RETRACTED ARTICLE: Regulatory effects of glycyrrhizae radix extract on DSS-induced ulcerative colitis

Glycyrrhizae Radix (GR) is a Korean traditional herb medicine that is widely-used in clinical health care. The clinical functions of GR include relief of toxicity, anti-cancer, regulating blood cholesterol and anti-inflammation. This study investigated the role of GR on ulcerative colitis in a dextran sulfate sodium (DSS)-induced mouse model of colitis. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) analyses were done on male BALB/c mice administered 5 % DSS during the experimental period. Ethanol extracts of GR were orally administered at same time daily to control mice. The severity of colitis was measured by body weight change and colon length. DSS-treated mice displayed weight loss and shortened colon length compared with control mice. Mice were administered GR showed less weight loss and longer colon length than the DSS-treated group. Inflammatory cytokines were decreased by GR treatment. Treatment also reduced DSS-induced microscopic damage to colon tissue. GR regulated the phosphorylation of transcription factors such as NF-κB p65 and IκB α. GR has beneficial effects in a colitis model. GR might be a useful herb medicine in the treatment of ulcerative colitis.


Background
Ulcerative colitis (UC) is a chronic and relapsing inflammatory disease characterized by dysregulation of the immune function response and imbalanced release of cytokines and unresolved inflammatory progress associated with intestinal mucosa [1,2]. The inflammation reaction may be initiated by proinflammatory cytokines [3]. Patients with bowel disease have increased interleukin-(IL) 6 in the intestinal mucosa and tumor necrosis factor-alpha (TNF-α) in the blood and colon tissue. Immune cells (T cells, intestinal epithelial cells, macrophages) secrete various cytokines including TNF-α, IL-1, IL-6, IL-8 and interferon-gamma (IFN-γ), which regulate the inflammatory response in UC. Elevated levels of cytokines have also been implicated in the pathogenesis of bowel disease [4,5].
Blocking the generation of inflammatory cytokines is an area of robust research in ulcerative colitis therapy [6]. In chronic colitis, inflammation extends into the colon muscle layer with acute manifestations, followed by an asymptomatic period and a relapse that occurs months or even years later [7]. UC affects the rectum and extends in a retrograde manner with extensive areas of superficial lesions [8]. The pathogenic factors of UC are unclear, but heredity, immune system dysfunction, and dietary habits could be influential [9].
Dextran sulfate sodium (DSS)-induced colitis is a suitable mice model characterized by the morphologically and biochemically. DSS induces bloody stools, ulcerations, epithelial injury and infiltration of inflammatory cells [10]. Histologically, DSS influences crypt abscesses and epithelioglandular hyperplasia in mice. DSS induced crypt architecture, disappearance of crypts and inflammatory infiltration. During DSS colitis, intercellular distance of mucosal cells in crypt and vascular endothelial cell was increased [11]. These colon conditions are similar to acute and chronic UC in humans.
DSS is toxic to gut epithelial cells of the basal crypts and affects the integrity of the mucosal barrier [12]. Thus, the DSS-induced colitis model is especially suitable to study mechanism of inflammatory colitis.
Cyclooxygenase is a constitutive enzyme that catalyzed the production of prostaglandins (PGs), which protect the stomach from damage. COX-2 is induced by inflammatory stimuli, such as cytokines, and also catalyzed PG production that contributes to inflammation-related swelling [13]. COX-2 and PGE2 levels are raised in the inflamed mucosa of patients with UC [14]. So, to elucidate reduction of COX-2 and PGE2 shows another pathway to ameliorate inflammation.
Glycyrrhiza Uralensis is a medical plant grown in Europe and Asia. It has been used as a herbal medicine respiratory system and gastrointestinal tract medicine [15]. Glycyrrhizae Radix (GR) has been traditionally used for treatment of cough, anti-allergy and bowel disease [16]. GR possesses various bioactive constitutents that include saponins, flavonoids and coumarins [17]. Constituents of GR have anti-inflammatory effects in lipopolysaccharide-stimulated macrophage. Glycyrrhizin suppressed nitric oxide, TNF-α and PGE 2 levels in LPS-stimulated macrophage. Glycyrrhetic acid and glycyrol have anti-inflammatory effect by regulation of NF-κB pathway in LPS-treated RAW264.7 macrophage through inhibition of inflammatory cytokines such as IL-6, IL-1β, and TNF-α. Also glycyrol regulates COX-2 and iNOS expression in LPS-treatment [18]. These results supposed that GR has antiinflammatory effects in vitro. Therapeutic activities attributed to GR include anti-cancer, anti-diabetes, antioxidant and anti-inflammation [19]. However, it is unknown whether GR or its constitutents can regulate intestinal inflammation.
To explore the potential of GR as a useful therapeutic in UC, we examined its effects on DSS-induced colitis in a mouse model. The goals were to evaluate the effect of GR on clinical signs including weight loss, colon shortening, diarrhea and gross bleeding, and to investigate the role of GR on inflammatory mediators in DSStreated mice.

Preparation of GR
GR purchased from Humanherb (kyuong San, Korea). GR extract was prepared by decocting with 70 % ethanol for 3 h (100 g/L). The solvent ethanol was filtered and allowed to evaporate using a rotary evaporator at a temperature of 40-45°C. The yield of dried extract was 3.022 %. The extract was diluted in 0.9 % saline and filtered through a 0.22 μm syringe filter (HYUNDAI Micro, Seoul, Korea).

Mice
Male BALB/c, 6-week-old mice obtained from SAMTACO (Osan, Kyungki-Do, Korea) were acclimatized in a specific pathogen-free environment under controlled conditions (22 ± 2°C with a 12 h light/dark cycle) for a week. The mice were housed in five colony cages with six mice per cage.

Experimental design
All experimental protocols (CBNU2015-089) were approved by the Committee on the Care of Laboratory Animal Resources, Chonbuk National University and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals. Acute colitis was induced by administering drinking water containing 5 % (w/v) DSS (MP Bio, Santa Ana, CA, USA) to mice for 10 days. Thirty mice were weighted before the experiment, and were divided into five groups with six mice per group. Group 1 comprised untreated mice. Mice in group 2 received DSS. In group 3 and group 4, mice with induced colitis were treated with GR 100 mg/kg or 50 mg/kg, respectively. In group 5, mice with induced colitis were treated with 50 mg/kg 5-aminosalicylic acid (5-ASA; Sigma-Aldrich, St. Louis, MO, USA) as the reference drug. Mice were checked daily for body weight, stool consistency and the presence of gross bleeding. GR was diluted with purified water and orally administered once a day during the 10 days of DSS treatment, after which the mice were sacrificed.

Disease Activity Index (DAI)
Intestinal disease activity was assessed based on the weight loss, presence of diarrhea accompanied by blood and mucus, and colonic shortening [20]. DAIs were calculated by scoring weight loss, diarrhea and rectal bleeding based on the scoring system shown in Table 1 that was previously described by Murthy et al. [21]. Weight loss was defined as the difference between initial and final weights, and diarrhea as the absence of fecal pellet formation and the presence of continuous fluid fecal material in the colon. Rectal bleeding was assessed based on the presence of diarrhea containing visible blood and on the presence of gross rectal bleeding. DAI values were calculated as {(weight loss score) + (diarrhea score) + (rectal bleeding score)}/4. The DAI was

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determined by three investigators blinded to the protocol. The clinical parameters used in the present study were chosen to represent the subjective clinical symptoms observed in human UC.

Cytokine assays
Levels of IL-6 and TNF-α in the serum and tissue were measured using an enzyme-linked immunosorbent assay (ELISA), as previously described [22].  Erosions and marked inflammatory cell infiltration and glyceraldehyde-phosphate dehydrogenase (GAPDH) (Thermo scientific, Waltham, MA, USA) and washed three times with PBST. Blots were incubated with secondary antibody for 1 h at room temperature. Antibody-specific proteins were visualized using an enhanced chemiluminescence detection system (Amersham, Newark, NJ, USA). Protein densities were quantified by densitometry.

Histological processing
Mice were sacrificed at the end of the experiment. The entire colon was dissected and flushed with ice-cold PBS. Sections of rectums were taken and fixed in 10 % neutralbuffered formalin (Sigma-Aldrich) for 24 h at room temperature and embedded in paraffin to provide sections for histological evaluation. Severity of colitis was evaluated in sections stained with Hematoxylin and Eosin (H&E; Muto Pere Chemicals, Tokyo, Japan and Sigma-Aldrich) by two independent observers blinded to the experimental

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conditions according to the modified criteria of Hamamoto et al. [23] summarized in Table 2. Mucosal damage was scored as 0-4 based on the loss of crypt (mucosa) and infiltration of inflammatory cells (maximum score = 4).

PGE 2 assay
Distal colons were homogenized in lysis buffer (iNtRON Biotech, South Korea), and centrifuged at 16,582 g for 5 min. PGE 2 levels were quantified using immunoassay kits according to the manufacturer's instructions (Enzo Life Science, East Farmingdale, NY, USA).

Identification of constituents of GR extract
GR extract lycyrrhizae was dissolved in methanol (1 mg/ml). Glycyrrhizin and glycyrrhetic acid (Tokyo Chemical Industry, Tokyo, Japan) were dissolved in methanol for high-performance liquid chromatography (HPLC) analysis. HPLC was performed on a Waters 2695 separation module (Waters, Milford, MA, USA) coupled with a model 2487 dual λ absorbance detector (Waters) under the following conditions: column, TSK-gel ODS-80Ts (4.6 mm × 150 mm; Tosoh Co., Tokyo, Japan); mobile phase, 0.1 % formic acid (solvent system A) and acetonitrile (solvent system B) in a gradient mode (B from 30 to 60 % in 15 min, B from 60 to 100 % from 15 to 30 min, B 100 % from 30 to 40 min); flow rate, 0.5 ml/min; temperature, 30°C and ultraviolet wavelength of 254 nm.

Statistical analysis
All results are presented as the mean ± S.E.M. Results were analyzed using Graph Pad Prism version 5.0 program (Graph Pad Software, Inc, La Jolla, CA, USA). One-way analysis of variance with Tukey post hoc test was used to determine statistically significant differences. P <0.05 was considered significant.

Effects of GR on clinical signs in DSS-induced colitis
DSS caused a decrease in body weight (Fig. 1a) and colon length ( Fig. 2a and b)

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respectively, compared to the control group. Both GR and 5-ASA alleviated the DSS effects on body weight loss and colon shortening (Figs. 1a, 2a and b). GR also attenuated the DSS-mediated increase in DAI scores (Fig. 1b).

Effect of GR on transcription factors in DSS-induced colitis
Nuclear factor-kappa B (NF-κB) is a important transcription factor of inflammation reactions [24]. The effect of GR on the activation of NF-κB was investigated. Phosphorylation of IκBα and NF-κB in the colon tissue was inhibited by GR treatment (Fig. 4).

Effect of GR on COX-2 and PGE 2 abundance in DSS-induced colitis
The effects of GR on COX-2 expression in colon tissues were explored using Western blot analysis. DSS markedly induced COX-2 expression in colon tissue versus control group, but increased COX-2 expression was significantly reduced by GR administration (Fig. 5a). Relative expression levels of COX-2 are presented in Fig. 5b. COX-2 catalyzed PGE 2 biosynthesis, and we examined whether GR

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affects PGE 2 levels. PGE 2 levels were enhanced by DSS, and this increase was significantly inhibited by GR administration (Fig. 5c).

Effects of GR on epithelial injury in DSS-induced colitis
Mucosal thickness is regarded as a parameter of normal mucosal condition. DSS caused epithelial injury and infiltration of inflammatory cells, including mast cells [25]. We observed histopathological changes of colon tissue between DSS-treatment and control groups. And then examined the effect of GR treatment. GR treatment attenuated these effects induced by DSS treatment (Fig. 6a). GR treatment also reduced the DSS-mediated microscopic damage to the colonic tissue (Fig. 6b).

Identification of GR constituents
When standards compounds were analyzed by HPLC, the retention times of glycyrrhizin and glycyrrhetic acid were 19.5 and 29.8 unit, respectively. The two compounds were detected in GR extracts (Fig. 7). Quantitative analysis of glycyrrhetic acid indicated a concentration of 0.027 mg/g in the extract.

Discussion
Inflammatory bowel disease (IBD) including Crohn's disease and UC is a chronic relapsing intestinal inflammatory disorder. Even if the pathogenic mechanism of IBD is barely understood, recent evidence suggests that deviant immune responses results in IBD symptoms [26][27][28][29]. UC symptoms can include weight loss and bloody diarrhea [30]. Immune modulators, such as sulfasalazine and glucocorticosteroids, have been broadly used for therapy for UC [31]. These therapies can cause adverse effects like vomiting, anemia and generalized edema. Thus, interest has grown in the use of traditional herbal medicines for

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inflammatory chronic disease [32]. Approximately 10 individuals per 100,000 people are diagnosed annually with UC [33]. There are over the 20 animal models of colitis. DSSinduced colitis is one of the most suitable experimental models [34]. Using this model, we presently demonstrate that GR alleviates clinical signs of UC (weight loss, colon shortening, diarrhea and occult/gross bleeding) (Figs. 1 and 2). IL-6, TNF-α and IFN-γ are pathogenic mediators of murine colitis [35,36]. In IBD, immune cells including T lymphocytes and macrophages secrete inflammatory

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cytokines in the area of inflammation. These activated cells modulate the balance between pro-and antiinflammatory cytokines. IL-6 and TNF-α expression is elevated in the rectal mucosa of UC patients [37]. Presently, GR suppressed the DSS-induced increase in IL-6 and TNF-α in mouse serum and tissues (Fig. 3).
In the inflammation reaction, COX-1 protein activity rarely changes, but COX-2 production dramatically increases, leading to the increased production of PGs [38]. Among the PGs, PGE 2 is increased in the intestines of IBD patients [39]. 5-ASA is used to treat IBD by inhibiting COX-2 activation [40]. COX-2 and PGE 2 play key role as mediators in UC. Presently, GR inhibited the DSS-induced activation of COX-2 and PGE 2 (Fig. 5). The results suggest that the anti-inflammatory effect of GR is attributable to the regulation of COX-2 in DSS-induces colitis. We also found that GR reduced epithelial injury and inflammatory cell infiltration into colon tissue (Fig. 6).
Pro-inflammatory mediators, such as inflammatory cytokines and chemokines are controlled by the activity of transcriptional factors. The activation of NF-κB is important in the pathogenesis of IBD [41][42][43]. DSSinduced IκBα phosphorylation and phosphorylation of NF-κB p65 in colonic tissues were suppressed by GR treatment. These results suggested that GR can inhibit the activation of transcription factors in UC.
Along with IL-6, TNF-α, COX-2 and NF-κB p65 phosphorylation, inflammation related factors including caspases and mitogen-activated protein kinases affect clinical signs [44]. Further investigation of GR effects on colitis is needed to clarify the discrepancy.
GR is used as a traditional herbal medicine in many Asian countries. Glycyrrhizin is the principal component of GR, and has been used for treatment of liver disease, allergic reaction and gastric ulcers [45]. Glycyrrhetic acid is another component, which has an anti-inflammatory effect on lipopolysaccharide-induced liver injury and inhibition of hepatic lipotoxicity [46]. These compounds might be useful for the prevention of inflammatory disease.
In summary, the anti-inflammatory activities of GR were as effective as those of 5-ASA. GR may inhibit inflammation by regulating the inflammatory mediators COX-2 and NF-κB. GR might be a useful therapeutic agent for the treatment of inflammatory diseases.

Conclusions
GR extract reduces the clinical signs and levels of inflammatory mediators in DSS-induced colitis in mice. GR extract suppresses PGE 2 production. This study provides experimental evidence to show that GR might be a useful therapy in the treatment of UC.