title: Midazolam anesthesia protects neuronal cells from oxidative stress-induced death via activation of the JNK-ERK pathway.
abstract: Midazolam is an anesthetic agent commonly used during clinical and surgical procedures, which has been shown to exert ROS‑suppressing and apoptosis‑modulating pharmacological activities in various cellular systems. However, the effects of midazolam on oxidative stress in neuronal cells require elucidation. The present study investigated the effects of midazolam on buthionine sulfoximine (BSO)‑ and hydrogen peroxide (H2O2)‑induced oxidative stress in primary cortical neuronal cells. In addition, the effects of midazolam on middle cerebral artery occlusion (MCAO) in mice and on ethanol‑induced neuroapoptosis in the brains of neonatal mice were determined. Subsequently, cell viability was detected using the MTT assay; intracellular reactive oxygen species (ROS) generation was determined using the 2',7'‑dichlorodihydrofluorescein diacetate method with confocal microscopy; terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was conducted to detect apoptotic cells; immunohistochemistry was performed to detect activated caspase‑3; neuronal deficit and infarct volume analyses were conducted; and quantitative polymerase chain reaction and western blotting were performed to detect the expression levels of genes and proteins associated with apoptosis and cell survival pathways. The results demonstrated that BSO (10 mM) and H2O2 (1 mM) suppressed proliferation of cortical neuronal cells by inducing apoptosis. These effects were suppressed following treatment with midazolam in a dose‑dependent manner. In addition, BSO and H2O2 induced ROS generation in neuronal cells; however, this was effectively suppressed by midazolam (100 µM). Beneficial synergistic effects were detected when midazolam was used in combination with the known antioxidant trolox. BSO and H2O2 also suppressed the protein expression levels of c‑Jun N‑terminal kinases (JNK), phosphorylated (p)JNK, extracellular signal‑regulated kinases (ERK)1/2, pERK1/2, AKT and nuclear factor‑κB; however, expression was recovered following treatment with midazolam. Midazolam also activated protein kinase C‑ε, which was suppressed by BSO, in cortical neuronal cells. In MCAO mice, midazolam post‑conditioning significantly suppressed infarct size and reduced the number of TUNEL‑positive cells. In addition, the expression levels of caspase‑3 and poly (ADP‑ribose) polymerase were suppressed in a dose‑dependent manner. In neonatal mice, midazolam reduced ethanol‑induced activated caspase‑3 staining and apoptotic TUNEL staining. The results of the present study demonstrated that midazolam may protect against neuronal degeneration and neuroapoptosis induced by physiological and oxidative stress.
authors.affiliation: Department of Anesthesiology, Dongying People's Hospital, Dongying, Shandong 257091, P.R. China.
authors.forename: Jing-Yu
authors.initials: JY
authors.lastname: Liu
authors.affiliation: Department of Anesthesiology, Dongying District People's Hospital, Dongying, Shandong 257091, P.R. China.
authors.forename: Feng
authors.initials: F
authors.lastname: Guo
authors.affiliation: Department of Anesthesiology, Kenli People's Hospital, Dongying, Shandong 257091, P.R. China.
authors.forename: Hong-Ling
authors.initials: HL
authors.lastname: Wu
authors.affiliation: Department of Anesthesiology, Dongying People's Hospital, Dongying, Shandong 257091, P.R. China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Anesthesiology, Dongying People's Hospital, Dongying, Shandong 257091, P.R. China.
authors.forename: Jin-Shan
authors.initials: JS
authors.lastname: Liu
mesh_terms: D000759:Adjuvants, Anesthesia / Q000494:pharmacology* / Q000627:therapeutic use; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D005260:Female; D020244:Infarction, Middle Cerebral Artery / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008807:Mice, Inbred BALB C; D008874:Midazolam / Q000494:pharmacology* / Q000627:therapeutic use; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018696:Neuroprotective Agents / Q000494:pharmacology* / Q000627:therapeutic use; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000759:Adjuvants, Anesthesia; D018696:Neuroprotective Agents; D017382:Reactive Oxygen Species; D008874:Midazolam