The Effect of Prim-O-Glucosylcimifugin on Tryptase-Induced Intestinal Barrier Dysfunction in Caco-2 Cells

Abstract

The intestinal barrier dysfunction is a critical pathological change in irritable bowel syndrome (IBS). The objective of this study was to evaluate the effect of Prim-O-glucosylcimifugin (POG) on intestinal barrier dysfunction and reveal possible molecular mechanisms. Human colon adenocarcinoma cell line (Caco-2) cell monolayers induced by tryptase (TRYP) were used to establish an intestinal barrier dysfunction model. Caco-2 cell monolayers from both functional and dysfunctional samples were treated with POG (30, 60 and 120 µg/mL) for 2, 8, 24, 36, 48 and 72 h. The Caco-2 cell monolayers were assessed by measurement of trans-epithelial electrical resistance (TEER) and percentage of fluorescein permeation (PFP). The expression of Protease Activated Receptor 2 (PAR-2) and myosin light chain kinase (MLCK) mRNA was analyzed by RT-PCR and the level of Zonula Occludens-1 (ZO-1) protein expression was determined by Western blot. In addition, the impact of POG on the distribution of the tight juction protein of Occludin was performed by immunofluorescence. Our results showed that POG elevated the TEER and decreased the PFP of the functional Caco-2 cell monolayers, as well as the dysfunctional Caco-2 cell monolayers. Furthermore, POG inhibited the expression of PAR-2 mRNA and MLCK mRNA and increased the level of ZO-1 protein expression in dysfunctional Caco-2 cells. The distribution of the Occludin proteins was ameliorated simultaneously. This study demonstrates that POG can enhance the intestinal barrier function of Caco-2 cell monolayers by inhibiting the expression of PAR-2 and MLCK and up-regulating the expression of ZO-1 protein, and ameliorated the distribution of Occludin protein.

The intestinal barrier plays an important role in the maintenance of the body’s health. It not only protects the intestinal tract from harmful substances, but also improves the immune function of the gut.^1,2) Disorders of the intestinal barrier are associated with the pathogenesis of Irritable Bowel Syndrome (IBS), caused gastrointestinal tract symptoms, such as diarrhea and abdominal pain.^3,4) Moreover, IBS is a chronic relapsing disease in which symptoms may vary over time, bringing great inconvenience to the life of the patients.

Tryptase (TRYP), a serine protease, is released by mast cells upon degranulation. An increased release of TRYP has been reported in patients with diarrheic IBS (D-IBS).⁵⁾ TRYP can activate the intestinal Protease Activated Receptor 2 (PAR-2), inducing intestinal barrier dysfunction and visceral pain sensitivity.⁶⁾ PAR-2 is a serine protease receptor that is highly expressed on intestinal epithelial cells. Then the activation of PAR-2 increases the expression of myosin light chain kinase (MLCK) and affects the tight junction proteins, such as Zonula Occludens-1 (ZO-1) protein and Occludin protein, that make up the intestinal epithelial barrier.⁷⁾ MLCK plays an important role in altering the intestinal epithelial tight junctions (TJs). MLCK transcription induces an increase in MLCK protein activity, which in turn promotes the phosphorylation of myosin light chain (MLC) and then changes the structure and distribution of TJ protein (ZO-1 protein and Occludin protein)^8–10) which also plays a pivotal role in the tight junctions of the intestinal epithelial barrier.¹¹⁾

Saposhnikovia divaricata (TURCZ.) SCHISCHK (SD, Apiaceae), called “FangFeng” in Chinese, is widely distributed in northeast China. SD has been found to exert multiple pharmacological effects, including analgesic, anti-diarrheal, anti-inflammatory, anti-oxidant, and anti-ulcerative effects, as well as to regulate intestinal motility.^12–15) What attracted our attention was that there have been reported that SD exerts anti-diarrheal and analgesic effects in rats with IBS.^16–18) And its therapeutic outcome have been attributed to the down-regulation of PAR-2 receptor expression and the reduction of the levels of substance P (SP), tumor necrosis factor-α (TNF-α) and interleukin (IL-6) in the colonic mucosa.¹⁹⁾ The effective substance remains unknown. Several kind of compounds have been isolated from SD, including chromones, coumarins, mannitol, glycoside, and polyacetylenes.^20,21) The major active constituents of SD are considered to be chromones and their glycosides.^20,22–24) Prim-O-glucosylcimifugin (POG) (Fig. 1) is the highest content chromone of SD.¹⁵⁾ Therefore, the present study was aimed to evaluate the effect of different doses of POG on intestinal barrier dysfunction and explore its possible molecular mechanisms.

Fig. 1. The Structural Formula of the Prim-O-Glucosylcimifugin

The effect of POG was examined on both a normal intestinal barrier and a dysfunctional intestinal barrier induced by TRYP. The possible molecular mechanisms of the effect of POG on the intestinal barrier were investigated. For this purpose, human colon adenocarcinoma cell line (Caco-2) cell monolayers of both functional and dysfunctional were treated with POG, at doses of 30, 60 and 120 µg/mL. The trans-epithelial electrical resistance (TEER) and percentage of fluorescein permeation (PFP) were measured at 2, 8, 24, 36, 48, 72 h after treatment POG. Additionally, the PAR-2 and MLCK mRNA expression levels in dysfunction Caco-2 cell monolayers were analyzed by RT-PCR, and the level of ZO-1 protein expression was measured by Western blot. Simultaneously, the expression and distribution of the Occludin protein were examined using immunofluorescence.

MATERIALS AND METHODS

Materials

Dulbecco’s modified Eagle medium (DMEM) and phosphate buffered saline (PBS) were obtained from Gibco (U.S.A.). Fetal bovine serum (FBS) and TRIzol Reagent were purchased from Invitrogen (U.S.A.). PAR-2 and MCLK PCR primers were synthesized by Sangon Biotech Co., Ltd. ZO-1, Occludin and β-actin antibodies were obtained from Abcam Inc. (U.K.). Tryptase, FSSRY-NH₂ and fluorescein were purchased from Sigma (U.S.A.). PrimeScript 1st-Strand cDNA Synthesis Kit and Primer Taq were purchased from TaKaRa (Japan). The total protein assay kit was obtained from Nanjing Jiancheng Bioengineering Institute (China). MEM Non-Essential Amino Acids (NEAA) Solution was purchased from Thermo Scientific (U.S.A.). Prim-O-glucosylcimifugin (98% pure) were supplied by Sichuan Weikeqi Biological Technology Co., Ltd. (China).

Cell Line and Culture Conditions

Caco-2 was obtained from the American Type Culture Collection (HTB037). Caco-2 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% NEAA at 37°C in an atmosphere with 95% air and 5% CO₂ and utilized for experimentation at approximately 70–80% confluence. And the Caco-2 cell monolayers were formed after 20–22 d-cultures, until the TEER value was more than 300 Ω·cm₂.

Cell Viability

The POG toxicity was determined using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) (Sigma) assay. Caco-2 cells were seeded into 96-well plates at a density of 10000/cell, with each well containing 200 µL, After adherence, the cells were treated with various concentrations of POG (10, 50, 100, 250, 500, 1000, 5000 and 10000 µg/mL) or dimethyl sulfoxide (DMSO, Sigma; control cells) for 24 h before 10 µL MTT in medium was added to each well. the cells were incubated at 37°C for 4 h. Then the absorbance was detected at 490 nm using a microtiter plate reader (Bioteck Instruments).

Culture of Caco-2 Cell Monolayers and Determination of Barrier Function

The Caco-2 cell monolayers were cultured, and the determination of barrier function was performed as previously described.²⁵⁾ Briefly, Caco-2 cells were transplanted on the apical compartment of collagen-coated polycarbonate membrane Transwell supports (transparent polyethylene terephthalate (PET) membrane: 1.0-cm² growth surface area, 0.4-µm pore size) in 24-well plates at 1×10⁵ cells/well. The apical and basolateral compartment respectively are 100 and 600 µL medium and incubated with appropriate culture conditions. The Caco-2 cells monolayers were grown on the Transwell, and the medium was replaced every 48 h. Recordings of TEER values were obtained every 24 h until they stabilized on 20–22 d using an Epithelial Voltohmmeter (World Precision Instruments) in each insert and multiplied by the membrane surface area, corrected by subtracting background resistance of the blank membrane (no cells). A 20–22-d model was used to study barrier functiion. It has been showen that after day 20–22 the cell monolayers were differentiated. The TEER values of the model Caco-2 cell monolayers was measured at 2, 8, 24, 36, 48 and 72 h after treatment with POG (30, 60 and 120 µg/mL). The same to the measurement of PFP values, 40 µg/mL fluorescein (dioxyfluoran, C₂₀H₁₂O₅) was added to the apical compartment, and the Transwells were incubated for 1 h. Then, 100 µL of medium from the apical and basal compartments was collected into 96-well plates, and the fluorescein was measured using a microplate reader at excitation and emission wavelengths of 485 and 540 nm, respectively. The monolayer permeability of fluorescein was quantified as the PFP from the apical to the basal compartment.

The dysfunctional Caco-2 cell monolayers from the intestinal barrier were induced by TRYP (Sigma) as previously described.²⁶⁾ Briefly, the model Caco-2 cell monolayers were incubated with TRYP (1.3 µmol/L) from the basal side for 72 h. To assess the effect of POG on intestinal barrier function, dyfunctional Caco-2 cell monolayers were incubated with POG (30, 60 and 120 µg/mL). The TEER and the PFP of the cell monolayers were determined as previously described.

RT-PCR Analysis of PAR-2 and MLCK mRNA Expression

The total RNA from the Caco-2 cells was extracted using the TRIzol reagent dissolved in DEPC-treated water according to the manufacturer’s instructions. RNA was reverse-transcribed to cDNA using oligo (dT) primers by PrimeScript 1st-Strand cDNA Synthesis Kit in a final volume of 20 µL under the conditions recommended by the supplier (TaKaRa). For PCR amplification of PAR-2 and β-actin, 1 µL of the cDNA template and the following specific primers were used: PAR-2 F, 5′-CCC TTT GTA TGT CGT GAA GCA GAC-3′; R, 5′-TTC CTG GAG TGT TTC TTT GAG GTG-3′ (452 bp); β-actin F, 5′-TTG TAA CCA ACT GGG ACG ATA TGG-3′; R, 5′-GAT CTT GAT CTT CAT GGT GCT AGG-3′ (754 bp). The reaction cycle conditions were the following: 5 min at 95°C and 35 amplification cycles consisting of denaturation at 95°C for 40 s, annealing at 60°C for 40 s, and extension at 72°C for 60 s. And for MLCK and β-actin, 1 µL of cDNA template and the following specific primers were used: MLCK F, 5′-TCT GAA CTC ACA ACG GTA GG-3′; R, 5′-GCT CAA AGT CCT CGT CAA-3′ (376 bp); β-actin F, 5′-TCA CCC ACA CTG TGC CCA TCT ACG A-3′; R, 5′-CAG CGG AAC CGC TCA TTG CCA ATG G-3′ (300 bp). The reaction cycle conditions: 5 min at 94°C and 30 amplification cycles consisting of denaturation at 94°C for 30 s, annealing at 54°C for 45 s, and extension at 72°C for 60 s.

The PCR products were resolved via 2% agarose gel electrophoresis and visualized through ethidium bromide staining. The PCR products of predicted size were stained using ethidium bromide and visualized by an UV transilluminator. Quantification of each band was performed using a Scion Image densitometry analysis software package.

Western Blot Analysis of Expression of ZO-1

Briefly, the cells were washed with pre-cooled PBS and subsequently lysed with RIPA lysis buffer. The protein concentration was determined through the bicinchoninic acid (BCA) assay reagent with the Bradford method. For Western blotting, protein samples (20 µg) of Caco-2 cells were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The samples were then transferred onto polyvinylidene difluoride (PVDF) membrane and incubated with primary antibody (anti-ZO-1 with dilution: 1 : 500 or anti-β-actin with dilution: 1 : 1200) overnight at 4°C and then with the secondary antibody (horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG)) for 2 h. The intensity of the immunoblot signal was determined by the ECL Western blotting detection reagent according to the manufacturer’s instructions and the expression of ZO-1 was analyzed quantitatively using the GeneTools software from Syngene.

Immunofluorescence Analysis of the Distribution of Occludin Proteins

The distribution occludin protein was analyzed with immunofluorescence microscopy. Cells (3×10⁶ cells/well) in 6-well plates were fixed with 4% paraformaldehyde at room temperature for 30 min after POG treatment for 36 h. Then cells were permeabilized with PBS containing 0.1% Triton and then blocked with blocking buffer containing 0.3% bovine serum albumin (BSA) for 30 min at room temperature. Cells were incubated with Occludin antibody (1 : 1000) overnight at 4 degree and then washed with PBS three times for 15 min and incubated with fluorescein isothiocyanate (FITC) marker Goat anti rabbit IgG (1 : 100) at room temperature for another 1 h. Observe and record the result as soon as possible with fluorescence microscopy at the condition of excitation wavelength 492 nm and emission wavelength 520 nm.

Statistical Analysis

All experimental data has been presented as the mean±standard deviation (S.D.), and the analyses of the significance of the differences were performed via one-way ANOVA followed by Student–Newman–Keuls test (S-N-K) using SPSS 17.0. Differences were considered significant if p<0.05.

RESULTS

The POG toxicity was illustrated in Fig. 2. The viability of Caco-2 cell incubated with POG (250, 500, 1000, 5000 and 10000 µg/mL) for 24 h was decreased compared with the control cells (p<0.05). In the range of POG concentration 10–250 µg/mL, the viability of Caco-2 cell was above 80%.

Fig. 2. The Viability of Caco-2 Cell Incubated with POG (10, 50, 100, 250, 500, 1000, 5000 and 10000 µg/mL) for 24 h (n=6)

Control: 0 µg/mL. * p<0.05 compared with the control cells; ^∆ p<0.01 compared to the control cells.

The TEER value of the normal Caco-2 cell monolayers that were incubated with POG (30, 60 and 120 µg/mL) for 8, 24, 36 and 48 h was significantly elevated compared with the control cells (p<0.05), contraried to the PFP (Figs. 3A, B). It can be seen in Figs. 4A, B that the TEER and PFP value of the dyfunctional Caco-2 cell monolayers induced by TRYP was obviously decreased and increased respectively compared with the control cells (p<0.05). And POG (30, 60 and 120 µg/mL) for 2, 8, 24, 36, and 48 h significantly increased the TEER and decreased PFP compared with the dyfunctional cell monolayers without treatment with POG (p<0.05). Our data showed that the addition of varying doses of POG produced a dose-dependent increase in the TEER and a decrease in the PFP on both functional and dyfunctional cell monolayers, suggesting that POG can promote the barrier function of Caco-2 cell monolayers in a dose-dependent manner.

Fig. 3. Effects of POG on Barrier Function in Normal Caco-2 Cells (n=9)

(A) Effect of POG on transepithelial electrical resistance (TEER) in Caco-2 cell monolayers. Control: 0 µg/mL POG; POG-30 µg/mL: 30 µg/mL POG; POG-60 µg/mL: 60 µg/mL POG; POG-120 µg/mL: 120 µg/mL POG. * p<0.05 compared with the control cells; ^∆ p<0.01 compared to the control cells. (B) Effect of POG on the percentage of fluorescein permeation (PFP) in Caco-2 cell monolayers. Control: 0 µg/mL POG; POG-30 µg/mL: 30 µg/mL POG; POG-60 µg/mL: 60 µg/mL POG; POG-120 µg/mL: 120 µg/mL POG. * p<0.05 compared with the control cells.

Fig. 4. Effect of POG on the Dysfunctional Caco-2 Cell Monolayer Induced by TRYP (n=9)

(A) Effect of POG on the transepithelial electrical resistance (TEER) in Caco-2 cell monolayers. Control: 0 µg/mL POG and 0 µmol/L tryptase; Tryptase: 0 µg/mL POG and 1.3 µmol/L tryptase; POG-30 µg/mL: 30 µg/mL POG and 1.3 µmol/L tryptase; POG-60 µg/mL: 60 µg/mL POG and 1.3 µmol/L tryptase; POG-120 µg/mL: 120 µg/mL POG and 1.3 µmol/L tryptase. ^□ p<0.01 compared with the control cells; * p<0.05 compared with the cells induced with tryptase; ^∆ p<0.01 compared with the cells induced with Tryptase. (B) Effect of POG on the percentage of fluoresce in permeation (PFP) in Caco-2 cell monolayers. Control: 0 µg/mL POG and 0 µmol/L tryptase; Tryptase: 0 µg/mL POG and 1.3 µmol/L tryptase; POG-30 µg/mL: 30 µg/mL POG and 1.3 µmol/L tryptase; POG-60 µg/mL: 60 µg/mL POG and 1.3 µmol/L tryptase; POG-120 µg/mL: 120 µg/mL POG and 1.3 µmol/L tryptase. ^□ p<0.01 compared with the control cells; * p<0.05 compared with the cells induced with tryptase; ^∆ p<0.01 compared with the cells induced with tryptase.

As shown in Fig. 5, the PAR-2 mRNA expression of the dysfunctional Caco-2 cell monolayers induced by TRYP was much more than the control cells (p<0.05). POG (30, 60 and 120 µg/mL) reduced the expression of PAR-2 mRNA compared with the dyfunctional cell monolayers (p<0.05). FSSRY-NH₂, which is the PAR-2 antagonist and was used as a positive control drug, distinctly affected the inhibition of the expression of PAR-2 mRNA compared with the dyfunctional cell monolayers (p<0.05).

Fig. 5. Effect of POG on PAR-2 mRNA Expression in the Dysfunctional Caco-2 Cell Monolayer Induced by TRYP

The Caco-2 cells were incubated with POG (30, 60 and 120 µg/mL) for 36 h. The expression of PAR-2 mRNA was detected via RT-PCR as described in the text. The figure represents one of four experiments with similar results. Control: control group; Tryptase: 1.3 µmol/L tryptase; POG-30: 30 µg/mL POG and 1.3 µmol/L tryptase; POG-60: 60 µg/mL POG and 1.3 µmol/L tryptase; POG-120: 120 µg/mL POG and 1.3 µmol/L tryptase; FSSRY-NH₂: 30 µmol/L FSSRY-NH₂ and 1.3 µmol/L tryptase. ^# p<0.05 compared with the control cells; * p<0.05 compared with the cells induced with tryptase.

The MLCK mRNA expression of the dysfunctional Caco-2 cell monolayers induced by TRYP was increased compared with the control cells (p<0.05) in Fig. 6. After treatment with POG (30, 60 and 120 µg/mL), the MLCK mRNA expression was decreased compared with the dyfunctional cell monolayers (p<0.05).

Fig. 6. Effect of POG on MLCK mRNA Expression in the Dysfunctional Caco-2 Cell Monolayer Induced by TRYP

The Caco-2 cells were incubated with POG (30, 60 and 120 µg/mL) for 36 h. The expression of MLCK mRNA was detected via RT-PCR as described in the text. The figure represents one of four experiments with similar results. Control: control group; Tryptase: 1.3 µmol/L tryptase; POG-30: 30 µg/mL POG and 1.3 µmol/L tryptase; POG-60: 60 µg/mL POG and 1.3 µmol/L tryptase; POG-120: 120 µg/mL POG and 1.3 µmol/L tryptase; FSSRY-NH₂: 30 µmol/L FSSRY-NH₂ and 1.3 µmol/L tryptase. ^# p<0.05 compared with the control cells; * p<0.05 compared with the cells induced with tryptase.

The ZO-1 protein expression in the dysfunctional Caco-2 cell monolayers induced by TRYP was decreased compared with the control cells (p<0.05). After treatment with POG (30, 60 and 120 µg/mL), the ZO-1 protein expression was significantly elevated compared with the dyfunctional cell monolayers (p<0.05). FSSRY-NH₂, applied as a positive control drug, significantly increased the expression of ZO-1 protein compared with the dyfunctional cell monolayers (p<0.05) (Fig. 7).

Fig. 7. Effect of POG on ZO-1 Protein Expression in the Dysfunctional Caco-2 Cell Monolayer Induced by TRYP

The Caco-2 cells were incubated with POG (30, 60 and 120 µg/mL) for 36 h. The protein expression of ZO-1 was detected via Western blotting as described in the text. The figure represents one of four experiments with similar results. Control: control group; Tryptase: 1.3 µmol/L tryptase; FSSRY-NH₂: 30 µmol/L FSSRY-NH₂ and 1.3 µmol/L tryptase; POG-120: 120 µg/mL POG and 1.3 µmol/L tryptase; POG-60: 60 µg/mL POG and 1.3 µmol/L tryptase; POG-30: 30 µg/mL POG and 1.3 µmol/L tryptase. ^# p<0.05 compared with the control cells; * p<0.05 compared with the cells induced with tryptase.

In Fig. 8. There was no significant difference in fluorescence intensity between each group. However, in the dysfunctional Caco-2 cell monolayers induced by TRYP, the occludin protein was loose, apparently serrated, or notched and fissured (Fig. 8B). Apparently, in POG (30, 60 and 120 µg/mL) treatment groups, the green fluorescence distribution showed obvious integrity and clear boundary. And the higher the dose, the more obvious the effect.

Fig. 8. The Distribution of Tight Junction Protein Occludin in the Dysfunctional Caco-2 Cell Monolayer Induced by TRYP (100*)

The Caco-2 cells were incubated with POG (30, 60 and 120 µg/mL) for 36 h. The protein location of Occludin was detected via Immunofluorescence as decribed in the text. A: control; B: 1.3 µmol/L tryptase; C: 30 µg/mL POG and 1.3 µmol/L tryptase; D: 60 µg/mL POG and 1.3 µmol/L tryptase; E: 120 µg/mL POG and 1.3 µmol/L tryptase; F: 30 µmol/LFSSRY-NH₂ and 1.3 µmol/L tryptase. (Color figure can be accessed in the online version.)

DISCUSSION

This study demonstrated that POG can promote intestinal barrier function and ameliorate intestinal barrier dysfunction in Caco-2 cells. These effects are possibly mediated through down-regulating the expression of PAR-2 and MLCK mRNA and enthance the level of ZO-1 protein expression, ameliorate the distribution of Occludin protein.

The intestinal barrier is a crucial component of gut homeostasis that defends against many pathogens, such as bacteria, toxins and food antigens.²⁷⁾ Caco-2 cells, which exhibit a well-differentiated brush border, tight junctions and intestinal proteins, have been widely used to investigate the drug’s effect on paracellular permeability and as an intestinal barrier model in vitro.^26,28) In the present study, our results demonstrated that POG can increase the TEER and reduce the PFP in Caco-2 cells, which shows that POG promoted epithelial barrier function in a dose-dependent manner (Fig. 3). In agreement with our present study, Elamin et al. used Caco-2 cells as an intestinal barrier model and found that short-chain fatty acids (SCFAs) can reinforce the barrier function in Caco-2 cells by measuring the TEER and paracellular permeability.²⁹⁾

Clinical data suggest that a high level of TRYP released by mast cells upon degranulation in D-IBS, is associated with alteration in intestinal barrier function.³⁰⁾ TRYP increases the permeability of colon tissue in diarrhea-predominant IBS patients.³¹⁾ A study conducted by Ludidi et al. revealed that TRYP can increase the permeation flux of 4-kDa fluorescein isothiocyanate-labelled dextran (FD4) in Caco-2 cells, indicating an effect of enhancing paracellular permeability and intestinal barrier dysfunction.³²⁾ In our study, we induced Caco-2 cell monolayers with TRYP, and this treatment reduced the TEER and increased the PFP. After treatment with POG, the TEER obviously increased, and the PFP decreased compared with the values obtained in the TRYP-induced intestinal barrier dysfunction Caco-2 cells (Fig. 4). The current evidence of the effect of POG on the intestinal barrier is limited. Two similar studies conducted showed that the active ingredients of SD affect the intestinal permeability and decrease the level of nitric oxide (NO) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals.³³⁾

The intestinal barrier dysfunction in IBS is associated with the disturbance of gut sensory and motor functions. PAR-2 activation is responsible for intestinal barrier dysfunction and the sensitization of sensory neurons in patients with IBS.³⁴⁾ PAR-2 belongs to the PARs family and is distributed throughout the gastrointestinal tract, localized in epithelial cells, myocytes and enteric neurons.³⁵⁾ The activation of PAR-2 on the intestinal epithelial barrier directly affects cytoskeletal contraction by triggering relevant paracellular pathways with subsequent changes in tight junction permeability. PAR-2 is activated by serine-proteases, such as trypsin and tryptase, which in turn brings great impact on epithelial migration, integrity, and barrier function.³⁶⁾ A study conducted by Piche et al. demonstrated that Caco-2 cells exposed to tryptase in the plasma of IBS-D patients elevates the expression of PAR-2 mRNA, leading to an increase in paracellular permeability and a reduction in TEER.³⁷⁾ In the present study, the results showed that the level of PAR-2 mRNA significantly elevated in dysfunctional Caco-2 cells and POG would down-regulated the expression of PAR-2 mRNA (Fig. 5).

The activation of MLCK is vital to the enhancement of paracellular permeability and is essential for tight junction barrier regulation in response to Na⁺-nutrient cotransport, inflammatory cytokines, or pathogenic bacteria.^38,39) MLCK can change the junctional actomyosin ring structure and tight junction morphology.⁴⁰⁾ The integrity of tight junctions depends on many tight junction proteins, which make up the normal intestinal architecture. ZO-1 and occludin proteins are essential for cytoskeletally-mediated barrier regulation.⁴¹⁾ Disruption and redistribution of the ZO-1 and occludin proteins could lead to tight junction barrier dysfunction. These changes in ZO-1 and occludin proteins are the result of the activation of MLCK.¹⁰⁾ In the dysfunctional Caco-2 cells, our data suggested that MLCK mRNA expression was significantly increased and ZO-1 protein expression was remarkably reduced. Although the expression level of occludin did not decrease obviously, the distribution of occludin protein changed remarkable. After treatment with POG, the expression of MLCK mRNA were down-regulated and the level of ZO-1 were enhanced. Beyond that, the POG would ameliorate the distribution of Occludin proteins (Figs. 6–8).

In conclusion, our research shows that POG has potential therapeutic efficacy in the treatment of IBS. Down-regulation of PAR-2 and MLCK expression, up-regulation of ZO-1 protein expression and ameliorated the distribution of Occludin proteins may be the possible molecular mechanism. Therefore, the beneficial effects of POG on intestinal barrier dysfunction suggest that POG may be a promising therapeutic drug for some diseases associated with intestinal barrier dysfunction, such as IBS.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 81673620) and the Joint Natural Sciences Fund of the Department of Science and Technology and the First Affiliated Hospital of Guangdong Pharmaceutical University (No. GYFYLH201328).

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Halpern MD, Denning PW. The role of intestinal epithelial barrier function in the development of necrotizing enterocolitis. Tissue Barriers, 3, e1000707 (2015).
• 2) Coskun M. Intestinal epithelium in inflammatory bowel disease. Front. Med., 1, 24 (2014).
• 3) Saha L. Irritable bowel syndrome: pathogenesis, diagnosis, treatment, and evidence based medicine. World J. Gastroenterol., 20, 6759–6773 (2014).
• 4) Beatty JK, Bhargava A, Buret AG. Post-infectious irritable bowel syndrome: mechanistic insights into chronic disturbances following enteric infection. World J. Gastroenterol., 20, 3976–3985 (2014).
• 5) Liang WJ, Zhang G, Luo HS, Liang LX, Huang D, Zhang FC. Tryptase and protease-activated receptor 2 expression levels in irritable bowel syndrome. Gut & Liver., 10, 382–390 (2016).
• 6) Lee JW, Park JH, Park DI, Park JH, Kim HJ, Cho YK, Sohn CI, Jeon WK, Kim BI. Subjects with diarrheapredominant IBS have increased rectal permeability responsive to tryptase. Dig. Dis. Sci., 55, 2922–2928 (2010).
• 7) Amadesi S, Bunnett N. Protease-activated receptors: protease signaling in the gastrointestinal tract. Curr. Opin. Pharmacol., 4, 551–556 (2004).
• 8) González-Mariscal L, Tapia R, Chamorro D. Crosstalk of tight junction components with signaling pathways. Biochim. Biophys. Acta, 1778, 729–756 (2008).
• 9) Shen L. Tight junctions on the move: molecular mechanisms forepithelial barrier regulation. Ann. N. Y. Acad. Sci., 1258, 9–18 (2012).
• 10) Yu D, Marchiando AM, Weber CR, Raleigh DR, Wang Y, Shen L, Turner JR. MLCK-dependent exchange and actin binding regiondependent anchoring of ZO-1 regulate tight junction barrier function. Proc. Natl. Acad. Sci. U.S.A., 107, 8237–8241 (2010).
• 11) Lee SH. Intestinal permeability regulation by tight junction: implication on inflammatory bowel diseases. Intest Res., 13, 11–18 (2015).
• 12) Chinese Pharmacopoeia Commission. 2010. Pharmacopoeia of the People’s Republic of China. Chinese Medical Science and Technology Press, Beijing. p. 140. (2010)
• 13) Zhao B, Yang X, Yang X, Zhang L. Biotransformation of prim-O-glucosylcimifugin by human intestinal flora and its inhibition on NO production and DPPH free radical. Chung Kuo Chung Yao Tsa Chih, 35, 1569–1572 (2010).
• 14) Chun JM, Kim HS, Lee AY, Kim SH, Kim HK. Anti-inflammatory and antiosteoarthritis effects of Saposhnikovia divaricata ethanol extract: in vitro and in vivo studies. Evidence-Based Complementray and Alternative Medicine, 2016, 1984238 (2016).
• 15) Kong X, Liu C, Zhang C, Zhao J, Wang J, Wan H, Zhu H, Zhang P, Chen W, Xiao Y, Lin N. The suppressive effects of Saposhnikovia divaricata (POG) chromone extract on rheumatoid arthritis via inhibition of nuclear factor-κB and mitogen activated proteinkinases activation on collagen-induced arthritis model. J. Ethnopharmacol., 148, 842–850 (2013).
• 16) Hu X, Xu D, Zhao Y, Yang X, Meng J, Shen H, Guo J. The alleviating pain effect of aqueous extract from tong-xie-yao-fang, on experimental visceral hypersensitivity and its mechanism. Biol. Pharm. Bull., 32, 1075–1079 (2009).
• 17) Tan X, Han Z, Wen Y, Liu D, Hu X. Effects of tongxie yaofang and radix saposhnikoviae on the barrier functions of human intestinal Caco-2 cell. Shenzhen J. Trad. Chi. Med., 24, 1–3 (2014).
• 18) Qi S, Cai J, Zhou L, Yang T, Tan X, Li M, Han B, Hu X. Effect of radix saposhnikoviae on intestinal flora and serine protease signal in rats with post-infection irritable bowel syndrome. Trad. Chi. Dru. Res. Cli. Pharmacol., 26, 790–796 (2015).
• 19) Hu X, Liao S, Wang Y, Gong M, Wang M, Liu S, Han B. Effect of Radix Saposhnikoviae in Tongxie Yaofang on PAR2 mRNA expression and inflammatory mediators in the colon of rats with postinfection irritable bowel syndrome. Trad. Chi. Dru. Res. Cli. Pharmacol., 24, 1–7 (2013).
• 20) Gui Y, Tsao R, Li L, Liu CM, Wang J, Zong X. Preparative separation of chromones in plant extract of Saposhnikovia divaricata by high-performance counter-current chromatography. J. Sep. Sci., 34, 520–526 (2011).
• 21) Chen L, Chen X, Su L, Jiang Y, Liu B. Rapid characterisation and identification of compounds in Saposhnikoviae Radix by high-performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight mass spectrometry. Nat. Prod. Res., 32, 898–901 (2018).
• 22) Scherübl R, Beggs A, Franz G, Heilmann J, Manns D. Analysis of chromone glycosides in saposhnikovia divaricata for the establishment of a monograph of the german pharmacopoeia. Planta Med., 78, PI149 (2012).
• 23) Jiang YY, Liu B, Shi RB, Tu GZ. Isolation and structure identification of chemical constituents from Saposhnikovia divaricata (Turcz.) Schischk. Yao Xue Xue Bao, 42, 505–510 (2007).
• 24) Li YY, Wang XX, Zhao L, Zhang H, Lv L, Zhou GC, Chai YF, Zhang GQ. High-performance liquid chromatography-electrospray ionization time-of-flight mass spectrometry analysis of radix saposhnikoviae for metabolomic research. J. Chromatogr. Sci., 51, 99–106 (2013).
• 25) Li N, Gu L, Qu L, Gong J, Li Q, Zhu W, Li J. Berberine attenuates pro-inflammatory cytokine-induced tight junction disruption in an in vitro model of intestinal epithelial cells. Eur. J. Pharm. Sci., 40, 1–8 (2010).
• 26) Bueno L, Fioramonti J. Protease-activated receptor 2 and gut permeability: a review. Neurogastroenterol. Motil., 20, 580–587 (2008).
• 27) Hidalgo IJ, Raub TJ, Borchardt RT. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology, 96, 736–749 (1989).
• 28) Sambuy Y, De Angelis I, Ranaldi G, Scarino ML, Stammati A, Zucco F. The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol. Toxicol., 21, 1–26 (2005).
• 29) Elamin EE, Masclee AA, Dekker J, Pieters HJ, Jonkers DM. Short-chain fatty acids activate AMP-activated protein kinase and ameliorate ethanol-induced intestinal barrier dysfunction in Caco-2 cell monolayers. J. Nutr., 143, 1872–1881 (2013).
• 30) Lee JW, Park JH, Park DI, Park JH, Kim HJ, Cho YK, Sohn CI, Jeon WK, Kim BI. Subjects with diarrhea-predominant IBS have increased rectal permeability responsive to tryptase. Dig. Dis. Sci., 55, 2922–2928 (2010).
• 31) Roka R, Rosztoczy A, Leveque M, Izbeki F, Nagy F, Molnar T, Lonovics J, Garcia-Villar R, Fioramonti J, Wittmann T, Bueno L. A pilot study of Fecal serine-protease activity: a pathophysiologic factor in diarrhea-predominant irritable bowel syndrome. Clin. Gastroenterol. Hepatol., 5, 550–555 (2007).
• 32) Ludidi S, Jonkers L, Elamin E, Pieters HJ, Schaepkens E, Bours P, Kruimel J, Conchillo J, Masclee A. The intestinal barrier in irritable bowel syndrome: subtype-specific effects of the systemic compartment in an in vitro model. PLOS ONE, 10, e0123498 (2015).
• 33) Zhao B, Yang XB, Yang XW, Liu JX. Biotransformation of prim-O-glucosylcimifugin by human intestinal flora and its inhibition on NO production and DPPH free radical. J. Asian Nat. Prod. Res., 14, 886–896 (2012).
• 34) Salim SY, Soderholm JD. Importance of disrupted intestinal barrier in inflammatory bowel diseases. Inflamm. Bowel Dis., 17, 362–381 (2011).
• 35) Cenac N, Coelho AM, Nguyen C, Compton S, Andrade-Gordon P, MacNaughton WK, Wallace JL, Hollenberg MD, Bunnett NW, Garcia-Villar R, Bueno L, Vergnolle N. Induction of intestinal inflammationin mouse by activation of proteinase-activated receptor-2. Am. J. Pathol., 161, 1903–1915 (2002).
• 36) Wilcz-Villega EM, McClean S, O’Sullivan MA. Mast cell tryptase reduces junctional adhesion molecule-A (JAM-A) expression in intestinal epithelial cells: implications for the mechanisms of barrier dysfunction in irritable bowel syndrome. Am. J. Gastroenterol., 108, 1140–1151 (2013).
• 37) Piche T, Barbara G, Aubert P, Bruley des Varannes S, Dainese R, Nano JL, Cremon C, Stanghellini V, De Giorgio R, Galmiche JP, Neunlist M. Impaired intestinal barrier integrity in the colon of patients with irritable bowel syndrome: involvement of soluble mediators. Gut, 58, 196–201 (2009).
• 38) Nusrat A, Turner JR, Madara JL. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells. Am. J. Physiol. Gastrointest. Liver Physiol., 279, G851–G857 (2000).
• 39) Shen L, Black ED, Witkowski ED, Lencer WI, Guerriero V, Schneeberger EE, Turner JR. Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure. J. Cell Sci., 119, 2095–2106 (2006).
• 40) Clayburgh DR, Barrett TA, Tang Y, Meddings JB, Van Eldik LJ, Watterson DM, Clarke LL, Mrsny RJ, Turner JR. Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo. J. Clin. Invest., 115, 2702–2715 (2005).
• 41) Furuse M, Itoh M, Hirase T, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S. Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions. J. Cell Biol., 127, 1617–1626 (1994).