Caffeic acid phenethyl ester protects against oxidative stress and dampens inflammation via heme oxygenase 1

Periodontal disease is associated with chronic oxidative stress and inflammation. Caffeic acid phenethyl ester (CAPE), which is a potent inducer of heme oxygenase 1 (HO1), is a central active component of propolis, and the application of propolis improves periodontal status in diabetic patients. Here, primary murine macrophages were exposed to CAPE. Target gene expression was assessed by whole-genome microarray, RT-PCR and Western blotting. The antioxidative and anti-inflammatory activities of CAPE were examined by exposure of the cells to hydrogen peroxide, saliva and periodontal pathogens. The involvement of HO1 was investigated with the HO1 inhibitor tin protoporphyrin (SnPP) and knockout mice for Nrf2, which is a transcription factor for detoxifying enzymes. CAPE increased HO1 and other heat shock proteins in murine macrophages. A p38 MAPK inhibitor and Nrf2 knockout attenuated CAPE-induced HO1 expression in macrophages. CAPE exerted strong antioxidative activity. Additionally, CAPE reduced the inflammatory response to saliva and periodontal pathogens. Blocking HO1 decreased the antioxidative activity and attenuated the anti-inflammatory activity of CAPE. In conclusion, CAPE exerted its antioxidative effects through the Nrf2-mediated HO1 pathway and its anti-inflammatory effects through NF-κB inhibition. However, preclinical models evaluating the use of CAPE in periodontal inflammation are necessary in future studies.


INTRODUCTION
Caffeic acid phenethyl ester (CAPE) is a central active component of propolis from honeybee hives. Propolis has multiple applications in dentistry. Systemic propolis improved the periodontal status in patients with type 2 diabetes, 1 and local propolis application reduced plaque accumulation, gingival inflammation 2 and oral mucositis during chemotherapy. 3 In preclinical models, CAPE protected against ligature-induced periodontitis 4 and systemic bone loss by cortisone, 5 and supported bone defect healing. 6 Therefore, CAPE is attracting attention in periodontology, 7,8 and interest is growing in uncovering the beneficial effects of CAPE in periodontal therapy.
Periodontal disease is characterised by chronic inflammation 9 and the concurrent challenge of oxidative stress, 10,11 which together culminate in oral tissue destruction and ultimately tooth loss. Therefore, CAPE is of potential clinical interest. For example, CAPE prevents damage of cells exposed to hydrogen peroxide, including neurite PC12 cells, 12 retinal 661W cells 13 and umbilical vein endothelial cells. 14 CAPE also exerts anti-inflammatory activity in gingival fibroblasts 7 and macrophages 8 exposed to endotoxins. Thus, CAPE supports the major defence mechanisms of cells challenged by oxidative stress and inflammation.
Nuclear factor erythroid 2-related factor 2 (Nrf2) is released from its suppressor Kelch-like ECH-associated protein 1 (Keap1) upon stimulation with CAPE. 15,16 Nrf2 translocates into the nucleus and initiates the transcription of heme oxygenase 1 (HO1). HO1, which is linked to orthodontic tooth movement 17,18 and inflammation in a periodontitis model, 10 is a major target of CAPE. For instance, CAPE increases HO1 expression in gingival fibroblasts 7 and macrophages. 8 In turn, HO1 activates the cellular defence mechanisms against oxidative stress, 19 including superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) expression. 20 In support of this mechanism, Nrf2 21 and HO1 22,23 regulate SOD expression.
Tin protoporphyrin IX dichloride (SnPP) blocks HO1 activity, which allows insights into the role of HO1 in anti-inflammatory activity in macrophages 24 and periodontal cells. 25 Moreover, Nrf2 activation participates in the inhibitory effect of CAPE on nuclear factor-κappa-B (NF-κB) signalling. 26 Independent of HO1, CAPE inhibits lipopolysaccharide (LPS)-induced interleukin (IL)-1β expression in RAW264.7 cells 8 and inflammation in epithelial cells. 27 However, CAPE requires HO1 to exert its anti-inflammatory activity, such as LPS-induced nitric oxide production, in RAW264.7 cells. 8 Thus, the data are heterogeneous with respect to the ability of HO1 to mediate the anti-inflammatory activity of CAPE.
Mitogen-activated protein kinase (MAPK) may contribute to the CAPE-induced increase in HO1 expression. For example, the p38 MAPK inhibitor SB203580 attenuates the HO1 expression induced by CAPE. 28 SB203580 also decreases HO1 expression in other in vitro models, including neuronal cells 29 and RAW264.7 macrophages. [30][31][32] Thus, the ability of CAPE to regulate HO1 and consequently to counteract oxidative stress and inflammation may involve p38 MAPK signalling.
The aim of the present study was to perform a genome-wide screening approach to evaluate CAPE-regulated genes and to use functional assays to better understand the role of HO1 in coordinating the cellular defence against oxidative stress and inflammation.

CAPE increases heat shock proteins
Based on a whole-genome microarray, treatment of three independent preparations of primary murine macrophages with CAPE caused expression changes of five characteristic stressresponse genes more than 10-fold: Hspa1a (HSP70), Hspa1b, Hsph1 (HSP105, member of the HSP70 family), growth arrest and DNAdamage-inducible protein GADD45 gamma (Gadd45g) 33 and adrenomedullin (ADM) 34 (Fig. 1a). Increased Hspa1a, Hspa1b, Hsph1, Gadd45g and ADM expression was confirmed by reverse transcription-PCR (RT-PCR) analysis (Fig. 1b). Considering that heat shock proteins include HO1 (Hsp32), which is frequently coexpressed, 35 and that CAPE is a known activator of HO1 expression in gingival fibroblasts 7 and macrophages, 8 we introduced SnPP, which is an inhibitor raised against HO1. 36 SnPP almost abolished the CAPE-induced increase in Hsp and Gadd45g expression (Fig. 1c).
CAPE increases HO1 expression Next, we confirmed that CAPE was capable of increasing HO1 expression in murine macrophages and gingival fibroblasts. HO1 expression was considerably increased by CAPE in primary macrophages, RAW264.7 cells and gingival fibroblasts (Fig. 2a). HO1 was also increased at the protein level in macrophages as determined by Western blotting analysis (Fig. 2b). In macrophages, blocking p38 MAPK with SB203580 but not JNK and ERK attenuated the increase in HO1 expression (Fig. 2c). Moreover, cells from Nrf2-knockout mice displayed reduced HO1 expression in response to CAPE (Fig. 2d). Time-response experiments showed a peak in HO1 gene expression changes after 3-12 hours (Fig. S1).

HO1 mediates the antioxidative activity of CAPE
To determine whether CAPE could support the ability of cells to cope with oxidative stress, various periodontal cell types were exposed to hydrogen peroxide. As predicted, 12-14 hydrogen peroxide decreased the viability of HSC-2, MC3T3-E1 and gingival fibroblasts, as indicated by the reduced capacity to form formazan crystals (Fig. 3a) and reduced trypan blue staining of the HSC-2 cells (Fig. 3b). Blocking HO1 activity with SnPP reversed the positive effects of CAPE in the various cell types (Fig. 3a, b). Moreover, SnPP counteracted the increased SOD expression induced by CAPE (Fig. 3c). A trend towards lower CAT and GST expression by SnPP was also observed but failed to reach the level of significance (Fig. 3c).
HO1 and inhibition of NF-κB subunit p65 control the antiinflammatory activity of CAPE In support of previous data, 37 primary macrophages showed a massive inflammatory response when exposed to human saliva (Fig. 4a). In the presence of CAPE, the strong proinflammatory response was almost abolished (Fig. 4a). Moreover, CAPE greatly suppressed the inflammatory response of macrophages exposed to supernatants of periodontal pathogens (e.g. Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia) (Fig. S2). Next, we sought to investigate whether HO1 mediated the anti-inflammatory response of CAPE. SnPP reversed the anti-inflammatory effect of CAPE on salivainduced IL-1α and IL-1β expression in primary macrophages but not in RAW264.7 cells (Fig. 4b, c). In support of previous findings, 8,38 CAPE inhibited the translocation of p65 into the nucleus in RAW264.7 cells exposed to inflammatory clues. As expected, inhibition of p65 translocation by CAPE in RAW264.7 cells occurred independent of SnPP ( Fig. 4d and    The data represent the mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001 in the two-tailed Mann-Whitney test

DISCUSSION
Chronic inflammation in periodontitis is accompanied by oxidative stress, which together culminate in the catabolic events that ultimately lead to tissue destruction and tooth loss. 39,40 One potential therapeutic strategy to regain periodontal health apart from scaling and root planing is to curtail inflammation and oxidative stress by local application of CAPE. Initial support for this hypothesis came from observations that propolis improved the periodontal status in type 2 diabetes patients 1 and that CAPE protected against ligature-induced periodontitis in rats. 4 CAPE also protects against other detrimental conditions, including peptidoglycan polysaccharide-induced colitis, 41 Cr(VI)-induced brain toxicity, 42 acetylsalicylic acid-induced lung damage, 43 cyclosporine A-induced nephrotoxicity, 44 cerulean-induced acute pancreatitis, 45 cholestatic liver injury induced by bile duct ligation 46 and acute myocardial ischaemia/reperfusion injury. 47 Therefore, CAPE reduces detrimental effects related to inflammation and also exerts cytotoxic activity. Our intention was to extend the existing knowledge of how CAPE helps cells in the periodontium cope with inflammatory cues and hydrogen peroxide. To screen for CAPE target genes, a microarray analysis was performed, which revealed a more than 10-fold increase in Hspa1a, Hspa1b, Hsph1, Gadd45g and ADM expression in murine macrophages. CAPE was reported to increase HspA5 expression in neuroblastoma cells 48 and Hspa1a/Hspa1b expression in endothelial cells. 49 With respect to possible relevance to periodontology, Hspa1a is upregulated in the periodontal ligament at the early stage of tooth movement in rats. 50 Hydrogen sulphide-exposed oral keratinocyte stem cells express Gadd45g 51 and ADM was detected in human gingival crevicular fluid. 52 Moreover, we can reasonably suggest that Hsps, Gadd45g and ADM play a role in defence of the periodontium against oral pathogens, because recombinant Hsp70 protects cells from oxidative stress 53 and reduces inflammation in sepsis models. 54 Gadd45 proteins are implicated in cell cycle checkpoints, apoptosis and DNA repair. 55 ADM not only regulates the vascular tonus but also has antioxidant and anti-inflammatory properties with therapeutic potential for myocardial infarction and inflammatory bowel diseases. 56 Hspa1a, Hspa1b, Hsph1 and Gadd45g expression was reduced by blocking HO1 activity with SnPP. Surprisingly, HO1 did not reach the 10-fold expression level in the gene array that it achieved in the RT-PCR. As expected, HO1 expression was increased by CAPE in all cell types investigated, including primary macrophages, RAW264.7 macrophages and gingival fibroblasts.
These findings support a role for CAPE as a potent agonist of HO1 expression in gingival fibroblasts 7 and macrophages. 8 However, we cannot state the percentage of HO1-positive cells, because no flow cytometry analysis was performed. To further explore the underlying signalling mechanism, the MAPKs were blocked. SB203580 attenuated the increased HO1 expression induced by CAPE. These findings are in support of the ability of p38 to regulate CAPE-induced HO1 expression in rat organotypic midbrain slice cultures. 28 MAPK signalling has been implicated in Nrf2 induction. However, whether this effect occurs directly through phosphorylation of Nrf2 or through indirect and less specific mechanisms is unclear. Although inhibition of p38 abolished Nrf2 activation in glial cells, 57 genetic deficiency or pharmacological inhibition of p38 increased HO1 expression through a Nrf2-dependent pathway. 58 Considering the role of Nrf2 as an upstream regulator of HO1, cells from Nrf2-knockout mice displayed reduced HO1 expression in response to CAPE. These findings are in agreement with the release of Keap1 upon stimulation with CAPE, 15,16 which allowed Nrf2 to initiate HO1 transcription.
In the present study, blocking HO1 with SnPP partially reversed the protective effects of CAPE on hydrogen peroxide damage similar to the other HO1 inducers quercetin 59 and baicalein. 60 In support of this functional assay, SnPP reduced CAPE-induced SOD expression. These findings were in line with reports showing that SnPP decreased SOD in smoke-induced emphysema rats. 61 Additionally, a moderate decrease was observed in CAT, which is the enzyme that catalyses the decomposition of hydrogen peroxide to water and oxygen, and GST in this setting. Together, the data suggested that CAPE increased HO1 expression, which in turn drove SOD expression and probably that of other detoxifying enzymes to avoid hydrogen peroxide-induced cell death.
Here, SnPP increased the expression of inflammatory cytokines in primary macrophages, suggesting that the anti-inflammatory activity of CAPE required HO1 activity. However, SnPP did not modulate the anti-inflammatory effects of CAPE in RAW264.7 cells. In support of previous findings, 8,38 CAPE inhibited translocation of p65 into the nucleus, thereby reducing the transmission of proinflammatory signals. Again, SnPP failed to reverse the effects of CAPE by blocking the translocation of p65 into the nucleus in RAW264.7 cells, suggesting that the anti-inflammatory activity of CAPE was independent of HO1 activity. Similar to our observations with primary macrophages, SnPP attenuated the inhibitory effects of flavonoids and hemin on LPS-induced inflammatory production in macrophages 24 Supplementary Fig. 3. The impact of MAPK signalling was investigated using the respective inhibitors (SP600125 for JNK, SB203580 for p38 and U0126 for ERK) (c), n = 3. HO1 gene expression was further investigated in the Nrf2 −/− mice (d), n = 3. Data represent the mean ± SD. *P < 0.05 and **P < 0.01 in the two-tailed Mann-Whitney test periodontal ligament cells. 25 However, CAPE dampened inflammation in epithelial cells 27 and LPS-induced IL-1β production in RAW264.7 cells 8 independent of HO1 signalling. Therefore, HO1 is not necessarily involved in mediating the anti-inflammatory activity of CAPE in vitro. Nevertheless, our cumulative in vitro data suggest that CAPE-induced HO1 may be a central regulator that helps cells cope with oxidative stress and inflammation. Support for this hypothesis came from studies in which SnPP reversed the beneficial effects of baicalein and other HO1 inducers. For example, SnPP abolished the baicalein-, tetramethylpyrazine-and cepharanthine-mediated protection against ischaemia/reperfusion injury in various models. 22,62,63 Future studies with CAPE should involve SnPP to reveal the role of HO1 in vivo.
The clinical relevance of the data presented here is based on revealing the molecular and cellular mechanisms of the beneficial effects of propolis on oral health. 1-3 Its major bioactive component (CAPE) protects against ligature-induced periodontitis 4 and systemic bone loss by cortisone 5 and supports bone defect healing. 6 Considering that periodontal disease is caused by chronic inflammation 9 and oxidative stress 10,11 and that CAPE suppresses inflammation and oxidative damage, our in vitro findings provide indirect support for HO1 as a target of periodontal therapy. Our data may also serve as a primer to extend this research to other fields in dentistry, including mucositis, periimplantitis and pulpitis. However, care should be taken concerning the possible side effects of CAPE. For example, CAPE can have a strong antimitogenic activity in lung cancer, prostate cancer, melanoma, 64 breast cancer 65 and oral cancer. 66 CAPE exerted cytotoxic effects on neck metastasis of gingiva carcinoma and tongue squamous cell carcinoma cells. 67 Hence, safety and efficacy studies are required prior to clinical topical application of CAPE in the periodontium.

CONCLUSION
Here we show that CAPE exerts its antioxidative effects through Nrf2-mediated HO1 expression and its anti-inflammatory activity through NF-κB inhibition.     Cell viability For the viability experiments, the cells were incubated with CAPE with or without SnPP for 30 min prior to exposure to 1% H 2 O 2 . After 3 h, the cell viability assay and trypan blue staining were performed. For cell viability, MTT (Sigma) solution at a final concentration of 0.5 mg·mL −1 was added to each well of a microtiter plate (CytoOne) and incubated for 2 h at 37°C. Then, the medium was removed, and the formazan crystals were solubilized with dimethyl sulphoxide. The optical density was measured at 570 nm. The data are expressed as percentages of the optical density in the treatment groups normalised to the unstimulated control values. Moreover, 50 μL of 0.4% trypan blue (Sigma) was added to each well and incubated for 5 min at room temperature. Then, the trypan blue solution was discarded, and the cells were examined by microscopy and photographed.
Cell inflammatory response For the inflammation experiments, the cells were incubated with CAPE with or without SnPP prior to exposure to 1% sterile saliva. After 24 h, the supernatant was harvested, and RNA was isolated. Whole human saliva was collected from two authors (R. G. and A.S.), who were non-smokers and gave their informed consent. Saliva flow was stimulated by chewing paraffin wax (Ivoclar Vivadent AG, Schaan, Liechtenstein) without eating and drinking for 1 h prior to collection. Immediately after collection, the saliva was centrifuged at 4 000 × g for 5 min. The saliva supernatant was passed through a filter with a 0. Bacterial cultures Bacterial cultures of P. gingivalis, T. denticola and T. forsythia were grown in Schaedler broth (Oxoid Basingstoke, GB) supplemented with 0.25 mg·L −1 of vitamin K (and 10 mg of N-acetyl muramic acid for T. forsythia) and modified mycoplasma broth (BD, Franklin Lake, NJ, USA) supplemented with 1 mg·mL −1 of glucose, 400 mg· mL −1 of niacinamide, 150 mg·mL −1 of spermine tetrahydrochloride, 20 mg·mL −1 of Na isobutyrate enriched with 1 g·mL −1 of cysteine and 5 mg·mL −1 of cocarboxylase. Fortyeight-hour broth cultures of the bacteria were centrifuged. The resulting supernatants were filtered through 400-μm membranes, and the sediments were exposed to intensive ultrasonication for 10 min.
Immunostaining Immunofluorescent analysis of NF-κB p65 was performed in RAW264.7 cells plated onto Millicell ® EZ slides (Merck KGaA, Darmstadt, Germany) and treated with 10 μmol·L −1 CAPE for 30 min prior to exposure to 1% saliva. The cells were fixed in paraformaldehyde and blocked with 1% bovine serum albumin and 0.3% Triton in PBS at room temperature for 1 h. Then, the cells were incubated with a NF-κB p65 primary antibody (1:100; Cell Signaling Technology, USA). An Alexa 488 secondary antibody (1:200, Santa Cruz Biotechnology, USA) was applied for 1 h. The cells were washed, the nuclei were stained with 4′,6-diamidino-2-phenylindole (100 ng·mL −1 ) and the cells were mounted onto glass slides. Fluorescent images were captured at ×100 in oil immersion using a Zeiss Axiovert 200M fluorescence microscope.

Statistical analysis
The primary culture experiments were performed at least three times. All experiments were repeated three to five times. The bars show the means and standard deviations of cumulative data from all experiments. The statistical analysis was based on the Mann-Whitney U test and Kruskal-Wallis test with Dunn's multiple comparisons correction. The analyses were performed using Prism v7 (GraphPad Software, La Jolia, CA, USA). Significance was set at P < 0.05.