PURPOSE: In the present work, we intended to explore the function of enhancer of zeste homolog 2 (EZH2) in modulated anesthetic reagent bupivacaine-induced neuronal apoptosis in spinal cord dorsal root ganglia (DRG). METHODS: Murine DRG explant was treated with 5 mM bupivacaine in vitro to induce neuronal apoptosis, which was examined by a TUNEL assay. Protein and mRNA expressions of EZH2 in bupivacaine-treated DRG were examined by western blot and qRT-PCR assays. EZH2 was downregulated by siRNA in bupivacaine-treated DRG. Its functional role in protecting bupivacaine-induced neuronal apoptosis was examined. In addition, apoptotic protein caspase-9 and EZH2-associated signaling pathway, and tropomyosin receptor kinase C (TrkC) were further investigated, by western blot assay, in EZH2-downregulated and bupivacaine-injured DRG. RESULTS: In vitro treatment of bupivacaine-induced DRG neuronal apoptosis, and upregulated EZH2 at both protein and mRNA levels. SiRNA transfection successfully downregulated EZH2, as confirmed by western blot and qRT-PCR assays. Examination of TUNEL assay showed that EZH2 downregulation rescued bupivacaine-induced DRG neuronal apoptosis. Moreover, in bupivacaine-injured DRG, EZH2 downregulation reduced caspase-9, whereas upregulated TrkC and phosphorylated-TrkC (p-TrkC). CONCLUSION: EZH2 is upregulated, whereas inhibiting EZH2 exerted rescuing effect in anesthetics (bupivacaine)-induced spinal cord DRG. The possible downstream target of EZH2 inhibition may interact with caspase and TrkC signaling pathways.
PURPOSE: In the present work, we intended to explore the function of enhancer of zeste homolog 2 (EZH2) in modulated anesthetic reagent bupivacaine-induced neuronal apoptosis in spinal cord dorsal root ganglia (DRG). METHODS:Murine DRG explant was treated with 5 mM bupivacaine in vitro to induce neuronal apoptosis, which was examined by a TUNEL assay. Protein and mRNA expressions of EZH2 in bupivacaine-treated DRG were examined by western blot and qRT-PCR assays. EZH2 was downregulated by siRNA in bupivacaine-treated DRG. Its functional role in protecting bupivacaine-induced neuronal apoptosis was examined. In addition, apoptotic protein caspase-9 and EZH2-associated signaling pathway, and tropomyosin receptor kinase C (TrkC) were further investigated, by western blot assay, in EZH2-downregulated and bupivacaine-injured DRG. RESULTS: In vitro treatment of bupivacaine-induced DRG neuronal apoptosis, and upregulated EZH2 at both protein and mRNA levels. SiRNA transfection successfully downregulated EZH2, as confirmed by western blot and qRT-PCR assays. Examination of TUNEL assay showed that EZH2 downregulation rescued bupivacaine-induced DRG neuronal apoptosis. Moreover, in bupivacaine-injured DRG, EZH2 downregulation reduced caspase-9, whereas upregulated TrkC and phosphorylated-TrkC (p-TrkC). CONCLUSION:EZH2 is upregulated, whereas inhibiting EZH2 exerted rescuing effect in anesthetics (bupivacaine)-induced spinal cord DRG. The possible downstream target of EZH2 inhibition may interact with caspase and TrkC signaling pathways.
Authors: Mark Verlinde; Markus W Hollmann; Markus F Stevens; Henning Hermanns; Robert Werdehausen; Philipp Lirk Journal: Int J Mol Sci Date: 2016-03-04 Impact factor: 5.923