Background & Aims

Bone cancer pain (BCP) is a debilitating illness that negatively impacted the living quality of patients with advanced-stage cancer. Remimazolam, a completely novel type of benzodiazepine sedative with ultra-short half-life, has yet to be proven to have a pain relief effect. Here, we investigated whether remimazolam has a relieving effect on rats BCP and its associated peripheral pathways.

Methods

Remimazolam was administered intraperitoneally 14 days post-inoculation of rat mammary carcinoma cells into the tibial medullary cavity?followed by the assessment of pain indicators. The interaction network of disease and drug targets was built using a network pharmacology technique. Thereafter, CytoHubba was used to discover hub genes through studies of GO and KEGG profiles based on shared targets of remimazolam and BCP. Validation of these genes was accomplished via molecular docking, Western blotting, and immunofluorescent staining.

Results

Initial results verified the pain-alleviating effects of remimazolam in a BCP rat model. The GO and KEGG analyses pinpointed anti-BCP targets mainly within the signaling pathway of phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) . Concurrently, molecular docking suggested high binding affinities between remimazolam and major constituents of the PI3K-Akt signal pathway – EGFR, PI3K, Akt, mTOR, Bcl-2, and Caspase-3. Moreover, remimazolam significantly downregulated the expression of these BCP-associated proteins in the dorsal root ganglion (DRG).

Conclusions

This study identifies and verifies the peripheral PI3K-Akt signaling pathway as an essential component in treating BCP with remimazolam by fusing network pharmacology with experimental validation.

References

[1] J.M. Jimenez-Andrade, W.G. Mantyh, A.P. Bloom, A.S. Ferng, C.P. Geffre, P.W. Mantyh, Bone cancer pain, Ann N Y Acad Sci 1198 (2010) 173-81.
[2] D.R. Clohisy, P.W. Mantyh, Bone cancer pain, Clin OrthopRelat Res (415 Suppl) (2003) S279-88.
[3] K. Wang, C.R. Donnelly, C. Jiang, Y. Liao, X. Luo, X. Tao, S. Bang, A. McGinnis, M. Lee, M.J. Hilton, R.R. Ji, STING suppresses bone cancer pain via immune and neuronal modulation, Nat Commun 12(1) (2021) 4558.
[4] X.Q. Zheng, Y.H. Wu, J.F. Huang, A.M. Wu, Neurophysiological mechanisms of cancer-induced bone pain, J Adv Res 35 (2022) 117-127.
[5] Y. Huh, R.R. Ji, G. Chen, Neuroinflammation, Bone Marrow Stem Cells, and Chronic Pain, Front Immunol 8 (2017) 1014.
[6] E.J. Chapman, Z. Edwards, J.W. Boland, M. Maddocks, L. Fettes, C. Malia, M.R. Mulvey, M.I. Bennett, Practice review: Evidence-based and effective management of pain in patients with advanced cancer, Palliat Med 34(4) (2020) 444-453.
[7] S.J. Keam, Remimazolam: First Approval, Drugs 80(6) (2020) 625-633.
[8] Q. Hu, X. Liu, C. Wen, D. Li, X. Lei, Remimazolam: An Updated Review of a New Sedative and Anaesthetic, Drug Des DevelTher 16 (2022) 3957-3974.
[9] G.J. Kilpatrick, M.S. McIntyre, R.F. Cox, J.A. Stafford, G.J. Pacofsky, G.G. Lovell, R.P. Wiard, P.L. Feldman, H. Collins, B.L. Waszczak, G.S. Tilbrook, CNS 7056: a novel ultra-short-acting Benzodiazepine, Anesthesiology 107(1) (2007) 60-6.
[10] H. Xie, F. Lu, W. Liu, E. Wang, L. Wang, M. Zhong, Remimazolam alleviates neuropathic pain via regulating bradykinin receptor B1 and autophagy, J Pharm Pharmacol 73(12) (2021) 1643-1651.
[11] H. Fang, Y. Zhang, J. Wang, L. Li, S. An, Q. Huang, Z. Chen, H. Yang, J. Wu, Z. Zeng, Remimazolam reduces sepsis-associated acute liver injury by activation of peripheral benzodiazepine receptors and p38 inhibition of macrophages, Int Immunopharmacol 101(Pt B) (2021) 108331.
[12] M. Shi, J. Chen, T. Liu, W. Dai, Z. Zhou, L. Chen, Y. Xie, Protective Effects of Remimazolam on Cerebral Ischemia/Reperfusion Injury in Rats by Inhibiting of NLRP3 Inflammasome-Dependent Pyroptosis, Drug Des DevelTher 16 (2022) 413-423.
[13] X. Liu, S. Lin, Y. Zhong, J. Shen, X. Zhang, S. Luo, L. Huang, L. Zhang, S. Zhou, J. Tang, Remimazolam Protects Against LPS-Induced Endotoxicity Improving Survival of Endotoxemia Mice, Front Pharmacol 12 (2021) 739603.
[14] W. Xu, J. Hu, W. Liu, Q. Zhu, X. Gong, P. Zhu, X. Yang, R. Xia, R. Xue, Remimazolan inhibits glioma cell growth and induces apoptosis through down-regulation of NF-?B pathway, IUBMB Life 73(2) (2021) 341-348.
[15] C. Nogales, Z.M. Mamdouh, M. List, C. Kiel, A.I. Casas, H. Schmidt, Network pharmacology: curing causal mechanisms instead of treating symptoms, Trends Pharmacol Sci 43(2) (2022) 136-150.
[16] X. Li, S. Wei, S. Niu, X. Ma, H. Li, M. Jing, Y. Zhao, Network pharmacology prediction and molecular docking-based strategy to explore the potential mechanism of HuanglianJiedu Decoction against sepsis, Comput Biol Med 144 (2022) 105389.
[17] L. Pinzi, G. Rastelli, Molecular Docking: Shifting Paradigms in Drug Discovery, Int J Mol Sci 20(18) (2019).
[18] L.G. Ferreira, R.N. Dos Santos, G. Oliva, A.D. Andricopulo, Molecular docking and structure-based drug design strategies, Molecules 20(7) (2015) 13384-421.
[19] X.T. He, X.F. Hu, C. Zhu, K.X. Zhou, W.J. Zhao, C. Zhang, X. Han, C.L. Wu, Y.Y. Wei, W. Wang, J.P. Deng, F.M. Chen, Z.X. Gu, Y.L. Dong, Suppression of histone deacetylases by SAHA relieves bone cancer pain in rats via inhibiting activation of glial cells in spinal dorsal horn and dorsal root ganglia, J Neuroinflammation 17(1) (2020) 125.
[20] X.F. Hu, X.T. He, K.X. Zhou, C. Zhang, W.J. Zhao, T. Zhang, J.L. Li, J.P. Deng, Y.L. Dong, The analgesic effects of triptolide in the bone cancer pain rats via inhibiting the upregulation of HDACs in spinal glial cells, J Neuroinflammation 14(1) (2017) 213.
[21] C. Yang, F. Kang, X. Huang, W. Zhang, S. Wang, M. Han, Z. Zhang, J. Li, Melatonin attenuates bone cancer pain via the SIRT1/HMGB1 pathway, Neuropharmacology 220 (2022) 109254.
[22] B. Remeniuk, D. Sukhtankar, A. Okun, E. Navratilova, J.Y. Xie, T. King, F. Porreca, Behavioral and neurochemical analysis of ongoing bone cancer pain in rats, Pain 156(10) (2015) 1864-1873.
[23] Y. Wang, S.H. Bryant, T. Cheng, J. Wang, A. Gindulyte, B.A. Shoemaker, P.A. Thiessen, S. He, J. Zhang, PubChem BioAssay: 2017 update, Nucleic Acids Res 45(D1) (2017) D955-d963.
[24] D. Gfeller, A. Grosdidier, M. Wirth, A. Daina, O. Michielin, V. Zoete, SwissTargetPrediction: a web server for target prediction of bioactive small molecules, Nucleic Acids Res 42(Web Server issue) (2014) W32-8.
[25] X. Wang, Y. Shen, S. Wang, S. Li, W. Zhang, X. Liu, L. Lai, J. Pei, H. Li, PharmMapper 2017 update: a web server for potential drug target identification with a comprehensive target pharmacophore database, Nucleic Acids Res 45(W1) (2017) W356-w360.
[26] Y. Wei, S. Zhang, S. Shang, B. Zhang, S. Li, X. Wang, F. Wang, J. Su, Q. Wu, H. Liu, Y. Zhang, SEA: a super-enhancer archive, Nucleic Acids Res 44(D1) (2016) D172-9.
[27] Y. Wang, S. Zhang, F. Li, Y. Zhou, Y. Zhang, Z. Wang, R. Zhang, J. Zhu, Y. Ren, Y. Tan, C. Qin, Y. Li, X. Li, Y. Chen, F. Zhu, Therapeutic target database 2020: enriched resource for facilitating research and early development of targeted therapeutics, Nucleic Acids Res 48(D1) (2020) D1031-d1041.
[28] J.S. Amberger, A. Hamosh, Searching Online Mendelian Inheritance in Man (OMIM): A Knowledgebase of Human Genes and Genetic Phenotypes, CurrProtoc Bioinformatics 58 (2017) 1.2.1-1.2.12.
[29] M. Safran, I. Dalah, J. Alexander, N. Rosen, T. Iny Stein, M. Shmoish, N. Nativ, I. Bahir, T. Doniger, H. Krug, A. Sirota-Madi, T. Olender, Y. Golan, G. Stelzer, A. Harel, D. Lancet, GeneCards Version 3: the human gene integrator, Database (Oxford) 2010 (2010) baq020.
[30] P. Shannon, A. Markiel, O. Ozier, N.S. Baliga, J.T. Wang, D. Ramage, N. Amin, B. Schwikowski, T. Ideker, Cytoscape: a software environment for integrated models of biomolecular interaction networks, Genome Res 13(11) (2003) 2498-504.
[31] UniProt: the universal protein knowledgebase in 2021, Nucleic Acids Res 49(D1) (2021) D480-d489.
[32] A. Jia, L. Xu, Y. Wang, Venn diagrams in bioinformatics, Brief Bioinform 22(5) (2021).
[33] D. Szklarczyk, A.L. Gable, K.C. Nastou, D. Lyon, R. Kirsch, S. Pyysalo, N.T. Doncheva, M. Legeay, T. Fang, P. Bork, L.J. Jensen, C. von Mering, The STRING database in 2021: customizable protein-protein networks, and functional characterization of user-uploaded gene/measurement sets, Nucleic Acids Res 49(D1) (2021) D605-d612.
[34] C.H. Chin, S.H. Chen, H.H. Wu, C.W. Ho, M.T. Ko, C.Y. Lin, cytoHubba: identifying hub objects and sub-networks from complex interactome, BMC Syst Biol 8 Suppl 4(Suppl 4) (2014) S11.
[35] W. Xie, G. Sun, J. Zhu, H. Wang, Z. Han, P. Wang, Anti-POSTN and Anti-TIMP1 Autoantibodies as Diagnostic Markers in Esophageal Squamous Cell Carcinoma, Front Genet 13 (2022) 860611.
[36] Y. Zhou, B. Zhou, L. Pache, M. Chang, A.H. Khodabakhshi, O. Tanaseichuk, C. Benner, S.K. Chanda, Metascape provides a biologist-oriented resource for the analysis of systems-level datasets, Nat Commun 10(1) (2019) 1523.
[37] T.R. Helgren, T.J. Hagen, Demonstration of AutoDock as an Educational Tool for Drug Discovery, J Chem Educ 94(3) (2017) 345-349.
[38] C. Gorgulla, S.S. Ç?naro?lu, P.D. Fischer, K. Fackeldey, G. Wagner, H. Arthanari, VirtualFlow Ants-Ultra-Large Virtual Screenings with Artificial Intelligence Driven Docking Algorithm Based on Ant Colony Optimization, Int J Mol Sci 22(11) (2021).
[39] D. Seeliger, B.L. de Groot, Ligand docking and binding site analysis with PyMOL and Autodock/Vina, J Comput Aided Mol Des 24(5) (2010) 417-22.
[40] Y. Zhang, W. Wang, Z. Gong, Y. Peng, X. Li, Z. Zhang, X. Zhang, X. You, J. Wu, Activation of the STING pathway induces peripheral sensitization via neuroinflammation in a rat model of bone cancer pain, Inflamm Res 72(1) (2023) 117-132.
[41] G.D. Panos, F.M. Boeckler, Statistical Analysis in Clinical and Experimental Medical Research: Simplified Guidance for Authors and Reviewers, Drug Des Dev Ther 17 (2023) 1959-1961.
[42] V. Bonalume, L. Caffino, L.F. Castelnovo, A. Faroni, S. Liu, J. Hu, M. Milanese, G. Bonanno, K. Sohns, T. Hoffmann, R. De Col, M. Schmelz, F. Fumagalli, V. Magnaghi, R. Carr, Axonal GABA(A) stabilizes excitability in unmyelinated sensory axons secondary to NKCC1 activity, J Physiol 599(17) (2021) 4065-4084.
[43] Y.E. De la Luz-Cuellar, E.J. Rodríguez-Palma, Ú. Franco-Enzástiga, A.B. Salinas-Abarca, R. Delgado-Lezama, V. Granados-Soto, Blockade of spinal ?(5)-GABA(A) receptors differentially reduces reserpine-induced fibromyalgia-type pain in female rats, Eur J Pharmacol 858 (2019) 172443.
[44] Y.E. De la Luz-Cuellar, E.J. Rodríguez-Palma, Ú. Franco-Enzástiga, A.B. Salinas-Abarca, R. Delgado-Lezama, V. Granados-Soto, Blockade of spinal ?(5)-GABA(A) receptors differentially reduces reserpine-induced fibromyalgia-type pain in female rats, Eur J Pharmacol 858 (2019) 172443.
[45] S.J.F. Cronin, S. Rao, M.A. Tejada, B.L. Turnes, S. Licht-Mayer, T. Omura, C. Brenneis, E. Jacobs, L. Barrett, A. Latremoliere, N. Andrews, K.M. Channon, A. Latini, A.C. Arvanites, L.S. Davidow, M. Costigan, L.L. Rubin, J.M. Penninger, C.J. Woolf, Phenotypic drug screen uncovers the metabolic GCH1/BH4 pathway as key regulator of EGFR/KRAS-mediated neuropathic pain and lung cancer, Sci Transl Med 14(660) (2022) eabj1531.
[46] J. Wei, W. Su, Y. Zhao, Z. Wei, Y. Hua, P. Xue, X. Zhu, Y. Chen, G. Chen, Maresin 1 promotes nerve regeneration and alleviates neuropathic pain after nerve injury, J Neuroinflammation 19(1) (2022) 32.
[47] H. Düzova, M. Naz?ro?lu, B. Çi?, P. Gürbüz, A.N. Akatl?, Noopept Attenuates Diabetes-Mediated Neuropathic Pain and Oxidative Hippocampal Neurotoxicity via Inhibition of TRPV1 Channel in Rats, Mol Neurobiol 58(10) (2021) 5031-5051.
[48] Y. Yang, Z. Chen, R. Hu, Y. Sun, L. Xiang, J. Yan, H. Jiang, Activation of the spinal EGFR signaling pathway in a rat model of cancer-induced bone pain with morphine tolerance, Neuropharmacology 196 (2021) 108703.
[49] J. Zhang, L. Wang, H. Wang, Z. Su, X. Pang, Neuroinflammation and central PI3K/Akt/mTOR signal pathway contribute to bone cancer pain, Mol Pain 15 (2019) 1744806919830240.
[50] J.R. Vane, R.M. Botting, The mechanism of action of aspirin, Thromb Res 110(5-6) (2003) 255-8.

Presenting Author

Yuxin Zhang

Poster Authors

Yuxin Zhang

medical doctor

Shanghai Chest Hospital

Lead Author

E-mail

Yuan Peng

shanghai chest hospital

Lead Author

E-mail

wei wang

shanghai chest hospital

Lead Author

E-mail

Zhihao GONG

shanghai chest hospital

Lead Author

E-mail

Zuojing ZHANG

shanghai chest hospital

Lead Author

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Biying LIU

shanghai chest hospital

Lead Author

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Xingji YOU

shanghai university

Lead Author

E-mail

Jingxiang WU

shanghai chest hospital

Lead Author

E-mail

Topics

  • Mechanisms: Biological-Molecular and Cell Biology