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ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)
International Journal of Oral Biology Vol.46 No.1 pp.51-59
DOI : https://doi.org/10.11620/IJOB.2021.46.1.51

Baicalin suppresses lipopolysaccharide-induced matrix metalloproteinase expression: action via the mitogenactivated protein kinase and nuclear factor κB-related protein signaling pathway

Seon-Yle Ko*
Department of Oral Biochemistry and Institute of Dental Science, Dankook University College of Dentistry, Cheonan 31116, Republic of Korea
*Correspondence to: Seon-Yle Ko, E-mail: seonyleko@dankook.ac.kr
February 15, 2021 March 18, 2021 March 19, 2021

Abstract


Periodontal disease is an inflammatory disease that affects the destruction of the bone supporting the tooth and connective tissues surrounding it. Periodontal ligament fibroblasts (PDLFs) induce overexpression of matrix metalloproteinase (MMP) involved in periodontal diseaseʼs inflammatory destruction. Osteoclasts take part in physiological bone remodeling, but they are also involved in bone destruction in many kinds of bone diseases, including osteoporosis and periodontal disease. This study examined the effect of baicalin on proteolytic enzymesʼ production and secretion of inflammatory cytokines in PDLFs and RAW 264.7 cells under the lipopolysaccharide (LPS)-induced inflammatory conditions. Baicalin inhibited the expression of the protein, MMP-1 and MMP-2, without affecting PDLFs’ cell viability, suggesting its possibility because of the inhibition of phosphorylation activation of mitogen-activated protein kinase’s p38, and the signal transduction process of nuclear factor κB (NFκB)-related protein. Also, baicalin reduced the expression of MMP-8 and MMP-9 in RAW 264.7 cells. This reduction is thought to be due to the inhibition of the signal transduction process of NFκB-related proteins affected by inhibiting p65RelA phosphorylation. Also, baicalin inhibited the secretion of nitric oxide and interleukin-6 induced by LPS in RAW 264.7 cells. These results suggest that baicalin inhibits connective tissue destruction in periodontal disease. The inhibition of periodontal tissue destruction may be a therapeutic strategy for treating inflammatory periodontal-diseased patients.


Baicalin , Scutellaria baicalensis , Matrix metalloproteinases , Cytokines

초록

    © The Korean Academy of Oral Biology. All rights reserved.

    This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Introduction

    Periodontal disease has characteristic of chronic inflammation caused by oral bacterial infections such as Porphyromonas gingivalis, which can lead to tooth loss due to periodontal destruction [1,2]. Lipopolysaccharide (LPS) permeates in the outer membrane of Gram-negative anaerobic bacteria such as P. gingivalis which causes periodontitis. It stimulates the secretion of the inflammatory mediators such as interleukin (IL)-1, nitric oxide (NO), and prostaglandin E2 [3,4]. These mediators play an important role in connective tissue destruction and osteoclastogenesis [5,6].

    In the inflammatory diseases including chronic periodontitis, proteolytic enzymes such as matrix metalloproteinases (MMPs) play an essential role in the process of connective tissue destruction. MMPs degrade the extracellular matrix components including collagen, fibronectin, and core proteins of proteoglycan [7] and if it is excessively produced, it stimulates degradation of the extracellular matrix of periodontal tissue, resulting in connective tissue destruction and alveolar bone loss, followed eventually by tooth loss.

    These proteolytic enzymes are collagenase, gelatinase, and stromelysin, and depending on the substrate specificity, are classified, MMP-1, MMP-8, and MMP-13 as collagenase, MMP-2 and MMP-9 as gelatinase, and MMP-3, MMP-10, and MMP-11 as stromelysin [8,9]. In many earlier studies on periodontitis patients [10-12], MMP-1, MMP-2, MMP-8, and MMP-9 levels were observed to be increased in gingival crevicular fluid, periodontal tissues and periodontal ligament (PDL) cells.

    It is a well-known fact that LPS stimulates macrophages, activates the signaling system for the mitogen-activated protein kinase (MAPK) as well as immunoreactive mediators such as NO. p38, one of the MAPK family, can be activated by phosphorylation of the subunit, which plays a vital role in the differentiation and proliferation of cell as well as inflammation [13]. Besides, LPS plays a vital role in the activation of the nuclear factor κB (NFκB), a protein belonging to the Rel protein group, which controls the transcription of the target gene by the formation of a dimer. RelA/p50 dimer is the most common form of transcription factor NFκB. When the signal transfer to a cell, inhibitory κB (IκB) is phosphorylated by IκB kinase (IKK) and then degraded, and NFκB isolated from IκB regulates their transcription. RelA could also phosphorylate, and RelA phosphorylation is known to give regulatory effects on transcriptional activation and protein stability [14,15].

    The PDL is a cell-connective tissue that connects cementum to the surrounding bone. The periodontal ligament fibroblasts (PDLFs) are the primary cell types of PDLs and play a role in immune response and supporting cells for PDL. It suggests that the location of these cells in pathological conditions of periodontal disease plays an important role in regulating the inflammatory response and amplifying the inflammatory signal [16].

    The osteoclast is a bone-resorbing cell derived from the monocyte/macrophage lineage, and the substances such as macrophage colony-stimulating factor and receptor activator of nuclear factor kappa-B ligand (RANKL) are essential for the osteoclast survival and differentiation [17]. RANKL, if it is bound to RANK, attracts adapter molecules such as tumor necrosis factor receptor-associated factor 6. This combination mediates a variety of signal transduction processes including NFκB, Jun N-terminal kinase (JNK), p38, extracellular signal-regulated kinase (ERK), and PI3K/AKT [18,19]. Bone destruction by osteoclasts plays a vital role in pathological bone destruction in inflammatory diseases as well as physiological bone resorption.

    Recently, with the increasing interest in oriental herbal medicines, many studies are underway to treat various inflammatory diseases. Natural compounds such as flavonoids are found in many plants, including edible plants [20]. Of these, baicalin (7-glucuronic acid, 5,6-dihydroxyflavone) is a bioactive flavonoid compound purified from the dried roots of the perennial plant, Scutellaria baicalensis. Baicalin, as a substance showing various biological features, is known to work on anti-inflammatory, analgesic and antimicrobial effects [21]. It has also been reported that baicalin stimulates osteoblast differentiation through Wnt/b-catenin signal [22]. The purpose of this study is to induce the inflammation reaction using LPS in human PDL cells and mouse mononuclear cells and identify baicalin effect on the expression of MMP enzyme and its mechanism.

    Materials and Methods

    1. Reagents

    LPS from Escherichia coli was purchased from Sigma-Aldrich (St. Louis, MO, USA). Baicalin (Sigma-Aldrich) was dissolved in dimethyl sulfoxide (1,000×). Antibodies against MMP-1, MMP-2, JNK, p38, ERK, IKK, and actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and antibodies against MMP-8 and MMP-9 were purchased from Abcam (Cambridge, UK). The antibodies against phospho-JNK, phospho- p38, phospho-ERK, phospho-IKK, p65RelA, phosphop65RelA, AKT, and phospho-AKT were purchased from Cell Signaling (Danvers, MA, USA).

    2. A culture of periodontal ligament fibroblasts and RAW 264.7 cells

    The human PDL tissue was taken from healthy teeth from three orthodontic patients. Dankook University Hospital Ethics Committee has approved it, and we had received consent before the extraction (IRB No. H-1009/006/002). After placing in a culture dish, the PDL tissue, scraped in a mid-third of the tooth root surface with a surgical blade, we cultured small dose of Dulbecco’s modified Eagle’s medium (DMEM) including 10% fetal bovine serum (FBS) and antibiotics (10,000 U/ mL penicillin G and supplemented with 10 μg/mL of streptomycin) (HyClone, South Logan, UT, USA). When we found out that the cells grew enough for the experiment while observing their growth from the outset, we removed the cells with 0.25% trypsin and 0.2% ethylenediaminetetraacetic acid and subcultured at a ratio of 1:5. Cells between the 5th and 9th generations were used.

    We cultured mouse monocyte/macrophage RAW 264.7 cells, obtained from the American Type Culture Collection (Manassas, VA, USA), in a cell incubator, maintained at the temperature of 37℃ and 5% CO2 atmosphere, with DMEM containing 10% FBS. Cell culture media were changed every 3 days.

    3. Cell survival test using methylthiazolyldiphenyltetrazolium bromide (MTT)

    We seeded PDLFs or RAW 264.7 cells in 96-well plates in DMEM containing 10% FBS at a density of 104 cells/well. We treated them, after 24 hours, with 1 μg/mL LPS and serially diluted baicalin. PDLFs were treated for 2 days, and RAW 264.7 cells were treated for 4 days. We tested cell survival rate with MTT analysis according to the manufacturer’s protocol (Sigma M5655) (Sigma-Aldrich). After the reaction, we removed the supernatant, dissolved the formazan granule in isopropyl alcohol, and measured the absorbance using a microplate absorbance reader.

    4. Test by western blot

    We plated PDLFs or RAW 264.7 cells in 6-well plates at a density of 5 × 105 cells/well. After 24 hours, we incubated the cells for 6 hours in serum-free medium, pretreated with serially diluted baicalin for 2 hours in medium containing 10% FBS, and then stimulated with 5 μg/mL LPS for 15 minutes or 2 hours. After incubation, we collected the cells and prepared protein samples using lysis solutions. Protein samples (30 μg/ lane) were electrophoresed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. We used as a block, Tris-buffered saline and Tween20, containing 5% bovine serum albumin, got reaction using such antibodies as MMP-1, MMP-2, MMP-8, MMP-9, phospho-ERK1/2 (Thr202/Tyr204), phospho-SAPK/ JNK (Thr183/Tyr185), phospho-p38, phospho-IKK, phosphop65 RelA, and phospho-AKT, and then reprobed with nonphospho antibodies corresponding to MAPK, IKK, p65RelA. The band was observed using X-ray film (Fuji film, Tokyo, Japan) exposure and chemiluminescence system (Amersham BioSciences, Buckinghamshire, UK).

    5. IL-6 secretion test using ELISA

    We plated RAW 264.7 cells in 96-well plates with 104 cells/ well and cultured them in α-minimum essential medium containing 10% FBS, to test the effect of baicalin on IL-6. One day later, we cultured the cells with 1 μg/mL of LPS and sequentially treated with diluted baicalin for 2 days. The amounts of IL-6 dissolved in the culture media were tested according to the manufacturer’s protocol, using an ELISA kit (R&D Systems, Minneapolis, MN, USA). Absorbance was detected at 450 nm, using a spectrophotometer.

    6. Nitrite test

    We evaluated the secretion of NO by measuring the level of nitrite in a stable oxidized form dissolved in culture media. To test the effect of baicalin on inducible nitric oxide synthase activation, we plated RAW 264.7 cells in 96-well plates at 104 cells/well and then cultured in α-MEM containing 10% FBS. After 24 hours, we cultured the cells with 1 μg/mL of LPS and diluted baicalin consecutively in the phenol red-free media for 72 hours. After incubation, we got a reaction from 100 μL of culture supernatant with 100 μL of Griess reagent (Sigma- Aldrich) in a 96-well reading plate and detected absorbance using a spectrophotometer at 550 nm.

    7. Statistical analysis

    For significance, Student’s t-test was used. All tests were conducted in triplicate. Each value represents the mean ± standard error. The difference was considered statistically significant at p ＜ 0.05 and p ＜ 0.01.

    Results

    1. Expression results of MMP-1 and MMP-2 in periodontal ligament cells

    To examine the effect of baicalin on the expression of MMP, we observed the expression of MMP-1 and MMP-2 from the PDL cells by a western blot method. First, we confirmed the cytotoxicity of baicalin. As a result, the cells treated with LPS and 1 and 10 μg/mL of baicalin showed no cytotoxicity (Fig. 1A). Therefore, we examined the expression of MMP-1 and MMP-2 in PDL cells and found that when treated with LPS, the protein expression of MMP-1 and MMP-2 increased, on the other hand, when treated with baicalin, was significantly reduced (Fig. 1B).

    2. Baicalin’s effect on MAPK activation in periodontal ligament cells

    MAPKs activation was observed by western blot analysis to examine the effect of baicalin on the signal transduction process in PDL cells. For this, protein samples were prepared 15 minutes after treatment with 1 and 10 μg/mL baicalin, and the activation of ERK, JNK, and p38 was observed as the degree of phosphorylation. Activation of JNK and p38 was observed at 15 minutes after the treatment of LPS with PDL cells, but ERK did not show any distinct change. Pretreated baicalin before stimulation with LPS inhibited phosphorylation of p38 (Fig. 2).

    3. An effect of baicalin on NFκB related activation in periodontal ligament cells

    Because it has been reported that NFκB could be a vital transcription factor in the expression of inflammatory cytokines by LPS, we investigated whether baicalin affects NFκB activation in PDL cells. After pretreatment with baicalin for 2 hours, stimulation with LPS was performed. We prepared protein samples and observed changes in phosphorylation of IKK. IKK phosphorylation was increased after 10 minutes of PDL cells treated with LPS, and quantitative changes were observed up to 30 minutes after treatment. IKK phosphorylation was inhibited within 15 minutes in all cases treated with LPS and baicalin (Fig. 3).

    Therefore, the changes of phosphorylation of IKK, p65RelA, and AKT were observed 15 minutes after treatment with LPS. As a result, phosphorylation of IKK, p65RelA, and AKT was increased in LPS-treated cells, and the increased phosphorylation was inhibited after pre-treatment with 10 μg/mL of baicalin (Fig. 4).

    4. Expression result of MMP-8 and MMP-9 in RAW 264.7 cells

    To examine the effect of baicalin on the expression of MMP, we used western blot method to observe the expression of MMP-8 and MMP-9 from RAW 264.7 cells. Treatment of LPS and 0.4 to 10 μg/mL baicalin did not show cytotoxicity in the culture of RAW 264.7 cells. At 50 μg/mL, baicalin showed a statistically significant 20% reduction in cell proliferation (Fig. 5A). Therefore, we found out that when treated with LPS, MMP-8 and MMP-9 protein expression increased and the increased MMP expression significantly reduced by pre-treatment with baicalin; from our examination of their expression in RAW 264.7 cells, treated with baicalin at a concentration of 10 μg/mL, which does not exhibit cytotoxicity (Fig. 5B).

    5. An effect of baicalin on MAPK activation in RAW 264.7 cells

    The effect of baicalin on the phosphorylation of MAPK was evaluated to examine which step of the signal transduction process is affected by baicalin in the osteoclast activation. RAW 264.7 cells were pretreated with baicalin for 2 hours and then induced with LPS for 15 minutes. The activation of MAPK molecules such as JNK, ERK1/2, and p38 was examined by western blot analysis. LPS induced phosphorylation of JNK, ERK1/2 and p38, but no change by baicalin showed up (Fig. 6).

    6. An effect of baicalin on NFκB-related activation in RAW 264.7 cells

    We investigated whether baicalin affects the activation of NFκB in RAW 264.7 cells. After pretreatment with baicalin for 2 hours, stimulation with LPS was performed. We prepared protein samples and observed changes in phosphorylation of IKK. The phosphorylation of IKK, p65RelA, and AKT was increased after 15 minutes treatment with LPS, and the increased phosphorylation was inhibited after pre-treatment with baicalin (Fig. 7).

    7. IL-6 and NO results

    To observe the effect of baicalin on the secretion of inflammatory mediators, we treated RAW 264.7 cells, in the culturing process, with LPS to promote dissolution of IL-6 and NO. As a result, we found out that the secreted amount of IL-6 and NO induced by LPS significantly decreased (Fig. 8).

    Discussion

    This study investigated effects of baicalin, one of the flavonoids, on the regulation of LPS-induced MMP expression and activation in human PDLFs and mouse monocyte/macrophage RAW 264.7 cells. Even though baicalin is present in many plants and herbal extracts and many studies have shown efficacy such as anti-inflammatory effects of baicalin under certain conditions, no effect on PDL cells has been reported.

    In chronic periodontitis patients, periodontal tissue destruction is related to MMP-1 and MMP-2 transcript levels, and the amount of MMP-1 and MMP-2 protein has been observed to be higher in periodontal patients than in healthy periodontal tissues [23]. Thus, it can be assumed that the expression of MMP-1 and MMP-2 in PDLFs is related to the level of connective tissue destruction in periodontal tissue. The results of this study showed that baicalin significantly decreased the expression of MMP-1 and MMP-2 in PDLFs compared to the control group treated with LPS alone.

    It has recently been reported that MMP-8 is produced not only in polymorphonuclear leukocytes [24] but also in non-epithelial karyotype cells such as gingival epithelial cells, gingival fibroblasts, and PDL cells [25,26]. Guan et al. [27] reported that Prevotella intermedia infection stimulates MMP-8 expression and activates this protein in PDL cells. Therefore, MMP-8 significantly increase, in case of patients with chronic periodontitis, in the gingival fluid, gingival tissue, and PDL cells, but it is remarkably inhibited after their surgical periodontal treatment, which indicates that MMP-8 is involved in pathological degradation of periodontal tissue. The results of this study show that baicalin inhibits the degradation of connective tissue by inhibiting the expression of MMP-8 and MMP-9 in RAW 264.7 cells. Thus, inhibition of MMP-8 and MMP-9 expression could be thought of as helpful in managing the progression of periodontal disease.

    In this study, we confirmed that baicalin not only inhibits the expression of MMP-1 and MMP-2 in PDLFs but also decreases the expression of MMP-8 and MMP-9 in mouse monocyte/macrophage. Furthermore, we examined the effect on intracellular signaling to determine which molecular mechanism was involved. We have found that baicalin inhibited the phosphorylation of p38 increased by LPS in PDLFs, while observing changes in the MAPKs activation after inducing an inflammatory response by LPS treatment. In addition, the effect of baicalin was observed concerning the NFκB-related signal transduction system. Regulation of phosphorylation of RelA is known to play an important role in activating NFκB pathway. It is also known that kinases such as casein kinase II, AKT, and IKK could induce phosphorylation of RelA [28]. This study showed that LPS treatment increased phosphorylation of IKK and AKT as well as RelA phosphorylation. When treated with baicalin in PDLFs and RAW 264.7 cells, the increased phosphorylation was suppressed. Since inhibition of RelA phosphorylation causes a decrease in DNA binding capacity, we can deduce that baicalin will decrease NFκB transcriptional activity.

    When periodontal tissue is exposed to bacteria, inflamma- tion occurs, and cytokine secretion is promoted. Therefore, we observed changes in cytokine secretion from monocytes after treatment with baicalin. IL-6 is an activator of bone destruction, and excessive production of this cytokine is involved in periodontal tissue destruction. In this study, baicalin inhibited LPS-activated IL-6 release in monocytes. NO, another inflammatory factor, is involved in bone loss and is produced in the salivary glands of the mouth and secreted into saliva. Microorganisms that induce inflammatory infections are known to stimulate immune cells such as macrophages to further promote NO production by oral tissues [29]. Recently, NO has been known to be a target for the development of therapeutic agents in periodontal disease patients because NO plays an important role in disease-related tissue degradation. In this study, baicalin reduced LPS-activated NO secretion in monocytes. Therefore, it is thought that baicalin may help to control the progression of periodontal disease by inhibiting the expression of the proteolytic enzyme as well as the production of an inflammatory cytokine.

    In conclusion, the results of this study show that baicalin has an inhibitory effect on connective tissue destruction of periodontal tissue, which indicates that it is shown by inhibition of MAPK and NFκB related proteins. The inhibition of periodontal tissue destruction may be recommended as a therapeutic strategy for the treatment of inflammatory periodontal disease patients. However, the results of this study suggest that it is necessary to study animal models of periodontitis to verify whether baicalin can benefit the clinical environment.

    Conflicts of Interest

    No potential conflict of interest relevant to this article was reported.

    Figure

    IJOB-46-1-51_F1.gif

    (A) Effect of baicalin on the viability of periodontal ligament fibroblasts (PDLFs). PDLFs were treated with baicalin and 1 μg/mL of lipopolysaccharide (LPS) for 2 days. After the reaction, the formazan granules were solubilized, and the absorbance was measured using a microplate absorbance reader. Cell viability is expressed as the absorbance ratio. Values are expressed as means ± standard error (n = 3). (B) Effect of baicalin on LPS-induced protein expression. The PDLFs were stimulated with 5 μg/mL of LPS for 2 hours. Protein expression levles were determined using western blot analysis.

    IJOB-46-1-51_F2.gif

    Effect of baicalin on lipopolysaccharide (LPS)-induced activation of mitogen-activated protein kinases. The periodontal ligament fibroblasts were pretreated with baicalin for 2 hours in a serum-starved state for 30 minutes, and stimulated with 1 μg/mL of LPS for 15 minutes. Protein levels of the indicated genes were determined using western blot analysis.

    JNK, Jun N-terminal kinase; ERK, extracellular signal-regulated kinase.

    IJOB-46-1-51_F3.gif

    Effect of baicalin on lipopolysaccharide (LPS)-induced activation of inhibitory κB kinase (IKK). The periodontal ligament fibroblasts were pretreated with baicalin for 2 hours in a serum-starved state for 30 minutes, and stimulated with 1 μg/mL of LPS for 10, 15, and 30 minutes. Protein levels of the indicated genes were determined using western blot analysis.

    IJOB-46-1-51_F4.gif

    Effect of baicalin on lipopolysaccharide (LPS)-induced activation of inhibitory κB kinase (IKK), p65RelA, and AKT. The periodontal ligament fibroblasts were pretreated with baicalin for 2 hours in a serum-starved state for 30 minutes, and stimulated with 1 μg/mL of LPS for 15 minutes. Protein levels of the indicated genes were determined using western blot analysis.

    IJOB-46-1-51_F5.gif

    (A) Effect of baicalin on RAW 264.7 cell viability. RAW 264.7 cells were treated with baicalin and 1 μg/mL of lipopolysaccharide (LPS) for 4 days. Cell viability was measured by an methylthiazolyldiphenyl-tetrazolium bromide assay and is expressed as the absorbance ratio. Values are expressed as means ± standard error (n = 3). (B) Effect of baicalin on LPS-induced protein expression. The RAW 264.7 cells were stimulated with 5 μg/mL of LPS for 2 hours. Protein levels of the indicated genes were determined using western blot analysis.

    MMP, matrix metalloproteinase.

    **p < 0.01.

    IJOB-46-1-51_F6.gif

    Effect of baicalin on lipopolysaccharide (LPS)-induced activation of mitogen-activated protein kinases. The RAW 264.7 cells were pretreated with baicalin for 2 hours in a serum-starved state for 30 minutes, and stimulated with 1 μg/mL of LPS for 15 minutes. Protein levels of the indicated genes were determined using western blot analysis.

    JNK, Jun N-terminal kinase; ERK, extracellular signal-regulated kinase.

    IJOB-46-1-51_F7.gif

    Effect of baicalin on lipopolysaccharide (LPS)-induced activation of inhibitory κB kinase (IKK), p65RelA, and AKT. The RAW 264.7 cells were pretreated with baicalin for 2 hours in a serum-starved state for 30 minutes, and stimulated with 1 μg/mL of LPS for 15 minutes. Protein levels of the indicated genes were determined using western blot analysis.

    IJOB-46-1-51_F8.gif

    Effects of baicalin on lipopolysaccharide (LPS)-induced interleukin -6 (IL-6) (A), and nitric oxide (NO) secretion (B). RAW 264.7 cells were cultured with 1 μg/mL of LPS for 2 days. After incubation, the cytokines were measured using an ELISA kit and Griess reagent. Values are expressed as means ± standard error (n = 3).

    *p < 0.05; **p < 0.01.

    Table

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