Professor Shilin Hu, a taxonomist at China Academy of Chinese Medical Sciences. A voucher specimen (Rh201005Z) was deposited in the institute. RTE was prepared according to the reported method with some modified steps [35]. 1 kg of rhubarb was powdered and extracted with 5 L of 60% ethanol for 30 min in a reflux condenser for three times and then was filtrated. The filtrate was merged and concentrated to 500 mL in vacuo in a rotary evaporator at 40°C. Then the concentrated solution was added into D-101 macroporous resin with 25% ethanol as the eluent. The solution was concentrated continually and then precipitated with 1% gelatin. The sediment was dissolved in acetone, recovered acetone, and dried in a vacuum oven as the total tannins extract of rhubarb. The total tannins extract was free of anthraquinones, according to the color reactions of anthraquinones with magnesium acetate methanol solution and tannins with ferric chloride solution. The yield of the total tannins from rhubarb was 3.40%. The total tannins extract of rhubarb was diluted with distilled water or cell culture medium to the proper concentration before usein vivo andin vitro experiments as follows.


### 2.2. High-Performance Liquid Chromatography (HPLC) Fingerprint of RTE

The authentic standards of gallic acid and epigallocatechin gallate were purchased from Jingchun Biological Technology Co. Ltd. (Shanghai, China). The standards of procyanidin-B1 and procyanidin-B2 were obtained from Tauto Biotech Co. Ltd. (Shanghai, China). Standards of catechin and epicatechin were supplied by National Institute for Food and Drug Control (Beijing, China). RTE and standard substance were dissolved in 50% methanol and then filtrated through 0.45μm filter membrane. HPLC was performed on a Shimadzu HPLC system (Shimadzu Corporation, Shimane, Japan) equipped with an LC-20AT binary pump, an SPD-M20A diode array detector, a CBM-20Alite system controller, an SIL-20A autosampler, a DGU-20A5 degasser, and a CTO-10ASvp column oven. An Ultimate XB-C18 column (150 × 4.6 mm, 5 μm particle size) was maintained at 30°C. Detection wave length was set at 280 nm. The mobile phase for fingerprint analysis consisted of solvent A (acetonitrile, Merck, NJ, USA) and solvent B (0.1% formic acid/water, v/v) at a flow rate of 0.8 mL/min. A gradient program was used as follows: 27–10% B at 0–25 min. The injected volume was 10 μL. Online UV spectra were recorded in the range of 190–800 nm. The HPLC fingerprint profile of RTE was established by optimizing the chromatographic separate conditions. The chromatogram of RTE at 280 nm could exhibit the whole chromatographic characteristic in Figure 1(a). By comparison of the retention time and online UV spectra of the reference standards, 6 compounds of tannins were identified as previously reported in rhubarb [39]. The structure of gallic acid, procyanidin-B1, catechin, procyanidin-B2, epicatechin, and epigallocatechin gallate were shown in Figure 1(b).HPLC profile (UV chromatograms at 280 nm) of rhubarb tannins extract (RTE). (a) HPLC fingerprint of RTE. (1)—RTE. (2)—standards. (b) Structure of identified components of RTE. (A)—gallic acid. (B)—procyanidin-B1. (C)—catechin. (D)—procyanidin-B2. (E)—epicatechin. (F)—epigallocatechin gallate.
(a)
(b)


### 2.3. Quantitative Analysis of RTE

The content of the RTE was determined using Shimadzu Visible Ultraviolet Spectrophotometer UV-1800 (Shimadzu Technologies) according to the method in Chinese Pharmacopoeia (Committee for the Pharmacopoeia of China, 2010) with gallic acid as reference substance. The mass of tannins in extracts was 55.69% equivalent of gallic acid.


### 2.4. Induction of MgSO4-Induced Diarrhoea Mouse Model and RTE Treatment

Thirty male ICR mice (18–22 g) were obtained from Laboratory Animal Center of Academy of Military Medical Sciences, Beijing, China (License No.: SCXK 2007-004). They were kept in a temperature controlled environment (22±2°C), 55±5% relative humidity with a 12 h : 12 h light-dark cycle and fed with standard chow, for at least one week before any manipulations. ICR mice were divided separately into 5 groups randomly with the equal number (n=6): the normal control group (Control), the MgSO4-induced diarrhoea model group (Vehicle), and the RTE- (125, 250, and 500 mg/kg, resp.) treated diarrhoea mice groups. Five groups of mice were orally administrated by syringe feeding with distilled water (20 mL/kg) or RTE daily for three days, respectively. Each mouse except for the normal group was orally administrated with MgSO4·7H2O (2 g/kg, Jinhuitaiya Chemical Industries, Tianjin, China) 1 h after the last administration to induce diarrhoea as previously reported [35]. The assay was repeated 3 times.


### 2.5. Fecal Water Content

Detailed procedures have been previously described [12]. Briefly, fecal samples from the mice were collected for up to 5 h after the administration of the MgSO4 solutions and were placed in silica gel followed by drying for 48 h in a desiccator. The fecal water content per gram of feces was calculated based on the difference between the wet and dry fecal weights. This was then used to calculate the percentage of water in the fecal samples. These percentages were averaged among six animals for each group and the assay was repeated 3 times.


### 2.6. Evacuation Index of Mice Defecation

The method as previously reported [35] was used to assess the defecation function of mice. A numerical score based on stool consistency was assigned: 1 = normal stool, 2 = semisolid stool, and 3 = watery stool. Each mouse received an evacuation index (EI) expressed according to the formula: EI = 1 × (number of normal stool) + 2 × (number of semi-solid stool) + 3 × (number of watery stool). The data were expressed as mean EI for each group (n=6). The assay was repeated 3 times.


### 2.7. Cell Culture

Human colon cancer HT-29 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences. HT-29 cells were maintained in sterile Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS, 100 U/mL penicillin, 100μg/mL streptomycin, and 2 mM Gln-glutamine at 37°C. Cells were plated on a 24-well plate, 96-well plate, or 100-mm dish at a density of 2 × 105 cells/cm2, incubated in a CO2 incubator at 37°C for 24 h, and then treated with compounds dissolved in culture medium. Experiments were done using cells that had previously been passaged five to fifteen times.


### 2.8. Cell Viability Assay

HT-29 cells were seeded in 96-well plates and incubated in serum free sterile DMEM (supplemented with 100 U/mL penicillin, 100μg/mL streptomycin, and 2 mM Gln-glutamine) for 24 h. Cells were then incubated with media containing MgSO4 (50 mM) and/or RTE (20, 40, and 80 μg/mL, resp.) for 24 h. After treatment, cells were washed twice with phosphate-buffered saline (PBS; pH 7.4), and then cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method using Cell Titer 96 Non-Radioactive Cell Proliferation Assay (Promega, Madison, USA) according to the manufacturer’s instructions. All absorbance at 570 nm was measured with a microplate reader. The experiments were carried out 3 times in triplicate measurements.


### 2.9. Immunolocalization

Mice were sacrificed by cervical dislocation under ether anesthesia at 2 h after the administration of the MgSO4 solutions, and the colons were removed. After washing the colons with PBS, the samples were fixed immediately for 3 h in 4% paraformaldehyde and embedded in paraffin and tissue sections (5 μm) of colon were prepared for immunofluorescence analysis.Forin vitro experiments, HT-29 cells were cultured in 24-well plates with medium containing MgSO4 and/or RTE for 24 h. The control was treated with the original medium. After washing with PBS twice, cells were fixed immediately for 15 min in 4% paraformaldehyde and prepared for immunofluorescence analysis. The experiments were done in triplicate for three times.Immunofluorescence analyses were carried out following the protocol of previous study [9, 40]. Tissue sections (5 μm) of colon and HT-29 cells were treated with 0.2% Triton in PBS prior to blocking with 5% normal goat serum (NGS) in TPBS-BSA (PBS containing 0.05% Tween-20 and 0.1% bovine serum albumin). The primary rabbit anti-AQP2 (1 : 50, Abcam, Cambridge, MA, UK) antibody or rabbit anti-AQP3 antibody (1 : 50, Abcam) in TPBS-BSA supplemented with 1% NGS was incubated on the tissue and cells overnight at 4°C. Then, the tissue and cells were washed extensively with the TPBS-BSA. After that, the sections and cells were exposed for 2 h at room temperature to a secondary goat anti-rabbit antibody conjugated with Alexa Fluor 488 (1 : 200, Zhongshanjinqiao, Beijing, China). Then, the tissue and cells were reacted with 4,6-diamidino-2-phenylindole (DAPI) solution (Beyotime, Jiangsu, China) in PBS at room temperature for 5 min, washed 3 more times with PBS, and then coverslipped with vectashield (Zhongshanjinqiao) and visualized using a Zeiss Lsm710 confocal microscope (Carl Zeiss AG, Oberkochen, German).


### 2.10. Preparation of Tissue and Cell Extraction for Western Blot Analysis

The colons were removed from mice. Large intestinal mucosa scraped with a slide glass was homogenized using dissecting buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, 8.5 mM leupeptin, and 1 mM phenylmethylsulfonyl fluoride; pH 7.2) on ice. The resulting suspension was centrifuged (800 ×g at 4°C for 15 min) and the supernatant was centrifuged (200,000 ×g at 4°C for 1 h). The precipitate was resuspended using dissecting buffer. Protein concentrations were measured by the Lowry method [41] using BSA as a standard. The protein expression levels of AQPs 2 and 3 in colons were determined.AQPs 2 and 3 were expressed in the plasma membrane and in intracellular vesicles. Thus, the crude fraction dominantly containing these fractions in HT-29 cells was prepared as previously reported [10]. Briefly, HT-29 cells were cultured in 100-mm dishes with media containing MgSO4 and/or RTE for further culture at 37°C for different times (3, 6, 12, and 24 h, resp.). The control was treated with the original medium. After washing with PBS, cells were recovered and suspended in dissecting buffer. The cell suspension was homogenized, and the homogenate was centrifuged (4000 ×g for 10 min). The supernatant was centrifuged (200,000 ×g for 1 h), and the resulting supernatant was discarded. Dissecting buffer was added to the pellets, and the suspension was homogenized using an ultrasonic homogenizer to make a crude fraction [42]. All procedures were carried out at 4°C.The phosphorylation of cyclic monophosphate (cAMP) response element-binding protein (CREB) occurred in the nucleus. Thus, the nuclear fraction, dominantly containing the nucleus, was prepared as previously reported [10], and the protein expression level of phosphorylated CREB (p-CREB) was determined. Briefly, isolated HT-29 cells were cultured with media containing MgSO4 and/or RTE for 24 h. After that, the cells were treated with extraction buffer (phosphosafe extraction reagent with 8.5 μM leupeptin and 1 mM phenylmethylsulfonyl fluoride). The suspension was left on ice for 5 min. After detaching and recovering cells with a cell scraper, the cells were then homogenized and centrifuged (4,000 ×g for 10 min). After the supernatant was discarded, extraction buffer was added to the pellets. The suspension was centrifuged (16,000 ×g for 30 min) to obtain the supernatant as a nuclear fraction [43, 44]. All procedures were carried out at 4°C.Meanwhile, HT-29 cells were treated with MgSO4 and/or RTE and then dissociated with lysis buffer (20 mM MOPS, 50 mM β-glycerophosphate, 50 mM sodium fluoride, 1 mM sodium vanadate, 5 mM EGTA, 2 mM EDTA, 1% NP-40, 1 mM DTT, 1 mM benzamidine, 1 mM PMSF, 10 μg/mL leupeptin, and 10 μg/mL aprotinin). After that, the cells were recovered and centrifuged (16,000 ×g for 15 min at 4°C). The supernatant was recovered, and the protein expression of cAMP-dependent protein kinase A (PKA) was determined by western blot.


### 2.11. Western Blot Analysis

The western blot protocol and semiquantitative analysis were carried out following the protocol of previous study [10]. The following primary antibodies were used: AQP2 (rabbit polyclonal antibody, dilution 1 : 200, Abcam), AQP3 (rabbit polyclonal antibody, dilution 1 : 200, Abcam) and PKA C-α (rabbit polyclonal antibody, dilution 1 : 1000, Cell Signaling, Boston, USA), p-CREB (ser133, rabbit polyclonal antibody, dilution 1 : 500, Thermo Fisher Scientific, Waltham, UK), and GAPDH (internal control, rabbit polyclonal antibody, dilution 1 : 1000, Beyotime, China). A goat-anti-rabbit or goat-anti-mouse antibody conjugated to horseradish peroxidase (1 : 2000, Thermo Fisher Scientific) was used as the second antibody. AQP2, AQP3, PKA, and p-CREB protein levels were normalized against GAPDH. All protein expressions are given as percentages compared to the control group (100%). All experiments were done for three times.


### 2.12. RNA Isolation and TaqManTM Real-Time RT-PCR

The colons were dissected from mice at 2 h after the administration of the MgSO4 solutions, snap-frozen in liquid nitrogen, ground into powder, and homogenized. This procedure was done under RNase-free conditions. The RNA isolation and real-time PCR assay were carried out following the protocol of previous study [11]. Briefly, total RNA was extracted with TRIzol reagent (Invitrogen, USA) from the tissue homogenates according to the manufacturer’s instructions. The total RNA (1 μg) was reverse transcribed to cDNA using the QuantiTect Reverse Transcription Kit (QIAGEN, Japan) according to the instruction manual. The specific transcripts were quantified by quantitative real-time PCR using TaqMan Universal Master Mix II (ABI, USA) and analyzed with ABI 7500 real-time PCR system (ABI). Gene-specific primers were used for AQP2 (Mm00437575_m1, Cat: 431182, ABI), AQP3 (Mm01208559_m1, Cat: 431182, ABI), and 18S rRNA (Mm00835095_g1, Cat: 431182, ABI). The mRNA levels of AQP2 and AQP3 were normalized to 18S rRNA level. PCR was performed as 40 cycles at 95°C for 15 s, 56°C for 30 s, and 72°C for 30 s. The relative mRNA expression was calculated with comparative CT method. All experiments were done in triplicate for three times.


### 2.13. Statistical Analysis

The software of SPSS version 16.0 for Windows (SPSS, Chicago, IL, USA) was used for statistical analysis. Values were expressed as means ± SD. Statistical differences were analyzed using One-Way-ANOVA method followed by Turkey’s multiple comparison test. Results withP<0.05 were considered to be significant.


## 2.1. Preparation of RTE

The dried root and rhizoma ofRheum palmatum, produced in Yushu county of Qinghai province of China, were purchased from the Yushu Pharmaceuticals Company and were identified by Professor Shilin Hu, a taxonomist at China Academy of Chinese Medical Sciences. A voucher specimen (Rh201005Z) was deposited in the institute. RTE was prepared according to the reported method with some modified steps [35]. 1 kg of rhubarb was powdered and extracted with 5 L of 60% ethanol for 30 min in a reflux condenser for three times and then was filtrated. The filtrate was merged and concentrated to 500 mL in vacuo in a rotary evaporator at 40°C. Then the concentrated solution was added into D-101 macroporous resin with 25% ethanol as the eluent. The solution was concentrated continually and then precipitated with 1% gelatin. The sediment was dissolved in acetone, recovered acetone, and dried in a vacuum oven as the total tannins extract of rhubarb. The total tannins extract was free of anthraquinones, according to the color reactions of anthraquinones with magnesium acetate methanol solution and tannins with ferric chloride solution. The yield of the total tannins from rhubarb was 3.40%. The total tannins extract of rhubarb was diluted with distilled water or cell culture medium to the proper concentration before usein vivo andin vitro experiments as follows.


## 2.2. High-Performance Liquid Chromatography (HPLC) Fingerprint of RTE

The authentic standards of gallic acid and epigallocatechin gallate were purchased from Jingchun Biological Technology Co. Ltd. (Shanghai, China). The standards of procyanidin-B1 and procyanidin-B2 were obtained from Tauto Biotech Co. Ltd. (Shanghai, China). Standards of catechin and epicatechin were supplied by National Institute for Food and Drug Control (Beijing, China). RTE and standard substance were dissolved in 50% methanol and then filtrated through 0.45μm filter membrane. HPLC was performed on a Shimadzu HPLC system (Shimadzu Corporation, Shimane, Japan) equipped with an LC-20AT binary pump, an SPD-M20A diode array detector, a CBM-20Alite system controller, an SIL-20A autosampler, a DGU-20A5 degasser, and a CTO-10ASvp column oven. An Ultimate XB-C18 column (150 × 4.6 mm, 5 μm particle size) was maintained at 30°C. Detection wave length was set at 280 nm. The mobile phase for fingerprint analysis consisted of solvent A (acetonitrile, Merck, NJ, USA) and solvent B (0.1% formic acid/water, v/v) at a flow rate of 0.8 mL/min. A gradient program was used as follows: 27–10% B at 0–25 min. The injected volume was 10 μL. Online UV spectra were recorded in the range of 190–800 nm. The HPLC fingerprint profile of RTE was established by optimizing the chromatographic separate conditions. The chromatogram of RTE at 280 nm could exhibit the whole chromatographic characteristic in Figure 1(a). By comparison of the retention time and online UV spectra of the reference standards, 6 compounds of tannins were identified as previously reported in rhubarb [39]. The structure of gallic acid, procyanidin-B1, catechin, procyanidin-B2, epicatechin, and epigallocatechin gallate were shown in Figure 1(b).HPLC profile (UV chromatograms at 280 nm) of rhubarb tannins extract (RTE). (a) HPLC fingerprint of RTE. (1)—RTE. (2)—standards. (b) Structure of identified components of RTE. (A)—gallic acid. (B)—procyanidin-B1. (C)—catechin. (D)—procyanidin-B2. (E)—epicatechin. (F)—epigallocatechin gallate.
(a)
(b)


## 2.3. Quantitative Analysis of RTE

The content of the RTE was determined using Shimadzu Visible Ultraviolet Spectrophotometer UV-1800 (Shimadzu Technologies) according to the method in Chinese Pharmacopoeia (Committee for the Pharmacopoeia of China, 2010) with gallic acid as reference substance. The mass of tannins in extracts was 55.69% equivalent of gallic acid.


## 2.4. Induction of MgSO4-Induced Diarrhoea Mouse Model and RTE Treatment

Thirty male ICR mice (18–22 g) were obtained from Laboratory Animal Center of Academy of Military Medical Sciences, Beijing, China (License No.: SCXK 2007-004). They were kept in a temperature controlled environment (22±2°C), 55±5% relative humidity with a 12 h : 12 h light-dark cycle and fed with standard chow, for at least one week before any manipulations. ICR mice were divided separately into 5 groups randomly with the equal number (n=6): the normal control group (Control), the MgSO4-induced diarrhoea model group (Vehicle), and the RTE- (125, 250, and 500 mg/kg, resp.) treated diarrhoea mice groups. Five groups of mice were orally administrated by syringe feeding with distilled water (20 mL/kg) or RTE daily for three days, respectively. Each mouse except for the normal group was orally administrated with MgSO4·7H2O (2 g/kg, Jinhuitaiya Chemical Industries, Tianjin, China) 1 h after the last administration to induce diarrhoea as previously reported [35]. The assay was repeated 3 times.


## 2.5. Fecal Water Content

Detailed procedures have been previously described [12]. Briefly, fecal samples from the mice were collected for up to 5 h after the administration of the MgSO4 solutions and were placed in silica gel followed by drying for 48 h in a desiccator. The fecal water content per gram of feces was calculated based on the difference between the wet and dry fecal weights. This was then used to calculate the percentage of water in the fecal samples. These percentages were averaged among six animals for each group and the assay was repeated 3 times.


## 2.6. Evacuation Index of Mice Defecation

The method as previously reported [35] was used to assess the defecation function of mice. A numerical score based on stool consistency was assigned: 1 = normal stool, 2 = semisolid stool, and 3 = watery stool. Each mouse received an evacuation index (EI) expressed according to the formula: EI = 1 × (number of normal stool) + 2 × (number of semi-solid stool) + 3 × (number of watery stool). The data were expressed as mean EI for each group (n=6). The assay was repeated 3 times.


## 2.7. Cell Culture

Human colon cancer HT-29 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences. HT-29 cells were maintained in sterile Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS, 100 U/mL penicillin, 100μg/mL streptomycin, and 2 mM Gln-glutamine at 37°C. Cells were plated on a 24-well plate, 96-well plate, or 100-mm dish at a density of 2 × 105 cells/cm2, incubated in a CO2 incubator at 37°C for 24 h, and then treated with compounds dissolved in culture medium. Experiments were done using cells that had previously been passaged five to fifteen times.


## 2.8. Cell Viability Assay

HT-29 cells were seeded in 96-well plates and incubated in serum free sterile DMEM (supplemented with 100 U/mL penicillin, 100μg/mL streptomycin, and 2 mM Gln-glutamine) for 24 h. Cells were then incubated with media containing MgSO4 (50 mM) and/or RTE (20, 40, and 80 μg/mL, resp.) for 24 h. After treatment, cells were washed twice with phosphate-buffered saline (PBS; pH 7.4), and then cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method using Cell Titer 96 Non-Radioactive Cell Proliferation Assay (Promega, Madison, USA) according to the manufacturer’s instructions. All absorbance at 570 nm was measured with a microplate reader. The experiments were carried out 3 times in triplicate measurements.


## 2.9. Immunolocalization

Mice were sacrificed by cervical dislocation under ether anesthesia at 2 h after the administration of the MgSO4 solutions, and the colons were removed. After washing the colons with PBS, the samples were fixed immediately for 3 h in 4% paraformaldehyde and embedded in paraffin and tissue sections (5 μm) of colon were prepared for immunofluorescence analysis.Forin vitro experiments, HT-29 cells were cultured in 24-well plates with medium containing MgSO4 and/or RTE for 24 h. The control was treated with the original medium. After washing with PBS twice, cells were fixed immediately for 15 min in 4% paraformaldehyde and prepared for immunofluorescence analysis. The experiments were done in triplicate for three times.Immunofluorescence analyses were carried out following the protocol of previous study [9, 40]. Tissue sections (5 μm) of colon and HT-29 cells were treated with 0.2% Triton in PBS prior to blocking with 5% normal goat serum (NGS) in TPBS-BSA (PBS containing 0.05% Tween-20 and 0.1% bovine serum albumin). The primary rabbit anti-AQP2 (1 : 50, Abcam, Cambridge, MA, UK) antibody or rabbit anti-AQP3 antibody (1 : 50, Abcam) in TPBS-BSA supplemented with 1% NGS was incubated on the tissue and cells overnight at 4°C. Then, the tissue and cells were washed extensively with the TPBS-BSA. After that, the sections and cells were exposed for 2 h at room temperature to a secondary goat anti-rabbit antibody conjugated with Alexa Fluor 488 (1 : 200, Zhongshanjinqiao, Beijing, China). Then, the tissue and cells were reacted with 4,6-diamidino-2-phenylindole (DAPI) solution (Beyotime, Jiangsu, China) in PBS at room temperature for 5 min, washed 3 more times with PBS, and then coverslipped with vectashield (Zhongshanjinqiao) and visualized using a Zeiss Lsm710 confocal microscope (Carl Zeiss AG, Oberkochen, German).


## 2.10. Preparation of Tissue and Cell Extraction for Western Blot Analysis

The colons were removed from mice. Large intestinal mucosa scraped with a slide glass was homogenized using dissecting buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, 8.5 mM leupeptin, and 1 mM phenylmethylsulfonyl fluoride; pH 7.2) on ice. The resulting suspension was centrifuged (800 ×g at 4°C for 15 min) and the supernatant was centrifuged (200,000 ×g at 4°C for 1 h). The precipitate was resuspended using dissecting buffer. Protein concentrations were measured by the Lowry method [41] using BSA as a standard. The protein expression levels of AQPs 2 and 3 in colons were determined.AQPs 2 and 3 were expressed in the plasma membrane and in intracellular vesicles. Thus, the crude fraction dominantly containing these fractions in HT-29 cells was prepared as previously reported [10]. Briefly, HT-29 cells were cultured in 100-mm dishes with media containing MgSO4 and/or RTE for further culture at 37°C for different times (3, 6, 12, and 24 h, resp.). The control was treated with the original medium. After washing with PBS, cells were recovered and suspended in dissecting buffer. The cell suspension was homogenized, and the homogenate was centrifuged (4000 ×g for 10 min). The supernatant was centrifuged (200,000 ×g for 1 h), and the resulting supernatant was discarded. Dissecting buffer was added to the pellets, and the suspension was homogenized using an ultrasonic homogenizer to make a crude fraction [42]. All procedures were carried out at 4°C.The phosphorylation of cyclic monophosphate (cAMP) response element-binding protein (CREB) occurred in the nucleus. Thus, the nuclear fraction, dominantly containing the nucleus, was prepared as previously reported [10], and the protein expression level of phosphorylated CREB (p-CREB) was determined. Briefly, isolated HT-29 cells were cultured with media containing MgSO4 and/or RTE for 24 h. After that, the cells were treated with extraction buffer (phosphosafe extraction reagent with 8.5 μM leupeptin and 1 mM phenylmethylsulfonyl fluoride). The suspension was left on ice for 5 min. After detaching and recovering cells with a cell scraper, the cells were then homogenized and centrifuged (4,000 ×g for 10 min). After the supernatant was discarded, extraction buffer was added to the pellets. The suspension was centrifuged (16,000 ×g for 30 min) to obtain the supernatant as a nuclear fraction [43, 44]. All procedures were carried out at 4°C.Meanwhile, HT-29 cells were treated with MgSO4 and/or RTE and then dissociated with lysis buffer (20 mM MOPS, 50 mM β-glycerophosphate, 50 mM sodium fluoride, 1 mM sodium vanadate, 5 mM EGTA, 2 mM EDTA, 1% NP-40, 1 mM DTT, 1 mM benzamidine, 1 mM PMSF, 10 μg/mL leupeptin, and 10 μg/mL aprotinin). After that, the cells were recovered and centrifuged (16,000 ×g for 15 min at 4°C). The supernatant was recovered, and the protein expression of cAMP-dependent protein kinase A (PKA) was determined by western blot.


## 2.11. Western Blot Analysis

The western blot protocol and semiquantitative analysis were carried out following the protocol of previous study [10]. The following primary antibodies were used: AQP2 (rabbit polyclonal antibody, dilution 1 : 200, Abcam), AQP3 (rabbit polyclonal antibody, dilution 1 : 200, Abcam) and PKA C-α (rabbit polyclonal antibody, dilution 1 : 1000, Cell Signaling, Boston, USA), p-CREB (ser133, rabbit polyclonal antibody, dilution 1 : 500, Thermo Fisher Scientific, Waltham, UK), and GAPDH (internal control, rabbit polyclonal antibody, dilution 1 : 1000, Beyotime, China). A goat-anti-rabbit or goat-anti-mouse antibody conjugated to horseradish peroxidase (1 : 2000, Thermo Fisher Scientific) was used as the second antibody. AQP2, AQP3, PKA, and p-CREB protein levels were normalized against GAPDH. All protein expressions are given as percentages compared to the control group (100%). All experiments were done for three times.


## 2.12. RNA Isolation and TaqManTM Real-Time RT-PCR

The colons were dissected from mice at 2 h after the administration of the MgSO4 solutions, snap-frozen in liquid nitrogen, ground into powder, and homogenized. This procedure was done under RNase-free conditions. The RNA isolation and real-time PCR assay were carried out following the protocol of previous study [11]. Briefly, total RNA was extracted with TRIzol reagent (Invitrogen, USA) from the tissue homogenates according to the manufacturer’s instructions. The total RNA (1 μg) was reverse transcribed to cDNA using the QuantiTect Reverse Transcription Kit (QIAGEN, Japan) according to the instruction manual. The specific transcripts were quantified by quantitative real-time PCR using TaqMan Universal Master Mix II (ABI, USA) and analyzed with ABI 7500 real-time PCR system (ABI). Gene-specific primers were used for AQP2 (Mm00437575_m1, Cat: 431182, ABI), AQP3 (Mm01208559_m1, Cat: 431182, ABI), and 18S rRNA (Mm00835095_g1, Cat: 431182, ABI). The mRNA levels of AQP2 and AQP3 were normalized to 18S rRNA level. PCR was performed as 40 cycles at 95°C for 15 s, 56°C for 30 s, and 72°C for 30 s. The relative mRNA expression was calculated with comparative CT method. All experiments were done in triplicate for three times.


## 2.13. Statistical Analysis

The software of SPSS version 16.0 for Windows (SPSS, Chicago, IL, USA) was used for statistical analysis. Values were expressed as means ± SD. Statistical differences were analyzed using One-Way-ANOVA method followed by Turkey’s multiple comparison test. Results withP<0.05 were considered to be significant.


## 3. Results

### 3.1. RTE Has the Antidiarrhoeal Effect in MgSO4-Induced Diarrhoea Mice

To investigate the effect of RTE on diarrhea, the MgSO4-induced diarrhoea model in mice was used. As shown in Figure 2(a), fecal water content in mice up to 5 h after MgSO4 administration increased significantly versus that in normal mice (P<0.01), and severe diarrhoea was observed. RTE dose-dependently interfered with the fecal water content in MgSO4-induced diarrhoea mice (all P<0.05). Consistent with the fecal water content, the assessment of EI of mice defecation also showed RTE to be highly effective (all P<0.05, Figure 2(b)). Additionally, macroscopic evidence of watery stool in colon was markedly observed in vehicle-treated diarrhoea mice, while dose of 125–500 mg/kg RTE significantly alleviated the watery stool of diarrhoea mice (Figure 2(c)). Meanwhile, the histological structure of colon in mice induced by MgSO4 appeared edematous while RTE treatment seemed to reduce edema (Figure 2(d)). Besides, we also investigated the effect of RTE on diarrhoea mice induced by castor oil (0.3 mL/mouse, p.o.), and the results are similar to above (data not shown).Rhubarb tannins extract (RTE) has the antidiarrhoeal activity in magnesium sulphate- (MgSO4-) induced diarrhoea mice. Mice were orally administrated with RTE (125, 250, and 500 mg/kg, resp.) or water daily for 3 days before MgSO4-induced diarrhoea. During the experiment, fecal water content and evacuation index (EI) of mice defecation were evaluated. (a) Doses of 125~500 mg/kg RTE significantly decreased the fecal water content dose-dependently compared with vehicle-treated diarrhoea mice. (b) Doses of 125~500 mg/kg RTE significantly decreased the EI of defecation in a dose-dependent manner compared with vehicle-treated diarrhoea mice. (c) Macroscopic evidence of watery stool in colon was markedly observed in vehicle-treated diarrhoea mice, while dose of 125~500 mg/kg RTE significantly alleviated the watery stool of diarrhoea mice. (d) Histological structure of colon in mice induced by MgSO4 appeared edematous while RTE treatment seemed to reduce edema. Data are represented as the mean ± SD. ###P<0.001 versus normal control group. P*<0.05, P**<0.01, and P***<0.001 versus vehicle group, respectively. n=6 in each group and each assay was repeated 3 times.
(a)
(b)
(c)
(d)Nextly, apparent adverse effects, including weight loss, alterations of physical appearance, and behavior changes, were not noted in mice treated with RTE (results not shown). Taken together, these observations indicate that systemic administration of RTE in mice has the antidiarrhoeal activity by inhibiting intestinal fluid accumulation without serious side effects.


### 3.2. RTE Inhibits the Production and Gene Expression of AQPs 2 and 3 BothIn Vivo andIn Vitro

AQPs 2 and 3, mainly localized in the colon, play a very important role in transepithelial fluid transport and involved in many kinds of diarrhoea [9–11]. As shown in Figure 3, significant amounts of AQPs 2 and 3 positive staining were present in both the apical and lateral mucosal epithelial cells in the colon tissues from vehicle-treated mice, while there was evident depression of AQPs 2 and 3 in the colon tissues from RTE-treated mice. As shown in Figures 4(a) and 4(b), compared with vehicle-treated diarrhoea mice, doses of 125~500 mg/kg RTE markedly reduced the protein levels of AQPs 2 and 3 in the colon tissues of diarrhoea mice by western blot analysis (all P<0.05). Additionally, findings were similar to the gene expression levels of AQPs 2 and 3 (all P<0.05, Figures 4(c) and 4(d)). These results suggest that RTE reduces the protein and gene expression of AQPs 2 and 3in vivo.Rhubarb tannins extract (RTE) reduces the protein expression of aquaporins (AQPs) 2 and 3 in the colons of magnesium sulphate-induced diarrhoea mice by fluorescent immunohistochemistry. ((a) and (b)) Localization of AQPs 2 and 3 in both the apical and lateral mucosal epithelial cells in the proximal colons of normal control, vehicle-treated diarrhea, and RTE- (500 mg/kg) treated diarrhoea mice. Negative controls in which the AQPs 2 and 3 antibodies were replaced with normal rabbit immunoglobulin (IgG) at identical concentrations resulted in a lack of specific staining. Tissue is colocalized with DAPI to demonstrate the location of cell nuclei (blue). The images in merge’ panel are the big magnification in merge panel, respectively. ((c) and (d)) Positive expression levels of AQPs 2 and 3 in the colons of normal control, vehicle-treated diarrhea, and RTE- (125, 250, and 500 mg/kg) treated diarrhoea mice. 10 microscopic fields were selected randomly and AQPs 2 and 3 positive cells were counted. The AQPs 2 and 3 positive expression levels of the control were taken as 100%. Data are represented as the mean ± SD.###P<0.001 versus normal control group. P*<0.05, P**<0.01, and P***<0.001 versus vehicle group, respectively. n=6 in each group and each assay was repeated 3 times.
(a)
(b)
(c)
(d)Rhubarb tannins extract (RTE) inhibits the protein and gene expression of aquaporins (AQPs) 2 and 3 in the colons of magnesium sulphate-induced diarrhoea mice by western blot and real-time Reverse Transcription-Polymerase Chain Reaction. ((a) and (b)) Expression levels of AQPs 2 and 3 protein in the colons of normal control, vehicle-treated diarrhea, and RTE- (125, 250, and 500 mg/kg) treated diarrhoea mice. ((c) and (d)) Expression levels of AQPs 2 and 3 mRNA in the colons of mice. Data are represented as the mean ± SD.###P<0.001 versus normal control group. P*<0.05, P**<0.01, and P***<0.001 versus vehicle group, respectively. n=6 in each group and each assay was repeated 3 times.
(a)
(b)
(c)
(d)HT-29 cells have been widely used in the study of the mechanisms of diarrhoea and laxative actions, since HT-29 cells represent the normal physiological condition of the colon, despite the fact that they are cancer cell lines derived from human colon cancer [45, 46]. In the study, the protein and gene expression of AQPs 2 and 3 were also investigated in HT-29 cells. As shown in Figure 5, significant amounts of AQPs 2 and 3 staining were present in MgSO4-treated HT-29 cells, while there was evident depression of AQPs 2 and 3 in HT-29 cells treated by RTE. Western blot analysis demonstrated that the expression levels of AQPs 2 and 3 proteins in HT-29 cells were reduced by RTE at a time-dependent manner (all P<0.05, Figure 6(a) for 6, 12, and 24 h; all P<0.05, Figure 6(b)) and a dose-dependent manner (all P<0.05, Figures 6(c) and 6(d)), respectively. These results suggest that RTE reduces the protein expression of AQPs 2 and 3in vitro.Rhubarb tannins extract (RTE) decreases the protein expression of aquaporins (AQPs) 2 and 3 in HT-29 cells induced by magnesium sulphate (MgSO4) by fluorescent immunohistochemistry. Cells were placed in 24-well plate or 100-mm dishes for 24 h in the presence of MgSO4 with or without RTE (control, MgSO4, and RTE- (20, 40, and 80 μg/mL) treatment groups, resp.). HT-29 cells were fixed, stained by immunofluorescence, and scanned in 10 random fields. ((a) and (b)) Immunolocalization of AQPs 2 (green) and 3 (red) in HT-29 cells. Negative controls in which the AQPs 2 and 3 antibodies were replaced with normal rabbit immunoglobulin (IgG) at identical concentrations resulted in a lack of specific staining. Nuclei were stained with DAPI (blue). ((c) and (d)) Positive expression levels of AQPs 2 and 3 in HT-29 cells. 10 microscopic fields were selected randomly and AQPs 2 and 3 positive cells were counted. The AQPs 2 and 3 positive expression levels of the control were taken as 100%. Data are represented as the mean ± SD. ###P<0.001 versus normal control group. P*<0.05, P**<0.01, and P***<0.001 versus vehicle group, respectively. n=3 in each group and each assay was repeated 3 times.
(a)
(b)
(c)
(d)Rhubarb tannins extract (RTE) reduces the protein expression of aquaporins (AQPs) 2 and 3 in magnesium sulphate- (MgSO4-) induced HT-29 cells by western blot. Cells were placed in 24-well plate or 100-mm dishes for 24 h in the presence of MgSO4 with or without RTE (control, MgSO4, and RTE- (20, 40, and 80 μg/mL) treatment groups, resp.). ((a) and (b)) Expression levels of AQPs 2 and 3 protein in HT-29 cells treated by RTE (80 μg/mL) for different time (0, 3, 6, 12, and 24 h, resp.). ((c) and (d)) Expression levels of AQPs 2 and 3 proteins in HT-29 cells treated by RTE at the dosages of 20, 40, and 80 μg/mL for 24 h, respectively. Data are represented as the mean ± SD. ###P<0.001 versus normal control group. P*<0.05, P**<0.01, and P***<0.001 versus vehicle group, respectively. n=3 in each group and each assay was repeated 3 times.
(a)
(b)
(c)
(d)


### 3.3. RTE Downregulates MgSO4-Induced Activation of PKA/p-CREB Downstream Pathway

In order to investigate whether the activation of MgSO4-induced PKA/p-CREB signal pathway was modulated by RTE, we further detected the expression levels of PKA and p-CREB protein in MgSO4-induced HT-29 cells. As shown in Figures 7(a) and 7(b), RTE markedly diminished the activation of MgSO4-induced PKA (all P<0.05) and p-CREB (all P<0.05), which are associated with the production of AQPs 2 and 3 in HT-29 cells.Rhubarb tannins extract (RTE) downregulates the expression of magnesium sulphate- (MgSO4-) activated PKA and p-CREB in HT-29 cells without effect on cell viability. Cells were placed in 100-mm dishes for 24 h in the presence of MgSO4 with or without RTE (control, MgSO4, and RTE- (20, 40, and 80 μg/mL) treatment groups, resp.). HT-29 cells were collected to detect the expression levels of PKA (a) and p-CREB (b) protein by western blot analysis. (c) No effect of RTE (20, 40, 80 μg/mL, resp.) on the cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Cell viability of the control was taken as 100%. Data are represented as the mean ± SD. ###P<0.001 versus normal control group. P*<0.05, P**<0.01, and P***<0.001 versus vehicle group, respectively. n=3 in each group and each assay was repeated 3 times.
(a)
(b)
(c)Nextly, we examined whether the above suppressive effect of RTE was due to its cytotoxicity. When confluent HT-29 cells were treated with RTE and/or MgSO4 for 24 h, the cytotoxicity was monitored by MTT assay. Our results showed that RTE did not exert any cytotoxic effects on HT-29 cells under the experimental conditions used in the present study (Figure 7(c)), suggesting that RTE might specifically suppress AQPs 2 and 3, PKA, and p-CREB expressionin vitro.


## 3.1. RTE Has the Antidiarrhoeal Effect in MgSO4-Induced Diarrhoea Mice

To investigate the effect of RTE on diarrhea, the MgSO4-induced diarrhoea model in mice was used. As shown in Figure 2(a), fecal water content in mice up to 5 h after MgSO4 administration increased significantly versus that in normal mice (P<0.01), and severe diarrhoea was observed. RTE dose-dependently interfered with the fecal water content in MgSO4-induced diarrhoea mice (all P<0.05). Consistent with the fecal water content, the assessment of EI of mice defecation also showed RTE to be highly effective (all P<0.05, Figure 2(b)). Additionally, macroscopic evidence of watery stool in colon was markedly observed in vehicle-treated diarrhoea mice, while dose of 125–500 mg/kg RTE significantly alleviated the watery stool of diarrhoea mice (Figure 2(c)). Meanwhile, the histological structure of colon in mice induced by MgSO4 appeared edematous while RTE treatment seemed to reduce edema (Figure 2(d)). Besides, we also investigated the effect of RTE on diarrhoea mice induced by castor oil (0.3 mL/mouse, p.o.), and the results are similar to above (data not shown).Rhubarb tannins extract (RTE) has the antidiarrhoeal activity in magnesium sulphate- (MgSO4-) induced diarrhoea mice. Mice were orally administrated with RTE (125, 250, and 500 mg/kg, resp.) or water daily for 3 days before MgSO4-induced diarrhoea. During the experiment, fecal water content and evacuation index (EI) of mice defecation were evaluated. (a) Doses of 125~500 mg/kg RTE significantly decreased the fecal water content dose-dependently compared with vehicle-treated diarrhoea mice. (b) Doses of 125~500 mg/kg RTE significantly decreased the EI of defecation in a dose-dependent manner compared with vehicle-treated diarrhoea mice. (c) Macroscopic evidence of watery stool in colon was markedly observed in vehicle-treated diarrhoea mice, while dose of 125~500 mg/kg RTE significantly alleviated the watery stool of diarrhoea mice. (d) Histological structure of colon in mice induced by MgSO4 appeared edematous while RTE treatment seemed to reduce edema. Data are represented as the mean ± SD. ###P<0.001 versus normal control group. P*<0.05, P**<0.01, and P***<0.001 versus vehicle group, respectively. n=6 in each group and each assay was repeated 3 times.
(a)
(b)
(c)
(d)Nextly, apparent adverse effects, including weight loss, alterations of physical appearance, and behavior changes, were not noted in mice treated with RTE (results not shown). Taken together, these observations indicate that systemic administration of RTE in mice has the antidiarrhoeal activity by inhibiting intestinal fluid accumulation without serious side effects.


## 3.2. RTE Inhibits the Production and Gene Expression of AQPs 2 and 3 BothIn Vivo andIn Vitro

AQPs 2 and 3, mainly localized in the colon, play a very important role in transepithelial fluid transport and involved in many kinds of diarrhoea [9–11]. As shown in Figure 3, significant amounts of AQPs 2 and 3 positive staining were present in both the apical and lateral mucosal epithelial cells in the colon tissues from vehicle-treated mice, while there was evident depression of AQPs 2 and 3 in the colon tissues from RTE-treated mice. As shown in Figures 4(a) and 4(b), compared with vehicle-treated diarrhoea mice, doses of 125~500 mg/kg RTE markedly reduced the protein levels of AQPs 2 and 3 in the colon tissues of diarrhoea mice by western blot analysis (all P<0.05). Additionally, findings were similar to the gene expression levels of AQPs 2 and 3 (all P<0.05, Figures 4(c) and 4(d)). These results suggest that RTE reduces the protein and gene expression of AQPs 2 and 3in vivo.Rhubarb tannins extract (RTE) reduces the protein expression of aquaporins (AQPs) 2 and 3 in the colons of magnesium sulphate-induced diarrhoea mice by fluorescent immunohistochemistry. ((a) and (b)) Localization of AQPs 2 and 3 in both the apical and lateral mucosal epithelial cells in the proximal colons of normal control, vehicle-treated diarrhea, and RTE- (500 mg/kg) treated diarrhoea mice. Negative controls in which the AQPs 2 and 3 antibodies were replaced with normal rabbit immunoglobulin (IgG) at identical concentrations resulted in a lack of specific staining. Tissue is colocalized with DAPI to demonstrate the location of cell nuclei (blue). The images in merge’ panel are the big magnification in merge panel, respectively. ((c) and (d)) Positive expression levels of AQPs 2 and 3 in the colons of normal control, vehicle-treated diarrhea, and RTE- (125, 250, and 500 mg/kg) treated diarrhoea mice. 10 microscopic fields were selected randomly and AQPs 2 and 3 positive cells were counted. The AQPs 2 and 3 positive expression levels of the control were taken as 100%. Data are represented as the mean ± SD.###P<0.001 versus normal control group. P*<0.05, P**<0.01, and P***<0.001 versus vehicle group, respectively. n=6 in each group and each assay was repeated 3 times.
(a)
(b)
(c)
(d)Rhubarb tannins extract (RTE) inhibits the protein and gene expression of aquaporins (AQPs) 2 and 3 in the colons of magnesium sulphate-induced diarrhoea mice by western blot and real-time Reverse Transcription-Polymerase Chain Reaction. ((a) and (b)) Expression levels of AQPs 2 and 3 protein in the colons of normal control, vehicle-treated diarrhea, and RTE- (125, 250, and 500 mg/kg) treated diarrhoea mice. ((c) and (d)) Expression levels of AQPs 2 and 3 mRNA in the colons of mice. Data are represented as the mean ± SD.###P<0.001 versus normal control group. P*<0.05, P**<0.01, and P***<0.001 versus vehicle group, respectively. n=6 in each group and each assay was repeated 3 times.
(a)
(b)
(c)
(d)HT-29 cells have been widely used in the study of the mechanisms of diarrhoea and laxative actions, since HT-29 cells represent the normal physiological condition of the colon, despite the fact that they are cancer cell lines derived from human colon cancer [45, 46]. In the study, the protein and gene expression of AQPs 2 and 3 were also investigated in HT-29 cells. As shown in Figure 5, significant amounts of AQPs 2 and 3 staining were present in MgSO4-treated HT-29 cells, while there was evident depression of AQPs 2 and 3 in HT-29 cells treated by RTE. Western blot analysis demonstrated that the expression levels of AQPs 2 and 3 proteins in HT-29 cells were reduced by RTE at a time-dependent manner (all P<0.05, Figure 6(a) for 6, 12, and 24 h; all P<0.05, Figure 6(b)) and a dose-dependent manner (all P<0.05, Figures 6(c) and 6(d)), respectively. These results suggest that RTE reduces the protein expression of AQPs 2 and 3in vitro.Rhubarb tannins extract (RTE) decreases the protein expression of aquaporins (AQPs) 2 and 3 in HT-29 cells induced by magnesium sulphate (MgSO4) by fluorescent immunohistochemistry. Cells were placed in 24-well plate or 100-mm dishes for 24 h in the presence of MgSO4 with or without RTE (control, MgSO4, and RTE- (20, 40, and 80 μg/mL) treatment groups, resp.). HT-29 cells were fixed, stained by immunofluorescence, and scanned in 10 random fields. ((a) and (b)) Immunolocalization of AQPs 2 (green) and 3 (red) in HT-29 cells. Negative controls in which the AQPs 2 and 3 antibodies were replaced with normal rabbit immunoglobulin (IgG) at identical concentrations resulted in a lack of specific staining. Nuclei were stained with DAPI (blue). ((c) and (d)) Positive expression levels of AQPs 2 and 3 in HT-29 cells. 10 microscopic fields were selected randomly and AQPs 2 and 3 positive cells were counted. The AQPs 2 and 3 positive expression levels of the control were taken as 100%. Data are represented as the mean ± SD. ###P<0.001 versus normal control group. P*<0.05, P**<0.01, and P***<0.001 versus vehicle group, respectively. n=3 in each group and each assay was repeated 3 times.
(a)
(b)
(c)
(d)Rhubarb tannins extract (RTE) reduces the protein expression of aquaporins (AQPs) 2 and 3 in magnesium sulphate- (MgSO4-) induced HT-29 cells by western blot. Cells were placed in 24-well plate or 100-mm dishes for 24 h in the presence of MgSO4 with or without RTE (control, MgSO4, and RTE- (20, 40, and 80 μg/mL) treatment groups, resp.). ((a) and (b)) Expression levels of AQPs 2 and 3 protein in HT-29 cells treated by RTE (80 μg/mL) for different time (0, 3, 6, 12, and 24 h, resp.). ((c) and (d)) Expression levels of AQPs 2 and 3 proteins in HT-29 cells treated by RTE at the dosages of 20, 40, and 80 μg/mL for 24 h, respectively. Data are represented as the mean ± SD. ###P<0.001 versus normal control group. P*<0.05, P**<0.01, and P***<0.001 versus vehicle group, respectively. n=3 in each group and each assay was repeated 3 times.
(a)
(b)
(c)
(d)


## 3.3. RTE Downregulates MgSO4-Induced Activation of PKA/p-CREB Downstream Pathway

In order to investigate whether the activation of MgSO4-induced PKA/p-CREB signal pathway was modulated by RTE, we further detected the expression levels of PKA and p-CREB protein in MgSO4-induced HT-29 cells. As shown in Figures 7(a) and 7(b), RTE markedly diminished the activation of MgSO4-induced PKA (all P<0.05) and p-CREB (all P<0.05), which are associated with the production of AQPs 2 and 3 in HT-29 cells.Rhubarb tannins extract (RTE) downregulates the expression of magnesium sulphate- (MgSO4-) activated PKA and p-CREB in HT-29 cells without effect on cell viability. Cells were placed in 100-mm dishes for 24 h in the presence of MgSO4 with or without RTE (control, MgSO4, and RTE- (20, 40, and 80 μg/mL) treatment groups, resp.). HT-29 cells were collected to detect the expression levels of PKA (a) and p-CREB (b) protein by western blot analysis. (c) No effect of RTE (20, 40, 80 μg/mL, resp.) on the cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Cell viability of the control was taken as 100%. Data are represented as the mean ± SD. ###P<0.001 versus normal control group. P*<0.05, P**<0.01, and P***<0.001 versus vehicle group, respectively. n=3 in each group and each assay was repeated 3 times.
(a)
(b)
(c)Nextly, we examined whether the above suppressive effect of RTE was due to its cytotoxicity. When confluent HT-29 cells were treated with RTE and/or MgSO4 for 24 h, the cytotoxicity was monitored by MTT assay. Our results showed that RTE did not exert any cytotoxic effects on HT-29 cells under the experimental conditions used in the present study (Figure 7(c)), suggesting that RTE might specifically suppress AQPs 2 and 3, PKA, and p-CREB expressionin vitro.


## 4. Discussion

Tannins, a unique group of phenolic metabolites with the property of precipitating proteins, are commonly found in plants such as apple fruit, pine bark, grape seed, tea, oak, and medicinal plants and possess a variety of biological effects, including anticarcinogenic, antimutagenic, antimicrobial, and antioxidative activities [30, 47]. Besides, tannins extracted from many medicinal plants also have exhibited an antidiarrhoeal activity (Table 1). The mechanisms of their antidiarrhoeal activity are those by inhibiting cystic fibrosis transmembrane conductance regulator protein chloride channels [39] and by generating protein-precipitating reaction to the gastrointestinal mucosa due to the protein-precipitating action [48]. However, their effects on AQPs are still unclear. In 2011, the total RTE has proved the antidiarrhoeal activity in different kinds of diarrhoea models for the first time [35]. In the study, we further investigated the effect of RTE on AQPs 2 and 3in vivo andin vitro. The results will help to elucidate the antidiarrhoeal action of tannins widely distributed in nature.Table 1
Traditional antidiarrhoeal plants with the composition of tannins.
Scientific name
Part used
Levels of tannins
Diarrhoea models
References
Libidibia ferrea (Mart. ex Tul.)L. P. Queiroz(Caesalpinia ferreaMart.)
Bark
6.24
—
[49]
Caesalpinia pyramidalisTul.
Bark
6.01
—
[49]
Croton blanchetianusBaill.
Bark
2.47
Castor oil
[49]
Eugenia uvalhaCambess.
Bark
1.68
—
[49]
Spondias tuberosaArruda.
Bark
1.51
—
[49]
Cedrela odorataL.
Bark
2.09
—
[49]
Endopleura uchi
Bark
—
—
[50]
Rhododendron arboreum
Flower
—
Castor oil, MgSO4
[51]
Green tea
Leaf
—
—
[52]
Heeria insignisO. Ktze(Anacardiaceae)
Leaf
—
Castor oil
[53]
Trilepisium madagascariense
Bark
—
Castor oil
[54]
Ixora coccineaLinn.(Rubiaceae)
Flower
—
Castor oil
[22]
Capparis zeylanica
Leaf
—
Castor oil
[55]
Rhubarb
Root
13.55
Castor oil, MgSO4
[35]
Note: —: not detected.Wet stool is one of the major characteristics of diarrhea, which is usually caused by altered motility and fluid accumulation in the intestine lumen. MgSO4, an osmotic-acting laxative, has been reported to induce diarrhoea by increasing the volume of intestinal content through prevention of reabsorption of water. In the present study, diarrhea characterized by intestinal fluid accumulation was developed in mice after the administration of MgSO4. Obvious watery stool in the colon, increased fecal water content, and increased EI of defecation were observed in vehicle-treated mice. While in RTE-treated mice we noticed that RTE attenuated the severity of diarrhea by reducing the degree of watery stool in the colon, the fecal water content, and EI of defecation in a dose-dependent manner, which is consistent with the data of the previous study [35]. The results suggest that RTE has an antidiarrhoeal activity in MgSO4-induced diarrhoea mice partly via reducing the colonic water secretion induced by MgSO4.In healthy individuals, about 1.5~2 L water is absorbed daily by the colon, while the maximal capacity of the human large intestine to absorb fluids may be as high as 5-6 L per day [56]. Theoretically, water crosses the colonic epithelia either by paracellular or transcellular routes driven by an osmotic gradient. Because the colon is a tight epithelium characterized by high electrical resistance, there is an underlying mechanism by which water can pass the colonic epithelium layer against considerable osmotic gradients [57]. The discovery of specific water channels called AQPs on epithelial cells in the gastrointestinal tract may give a reasonable explanation. AQPs are a family of highly conserved transmembrane channel proteins mainly responsible for rapid water movements. There are currently thirteen known types of AQPs in humans, AQP0 through AQP12, that are expressed in a variety of tissues [58, 59]. In intestine, various subtypes of AQP are localized (AQPs 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10) [60–63]. Of these, the main types expressed in mucosal epithelial cells in colon are AQPs 2, 3, 4, and 8 [9, 62]. In this study, we focused on AQPs 2 and 3 because the mice deficient in AQPs 4 and 8 demonstrated no alteration in stool water content [64, 65]. We found that AQPs 2 and 3 were obviously expressed in both the apical and lateral mucosal epithelial cells in the colons of MgSO4-induced diarrhoea mice, but when mice were treated with RTE, the expression levels of AQPs 2 and 3 were significantly decreased, which was confirmed by immunofluorescence. Similarly, RTE could also reduce both the mRNA and protein expression levels of AQPs 2 and 3 in the colons of MgSO4-induced diarrhoea mice by real-time RT-PCR and western blot analysis. Furthermore, to confirm these findings, the HT-29 cells, which have been widely used in the study of the mechanisms of diarrhoea and laxative actions, were employed. Compared with HT-29 cells treated with MgSO4, we found that the protein levels of AQPs 2 and 3 were markedly decreased in HT-29 cells cotreated with MgSO4 and RTE. These results indicate that RTE has the ability to decrease the expression of AQPs 2 and 3, which partly accounts for reducing the colonic water secretion induced by MgSO4 involved in the antidiarrhoeal activity of RTE.In addition to these findings, we further explored the precise mechanism involved in the inhibition of AQPs 2 and 3 of RTE in diarrhoea induced by MgSO4. Previous study has demonstrated that MgSO4 as a laxative may increase the AQP3 expression level in HT-29 cells via the following PKA/p-CREB signal pathway [10]. First, an increase in the intracellular Mg2+ concentration causes the activation of adenylate cyclase, which leads to an increase in cAMP production. Then, the increase in the cAMP concentration in turn causes PKA activation, which promotes CREB phosphorylation. Meanwhile, the increased expression of AQP3 in HT-29 cells by vasoactive intestinal polypeptide and natriuretic peptide is correlated with the activation of PKA [16, 30]. Another study has reported that the AQP2 expression induced by arginine-vasopressin is also mediated by the increase of cAMP [66], although the relationship between AQP2 expression and the PKA/p-CREB pathway in MgSO4-induced HT-29 cells is still unclear. In the present study, our data showed that RTE significantly reduced the expression levels of PKA and CREB phosphorylation in MgSO4-induced HT-29 cells in a dose-dependent manner, suggesting that the inhibitory effect of RTE on AQPs 2 and 3 is partly by the downregulation PKA/p-CREB signal pathway.In conclusion, our data offered the convincing evidence for the first time that RTE inhibit AQPs 2 and 3 expressionin vivo andin vitro via downregulating PKA/p-CREB signal pathway, which partially accounts for the antidiarrhoeal effect of RTE. This observation disclosed a novel function of RTE on the management of diarrhoea.



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*Source: 619465-2014-08-18.xml*