br Introduction Local anesthetics LAs
Introduction Local anesthetics (LAs) are a set of common medicines used for pain management in clinical settings. Up to now, LAs have been extensively used for peripheral nerve blocks, interventional spinal procedures, and epidural anesthesia . In addition to their anesthetic and analgesic effects, LAs can interact with structural proteins and affect intracellular signaling pathways . LAs also possess the capacity to exert different toxic actions in various tissues . The most striking and consistent findings regarding the cytotoxic effects of LAs involve neuronal toxicity . Ropivacaine is a member of a new generation of amide-type local anesthetics. Various neuronal cellular activities of ropivacaine have been reported to induce cytotoxic effects and apoptosis . A recent study demonstrated that in vitro treatment with ropivacaine hydrochloride significantly reduced cell viability and increased apoptosis in dorsal root ganglion neurons (DRG) by regulating the expression of CaMK II γ . Additionally, long-term exposure to ropivacaine has been found to cause oxidative stress by increasing intracellular levels of ROS and mitochondrial dysfunction by reducing mitochondrial membrane potential. Inhibition of ROS generation by N-acetyl-L-cysteine (NAC) prevented ropivacaine-induced apoptosis . However, the underlying molecular mechanisms remain unclear and need to be elucidated. Heme oxygenase-1 (HO-1) was originally identified as an enzyme that catalyzes the degradation of heme . HO-1 is ubiquitously expressed in various tissues . It is well-documented that HO-1 is highly induced and serves as a cytoprotective factor in response to cellular stresses, including hypoxia, inflammation, UV irradiation, and oxidative stress . Toxic agents initiate intracellular signaling pathways and activate transcriptional factors, including the transcription factor NFE2-related factor 2 (Nrf2) and nuclear factor-κ B (NF-κB) . Interestingly, HO-1 is highly expressed in the central nervous system (CNS). The expression levels of HO-1 are up-regulated during gdc-0980 injury in a variety of neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) . Similarly, both in vivo and in vitro studies have shown that HO-1 expression is upregulated by different types of stimuli, including chemicals, cytokines, and drugs . However, little information regarding the physiological functions of HO-1 in ropivacaine-induced neurotoxicity has been reported before.
Materials and methods
Results Elevated expression of HO-1 plays a key role in maintaining redox homeostasis. Therefore, we first investigated the effects of ropivacaine on the expression of HO-1 in human SHSY5Y cells. Our results indicated that exposure to ropivacaine for 1 h induced elevation of HO-1 at the mRNA level in a dose-dependent manner from 0.68 mM to 2.72 mM (Fig. 1A). Similarly, treatment with ropivacaine for 18 h markedly increased the expression of HO-1 at the protein level (Fig. 1B). Using an LDH assay, we found that the IC50 value of ropivacaine in human SHSY5Y cells was 3.2 mM. Treatment with ropivacaine at the concentration of 1.36 mM led to mild cell death in human SHSY5Y cells (Fig. 7). Therefore, 1.36 mM ropivacaine was used to treat SHSY5Y cells for different time durations. The results indicate that HO-1 mRNA (Fig. 1C) and protein (Fig. 1D) levels peaked at 1 h and 18 h, respectively, in response to ropivacaine treatment. Ropivacaine treatment for 18 h increased HO-1 activity in a dose-dependent manner (Fig. 2). Numerous signaling pathways are associated with the induction of HO-1 expression, including the p38 MAPK pathway . We found that ropivacaine treatment significantly increased phosphorylation of p38 (p-p38) (Fig. 3A). Importantly, blockage of the p38 pathway with its specific inhibitor SB203580 (1, 2 nM) significantly reduced the expression of HO-1 induced by ropivacaine treatment (Fig. 3B). To further confirm the involvement of p38 in the induction of HO-1, the expression of p38 was silenced by transfection with p38 siRNA. Successful knockdown of p38 is shown in Fig. 3C. As expected, inhibition of p38 restrained ropivacaine-stimulated HO-1 expression (Fig. 3D). Expression of HO-1 is mainly regulated through the Nrf2/Keap1 transcription factor system via binding to ARE in the nucleus . Here, we investigated whether ropivacaine could trigger nuclear translocation of Nrf2 and initiate expression of HO-1. Our findings indicate that ropivacaine treatment significantly increased nuclear translocation of Nrf2 (Fig. 4A). Importantly, we found that silencing of Nrf2 by transfection with Nrf2 siRNA (Fig. 4B) eliminated ropivacaine-induced expression of HO-1 (Fig. 4C). A luciferase reporter assay was used to assess ARE promoter activity. SHSY5Y cells were transfected with ARE luciferase reporter plasmids and incubated with ropivacaine for 5 h. Indeed, our results indicate that ropivacaine treatment markedly increased ARE luciferase activity in a dose-dependent manner (Fig. 5). These findings suggest that Nrf2/ARE is responsible for ropivacaine-induced HO-1 expression.