Berberine’s Effect on Periodontal Tissue Degradation by
Matrix Metalloproteinases: an In Vitro and In Vivo
Experiment
Hsiao-Pei Tu a, b, Martin MJ Fu c, Po-Jan Kuo a, Yu-Tang China, Cheng-Yang Chiang a, Cheng-Long Chung a, Earl Fu a
a Department of Periodontology, School of Dentistry, National Defense Medical
Center and Tri-Service General Hospital, Taipei, Taiwan, ROC
b Department of Dental Hygiene, China Medical University, Taichung, Taiwan, ROC. c Resident, Division of Periodontology, Department of Oral Medicine, Infection and
Immunity, Harvard School of Dental Medicine, Boston, MA, USA
Correspondence to: Dr. Earl Fu
Department of Periodontology School of Dentistry
National Defense Medical Center
PO Box 90048-507, Taipei, Taiwan, ROC Tel: +886-2-87927150; Fax: +886-2-87927145 Email: [email protected]
Running title: Berberine ameliorates periodontal degradation
Key words: berberine, periodontitis, matrix metalloproteinase, fibroblast, macrophage Abbreviations: Matrix metalloproteinase: MMP, porphyromonas gingivalis: P.g.; lipopolysaccharide: LPS; Micro Computed Tomography: micro-CT; cemento-enamel junction: CEJ; immunohistochemistry: IHC; connective tissue: CT; human gingival fibroblasts: HGF.
Abstract
Periodontal disease involves tissue destruction caused by complex interactions among bacterial antigens and inflammatory mediators including matrix metalloproteinases (MMPs)-2 and -9. Berberine, an isoquinoline alkaloid isolated from many medicinal herbs, can inhibit has an inhibitory effect on the degradative action of extracellular MMPs. In this study, the The effect of berberine on the periodontal expression of MMPs-2 and -9 was examined in vitro and in vivo. Gelatinolytic activity of pro-MMP-2, pro-MMP-2, and MMP-9 in the supernatants of the cultures of human gingival fibroblast (HGF) and/or U-937 was compared after treatment with Porphyromonas
gingivalis lipopolysaccharide (P.g. LPS) in four medias containing 0, 1, 10 and 100 µM of berberine each without and with berberine (1, 10 and 100 µM). Twelve animals were divided into three groups for the study: (A) non-ligation, (B) ligation, and (C) ligation-plus-berberine (75 mg/kg berberine by gastric lavage daily); and the The effect of berberine on periodontal destruction was evaluated in the ligature-induced periodontitis in rats for 8 days by micro computerized tomography (micro-CT), histology and immunohistochemistry (IHC). Twelve animals were equally divided into groups A, B, and C with the following designation respectively: non-ligation, non-ligation, and ligation-plus-berberine (75 mg/kg berberine by gastric lavage daily). Animal were sacrificed on the eighth day of the study and the tissue samples
enhanced MMP activitieswas identified, with; however, a greater such enhancement effect on fibroblasts /U937 co-culture of HGF fibroblasts and U937 than on either culture alone. On the contrary, When when berberine was added to the such LPS-treated cultures, the activities of MMPs (pro-MMP-2, pro-MMP-9 and/or MMP-2) were significantly reduced in dose-dependent manner. In the animals model, the trends of the following several parameters were compared. 1. Micro-CT distances between cemento-enamel junction (CEJ) and dental alveolar bone crest: B > C > A. 2. Histometrically measured crest bone levels: B > C > A. 3. Amount of collagen deposited in connective tissue areas: A > C > B. 4. Attachment loss: B > C ≈ A. 5. Connective tissue (CT) attachment: B > either A or C. 6. Expression of cells stained positive for MMP-2 and -9 by IHC: B > C > A. In conclusion, berberine demonstrated
in vitro an inhibitory effect on P.g. LPS-enhanced MMP activities of HGF and U937
macrophages, reducing in vivo gingival tissue degradation in periodontitic rats. We thus propose that berberine may slow periodontal degradation through the regulation of MMPs in periodontitis induced by bacterial plaque.
Introduction
Periodontal disease is an inflammatory disorder in which inflammatory reaction may damage surrounding cells and tissue structures including the alveolar bone, causing tooth loss. In the inflamed periodontal tissues, dental plaque bacteria and their products induce polymorphonuclear leukocyte infiltration, edema and vascular dilatation . Complex interactions among various inflammatory mediators and tissue degradation have been carefully examined in vitro and in vivo and suggested to may be involved in the pathogenic mechanisms of periodontitis. In order to examine the interactions between fibroblasts and inflammatory cells, the in vitro fibroblast and macrophage cultures, either individual culture or co-culture, have been selected and used , and the results of increased cytokine expressions or protease activities were observed. The major component of the outer membrane of gram-negative bacteria, lipopolysaccharide (LPS), can penetrate gingival connective tissue and induce the local inflammatory response that leads to periodontal bone resorption . In vivo, the periodontal disease can be induced by introduction of pathogenic microorganisms , placement of a ligature that acts as a site for bacterial colonization or by injection of bacterial LPS .
Berberine, an isoquinoline alkaloid, can be isolated from many medicinal herbs, such as Rhizoma coptidis (Huanglian), Hydrastis canadensis (goldenseal) and Cortex
phellodendri (Huangbai) . Berberine-containing plants are used medicinally in many
traditional medical systems, including Chinese herbal and Ayurvedic herbal medicine . Studies have indicated that berberine has multiple pharmacological activities including anti-inflammatory, anti-cyclooxygenase, and anti-inducible nitric oxide synthases effects . Recently, the effect of berberine against the extracellular matrix degradation through the regulation of the matrix metalloproteinase (MMP) has been evaluated . MMPs are a family of structurally related proteins that degrade most components of the extracellular matrix and basal membranes in a zinc-dependent manner at physiological pH . They have been implicated in extracellular matrix remodeling in embryonic development, inflammation, tumor invasion, metastasis and fibrosis . The activation and over-expression of host MMPs caused by periodontal pathogens such as Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, and by inflammatory cytokines have been reported . The increased activity/levels of proteinases during experimental gingivitis and their decrease after periodontal therapy further suggest the involvement of certain MMPs in periodontal tissue destruction and degradation.
Berberine can in vitro effectively suppress the phorbol 12-myristate 13-acetate induced MMP-9 expressions in the induced macrophages . In human keratinocytes, berberine prevents skin inflammation and degradation of extracellular matrix proteins,
including collagen, by MMPs . In order to understand the effect of berberine on local degradation caused by periodontitis, the expression of MMP-2 and -9 in periodontium was in vitro and in vivo evaluated. The expression of MMPs in human gingival fibroblasts, as well as the co-culture with U-937 human monocytes, after Porphyromonas gingivalis (P.g.) LPS treatment was analyzed as the in vitro model, whereas the expression in a ligature-induced experimental periodontitis of rats was evaluated as the in vivo model.
Materials and methods
The in vitro experiment
Human gingival fibroblasts (HGF) were obtained as described elsewhere . In brief, the gingival specimens were immersed in Leibovitz L-15 medium containing 2 mg/mL dispase II (Roche Diagnostics, Indianapolis, IN, USA) and 10% fetal bovine serum at 4 °C for 2 d. After separation from the outer epithelial layer, the connective tissue was minced and digested in medium containing 10% FBS and 2 mg/mL collagenase for 24 h. The tissue was then placed in flasks containing 10% FBS in DMEM/F-12 media to allow the cells to migrate from the explants. Confluent fibroblasts were starved in serum-free medium for 24 h before experiments. Various concentrations of P.g. LPS (InvivoGen, San Diego, CA, USA) between 0 and 100 ng/mL were adjusted for the individual cultures and co-cultures. The gelatinolytic activity of proMMP-2, MMP-2, and MMP-9, in supernatants of the cultures treated with LPS (100 ng/mL) or berberine, at the concentrations of 0, 1, 10, and 100 μM, for 24 and/or 48 hours was investigated using zymography. Similar to our previous study , the U-937 macrophages at concentration of 105 per mL were also selected and
used. The serum free RPMI-1640 media (InvitroGen, Grand Island, NY, USA) was used for the U937 cells. In the co-cultures of the U937 and HGF, the same condition for the media was maintained for the fibroblast cultures throughout the experiment. In
the in vitro experiments, all the HGFs or/and U937 cells were incubated in a humidified atmosphere of 5% CO2 at 37°C, whereas the HGFs used were from the
third to seventh passages.
Gelatin zymography
The release of MMPs (proMMP-2, MMP-2, and MMP-9) from cell cultures were evaluated using gelatin zymography. The proteins in the medium in which the cells were incubated were separated under nonreducing conditions using a 10% SDS-polyacrylamide gel containing 0.1% gelatin. Equal amounts of protein, measured using the BCATM protein assay (Pierce, Rockford, IL, USA), were loaded into each lane of the gel. After electrophoresis, the gel was shaken gently in renaturing buffer (2.5% Triton-X100) to remove SDS and then incubated in developing buffer for 16 h. The gel was stained with 2.5% Coomassie Brilliant Blue. The latent and active forms of MMP-2 were identified as the 72 kDa and 62 kDa bands, respectively, while MMP-9 was detected as the 92 kDa band. The gel images were scanned directly (Transilluminator/SPOT, Diagnostic Instruments, Sterling Heights, MI, USA).
In vivo experiment
periodontis caused by ligation around molars was evaluated. After being approved by the Institutional Animal Care and Use Committee, National Defense Medical Center, Taipei, Taiwan (IACUC-12-147), twelve male Sprague Dawley (SD) rats, six-week-old and weighing 180–230g, were randomly divided into three groups (four rats per group). Rats in the ligation group had 3-O silk (surgical silk sutures; UNIK, Taiwan) placed on the cervices of the second maxillary and first mandibular molars as that described in our previous study . Rats in these two groups were fed daily with the solvent, the mixture of dimethyl sulfoxide (DMSO) (SIGMA-ALORICH, Inc., MO, USA) with normal saline (1/3 by volume). Rats in the ligation-plus-berberine group were given the same silk ligations as rats in the ligation group, with daily doses of berberine (75 mg/kg in the solvent of DMSO) from the day before the ligation by gastric lavage. All animals were caged in clear plastic cages and kept in an environmentally controlled room maintained at 23°C, relative humidity 55%, and a light-dark cycle of 12 hours/12 hours in the Animal Center of National Defense Medical Center, Taipei, Taiwan. On day 8 of experiment, all rats were sacrificed by carbon dioxide inhalation. The maxillary and mandibular specimens (including gingivae, teeth and bones) were taken and fixed in 4% paraformaldehyde and prepared for micro computerized tomography (micro-CT) and histological examination.
Micro computed tomography imaging
Twelve jaw biopsies, four from each group, were subjected to micro-CT imaging using a multimodality preclinical imaging system (FLEX Triumph; Gamma Medica-Ideas, Northridge, CA, US) equipped with an X-O CT. The x-ray tube was operated at an accelerated potential of 75 kVp with a beam current of 120 A. The field-of-view for micro-CT was fixed at 61.44 mm leading to 2 magnification of images. The micro-CT images were taken under fly mode with 1024 projections and one frame per projection to achieve a voxel size of 120 120 120 m3. Micro-CT data were acquired and reconstructed using Triumph XO
software (Gamma Medica-Ideas) and then visualized and analyzed using VIVID software (Gamma Medica-Ideas). This enabled us to observe the morphology around the tooth and dental alveolar bone in all dimensions, including the cemento–enamel junction (CEJ), root surface and dental alveolar crest, as well as the relationships between these areas, and assess the distance between the CEJ and the coronal level of the alveolar bone crests (the micro-CT bone levels) at 12 sites, including the mesio-and disto-buccal sites, the mesio- mesio-and disto-palatal sites of the right mesio-and left second maxillary molars, and the disto-buccal and disto-lingual sites of the right and left first mandibular molars on reconstructed three-dimensional micro-CT images.
Histology, histometry and immunohistochemistry
In this study, the maxillary specimens were prepared for histological examination in the same way as our previous study . In brief, the palatal specimens were sectioned buccopalatally into 4-m-thick sections after EDTA decalcification, dehydration and paraffin embedding, and then prepared for hematoxylin and eosin (H&E) staining. On the mesial surfaces of the second molars in each rat, the following histometric measurements were performed: the distance of the CEJ to the coronal level of epithelial cells (JEc) (attachment loss); the distance of the CEJ to the alveolar bone crest (ABC) (the alveolar crest bone level); the distance of the apical level of epithelial cells (JEa) to the ABC (the CT attachment); and the region of collagen deposited connective tissue in a zone of 0.14 mm2 of sub-epithelial gingiva on the
mesial surface of the second maxillary molar in each rat as that in our previous study . In this study, the protein expression of MMP-2 and MMP-9 in gingival tissue was further evaluated by IHC. After fixation in acetone, tissue sections were incubated in 0.1% hydrogen peroxide in distilled water to quench endogenous peroxidase activity. They were then incubated for 2 h with unconjugated primary polyclonal antibodies against MMP-2 and -9 (mouse IgG) (Chemicon International Inc., Temecula, CA, USA), followed by incubation with biotinylated secondary antibody,
streptavidin-conjugated horseradish peroxidase complexes and 3-amino-9-ethyl carbazole solution for 4 min, 20 min, and 20 min, respectively. The specimens were then washed with distilled water and counterstained with hematoxylin. The tissue sections were dehydrated and mounted for microscopic observation. Positively stained MMP-2 and -9 cells were detected using a microscope as our previous study described.
Statistical Analysis
A one-way ANOVA and Duncan’s test for post hoc analysis was used to evaluate the differences of the gelatinolytic activity of pro-MMP-2, MMP-2 and proMMP-9 among the independent cultures of U937 macrophages and human gingival fibroblasts, as well as their co-cultures. Regression analysis was used to determine the dose effect of LPS or CsA on the MMP activities. Repeated-measures analysis of variance (ANOVA) was used to evaluate the influence of the inter-subject factor (berberine or ligation treatment), as well as the intra-subject factors (including the maxillary or mandibular jaw, the left or right side, the buccal or palatal/lingual site, and mesial or distal end) on the location of dental alveolar bone crest measured by micro-CT. One-way ANOVA, with Duncan’s test for post hoc analysis, was used to determine the effect of berberine on the histometric measurements. P < 0.05 was considered significant.
Results
P.g. LPS significantly increased the activities of MMPs in all independent
cultures and co-culture of U937 and HGF (Figure 1). In the co-cultures, the activities of MMPs were increased if compared with those in the independent cultures. The activity of pro-MMP-9 is mainly present in the independent cultures of U-937, while the activities of pro-MMP-2 and MMP-2 were mainly expressed in the independent culture of HGF.
The berberine treatments significantly decreased, in dose dependent manner, the activities of MMPs in the cultures treated with P. gingivalis LPS, regardless the type of culture (Figure 2).
In the animal study, the distance of CEJ to bone (the micro-CT bone levels) was significantly different among the experimental groups (Figure 3A). The distance was shortest in the non-ligation group, longest in the ligation group, and intermediate in the ligation-plus-berberine group although it was influenced by other factors such as jaw location (maxillae or mandible), buccal or palatal/lingual surface, and mesial or distal site (Figure 3B-C).
Periodontal destruction was further histologically evaluated (Figure 4A). By histometry, t he level of bone crest in the ligation-plus-berberine group was coronally positioned if compare with that in the ligation group, but it was apically positioned
connective tissue areas in the ligation-plus-berberine group significantly greater than that of the ligation group but lesser than that of the non-ligation group. The attachment loss (CEJ to JEc) in the ligation and the ligation-plus-berberine groups was significantly greater than that in the non-ligation group. The CT attachment in the ligation group was significantly greater than that in the non-ligation and the ligation-plus-berberine groups. By IHC, the gingiva from the ligation group contained greater number of cells stained positive for MMP-2 and MMP-9 than that from the non-ligation group; and that from the non-ligation-plus-berberine group showed an intermediate value (Figure 5).
Discussion
The effects of berberine on periodontal MMPs, including MMP-2 and MMP-9, expressions were examined in vitro and in vivo in the present study. In the in vivo model, periodontitis in rats was induced by ligature (Figures 3 and 4), whereas in the
in vitro model the co-culture of human gingival fibroblasts and U-937 human
monocytes cell line was given Porphyromonas gingivalis (P. gingivalis) LPS treatment to simulate local inflammation in periodontium (Figures 1 and 2).
In the present study, the inhibitory effect of berberine was first in vitro tested in the individual cultures or co-cultures of fibroblasts and macrophages as in our previous study . The P. gingivalis LPS treatment significantly increased the activities of MMPs in the independent cultures of HGF and U937 macrophages and those in the co-culture (Figure 4) in this and our previous study. In a study by Sundararaj and colleagues, the co-cultures of gingival fibroblasts and macrophages were tested for normal and high glucose conditions to investigate the hyperglycemic effect on MMP expression via intercellular communications . In the result, the augmentation of MMP-1 expression was observed in the co-cultures of fibroblasts and HGF and this augmentation was further enhanced by high glucose concentration. Because interleukin-6 released by gingival fibroblasts was essential for the augmentation of MMP-1 expression by the macrophages, intercellular communication between the two
cell types was implicated. In another co-culture study, interleukin-6 release increased 100-fold of that of independent cultures of macrophages . The exact mechanism of intercellular communication between fibroblasts and U937 remains obscure and further investigation is indicated.
In our study, the processes of periodontal destruction and osteoclastic bone resorption were induced in vivo by silk ligation around the molars of rats in similar methods to our previous study. In the present study, the destruction of dental alveolar bone was evaluated by micro-CT and histologic parameters. In terms of micro-CT bone level (the distance from CEJ to bone crest), rats in the ligation group showed greater length than the non-ligation group while the ligation-plus-berberine group measures in between the two (Figure 3). With this finding we formulated that dental alveolar bone level could be deduced from the micro-CT observations as well as histologic estimation, but this may not be the precise representation of the periodontal soft tissue loss. In this study, the attachment losses (CEJ to JEc) in the ligation and the ligation-plus-berberine groups were similar (both significantly greater than in the non-ligation group). In terms of the amount of collagen deposit, the non-ligation-plus-berberine group showed less increase than the ligation group and significantly greater increase than the non-ligation group. The observed finding reflected reduced inflammatory condition by berberine.
In addition, the development of periodontitis involves complex mechanisms associated with bacteria and immune modulations, thus our experimental periodontitis in rats still faces certain limitations . For the purpose of bacterial retention, the periodontitis in the experiment was induced by inserting the ligature around the tooth neck. This artificial plaque retention, however, inflicted an inevitable trauma to the local gingival tissue . Besides, the tissue observation was limited to a short period of 7 days. Longer observational period is implicated from the chronic patter of periodontal disease.
In the present study, our in vitro results showed that the berberine treatments significantly decreased, in dose dependent manner, the activities of MMP-9, pro-MMP-2 or pro-MMP-2 in the cultures of hGF, U937 and hGF/U937 cells received the P.
gingivalis LPS treatment (Figure 2). In the rats with experimental periodontitis, by
ligature placement for 8 days, the berberine treatment not only attenuated the degradation process of periodontal tissue but also reduced the number of gingival cells stained positive for MMP-2 and MMP-9 (Figures 4-5). The precise mechanism remains to be elucidated; however, berberine attenuated the degradation process of periodontal tissue through inhibition of matrix metalloproteinases was suggested. In recent researches, the extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein extensively glycosylated to the plasma membrane, was identified and its
expression is considered to induce the fibroblasts to secrete MMPs . EMMPRIN (CD147) contains two immunoglobulin superfamily domains, a transmembrane domain, and a stoplasmic domain and was originally identified on the surfaces of tumor cells. Its expression on tumor cells may enhance tumor progression and invasion by triggering the production and release of MMPs by fibroblasts and endothelial cells . The ability of EMMPRIN to stimulate MMP production implies that this molecule may be associated with several physiological and pathological tissue modulatory processes and tissue remodeling . We examined the expression of EMMPRIN in gingiva with the ligature-induced experimental periodontitis by IHC and an increased expression was observed; however, the expression was attenuated by berberine administration (un-published data). Nevertheless, the role of EMMPRIN in the berberine-inhibited matrix metalloproteinases requires further investigation.
Berberine is commonly known as an important compound in traditional Chinese medicine . Berberine has been thought to be a candidate for preventing various diseases because of the discoveries of its therapeutic biological effects, including anti-inflammation and anti-matrix-degradation . In this study, we provided the in vitro and
in vivo evidences showing that berberine might have a preventive potential in the
periodontal tissue degradation during inflammation. Because the primary etiology of periodontitis is the microbial pathogens, the removal of pathogens, as well as the
prevention of microbial recurrence, is the principal strategy of treatment. However, the chemotherapies and the host modulations are still be used as the adjunctive therapies due to the complex interactions among inflammatory mediators and tissue degradation are involved in the pathogenic mechanisms of periodontitis .
In conclusion, this was the first study to in vitro and in vivo demonstrate that berberine could inhibit the P.g. LPS-enhanced MMP activities in the cultures of HGFs and U937 macrophages and attenuate the tissue matrix degradation in the experimental periodontitis of rats. We therefore suggest that berberine may attenuate the periodontal tissue degradation through the regulation of MMPs during the progression of periodontitis.
Acknowledgment
This study was partially supported by the research grants from the Department of National Defence (DOD-C101-63) and the C.Y. Foundation for Advancement of Education, Sciences and Medicine.
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Legends
Figure 1. Effect of LPS on the activities of pro-MMP-9, pro-MMP-2 and MMP-2 in the independent cultures of HGFs and U937, and the co-cultures. (A: the images of zymography, and B: the statistical comparisons of the MMP activities) The upper part of the figure (A) presented the images of zymography. The lower plots (B) showed the comparison of the MMP activities between the culture techniques (U937 or HGF independent culture and their co-culture), and between the treatments with and without LPS (100 ng/mL). Data are expressed as means and standard errors of five separate experiments. (*: significant difference vs that of no LPS treatment, #: significant difference vs that of independent cultures of U937 and HGF.).
Figure 2. Effect of berberine on the activities of MMP-9, pro-MMP-2 and MMP-2 in independent cultures and the co-cultures of HGFs and U937 macrophages received the P.g LPS treatment. (A: the images of zymography, and B: the dose effect of berberine on the MMP activities in the cultures received LPS) The upper part of the figure (A) presented the images of zymography. The lower plot shows the dose effect of berberine on the relative MMP intensities in the cultures received the LPS (100 ng/mL) stimulation. (Data are expressed as means and standard errors of five separate experiments, *: significant difference at p < 0.05)
Figure 3. Micro-CT bone levels among the non-ligation, ligation and ligation-plus-berberine groups. (the top: the reconstructed 3-dimensional CT images of the molars
viewed on buccal; the middle: the micro-CT bone levels recorded; and the bottom: the statistical evaluation of the effects of the ligation/berberine treatment and the examining factors, including the location, the buccal/palatal/lingual site, and the right /left side, on the bone levels) Reconstructed three-dimensional CT images for maxillae viewed from the buccal side in the three groups of rats on experimental day 8 (the X indicate the molars with ligation) (top part of figure). The bone levels recorded at the 16 sites of second maxillary molars and first mandibular molars on the examined micro-CT images (the plot in bottom left). The influence of the ligation or berberine treatment (the inter-subject factor), the location at the maxillary or mandibular molars, the buccal or palatal/lingual site , and the right or left side (the intra-subject factors) on bone levels, the distance of CEJ to bone, revealed by micro-CT with repeated measures analysis of variance (*: significant difference, p < 0.05; R: right side, L: left side; M: mesial site, D: distal site; B: buccal location, P/L: palatal or lingual location; U: maxillae, L: mandible; N-L: non-ligation group, Lig: ligation group, and L + Berb: ligation-plus-berberine group) (the plot in bottom right).
three animal groups nonligation, ligation and ligation-plus-berberine groups. Histographs in the first row show histological images, including dentin (d), gingiva (g), alveolar bone (ab), and periodontal ligament (pl). The histographs micrographs in the second row represent higher magnification of images in the first row (arrowhead: indicates the level of CEJ; and arrows: indicate the crest of dental alveolar bone) (H&E stain, magnifications of 40 and 100 for micrographs in the first and second rows, respectively). Comparisons of alveolar crest bone level, attachment loss, CT attachment, and regions with collagen deposits among the three animal groups are summarized in the legends in the third and fourth rows.
Figure 5. Microphotographs showed the IHC staining for MMP-9 (upper raw) and MMP-2 (lower raw) in gingival tissues from a non-ligation, ligation and ligation-plus-berberine rat. (arrow indicates the cell stained positive by IHC) (original
