In Vivo and In Vitro Antinociceptive Effect of Fagopyrum cymosum (Trev.) Meisn Extracts: A Possible Action by Recovering Intestinal Barrier Dysfunction

Fagopyrum cymosum (Trev.) Meisn (Fag) is a herb rhizome which has been widely used to treat diseases. To investigate the effects and mechanisms of the Fag on irritable bowel syndrome (IBS), in vivo neonatal pups maternal separation (NMS) combined with intracolonic infusion of acetic acid (AA) was employed to establish IBS rat models. Fag reduced their visceral hyperalgesia and the whole gut permeability, ameliorated colonic mucosa inflammation and injury, and upregulated the expression of decreased tight junction proteins (TJs) of claudin-1, occludin, and ZO-1 (except ZO-2) in colonic epithelium. Caco-2 monolayer cells were incubated with TNF-α and IFN-γ   in vitro to establish an epithelial barrier dysfunction model whose transepithelial electrical resistance (TER) depended more on dose of Fag than that of the controls, and whose TJs levels were lower than those of the controls. Fag upregulated the NP-40 insoluble and soluble components of the four TJs markedly in a dose-dependent manner. These data suggest that Fag alleviated the hyperalgesia of IBS rats by reducing intestinal inflammation and enhancing mucosal epithelial function after regulating the structure and function of TJs.


Introduction
Irritable bowel syndrome (IBS) is a chronic functional bowel disorder featured in abdominal pain and disturbed bowel habits. One of the pathomechanism is gastrointestinal motility dysfunction accompanied by visceral hyperalgesia [1]. Recent studies have found that chronic low-grade mucosal inflammation potentially led to IBS [2], as well as coexistent immune abnormalities. The T lymphocytes in descending colon mucosa significantly increased in the case of postinfectious IBS (PI-IBS) and non-PI-IBS [3]. Furthermore, the cytokine concentrations in the colonic mucosa of IBS patients increased due to activated immune system [4], and expression of mast cells adjacent to intestinal plexus in non-PI-IBS patients was highly expressed, which [2] resulted in hyperalgesia.
It has been reported that the intracolonic infusion of supernatants from the colonic biopsies of IBS patients led to somatic and visceral hyperalgesia [5] as well as impaired colonic permeability and sensitivity of mice [6]. Repeated stress increased CD4 + /CD8T + cells [7] and IFN-γ, which directly affected the tight junction proteins (TJs), and altered colonic mucosal barrier functions eventually [8]. However, adding supernatants of the culture from human colonic biopsies to Caco-2 cells reduced transepithelial resistance (TER), decreased ZO-1 mRNA in Caco-2 evidently, and increased paracellular permeability (PP) which correlated with abdominal pain [9]. Dunlop [10] and Spiller [11] have demonstrated that the intestinal permeability in PI-IBS and non-PI-IBS subgroups of diarrhea-predominant IBS elevated. Kong et al. [12] also showed that downregulated 2 Evidence-Based Complementary and Alternative Medicine claudin-1 and -4 expressions were associated with changed defecation habits of D-IBS patients [13]. Finally, intestinal mucosal inflammation may contribute to altering TJs structures and PPs in IBS patients.
Fagopyrum cymosum (Trev.) Meisn (Fag), which is a herb rhizome of the Polygonaceae family and buckwheat species, has been widely used to treat bacterial dysentery, menorrhalgia and abdominal pain in Chinese medicine. Our previous clinical practices have revealed that Fag effectively mitigated abdominal pain, diarrhea, and bloating of IBS, and we have a Chinese authorized patent on Fag treatment of IBS. We assumed that Fag alleviates hyperalgesia of IBS by preventing intestinal mucosal immune disorders or decreasing colonic permeability via affecting TJs. Thereby motivated, we extracted the active ingredients of Fag and established animal and cell models to verify the hypothesis and to clarify the pharmacological targets and mechanisms.

Preparation of Fag Extracts.
Fag was provided by Nantong Jinghua Pharmaceutical Co., Ltd., Jiangsu Province of China, and was extracted by conventional refluxing in 3 fold of 50% ethanol for 3 h, 2 fold of 50% ethanol for 2 h, and 1.5 fold of 50% ethanol for 1 h, respectively. Then the product was decompressively concentrated and then spray-dried. The resulting powders were dissolved in 1% NaOH, the pH of which was then adjusted to 7.4 by HCl. The product was then diluted to a constant volume by saline and finally filtered prior to sterilization. The samples were identified by the Jiangsu Provincial Institute for pharmaceutical inspection of China.

Animals and Neonatal IBS Modeling.
Neonatal Sprague-Dawley rats weighing 5-6 g in average were provided by the Animal Center of Nanjing University of Chinese Medicine. Experiments were conducted in accordance with the standards of Animal Ethics Committee of Nanjing University of Chinese Medicine and the regulations of animal welfare of NIH in the USA. IBS rat models were established referring to neonatal maternal separation (NMS) pups [14] plus intracolonic infusion of 0.5% acetic acid [15]. Postnatal days 4 and 21 pups were subjected to maternal deprivation for 3 h (from 9:00 to 12:00 am), during which they were transferred from the home cage to a new plastic cage individually equipped with constant-temperature bedding (37 ± 0.5 • C, heat source: a hot plate). During NMS, pups were intracolonically infused with 0.2-0.5 mL of 0.5% acetic acid daily at the same time by an angioplasty catheter (3 mm in diameter and 20 mm in length, Cordis Inc., USA) that was inserted from anus to descending colon (2 cm from anus). The control pups were left undisturbed and intracolonically infused with the same amount of saline. The pups were weaned on 22nd day. The males were selected and housed in the same cage undisturbed until they grew to 160 g (6-8 weeks old). The adult model rats in drug groups were treated with Fag (6 g/kg, 24 g/kg, resp.) and VLS#3 (VLS Pharmaceuticals. Ft, Lauderdale, FL, USA. 0.08 g/kg, 3.0 g of each tablet containing 450 billion freeze-dried bacteria) orally once daily for two weeks. The normal rats and model rats were only treated with saline.

Evaluation of Viscera Hyperalgesia.
Intense colorectal distension (CRD) is a nociceptive stimulus which enhances the contractile activities of abdomen and lowers those of limb muscles. Abdominal withdrawal reflex (AWR) evaluates the visceral sensitivity of IBS model during CRD. Rats were anaesthetized by ethyl ether, and a catheter-balloon assembly (made from latex glove; 2.5 cm in diameter and 4.0 cm in length, attached to 6 F catheter) was inserted into their descending colon (1 cm away from anus; exterior catheter was connected to a sphygmomanometer via a three-way pipe) with paraffin oil lubricant. Then the rats were put into transparent Lucite Cubicles (18 cm × 5 cm × 7 cm) and prevented from turning around. Thirty minutes after the rats' regained consciousness, air was injected into the balloon to produce the pressures of 20, 30, 40, 50, 60, 70, and 80 mmHg, respectively. Each expansion was lasted for 20 s with the intermittent of 2 min. The experiments were performed in triplicate, and the average values were recorded. Al-Chaer's method [16] was utilized as the AWR evaluation standard: 0 for no behavior responses, 1 for action pause followed by short head movement after stimulation, 2 for visible abdominal muscles without abdomen lifting off the platform, 3 for abdominal lifting off the platform, and 4 for body arching and pelvic structures or scrotum lift off. 51 Cr-Ethylene diamine tetraacetic acid ( 51 Cr-EDTA, Perkin Elmer Life Science, Paris, France) was used as the selective paracellular permeation marker according to the Barreau's method, aiming to determine the total gut permeability towards large molecules [14]. 0.7 μ Ci of 51 Cr-EDTA diluted in 500 μL of saline was slowly administered orally. Then the animals were placed in metabolic cages, the faeces and urine of which were collected for 24 h. 1 mL of the collected urine and 1 mL of the 100 times diluted standard were subjected to γ counting measurements for 1 min to calculate the 51 Cr-EDTA excretory rate: 51Cr − EDTA excretory rate of urine during 24 h (%) = counts of urine sample − background × urine volume counts of diluted standard sample − background × dilution multiples × 100%.

Total Gut PP.
(1)  [17]. Frozen pieces of distal colon (5 cm from anus) were homogenized in the phosphate buffer (in 50 mM/L, pH = 6) containing hexadecyl trimethyl ammonium bromide (0.5% w/v). Homogenates were subjected to 3 cycles of freezing and thawing (−196 • C, 1 min and 37 • C, 10 min) and then were further disrupted with a sonicator (Kunshan Hechuang Ultrasonic CO., LTd., China) and then centrifuged (6000 g at 4 • C for 15 min). The supernatants were collected for MPO activity measurements. Protein concentrations were determined according to the modified Lowry's method (Bio Rad DC Protein Assay, France) and MPO activities were expressed as units/per gram of protein. In IFN-γ and TNF-α assay, distal colon tissues were homogenized in ice buffer solution (1mL/0.1 g) and then centrifuged for 10 min (600 g at 4 • C). The supernatants were collected and detected using an ELISA kit (BIO-RAD Laboratories, Inc., USA) according to the instructions. The absorbance was measured at 492 nm.

Immunofluorescence Staining of ZO-1 and Occludin.
Rat distal colon samples were embedded in paraffin, cut into sections (thickness: 4 μM), deparaffinized in dimethyl benzene, and then dehydrated in 100%, 95%, and 70% alcohol for 5 min. The antigens of the sections were restored by being placed on a metal staining rack in a pressure kettle containing boiling water to which was added phosphate buffer (0.01 mol/L, pH 6.0). The samples were processed for 5 min by gradually increasing pressure, removed, and quickly washed with cool water, followed by an additional wash 3 times with PBS (0.01 mol/L PH7.4) for 2 min. Goat serum was added to the samples, which was incubated for 20 min at 37 • C, and the residual fluid was discarded. Then the samples were incubated with primary antibody (1 : 1000, rabbit anti-ZO-1, Santa Cruz, Inc. America; rabbit antioccludin, abcam, Inc., USA) overnight at 4 • C, then washed 3 times with PBS for 2 min. Thereafter, the samples were incubated with secondary antibody (1 : 200, TRITC or FITC goat anti-rabbit Ig G (H + L) conjugate, Invitrogen Inc., USA) at 37 • C and then washed 3 times with PBS for 2 min. 50% glycerin was added to seal up the sections, which were then fluorescently visualized using a laser scanning confocal microscope (LSCM, Nikon Inc., Japan).
The resistance values (RV) were measured consecutively at least 3 times. TER was determined according to: where R total represents the measured RV; R blank represents the RV without cells; A represents the membrane surface area [20].   Figure 1: Sensitivities of the rats to CRD. (a) AWR scores of the rats in response to graded CRD. * P < 0.05, * * P < 0.01, NMS + AA rats (model group) versus controls. (b) AWR scores of NMS + AA rats treated with Fag (6 g/kg, 24 g/kg) and VLS#3 a P < 0.05, Fag (6 g/kg) treated rats versus NMS + AA rats; b P < 0.05, Fag (24 g/kg) treated rats versus NMS + AA rats; c P < 0.05, VLS#3 treated rats versus NMS + AA rats. The data are expressed as mean ± SEM (n = 8-10 in each group).

Statistical Analysis.
All values were expressed as mean ± SEM or mean ± SD. The statistics of the two groups were compared by t-test or Mann-Whitney U-test using Graph-Pad-InStat, version 5.01, and P < 0.05 was considered as statistically significant.

Assessment of Visceral Sensitivity to CRD.
The adult NMS + AA rats responded to graded CRD (20, 30, 40, 50, 60 mmHg) were significantly different from the controls (P < 0.05 or <0.01) (Figure 1(a)). The responses of NMS + AA and 6 g/kg Fag rats to CRD did not differ significantly except AWR decreased when CRD = 50 mmHg. The AWR score of 24 g/kg Fag rats was significantly lower (when CRD at 30, 40, 50, 80 mmHg, P < 0.05) than that of NMS+AA rats. Besides, the AWR score of VSL#3 rats was effectively lower (P < 0.05) than that of the controls (Figure 1(b)).

Effect of Fag on Gut PP and Colonic Damage/Inflammation.
In NMS + AA rats, 51 Cr-of EDTA excreted in urine over 24 h (GPP) significantly increased compared to that in control rats (P < 0.001), while gut PP in 6 g/kg, 24 g/kg Fag and VLS#3 rats decreased significantly (P < 0.05, P < 0.01) (Figure 2(a)). The colonic MPO activity of control rats was 157.6 ± 95 U/g of protein, which was significantly lower (P < 0.001) than that of NMS rats. The activities of 24 g/kg Fag and VLS#3 rats decreased (P < 0.01) (Figure 2(b)). The levels of TNF-α and IFN-γ in the colon of NMS + AA rats were significantly higher (P < 0.01) than those of the controls. TNF-α and IFN-γ in Fag treated rats exhibited a dose-dependent recovery to normal significantly compared with those in NMS rats did. VLS#3 decreased the contents of both TNF-α and IFN-γ (Figures 2(c) and 2(d)). These data demonstrated that Fag reduced gut PP by mitigating intestinal damage and inflammation.
Evidence-Based Complementary and Alternative Medicine

Effect of Fag on Colonic Lamina Propria Inflammatory
Cell Counts. HE staining shows a small number of lamina propria inflammatory cells infiltrated in the distal colon of the controls, but there were numerous inflammatory cells in NMS+AA rats (Figure 3(a)), which differed significantly (P < 0.01) from those in the controls. Compared with NMS + AA rats, the cell counts in Fag 24 g/kg (P < 0.05) and VLS#3 rats (P < 0.01) decreased significantly, whereas those in Fag 6 g/kg rats only decreased slightly (P > 0.05) (Figure 3(b)).

TJs Expression of Colon Tissues.
The colonic TJs of claudin-1, occludin, ZO-1, ZO-2 were analyzed by western blotting (Figure 4(a)). Compared with the controls (100%), the total protein of the four TJs in NMS + AA rats significant decreased (P < 0.05). Besides, the TJs of occludin (P < 0.05), and ZO-1 (P < 0.05) in Fag-treated rats as well as the TJs of claudin-1, occludin and ZO-1 in VSL#3 rats (P < 0.01) were significantly higher than those in NMS + AA rats (Figure 4(b)). Moreover, we examined the expression and distribution of TJs by immunofluorescence. The four types of TJs were localized at both the surface and crypts connecting colonic cells, which is consistent with TJs distribution. However, the TJs of occludin, ZO-1 in NMS + AA rats drastically reduced. The membrane fluorescence intensities and discontinuities of NMS + AA rats declined or even, lost compared to those of the controls. In addition, 24 g/kg Fag and VLS#3 considerably prevented the loss of occludin and ZO-1, the densities of which were remarkably higher than those in NMS + AA rats ( Figure 5).

Effect of Fag on
Immunofluorescence of Claudin-1 in Caco-2 Cells. The claudin-1 in the cells incubated with Fag (15, 30 μg/mL) without cytokines (100 ng/mL TNF-α and 100 ng/mL IFN-γ) for 24 h exhibited more intense immunofluorescence than the control monolayers did (Figure 8(a)). However, the fluorescence intensity decreased after being treated with cytokines. Therefore, Fag raised the fluorescence intensity of the inflammatory monolayers (Figure 8(b)).

Discussion
Previous studies have shown that NMS predisposed adult rats to colonic barrier dysfunction in response to mild stress [22] and altered the long-term colonic sensitivity to rectal distension [23]. Barreau et al. [14] found that NMS continuously altered the colonic epithelial barrier as a stress factor owing to the exaggerated expression of cytokines. Winston et al. [15] infused ten-day-old rat pups with saline containing 0.5% acetic acid intracolonically, which resulted in higher sensitivities and IFN-γ levels in the proximal colon in adult rats compared to those of the controls. In our study, a new IBS model of intestinal barrier dysfunction was established via repeatedly stimulating AA based on NMS, which excessively activated the intracolonic immunity. The results show that adult rats had visceral hypersensitivity and high permeability of the colonic mucosa, which are associated with the increased colonic MPO activity, lamina propria inflammatory cells, and cytokine expression, as well as the declined expressions of colonic epithelial claudin-1, occludin, ZO-1, and ZO-2.
We assume that the combination of NMS with AA activated the hypothalamic-pituitary-adrenal (HPA) axis, which may account for the increased translocation of pathogenic bacteria in gut rather than the reduced probiotic bacteria protection [7,24]. As a result, intestinal immunity was interfered, mucosa was destructed, and PP increased ultimately [25].
Complicatedly structured TJs, which comprise over 50 proteins, form plasma membrane-crossing fibrils and interact with proteins in the adjoining cells and the adherens junctions that are linked to the perijunctional actomyosin ring relating to the assembly of TJs and paracellular permeability [26,27]. TJs are controlled by various signaling pathways, including protein kinase C (PKC), mitogen-activated protein kinases (MAPK), myosin light chain kinase (MLCK), and the Rho family of small GTPases. Phosphorylated TJs are active and exhibit augmented epithelial barrier function. The phosphorylation of threonine residues in occludin plays a crucial role in the assembly and maintenance of TJs in Caco-2 and MDCK cell monolayers [28]. The assembly and integrity of adherens junctions and the level of TER depend on the phosphorylation of tyrosine residues in Caco-2 cells [29].  Figure 6: TER of Caco-2 cell monolayers (Ω·cm 2 ). (a) Effects of treatment time of cytokines: measured at 0, 12, 24, 36, 48, and 72 h after incubating the cells with or without cytokines (100 ng/mL TNF-α and 100 ng/mL IFN-γ). All data are expressed as mean ± SD, n = 6. a P < 0.05, controls without cytokines: 72 h versus 0 h; bbb P < 0.001, at the same time: cells with cytokines versus controls without cytokines.
Nonphosphorylated occludin concentrates on the basolateral membranes while phosphorylated occludin is mainly distributed on the membranous surface as NP-40-insoluble form, almost whole cell occludin is NP-40 soluble though [30]. Fujibe et al. [31] also confirmed that phosphorylated claudin-1 was one of the main detergent-insoluble constituent in rat lung endothelial cell line RLE. TNFα upregulated MLCK through initiating NF-κB-mediated response, leading to ZO-1 downregulation and increased colonic epithelial permeability [32,33]. IFN-γ increased the responses of epithelial monolayers to TNF-α, thus they synergetically disrupted TJs morphology and barrier function via MLCK up-regulation and myosin light chain (MLC) phosphorylation [34,35]. Sappington [36] and Han [37] found that the ZO-1, ZO-3, occludin protein and ZO-1 mRNA levels decreased after exposing enterocytes to the proinflammatory mixture of TNF-α, IFN-γ and IL-1β. VSL#3 treatment significantly lowers the visceral hypersensitivity and epithelial permeability of IBS rats, and inhibits the decreased expression of TJs of occludin and ZO-1  (b1) and (b2) were incubated with or without Fag; (b3) and (b4) were incubated with or without Fag and cytokines (100 ng/mL TNF-α and 100 ng/mL IFN-γ). The density values are normalized to those of the corresponding controls. The values are expressed as mean ± SD (n = 3). * P < 0.05, * * P < 0.01, * * * P < 0.001, the symbols indicate the differences from the controls (Fag = 0); a P < 0.05, aa P < 0.01, aaa P < 0.001, b P < 0.05, bb P < 0.01, "a" represents TJs in the cells incubated with only cytokines versus controls (TNF-α 0 + IFN-γ 0 + Fag 0); "b" represents TJs in the cells incubated with Fag and cytokines versus those incubated with cytokines alone.  [38]. Moreover, probiotics facilitate the redistribution of TJs from the cytoplasm to the membrane [39] and TJs gene expressions [40], compete for adhesion space with intestinal pathogens [41], and antagonize cytokines-induced epithelial barrier dysfunction [42]. In this study, we found that Fag and positive control drug VLS#3 not only relieved the hyperalgesia of IBS rats, but also decreased the levels of TNFα and IFN-γ, and promoted ZO-1, occluding or claudin-1 expression, which reduced the overall gut PP in a dose-dependent manner. In other words, Fag and VLS#3 integrated the intestinal barrier in IBS rats.
To further investigate the Fag mechanism, we found that in vitro TER decreased depending on time during the 24 h of incubation of the monolayer cells with TNF-α and IFN-γ, suggesting the intestinal epithelial permeability was proportional to the duration in which cells were incubated with cytokines. Moreover, cytokines downregulated four target insoluble components of TJs, indicating inhibited phosphorylation and membrane localization. We assumed that cytokines may influence the claudin-1 and ZO-1 of monolayers at the transcriptional level due to the decreased NP-40 soluble components that represent the whole cell protein level. Nevertheless, the total levels of TJs in IBS models all decreased owing to cytokines. The immunoblotting alteration of TJs in vitro was not completely consistent with that in vivo, which may be attributed to the longer-preserved and more sophisticated colonic inflammatory microenvironment in vivo than those in vitro. The underlying mechanisms need to be further explored.
The in vitro experiments show that Fag increased TER of the monolayer cells depending on concentration, and upregulated detergent-insoluble components in claudin-1, occludin, ZO-1 and ZO-2 with or without cytokines' intervention. We speculated that flavonoids such as quercetin directly intensified the phosphorylation of TJs, via integration, benefited the membrane assembly of TJs and improved intestinal barrier function. Fag might promote the expression of TJs at transcriptional level because it could sustainably restore the epithelial barrier dysfunction caused by combination treatment with TNF-α and IFN-γ and dose-dependently increase the detergent soluble components components of TJs. Probably, tannins in Fag indirectly protected the intestinal barrier from impairing by immunomodulation and prevented TJs from blocking by cytokines. The reason for the untouched total protein levels of claudin-1 and ZO-1 by Fag in the absence of cytokines is still unclear and merits further studies. So we speculated Fag may exert the direct and indirect role in reducing the colonic epithelial barrier permeability in vivo. Fag perhaps further avoided the exposure of intraepithelial nerve plexus and restored sensitizing process due to inflammation, which thus alleviated the visceral hypersensitivity of IBS rats. In a word, Fag may treat IBS via multi-target and multi-channel analgesia.
In summary, our study has demonstrated that Fag soothed the visceral hypersensitivity of model rats, possibly by inhibiting the colonic epithelium inflammation and injury, as well as by facilitating membrane localization and expressions of colonic epithelial TJs which strengthened the colonic barrier. This study herein provides a theoretical basis for Fagtreated IBS due to its multi-target and multichannel pharmacological values.