Acer palmatum thumb. Ethanol Extract Alleviates Interleukin-6-Induced Barrier Dysfunction and Dextran Sodium Sulfate-Induced Colitis by Improving Intestinal Barrier Function and Reducing Inflammation

Ulcerative colitis is one inflammatory bowel disease (IBD) and is caused by diverse factors, including the extent and duration of intestinal inflammation. We investigated the effect of Acer palmatum thumb. ethanol extract (KIOM-2015E) on the expression of tight junction proteins and the levels of inflammation in the cell model induced with interleukin-6- (IL-6-) and mouse model of dextran sodium sulfate (DSS) induced with acute colitis. KIOM-2015E (100 mg/kg) was orally administered once per day to BALB/C mice with colitis induced by administration of 5% DSS in drinking water. KIOM-2015E did not affect viability in Caco-2 cells. Also, KIOM-2015E repaired the IL-6-induced intestinal barrier dysfunction in Caco-2 cells. Furthermore, KIOM-2015E recovered the loss of body weight and the abnormally short colon lengths in the DSS-induced model of acute colitis. Moreover, KIOM-2015E significantly inhibited the decrease of zonula occluden-1 and occludin in colonic tissue relative to the DSS-treated control group. KIOM-2015E also significantly inhibited the expression of IL-6 and tumor necrosis factor-α in the level of serum relative to the control group. Collectively, these data suggest that KIOM-2015E protects colitis principally by improving intestinal barrier function and promoting anti-inflammatory responses. In turn, these effects inhibit macrophage infiltration into the colon and thus may be a candidate treatment for IBD.


Introduction
Inflammatory bowel disease (IBD) has two main forms including ulcerative colitis (UC) and Crohn's disease (CD) in human [1]. Symptoms of UC and CD include abdominal pain, diarrhea, and/or fever [2]. Also, IBD is caused by environmental interactions and genetic and lifestyle factors [3]. Many evidence demonstrated that one of the most typical character of IBD is the abnormal immune response within the intestinal wall by luminal bacterial antigens following epithelial barrier disruption [2]. The epithelial junction complex comprised tight junction (TJ) proteins of epithelial cells, such as zonula occluden-1 (ZO-1), occludin, and claudins, which are related with intracellular complex and cytoskeleton [4,5]. These TJ proteins play an important role in forming and maintaining the paracellular barrier, but destruction of these functions was associated with initiation and acceleration of the inflammation of IBD [6,7]. Recently, therapies of IBD have used anti-inflammatory drugs, immune modulators, and surgery. However, these therapies focused on controlling the immune cell response, including the cytokine pathway, but have many serious side effects including allergies and carcinoma [8]. Therefore, several researchers have been focusing on reliable data regarding the efficacy and safety to improved intestinal barrier function using natural or traditional herbs [9].
Acer palmatum Thumb is widely distributed as ornamentals in diverse regions including Asia, North America, North Africa, and Europe, and it has been used in traditional medication in East Asia [10,11]. The roots, leaves, husks, stems, and fruits of maple are used as herbal medicines for treatment with detoxification, relieving rheumatism and bone pain, reducing sore throat, improving eyesight, and reducing digestion [11]. Especially, stems and roots of maple has been used for the treatment of arthritis, bruises, pain, fractures, and liver diseases and the leaves are used for the treatment of inflammation of the eye [10,11]. However, the intracellular mechanism of Acer palmatum Thumb underlying the efficacy of colitis, inflammation, and damage to tight junctions remains unclear. Therefore, we investigated whether ethanol extract of Acer palmatum Thumb (KIOM-2015E) can alleviate acute colitis and evaluated the protective effects using the DSS-induced mouse colitis model.
2.3. Cell Viability. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies Inc., Rockville, MD, USA). Briefly, Caco-2 cells were plated at 1 × 10 3 cells/well in a 96-well plate. Cells were pretreated with KIOM-2015E for 1 h and then treated with IL-6 for an additional 24 h. CCK-8 assay was then performed in accordance with the manufacturer's instructions. Absorbance was determined at 450 nm on a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA). Cell viability was calculated relative to untreated controls (i.e., viability (% control) = 100 × absorbance of treated sample/absorbance of control).

Measurement of Transepithelial Electrical Resistance
(TER). Caco-2 cells were seeded at 1 × 10 5 cells/insert in 0.33 cm 2 polyethylene terephthalate membrane inserts with 0.4 μm pores (Millipore, Bedford, MA). The medium was changed every 2 days until complete differentiation. Cells were pretreated with KIOM-2015E for 1 h and then treated with IL-6. The electrical resistance was measured for three independent measurements using by Millicell ERS-2 Voltohmmeter (Millipore). TER was obtained at 18 and 24 h and presented as Ohm·cm 2 .
2.5. Epithelial Paracellular Permeability. Caco-2 cells were seeded at 1 × 10 5 cells/insert in 0.33 cm 2 polyethylene terephthalate membrane inserts with 0.4 μm pores (Milipore). The medium was changed every 2 days until complete differentiation. Cells were pretreated with KIOM-2015E for 1 h and then treated with IL-6 for an additional 24 h. The paracellular permeability was evaluated using a nonabsorbable, FITC-conjugated dextran probe (FD-4). Following pretreatment, the apical and basolateral sections were washed with PBS and then added to 1 mg/ml FD-4 in the apical side and PBS in the basolateral side. Following 1 h of incubation at 37°C, 100 μl of media from the basolateral side was plated to a 96-well plate The excitation and emission wavelengths were determined at 490 and 520 nm on a VersaMax microplate reader (Molecular Devices).

Large
Intestine Endoscopy and Histological Analysis. On 8 days of the experiment, we performed endoscopy using a mini-endoscope (OLYMPUS, Tokyo, Japan; 670 mm length and 2.8 mm diameter) with a visible light source, to investigate the colons of mice anesthetized with isoflurane, and we obtained high-resolution images. After the endoscopy procedure, whole blood was obtained from the mice abdominal vein. And the animals were then sacrificed for intestinal tissue collection. The isolated colons were fixed with 4% paraformaldehyde solution, embedded in paraffin block, and sectioned using a microtome. Histological sections were stained with hematoxylin and eosin (H&E), alcian blue, or PAS solution or incubated with antibodies to detect the macrophage marker, F4/80, and the tight junction protein, ZO-1.

Cell
Culture and Establishment of a Stable Cell Line. The murine macrophage Raw264.7 cell line was obtained from ATCC (Manassas, VA, USA). Cells were cultured at 37°C in a humidified atmosphere with 5% CO 2 in Dulbecco's modified Eagle's medium (HyClone) containing 10% fetal bovine serum (HyClone), 100 unit/ml penicillin, and 100 μg/ml streptomycin (HyClone). Cells were lentivirally transfected to express with both human sodium iodide symporter (hNIS) as a single nuclear reporter gene and green fluorescent protein (GFP) genes. The GFP-positive cells were sorted by flow cytometry (FACSaria; BD Biosciences, San Jose, CA, USA). And then cells were further retrovirally transduced to express both enhanced firefly luciferase (effluc) and Thy1.1 gene to stabilize expressed hNIS. Thy1.1-positive cells were sorted using CD90.1 microbeads (Miltenyi Biotec, Auburn, CA, USA) and sorted by flow cytometry to determine the stable expression of the GFP and hNIS genes using a PercPconjugated Thy1.1 antibody (BD Biosciences). These cells were named the Raw264.7/NF cell line in the present study [13].
2.9. In Vivo Image for Macrophage Infiltration Using Bioluminescence Imaging (BLI). To monitor for infiltration of macrophage in DSS-induced colitis mice, we performed in vivo imaging systems (IVIS). On the 7th day of experiment, the mice were injected in the tail intravenously with 100 μl of Raw264.7/NF cells (3 × 10 6 cells/mouse). On the 8th day of experiments, the mice were injected intraperitoneally with 100 μl of D-luciferin (3 mg/mouse). For the acquisition of BLI, the mice were placed individually in the specimen chamber of the IVIS system and the light intensity at 10 min after luciferin administration was measured for 1 min. BLI images were analyzed using Living Image (version 2.12, Xenogen, Alameda, CA, USA) and IGOR Image Analysis FX software (WaveMetrics, Portland, OR, USA). BLI signals were evaluated by drawing uniformly sized ROIs throughout the whole experiment and indicated in units of photons per square centimeter per second per steradian [13].

2.10.
Enzyme-Linked Immunosorbent Assay for Myeloperoxidase (MPO) Activity, IL-6, and TNF-α. Collected whole blood was incubated at 4°C overnight and then centrifuged (3000 ×g at 4°C) for 15 min. The separated serum was stored at −80°C until use. The levels of IL-6 and TNF-α in the serum were determined using ELISA kits according to the manufacturer's protocol. In addition, MPO, a marker for neutrophil infiltration, was determined using MPO activity assay kit according to the manufacturer's protocol.
2.11. Western Blot Analysis. Caco-2 cells and mouse intestinal protein were extracted using RIPA lysis buffer with phosphatase and protease inhibitor cocktails. Equal proteins of quantification with a BCA kit were electrophoretically separated by 8% and 12% SDS-PAGE gels and then transferred to PVDF membranes. After blocking, the membranes were incubated with a 1 : 1000 dilution of ZO-1 or occludin antibody at 4°C overnight. The following day, the membranes were incubated with secondary antibodies and detected using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA). The band density was then normalized to the β-actin reference.
2.12. Statistical Analysis. All statistical analyses were performed with SPSS version 18, and graphs were drawn with GraphPad Prism version 5. Experimental values are given as the means ± standard error of the mean. The significant difference was determined by one-way ANOVA test. p values less than 0.05 were regarded as statistically significant.  be decreased in TER (increased in permeability) in 50 ng/ml, with a maximal decrease in TER at 24 h [14]. Pretreatment with KIOM-2015E resulted in a significant increase in TER in a time-and dose-dependent manner compared with IL-6 treatment (Figures 2(a) and 2(b)). Also, pretreatment with KIOM-2015E decreases the FD-4 permeability in a dose-dependent manner compared with IL-6 treatment (Figure 2(c)). Furthermore, treatment with IL-6 decreased the tight junction-associated protein expression, namely, ZO-1and occludin; however, pretreatment with KIOM-

Effects of KIOM-2015E on DSS-Induced Loss of Body
Weight, Colon Length, and MPO Activity. We verified the therapeutic effect of orally administered KIOM-2015E on the severity of DSS-induced colitis. Compared with the vehicle control group, DSS-treated mice were significantly reduced body weight (Figure 3(a)). However, the administration of KIOM-2015E obviously improved the body weight loss as compared with the DSS-treated group (Figure 3(a)). Furthermore, consistent with body weight results, colon length shortened was markedly increased by administration of KIOM-2015E compared with that of the DSS-treated group (Figures 3(b) and 3(c)). In addition, DSS-treated mice had significantly higher MPO activity than those of the KIOM-2015E-treated group and the control group (Figure 3(d)).  The body weights were monitored before KIOM-2015E or 5-ASA treatment during the experimental periods. The colonic lengths of mice were measured after being isolated from the sacrificed mice. The MPO activity was measured using MPO activity assay kit according to the manufacturer's protocol. The results represent the mean ± standard error of the mean values of each mouse in the same group. * * * p < 0 001 versus the control group; # p < 0 05 and ## p < 0 01 versus the DSS-treated group.

Effects of KIOM-2015E on Intestinal Barrier Function in DSS-Induced Colitis Mice.
Intestinal epithelial tight junctions have an important role in protecting against inflammation. Also, tight junction regulated the paracellular permeability by the forming of apical junction complex. Dysfunction of tight junction causes the disruption of the intestinal barrier and induced inflammation, following the pathogenesis of colitis [15]. Therefore, the present study evaluated the effects of KIOM-2015E on the protein expression of ZO-1 and occludin by immunofluorescence and Western blot analysis. ZO-1 expression was reduced after DSS treatment, and this downregulation was increased by treatment with KIOM-2015E ( Figure 5(a)). Moreover, the decreased expression of ZO-1 and occludin in DSS-induced colitis was recovered by KIOM-2015E treatment (Figures 5(b)-5(d)). Together, these results indicated that KIOM-2015E prevented the downregulation of tight junction proteins in the DSS-induced acute colitis model and it might be relative with the preventive mechanism of colitis.

Effects of KIOM-2015E on Infiltration of Macrophages and Serum Levels of Proinflammatory Cytokines in DSS-Induced Colitis Mice.
A previous study showed that DSS induced the dysfunction of the intestinal barrier and aggravated the inflammatory process, following the lead to macrophage infiltration into the colon [16]. Therefore, we ). Furthermore, consistent with IVIS results, macrophage as stained by F4/80 antibody was significantly increased in the DSS-treated group compared with the control group (Figure 6(c)). However, KIOM-2015E obviously reduced infiltration of macrophage compared with the DSS-treated group (Figure 6(c)). Several reports showed that proinflammatory cytokines into the serum increased secretion in DSS-induced colitis [17,18]. Thus, we measured serum cytokine levels of IL-6 and TNF-α. Both levels of IL-6 and TNF-α were significantly higher in the DSS-treated group than in the control group (Figures 7(a) and 7(b)). However, both levels of IL-6 and TNF-α in mice treated with KIOM-2015E were lower than those in the DSS-treated group (Figures 7(a) and 7(b)).

Discussion
In the present study, we demonstrated that KIOM-2015E inhibited the downregulation of tight junction proteins such as ZO-1 and occludin and improved the dysfunction of the intestinal barrier induced by IL-6 and DSS. Furthermore, we showed that the loss of body weight, intestinal shortening, and crypt damage were reduced by treatment with KIOM-2015E. Increasing productions of proinflammatory cytokine are associated with DSS-induced colitis [17,18], which produce various proinflammatory cytokines, including TNF-α, IFN-γ, IL-6, IL-8, IL-12, and IL-17 [17,19,20]. Many studies showed the regulation of cytokines as potential targets in therapeutic approaches [21,22]. Previous reports showed that major compounds of KIOM-2015E identified orientin, isoorientin, and vitexin [23]. Especially, vitexin has anti-inflammatory [24,25], anticancer [26,27], and antioxidant properties [28]. Our study showed that KIOM-2015E also reduced the levels of proinflammatory factors TNF-α and IL-6 in serum. Moreover, KIOM-2015E inhibited oxidative stress by decreasing MPO activity in colon tissue. Collectively, these results indicated that KIOM-2015E alleviates DSS-induced colitis though regulation of intestinal permeability and inflammation considering the anti-inflammatory and antioxidant effects of vitexin. Dysfunction of the intestinal epithelial barrier has been one of the major symptoms of IBD, including UC and CD. Also, defective intestinal barrier function has been known as a critical pathogenic factor of inflammatory conditions in IBD [29]. These dysfunctions increased intestinal permeability in a patient of IBD. In this study, we confirmed the expression of ZO-1 and occludin as the representative proteins of tight junctions. It was found that KIOM-2015E repaired the expression of tight junctions and its associated protein ZO-1 and occludin in the treatment and prevention of colitis diseases. Several reports showed that lymphocytes, macrophages, and neutrophils markedly increased in number in DSS-induced acute colitis [17,20]. Especially, mainly macrophages could contribute to abnormal production of inflammatory cytokines and mediators [30]. Also, macrophages have an important role in immune responses, in which recruited macrophages are known to be associated with the secretion of TNF-α, IL-6, and IL-1β [31]. The present study confirmed in the IVIS image that the infiltration of macrophage with bioluminescent signals was increasing in DSS-treated mice, whereas KIOM-2015E significantly decreased the infiltration of macrophage. Furthermore, immunohistochemistry data showed that the macrophages with the F4/80 marker were increased in DSS-treated mice and KIOM-2015E inhibited macrophage infiltration into the colonic mucosa in DSS-induced mice in consistency with IVIS data. Taken together, our results verified that KIOM-2015E relieved inflammation by inhibiting macrophage infiltration into the intestinal crypts in DSS-induced colitis mice.

Conclusion
This present study proved that KIOM-2015E exerted useful effects by improving intestinal barrier function and inhibiting inflammation in DSS-induced colitis mice. It was also indicated that due to the beneficial effects of KIOM-2015E, its inhibition of macrophage infiltration into the intestinal epithelium is likely. In conclusion, KIOM-2015E is a potentially new therapeutic agent for colitis through The acquisition of BLI intensity measured using IVIS. (c) Immunohistochemistry for F4/80 antibody. Collected intestinal tissues were fixed with 4% paraformaldehyde solution, embedded in paraffin block, and sectioned using a microtome. Histological sections were stained with anti-F4/80 antibody. The total photon efflux results represent the mean ± standard error of the mean values of each mouse in the same group. * p < 0 05 versus the control group; # p < 0 05 versus the DSS-treated group.
anti-inflammatory activity and maintenance of the colonic epithelial barrier.

Data Availability
All the data supporting the results are shown in the article and can be requested from the corresponding author.