Bupivacaine at clinically relevant concentrations induces toxicity in human intervertebral disc cells via the induction of autophagy in vitro

Yang,Ge; Li,Zhuoyang; Mei,Haibo; Ye,Weihua; Huang,Shengxiang; Liu,Kun; Tan,Qian

doi:10.3892/mmr.2019.10279

Bupivacaine at clinically relevant concentrations induces toxicity in human intervertebral disc cells via the induction of autophagy in vitro

Authors:
- Ge Yang
- Zhuoyang Li
- Haibo Mei
- Weihua Ye
- Shengxiang Huang
- Kun Liu
- Qian Tan
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Affiliations: Department of Orthopedics, Hunan Children's Hospital, The Pediatric Academy of University of South China, Changsha, Hunan 410007, P.R. China, Department of Orthopedics, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China
Published online on: May 22, 2019 https://doi.org/10.3892/mmr.2019.10279
Pages: 837-843

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Abstract

It has been reported that bupivacaine, the most widely used local anesthetic to relieve discogenic back pain in clinical settings, is cytotoxic to intervertebral disc (IVD) cells in vitro; however, the precise mechanisms of cytotoxicity induced by bupivacaine remain unclear. Autophagy is an intracellular lysosomal degradation process that is important for cellular survival. The present study investigated the role of autophagy in the survival of IVD cells subjected to bupivacaine treatment. Human nucleus pulposus (NP) cells isolated from IVD cells were exposed to various concentrations of bupivacaine for 2, 6 and 12 h, and analyzed for cellular viability using MTT assay and western blotting. Additionally, autophagosome formation and autophagy‑associated biomarkers were evaluated by electron microscopy and western blotting to determine the autophagic activity and signaling alterations in NP cells under bupivacaine treatment. Furthermore, autophagic activity was inhibited in vitro using 3‑methyladenine to further analyze the association between autophagy and apoptosis in bupivacaine‑treated NP cells. Bupivacaine exhibited time‑ and dose‑dependent cytotoxic effects on human IVD cells at clinically relevant concentrations. Bupivacaine increased autophagic activity by promoting autophagosome formation, and LC3‑II and Beclin‑1 production. Additionally, bupivacaine inhibited protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/S6 kinase (S6K) signaling, which is a negative regulator of autophagic activity. Of note, pharmacological inhibition of autophagy alleviated bupivacaine‑induced cytotoxicity of IVD cells. The findings indicated that application of clinically relevant concentrations of bupivacaine upregulated autophagic activity via inhibition of Akt/mTOR/S6K signaling. In addition, the inhibition of autophagic activation served as a protective mechanism against bupivacaine‑induced cytotoxicity. Collectively, these findings may provide novel insight into the mechanisms underlying cytotoxicity induced by bupivacaine when controlling spine‑associated pain.

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1	Deyo RA and Weinstein JN: Low back pain. N Engl J Med. 344:363–370. 2001. View Article : Google Scholar : PubMed/NCBI
2	de Schepper EI, Damen J, van Meurs JB, Ginai AZ, Popham M, Hofman A, Koes BW and Bierma-Zeinstra SM: The association between lumbar disc degeneration and low back pain: The influence of age, gender, and individual radiographic features. Spine (Phila Pa 1976). 35:531–536. 2010. View Article : Google Scholar : PubMed/NCBI
3	Friedly J, Chan L and Deyo R: Increases in lumbosacral injections in the Medicare population: 1994 to 2001. Spine (Phila Pa 1976). 32:1754–1760. 2007. View Article : Google Scholar : PubMed/NCBI
4	Kotilainen E, Muittari P and Kirvelä O: Intradiscal glycerol or bupivacaine in the treatment of low back pain. Acta Neurochir (Wien). 139:541–545. 1997. View Article : Google Scholar : PubMed/NCBI
5	Ohtori S, Inoue G, Orita S, Eguchi Y, Ochiai N, Kishida S, Kuniyoshi K, Nakamura J, Aoki Y, Ishikawa T, et al: No acceleration of intervertebral disc degeneration after a single injection of bupivacaine in young age group with follow-up of 5 years. Asian Spine J. 7:212–217. 2013. View Article : Google Scholar : PubMed/NCBI
6	Huang YH, Tsai PS and Huang CJ: Bupivacaine inhibits COX-2 expression, PGE2, and cytokine production in endotoxin-activated macrophages. Acta Anaesthesiol Scand. 52:530–535. 2008. View Article : Google Scholar : PubMed/NCBI
7	Yanagidate F and Strichartz GR: Bupivacaine inhibits activation of neuronal spinal extracellular receptor-activated kinase through selective effects on ionotropic receptors. Anesthesiology. 104:805–814. 2006. View Article : Google Scholar : PubMed/NCBI
8	Iwasaki K, Sudo H, Yamada K, Ito M and Iwasaki N: Cytotoxic effects of the radiocontrast agent iotrolan and anesthetic agents bupivacaine and lidocaine in three-dimensional cultures of human intervertebral disc nucleus pulposus cells: Identification of the apoptotic pathways. PLoS One. 9:e924422014. View Article : Google Scholar : PubMed/NCBI
9	Quero L, Klawitter M, Nerlich AG, Leonardi M, Boos N and Wuertz K: Bupivacaine-the deadly friend of intervertebral disc cells? Spine J. 11:46–53. 2011. View Article : Google Scholar : PubMed/NCBI
10	Cai XY, Xiong LM, Yang SH, Shao ZW, Xie M, Gao F and Ding F: Comparison of toxicity effects of ropivacaine, bupivacaine, and lidocaine on rabbit intervertebral disc cells in vitro. Spine J. 14:483–490. 2014. View Article : Google Scholar : PubMed/NCBI
11	Hale AN, Ledbetter DJ, Gawriluk TR and Rucker EB III: Autophagy: Regulation and role in development. Autophagy. 9:951–972. 2013. View Article : Google Scholar : PubMed/NCBI
12	Ma KG, Shao ZW, Yang SH, Wang J, Wang BC, Xiong LM, Wu Q and Chen SF: Autophagy is activated in compression-induced cell degeneration and is mediated by reactive oxygen species in nucleus pulposus cells exposed to compression. Osteoarthritis Cartilage. 21:2030–2038. 2013. View Article : Google Scholar : PubMed/NCBI
13	Li R, Ma H, Zhang X, Li C, Xiong J, Lu T, Mao Y, Dai J, Liu L and Ding Z: Impaired autophagosome clearance contributes to local anesthetic bupivacaine-induced myotoxicity in mouse myoblasts. Anesthesiology. 122:595–605. 2015. View Article : Google Scholar : PubMed/NCBI
14	Alers S, Löffler AS, Wesselborg S and Stork B: Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: Cross talk, shortcuts, and feedbacks. Mol Cell Biol. 32:2–11. 2012. View Article : Google Scholar : PubMed/NCBI
15	Yang GE, Duan X, Lin D, Li T, Luo D, Wang L and Lian K: Rapamycin-induced autophagy activity promotes bone fracture healing in rats. Exp Ther Med. 10:1327–1333. 2015. View Article : Google Scholar : PubMed/NCBI
16	Beigh MA, Showkat M, Bashir B, Bashir A, Hussain Mu and Andrabi KI: Growth inhibition by bupivacaine is associated with inactivation of ribosomal protein S6 kinase 1. Biomed Res Int. 2014:8318452014. View Article : Google Scholar : PubMed/NCBI
17	Pfirrmann CW, Metzdorf A, Zanetti M, Hodler J and Boos N: Magnetic resonance classification of lumbar intervertebral disc degeneration. Spine (Phila Pa 1976). 26:1873–1878. 2001. View Article : Google Scholar : PubMed/NCBI
18	Hou H, Zhang Y, Huang Y, Yi Q, Lv L, Zhang T, Chen D, Hao Q and Shi Q: Inhibitors of phosphatidylinositol 3′-kinases promote mitotic cell death in HeLa cells. PLoS One. 7:e356652012. View Article : Google Scholar : PubMed/NCBI
19	Lee H, Sowa G, Vo N, Vadala G, O'Connell S, Studer R and Kang J: Effect of bupivacaine on intervertebral disc cell viability. Spine J. 10:159–166. 2010. View Article : Google Scholar : PubMed/NCBI
20	Roelofs PD, Deyo RA, Koes BW, Scholten RJ and van Tulder MW: Nonsteroidal anti-inflammatory drugs for low back pain: An updated Cochrane review. Spine (Phila Pa 1976). 33:1766–1774. 2008. View Article : Google Scholar : PubMed/NCBI
21	Gump JM and Thorburn A: Autophagy and apoptosis: What is the connection? Trends Cell Biol. 21:387–392. 2011. View Article : Google Scholar : PubMed/NCBI
22	Jain MV, Paczulla AM, Klonisch T, Dimgba FN, Rao SB, Roberg K, Schweizer F, Lengerke C, Davoodpour P, Palicharla VR, et al: Interconnections between apoptotic, autophagic and necrotic pathways: Implications for cancer therapy development. J Cell Mol Med. 17:12–29. 2013. View Article : Google Scholar : PubMed/NCBI
23	Chaabane W, User SD, El-Gazzah M, Jaksik R, Sajjadi E, Rzeszowska-Wolny J and Los MJ: Autophagy, apoptosis, mitoptosis and necrosis: Interdependence between those pathways and effects on cancer. Arch Immunol Ther Exp (Warsz). 61:43–58. 2013. View Article : Google Scholar : PubMed/NCBI
24	Matsunaga K, Saitoh T, Tabata K, Omori H, Satoh T, Kurotori N, Maejima I, Shirahama-Noda K, Ichimura T, Isobe T, et al: Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages. Nat Cell Biol. 11:385–396. 2009. View Article : Google Scholar : PubMed/NCBI
25	Afanas'ev I: Signaling and damaging functions of free radicals in aging-free radical theory, hormesis, and TOR. Aging Dis. 1:75–88. 2010.PubMed/NCBI
26	Farrand L, Oh SW, Song YS and Tsang BK: Phytochemicals: A multitargeted approach to gynecologic cancer therapy. Biomed Res Int. 2014:8901412014. View Article : Google Scholar : PubMed/NCBI
27	Lv D, Bai Z, Yang L, Li X and Chen X: Lipid emulsionreverses bupivacaine-induced apoptosis of h9c2 cardiomyocytes: PI3K/Akt/GSK-3β signaling pathway. Environ Toxicol Pharmacol. 42:85–91. 2016. View Article : Google Scholar : PubMed/NCBI
28	Fan YL, Li HC, Zhao W, Peng HH, Huang F, Jiang WH and Xu SY: Curcumin attenuated bupivacaine-induced neurotoxicity in SH-SY5Y cells via activation of the Akt signaling pathway. Neurochem Res. 41:2425–2432. 2016. View Article : Google Scholar : PubMed/NCBI
29	Jiang L, Jin Y, Wang H, Jiang Y and Dong J: Glucosamine protects nucleus pulposus cells and induces autophagy via the mTOR-dependent pathway. J Orthop Res. 32:1532–1542. 2014. View Article : Google Scholar : PubMed/NCBI
30	Hsin IL, Sheu GT, Jan MS, Sun HL, Wu TC, Chiu LY, Lue KH and Ko JL: Inhibition of lysosome degradation on autophagosome formation and responses to GMI, an immunomodulatory protein from Ganoderma microsporum. Br J Pharmacol. 167:1287–1300. 2012. View Article : Google Scholar : PubMed/NCBI