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. 2019 Nov 25;29(1):131–140. doi: 10.1007/s10068-019-00711-8

Perilla frutescens Britton var. frutescens leaves attenuate dextran sulfate sodium-induced acute colitis in mice and lipopolysaccharide-stimulated angiogenic processes in human umbilical vein endothelial cells

Yuna Lee 1, Jungjae Lee 1, Jihyeung Ju 1,✉
PMCID: PMC6949358  PMID: 31976135

Abstract

The aim of the current study was to investigate whether the leaves of Perilla frutescens Britton var. frutescens (PL), a frequently consumed vegetable in Korea, attenuate dextran sulfate sodium (DSS)-induced acute colitis in mice and lipopolysaccharide (LPS)-stimulated angiogenic processes in human umbilical vein endothelial cells (HUVEC). In DSS-treated mice, dietary supplementation with PL mitigated DAI and colon shortening. The dietary PL also reduced colonic levels of inflammatory and angiogenic mediators, such as interleukin-1β, interleukin-6, monocyte chemoattractant protein-1, macrophage inflammatory protein-2, leukotriene B4, inducible nitric oxide synthase, cyclooxygenase-2, basic fibroblast growth factor, and intercellular adhesion molecule-1 (ICAM-1). Treatment of HUVEC with ethanol extract of PL attenuated LPS-stimulated increases in ICAM-1 levels, monocyte adhesion, invasion, and tube formation. This study suggests that dietary PL effectively inhibited DSS-induced acute colitis in mice, and its anti-angiogenic activities may partially contribute to the inhibition.

Keywords: Perilla frutescens Britton var. frutescens leaf, Colitis, Inflammatory mediator, Angiogenesis

Introduction

Ulcerative colitis (UC) is a major form of chronic inflammatory bowel disease and characterized by relapsing inflammation in colonic mucosa with continuous lesions (Cabre and Domenech, 2012). UC is most common in Western countries, and the prevalence is growing rapidly in Asian countries, such as Japan, Hong Kong, and South Korea (Prideaux et al., 2012). The etiopathogenesis of UC remains uncertain, but the environmental factors are thought to play an important role (Cabre and Domenech, 2012). Diet has long been purported to modify the onset and progress of UC. Total fat, omega-6 fatty acids, and meat consumption have been associated with an increased risk of UC, whereas vegetable consumption has been inversely associated with risk of UC (Hou et al., 2011). Since many therapeutic approaches for UC provide limited beneficial action and produce toxic side effects (Arora and Shen, 2015), great efforts have been made for identifying safe dietary components that possess inhibitory activities against UC.

Angiogenesis is defined as the formation of new blood vessel growth from existent micro vessels and involves several key processes, including proliferation, adhesion, invasion, and tube formation of endothelial cells (Pousa et al., 2008). Intestinal damage is initially followed by a physiological angiogenesis, which is essential for tissue repair and organ regeneration. The abnormal balance of pro- and anti-angiogenic molecules and changes of vascular cell types, however, induce pathogenic angiogenesis, which plays an integral role in establishing, sustaining, and exacerbating UC (Chidlow et al., 2007). Since pathogenic angiogenesis is closely related to the disease severity of UC, it is regarded as a hallmark of active status of UC in humans and animal models (Chidlow et al., 2007).

Perilla frutescens Britton belongs to Lamiaceae family, and the leaves are used for gourmet foods and complementary medicines in Asian countries (Seo and Baek, 2009). The plant encompasses different varieties, including var. frutescens and var. crispa. The leaves of var. frutescens, called ‘kkaennip’, are one of the most commonly consumed vegetables in Korea, which are distinct to those of var. crispa, called ‘zisu’ and ‘shiso’, consumed mainly as herb in China and Japan. P. frutescens Britt. leaves have been reported to possess different biological activities, including anti-oxidant (Meng et al., 2009), anti-inflammatory (Huang et al., 2014; Lee and Han, 2012; Park et al., 2017; Ueda and Yamazaki, 1997; Ueda and Yamazaki, 2001; Urushima et al., 2015), anti-cancer (Kwak et al., 2009; Kwak and Ju, 2015; Lin et al., 2007; Ueda et al., 2003) activities and inhibitory activities against hepatotoxicity (Kim et al., 2007), allergy (Liu et al., 2013; Makino et al., 2001; Makino et al., 2003), and obesity (Kim and Kim, 2009). However, most studies have been conducted with the leaves of var. crispa, and only a few studies have shown in vitro antioxidant, anti-inflammatory, and anti-cancer activities of the leaves of var. frutescens (Kwak and Ju, 2015; Lee and Han, 2012; Meng et al., 2009).

The aim of the present study was to investigate the efficacy of dietary supplementation with P. frutescens Britt. var. frutescens leaves (PL) in a mouse model of dextran sulfate sodium (DSS)-induced colitis. Anti-angiogenic effects of the ethanol extract of PL (PLE) were also evaluated in vitro using lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVEC).

Materials and methods

Preparation of perilla leaf

PL was purchased from a local retail store (Cheongju, Korea) and cleaned with water. The leaves were then freeze-dried (PH1316, IshinBioBase, Yangju, Korea) and used for either formulating animal diet or preparing ethanol extract. The dried PL were mixed with 70% Ethanol (10-fold volume), stirred for 4 h at room temperature, and centrifuged at 3000×g for 3 min (A320101, Gyrozen, Daejeon, Korea). The ethanol solvent was removed from the supernatant via a rotary evaporator (NB-503CIR, N-bioteck, Bucheon, Korea). The resulting residue was retained at − 70 °C for further use for cell studies. The extraction yield was 17.6 ± 2.3% on a dry weight basis.

Animal care and experimental design

Animal study was carried out in a strict accordance with guidelines of the Institutional Animal Care and Use Committee and the protocol approved by the Committee on the Ethics of Animal Experiments of Chungbuk National University (CBNUA-640-10-01). Mice were kept under semi-SPF conditions (21.2 ± 2 °C, 30–70% relative humidity, 12 h dark/light cycles) providing free access to food and fluid. Five-week old male ICR mice weighing 27–29 g were purchased (Samtako, Osan, Korea) and acclimated on a standardized AIN93G rodent diet (Doo Yeol Biotech, Seoul, Korea) and sterile water for 1 week. Mice were then randomly divided into four groups (N = 7–8/group) and maintained on AIN93G diet or the same basal diet supplemented with freeze-dried PL at the level of 10% (Doo Yeol Biotech) for 15 days (day 0–15). At the 7th day after the diet initiation (day 7), mice were given 1.5% (w/v) DSS (Mw 36,000–44,000; MP Biomedicals, Seoul, Korea) in their drinking water for 8 consecutive days until the experiment was terminated at day 15. Water-treated mice maintained on AIN93G diet were also included as a negative control group (N = 4). Body weight, food intake, fluid intake, and general health status were monitored every 2–3 days prior to DSS treatment (day 0–7) and then everyday during DSS treatment (day 7–15). On day 15, mice were anesthetized using diethyl ether (Sigma-Aldrich, St. Louis, MO, US) and euthanized by exsanguination via cardiac puncture. Serum samples were obtained by centrifugation at 3000×g for 15 min. At necropsy, colons were harvested, rinsed with ice-cold saline, flattened on filter paper for the measurement of length, and then frozen at − 70 °C for further biochemical analyses. Liver, spleen, and kidney were dissected, rinsed, and weighed.

Evaluation of acute colitis severity

The disease activity index (DAI) was determined by summing up the scores of three individual parameters, body weight loss, stool consistency, and gross bleeding (Cooper et al., 1993; Kumar et al., 2011). Each parameter was scored in a scale of 0–4 during the 8 days of DSS treatment as described previously (Cooper et al., 1993; Kumar et al., 2011). The length of colon from the ileocecal junction to rectum of each mouse was measured as a further indicator of disease severity (Kumar et al., 2011).

Enzyme immunoassay

Frozen colon tissues were placed into ice-cold RIPA buffer (Rockland Immumochemicals Inc., Gilbertsville, Germany) containing protease inhibitor cocktails (Sigma-Aldrich) and then homogenized using a Dounce tissue grinder (Wheaton, Millville, NJ, USA). After centrifugation for 15 min at 13,500×g, the supernatants were obtained as colon homogenates. Levels of interleukin (IL)-1β, IL-6, and leukotriene B4 (LTB4) in the colon homogenates were measured using enzyme immunoassay (EIA) kits according to the procedure provided by the manufacturers. IL-1β and IL-6 kits were purchased from Koma Biotech Inc. (Seoul, Korea), and LTB4 kit was purchased from Cayman Chemical (Ann Arbor, MI, USA). Prior to ELISA for LTB4, solvent extraction was conducted. In brief, ethyl acetate was added to the colon homogenates, vortexed for 30 min, and centrifuged at 10,000×g for 20 min. After evaporation of the organic layer using a rotary evaporator (NB-503CIR, N-bioteck), the dried samples were reconstituted in the buffer provided in the kit (Cayman Chemical). Monocyte chemoattractant protein-1 (MCP-1), macrophage-inflammatory protein-2 (MIP-2), basic fibroblast growth factor (bFGF), and intercellular cell adhesion molecule-1 (ICAM-1) were measured using Luminex Performance Assay kits (R&D systems, Minneapolis, MN, USA) according to the procedure provided by the manufacturers. Colonic levels of inflammatory markers were normalized by the amount of protein quantified using bicinchonic acid protein assay kit (Thermo Scientific, Logan, UT, US).

Western blot analysis

Colon homogenates were combined with Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA). Denatured protein samples at 95 °C for 5 min were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide (12%) gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). The membranes were then probed with primary antibodies against inducible nitric oxide synthase (iNOS), cyclooxyagenase-2 (COX-2), or β-actin at 4 °C overnight. After washing with Tris-buffered saline containing 0.1% Tween-20 three times, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (Santa Cruz, CA, USA). Bands were visualized using enhanced chemiluminescence (ECL, Thermo Scientific). The densities of the bands were quantified using Image J program (NIH, Bethesda, MD, USA).

Assessment of liver and kidney function

Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine were determined using commercially available spectrophotometric assay kits (YD-diagnostics, Gyeonggi, Korea for ALT; Bioassay Systems, Hayward, CA, USA for AST and creatinine) according to the manufacturer’s instructions.

Cell culture

HUVEC were purchased from Lonza (Walkersville, MD, USA) and maintained on endothelial basal medium (EBM-2, CC-3156, Lonza) containing growth supplements (CC-4176, Lonza). HUVEC at passages ≤ 5 were used for all of the experiments. THP-1 human monocytes were purchased from Korean Cell Line Bank (Seoul, Korea) and maintained on RPMI medium (Gibco Co., Rockville, MD, USA) containing 10% fetal bovine serum (FBS; Thermo Scientific), 100 units/mL penicillin, and 0.1 mg/mL streptomycin (Welgene Inc., Daegu, Korea). Cells were cultured at 37 °C in 95% humidity and 5% CO2.

Cell viability assay

Cell viability was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT; Sigma-Aldrich) assay. The ethanol extract of PL (PLE) was dissolved initially in dimethyl sulfoxide (DMSO; Biosesang Inc., Seongnam, Korea) and then further diluted using the corresponding cell culture medium right before the cells were treated. The final concentration of DMSO in the culture medium was adjusted to be less than 0.2% (v/v). HUVEC (1 × 105 cells per well) were seeded in 96-well plates (Corning Inc., New York, NY, USA). After 24 h, HUVEC were pretreated with PLE at the concentrations of 0, 5, 10, and 20 μg/mL for 2 h and stimulated with 10 μg/mL LPS for another 22 h. The medium was collected for further determination of bFGF and ICAM-1 levels, and the cells attached on the bottom of well were incubated with MTT at 0.5 μg/mL. After 4 h, MTT-containing media were removed. The reduced formazan precipitate was dissolved by adding DMSO and determined spectrophotometrically at 540 nm using a plate reader (Bio-Rad Laboratories). Levels of bFGF and ICAM-1 in the cell culture medium were measured using enzyme immunoassay (EIA) kits (Koma Biotech Inc.) according to the procedure provided by the manufacturers.

Monocyte adhesion assay

To determine the effect of PLE on the adhesion of monocyte to HUVEC, an assay using THP-1 cells was performed as previously described (Bian et al., 2017) with slight modification. Briefly, HUVEC (5 × 104 cells/well) were seeded in 96-well plates (Corning Inc.). After 24 h, HUVEC were pretreated with PLE at the concentration of 20 μg/mL in the absence of LPS for 2 h and then in the presence of 10 μg/mL LPS for another 22 h. THP-1 cells were labeled with a fluorescent dye, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF, Sigma-Aldrich), for 30 min and then added onto the HUVEC-containing plates. After 1 h, unattached THP-1 cells were washed out with PBS, and THP-1 cells attached to HUVEC were quantified using a fluorescence plate reader (FL × 800, Biotek Instruments, Inc., Winooski, VT, USA).

Invasion assay

To determine the effect of PLE on cell invasion, a transwell assay was performed. Briefly, matrigel (20 µg/well, Sigma-Aldrich) was added on transwell inserts (8 µm pore size; Corning Inc.). After incubation for 30 min at 37 °C, HUVEC (1 × 105 cells/well) were loaded on the matrigel-coated transwell insert containing serum free Dulbeco’s Modified Eagle’s media (DMEM; Gibco Co.) with PLE (0–20 µg/ml) in the absence of LPS for 2 h and then in the presence of 10 μg/mL LPS for another 22 h. After removing the non-invaded cells using cotton swabs, the invaded cells were stained with 0.5% crystal violet in 4% buffered formalin (Sigma-Aldrich) for 15 min. The dye was dissolved using 1% SDS (Sigma-Aldrich) and quantified spectrophotometrically using a plate reader (Bio-Rad Laboratories) at 540 nm.

Tube formation assay

To determine the effect of PLE on the formation of capillary-like structure of HUVEC, a tube formation assay was performed. Briefly, matrigel (20 µg/well, Sigma-Aldrich) was coated in 96-well plates for 30 min at 37 °C. HUVEC (1.5 × 104 cells/well) were then loaded in the matrigel-coated plates containing serum free DMEM (Gibco Co.) with PLE (0–20 µg/ml) in the absence of LPS for 2 h and then in the presence of 10 μg/mL LPS for another 22 h. After 4 h. the images of tubular structure were captured by iSolution Lite software (IMT i-solution, Burnarby, Canada). Total length of tubes and the number of branches were determined using Image-J software (NIH).

Statistical analyses

All analyses were performed using SPSS software (version 22.0; SPSS Inc., Chicago, IL, USA). Student’s t test was used for comparing between two groups. One-way ANOVA combined with Duncan test was used for comparing among multiple groups. Regression analysis was used for assessing a dose-response relationship. All of the analyses, a probability value of p < 0.05 was considered significant.

Results and discussion

Dietary administration with PL alleviated DSS-induced acute colitis in mice without causing toxicity

We first investigated whether dietary PL supplementation alleviates DSS-induced colitis in mice. DSS is a synthetic sulfated polysacharride that induces colitis in rodents. DSS-treated rodent model closely mimics several clinical and histological characteristics of human UC, including diarrhea, bloody stool, shortened colon, and mucosal ulceration (Cooper et al., 1993) and therefore has been widely used for both studying the pathology of human UC and identifying preventive agents against UC. The PL level on diet used in the current study (100 mg/g diet) corresponds to human intake of 50 g/day for a person requiring 2000 kcal based on the concept of calorie density (Newmark, 1987), which is approximately 10-fold of estimated daily intake of PL in Korea (~ 5 g/day) (Korea Rural Economic Institute, 2011).

Two different parameters of colitis were used in this study, DAI as a clinical symptomatic parameter (Cooper et al., 1993) and colon length as a morphological parameter (Kumar et al., 2011). DAI was determined using three individual parameters, body weight loss, stool consistency, and gross bleeding. As shown in Fig. 1A, DSS-treated control mice began to lose their body weight on day 12 (the 5th day of DSS treatment) and exhibited substantial weight loss (approximately 8% of their body weight) on day 15. DSS-treated mice that received 10% PL on diet exhibited minimal weight loss until day 14 (≤ 1%), and their weight loss at the termination of experiment on day 15 (~ 4%) was still significantly lower than that of DSS-treated control group (p < 0.05). As shown in Fig. 1B, C, DSS-treated control mice began to show both inconsistent stool and gross bleeding on day 12, and those symptoms were exacerbated until day 15. The scores of stool consistency and gross bleeding of DSS-treated mice on PL diet were significantly lower than those of DSS-treated control mice (0–0.7 vs. 3.0; p < 0.05). As shown in Fig. 2D, final DAI of DSS-treated mice on PL diet were significantly lower than that of DSS-treated control mice (1.1–4.5 vs. 9.1; p < 0.05). As shown in Fig. 2, the colons of DSS-treated control mice were shorter than those of water-treated control mice (7.0 ± 0.3 cm vs. 8.5 ± 0.5 cm, p < 0.05), indicating that DSS-induced shortening of colon length. In the DSS-treated mice, PL resulted in longer colon (9.1 ± 0.5 cm, respectively), which were comparable to the length of water-treated control mice. These findings indicate that the dietary PL ameliorated DSS-induced colitis in mice.

Fig. 1.

Fig. 1

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Effects of dietary supplementation with PL on DSS-induced acute colitis in mice. (A) Body weight loss, (B) stool consistency, (C) gross bleeding, and (D) disease activity index. *p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 2.

Fig. 2

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Effects of dietary supplementation with PL on DSS-induced colon shortening in mice. (A) Colon length and (B) representative colons. Different superscripts (a, b) mean p < 0.05

Liver, spleen, kidney weights of DSS-treated mice on PL diet (51.2 ± 3.7 mg/g bw, 4.6 ± 0.5 mg/g bw, 16.2 ± 1.3 mg/g bw, respectively) did not differ from those of DSS-treated control mice (47.2 ± 2.0 mg/g bw, 5.1 ± 0.5 mg/g bw, 16.0 ± 0.4 mg/g bw, respectively). Levels of serum ALT and AST, which reflect hepatic function (Inoue et al., 2013), were not statistically different among groups (21–49 unit/L and 19–31 unit/L, respectively). Levels of serum creatinine, which reflects renal function (Inoue et al., 2013), were also similar among groups (0.5–0.6 mg/dL). These results indicate that the dietary PL did not induce any apparent liver and kidney toxicity.

Dietary PL reduced levels of inflammatory mediators in DSS-treated mice

We next determined whether dietary supplementation with PL suppressed the colonic levels of inflammatory cytokines, chemokines, lipid, and enzymes in the DSS-treated mice. Pro-inflammatory cytokines, IL-1β and IL-6, are produced by resident macrophages in response to harmful stimuli such as DSS, leading to amplified inflammatory responses and further development of the clinical signs of inflammation (Soufli et al., 2016). MCP-1, also known as CCL2, and MIP-2, also known as CXCL2, are pro-inflammatory chemokines that recruit monocytes, neutrophils, and lymphocytes to the sites of inflammation (Soufli et al., 2016). LTB4, is a major metabolite of arachidonic acid that recruits and activates a range of inflammatory cells including neutrophils, eosinophils and dendritic cells (Lobos et al., 1987). iNOS is aberrantly expressed in macrophages in response to different stimuli including IL-1β and IL-6, leading to overproduction of NO, generation of reactive nitric oxygen species, tissue damage, and colonic inflammation (Soufli et al., 2016). Aberrant expression of COX-2 in epithelial, endothelial, and inflammatory cells leads to excessive production of prostaglandin E2, resulting in inflammatory cell infiltration, colonic mucosal inflammation, tissue damage, and ulceration (Mitchell et al., 1995). These inflammatory mediators are significantly implicated in the pathogenesis of UC in humans and experimental animals (Lobos et al., 1987; Soufli et al., 2016).

As shown in Fig. 3A–E, IL-1β, IL-6, MCP-1, MIP-2, and LTB4 levels in the DSS-treated mice were significantly higher than those in water-treated mice (IL-1β, MCP-1, MIP-2, and LTB4: 6.1-fold, 1.5-fold, 100-fold, and 3.9-fold of the water-treated mice, respectively). In DSS-treated mice, dietary supplementation with PL reduced the levels of IL-1β (to 42% of the DSS-treated control), IL-6 (to 7.7% of the DSS-treated control), MCP-1 (to 64% of the DSS-treated control), MIP-2 (to 5% of the DSS-treated control), and LTB4 (to 67.3% of the DSS-treated control). As shown in Fig. 3F, G, the protein levels of iNOS and COX-2 of DSS-treated mice were 2.7-fold and 3.2-fold higher than those of water-treated mice, respectively, and the DSS-induced increases were normalized by PL supplementation (iNOS and COX-2 levels down to 39% and 48% of DSS-treated control, respectively; Fig. 3F–H). These results indicate that the inhibitory activity of dietary PL against DSS-induced colitis in mice is attributed to the reduction of these inflammatory mediators.

Fig. 3.

Fig. 3

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Effect of dietary supplementation with PL on the levels of inflammatory mediators in the colon homogenates of DSS-treated mice. Levels of (A) IL-1β, (B) IL-6, (C) MCP-1, (D) MIP-2, (E) LTB4, (F) iNOS, (G) COX-2 and (H) representative blots. Different superscripts (a–c) mean p < 0.05. ***p < 0.001. ND: not detected

Dietary PL reduced colonic levels of angiogenic mediators in DSS-treated mice

DSS-treated mice manifest significant pathogenic angiogenesis during the progression of disease (Chidlow et al., 2007). In order to assess whether dietary supplementation with PL mitigated such angiogenesis in the DSS-treated mice, levels of angiogenic mediators, bFGF and ICAM-1, were measured in the colon homogenates. bFGF is one of the most potent angiogenic growth factors liberated from extracellular matrix and contributes to the proliferation of mesenchymal and endothelial cells through a paracrine or autocrine way (Pousa et al., 2008). ICAM-1 is a cellular adhesion protein upregulated in endothelial cells by the stimulation of different cytokines, including IL-1β, and mediates the binding of leukocytes to endothelial cells resulting in the transmigration of leukocytes into inflamed tissues (Pousa et al., 2008). bFGF and ICAM-1 levels have been reported to be elevated in UC of humans and experimental animals (Chidlow et al., 2007).

As shown in Fig. 4A, B, bFGF and ICAM-1 levels in the DSS-treated mice were 3.9-fold and 3.8-fold higher than those in water-treated mice, respectively. In DSS-treated mice, dietary supplementation with PL resulted in substantially reduced levels of bFGF (to 31.3% of the DSS-treated control) and ICAM-1 (to 42.3% of the DSS-treated control). Since IL-1β, MCP-1, and COX-2 are also known to be involved in angiogenesis (Chidlow et al., 2007; Pousa et al., 2008) and these levels were found to be reduced by the dietary PL (Fig. 3A, C, G), our results collectively indicate that dietary PL supplementation alleviated not only inflammation but also angiogenesis in the colon of DSS-treated mice. To our knowledge, this is the first report showing such effects of dietary PL.

Fig. 4.

Fig. 4

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Effect of dietary supplementation with PL on the levels of angiogenic mediators in the colon homogenates of DSS-treated mice. Levels of (A) bFGF and (B) ICAM-1. Different superscripts (a–c) mean p < 0.05

Treatment with PLE inhibited LPS-stimulated angiogenic processes in HUVEC

We further evaluated anti-angiogenic activities of PLE using LPS-stimulated HUVEC. LPS, the major component of the outer membrane of gram-negative bacteria, is a well characterized inflammation inducer (Willeaume et al., 1995) and angiogenesis promoter (Li et al., 2017; Ni et al., 2014). LPS-stimulated HUVEC model was chosen because it enabled us to investigate the effect of PLE on angiogenesis associated with inflammation, as DSS-treated mouse model did.

As shown in Fig. 5A, PLE at the concentrations of 5–20 μg/mL did not affect cell growth in LPS-treated HUVEC, and therefore, this range of concentrations was selected for the subsequent assessment of anti-angiogenic efficacy of PLE. As shown in Fig. 5B, C, treatment of HUVEC with LPS elevated levels of bFGF slightly (to 1.1-fold) and ICAM-1 substantially (to 3.7-fold), and the treatment with PLE reduced the LPS-stimulated increases in the levels of bFGF (to 91.0% of the control at the concentration of 20 μg/mL) and ICAM-1 (to 67.4–80.3% of the control at the concentration of 5–20 μg/mL). Regression analysis showed a significant inverse linear relationship between the concentrations of PLE and the levels of ICAM-1, indicating a dose-response relationship (R2 ≥ 0.9, p < 0.001). Changes of bFGF in HUVEC by either LPS or PLE were much less prominent than those in mice by either DSS or dietary PL (Fig. 4A), suggesting that other angiogenic factors are more importantly involved in the LPS-stimulated angiogenesis of HUVEC.

Fig. 5.

Fig. 5

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Effects of PLE on cell viability and the levels of bFGF and ICAM-1 in LPS-stimulated HUVEC. (A) Cell viability and levels of (B) bFGF and (C) ICAM-1 in the cell culture medium. Different superscripts (a–c) mean p < 0.05

We subsequently investigated effects of PLE on key processes of angiogenesis, such as monocyte adhesion, invasion, and tube formation (Pousa et al., 2008), in LPS-stimulated HUVEC. As shown in Fig. 6, treatment with LPS increased the adhesion of monocytes to HUVEC (to 1.2-fold), invasion (to 1.9-fold), and tube formation (to 1.6-fold in the length and to 1.4-fold in the number of branches). The treatment with PLE significantly inhibited the LPS-stimulated monocyte adhesion (by 20.4%, at the concentration of 20 μg/mL), invasion (by 19.2–44.0%, at the concentrations of 10, 20 μg/mL), and tube formation (by 32.1% in total length at the concentration of 20 μg/mL, by 18.7–35.6% in the number of branches at the concentrations of 10, 20 μg/mL). The inhibition of monocyte adhesion of PLE is apparently due to the reduction of ICAM-1 (Fig. 5C). To our knowledge, this is the first report on anti-angiogenic activities of PLE. Since angiogenesis is significantly implicated in the pathogenesis of UC and has been reported to well reflect the degree of inflammation in the DSS model (Chidlow et al. 2007; Pousa et al., 2008), our results from animal and cells studies collectively suggest that the inhibitory activity of dietary PL against DSS-induced colitis in mice were associated at least partially with the reduction of angiogenesis.

Fig. 6.

Fig. 6

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Effects of PLE on monocyte adhesion, invasion, and tube formation in LPS-stimulated HUVEC. (A) Adhesive THP-1 cells to HUVEC, (B) invasive cells, and (C) total length of tubes (upper panel), the number of branches (lower panel), and representative images of tube structure (right panel). Different superscripts (a–c) mean p < 0.05

Rosmarinic acid (Osakabe et al., 2004, Sanbongi et al., 2004) and luteolin (Jeon et al., 2014; Ueda et al., 2002; Ueda et al., 2003) have been suggested as active constituents for the anti-inflammatory activities of leaves of other varieties of P. frutescens. Rosmarinic acid also has recently been shown to suppresses DSS-induced colitis (Jin et al., 2017) and angiogenesis in vitro (Huang and Zheng, 2006). Dietary fibers are ubiquitously present in plants, including PL, and inulin and pectin have been shown to reduce the severity of colitis in rats (Mao et al., 1996; Schrenk, 2009). Dried PL used in the present study is a complex mixture containing various bioactive phytochemicals. It needs to be clarified whether a single compound or different compounds in combination are responsible for the inhibitory activities of PL against DSS-induced colitis found in our study. In addition, since we cannot rule out the possibility that certain components of the mixture might interfere with the action of DSS directly, the inhibitory activities of PL against colitis need to be validated in other experimental models, such as genetic models.

In summary, dietary supplementation with PL attenuated DSS-induced acute colitis in mice without causing toxicity, and the inhibitory activity was attributed to the reduction of inflammatory and angiogenic mediators. Treatment of HUVEC with PLE mitigated LPS-stimulated increases in ICAM-1 levels, monocyte adhesion, invasion, and tube formation. These results suggest that dietary PL supplementation is safe and effective for the inhibition of DSS-induced acute colitis in mice, and the anti-angiogenic activities of PL may, at least partially, contribute to the inhibition. The present results provide preclinical data, and it remains to be determined whether such effects are reproduced in humans. More studies are needed to better understand the underlying mechanism for the observed inhibitory action of PL against colitis and inflammation-associated angiogenesis.

Acknowledgements

This work was supported by National Research Foundation of Korea funded by the Korea government (NRF-2010-0025311 & NRF-2015R1D1A1A01059139) and conducted during the research year of Chungbuk National University in 2017.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Footnotes

Publisher's Note

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Contributor Information

Yuna Lee, Email: rnfk9802@naver.com.

Jungjae Lee, Email: dlwndwo41@naver.com.

Jihyeung Ju, Email: jujihg@chungbuk.ac.kr.

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