沙漠研究 19-1, 89- 92 (2009 )Journal of Arid Land Studies“Desert Technology IX ”
Refereed Paper

Effect of Capsaicin on the Tight Junctional Permeabilityof the Human Intestinal Cells
Rie UCHIDA1, Junkyu HAN1, Hiroko ISODA*1
Abstract: In arid and semi-arid lands, people eats many kinds of spices. Capsaicin is
one effect active component of spices. Previously we demonstrated that capsaicin induced
tight junction (TJ) opening in human intestinal Caco-2 cells. We showed that the increase ofTJ permeability was involved in actin polymerization. However the underlying mechanisms
are unclear. The major aims of this study were to determine the effect of capsaicin on
intestinal epithelial TJ permeability and to elucidate the mechanisms involved in this processusing an established in vitro intestinal epithelial model system consisting of Caco-2 intestinal
epithelial monolayer. Treatment Caco-2 cells with capsaicin increased TJ permeability as
measured by transepithelial electrical residence (TER). However, TJ was recovered soon.
Hence, in this study, we tested our hypothesis that influence on Caco-2 cells by the capsaicin isdifferent depending on time moment. Our data suggest that capsaicin induced different
influence depending on the time moment.
Keywords: Capsaicin, Claudin-1, Occludin, Tight Junction (TJ), Transepithelial electricalresidence (TER)

1. Introduction
Capsaicin is a major constituent of red pepper, may affect the function and permeability of the humanintestinal epithelial Caco-2 cells in vitro. In previous studies, we showed that TJ permeability increasedin capsaicin-treated human intestinal epithelial Caco-2 cells, and this mechanism involved actin
polymerization (Han et al, 2002). There is many evidence that member of the small GTPase protein
family of molecular “switches” such as Ras, Rac, RhoA, have functional roles in adhesions and tightjunction assembly and maintenance (Braga, 2002; Rubenstein et al., 2005; Giesemann et al., 2008).
Caco-2 cells are often used as an in vitro model of the intestinal epithelium. In these cells the mostapical cell-cell junction is TJ, which permeability is regulated by various factors, such as food factors andchemicals. Measurement TER value of TJ is known to be correlated with change in the paracellularpermeability of the cell monolayer (Hashimoto et al., 1997). The TJ forms a paracellular barrier on thelateral membranes of adjacent cells and acts as a structural barrier to the paracellular passage ofwater-soluble molecules bacteria (Tsukura et al., 2007). Disruption of the TJ barrier allows paracelllular
penetration of toxic luminal substances such as anti-inflammatory drugs, which promote gastrointestinal
mucosal injury. A number of proteins associated with the cytoplasmic surface of TJ have been identified,including ZO-1, ZO-2, as well as two transmembrane proteins, occludin and claudin-1 which has beensuggested to function in creating the intercellular seal (Christina et al., 1997).
In this study, we have measured the TER and paracellular flux analysis to elucidate TJ opening. The
level of polymerized actin was determined by flow cytometry. We also have performed western blot
analysis to determine the possible mechanism behind the TJ opening induced by capsaicin in Caco-2 cells.
We found that capsaicin effectively induces change expression of occludin, claudin-1 and RhoA, Rac1.
In short time treatment, capsaicin induced the same change of occludin and claudin-1 protein expression.
Although in long time treatment, capsaicin down regulated only occludin protein expression level.
2. Materials and Methods
2.1. Cell culture
Human intestinal epithelial Caco-2 cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM) supplement with 10% fetal bovine serum, 1% non-essential amino acid, 1% penicillin
streptomycin. They were incubated in an atmosphere of 5% CO2 at 37˚C. Cells from each flask were
1Graduate School of Life and Enviromentak Science, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki
305-8572, Japan*Corresponding author. Fax: +81 29 853 5776; E-mail address: isoda@sakura.cc.tsukuba.ac.jp (H. Isoda).

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detached trypsin, resuspended in fresh medium, and cultured on collagen coated dish for TER assay anddetermination of F-actin and western blot assay. The cells were seeded 1 x 105cells/ml for every assay.
2.2. TER measurement
To study the time-course effect of 100 μM capsaicin on TJ opening, TER measurement wasperformed. For the TER experiments, cells were seeded in 24 well transwell coated with collagen. Thecells were cultured about 10 days to establish monolayer integrity. Medium of the cells were changed
every other day. And the cells were treated with 100 μM capsaicin for varying time periods. Then TER
measurements were performed following the methods of Han et al. (2002).
2.3. Paracellular flux
To assess paracellular permeability, cell monolayer was established on 12 well transwell coated withcollagen. The cells were cultured under the same condition as for TER measurement condition. And thecells were incubated with 100 μM capsaicin for varying time periods in the presence of Lucifer yellow CH
dilithium salt. At given time, sample from basal side were taken and placed in 96 well plates.
Fluorescence was measured with plate reader (POWERSCAN HT; Dainippon pharmaceutical) for
excitation at 485 nm and emission at 530 nm.
2.4. Detemination of cellular F-actinDetermination the levels of polymerized actin was performed as described. Briefly Caco-2 cellmonolayers that had been incubated on the dish for 5 days were rinsed with phosphate-buffered saline(PBS) (-) and then incubated for varying time periods with or without capsaicin (100 μM). Afterincubation, the cell monolayers were detached by trypsin and cell-suspension was made. The cell lysates
were centrifuged to yield a clear lysate. Supernatant was removed, and 3.7% formealdehyde was added,
with incubatation for 15 min, vortexed and centrifuged at 8000 x g, 4˚C for 5 min. The supernatant wasremoved, and then washed with PBS (-). The cells were fixed with 0.2% TritonX and incubated for 5 min,centrifuged at 8000 x g, 4˚C for 5min and the supernatant was removed. The cells were washed by PBS(-), stained with 70 nM rhodamine-phalloidin, and washed by PBS (-). Stained cells were measured byflow cytometry (GE Healthcare).
2.5. Western blot analysis
To study the time-course effect of 100 μM capsaicin on TJ proteins and small GTPases protein
expression, Caco-2 monolayers were treated with 100 μM capsaicin for varying time periods. Aftercapsaicin treatment, Caco-2 monolayers were immediately rinsed with ice-cold PBS, and then lysed in 1 mlof cold RIPA buffer (50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% lgepal CA-630(NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) according to the manufacture’sinstruction. The cell lysates were centrifuged to yield a clear lysate. Supernatant was collected andprotein measurement was performed using 2-D Quant Kit from GE Healthcare. Loading buffer wasadded to the lysate containing 15 μg of protein and boiled for 5 min, after which proteins were separated ona SDS-PAGE gel. Proteins from the gel were transferred on a membrane (Nitrocellulose membrane).
The membrane was incubated with blocking solution (5% skimmilk, Brock ace) for 1 hour. Themembrane was incubated with appropriate primary anti-bodies in blocking solution. After being washed
in TBS-1% Tween 20 buffer, the membrane was incubated in appropriate secondary anti-bodies anddeveloped using the ECL plus Western Blotting Detection System (GE Healthcare) on Polaroid 667 film(Polaroid) using ECL TM Mini-camera (GE Healthcare).
3. Results and Discussion
Capsaicin was not cytotoxic at concentration up to 100 μM as determined by the MTT assay (data notshown). The permeability of Caco-2 cells was assessed by measuring TER and the flux of the Lucifer
yellow. TER is direction indicator to check whether TJ is tightness of the paracellular seal or not. TER
value was decreased, and after increased significantly. This indicated that TJ permeability was recovered.
The result of paracellular flux measurement also confirmed the same effect. However after TJ recovery,

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(B)

(A)

Fig.1. (A) Capsicum annum: local name is chill, red pepper, paprika, capsicum, (B) Structure of capsaicin.

(A)

MILLICELL-ERS(B)
electrode
LYmeasureCaco-2 cells
mediumCaco-2 cells

Fig.2. (A) Illustration of TER measurement. To evaluate the state of TJ is measuring the electrical resistance valueof the cell layer by an electrode. TER is decided on a cell part and permeability of the intercellular space part by
the ion. However, TER value expresses intercellular space namely a state of TJ because the latter is much bigger.
(B) Illustration of measuring of paracellular flux. This supports the TER measurement.

TER decreased slowly which indicated that TJ is open. And we confirmed that actin polymerization
regulated TJ opening.
To examine the expression of TJ proteins and small GTPases involved in TJ opening induced bycapsaicin, we performed a western blot analysis on capsaicin-treated Caco-2 cells. Since we observed TJ
opening happened two time course (long and short time) during capsaicin treatment we investigated these
proteins expression in both condition.
The intestinal epithelial TJ plays a very important role in absorption and barrier function. However,the defective intestinal epithelial TJ barrier has been proposed as an important pathogenic factor leading to
the intestinal inflammation (Welcker et al, 2004). In previous study, we demonstrated that capsaicininduced increase in Caco-2 cells TJ permeability recovery (Isoda et al., 2001; Han et al., 2005).
Clarification of the mechanism may give possiblity to regulate of absorbance and barrier function without
inflammations. The time course of capsaicin effect on Caco-2 TJ permeability suggested that thecapsaicin effect was due to acute intracellular signaling process by capsaicin binding to capsaicin receptor
TRPV1. In previous study, we clarified that capsaicin-induced increase of capsaicin receptor TRPV1
protein level and increase of Caco-2 TJ permeability (Isoda et al., 2001). The initial increase in Caco-2
TJ permeability occurred within 30min following capsaicin exposure with subsequent recovery.
Moreover, after recovery TER slowly decreased over time. In this study, we examined some of theexpression of proteins that mediated the capsaicin modulation of Caco-2 TJ barrier, TJ proteins and small
GTPases. In previous study, we indicated that capsaicin-induced increase in TJ permeability is associatedwith the cytoskeleton reorganization of the actin filaments by determining the cellular F-actin amount inCaco-2 cells (Han et al., 2002). Cofilin dephosphorylation can directly induce actin reorganization,
which can result in TJ opening (Nagumo et al., 2007). We investigated protein expression of Rac1,α-actinin and RhoA, because they were well-known interaction with actin. On the other hand, the preciserole of TJ proteins in the modulation of intestinal TJ barrier function remains unclear. Reference
suggested a correlation between occludin expression and TJ barrier function. Increase expression ofintestinal occludin and claudins cooperate in the regulation of selective paracellular permeability in MDCK

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cells (Balda et al., 2000). Moreover, a number of studies indicated a correlation between an increase inepithelial TJ permeability and a decrease in occludin expression.
4. Conclusions
In this study for the first time, we showed that 100μM capsaicin concentration increased thepermeability in Caco-2 cells. We confirmed different changes of the expression of TJ proteins and small
GTPases protein expression.
Acknowledgement
This study was supported in part by funds from the Grant-in-Aid for Scientific Research (Grant No.
17255011).
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