Protective Effect of Iridoid Glycosides of the Leaves of Syringa oblata Lindl. on Dextran Sulfate Sodium-Induced Ulcerative Colitis by Inhibition of the TLR2/4/MyD88/NF-κB Signaling Pathway.

Iridoid glycoside (IG) is the major active fraction extracted from the leaves of Syringa oblata Lindl. In view of its antimicrobial and antidiarrheal potential, it could be beneficial for the treatment of ulcerative colitis (UC). In the present study, IG (20, 40, and 80 mg/kg) was administered orally for 14 days to dextran sulfate sodium- (DSS-) induced colitis rats. The anti-inflammatory effects of IG on DSS-induced UC were evaluated by comparing observations in DSS-induced colitis and drug-treated groups using disease activity index (DAI), macroscopic score, histological analysis, and apoptosis assay. To elucidate the antioxidant mechanisms of IG on NOX-dependent ROS production, the activities of 8-OHdG, NOX1, and NOX2 in DSS-induced colitis were determined. The levels of proinflammatory cytokines such as IL-2, IL-4, IL-5, IL-12p40, and IL-13 were detected. The inflammation-associated protein and mRNA expressions of TLR-2, TLR-4, MyD88, and NF-κBp65 were assessed by immunohistochemistry and real-time quantitative PCR, respectively. The results suggested that IG treatment significantly reduced DAI, macroscopic score, and histological damage compared to untreated animals (p < 0.01), whereas administration of IG remarkably attenuated the upregulation of 8-OHdG, NOX1, and NOX2 and the expression of proinflammatory cytokines such as IL-2, IL-4, IL-5, IL-12p40, and IL-13 in DSS-treated rats in a concentration-dependent manner. In addition, IG treatment could dose dependently suppress the protein and mRNA levels of TLR-2, TLR-4, MyD88, and NF-κBp65. The dose of IG that produced the most significant protective effect was 80 mg/kg. The above results demonstrate that IG exerts its inhibitory effect on cell apoptosis, oxidative stress, and proinflammatory cytokines in DSS-induced colitis through modulation of the TLR2/4/MyD88/NF-κB signaling pathway.


Introduction
Inflammatory bowel disease (IBD) is becoming a global issue with accelerating incidence in newly industrialized countries during the past three decades [1]. Ulcerative colitis (UC), the major form of IBD, is a common inflammatory disease of the gastrointestinal tract, which is characterized by nonspecific, severe chronic relapsing course with clinically quiescent periods followed by bouts of severe intestinal inflammation, which are characterized by abdominal pain, diarrhea, weight loss, and fecal blood [2,3]. Although the exact pathogenesis of UC has not been ascertained up to now, the abnormal mucosal immunity and colonic inflammation have been demonstrated to be major mechanisms involved in the pathophysiology of UC [3,4]. The abnormal activity of the host immune system and inflammatory response are predominantly characterized by increased production of proinflammatory cytokines and the activation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) [5,6]. In this regard, modulation of both abnormal mucosal immunity and inflammatory response could be an important therapeutic modality for UC.
Increasing studies have suggested that the TLRs/MyD88/NF-κB axis is a crucial inflammatory signaling pathway in the progression of UC and is involved in the process of immunization, colonic inflammation, oxidative stress, apoptosis, tumor occurrence, and other biological processes [7]. NF-κB plays an important role in regulating the expression of inflammatory cytokines, chemokines, growth factors, COX-2, iNOS, and so on [3,8]. Once activated, NF-κB can translocate to the nucleus and tie in DNA binding sites, which induces the production of important immune medi-ators such as proinflammatory cytokines, ROS, and RNS [9,10]. Oxidative stress in the intestinal tract is considered a major factor that contributes to the pathogenesis and progression of IBD, and NADPH oxidases (NOXs) are the main sources of ROS. The recent data suggest a positive correlation between upregulated NADPH oxidase (NOX) expression and gastrointestinal inflammation [11]. NOX possesses the special function of producing reactive oxygen. The previous studies also indicated that the epithelial NOX homologs, NOX1 and DUOX2, could produce a higher level of superoxide in the colon compared with phagocyte NOX2 [12]. Specially, the expression of NOX1 in human colonic epithelial cells is higher, and lymphocytes in lesions of CD and UC showed high levels of NOX1 expression [13]. In addition, oxidative stress contributes to the accumulation of oxidative damage products in the colon tissue such as 8-hydroxydeoxyguanosine (8-OHdG) [5]. Recent studies reveal that toll-like receptors (TLRs), such as TLR2, TLR4, and TLR6, are activated in the progression of UC [14]. TLR activation often stimulates the expression of  Figure 1: Chemical structure of syringopicroside, the major ingredient in iridoid glycoside (IG) fraction. HPLC chromatogram of IG fraction purified using D141 macroporous adsorption resin is measured at 221 nm. 2 BioMed Research International proinflammatory cytokines, such as interleukin-(IL-) 2, IL-4, IL-5, IL-12p40, and IL-13 [5,15]. Therefore, targeted inhibition of TLRs/MyD88/NF-κB by removal or deactivation of inflammatory mediators and oxidative stress could be an important protective and therapeutic treatment for UC. Syringa oblata Lindl., a green plant with the major fraction iridoid glycosides (IG), is widely used in China as a traditional Chinese medicine to treat intestinal inflammations [3]. As the most abundant active fraction extracted from the leaves of Syringa oblata Lindl., IG contain the high content of an active ingredient, syringopicroside [16]. In previous preclinical studies, we have evaluated their various potential applications such as antioxidative, anti-inflammatory, and immunomodulatory [3,16]. However, the precise mechanism of anti-inflammatory effects by IG in dextran sulfate sodium-(DSS-) induced colitis is still limited, and few studies have described the antioxidative and antiapoptotic effects of IG in DSS-induced colitis.
Herein, our hypothesis of the immunoregulatory and anti-inflammatory effects of IG might associate with intervention on the TLRs/MyD88/NF-κB signaling pathway. In the follow-up study, we investigated the protective effect of IG on DSS-induced UC in rat by assessing disease activity index (DAI), macroscopic scores, colon lengths, and histological changes. In vivo cytokine levels of IL-2, IL-4, IL-5, IL-12p40, and IL-13 were measured by an enzyme-linked immunosorbent assay (ELISA). The antioxidative role of IG was assessed by determining the activity of NOX1, NOX2, and 8-OHdG. Furthermore, the antiapoptosis role of IG was evaluated by TUNEL staining. The anti-inflammatory mechanisms of IG against the TLR2/4/MyD88/NF-κB signaling pathway were further elucidated by detecting protein and  Figure 2: Iridoid glycosides attenuated changes in DAI score, macroscopic score, and body weight loss in DSS-induced colitis. Rats were treated orally at different doses of IG 20, 40, and 80 mg/kg once for 14 days after administration of DSS. The severity of colonic injury and the clinical evaluation were measured by (a) DAI score, (b) macroscopic score, and (c) body weight. IG administration dose dependently attenuated these pathological parameters. Data are presented as the mean ± SD (n = 6 per group). ## p < 0:01 vs. the normal control group, * * p < 0:01 vs. the DSS-induced colitis group, § § p < 0:01 vs. the IG (20 mg/kg)+DSS group, and ★★ p < 0:01 vs. the IG (40 mg/kg)+DSS group.

Purification and Identification of Iridoid Glycosides.
Iridoid glycoside (IG) was purified using D-141 macroporous adsorption resin column from the leaves of Syringa oblata Lindl. under the guidance of a previously established procedure [17]. Briefly, the dried leaves of Syringa oblata Lindl. (500 g) were pulverized to a powder and passed through a 20-mesh sieve. The powder was extracted for 120 min by refluxing with deionized water at 90°C and repeated two times. The filtered solution was concentrated under vacuum The HPLC analysis of IG fraction was performed by a previously described method [16]. 1 H-NMR and 13 C-NMR spectra of the most abundant active ingredient in IG fraction, syringopicroside, were recorded on a Bruker AVANCE 500 MHz NMR spectrometer (Switzerland) using tetramethylsilane (TMS) as an internal standard. The samples were dissolved in deuterated methanol (CD 3 OD) before NMR analysis. Fast atom bombardment-mass spectrometry (FAB-MS) was recorded on a Micromass Autospec Ultima ETOFJEOL mass spectrometer.

Induction of Colitis and Evaluation.
Rats were randomly divided into five groups (n = 6 each group). Acute colitis was induced using a dose of 4% (w/v) DSS in drinking water for 7 days [3]. Rats in the normal group (I) received 0.9% saline solution only; by contrast, rats in the model group (II) received 4% DSS in drinking water. Following 7 days of DSS administration, rats in groups III~V received IG orally (20,40, and 80 mg/kg, respectively) during DSS treatment once per day for 14 days. In the experimental period, weights of rats were recorded daily. DAI and macroscopic scores were evaluated based on the previously established scoring system [3,16]. At the end of day 14, rats were sacrificed, and the colon was excised and measured. The colon tissues were fixed in 4% paraformaldehyde, then embedded in paraffin, and finally sectioned in 4 μm sections. The samples were stained with hematoxylin and eosin (H&E) according to the standard procedures for histological evaluation [16].

Apoptosis
Assay. The colonic cell apoptosis was assessed using the terminal deoxynucleotidyl transferase-(TdT-) mediated dUTP-biotin nick end labelling (TUNEL) kit. TUNEL-positive epithelial cells in colonic tissue were clearly identified as brown-stained nuclei, which suggested DNA fragmentation due to apoptosis. TUNEL-positive expression was detected via 1000 cells in random fields. 5 BioMed Research International of NOX1 and NOX2 were detected as previously reported [18]. In brief, a 20 μl supernatant of homogenized and centrifuged colon samples was added into a 96-well luminescence plate, then mixed with 80 μl PBS and 6.25 μl l M lucigenin. NADPH was added to start the reaction, and photoemission was determined by the absorbance at 340 nm, which is monitored every 30 s for 5 min.
2.7. Determination of Inflammatory Cytokine. The colon tissues were homogenized in ice-cold physiological saline at the final concentration of 10% (w/v). Cytokine levels of IL-2, IL-4, IL-5, IL-12p40, and IL-13 in colon tissue homogenates (1/5 dilution) were quantified using ELISA kits according to the manufacturer's instructions.
2.8. Immunohistochemical Staining. The protein expressions of TLR2, TLR4, MyD88, and NF-κBp65 were detected according to a method described previously [3]. Briefly, 4 μm colon sections were first treated with 3% hydrogen peroxidase for 10 min to block endogenous peroxidase, then incubated with the polyclonal primary antibody of TLR2, TLR4, MyD88, and NF-κBp65 (diluted to 1 : 100) overnight at 4°C. The colon sections were then washed with phosphate-buffered saline (PBS) and incubated with polyclonal rabbit anti-mouse biotinylated secondary antibody (Dako, CA, USA). After that, colon sections were incubated with 3,3′-diaminobenzidine solution (Sigma-Aldrich, St. Louis, MO, USA) and then stained with hematoxylin. Finally, images were observed under an Olympus BH-2 microscope (Tokyo, Japan).

Identification and Quantification of Iridoid Glycosides.
HPLC analysis confirmed that the major ingredient in IG fraction is syringopicroside (Figure 1). The content of syringopicroside in the iridoid glycosides fraction reached 57.83%, which was 25-fold to that in the crude extracts. 1

Iridoid Glycosides Ameliorate DSS-Induced Colitis.
Severe DSS-induced colitis was observed and characterized by obvious hyperemia, edema, stool consistency, and ulceration. In this study, DAI and macroscopic scores in the DSS-induced colitis group were higher than those in the normal control group. However, IG treatment significantly attenuated DAI and macroscopic scores during experimental colitis in comparison with the DSS group (Figures 2(a) and 2(b)). The difference among the three dose groups of IG was statistically significant (p < 0:05). Moreover, DSS intake obviously induced colonic shortening and weight loss. The mean colon length (63 ± 2:9 mm) in the DSS-induced model group was lower than that in the normal group (92 ± 6:3 mm, p < 0:01). Animals show significant weight loss accompanied with obvious diarrhea in the DSS model group compared to the normal control group. The decrease in colon length and body weight after DSS administration was gradually reversed by IG in a dose-dependent manner (p < 0:01, Figures 2(c) and 3). As described above, treatment with IG dose dependently improved these pathological symptoms.

Iridoid Glycosides Suppressed Histopathological Damage
and Apoptosis. H&E staining showed that DSS administration distorted glandular formation and led to the recruitment of inflammatory cells into the submucosal layer, leading to necrosis, hyperemia, and mucosal destruction (Figure 4(b)).
On the other hand, the histologic damage score in the DSS-induced colitis group was higher than that in the control group (Figure 4(f)). IG treatment remarkably attenuated these pathologic changes with a lower histologic damage score in a dose-dependent manner (p < 0:01, Figures 4(c)-4(f)).
The results are shown in Figure 4. Additionally, TUNEL staining indicated that colon tissues exhibited a significant increase of brown apoptotic cells and intercellular apoptotic fragments after treatment with DSS (p < 0:01, Figure 5(b)), whereas few TUNEL staining-positive cells were observed in the normal group ( Figure 5(a)). In contrast, a low-dose group of IG (20 mg/kg) reduced the number of apoptotic epithelial cells (Figure 5(c)).  Figure 5(d)). In particular, IG in a high dose of 80 mg/kg was the most effective in suppressing intestinal epithelial cell apoptosis (p < 0:01, Figure 5(e)). The results are shown in Figure 5.

Iridoid Glycosides Attenuated Oxidative Stress.
To evaluate the antioxidant effects of IG in a DSS-induced colitis model, we analyzed the levels of 8-OHdG, NOX1, and NOX2. In the DSS-induced colitis group, the expression levels of 8-OHdG, NOX1, and NOX2 in colon tissue were significantly higher than those in the normal control group. In addition, IG treatment could markedly suppress this colitis-induced increase in the levels of these three oxidative stress indicators (p < 0:01). The results suggest a negative correlation between the increased dose of IG and the expression of 8-OHdG and NADPH oxidase (NOX), as shown in Figures 6(a)-6(d).
3.5. Iridoid Glycosides Alleviate Inflammatory Cytokine. The levels of inflammatory cytokine in colon tissue were significantly upregulated in the DSS-induced colitis group compared to the normal control group (p < 0:01). In addition, IG treatment could remarkably reverse the levels of IL-2, IL-4, IL-5, IL-12p40, and IL-13 in a dose-dependent manner (p < 0:01). The maximum inhibition effect was observed with IG at a dose of 80 mg/kg, as shown in Figures 7(a)-7(e).

3.6.
Iridoid Glycosides Inhibited DSS-Induced TLR2/4/MyD88/NF-κB Signaling Pathway Activation. The TLR2/4/MyD88/NF-κB signaling pathway is an important signaling pathway in regulating inflammation. Its activation has been demonstrated to be related to the development of UC [14]. The activation of the TLR2/4/MyD88/NF-κB signaling pathway enhances the production of various proinflammatory cytokines and the induction of oxidative stress and apoptosis [10,14]. To explore the inhibitory mechanism of IG on oxidative stress and cytokines, the key protein expressions in the TLR2/4/MyD88/NF-κB signaling pathway were detected using real-time PCR and immunohistochemical staining. The TLR2/4/MyD88/NF-κB signaling pathway was significantly activated in the DSS-induced colitis group. The mRNA and protein levels of TLR2, TLR4, MyD88, and NF-κBp65 were all increased compared to the normal control group (p < 0:01). As expected, IG dose dependently suppressed the mRNA and protein levels of TLR2, TLR4, MyD88, and NF-κBp65 in comparison with the DSS group (p < 0:01). These data verified our hypothesis that IG might have an anti-inflammatory effect by blocking TLR2/4/MyD88/NF-κB activation. The results are shown in Figures 8-11. the intestine, leading to the dissemination of proinflammatory intestinal contents including ROS production, bloody diarrhea, weight loss, colon shortening, and mucosal ulceration [5,19]. Hence, DSS-induced colitis models are commonly used to study for IBD. DAI, macroscopic score, colon length shortening, and body weight loss are the main parameters in the evaluation of the severity of UC. In the present study, DAI, macroscopic score, colon length shortening, and weight loss were significantly increased after DSS treatment. From histological analysis and TUNEL staining, we also found that DSS intake caused remarkably inflamed tissue and had marked necrosis, hyperemia, numerous granulocytes, and high levels of apoptosis. Our findings demonstrated that administration of IG attenuated DSS-induced colonic injury dose dependently as assessed by these pathological parameters and the histologic damage. In addition, we indicated that IG treatment markedly suppressed DSS-induced high levels of apoptosis in a dosedependent manner. Excessive reactive oxygen species (ROS) in the physiopathology of UC contributes to oxidative damage and inflammatory cascade, and NADPH oxidases (NOXs) are the main sources of ROS [6]. Increasing evidence shows that oxidative stress leads to the accumulation of 8-OHdG in colon tissue [5]. Our previous reports have demonstrated that IG treatment could lead to a significant reduction in oxidative stress levels by virtue of decreasing levels of malondialdehyde (MDA) and NO in a trinitrobenzene sulfonic acid-induced colitis rat model [16]. However, few studies have investigated whether IG can suppress NOX-dependent ROS production in DSS-induced colitis. Our results confirmed experimental colon injury significantly induced the high expression of 8-OHdG, NOX1, and NOX2 compared to the normal control group. We also found IG was able to inhibit the upregulation of OHdG, NOX1, and NOX2 in a dosedependent manner.

Discussion
Recent studies have demonstrated increased production of proinflammatory cytokines including TNF-α, IL-2, IL-4, IL-5, IL-12, and IL-13 in IBD that are known to play a key role in the modulation of the intestinal immune system [3,15]. Our previous studies suggested that IG could obviously reduce the levels of proinflammatory cytokines such as TNF-α, IL-6, and IL-8 [3,16]. To further understand the protective roles of IG in DSS-induced colonic injury, we investigated the effects of IG on inflammation-related cytokines and the levels of IL-2, IL-4, IL-5, IL-12p40, and IL-13. In this study, DSS treatment dramatically enhanced the high expression of IL-2, IL-4, IL-5, IL-12p40, and IL-13. In our in vitro study, administration of IG significantly attenuated the expression of proinflammatory cytokines such as IL-2, IL-4, IL-5, IL-12p40, and IL-13 in DSS-treated rats in a concentration-dependent manner at the dose of 20-80 mg/kg. Hence, these data strongly indicated that IG could modulate the inflammatory process by inhibition of multiple inflammation-associated cytokines in experimental colitis.