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Metformin’in Insan Gingival Fibroblastları Üzerindeki Sitotoksisitesinin In Vitro Değerlendirilmesi

Yıl 2023, Cilt: 33 Sayı: 2 - 2023, 33:2, 73 - 78, 14.07.2023
https://doi.org/10.5152/CRDS.2023.2270115

Öz

Amaç: Bu çalışmanın amacı; antiapoptotik, antienflamatuar ve antioksidan etkileri olan, osteoblastik hücre farklılaşması ve mineralizasyon nodülü oluşumunu indükleyen metformin adlı ilacın Primer İnsan Gingival Fibroblast (HGF) hücreleri üzerindeki doz bağımlı etkisinin in-vitro olarak incelenmesidir. Bu çalışma daha ileri çalışmalara ve olası insan kullanımlarına ışık olması adına planlanmıştır
Yöntemler: HGF hücreleri besiyerlerine eklenen 11 farklı metformin dozu ile 24 saat kültüre edilmiştir. Her metformin dozu için 3 farklı kuyucuk kullanılmıştır. Bu değerler MTT testi ile ölçülmüştür.
Bulgular: Metformin düzeyleri ile fibroblast canlılığı ve üremesi arasında ters bir orantı olduğu söylenebilir. Ayrıca, metformin düzeylerinin 2 mM ve altındaki değerleri için fibroblast ölçümleri hemen hemen aynı kalmaktadır. Metforminin 2’den büyük değerleri için fibroblast canlılığı ve üremesinin önemli derecede azaldığı görülmektedir.
Sonuç: Faz 2 tedavi ihtiyacını azaltacak, iyileşme periyodunu kısaltacak ve antibakteriyel etki sağlayacak etkili bir lokal ajan bulunması için yapılan çalışmalar sürekli güncelliğini korumaktadır. Rejenerasyona pozitif katkı sağlama ihtimali olan metforminin uygulanacağı bölgedeki öncül hücrelerden olan fibroblastlara etkisinin doz bağımlı değişiminin 2 mM konsantrasyonuna kadar anlamlı bir etki yapmadığı ancak 2 mM’den daha yoğun konsantrasyonlarda sitotoksisite oluşturduğu çalışmamızda gösterilmiştir.
Anahtar Kelimeler: Fibroblast, metformin, sitotoksisite
ABSTRACT
Objective: The aim of this study is to investigate the dose-dependent effect of metformin on Primary Human Gingival Fibroblast (HGF) cells in vitro, which has antiapoptotic, antienflamatuar and antioxidant effects, can promote osteoblastic differantitation and mineralization nodules. This study designed to be a precursor for further and possible human studies.
Methods: HGF cells were cultured for 24 hours with 11 different metformin dose added to the medium. 3 different applications were made for each metformin dose. These values were measured with the MTT test.
Results: İn our research, it is shown that there is an inverse relationship between metformin levels and fi- broblast viability and reproduction. Fibroblast vitality measurements didn't show any significant difference on metformin levels, below 2 mM. Fibroblast viability and proliferation appear to be significantly reduced for metformin values of 3 mM and greater than 3 mM.
Conclusion: Studies to find an effective local agent that reduces the need for The Phase 2 treatment, shorten the recovery period and provide an antibacterial effect are constantly up to date. It has been shown in our study that the dose-dependent change of the effect of metformin, which is likely to contribute positively to regeneration, of fibroblasts, which are the precursor cells in the area where it will be applied, does not have a significant effect up to a concentration of 2 mM, but creates cytotoxicity at concentrations of 3 mM and more dense than 3 mM.
Keywords: metformin, cytotoxicity, fibroblast

Kaynakça

  • 1. Page RC, Offenbacher S, Schroeder HE, Seymour GJ, Kornman KS. Advances in the pathogenesis of periodontitis: summary of develop- ments, clinical implications and future directions. Periodontol 2000. 1997;14(1):216-248.
  • 2. Pradeep AR, Patnaik K, Nagpal K, et al. Efficacy of locally-delivered 1% metformin gel in the treatment of intrabony defects in patients with chronic periodontitis: a randomized, controlled clinical trial. J Inves- tig Clin Dent. 2016;7(3):239-245.
  • 3. McCulloch CA. Basic considerations in periodontal wound healing to achieve regeneration. Periodontol 2000. 1993;1(1):16-25.
  • 4. Takeuchi Y, Umeda M, Sakamoto M, Benno Y, Huang Y, Ishikawa I. Tre- ponema socranskii, Treponema denticola, and Porphyromonas gin- givalis are associated with severity of periodontal tissue destruction. J Periodontol. 2001;72:1354-1363.
  • 5. Kang W, Wang T, Hu Z, Liu F, Sun Y, Ge S. Metformin Inhibits Porphyromonas gingivalis Lipopolysaccharide-Influenced Inf- lammatory Response in Human Gingival Fibroblasts via Regula- ting Activating Transcription Factor-3 Expression. J Periodontol. 2017;88(10):e169-e178.
  • 6. Mealey BL, Ocampo GL. Diabetes mellitus and periodontal disea- se. Periodontol 2000. 2007;44:127-153.
  • 7. Wang C, Liu C, Gao K, et al. Metformin preconditioning provide neuroprotection through enhancement of autophagy and suppres- sion of inflammation and apoptosis after spinal cord injury. Biochem Biophys Res Commun. 2016;477(4):534-540.
  • 8. Ismaiel AA, Espinosa-Oliva AM, Santiago M, et al. Metformin, besi- des exhibiting strong in vivo anti-inflammatory properties, increa- ses mptp-induced damage to the nigrostriatal dopaminergic sys- tem. Toxicol Appl Pharmacol. 2016;298:19-30.
  • 9. Grahame Hardie D. AMP-activated protein kinase: a key regulator of energy balance with many roles in human disease. J Intern Med. 2014;276(6):543-559.
  • 10. Hattori Y, Suzuki K, Hattori S, Kasai K. Metformin inhibits cytoki- ne-induced nuclear factor kappaB activation via AMP-activated protein kinase activation in vascular endothelial cells. Hypertension. 2006;47(6):1183-1188.
  • 11. Hyun E, Ramachandran R, Hollenberg MD, Vergnolle N. Mechanisms behind the anti-inflammatory actions of insulin. Crit Rev Immunol. 2011;31(4):307-340.
  • 12. Monnier L, Hanefeld M, Schnell O, Colette C, Owens D. Insulin and at- herosclerosis: how are they related? Diabetes Metab. 2013;39:111-117.
  • 13. Valko M, Morris H, Cronin MT. Metals, toxicity and oxidative stress. Curr Med Chem. 2005;12(10):1161-1208.
  • 14. Cho JG, Song JJ, Choi J, Im GJ, Jung HH, Chae SW. The suppressive effects of metformin on inflammatory response of otitis media model in human middle ear epithelial cells. Int J Pediatr Otorhinolaryngol. 2016;89:28-32.
  • 15. Diniz Vilela D, Gomes Peixoto L, Teixeira RR, et al. The Role of Metfor- min in Controlling Oxidative Stress in Muscle of Diabetic Rats. Oxid Med Cell Longev. 2016;2016:6978625. 16. Kelly B, Tannahill GM, Murphy MP, O’Neill LA. Metformin inhibits the production of reactive oxygen species from NADH: ubiquinone oxi- doreductase to limit induction of interleukin-1β (IL-1β) and boosts interleukin-10 (IL10) in lipopolysaccharide (LPS)-activated macrop- hages. J Biol Chem. 2015;290(33):20348-59.
  • 17. Batchuluun B, Inoguchi T, Sonoda N, et al. Metformin and liraglutide ameliorate high glucose-induced oxidative stress via inhibition of PKC-NAD(P)H oxidase pathway in human aortic endothelial cells. At- herosclerosis. 2014;232(1):156-164.
  • 18. Markowicz-Piasecka M, Sikora J, Szydłowska A, Skupień A, Mikici- uk-Olasik E, Huttunen KM. Metformin - a Future Therapy for Neuro- degenerative Diseases : Theme: Drug Discovery, Development and Delivery in Alzheimer’s Disease Guest Editor: Davide Brambil- la. Pharm Res. 2017;34(12):2614-2627.
  • 19. Mummidi S, Das NA, Carpenter AJ, et al. Metformin inhibits aldoste- rone-induced cardiac fibroblast activation, migration and proliferati- on in vitro, and reverses aldosterone+salt-induced cardiac fibrosis in vivo. J Mol Cell Cardiol. 2016;98:95-102.
  • 20. Bułdak Ł, Łabuzek K, Bułdak RJ, et al. Metformin affects macropha- ges’ phenotype and improves the activity of glutathione peroxida- se, superoxide dismutase, catalase and decreases malondialdehyde concentration in a partially AMPK-independent manner in LPS-sti- mulated human monocytes/macrophages [published correction ap- pears in Pharmacol Rep. 2017 Jun;69(3):594] [published correction appears in Pharmacol Rep. 2019 Oct;71(5):981-982]. Pharmacol Rep. 2014;66(3):418-429.
  • 21. Dai J, Liu M, Ai Q, et al. Involvement of catalase in the protective be- nefits of metformin in mice with oxidative liver injury. Chem Biol In- teract. 2014;216:34-42.
  • 22. Kanazawa I, Yamaguchi T, Yano S, Yamauchi M, Sugimoto T. Metfor- min enhances the differentiation and mineralization of osteoblastic MC3T3-E1 cells via AMP kinase activation as well as eNOS and BMP-2 expression. Biochem Biophys Res Commun. 2008;375(3):414- 419.
  • 23. Cortizo AM, Sedlinsky C, McCarthy AD, Blanco A, Schurman L. Oste- ogenic actions of the anti-diabetic drug metformin on osteoblasts in culture. Eur J Pharmacol. 2006;536(1-2):38-46.
  • 24. Wang P, Ma T, Guo D, et al. Metformin induces osteoblastic differenti- ation of human induced pluripotent stem cell-derived mesenchymal stem cells. J Tissue Eng Regen Med. 2018;12(2):437-446.
  • 25. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Met- hods. 1983;65(1-2):55-63.
  • 26. Kumar P, Nagarajan A, Uchil PD: Analysis of Cell Viability by the MTT Assay. Cold Spring Harb Protoc 2018, 2018(6).
  • 27. Pradeep AR, Rao NS, Naik SB, Kumari M. Efficacy of varying concent- rations of subgingivally delivered metformin in the treatment of ch- ronic periodontitis: a randomized controlled clinical trial. J Periodon- tol. 2013;84(2):212-220.
  • 28. Pradeep AR, Nagpal K, Karvekar S, Patnaik K, Naik SB, Guruprasad CN. Platelet-rich fibrin with 1% metformin for the treatment of intra- bony defects in chronic periodontitis: a randomized controlled clini- cal trial. J Periodontol. 2015;86(6):729-737.
  • 29. Pradeep AR, Patnaik K, Nagpal K, Karvekar S, Guruprasad CN, Kuma- raswamy KM. Efficacy of 1% Metformin Gel in Patients With Moderate and Severe Chronic Periodontitis: A Randomized Controlled Clinical Trial. J Periodontol. 2017;88(10):1023-1029.
  • 30. Kurian IG, Dileep P, Ipshita S, Pradeep AR. Comparative evaluati- on of subgingivally-delivered 1% metformin and Aloe vera gel in the treatment of intrabony defects in chronic periodontitis pa- tients: A randomized, controlled clinical trial. J Investig Clin Dent. 2018;9(3):e12324

In Vitro Evaluation of the Cytotoxicity of Metformin on Human Gingival Fibroblasts

Yıl 2023, Cilt: 33 Sayı: 2 - 2023, 33:2, 73 - 78, 14.07.2023
https://doi.org/10.5152/CRDS.2023.2270115

Öz

Amaç: Bu çalışmanın amacı; antiapoptotik, antienflamatuar ve antioksidan etkileri olan, osteoblastik hücre farklılaşması ve mineralizasyon nodülü oluşumunu indükleyen metformin adlı ilacın Primer İnsan Gingival Fibroblast (HGF) hücreleri üzerindeki doz bağımlı etkisinin in-vitro olarak incelenmesidir. Bu çalışma daha ileri çalışmalara ve olası insan kullanımlarına ışık olması adına planlanmıştır
Yöntemler: HGF hücreleri besiyerlerine eklenen 11 farklı metformin dozu ile 24 saat kültüre edilmiştir. Her metformin dozu için 3 farklı kuyucuk kullanılmıştır. Bu değerler MTT testi ile ölçülmüştür.
Bulgular: Metformin düzeyleri ile fibroblast canlılığı ve üremesi arasında ters bir orantı olduğu söylenebilir. Ayrıca, metformin düzeylerinin 2 mM ve altındaki değerleri için fibroblast ölçümleri hemen hemen aynı kalmaktadır. Metforminin 2’den büyük değerleri için fibroblast canlılığı ve üremesinin önemli derecede azaldığı görülmektedir.
Sonuç: Faz 2 tedavi ihtiyacını azaltacak, iyileşme periyodunu kısaltacak ve antibakteriyel etki sağlayacak etkili bir lokal ajan bulunması için yapılan çalışmalar sürekli güncelliğini korumaktadır. Rejenerasyona pozitif katkı sağlama ihtimali olan metforminin uygulanacağı bölgedeki öncül hücrelerden olan fibroblastlara etkisinin doz bağımlı değişiminin 2 mM konsantrasyonuna kadar anlamlı bir etki yapmadığı ancak 2 mM’den daha yoğun konsantrasyonlarda sitotoksisite oluşturduğu çalışmamızda gösterilmiştir.
Anahtar Kelimeler: Fibroblast, metformin, sitotoksisite
ABSTRACT
Objective: The aim of this study is to investigate the dose-dependent effect of metformin on Primary Human Gingival Fibroblast (HGF) cells in vitro, which has antiapoptotic, antienflamatuar and antioxidant effects, can promote osteoblastic differantitation and mineralization nodules. This study designed to be a precursor for further and possible human studies.
Methods: HGF cells were cultured for 24 hours with 11 different metformin dose added to the medium. 3 different applications were made for each metformin dose. These values were measured with the MTT test.
Results: İn our research, it is shown that there is an inverse relationship between metformin levels and fi- broblast viability and reproduction. Fibroblast vitality measurements didn't show any significant difference on metformin levels, below 2 mM. Fibroblast viability and proliferation appear to be significantly reduced for metformin values of 3 mM and greater than 3 mM.
Conclusion: Studies to find an effective local agent that reduces the need for The Phase 2 treatment, shorten the recovery period and provide an antibacterial effect are constantly up to date. It has been shown in our study that the dose-dependent change of the effect of metformin, which is likely to contribute positively to regeneration, of fibroblasts, which are the precursor cells in the area where it will be applied, does not have a significant effect up to a concentration of 2 mM, but creates cytotoxicity at concentrations of 3 mM and more dense than 3 mM.
Keywords: metformin, cytotoxicity, fibroblast

Kaynakça

  • 1. Page RC, Offenbacher S, Schroeder HE, Seymour GJ, Kornman KS. Advances in the pathogenesis of periodontitis: summary of develop- ments, clinical implications and future directions. Periodontol 2000. 1997;14(1):216-248.
  • 2. Pradeep AR, Patnaik K, Nagpal K, et al. Efficacy of locally-delivered 1% metformin gel in the treatment of intrabony defects in patients with chronic periodontitis: a randomized, controlled clinical trial. J Inves- tig Clin Dent. 2016;7(3):239-245.
  • 3. McCulloch CA. Basic considerations in periodontal wound healing to achieve regeneration. Periodontol 2000. 1993;1(1):16-25.
  • 4. Takeuchi Y, Umeda M, Sakamoto M, Benno Y, Huang Y, Ishikawa I. Tre- ponema socranskii, Treponema denticola, and Porphyromonas gin- givalis are associated with severity of periodontal tissue destruction. J Periodontol. 2001;72:1354-1363.
  • 5. Kang W, Wang T, Hu Z, Liu F, Sun Y, Ge S. Metformin Inhibits Porphyromonas gingivalis Lipopolysaccharide-Influenced Inf- lammatory Response in Human Gingival Fibroblasts via Regula- ting Activating Transcription Factor-3 Expression. J Periodontol. 2017;88(10):e169-e178.
  • 6. Mealey BL, Ocampo GL. Diabetes mellitus and periodontal disea- se. Periodontol 2000. 2007;44:127-153.
  • 7. Wang C, Liu C, Gao K, et al. Metformin preconditioning provide neuroprotection through enhancement of autophagy and suppres- sion of inflammation and apoptosis after spinal cord injury. Biochem Biophys Res Commun. 2016;477(4):534-540.
  • 8. Ismaiel AA, Espinosa-Oliva AM, Santiago M, et al. Metformin, besi- des exhibiting strong in vivo anti-inflammatory properties, increa- ses mptp-induced damage to the nigrostriatal dopaminergic sys- tem. Toxicol Appl Pharmacol. 2016;298:19-30.
  • 9. Grahame Hardie D. AMP-activated protein kinase: a key regulator of energy balance with many roles in human disease. J Intern Med. 2014;276(6):543-559.
  • 10. Hattori Y, Suzuki K, Hattori S, Kasai K. Metformin inhibits cytoki- ne-induced nuclear factor kappaB activation via AMP-activated protein kinase activation in vascular endothelial cells. Hypertension. 2006;47(6):1183-1188.
  • 11. Hyun E, Ramachandran R, Hollenberg MD, Vergnolle N. Mechanisms behind the anti-inflammatory actions of insulin. Crit Rev Immunol. 2011;31(4):307-340.
  • 12. Monnier L, Hanefeld M, Schnell O, Colette C, Owens D. Insulin and at- herosclerosis: how are they related? Diabetes Metab. 2013;39:111-117.
  • 13. Valko M, Morris H, Cronin MT. Metals, toxicity and oxidative stress. Curr Med Chem. 2005;12(10):1161-1208.
  • 14. Cho JG, Song JJ, Choi J, Im GJ, Jung HH, Chae SW. The suppressive effects of metformin on inflammatory response of otitis media model in human middle ear epithelial cells. Int J Pediatr Otorhinolaryngol. 2016;89:28-32.
  • 15. Diniz Vilela D, Gomes Peixoto L, Teixeira RR, et al. The Role of Metfor- min in Controlling Oxidative Stress in Muscle of Diabetic Rats. Oxid Med Cell Longev. 2016;2016:6978625. 16. Kelly B, Tannahill GM, Murphy MP, O’Neill LA. Metformin inhibits the production of reactive oxygen species from NADH: ubiquinone oxi- doreductase to limit induction of interleukin-1β (IL-1β) and boosts interleukin-10 (IL10) in lipopolysaccharide (LPS)-activated macrop- hages. J Biol Chem. 2015;290(33):20348-59.
  • 17. Batchuluun B, Inoguchi T, Sonoda N, et al. Metformin and liraglutide ameliorate high glucose-induced oxidative stress via inhibition of PKC-NAD(P)H oxidase pathway in human aortic endothelial cells. At- herosclerosis. 2014;232(1):156-164.
  • 18. Markowicz-Piasecka M, Sikora J, Szydłowska A, Skupień A, Mikici- uk-Olasik E, Huttunen KM. Metformin - a Future Therapy for Neuro- degenerative Diseases : Theme: Drug Discovery, Development and Delivery in Alzheimer’s Disease Guest Editor: Davide Brambil- la. Pharm Res. 2017;34(12):2614-2627.
  • 19. Mummidi S, Das NA, Carpenter AJ, et al. Metformin inhibits aldoste- rone-induced cardiac fibroblast activation, migration and proliferati- on in vitro, and reverses aldosterone+salt-induced cardiac fibrosis in vivo. J Mol Cell Cardiol. 2016;98:95-102.
  • 20. Bułdak Ł, Łabuzek K, Bułdak RJ, et al. Metformin affects macropha- ges’ phenotype and improves the activity of glutathione peroxida- se, superoxide dismutase, catalase and decreases malondialdehyde concentration in a partially AMPK-independent manner in LPS-sti- mulated human monocytes/macrophages [published correction ap- pears in Pharmacol Rep. 2017 Jun;69(3):594] [published correction appears in Pharmacol Rep. 2019 Oct;71(5):981-982]. Pharmacol Rep. 2014;66(3):418-429.
  • 21. Dai J, Liu M, Ai Q, et al. Involvement of catalase in the protective be- nefits of metformin in mice with oxidative liver injury. Chem Biol In- teract. 2014;216:34-42.
  • 22. Kanazawa I, Yamaguchi T, Yano S, Yamauchi M, Sugimoto T. Metfor- min enhances the differentiation and mineralization of osteoblastic MC3T3-E1 cells via AMP kinase activation as well as eNOS and BMP-2 expression. Biochem Biophys Res Commun. 2008;375(3):414- 419.
  • 23. Cortizo AM, Sedlinsky C, McCarthy AD, Blanco A, Schurman L. Oste- ogenic actions of the anti-diabetic drug metformin on osteoblasts in culture. Eur J Pharmacol. 2006;536(1-2):38-46.
  • 24. Wang P, Ma T, Guo D, et al. Metformin induces osteoblastic differenti- ation of human induced pluripotent stem cell-derived mesenchymal stem cells. J Tissue Eng Regen Med. 2018;12(2):437-446.
  • 25. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Met- hods. 1983;65(1-2):55-63.
  • 26. Kumar P, Nagarajan A, Uchil PD: Analysis of Cell Viability by the MTT Assay. Cold Spring Harb Protoc 2018, 2018(6).
  • 27. Pradeep AR, Rao NS, Naik SB, Kumari M. Efficacy of varying concent- rations of subgingivally delivered metformin in the treatment of ch- ronic periodontitis: a randomized controlled clinical trial. J Periodon- tol. 2013;84(2):212-220.
  • 28. Pradeep AR, Nagpal K, Karvekar S, Patnaik K, Naik SB, Guruprasad CN. Platelet-rich fibrin with 1% metformin for the treatment of intra- bony defects in chronic periodontitis: a randomized controlled clini- cal trial. J Periodontol. 2015;86(6):729-737.
  • 29. Pradeep AR, Patnaik K, Nagpal K, Karvekar S, Guruprasad CN, Kuma- raswamy KM. Efficacy of 1% Metformin Gel in Patients With Moderate and Severe Chronic Periodontitis: A Randomized Controlled Clinical Trial. J Periodontol. 2017;88(10):1023-1029.
  • 30. Kurian IG, Dileep P, Ipshita S, Pradeep AR. Comparative evaluati- on of subgingivally-delivered 1% metformin and Aloe vera gel in the treatment of intrabony defects in chronic periodontitis pa- tients: A randomized, controlled clinical trial. J Investig Clin Dent. 2018;9(3):e12324
Toplam 29 adet kaynakça vardır.

Ayrıntılar

Birincil Dil İngilizce
Konular Periodontoloji
Bölüm Araştırma Makalesi
Yazarlar

Gökay Tekçam Bu kişi benim

Adil Başman Bu kişi benim

Emin Ümit Bağrıaçık Bu kişi benim

Necla Gündüz Bu kişi benim

Yayımlanma Tarihi 14 Temmuz 2023
Gönderilme Tarihi 21 Ekim 2022
Yayımlandığı Sayı Yıl 2023 Cilt: 33 Sayı: 2 - 2023, 33:2

Kaynak Göster

AMA Tekçam G, Başman A, Bağrıaçık EÜ, Gündüz N. In Vitro Evaluation of the Cytotoxicity of Metformin on Human Gingival Fibroblasts. Curr Res Dent Sci. Temmuz 2023;33(2):73-78. doi:10.5152/CRDS.2023.2270115

Current Research in Dental Sciences is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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