İndigo Naturalis’in İnsan Periodontal Ligament Fibroblast Hücrelerinin Canlılığı Üzerine Etkisi: Bir İn Vitro Çalışma

Abstract

Amaç: Bu in vitro araştırmada, insan periodontal ligament fibroblast (İPDLF) hücrelerine farklı konsantrasyonlarda İndigo naturalis (İN) uygulanarak hücrelerin canlılık düzeylerinin araştırılması hedeflenmiştir. Gereç ve Yöntemler: Çalışmamız dahilinde İndigo naturalis DMSO ile çözülerek 5 µg/ml, 10 µg/ml ve 15 µg/ml olacak şekilde üç farklı konsantrasyon hazırlandı. Hücre canlılığını değerlendirmek amacıyla, 96 kuyucuklu plakalara her konsantrasyon için 4 defa tekrarlanacak şekilde hücrelerin ekimi gerçekleştirildi. Hücrelere 24 saat süreyle İndigo Naturalis uygulandı. Canlılık değerlendirmesi için 3-(4,5-dimetiltiyazol-2-il)-2-5-difeniltetrazolyum bromür (MTT) deney protokolü izlendi. Verilerin istatistiksel analizleri SPSS paket programı kullanılarak değerlendirildi. İstatistiksel anlamlılık p<0.05 olarak kabul edildi. Bulgular: İN uygulamasının kontrol grubuna kıyasla test gruplarında anlamlı değişiklik olmadığı görülürken 15 μg/ml ve 5 μg/ml konsantrasyon arasında anlamlı fark tespit edilmiştir. Sonuç: Bu çalışmanın sınırları içerisinde sağlanan bulgular, İN’nin iPDLF hücrelerinin canlılığı üzerine farklı konsantrasyonlarda benzer etki gösterdiğini ortaya koymaktadır. İN’nin daha farklı konsantrasyonlarda hazırlanarak ve daha uzun süre aralıklarında takip edilerek araştırıldığı çalışmalara gereksinim vardır.

Keywords

• İndigo naturalis
• periodontal ligament fibroblastı
• hücre canlılığı

Effect of Indigo Naturalis on the Viability of Human Periodontal Ligament Fibroblast Cells: An In Vitro Study

Abstract

Objective: In this in vitro research, it was aimed to examine the viability levels of human periodontal ligament fibroblast (hPDLF) cells by applying different concentrations of Indigo naturalis (IN). Materials and Methods: Within the scope of our study, IN was dissolved in DMSO and three different concentrations were prepared:, 5 µg/ml, 10 µg/ml and 15 µg/ml. In order to evaluate cell viability, cells were seeded in 96-well plates, repeated 4 times for each concentration. Indigo Naturalis was applied to the cells for 24 hours. 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) experimental protocol was followed for viability assessment. Statistical analyzes of the data were evaluated using the SPSS package program. Statistical significance was accepted as p<0.05. Results: While it was observed that there was no significant change in the test groups with IN application compared to the control group, a significant difference was detected between 15 μg/ml and 5 μg/ml concentration. Conclusion: The findings obtained within the limits of this study reveal that IN has similar effects at different concentrations on the viability of hPDLF cells. There is a need for studies in which IN is investigated by preparing it in different concentrations and monitoring it for longer periods of time.

Keywords

• Indigo naturalis
• cell viability
• periodontal ligament fibroblast

References

1. Benatti BB, Silvério KG, Casati MZ, Sallum EA, Nociti Jr FH. (2007). Physiological features of periodontal regeneration and approaches for periodontal tissue engineering utilizing periodontal ligament cells. JOBB, 2007;103(1): 1-6.
2. Bonewald LF, Johnson ML. Osteocytes, mechanosensing and Wnt signaling. Bone. 2008;42(4): 606-615.
3. Cheon SS, Cheah AY, Turley S, Nadesan P, Poon R, Clevers H, Alman BA. Beta-catenin stabilization dysregulates mesenchymal cell proliferation, motility, and invasiveness and causes aggressive fibromatosis and hyperplastic cutaneous wounds. Proc Natl Acad Sci USA. 2002;99(10): 6973-6978.
4. Cheon SS, Nadesan P, Poon R, & Alman BA. Growth factors regulate beta-catenin-mediated TCF-dependent transcriptional activation in fibroblasts during the proliferative phase of wound healing. Exp Cell Res. 2004;293(2): 267-274.
5. Choe Y, Yu JY, Son YO, Park SM, Kim JG, Shi X, Lee JC. Continuously generated H2O2 stimulates the proliferation and osteoblastic differentiation of human periodontal ligament fibroblasts. J Cell Biochem. 2012;113(4): 1426-1436.
6. Correia VdeF, Caldeira CL, Marques MM. Cytotoxicity evaluation of sodium alendronate on cultured human periodontal ligament fibroblasts. Dent Traumatol. 2006;22(6): 312-317.
7. Gaitanis G, Magiatis P, Velegraki A, Bassukas I. A traditional Chinese remedy points to a natural skin habitat: indirubin (indigo naturalis) for psoriasis and the Malassezia metabolome. British Journal of Dermatology. 2018;179(3): 800-800.
8. Graziani F, Karapetsa D, Alonso B, Herrera D. Nonsurgical and surgical treatment of periodontitis: how many options for one disease? Periodontol 2000. 2017;75(1): 152-188.

9. Griffin MF, Huber J, Evan FJ, Quarto N, Longaker MT. The role of Wnt signaling in skin fibrosis. Med Res Rev. 2022;42(1): 615-628.
10. Han P, Ivanovski S, Crawford R, Xiao Y. Activation of the Canonical Wnt Signaling Pathway Induces Cementum Regeneration. J Bone Miner Res. 2015;30(7): 1160-1174.
11. Han P, Wu C, Chang J, Xiao Y. The cementogenic differentiation of periodontal ligament cells via the activation of Wnt/β-catenin signalling pathway by Li+ ions released from bioactive scaffolds. Biomaterials. 2012;33(27): 6370-6379.
12. Heo JS, Lee SY, Lee JC. Wnt/β-catenin signaling enhances osteoblastogenic differentiation from human periodontal ligament fibroblasts. Mol Cells. 2010;30(5): 449-454.
13. Hsu YH, Kiel DP. Clinical review: Genome-wide association studies of skeletal phenotypes: what we have learned and where we are headed. J Clin Endocrinol Metab. 2012;97(10): E1958-1977.
14. Kikuchi A, Yamamoto H, Sato A, Matsumoto S. New insights into the mechanism of Wnt signaling pathway activation. Int Rev Cell Mol Biol. 2011;291: 21-71.
15. Klapholz-Brown Z, Walmsley GG, Nusse YM, Nusse R, Brown PO. Transcriptional program induced by Wnt protein in human fibroblasts suggests mechanisms for cell cooperativity in defining tissue microenvironments. PLoS One. 2007;2(9): e945.
16. Kook SH, Heo JS, Lee JC. Crucial roles of canonical Runx2-dependent pathway on Wnt1-induced osteoblastic differentiation of human periodontal ligament fibroblasts. Mol Cell Biochem. 2015;402(1-2): 213-223.
17. Kook SH, Lee D, Cho ES, Heo JS, Poudel SB, Ahn YH, Hwang JW, Ji H, Kim JG, Lee JC. Activation of canonical Wnt/β-catenin signaling inhibits H2O2-induced decreases in proliferation and differentiation of human periodontal ligament fibroblasts. Mol Cell Biochem. 2016;411(1-2): 83-94.
18. Kumar P, Nagarajan A, Uchil PD. Analysis of Cell Viability by the MTT Assay. Cold Spring Harb Protoc 2018(6).
19. Leclerc S, Garnier M, Hoessel R, Marko D, Bibb JA, Snyder GL, Greengard P, Biernat J, Wu YZ, Mandelkow EM. Indirubins Inhibit Glycogen Synthase Kinase-3β and CDK5/P25, Two Protein Kinases Involved in Abnormal Tau Phosphorylation in Alzheimer's Disease: A PROPERTY COMMON TO MOST CYCLIN-DEPENDENT KINASE INHIBITORS?. J Biol Chem. 2001;276(1): 251-260.
20. Lin YK, Chen HW, Leu YL, Yang YL, Fang Y, Su Pang JH, Hwang TL. Indigo naturalis upregulates claudin-1 expression in human keratinocytes and psoriatic lesions. J Ethnopharmacol. 2013;145(2): 614-620.
21. Lin YK, Chen HW, Yang SH, Leu YL, Huang YH, Yen HC. Protective effect of indigo naturalis extract against oxidative stress in cultured human keratinocytes. J Ethnopharmacol. 2012;139(3): 893-896.
22. Lin YK, Leu YL, Huang TH, Wu YH, Chung PJ, Su Pang JH, Hwang TL. Anti-inflammatory effects of the extract of indigo naturalis in human neutrophils. J Ethnopharmacol. 2009;125(1): 51-58.
23. Liu F, Millar SE. Wnt/beta-catenin signaling in oral tissue development and disease. J Dent Res. 2010;89(4): 318-330.
24. Melcher AH. On the repair potential of periodontal tissues. J Periodontol. 1976;47(5): 256-260.
25. Minear S, Leucht P, Jiang J, Liu B, Zeng A, Fuerer C, Nusse R, Helms JA. Wnt proteins promote bone regeneration. Sci Transl med. 2010;2(29); 29ra30.
26. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1-2): 55-63.
27. Naganuma M. Treatment with indigo naturalis for inflammatory bowel disease and other immune diseases. Immunol Med. 2019;42(1): 16-21.
28. Narayanan AS, Page RC. Connective tissues of the periodontium: a summary of current work. Coll Relat Res. 1983;3(1): 33-64.
29. Neves VC, Babb R, Chandrasekaran D, Sharpe PT. Promotion of natural tooth repair by small molecule GSK3 antagonists. Sci Rep. 2017;7: 39654.
30. Newman MG, Takei H, Klokkevold PR, Carranza FA. Carranza's clinical periodontology. Birleşmiş Milletler: Elsevier Health Sciences; 2011.
31. Patel M, Post Y, Hill N, Sura A, Ye J, Fisher T, Suen N, Zhang M, Cheng L, Pribluda A, Chen H, Yeh WC, Li Y, Baribault H, Fletcher RB. A WNT mimetic with broad spectrum FZD-specificity decreases fibrosis and improves function in a pulmonary damage model. Respir Res. 2024;25(1): 153.
32. Qi-Yue Y, Ting, Z, Ya-Nan H, Sheng-Jie H, Xuan D, Li H, Chun-Guang X. From natural dye to herbal medicine: A systematic review of chemical constituents, pharmacological effects and clinical applications of indigo naturalis. Chin Med. 2020;15: 1-13.
33. Somanader DVN, Zhao P, Widdop RE, Samuel CS. The involvement of the Wnt/β-catenin signaling cascade in fibrosis progression and its therapeutic targeting by relaxin. Biochem Pharmacol. 2024;223: 116130.
34. Sun X, Lv G, Zhu C. Research on Application of Qingdai (Natural Indigo) in History. Acta Chin Med. 2020;35: 1653-1655.
35. Sun Q, Leng J, Tang L, Wang L, Fu C. A comprehensive review of the chemistry, pharmacokinetics, pharmacology, clinical applications, adverse events, and quality control of Indigo Naturalis. Front Pharmacol. 2021;12: 664022.
36. Taba MJr, Jin Q, Sugai JV, Giannobile WV. Current concepts in periodontal bioengineering. Orthod Craniofac Res. 2005;8(4): 292-302.
37. Tanır H, Demirezen Ş. Wnt sinyal yolunun biyolojisi ve bu yolda görev alan biyomoleküller. Turkiye Klinikleri J Med Sci. 2009;29: 1292-1297.
38. Tian Y, Chen J, Huang W, Ren Q, Feng J, Liao J, Fu H, Zhou L, Liu Y. Myeloid-derived Wnts play an indispensible role in macrophage and fibroblast activation and kidney fibrosis. Int J Biol Sci. 2024;20(6): 2310-2322.
39. Tonetti MS, Greenwell H, Kornman KS. Staging and grading of periodontitis: Framework and proposal of a new classification and case definition. J Periodontol 2018;89 Suppl 1: S159-S172.
40. Whyte JL, Smith AA, Helms JA. Wnt signaling and injury repair. Cold Spring Harbor Perspect Biol. 2012;4(8): a008078.
41. Xu Y, Lin C, Tan Hy, Bian Zx. The double-edged sword effect of indigo naturalis. Food Chem Toxicol. 2024;185: 114476.
42. Yu H, Li TN, Ran Q, Huang QW, Wang J. Strobilanthes cusia (Nees) Kuntze, a multifunctional traditional Chinese medicinal plant, and its herbal medicines: A comprehensive review. J Ethnopharmacol. 2021;265: 113325.
43. Zhang R, Oyajobi BO, Harris SE, Chen D, Tsao C, Deng HW, Zhao M. Wnt/β-catenin signaling activates bone morphogenetic protein 2 expression in osteoblasts. Bone. 2013;52(1): 145-156.
44. Zhang, Y, Lu F. Molecular mechanism of triptolide in myocardial fibrosis through the Wnt/β-catenin signaling pathway. Scand Cardiovasc J. 2024;58(1): 2295785.