pp 1–11 | Cite as

Chloroquine and 3-Methyladenine Attenuates Periodontal Inflammation and Bone Loss in Experimental Periodontitis

  • Shasha He
  • Qian Zhou
  • Binyan Luo
  • Bin Chen
  • Lingjun Li
  • Fuhua YanEmail author
Original Article


Periodontitis is an inflammation characterized by alveolar bone resorption caused by imbalance in bone homeostasis. It is known that autophagy is related to inflammation and bone metabolism. However, whether autophagy inhibitors could be used for periodontitis in animal models remains unknown. We investigated the role of two classical autophagy inhibitors, 3-methyladenine (3-MA) and chloroquine (CQ), on the development of rat experimental periodontitis in terms of the bone loss (micro-CT), the number of inflammatory cells (hematoxylin and eosin staining), and the osteoclastic activity (tartrate-resistant acid phosphatase staining). Expression of autophagy-related genes and nuclear factor kappa B p65 (NF-κB p65) were assessed by immunohistochemistry. Expression of Beclin-1 and microtubule-associated proteins 1A/1B light chain 3 (LC3) were analyzed by Western blot. To further observe the effect of autophagy inhibitors on osteoclasts (OCs) in vitro, bone marrow–derived mononuclear macrophages were used. Together, these findings indicated that topical administration of 3-MA or CQ reduced the infiltration of inflammatory cells and alveolar bone resorption in experimental periodontitis. Furthermore, 3-MA and CQ may attenuate activation of OCs by autophagy. Therefore, 3MA and CQ may have prophylactic and therapeutic potential for inflammation and alveolar bone resorption in periodontitis in the future.


experimental periodontitis autophagy chloroquine 3-methyladenine osteoclasts 



The authors thank Central Laboratory of Stomatology, Nanjing Stomatological Hospital, Medical School of Nanjing University.

Funding Information

This study was financially supported by the National Natural Science Foundation Project (No. 81400521, 81771078) and the Nanjing Medical Science and Technique Development Foundation (QRX17177).

Compliance with Ethical Standards

All experimental procedures described in this study have been approved by the Animal Ethics Committee of Nanjing University and were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals.

Conflict of Interest

The authors declare that they have no conflicts of interest.


  1. 1.
    Hajishengallis, G. 2015. Periodontitis: from microbial immune subversion to systemic inflammation. Nature Reviews Immunology 15 (1): 30–44. Scholar
  2. 2.
    Pihlstrom, B.L., B.S. Michalowicz, and N.W. Johnson. 2005. Periodontal diseases. Lancet 366 (9499): 1809–1820. Scholar
  3. 3.
    Schwartz, Z., J. Goultschin, D.D. Dean, and B.D. Boyan. 1997. Mechanisms of alveolar bone destruction in periodontitis. Periodontol 2000 (14): 158–172.CrossRefGoogle Scholar
  4. 4.
    Boyle, W.J., W.S. Simonet, and D.L. Lacey. 2003. Osteoclast differentiation and activation. Nature 423 (6937): 337–342. Scholar
  5. 5.
    Kobayashi, Y., S. Uehara, M. Koide, and N. Takahashi. 2015. The regulation of osteoclast differentiation by Wnt signals. BoneKEy Reports 4: 713. Scholar
  6. 6.
    Zhang, L., Y.F. Guo, Y.Z. Liu, Y.J. Liu, D.H. Xiong, X.G. Liu, L. Wang, T.L. Yang, S.F. Lei, Y. Guo, H. Yan, Y.F. Pei, F. Zhang, C.J. Papasian, R.R. Recker, and H.W. Deng. 2010. Pathway-based genome-wide association analysis identified the importance of regulation-of-autophagy pathway for ultradistal radius BMD. Journal of Bone and Mineral Research 25 (7): 1572–1580. Scholar
  7. 7.
    Levine, B., and G. Kroemer. 2008. Autophagy in the pathogenesis of disease. Cell 132 (1): 27–42. Scholar
  8. 8.
    Bullon, P., M.D. Cordero, J.L. Quiles, C. Ramirez-Tortosa Mdel, A. Gonzalez-Alonso, S. Alfonsi, R. Garcia-Marin, M. de Miguel, and M. Battino. 2012. Autophagy in periodontitis patients and gingival fibroblasts: unraveling the link between chronic diseases and inflammation. BMC Medicine 10: 122. Scholar
  9. 9.
    Lin, N.Y., C. Beyer, A. Giessl, T. Kireva, C. Scholtysek, S. Uderhardt, L.E. Munoz, C. Dees, A. Distler, S. Wirtz, G. Krönke, B. Spencer, O. Distler, G. Schett, and J.H. Distler. 2013. Autophagy regulates TNFalpha-mediated joint destruction in experimental arthritis. Annals of the Rheumatic Diseases 72 (5): 761–768. Scholar
  10. 10.
    Cejka, D., S. Hayer, B. Niederreiter, W. Sieghart, T. Fuereder, J. Zwerina, and G. Schett. 2010. Mammalian target of rapamycin signaling is crucial for joint destruction in experimental arthritis and is activated in osteoclasts from patients with rheumatoid arthritis. Arthritis and Rheumatism 62 (8): 2294–2302. Scholar
  11. 11.
    Ti, Y., L. Zhou, R. Wang, and J. Zhao. 2015. Inhibition of microtubule dynamics affects podosome belt formation during osteoclast induction. Cell Biochemistry and Biophysics 71 (2): 741–747. Scholar
  12. 12.
    Kim, W.K., K. Ke, O.J. Sul, H.J. Kim, S.H. Kim, M.H. Lee, H.J. Kim, S.Y. Kim, H.T. Chung, and H.S. Choi. 2011. Curcumin protects against ovariectomy-induced bone loss and decreases osteoclastogenesis. Journal of Cellular Biochemistry 112 (11): 3159–3166. Scholar
  13. 13.
    Petiot, A., E. Ogier-Denis, E.F. Blommaart, A.J. Meijer, and P. Codogno. 2000. Distinct classes of phosphatidylinositol 3’-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. The Journal of Biological Chemistry 275 (2): 992–998. Scholar
  14. 14.
    Ito, S., N. Koshikawa, S. Mochizuki, and K. Takenaga. 2007. 3-Methyladenine suppresses cell migration and invasion of HT1080 fibrosarcoma cells through inhibiting phosphoinositide 3-kinases independently of autophagy inhibition. International Journal of Oncology 31 (2): 261–268.PubMedGoogle Scholar
  15. 15.
    Yang, A., N.V. Rajeshkumar, X. Wang, S. Yabuuchi, B.M. Alexander, G.C. Chu, D.D. Von Hoff, A. Maitra, and A.C. Kimmelman. 2014. Autophagy is critical for pancreatic tumor growth and progression in tumors with p53 alterations. Cancer Discovery 4 (8): 905–913. Scholar
  16. 16.
    Ke, X., L. Lei, H. Li, H. Li, and F. Yan. 2016. Manipulation of necroptosis by Porphyromonas gingivalis in periodontitis development. Molecular Immunology 77: 8–13. Scholar
  17. 17.
    Bugueno, I.M., F. Batool, L. Korah, N. Benkirane-Jessel, and O. Huck. 2018. Porphyromonas gingivalis differentially modulates apoptosome apoptotic peptidase activating factor 1 in epithelial cells and fibroblasts. The American Journal of Pathology 188 (2): 404–416. Scholar
  18. 18.
    Wada-Mihara, C., H. Seto, H. Ohba, K. Tokunaga, J.I. Kido, T. Nagata, and K. Naruishi. 2018. Local administration of calcitonin inhibits alveolar bone loss in an experimental periodontitis in rats. Biomedicine & Pharmacotherapy 97: 765–770. Scholar
  19. 19.
    Ni, C., J. Zhou, N. Kong, T. Bian, Y. Zhang, X. Huang, Y. Xiao, W. Yang, and F. Yan. 2019. Gold nanoparticles modulate the crosstalk between macrophages and periodontal ligament cells for periodontitis treatment. Biomaterials 206: 115–132. Scholar
  20. 20.
    Wu, Y.H., R. Kuraji, Y. Taya, H. Ito, and Y. Numabe. 2018. Effects of theaflavins on tissue inflammation and bone resorption on experimental periodontitis in rats. Journal of Periodontal Research 53 (6): 1009–1019. Scholar
  21. 21.
    Yang, D., R. Liu, L. Liu, H. Liao, C. Wang, and Z. Cao. 2017. Involvement of CD147 in alveolar bone remodeling and soft tissue degradation in experimental periodontitis. Journal of Periodontal Research 52 (4): 704–712. Scholar
  22. 22.
    Gyongyosi, A., K. Szoke, F. Fenyvesi, Z. Fejes, I.B. Debreceni, B. Nagy Jr., A. Tosaki, and I. Lekli. 2019. Inhibited autophagy may contribute to heme toxicity in cardiomyoblast cells. Biochemical and Biophysical Research Communications 511 (4): 732–738. Scholar
  23. 23.
    Bostanci, N., and G.N. Belibasakis. 2012. Porphyromonas gingivalis: an invasive and evasive opportunistic oral pathogen. FEMS Microbiology Letters 333 (1): 1–9. Scholar
  24. 24.
    Zhou, R., L. Shen, C. Yang, L. Wang, H. Guo, P. Yang, and A. Song. 2018. Periodontitis may restrain the mandibular bone healing via disturbing osteogenic and osteoclastic balance. Inflammation 41 (3): 972–983. Scholar
  25. 25.
    Tong, X., J. Gu, R. Song, D. Wang, Z. Sun, C. Sui, C. Zhang, X. Liu, J. Bian, and Z. Liu. 2018. Osteoprotegerin inhibit osteoclast differentiation and bone resorption by enhancing autophagy via AMPK/mTOR/p70S6K signaling pathway in vitro. Journal of Cellular Biochemistry. Scholar
  26. 26.
    Sul, O.J., H.J. Park, H.J. Son, and H.S. Choi. 2017. Lipopolysaccharide (LPS)-induced autophagy is responsible for enhanced osteoclastogenesis. Molecular Cell 40 (11): 880–887. Scholar
  27. 27.
    Giampieri, F., S. Afrin, T.Y. Forbes-Hernandez, M. Gasparrini, D. Cianciosi, P. Reboredo-Rodriguez, A. Varela-Lopez, J.L. Quiles, and M. Battino. 2019. Autophagy in human health and disease: novel therapeutic opportunities. Antioxidants & Redox Signaling 30 (4): 577–634. Scholar
  28. 28.
    An, Y., W. Liu, P. Xue, Y. Zhang, Q. Wang, and Y. Jin. 2016. Increased autophagy is required to protect periodontal ligament stem cells from apoptosis in inflammatory microenvironment. Journal of Clinical Periodontology 43 (7): 618–625. Scholar
  29. 29.
    Carloni, S., S. Girelli, C. Scopa, G. Buonocore, M. Longini, and W. Balduini. 2010. Activation of autophagy and Akt/CREB signaling play an equivalent role in the neuroprotective effect of rapamycin in neonatal hypoxia-ischemia. Autophagy 6 (3): 366–377. Scholar
  30. 30.
    Morgan, M.J., G. Gamez, C. Menke, A. Hernandez, J. Thorburn, F. Gidan, L. Staskiewicz, S. Morgan, C. Cummings, P. Maycotte, and A. Thorburn. 2014. Regulation of autophagy and chloroquine sensitivity by oncogenic RAS in vitro is context-dependent. Autophagy 10 (10): 1814–1826. Scholar
  31. 31.
    Zou, Y., Y.H. Ling, J. Sironi, E.L. Schwartz, R. Perez-Soler, and B. Piperdi. 2013. The autophagy inhibitor chloroquine overcomes the innate resistance of wild-type EGFR non-small-cell lung cancer cells to erlotinib. Journal of Thoracic Oncology 8 (6): 693–702. Scholar
  32. 32.
    Shin, H., S. Choi, and H.J. Lim. 2014. Relationship between reactive oxygen species and autophagy in dormant mouse blastocysts during delayed implantation. Clinical and Experimental Reproductive Medicine 41 (3): 125–131. Scholar
  33. 33.
    Zhao, Y., G. Chen, W. Zhang, N. Xu, J.Y. Zhu, J. Jia, Z.J. Sun, Y.N. Wang, and Y.F. Zhao. 2012. Autophagy regulates hypoxia-induced osteoclastogenesis through the HIF-1alpha/BNIP3 signaling pathway. Journal of Cellular Physiology 227 (2): 639–648. Scholar
  34. 34.
    Wu, Y.T., H.L. Tan, G. Shui, C. Bauvy, Q. Huang, M.R. Wenk, C.N. Ong, P. Codogno, and H.M. Shen. 2010. Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. The Journal of Biological Chemistry 285 (14): 10850–10861. Scholar
  35. 35.
    Lin, N.Y., A. Stefanica, and J.H. Distler. 2013. Autophagy: a key pathway of TNF-induced inflammatory bone loss. Autophagy 9 (8): 1253–1255. Scholar
  36. 36.
    Hou, C.H., Y.C. Fong, and C.H. Tang. 2011. HMGB-1 induces IL-6 production in human synovial fibroblasts through c-Src, Akt and NF-kappaB pathways. Journal of Cellular Physiology 226 (8): 2006–2015. Scholar
  37. 37.
    Min, Y., M.J. Kim, S. Lee, E. Chun, and K.Y. Lee. 2018. Inhibition of TRAF6 ubiquitin-ligase activity by PRDX1 leads to inhibition of NFKB activation and autophagy activation. Autophagy 14 (8): 1347–1358. Scholar
  38. 38.
    Levine, B., M. Packer, and P. Codogno. 2015. Development of autophagy inducers in clinical medicine. The Journal of Clinical Investigation 125 (1): 14–24. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Periodontology, Nanjing Stomatological HospitalMedical School of Nanjing UniversityNanjingChina
  2. 2.Department of Endodontics, Nanjing Stomatological HospitalMedical School of Nanjing UniversityNanjingChina

Personalised recommendations