Activation of p47phox as a Mechanism of Bupivacaine-Induced Burst Production of Reactive Oxygen Species and Neural Toxicity

Abstract

Bupivacaine has been shown to induce neurotoxicity through inducing excessive reactive oxygen species (ROS), but the underlying mechanism remains unclear. NOX2 is one of the most important sources of ROS in the nervous system, and its activation requires the membrane translocation of subunit p47phox. However, the role of p47phox in bupivacaine-induced neurotoxicity has not been explored. In our in vitro study, cultured human SH-SY5Y neuroblastoma cells were treated with 1.5 mM bupivacaine to induce neurotoxicity. Membrane translocation of p47phox was assessed by measuring the cytosol/membrane ratio of p47phox. The effects of the NOX inhibitor VAS2870 and p47phox-siRNA on bupivacaine-induced neurotoxicity were investigated. Furthermore, the effect of VAS2870 on bupivacaine-induced neurotoxicity was assessed in vivo in rats. All these changes were reversed by pretreatment with VAS2870 or transfection with p47phox-siRNA in SH-SY5Y cells. Similarly, pretreatment with VAS2870 attenuated bupivacaine-induced neuronal toxicity in rats. It is concluded that enhancing p47phox membrane translocation is a major mechanism whereby bupivacaine induced neurotoxicity and that pretreatment with VAS2870 or local p47phox gene knockdown attenuated bupivacaine-induced neuronal cell injury.

Figure 1

Bupivacaine-induced cell injury in vitro and in vivo. (a, b) The protein expression level of cleaved caspase-3 and phospho-γ-H2A.x after bupivacaine incubation at different time points. (c, d) TUNEL staining indicated the ratio of apoptotic cells after bupivacaine (1.5 mM) incubation for 24 h in SH-SY5Y cells. Cells tagged by white arrows were TUNEL positive. (e, f) Schematic diagram indicates the area of tested sections in the spinal dorsal horn. Cell apoptosis in the spinal dorsal horn was assessed by TUNEL staining. Cells tagged by white arrows were TUNEL staining positive. Data represent mean ± SD of at least 3 independent experiments or for 6 rats each group. Scale bar: 50 μm.^∗P < 0.05 versus control (Con) group.

Figure 2

NOX inhibition protected cells and rats from bupivacaine-induced toxicity via reducing excessive production of ROS. SH-SY5Y cells were pretreated with VAS2870 (10 μM) 30 min prior to bupivacaine treatment. (a) SH-SY5Y cells were treated with bupivacaine (1.5 mM) for 1 h, 2 h, and 3 h; the production of cytoplasmic peroxide (DCFH-DA) and O₂^.− (DHE) was measured by a microplate reader. (b, c) SH-SY5Y cells were treated with bupivacaine for 3 h. The level of cytoplasmic peroxide (DCFH-DA, green) and O₂^.− (DHE, red) was measured by microscopy. (d) The production of cytoplasmic peroxide (DCFH-DA) and O₂^.− (DHE) was measured by a microplate reader. (e, f) The protein level of cleaved caspase-3 and phospho-γ-H2A.x after bupivacaine treatment. (g, h) The ratio of apoptotic cells was detected by TUNEL staining. Cells tagged by white arrows were TUNEL positive. (i) The basic lines of paw mechanical thresholds were tested before treatment. Paw mechanical threshold was tested by the electronic von Frey system after different treatments. (j, k) Schematic diagram indicates the area of tested sections in the spinal dorsal horn. The level of cytoplasmic O₂^.− (DHE, red) was measured by microscopy. (l, m) The ratio of apoptotic cells was detected by TUNEL staining. Cells tagged by white arrows were TUNEL positive. (n, o) The protein level of cleaved caspase-3 was measured by Western blot. Data represent mean ± SD of at least 3 independent experiments or for 6 rats each group. Scale bar: 50 μm. ^∗P < 0.05 versus control group. ^#P < 0.05 versus Bup group. ^&P < 0.05 versus VAS group.

Figure 3

Membrane translocation of p47phox was increased after bupivacaine exposure, which was inhibited by NOX inhibition. (a, b) The protein level of total p47phox after bupivacaine incubation at different time points was measured by Western blot. The level of p47phox membrane translocation was detected by the ratio of p47phox expressed in the cytosol and membrane. (c, d) SH-SY5Y cells were treated with 1.5 mM bupivacaine for 3 h and incubated with DH10 medium until 24 h. SH-SY5Y cells were pretreated with VAS2870 (10 μM) 30 min prior to bupivacaine treatment. The level of p47phox translocation in SH-SY5Y cells with different treatments. (e, f) The level of p47phox translocation in animal models. (g) The level of p47phox membrane translocation was detected by immunofluorescent staining. Pan-cadherin is a widely used membrane marker and was stained green, targeted protein p47phox was stained red, and nucleus was stained with DAPI (blue). Data represent mean ± SD of at least 3 independent experiments or for 6 rats each group. Scale bar: 10 μm.^∗P < 0.05 versus Con group. ^#P < 0.05 versus Bup group. ^&P < 0.05 versus VAS group.

Figure 4

p47phox gene knockdown reduced bupivacaine-induced ROS overexpression and cell injury. (a, b) The level of cytoplasmic peroxide (DCFH-DA, green) and O₂^.− (DHE, red) was measured by microscopy. (c) The production of cytoplasmic peroxide (DCFH-DA) and O₂^.− (DHE) was measured by a microplate reader. (d, e) The ratio of apoptotic cells was detected by TUNEL staining. Cells tagged by white arrows were TUNEL positive. (f, g) The protein level of cleaved caspase-3 and phospho-γ-H2A.x was measured by Western blot. (f, h) The translocation of p47phox was measured by Western blot. Data represent mean ± SD of at least 3 independent experiments. Scale bar: 50 μm. ^∗P < 0.05 versus control (NC) group. ^#P < 0.05 versus Bup group. ^&P < 0.05 versus p47si group.