140 J Contemp Med Sci | Vol. 7, No. 3, May-June 2021: 140–144 Original Eremostachys binalodensis, a potential therapeutic choice for gingival inflammatory wounds Armin S. Hariri1, Sevda Shayesteh2, Parina Asgharian3, Mohsen Chamanara4,5, Maryam-Sadat Sadrzadeh-Afshar6* 1Oral and Maxillofacial Medicine Department, Faculty of Dentistry, Aja University of Medical Sciences, Tehran, Iran 2 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Alborz University of Medical Sciences, Karaj, Iran 3Department of Pharmacognosy, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran 4Department of Pharmacology, Faculty of Medicine, Aja University of Medical Sciences, Tehran, Iran 5 Toxicology Research Center, Aja University of Medical Sciences, Tehran, Iran 6Oral and Maxillofacial Medicine Department, Faculty of Dentistry, Aja University of Medical Sciences, Tehran, Iran *Correspondence to: Maryam-Sadat Sadrzadeh-Afshar, m_sadrzade@alumnus.tums.ac.ir (Submitted: 07 April 2021 – Revised version received: 18 April 2021 – Accepted: 27 April 2021 – Published online: 26 June 2021) Introduction Gingiva is the first tissue destructed in periodontal diseases and gingivitis leads to oral cavity complications if remained untreated. The healing process includes 4 main phases hemo- stasis, inflammation, proliferation and remodeling; and prolif- eration and migration of fibroblasts, play crucial role in healing process.1 Despite a rapid healing process, the presence of bac- teria in oral cavity accounts for the complicated wounds accompanied with inflammation.2 Chronic increased levels of pro-inflammatory factors such as interleukin (IL-1β) and Tumor necrosis factor (TNF-a) activates apoptotic pathways in fibroblasts as well as inducing the production of IL-6 which leads to a positive inflammatory cycle.3,4 In addition, inflam- mation diminishes the migration and proliferation of the cells, resulting in delayed wound healing.5 Moreover, enhanced inflammatory response leads to loss of connective tissue attachments and alveolar bone destruction.6 On the other hand, the administration of anti-inflammatory drugs such as corticosteroids, decelerate the healing process by reducing the proliferation rate of the fibroblasts as well as decreasing col- lagen and glycosaminoglycan synthesis.7 Therefore, investi- gating anti-inflammatory agents not impairing the healing process is of great importance for gingiva wounds treatment. Eremostachys binalodensis is an Iranian endemic specie of Eremostachys genus from Lamiaceae family. It is mostly grown in middle-east and west Asia, having thick roots.8 Several bioactive compounds have been isolated from the Eremostachys species including: terpenoids, mono- and sesqui- tepenes, linear and branched hydrocarbons and derivatives.9 This plant was topically used for wound healing of snake bites and rheumatism joint pains.10 The rhizomes, rich in iridoid glycosides, are known for analgesic effects.11 In addition, the anti-inflammatory effects of Eremostachys rhizomes have been reported in allergies and auto-immune diseases.12 It has been suggested that, attenuating the prostaglandins forma- tion is responsible for Eremostachys anti-inflammatory effects.13 Besides mentioned properties, the anti-bacterial effects of Eremostachys on Escherichia coli and Staphylo- coccus aureus species suggests it for oral wounds and infec- tions which have not been studied yet.14 Taking this information into consideration, the purpose of this study was to evaluate the effectiveness of E. Binalodensis on gingival wound healing through altering fibroblast prolifer- ation and secretion of three important inflammatory cytokines. Materials and Methods E. binalodensis extract preparation The rhizomes of E. binalodensis were collected on July from Binalud mountains, Mashhad Iran [(3 36° 17’ 60.00” N) lati- tude (58° 32’ 60.00” E) longitude and altitude 1950 m above sea level]. A voucher specimen has been deposited in the her- barium of the Faculty of Pharmacy, Tabriz University of Med- ical Sciences, Iran under the accession code TBZ-FPH 4033. Air-dried rhizomes of E. binalodensis (100 g each) were extracted with methanol. All obtained extracts were separately concentrated using a rotary evaporator at a maximum temper- ature of 45°C. Cell culture Human gingival fibroblasts (hGFs) (HGF1-PI 1) were obtained from Pasteur institute, Tehran, Iran. Cells were cultured in Abstract Objectives In this study we aimed to evaluate the effect of E. binalodensis on gingival inflammatory wounds. Materials and Methods In-vitro wound was induced by scratching the surface layer of human gingival fibroblasts (hGFs). Cells were retreated with 1,10,100,1000 µg/ml of E. binalodensis methanol extract prior to 1 µg/ml LPS stimulation. hGFs proliferation was assessed by MTT test. Also levels of critical inflammatory cytokines such as IL-1b, IL-6 and TNF-a were determined by enzyme-linked immunosorbent assay (ELISA). Results Wound induction was associated with secretion of IL-1β, IL-6 and TNF-a from hGFs. E. binalodensis enhanced the hGFs proliferation besides reducing the level of IL-1β, IL-6 and TNF-a in LPS-scratch-stimulated hGFs. Conclusions Regarding anti-inflammatory and proliferative effects of E. binalodensis on hGFs, availability and safety of it, it is suggested for enhancing the wound healing process in gingival inflammatory wounds. Key words Eremostachys, gingivitis, fibroblast, inflammation, wound ISSN 2413-0516 141J Contemp Med Sci | Vol. 7, No. 3, May-June 2021: 140–144 A. S. Hariri et al. Original Eremostachys binalodensis, a potential therapeutic choice for gingival inflammatory wounds Dulbecco’s modified Eagle medium (Sigma-Aldrich, Germany) including 10% fetal bovine serum, 2 mM L-glutamine, 100 mM L-ascorbate-2-phosphate, 50 U/mL streptomycin, 50 U/mL penicillin and 1 mM sodium pyruvate.15 Cells were seeded (1 ´ 105 cells/well) in 24-well plates and incubated for 24 hours in 37 ˚C, 5% CO2 and 95% humidity. In-vitro wound and LPS induction In-vitro wound was induced by scrapping the 2-3 mm of sur- face layer of hGFs creating a scratch to the cells followed by mentioned incubation method.16 In order to induce inflamma- tion, 1 µg/ml bacterial endotoxin (Lipopolysaccharides (LPS)) was added 1 hour after E. binalodensis extract application on cells. Experimental groups First, the effective concentration of E. binalodensis on hGFs’ proliferation was determined by 3-(4,5-dimethylthi- azol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) test. 1, 10, 100 and 1000 µg/ml of the methanolic E. binalodensis extracts in 1% Dimethyl sulfoxide (DMSO) were selected for MTT test. Regarding the results of MTT test, the work was performed in five groups: 1. Control, 2. Scratch, 3. Scratch + Extract, 4. Scratch + LPS, 5. Scratch + LPS + Extract. Groups 1, 2, 4 were pretreated with the same amount of DMSO. Extract was per- formed 1 hour prior to LPS and scratch and cells were incubated for 48 hours. MTT assay The effect of E. binalodensis on hGFs proliferation was assessed by MTT test. MTT assay is based on the reduction of MTT to formazan crystals by mitochondria of viable cells.17 First, cells which were 48 hours incubated with 1, 10, 100 and 1000 µg/ml of the methanolic extracts of E. binalodensis, were seeded at a density of 1 × 104 cells/well in a 96-well plate. Second, the medium was removed and 10 µL MTT was added to the wells and incubated for 4 hours. After observing formazan crystals, 200 µL DMSO was added to dissolve the crystals. Finally, the absorbance of the plate was read at 450 nm by spectrophotom- eter (Specord 250, Analytik Jena), cell viability was expressed relative to the control group which was regarded as 100%. The test was repeated three times. ELISA assay of inflammatory markers In order to evaluate the main inflammatory markers involved in periodontal diseases, the protein levels of IL-1β, IL-6 and TNF-a in culture supernatants were measured by enzyme- linked immunosorbent assay (ELISA). Human IL-1β ELISA Kit (ab100562), Human TNF alpha ELISA Kit (ab181421), Human IL-6 ELISA Kit (ab46027) were purchased. For this purpose, 50 µL of cells were added to a 96-well plate. 50 µL of the antibody cocktail, regarding the type of measured marker, was added to each well and incubated for 2 hours on a plate shaker with 400 rpm. After washing the plate regarding the kit instruction, 100 µL 3,3',5,5'-Tetramethylbenzidine (TMB) development solution was added and incubated. Finally, after washing and adding the stop solution, the plate absorbance was read at 450 nm by spectrophotometer. The levels of IL-1b, L-6 and TNF-a in cell culture supernatants, expressed as pg/mL, were quantified based on each corresponding standard curve. Each experiment was repeated three times. Statistical analysis The data was analyzed by GraphPad Prism 6 through one-way analysis of variance with post-hoc Tukey tests. In this study, P < 0.05 was considered a significant difference. Results Effect of E. binalodensis on hGFs proliferation Effects of E. binalodensis on hGFs proliferation was evaluated by MTT assay. As shown in Figure 1, the cell viability did not change by 1 and 10 µg/ml of extract; However, 100 and 1000 µg/ml E. binalodensis extract enhanced the hGFs proliferation significantly (P < 0.001, P < 0.0001). The results illustrated the proliferative effect of E. binalodensis on hGFs. Considering MTT test results, 100 µg/ml of E.binalodensis, the lowest pro- liferative concentration in this research, was selected for fur- ther ELISA tests. Effect of E. binalodensis on production of IL-1β in LPS-stimulated hGFs As presented in figure 2, scratch induction raised the IL-1β significantly compared to control group (P < 0.01). Also, LPS application (scratch + LPS group) induced 1L-1β significantly in comparison with scratch group (P < 0.0001). However, E. binalodensis administration significantly reduced the IL-1β level in comparison with scratch + LPS group (P < 0.0001). Effect of E. binalodensis on production of IL-6 in LPS-stimulated hGFs Regarding figure 3, IL-6 elevated in scratch group compared to control group (P < 0.05). In addition, E. binalodensis extract application reduced the IL-6 level in comparison with scratch Fig. 1 The effect of E. binalodensis on hGFs proliferation. E: Eremostachys Binalodensis. (*** P < 0.001, **** P < 0.0001 compared to control group). Data are presented as Mean ± SD. Fig. 2 Effect of E. binalodensis on production of IL-1β in LPS- stimulated hGFs. Data are presented as Mean ± SEM. (**P < 0.01 compared to control group, ####P < 0.0001 compared to scratch group, &&&&P < 0.0001 compared to scratch + LPS group). 142 J Contemp Med Sci | Vol. 7, No. 3, May-June 2021: 140–144 Eremostachys binalodensis, a potential therapeutic choice for gingival inflammatory wounds Original A. S. Hariri et al. group (P < 0.05). LPS induction (scratch + LPS group), raised the IL-6 level significantly compared to scratch group (P < 0.0001). In contrast, E. binalodensis extract administra- tion significantly decreased the IL-6 level compared to scratch + LPS group (P < 0.0001). Effect of E. binalodensis on production of TNF-a in LPS-stimulated hGFs As shown in figure 4, TNF-a significantly increased in scratch-applied group in comparison with control group (P < 0.05, P < 0.0001). In group receiving E. binalodensis extract, TNF-a level declined significantly in comparison with scratch group (P < 0.05). Similarly, LPS administration (scratch + LPS group) induced TNF-a compared to scratch group (P < 0.0001). Contrarily, E. binalodensis extract admin- istration reduced the TNF-a level when compared to scratch + LPS group (P < 0.0001). Discussion In the present study, we evaluated the possible effect of E. bin- alodensis on gingival inflammatory wounds. Gingival wounds, leading in gingivitis, are the first stage of periodontal diseases. In comparison with dermal or mucosal wounds, gingival wounds are thought to be more complicated due to presence of microbial plaques, low accessibility of mouthwashes and anti- microbial agents to the bacteria and pH variations.18 The untreated gingivitis can lead to periodontal disease accompa- nied with sever medical conditions such as cardiovascular dis- ease, diabetes, and adverse pregnancy outcomes.19 Inflammation is regarded as the first stage of healing pro- cess of gingival wounds followed by fibroblast-mediated tissue formation and remodeling.20 It starts with infiltration of leuko- cytes to the wound site, resulting in pathogen combating, tissue degradation and regeneration.21 However, excessive inflammatory response diminishes mitogenic cell activity and tissue remodeling.22 In this study, it was observed that IL-1β was elevated in scratched fibroblasts, confirming fibroblasts as one of the major sources of IL-1β. It has been reported that, besides fibroblasts the accumulated neutrophils post-injury, produce pro-inflammatory cytokines such as IL-1β and TNF-a.23 LPS or pathogen-activated Toll-like receptors induce Nuclear factor kappa B (NF-kB) expression which results in pro IL-1β production.24 IL-1β increases neutrophil infiltration and triggers inflammatory cascades in wound area. Also, ele- vated levels of IL-1β induces prostaglandin E2 formation, together inducing collagenase which leads to periodontal attachment loss.25 Moreover, IL-1β stimulated matrix metal- loproteinases, contribute in extracellular matrix degradation and tissue destruction.26 In contrast, our results showed that E. binalodensis markedly decreases the scratch + LPS- induced IL-1β level. It has been reported that there is a strong relationship between increasing gingival cervical fluid levels of IL-1β and severity of gingivitis and blocking IL-1β activity or reducing it by anti-inflammatory agents, have improving effects on gingivitis.27,28 Similarly, in this research TNF-a was elevated post- scratch in fibroblasts. TNF-a is involved in late steps of wound healing, shifting the cells to tissue remodeling phase; however, increased levels of it results in tissue damaging by impairing fibroblast activity and stimulating osteoclastogen- esis.29 Also TNF-a induces fibroblast apoptosis through Foxo signaling pathway.30 Although increase of TNF-a level was observed following scratch + LPS application, pretreatment with E.binalodensis attenuated the TNF-a rise in fibroblasts. Regarding the synergistic effects of IL-1β and TNF-a, it has been reported that administration of antagonists of both cytokines has more beneficial effects on gingivitis.27 Moreover, the level of IL-6, one of the predictors of per- iodontal disease initiation, was evaluated in this study. The results showed that, both scratch and LPS induction increases the IL-6 level. It is suggested that, enhanced levels of IL-1β and TNF-a are involved in stimulating fibroblast-secretion of IL-6 through mitogen-activated protein kinase (MAPK) pathway, resulting in positive inflammatory feedback.31 IL-6 is highly associated with the pocket depth and severity of gin- givitis.32 Also the differentiation of CD4 to T cells is impaired by high levels of IL-6, resulting in reduced bacterial inhibi- tion in oral cavity.33 In addition, IL-6, known as stimulator of osteoclasts, is responsible for bone resorption followed by gingivitis.34 Our results showed that E.binalodensis reduced the IL-6 level post scratch as well as post LPS application. Similarly, it has been shown that, agents reducing IL-6, decrease bone loss in inflamed gingival mucosa. Also Tocili- zumab (IL-6 receptor inhibitor) administration have been reported to improve gingivitis and reduce bleeding sites.35 Therefore, E. binalodensis inhibits inflammation-induced complications by reducing the secretion of three critical pro-inflammatory cytokines. The potential anti-bacterial effects of Eremostachys genius, have been reported in pre- vious studies.14 Besides inhibiting inflammation, anti-bacterial effects of E. binalodensis may enhance the gingival wound healing process. Fig. 3 Effect of E. binalodensis on production of IL-6 in LPS- stimulated hGFs. Data are presented as Mean ± SEM. (*P < 0.05 compared to control group, #P < 0.05 and ####P < 0.0001 com- pared to scratch group, &&&&P < 0.0001 compared to scratch + LPS group). Fig. 4 Effect of E. binalodensis on production of TNF-a in LPS- stimulated hGFs. Data are presented as Mean ± SEM. (**P < 0.01 compared to control group, ##P < 0.05 and ####P < 0.0001 compared to scratch group, &&&&P < 0.0001 compared to scratch + LPS group). 143J Contemp Med Sci | Vol. 7, No. 3, May-June 2021: 140–144 A. S. Hariri et al. Original Eremostachys binalodensis, a potential therapeutic choice for gingival inflammatory wounds The second stage in reformation of periodontal tissue centrally involves fibroblasts, which generate and organize the collagen fibers that attach the alveolar bone and gingiva to the cementum covering the tooth root.36 In this study, it was observed that E.binalodensis extract, not only reduces the inflammatory cytokines, but also enhances the fibroblast proliferation. This is the remarkable feature of proposing E. binalodensis for gingival wounds. The increased rate of gingival fibroblast proliferation, results in enhanced wound healing process. Our results are similar to Liao et al. study indicating that, traditional medicine inhibiting IL-6 and inducing fibroblast proliferation are suggested for gingival wound healing.37 The treatment choices for gingivitis include anti-inflammatory drugs which usually impair fibroblast activation.7 Regarding the crucial role of fibroblasts in gin- gival wound healing, it is of a great importance to administer agents that prevent inflammation without attenuating fibro- blast proliferation and activity. Regarding the previous studies, it is suggested that the anti-inflammatory effects of E. binalodensis is related to the presence of iridoid glycosides in this plant.13 Taken together, it can be concluded that, the methanol extract of E. binalodensis has improving effects on inflammatory gingival wounds. Also availability of this plant and previously- confirmed safety of it, reduces the cost of treatment besides enhancing the healing process. Further in-vivo investigations can be applied for evaluating the effects of E. binalodensis in details. Funding Sources This study was supported by Aja University of Medical Sciences (Grant:97001404). Acknowledgment The authors would like to thank Sara laboratory (Tabriz, Iran) and Pharmacognosy department of Tabriz University of Med- ical Sciences for their contribution in this study. Conflicts of Interest The authors have no conflicts of interest to disclose.  References 1. Smith PC, Cáceres M, Martínez C, Oyarzún A, Martínez J. Gingival Wound Healing: An Essential Response Disturbed by Aging? Journal of Dental Research. 2015;94:395–402. 2. Bhattacharya R, Xu F, Dong G, Li S, Tian C, Ponugoti B, et al. Effect of bacteria on the wound healing behavior of oral epithelial cells. PloS one. 2014;9:e89475. 3. Palmqvist P, Lundberg P, Lundgren I, Hänström L, Lerner U. IL-1β and TNF-a regulate IL-6-type cytokines in gingival fibroblasts. Journal of dental research. 2008;87:558–63. 4. 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The Kampo medicine Rokumigan possesses antibiofilm, anti-inflammatory, and wound healing properties. BioMed research international. 2014;2014. This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. Doi: https://doi.org/10.22317/jcms.v7i3.976