6-Shogaol Inhibits Advanced Glycation End-Products-Induced IL-6 and ICAM-1 Expression by Regulating Oxidative Responses in Human Gingival Fibroblasts

Advanced glycation end-products (AGEs) cause diabetes mellitus (DM) complications and accumulate more highly in periodontal tissues of patients with periodontitis and DM. AGEs aggravate periodontitis with DM by increasing the expression of inflammation-related factors in periodontal tissues. 6-Shogaol, a major compound in ginger, has anti-inflammatory and anti-oxidative activities. However, the influence of shogaol on DM-associated periodontitis is not well known. In this study, the effects of 6-shogaol on AGEs-induced oxidative and anti-oxidative responses, and IL-6 and ICAM-1 expression in human gingival fibroblasts (HGFs) were investigated. When HGFs were cultured with 6-shogaol and AGEs, the activities of reactive oxygen species (ROS) and antioxidant enzymes (heme oxygenase-1 [HO-1] and NAD(P)H quinone dehydrogenase 1 [NQO1]), and IL-6 and ICAM-1 expressions were investigated. RAGE expression and phosphorylation of MAPKs and NF-κB were examined by western blotting. 6-Shogaol significantly inhibited AGEs-induced ROS activity, and increased HO-1 and NQO1 levels compared with the AGEs-treated cells. The AGEs-stimulated expression levels of receptor of AGE (RAGE), IL-6 and ICAM-1 and the phosphorylation of p38, ERK and p65 were attenuated by 6-shogaol. These results suggested that 6-shogaol inhibits AGEs-induced inflammatory responses by regulating oxidative and anti-oxidative activities and may have protective effects on periodontitis with DM.


Introduction
Diabetes mellitus (DM) is a major risk factor of periodontal diseases [1]. Hyperglycemia occurs in DM and induces high glycation of proteins by a Maillard reaction, and then glycated proteins result in advanced glycation end-products (AGEs) [2]. AGEs accelerate diabetic complications, including diabetic nephropathy, diabetic cataract, diabetic peripheral neuropathy, and cardiovascular disorders [3]. AGEs bind to a receptor for AGE (RAGE) and increase the expression of pro-inflammatory molecules such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and vascular cell adhesion molecule-1 in some cells [4]. AGEs accumulate excessively in periodontal tissues of DM patients compared with non-DM individuals [5] and were detected in epithelial cells, fibroblasts, endothelial cells, and inflammatory cells in periodontal tissues of DM patients [6]. AGEs levels in serum from diabetic In the present study, we investigated the effects of 6-shogaol on AGEs-induced oxidative and anti-oxidative responses and on AGEs-upregulated IL-6 and ICAM-1 expression in HGFs.

6-Shogaol Increases the Levels of HO-1 and NQO1 in HGFs
The effect of 6-shogaol on anti-oxidative factor levels was investigated in cultures with AGEs (500 µg/mL) for 12 h. Although AGEs had no effect on HO-1 production in HGFs, 5 µM 6-shogaol in the presence of AGEs significantly increased HO-1 levels, and 10 µM 6-shogaol elevated HO-1 levels to approximately 4.2-fold compared with in cells cultured with only AGEs ( Figure 3A). Although AGEs (500 µg/mL) significantly inhibited NQO1 activity, 6-shogaol (5 and 10 µM) significantly restored AGEs-downregulated NQO1 activity, and its activity returned to the control level (bovine serum albumin [BSA]) ( Figure 3B). Effects of AGEs and 6-shogaol on antioxidant enzymes production. HGFs were cultured with AGEs (500 µg/mL) or BSA (500 µg/mL) for 12 h after pre-treatment with 6-shogaol (5 or 10 µM) for 1 h. (A) HO-1 levels in the cell lysate of treated HGFs were assessed using ELISA. (B) NQO1 levels in the cell lysate were assessed using an NQO1 activity assay kit, as described in the Materials and Methods section. Data are expressed as the mean ± SD of 6 (A) and 5 (B) independent experiments (* p < 0.05, ** p < 0.01).

Effects of 6-Shogaol on AGEs-Induced RAGE Expression and Antivations of MAPKs and NF-κB Signaling Pathways in HGFs
AGEs (500 µg/mL) elevated RAGE expression in HGFs, and 6-shogaol canceled AGEs-upregulated RAGE expression in HGFs ( Figure 4A). When the effects of 6-shogaol and AGEs on the phosphorylation of MAPKs (p38 and ERK) and NF-κB p65 were investigated, AGEs (500 µg/mL) upregulated the phosphorylation of p38 and ERK MAPKs and NF-κB p65 ( Figure 4B-D). This effect of AGEs was similar to our previous study [11]. 6-Shogaol (10 µM) decreased AGEs-stimulated phosphorylation of p38 and ERK MAPKs and NF-κB p65 ( Figure 4B-D). When the intensity of each band of three separate samples was analyzed by densitometric measurement, AGEs significantly increased the phosphorylation levels of p38 and ERK MAPKs and NF-κB p65, and 6-shogaol significantly decreased AGEs-upregulated phosphorylation levels. Sub-confluent HGFs were cultured with AGEs (500 µg/mL) or BSA (500 µg/mL) for 24 h after pre-treatment with 6-shogaol (10 µM) for 1 h. Cell lysates were used for western blotting for RAGE as described in the Materials and Methods section. (B-D) Sub-confluent HGFs were pre-treated with 6-shogaol (10 µM) and then stimulated with AGEs (500 µg/mL) or BSA (500 µg/mL) for 30 min. Cell lysates were analyzed by western blotting using antibodies against MAPKs (p38, phospho-p38, ERK and phospho-ERK) (B,C) and NF-κB (p65 and phospho-p65) (D) as described in the Materials and Methods section. Data are expressed as the mean ± SD of 3 independent experiments. The density of signal bands was determined by densitometric analysis and normalized to that of the BSA group. (* p < 0.05, ** p < 0.01).

Figure 5.
Effects of 6-shogaol on AGEs-induced production of IL-6 and ICAM-1. Sub-confluent HGFs were pre-treated with 6-shogaol (5 or 10 µM) for 1 h and cultured with AGEs (500 µg/mL) or BSA (500 µg/mL) for 48 h. NAC (1 mM) was also added into the HGF culture with AGEs. The amounts of IL-6 (A) in the supernatant and ICAM-1 (B) in the cell lysate were determined using their respective ELISA kits as described in the Materials and Methods section. Data are expressed as the mean ± SD of 6 independent experiments (** p < 0.01).

Discussion
AGEs, a major factor in DM complications, is thought to influence DM-associated periodontitis by regulating oxidative stress and IL-6, and ICAM-1 expression levels in HGFs [12]. In the present study, 6-shogaol inhibited AGEs-induced inflammation-related responses including ROS, IL-6, and ICAM-1. Ginger components such as 6-shogaol and 6-gingerol have been investigated as therapeutic treatment agents for diabetes-associated complications [40][41][42]. 6-Shogaol and 6-gingerol trapped methylglyoxal (MGO), inhibiting the formation of MGO-induced AGEs [43], and the combination of 6-shogaol and epicatechin more strongly trapped MGO in mouse urine [44], suggesting that 6-shogaol not only suppressed AGEs-induced ROS production and IL-6 expression, but also inhibited a formation of AGEs by trapping MGO.
This component in ginger suppressed ROS production in several cells such as PMNs [36], endothelial cells [45], and epidermal keratinocytes [46], whereas 6-shogaol increased the levels of antioxidants. When human epidermal keratinocytes were cultured with 6-shogaol (10 µM), Nrf2 was positively translocated into the nucleus, NQO1 levels were elevated, and dexamethasone-suppressed HO-1 levels were restored [46]. Another study also showed that 6-shogaol and a 6-shogaol-rich extract from ginger increased the translocation of Nrf2 into the nucleus and enhanced HO-1 levels in a human hepatoma cell line [38]. Considering our study and others' reports, 6-shogaol appears to inhibit AGEs-induced IL-6 and ICAM-1 expression by regulating oxidative and anti-oxidative responses in HGFs.
The effects of 6-shogaol on oxidative stress and inflammatory responses are similar to those of several plants and their extracts. Green and black tea extracts significantly inhibited AGEs-induced ROS production and IL-6 secretion in 3T3-L1 preadipocytes [47], and Moutan Cortex extract suppressed AGEs-induced IL-6 and monocyte MCP-1 levels in vivo and in vitro [48], and Ginkgo biloba extract suppressed AGEs-induced ROS production in rat kidney fibroblasts [49]. These plant extracts are thought to have anti-diabetic potentials via their anti-oxidative activities [47][48][49]. We speculate that 6-shogaol may also contribute to the treatment for diabetes-associated complications because 6-shogaol has similar effects to other plant extracts.
Although ginger components show several anti-diabetic actions, such as reducing the levels of blood glucose and HbA1c, and decreasing the production of ROS, IL-6, and TNF-α [36,[39][40][41], their mechanisms of action are not well known. 6-Shogaol inhibited AGEs-induced RAGE expression in HGFs in the present study, and it inhibited MGO-induced RAGE expression and increased HO-1 expression in human retinal epithelial cells [50], suggesting that 6-shogaol partially functions in a regulator of RAGE expression. We showed previously that AGEs increased the expression levels of IL-6 and ICAM-1 via the RAGE, MAPKs (ERK and p38) and NF-κB pathways in HGFs [12]. When the effects of 6-shogaol on the phosphorylation of p38 and ERK MAPKs and NF-κB p65 were investigated in HGFs, 6-shogaol inhibited the AGEs-induced phosphorylation of these proteins. 6-Shogaol also increased the phosphorylation of p38 and ERK MAPKs, and slightly elevated JNK phosphorylation in human breast cancer cell [51], and specific inhibitors of p38 (SB202190) and PI-3K (LY294002) suppressed 6-shogaol-induced Nrf2 translocation into the nucleus in human colon cancer cells [52]. In the present study, 6-shogaol significantly decreased AGEs-induced ROS production in HGFs, however, its inhibition was not complete. Park et al. [46] also showed that 6-shogaol partially inhibited TNF-α+INF-γ-induced ROS production in human keratinocytes. We did not know the exact reason of partial inhibitory effect of 6-shogaol on ROS production. Since the regulation of ROS production by AGEs and 6-shogaol are associated with multiple factors including MAPK (p38, ERK, JNK), NF-κB and PI3K [12,[51][52][53], 6-shogaol may partially inhibit AGEs-induced ROS production. Ginger extract inhibited NF-κB activity and decreased IL-8 and vascular endothelial growth factor secretion in an ovarian cancer cell line [54], and Zingiber officinale decreased nuclear NF-κB levels in the livers of high-fat diet-fed rats and IL-1β-induced IL-6 and IL-8 expression levels by downregulation of NF-κB activity in human hepatocytes [55]. Ginger extracts show anti-inflammatory effects through the RAGE, MAPKs, and NF-κB pathways in metabolic diseases and cancers [12,51,52,54,55]. In contrast, oxidative and anti-oxidative pathways are related to the shogaol-induced anti-inflammatory responses, because 6-shogaol and ginger extracts suppressed ROS production and elevated the levels of anti-oxidative factors such as Nrf2, HO-1, and NQO1 levels [36][37][38]45,46]. Kim et al. [37] showed that 6-shogaol exerts antioxidant activity via signaling pathways related to HO-1, Nrf2, and γ-glutamylcysteine synthetase. 6-Shogaol inhibited ROS production and activated HO-1, NQO1, and Nrf2 in vitro and ameliorated experimental allergic dermatitis-like skin lesions in vivo [46], and it suppressed Nrf2 expression in the kidney and decreased the levels of blood glucose and HbA1c in diabetic mice [40]. In HGFs, 6-shogaol inhibited AGEs-induced IL-6 and ICAM-1 expression levels, and NAC, a ROS inhibitor, also showed similar inhibitory effects. 6-Shogaol may produce anti-inflammatory and anti-diabetic actions by modulating oxidative and anti-oxidative responses in periodontal tissues.
Ginger extracts and 6-shogaol exert anti-diabetic effects including a decrease in blood glucose and HbA1c levels, and have preventive potential for DM therapy and amelioration of diabetic complications [39][40][41]. Ginger extracts may have therapeutic effects on several inflammatory diseases: 6-shogaol reduced chronic inflammation in the knees [56] and attenuated ulcerative colitis in a murine model [57], and gingerol prevented both joint inflammation and destruction in experimental rheumatoid arthritis [58]. Furthermore, mouthwash containing ginger extracts showed significant improvement of modified gingival index and gingival bleeding index in patients with gingivitis [59], and 10-and 12-gingerols showed effective anti-bacterial activity for P.gingivalis and Prevotella intermedia [60].
Taken together, 6-shogaol inhibited AGEs-induced inflammation-related factors by regulating oxidative and anti-oxidative activities in HGFs. Ginger extracts including 6-shogaol may be effective for the prevention and treatment of DM-associated periodontitis.

AGEs and Reagents
AGEs were prepared in accordance with the modified method of Takeuchi et al. [61]. Briefly, 50 mg/mL of BSA (Sigma-Aldrich, St. Louis, MO, USA) and 0.1 mol/L of dl-glyceraldehyde (Sigma-Aldrich) were mixed in sterile phosphate-buffered saline (PBS, 0.2 mol/L, pH 7.4) with penicillin (100 U/mL) and streptomycin (100 µg/mL), and incubated at 37 • C for 7 days. The mixture was dialyzed against PBS (pH 7.4) at three times for 3 days to remove low-molecular-weight reactants and free glyceraldehyde. Non-glycated BSA was prepared as a control solution from the mixture without glyceraldehyde in the same conditions. AGE activity was assayed by determining the fluorescence of AGE and non-glycated BSA solutions at excitation/emission wavelengths of 370/440 nm with reference to the method of Huang et al. [62], and AGE solution showed 45-fold stronger fluorescence intensity than that of control non-glycated BSA solution was used in the present study.

Cell Culture
The HGF cell line, CRL-2014 ® , was obtained from ATCC (Manassas, VA, USA). HGFs were seeded at 4,800 cells/cm 2 and cultured in DMEM supplemented with 10% FBS for 5 days and reached sub-confluency. HGFs were cultured using a 96-well plate (SUMITOMO BAKELITE, Tokyo, Japan) for 5 days and used for cell viability assays. In ROS assays, HGFs were cultured using a 96-well black plate (Nunclon ™ , Thermo Scientific ™ , Waltham, MA, USA) for 5 days, and then treated for ROS assays.
In other experiments, the sub-confluent HGFs were pre-treated with 2.5-15 µM 6-shogaol for 1 h and cultured with 500 µg/mL of AGEs or BSA for 0.5-48 h and used for a western blotting analysis and enzyme-linked immunosorbent assays (ELISAs).

Cell viability Assay and Observation of Cellular Morphology
Cell viability was examined using a Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) in accordance with the manufacturer's instructions. Briefly, HGFs were cultured with 2.5-15 µM 6-shogaol for 48 h after reaching sub-confluency, and then incubated with 10 µL CCK-8 solution at 37 • C for 2 h in a humid atmosphere with 5% CO 2 . The absorbance of each well was measured at 450 nm using a microplate reader (iMark™; Bio-Rad, Hercules, CA, USA). The morphologies of cells treated with 6-shogaol (2.5-15 µM) for 48 h were observed using a phase contrast microscope at 40-fold magnification.

ROS Measurement
ROS was measured using an OxiSelect™ Intracellular ROS Assay kit (Cell Biolabs, San Diego, CA, USA) in accordance with our previously reported method [12]. Briefly, HGFs were cultured in a 96-well black plate for 5 days and incubated with 2 ,7 -dichlorofluorescin diacetate (DCF, 20 µmol/L) at 37 • C for 1 h. Cells were washed in PBS and stimulated with 2.5-15 µM 6-shogaol in the presence of AGEs (500 µg/mL) or BSA (500 µg/mL) for 12-48 h. The fluorescence intensity of DCF in the cultured cells was determined at 480/530 nm using the Varioskan™ Flash Multimode Reader (Nunclon ™, Thermo Scientific™).

HO-1 and NQO1 Measurement
Sub-confluent HGFs were pre-treated with 6-shogaol (5 or 10 µM) for 1 h and cultured with AGEs (500 µg/mL) or BSA (500 µg/mL) for 12 h. HO-1 and NQO1 levels were determined using a Human Heme Oxygenase 1 (HO 1) Simple Step ELISA Kit (Abcam) and an NQO1 activity assay Kit (Abcam), respectively, in accordance with the manufacturer's instructions. Briefly, the lysate from treated cells was collected in cell extraction buffer from the HO-1 kit, incubated on ice for 20 min, and centrifuged at 18,000× g for 20 min at 4 • C. HO-1 in the supernatant responded with the antibodies and then with TBS substrate, and the absorbance of the reaction solution was measured at 450 nm. HO-1 levels were normalized to the total cell protein amount, which was measured using Bio-Rad protein assay reagent (Bio-Rad). NQO1 activity in the cell lysate samples was determined by following the reduction of menadione and the simultaneous reduction of water soluble tetrazolium salts (WST1), and expressed as dicoumarol-sensitive activity using the NQO1 kit. Briefly, the treated HGFs were solubilized in the extraction buffer from the kit, incubated on ice for 15 min and centrifuged at 18,000 × g for 20 min at 4 • C. NQO1 activity in the supernatant samples was determined and expressed as a percentage of activity in the BSA group.

Enzyme-Linked Immunosorbent Assay
Sub-confluent HGFs were pre-treated with 6-shogaol (5 or 10 µM) for 1 h and cultured with AGEs (500 µg/mL) or BSA for 48 h. NAC (1 mM) was also added to HGF cultures with AGEs. The conditioned medium was collected and mixed with protease inhibitor cocktail, and cell lysates were extracted in lysis buffer including 10 mmol/l Tris-HCl, pH 7.4, 50 mmol/l NaCl, 5 mmol/l EDTA, 1 mmol/l sodium orthovanadate, 1% NP-40 and protease inhibitor cocktail. IL-6 in the conditioned medium was measured using a Human IL-6 ELISA kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer's instructions. ICAM-1 in the cell lysate was determined using a ICAM1 Human ELISA kit (Invitrogen, Carlsbad, CA, USA), and the amount of ICAM-1was normalized to total cell protein amount.

Statistical Analysis
All statistical analyses were performed with SPSS Statistics version 20 (IBM, Chicago, IL, USA). The significant differences among multiple groups were performed using one-way analysis of variance followed by Tukey's HSD. p < 0.05 was considered significant.