Anti-Inflammatory Effect of the Isatis tinctoria L. Root Extract on Lipopolysaccharide-Induced Periodontitis in Rats

Objectives: Isatis tinctoria L., clary, is an herbal plant traditionally used in folk medicine for the treatment of various diseases and conditions. Although it has been primarily used as an antimicrobial and antifungal, there are data on traditional use of I. tinctoria as an agent against antiallergic, an-ti-thrombocytosis. The aim of the study was to examine the effect of the I. tinctoria root extract on the lipopolysaccharide (LPS)-induced periodontitis in rats on osteoclast associated bone resorptive activity, cell death including apoptosis, and inflammation in a rat of disease model of periodontitis. Materials and Methods: Periodontitis, acute or chronic inflammatory status in periodontal tissue in rats could be induced by repeated injections of LPS from Escherichia coli into the periodontal pocket area between the first and second right maxillary molars. Eighteen male rats were distributed among the fol-lowing treatment groups: 1) I. tinctorial root extract (Antifect) 200 mg/kg body weight, 2) acetylsalicylic acid (ASA), 20 mg/kg body weight and 3) Phosphate buffered saline (PBS) treatment used as a control. After 15 days, maxilla, alveolar bone, molar teeth and associated periodontal tissues were harvested. Inflammatory (IL-6) and tumor necrosis factor-α (TNF-α) of gingival tissues and descriptive analysis of histological sections of periodontal. Results: Treatment with Antifect, compared to the control group, significantly diminished the process of inflammation decreasing the levels of IL-Iβ, IL-6 and TNF-α, reducing the gingival tissue lesions and preserving bone alveolar resorption. Considerably a smaller number of inflammatory cells and a larger number of fibroblasts were noticed. Also, μCT analysis showed that only Antifect treated group reduced bone resorption and the number of TRAP-positive multinucleated cells (osteoclasts), also, significantly reduced the number of apoptotic cells in the gingival tissues and of osteocytes in the alveolar bone crest. Conclusion: Antifect manifested anti-inflammatory elect and reducing alveolar bone resorption in LPS-induced periodontitis suggest that it may have a role as a therapeutic agent in periodontal diseases.


Introduction
One major actor of the oral ecosystem is the dental plaque which develops naturally on hard and soft tissues of the mouth. This biofilm has a very complex organization that remains relatively stable with time despite regular environmental changes [1]. Periodontal disease is a chronic infective disease of the periodontal caused by periodontopathic bacteria accumulated on the tooth surface, characterized by destruction of the tooth supporting tissues including alveolar bone resorption [2] [3]. Subgingival Gram-negative organisms containing lipopolysaccharides (LPS) cause inflammation of the periodontal tissues inducing a local response, which implies a polymorphonuclear leukocyte infiltration, a production of reactive oxygen species (ROS) and inflammatory mediators such as cytokines and prostaglandins, amplification of lytic enzymes and activation of osteoclasts, edema and vascular dilatation [4] [5]. Recent investigations have shown that the gingival tissue from the inflamed area contains increased levels of some cytokines, primarily interleukin-lβ (IL-Iβ), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), implying that these cytokines mainly participate in the pathogenesis of periodontitis [6] [7] [8]. The culminating stadium of damaging effects of the produced cytokines and ROS is destruction of tooth-supported tissues including connective and mineralized tissue [9].
A remarkable number of studies have demonstrated the use of plants to be a new trend in the prevention and treatment of periodontal diseases with less adverse effects on humans. Some drugs for oral inflammations treatment can change oral microbiota and cause teeth discoloration [10] [11] [12]. In addition, clinical use of some phytochemicals is limited because of its poor absorption in the gastrointestinal tract, short plasma half-life and low bioavailability after oral S. W. Lee [13]. Therefore, the search for natural products or phytochemicals is growing increasingly with prospects to become the acceptable alternatives or complementary agents for oral inflammations [14]. Bioactive phytochemicals from herbs such as phenolic acids, flavonoids, tannins, terpenoids, alkaloids etc.
have been found to be particularly helpful in periodontitis due to their potent antimicrobial and anti-inflammatory activities [15].  [21]. Inhibition of aggregation of thrombocytes has been reported [22].
Although there are data on traditional medicinal use of woad, there have been no studies concerning its inhibiting impact on the development of periodontitis on an animal model; in addition this is the first experimental research focusing on antiinflammatory properties of the I. tinctoria root extract (here in "Antifect"). In this pilot study, we investigated the effectiveness of the Antifect in suppressing the inflammation of the rats periodontal caused by LPS, by determining the levels of proinflammatory factors and histopathological analysis, as well as the chemical composition and antioxidant activity of the same extract.

Experimental Design
All animal experiments were conducted after obtaining the approval of the Seoul National University Institute of Laboratory Animal Resources and Use Committee. Ten weeks old (247.5 ± 54.16 g), were kept in wire cages, in a room with proper, controlled conditions of temperature (21˚C -23˚C) and humidity (65% -70%) with 12 h light/dark cycle. The animals were fed with milled pellets, and had free access to water during the experiment. Lipopolysaccharide, derived from Escherichia coli (055:B5) and diluted in a sterile saline solution, was used for induction of periodontitis. One microliter of LPS solution (10 µg/µL) was slowly injected into interdental papilla between the first and second right maxillary molar [23], two times for fifteen days, using a Hamilton microsyringe. Rats were randomly divided into five experimental groups with six animals in each: Journal of Biosciences and Medicines condition were subjected to 10% buffered formalin fixation and subsequent histological processing for paraffin embedding (as described below) and used in histological and immunohistochemical analysis.

Plant Material and Extraction
Fresh and dried roots of woad (Isatis tinctoria L.) were provided in July 2018 by

Microcomputer Tomography
All hemi-maxillae (with and without soft tissues) were scanned on a microcomputer tomograph (Skyscan 1176, SkyScan Aartselaar, Belgium) using 18 μm slices. Digital radiographic images of each sample were reconstructed into a three dimensional model and a standardized gray scale value was set to distinguish mineralized from non-mineralized tissues. These three dimensional images were re-oriented on the sagittal, coronal and transversal planes in a standardized manner using anatomical landmarks on molar teeth. A standardized region of interest of 9.72 mm 3 was defined, including the first molar, the anterior half of the second molar and extending medially (towards the center of the palate) approximately 1 mm from the most palatal aspect of the crown of the first molar.
All image reconstruction, reorientation and analysis of bone volume/total volume (BV/TV fraction) in the region of interest was performed using the software package of the scanning equipment software.

Histopathological Analysis
The material, molars with the surrounding gingival tissues, was rinsed in saline and fixed in 10% formaldehyde solution for up to 7 days and then subjected to a process of demineralization, due to the presence of bone elements and teeth, for 15 days in a 18% solution of disodium ethylenediaminetetraacetate (EDTA). After the completion of decalcination, the material was cut into thin parts in the mesial-distal direction and treated by conventional histological procedures to paraffin molds. The final preparations were 4 µm thick stained with hematoxylin and eosin (H&E).

Osteoclast Count
Osteoclasts were identified by their morphology, location and positivity for the

Apoptosis Analysis
Detection of active (cleaved) caspase-3 in the cells of the gingival tissues was performed by immunohistochemistry. The same general protocol described for detection of TRAP was used, with the inclusion of an antigen retrieval step, per-

Measurements of the Factors of Inflammation
The levels of the factors of inflammation, TNF-α, IL-1β, and IL-6, were determined using supernatants of 5% homogenate made up of fresh gingival tissues with deionized water. The measurement of cytokines was performed by enzyme-linked immunosorbent assay (ELISA) kits specific for rats in accordance

Statistical Analysis
Data obtained from each experiment were analyzed using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). The objective of the analysis was to compare the results of each outcome according to the different tested compounds. The Shapiro-Wilk test was used to verify that the data had a normal distribution. Pairwise comparisons were performed by unpaired Student t-tests with Welch's correction for unequal variances and by one-way analysis of variance (ANOVA) followed by Tukey test for post-hoc pairwise comparisons. We adopted 5% as significant level (p < 0.05).

Effects of Antifect on Histopathological Characteristics of Periodontitis in Rats
Morphological changes in HE preparations are not visible in the Antifect treated group compare to the control. Figure 1

Oral Administration of Antifect Significantly Reduces Alveolar Bone Loss and Osteoclastogenesis Associated with LPS Induced Periodontal Disease
Microcomputer tomography analysis indicates that attenuated inflammatory bone resorption in antifect treatment group (Figure 2). Group I and II had no   (Figure 2(a)). Long-term ASA administration reported recovery of inflammatory symptom of periodontitis patient [24], but gastric-intestinal irritation limits its long-term treatment. Antifect treatment group (IV) consistently showed decrease of alveolar bone loss similar with ASA treatment group (V) (Figure 2(b)).
Of primary importance, the number of TRAP-positive multinucleated cells, identified by immunohistochemistry, was significantly decreased only in the group IV even not in the group V, which supports the interpretation that the bone-sparing effects of this compound observed in the μCT analysis are due, at least in part, to the inhibition of osteoclastogenesis in vivo. ASA treatment had no effect on the number of osteoclasts present in the periodontal and gingival tissue (Figure 3).

Effects of Antifect on the Gingival Concentrations of TNF-α, IL-1β and IL-6 of Rats
The treatment with Antifect and ASA (IV and V group) significantly decreased the level of TNF-α, IL-6 and IL-1β in the gingival tissue with LPS-induced periodontitis when compared to the untreated group (III). There was no statistically significant difference of the levels of cytokines between groups which did not receive LPS (I and II). ASA treated group (V group) significantly reduced the levels of IL-1β, but not IL-6 and TNF-α, in comparison to the group that treated antifect (IV) ( Figure 5).

Discussion
Wistar rats were chosen to be used in the study as a model for periodontal disease since it has been shown that periodontal anatomy of molar region in rats are similar to that in humans. It has been proven that both, topical application, as well as the injection of LPS in mouse or rat periodontal tissue and gingiva develops common inflammatory changes, such as junctional epithelial disruption, infiltration of leukocytes and edema of the subepithelial connective tissue due to inflammatory cell infiltration. Additionally, LPS injection in rats periodontal tissue and gingiva shows significant alveolar bone loss starting at second day after periodontitis induction, reaching a top at seventh day [23]. Our histopathological findings in periodontal tissue and gingiva, obtained from the third group of experimental animals, show similar changes in these tissues. Intragingival   the group II, as it was also shown by immunoassay where there was no statistically difference in the level of cytokines between these groups. But, bacterial stimulation, especially by LPS, has been described to increase the secretion of proinflammatory cytokines by gingival inflammatory cells, especially key cytokines such as TNF-α, IL-1β and IL-6 [26]. These cytokines play a relevant role in the initiation and development of periodontitis amplifying the immune response and activating of immune and nonimmune cells [27]. They are also able to provoke remarkable tissue damage by induction of collagenase in fibroblasts and osteoclasts activation [28]. The synergism between IL-1β and TNF-α and between IL-6 and TNF-α have been found, whereby TNF-α is likely more important cytokine. It has also been noticed that healthy gingival tissue may contain inflammatory cytokines including the ones mentioned above present in low amounts [29]. This implies that cytokines are actually considerable factors for the maintenance of normal tissue homeostasis. Previous report about anti-allergic effects of Antifect was related to activity of its polyphenols which conspicuously reduced inflammatory process in mice induced by tryptanthrin and histamine [30]. Our study has shown that Antifect manifests anti-inflammatory effects in rats by significantly decreasing the levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α ( Figure 5) with visible histological changes. It is evident that the administration of the extract in IV and V groups of rats modulated host response with extensive reduction of inflammatory infiltrate in comparison to III group. Immunoassay revealed that there were not statistically significant differences in the levels of the factors of inflammation IL-6 and TNF-α in IV and V groups of the rats. Because, the COX-2 inhibitory activity also reported in I. tinctoria extract [19]. Generally, there were no differ- anti-apoptotic effect and inhibition of bone resorption significantly increased in group IV. Usually occur of tissue destruction and cell apoptosis under acute inflammatory condition due to ROS (reactive oxygen species) stress [9]. I. tinctoria extracts known as contained high antioxidative molecules such as polyphenol, indoles also. And antiviral [31], antimicrobial effects [32] of I. tinctoria also assist inhibitory effect of periodontal inflammatory tissue destruction.

Conclusion
This study demonstrated that the oral administration of the extract from I. tinctorial Antifect had a suppressive effect on the LPS-induced inflammation of the rats' gingival tissue. The decrease of bony resorption, tissue destruction and cytokines production IL-6, IL-1β and TNF-α was in correlation with the reduction of the inflammatory process confirmed by histopathological analysis. In general, the results suggest that Antifect could act as a potential therapeutic and complementary agent in the treatment of periodontal disease. However, further studies should be conducted in order to confirm and define the efficiency of humans in certain clinical researches.