Thymol exerts anti-inflammatory effect in dextran sulfate sodium-induced experimental murine colitis

Purpose: To investigate the effect of thymol on dextran sulfate sodium (DSS)-induced experimental colitis in mice. Methods: Colitis in mice was induced by drinking water with 2.5 % (w/v) DSS. The levels of myeloperoxidase (MPO), malondialdehyde (MDA), glutathione (GSH) and superoxide (SOD) in colonic tissues were determined using commercial kits. Histological changes were evaluated by hematoxylin and eosin staining. The production of nitric oxide (NO) was measured by Griess assay. The levels of pro-inflammatory cytokines including TNF-α, IL-1β and IL-6 were assessed by quantitative PCR and ELISA kits. Nuclear factor-kappaB (NF-κB) pathway activation was determined by Western blot. Results: Thymol markedly reduced disease activity index (DAI) scores, and recovered the colon length. Histological damage and MPO levels in the colonic tissues were markedly inhibited (p < 0.05) by thymol, which also reduced mRNA expressions of TNF-α, IL-1β and IL-6 in the colon. In addition, it downregulated MDA level but elevated GSH and SOD levels. Moreover, in vitro data showed that thymol significantly inhibited (p < 0.05) lipopolysaccharide-induced secretion of NO, TNF-α, IL-1β and IL-6 in macrophages, and suppressed (p < 0.05) the activation of NF-κB pathway. Conclusion: Thymol attenuates experimental colitis by down-regulating the activation of NF-κB pathway. Therefore, thymol is a potential candidate drug for the management of ulcerative colitis.


INTRODUCTION
Ulcerative colitis (UC) is a chronic inflammatory disorder in the colon, which not only harms the life quality of patients, but elevates the probability of colorectal cancer [1,2].The precise cause of UC is poorly understood.However, bowel inflammation could be elicited by dysregulated immune response to commensal gut flora in individuals with genetic susceptibility [3].Dextran sulfate sodium (DSS)-induced colitis in mice has been extensively applied for UC study, which is characterized by many symptoms mimic some clinical features in human UC such as body weight loss, diarrhea, blood in stool and mucosal ulceration [4].
Traditional therapies of UC are 5-
Thymol, a natural monoterpene compound, is one of the active components of Chinese herb Thymus vulgaris, and the constituent of other plants such as Monarda punctate and Origanum vulgare spp [7].Previous studies showed that thymol had various bioactivities, such as antiinflammatory [8,9], anti-bacterial [10], anticancer and antioxidant properties [11].Although thymol had wide-ranging pharmacological actions, the anti-inflammatory ability of thymol on experimental colitis has not been studied.The present study was to investigate the effect of thymol on DSS-induced colitis in mice.The mechanisms of how the thymol inhibits oxidative stress, pro-inflammatory mediator's production and NF-κB signaling pathway were explored.

Animals
Male C57BL/6 mice, 6-8 weeks old, were used (Beijing Vital River Laboratory Animal Tech, Beijing, China).The study protocol was approved by the Ethics Committee of Chengdu University of Traditional Chinese Medicine (no.2014 DL-023) in accordance to "Principles of Laboratory Animal Care" (NIH publication no.85-23, revised 1985) [12].

Colitis induction
DSS-induced colitis was performed as described previously [13].Mice were randomly assigned into five groups: normal, model, thymol (30 mg/kg), thymol (60 mg/kg) and 5-ASA (100 mg/kg).In addition to mice in normal group, other mice were provided with drinking water with 2.5 % (w/v) DSS for seven days ad libitum, from day 0 to day 6, and then with water from day 7 to day 9. Thymol (30, 60 mg/kg) and 5-ASA (100 mg/kg) were orally administered for 10 consecutive days, from day 0 to day 9.
The mice were observed and recorded daily for body weight, diarrhea and gross bleeding in feces.Disease activity index (DAI) score = (body weight + diarrhea + gross bleeding) / 3 [14].Mice were euthanized by CO 2 inhalation at day 9 after DSS induction.The colon was harvested and the length was determined.

Histological assessment
The colon tissues were fixed in paraformaldehyde and further embedded in paraffin.Then, the slides of colon tissues stained with hematoxylin and eosin.Histological score was carried out by analyzing the inflammation severity, inflammation extent and crypt damage as previously described [15].

Evaluation of MDA, GSH, SOD and MPO
Levels of MDA, GSH, SOD and MPO in colon homogenate were determined according to the manufacturer's protocol.Total protein was measured by a bicinchoninic acid protein kit (Beyotime Biotech, Shanghai, China).

Real-time PCR analysis
Total RNA was extracted from colon tissue using TRIzol reagent and reverse transcribed to cDNA.The mRNA expression were performed on a CFX96 Real-time PCR Detection System (Biorad) according to the instructions of HiScript Q RT SuperMix and AceQ qPCR SYBR Green Master Mix.The primer sequences are listed in Table 1.The expression of GAPDH was used for the normalization.

Cell culture
RAW 264.7 cells were obtained from Shanghai Institute of Cell Biology (Shanghai, China).RAW

Measurement of nitric oxide (NO) and cytokines
RAW 264.7 cells were plated in 96-well plate at a density of 2 × 10 5 cells per well, and stimulated with 1 μg/mL LPS in the presence of thymol (50, 100 and 200 μM) for 24 h.NO level in supernatant was evaluated as described before [17].The absorbance at 550 nm was measured using a microplate reader (Thermo).According to the manufacture's protocol, the levels of TNF-α, IL-6 and IL-1β in cell supernatant were determined using specific ELISA kits.

Statistical analysis
Data are shown as mean ± SEM.One-way ANOVA followed by Dunnett's test were used to determine the level of significance, and p < 0.05 was considered statistically significant.The data were analyzed using GraphPad Prism 5 (La Jolla, USA).

Effect of thymol on colitis
To determine the effect of thymol on experimental colitis, colitis was induced by adding DSS into drinking water in mice.DSS markedly induced DAI elevation in mice.Thymol (60 mg/kg) as well as 5-ASA decreased the DAI score when compared to the DSS group (Figure 1A). Figure 1B shows that thymol (60 mg/kg) significantly recovered the colon length when compared with the DSS group, so as 5-ASA did. Figure 1C shows that the activities of MPO in colons were also decreased in thymol (60 mg/kg) and 5-ASA.

Effect of thymol on colonic pathological changes
Histological analysis of colons from DSS-induced mice demonstrated severe damage to the epithelium, goblet cells lost, inflammatory cells infiltration, and severe damages in mucosal and submucosal.However, these pathological changes were much attenuated by thymol (60 mg/kg) and 5-ASA (Figure 2A).The histological scores of each group were shown in Figure 2B.

Effect of thymol on the production of proinflammatory cytokines in colons
To analyze the effect of thymol on the proinflammatory cytokines involved in the induction of UC, the mRNA expressions of TNF-α, IL-6 and IL-1β in colon tissues were determined by realtime PCR.In DSS group, the mRNA expressions of TNF-α, IL-6 and IL-1β were markedly increased when compared with normal group.Thymol (60 mg/kg) and 5-ASA treatments significantly reduced the level of TNF-α, IL-6 and IL-1β in colon tissues (Figure 3A-C).

Effect of thymol on oxidative stress in colon tissues
To determine the effect of thymol on oxidative stress involving in the inflammatory response and intestinal damage, the levels of MDA, GSH and SOD in colon tissues were measured.The DSS administration increased MDA level, decreased levels of GSH and SOD in colon tissues.However, thymol (60 mg/kg) treatment

Effect of thymol on the cell viability in macrophages
Macrophages play key roles in the secretion of pro-inflammatory cytokines.Firstly, the effect of thymol on the cell viability in the presence or absence of LPS in RAW264.7 cells was investigated.Figure 5 A and B showed thymol had no significant cytotoxicity on RAW 264.7 cells at concentrations below 400 μM.

Effect of thymol on levels of inflammatory mediators in LPS-induced macrophages
The effect of thymol on the secretion of inflammatory mediators, such as NO, TNF-α, IL-6 and IL-1β in LPS-induced macrophages was evaluated.LPS stimulation markedly increased the production of NO, TNF-α, IL-6 and IL-1β in RAW264 cells.However, thymol (100 and 200 μM) inhibited the LPS-induced increases in levels of NO, TNF-α, IL-6 and IL-1β in a concentration-dependent manner (Figure 6 A-D).

Effect of thymol on activation of NF-κB pathway in LPS-induced macrophages
In general, LPS binds to toll-like receptors and activates many downstream signaling pathways, such as NF-κB pathway [18].To address the mechanism for thymol inhibiting the levels of proinflammatory mediators, effect of thymol on the activation of NF-κB signaling by using Western blot was determined.Figure 7 showed that LPS treatment markedly increased the level of phosphorylation of IκBα and NF-κB-p65 in RAW264.7 cells.Thymol treatment significantly down-regulated the activation of NF-κB signaling pathway.

DISCUSSION
It is indispensable to optimize the therapeutic options for UC treatment.The present study aimed to find a potent drug candidate for the treatment of UC.The study report that thymol attenuates colitis induced by DSS in mice by inhibiting pro-inflammatory mediator production from macrophages via down-regulation of activation of NF-κB pathways.
In the clinic, UC is often associated with the goblet cell depletion, destruction of crypt architecture, inflammatory cells infiltration, and crypt inflammation [19].In the results, thymol treatment inhibited inflammatory damage such as loss of goblet cell, mononuclear cell infiltration.The level of MPO indicated that infiltration of neutrophils in the colon tissues, and thymol showed a markedly reduction in MPO levels in the colon.Previous studies showed that overproduction of inflammatory mediators from macrophages, such as pro-inflammatory cytokines and reactive oxygen metabolites (e.g., NO), is mostly involved in the inflammation cascade reaction and subsequent colon damage in UC [20].Macrophages secrete TNF-α, IL-6 and IL-1β, which are considered as crucial inflammatory mediators.In the colon mucosa of patients with UC, their levels are markedly increased [21].The results demonstrated that thymol inhibited production of pro-inflammatory mediators in the colons of colitis mice, as well as in LPSstimulated macrophages.
Previous studies showed that oxidative stress kept the inflammation response active in the colon mucosa.The lipid peroxidation product MDA cause tissue damage.In parallel, reduction of GSH and SOD, the main intracellular antioxidant, have been detected in the colonic mucosa of UC patients [22].The results showed that treatment with DSS markedly reduced the levels of GSH and SOD, and enhanced MDA level in the colonic tissues, which is in consistent with the previous finding [23].The results shows that thymol significantly suppressed oxidative stress induced by DSS.
LPS are recognized by mucosal macrophages via TLR4.Then macrophages have an elevated ability to produce TNF-α, IL-6 and IL-1β, causing the activation of NF-κB [24].Hence, it is speculated that the anti-inflammatory effect of thymol was due to the suppression of NF-κB pathway, regulating the expression of numerous inflammatory factors.The inducible form of NF-κB is typically a heterodimer composed of a p50 and p65 subunits, present in cytoplasm as an inactive complex associated with inhibitory protein IκB.The activation of this complex by various stimuli lead to the phosphorylation, ubiquination and degradation of IκB proteins.Then, NF-κB heterodimer (p65/p50) will rapidly translocate into the nucleus, regulate the transcriptions of a large number of target genes, such as pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) [25,26].Although thymol inhibited NF-κB-p65 phosphorylation in mouse epithelial cells [27], the results demonstrated it can inhibit LPSinduced NF-κB activation in macrophages.

CONCLUSION
Thymol shows protective effects against DSSinduced colitis, and reduces the production of pro-inflammatory mediators and oxidative damage in colon tissues.The mechanism involves the inhibition of the NF-κB activation.Thus, thymol is a potential candidate drug for UC treatment.

Figure 4 :
Figure 4: Effect of thymol on oxidative stress in colon tissues.The levels of (A) MDA, (B) GSH and(C) SOD in the colon tissues were measured by commercial kits (n = 6).## P < 0.01 vs normal, p < 0.05, **p < 0.01 vs DSS attenuated MDA level, improved levels of GSH and SOD in colon tissues (Figure 4A-C).

Figure 5 :
Figure 5: Effect of thymol on the cell viability in macrophages.Cell viability was determined by using MTT assay.RAW264.7 cells were treated with various concentrations of thymol (25, 50, 100, 200 and 400 μM) (A) in the absence or (B) presence of LPS (1 μg/mL) for 24 h.Data are representative of three independent experiments; **p < 0.01 vs normal

Figure 6 :
Figure 6: Effects of thymol on levels of inflammatory mediators in LPS-induced macrophages.RAW264.7 cells were cultured with thymol (50, 100 and 200 μM) in the presence or absence of 1 μg/mL LPS.After treatment with 24 h, the culture supernatants were collected.(A) NO level was measured by using Griess assay.The levels of (B) TNF-α, (C) IL-6 and (D) IL-1β were determined by using ELISA kits.Data are representative of three independent experiments; ## p < 0.01 vs normal, *p < 0.05, **p < 0.01 vs model

Table 1 :
Prime sequences of target genes