Angelica gigas ameliorates the destruction of gingival tissues via inhibition of MMP-9 activity

Angelica gigas (AG) has been used for periodontal diseases in traditional Korean medicine. However, the effects of AG on periodontitis have not been clarified yet. In this study, we investigated the ameliorative effects of AG against ligature-induced periodontitis. Sprague-Dawley rats were divided into four groups; non-ligatured (normal), ligatured and treated with vehicle (ligatured), ligatured and treated with 1 mg mL−1 AG (AG1), and ligatured and treated with 100 mg mL−1 AG (AG100). 70% ethanol extracts of AG were topically applied onto both sides of the first molar daily for 14 days. In addition, human dermal fibroblast cells were treated with 1, 10 and 100 μg mL−1 AG to characterize the expression of matrix metallopeptidase 9 (MMP-9). Topical AG treatment reduced alveolar bone resorption, as assessed by methylene blue staining. The structures of soft gingival tissues (periodontal pocket) were recovered in the AG-treated groups. The expression of MMP-9 was decreased, and that of type 1 collagen was significantly increased in AG-treated gingival tissues. In addition, AG treatment inhibited the activity of MMP-9 in LPS-treated human dermal fibroblast cells. This study reveals that topical AG treatment has the potential to ameliorate the destruction of gingival tissues by inhibiting MMP-9 activity. AG may be a candidate for the treatment of periodontitis.


Introduction
Periodontal disease is caused by specic oral microbes that infect up to 20% of the global adult population. 1 Typical consequences of periodontitis include alveolar bone resorption, gingival inammation, periodontal pocket formation and the breakdown of supporting connective tissues, ultimately resulting in tooth loosening from the jaw bone. 2 To date, several periodontal treatments have focused on modulation of the host immune response. Subantimicrobial dose doxycycline, a host response modier, is an approved systemic therapy that decreases prostaglandins and inammatory cytokines. However, accelerated bone loss sometimes occurs aer cessation of treatment. 3,4 Recently, a combination of host modulatory therapy involving implantation, chemical root conditioning and growth factors has been used in clinical trials to reconstruct periodontal tissues. 5 However, these therapies have limitations related to adverse effects and high costs; therefore, identication of alternative treatments for periodontal diseases is still needed.
Traditionally, the dried roots of A. gigas Nakai (Apiaceae) have been used as a functional food and herbal medicine to enrich and tonify the blood for thousands of years. 6 Several studies have demonstrated its pharmacological activities, which include inhibition of cancer, bacterial and nematode infection, platelet aggregation and oxidation. 7 Especially, A. gigas was reported to be used as either formula or single for dental health based on the classic book. 8 Previous studies demonstrate that A. gigas includes various chemical components such as decursin, decursinol, decursinol angelate, nodakenin, n-butylidenephthalide, and umbelliferoneate. Decursin has been used as a standard of identication of A. gigas, as a main bioactive compound of AG. 9 Recently, decursin has the potential to inhibit ultraviolet B-induced matrix metalloproteinase (MMP)-1 and MMP-3 expression in human dermal broblast (HDF) cells. 10 Decursin has also been shown to ameliorate bone loss by inhibiting osteoclastogenesis. 11 However, there is still a lack of study on the effects and underlying mechanism of A. gigas on periodontitis. In this study, we investigated the effects of A. gigas on periodontitis and potential mechanism in rats.

Preparation of AG extract
Thirty grams of dried roots of A. gigas Nakai (Jung-do Herb; Seoul, Korea) was extracted with 300 mL of 70% ethanol for 24 h at room temperature. The extract was ltered with Whatman lter paper no. 3 (Whatman, Maidstone, Kent, England), concentrated in a rotary vacuum evaporator and freeze-dried. The weight of nal dried powder (named AG) was 11.27 g (yield: 37.6%). A voucher specimen (AG070, 70% ethanol extract of A. gigas) was deposited at our laboratory.
Decursin, which is main component of A. gigas, was used to identify AG by High-Performance Liquid Chromatography-Evaporative Light Scattering Detector (HPLC-ELSD Agilent 1100 series). The extract was dissolved in 70% methanol and sonicated 30 min. Aer ltering, aliquot mixed with methanol was injected in HPLC analysis. The used column was SHISEIDO CAPCELL PAK C18 (250 Â 4.6 mm, 5 mm). The mobile phase consisted of 30 mM ammonium acetate and acetonitrile (20 : 80) with 1.0 mL min À1 of ow rate at 30 C. The peak of decursin in AG was synchronized with standard decursin.

Experimental design
Twenty-eight male Sprague Dawley rat aged 7 weeks (RaonBio Inc., Yongin, Korea) were adapted for 1 weeks in an airconditioned room under a 12 h light/dark cycle with food and water freely available. Appropriate temperature and humidity were maintained for animal's convenience. All experiments were approved by Committee on Care and Use of Laboratory Animals of the Kyung Hee University (KHUASP(SE)-14-029).
Twenty one rats were ligatured with sterilized 3-0 nylon into the subgingival sulcus around the both sides of rst mandibular molar of rats while the rest of rats were non-ligatured, and randomly divided into 4 groups (n ¼ 7, respectively); (normal) no ligature placement and non-treatment, (ligatured) ligature placement and administration of vehicle, (AG1) ligature placement and administration of AG 1 mg mL À1 and (AG100) ligature placement and administration of AG 100 mg mL À1 . For reliability of topical application, 1% carboxymethylcellulose was added to AG solution in distilled water based on previous report. 12 AG was administered once daily in 100 mL of volume at 1 and 100 mg mL À1 for consecutive 14 days. Then, rats were sacriced.

Measurement of mandible bone loss
The right side of the mandibles were collected and immersed in 1% methylene blue aqueous solution (Sigma, MO, USA) for 1 min at room temperature and 20 AE 5% humidity. Aer drying with compressed air, the specimens were photographed with 100 mm macrolens Canon digital camera (Canon, Tokyo, Japan). The length of three root surfaces from rst mandibular molar were measured by a computerized densitometry system Image J (NIH, Bethesda, MD, USA). The score of alveolar bone loss was determined by sum of three values of the length of three root surfaces from rst mandibular molar.

Evaluation of histological changes
The le side of the mandibles were xed in 10% neutral buffered formalin for 18 h and demineralized in a solution of 0.1 M ethylene diamine tetraacetic acid for 2 months. Aer dehydration with ethanol and xylene, the specimens were embedded in paraffin. Serial sections of 7 mm thickness were obtained and stained with hematoxylin and eosin (H&E). The digital images were obtained from Leica Application Suite (LAS) microscope soware (Leica Microsystems, Buffalo Grove, IL, USA) with the Â40 magnication.

Measurement of pro-collagen type 1 and MMP-9
The gingival tissues around the ligature placement were excised. Total RNA from gingival tissues was extracted by Trizol methods. Complementary DNA (cDNA) was synthesized by commercially available cDNA synthesis kits (Invitrogen, Carlsbad, CA, USA) at 45 C for 60 min and then at 95 C for 5 min. Reverse transcription polymerase chain reaction (RT-PCR) was performed using 10 mg of cDNA with pre-mixed PCR-kit (Invitrogen). The following primers were used: type 1 collagen, 5 0 -TCT ACT GGC GAA ACC TGT ATC CG-3 0 (forward) and 5 0 -CAA GGA AGG GCA GGC GTG AT-3 0 (reverse). MMP-9, 5 0 -GGG ACG CAG ACA TCG TCA TC-3 0 (forward) and 5 0 -TCG TCA TCG TCG AAA TGG GC' (reverse). GAPDH, 5 0 -CCA TCA CCA TCT TCC AGG AG -3 0 (forward) and 5 0 -CCT GCT TCA CCA CCT TCT TG-3 0 (reverse). The amplication program was comprised of the initial denaturation step 35 cycles. Relative quantication on gene expression was performed in relation to GAPDH mRNA expression by a computerized densitometry system Image J.

Determination of MMP-9 release level
HDF cells were grown in Dulbecco's modied Eagle's medium supplemented with 10% fetal bovine serum and 1% antibiotics (Gibco-BRL, Grand Island, NY, USA) at 5% CO 2 and 95% humidity condition. Seeded cells in 6 well plates were infected with 15 mg mL À1 of lipopolysaccharide (LPS) and co-treated with 1, 10 and 100 mg mL À1 of AG for 24 h. The supernatants were collected and centrifuged. Commercial Human MMP-9 Quantikine enzyme-linked immunosorbent assay kit (R&D systems, USA) was used to analyze the MMP-9 concentration.

Statistical analysis
Signicance was determined by one-way analysis of variance (ANOVA) and Dunnett's multiple comparison tests using a GRAPHPAD PRISM 5 soware (Ver. 5, GraphPad Soware, Inc., CA, USA). In all analyses, p < 0.05 was taken to indicate statistical signicance.

Results and discussion
Periodontal disease can lead to periodontitis and gingivitis depending on the disease stage. 13 Gum tissue and connective tissue becomes inamed (gingivitis) at early stage, and further advances to periodontitis such as alveolar bone loss. 1 Accordingly, recent therapies for periodontal disease are required to target not only alveolar bone resorption, but also so tissue degradation. 14 In the present study, a signicant increase of alveolar bone loss was appeared (normal ¼ 2.495 AE 0.34 mm; ligatured ¼ 3.338 AE 0.172 mm) in ligature-induced periodontitis group. Compared to ligatured group, AG 100 mg mL À1 treated group showed a signicant decrease in alveolar bone loss (AG1 ¼ 3.203 AE 0.167 mm; AG100 ¼ 2.925 AE 0.275 mm; p < 0.05, Fig. 1). In addition, severe destruction of gingival tissues in space between rst and second mandibles regarded as periodontal pocket was observed in ligatured group in comparison with normal group. Treatment of 1 and 100 mg mL À1 of AG recovered the collapsed gingival tissues compared with ligatured group (Fig. 2). Especially, 100 mg mL À1 of AG-treated group showed an almost complete recovery nearby normal group. Taken together, AG treatment ameliorated the formation of periodontal pockets resulting from so tissue destruction, as well as alveolar bone loss, in a ligature-induced periodontitis rat model.
Fibroblasts, which produce a collagen-rich extracellular matrix, are the predominant components of gingival tissues. 15 Well-organized type 1 collagen is responsible for the adherence of the gingiva to the teeth. 16 It is well established that gingival inammation (i.e., gingivitis) accompanies the development of periodontitis. In periodontitis, the destruction of gingival tissues results in the degradation of collagen, including type 1 collagen. 17 AG treatment recovered the destruction of gingival tissue, which is affected by periodontitis. These improvement of so tissue inammation was accompanied with an increase of type 1 collagen mRNA expression by AG treatment. Gingival tissue from ligatured group exhibited a signicant lower mRNA expression of type 1 collagen (p < 0.05) compared with normal group. The mRNA expressions of type 1 collagen were increased dose-dependently by AG treatment (Fig. 3A).
To clarify the protective effects of AG on collagen degradation, collagenase activity was investigated in gingival tissue. MMPs are responsible for the degradation of denatured interstitial collagens in damaged tissue. [18][19][20] As described in previous reports, MMP-9 distributed in broblasts, keratinocytes, endothelial cells and osteoblasts could be a key enzyme to disassemble the type 1 collagen. Therefore, inhibition of MMP-9 activity might be involved in ability to attenuate the degradation of gingival tissues and the expression of MMP-9 is seriously increased in impaired gingiva by periodontal inammation. 21 The present study shows that the activity of MMP-9 in gingival tissue was signicantly inhibited by AG treatment. The mRNA expression of MMP-9 was clearly increased in ligatured group. Treatment of AG 100 mg mL À1 reduced the mRNA expression of MMP-9 (Fig. 3A). To conrm the effect on MMP-9 expression by AG treatment, collagenase activity was analyzed in vitro. Approximately 7.25 times of increase of MMP-9 level was found in LPS-induced HDF cells. Following treatment with 1, 10 and 100 mg mL À1 of AG, elevated MMP-9 concentration were suppressed in dose-dependent manner (30.22, 54.84 and 75.83%, respectively, Fig. 3B). These results suggest that the restoration of gingival tissue by AG treatment is positively correlated with the inhibition of MMP-9 activity.

Conclusion
In conclusion, topical AG treatment inhibited the alveolar bone loss in ligature-induced periodontitis rat. AG improved the periodontal pocket formation by recovery of the destruction of gingival tissues. The expressions of type 1 collagen and MMP-9 in gingival tissue were signicantly regulated by AG administration. The present study revealed that topical AG treatment ameliorates alveolar bone loss and gingiva tissue degradation by inhibiting MMP-9 activity (Fig. 4). These results suggest that AG may help regenerate impaired gingiva in periodontitis.

Conflicts of interest
The authors declare no conict of interest.