CAJ | 학술논문

目的探讨地塞米松对布比卡因诱导小鼠神经母细胞瘤株(N2a)细胞毒性的影响.方法 N2a细胞悬液(105/ml)随机分为4组:对照组(C组)、布比卡因组(Bup组)、地塞米松组(Dex组)和地塞米松+布比卡因组(Dex+Bup组).各组N2a细胞分别接种于24孔培养板(0.5 ml/孔)和直径10 cm的培养皿中(7 ml/皿),每组24孔和4皿.C组不行干预;Bup组加入含900μmol/L布比卡因的MEM培养基500μl;Dex组加入含1μmol/L地塞米松的MEM培养基500μl;Dex+Bup组加入含1/μmol/L地塞米松的MEM培养基500μl孵育12 h后加入布比卡因900 μmol/L.于24孔板中孵育5 h时,测定线粒体跨膜电位(△Ψm);于24孔板中孵育9 h时观察细胞形态,并计算LDH释放率和细胞核固缩率;于培养皿中孵育5 h时测定磷酸化蛋白激酶B(p-Akt)和磷酸化胞外信号调节激酶(p-ERKs)的表达.结果 C组细胞多边形,胞体折光性强,突触伸展良好;Dex组细胞形态学与C组相似;Bup组细胞扁平,突触缩短或断裂,胞体折光性弱;Dex+Bup组扁平细胞减少,突触缩短或断裂减少,胞体折光性略低.与C组比较,Bup组LDH释放率和细胞核固缩率升高,△Ψm降低,p-ERKs和p-Akt表达下调,Dex+Bup组细胞核固缩率升高,△Ψm降低,p-ERK表达下调,p-AKt表达上调,Dex组p-ERKs和p-Akt表达上调(P<0.01).与Bup组比较,Dex+Bup组LDH释放率和细胞核固缩率降低,△Ψm增加,p-ERKs和p-Akt表达上调(P<0.01).结论地塞米松可减轻布比卡因诱导N2a细胞毒性,其机制可能与恢复线粒体膜电位、抑制Akt和ERKs脱磷酸化有关.
목적 탐토지새미송대포비잡인유도소서신경모세포류주(N2a)세포독성적영향.방법 N2a세포현액(105/ml)수궤분위4조:대조조(C조)、포비잡인조(Bup조)、지새미송조(Dex조)화지새미송+포비잡인조(Dex+Bup조).각조N2a세포분별접충우24공배양판(0.5 ml/공)화직경10 cm적배양명중(7 ml/명),매조24공화4명.C조불행간예;Bup조가입함900μmol/L포비잡인적MEM배양기500μl;Dex조가입함1μmol/L지새미송적MEM배양기500μl;Dex+Bup조가입함1/μmol/L지새미송적MEM배양기500μl부육12 h후가입포비잡인900 μmol/L.우24공판중부육5 h시,측정선립체과막전위(△Ψm);우24공판중부육9 h시관찰세포형태,병계산LDH석방솔화세포핵고축솔;우배양명중부육5 h시측정린산화단백격매B(p-Akt)화린산화포외신호조절격매(p-ERKs)적표체.결과 C조세포다변형,포체절광성강,돌촉신전량호;Dex조세포형태학여C조상사;Bup조세포편평,돌촉축단혹단렬,포체절광성약;Dex+Bup조편평세포감소,돌촉축단혹단렬감소,포체절광성략저.여C조비교,Bup조LDH석방솔화세포핵고축솔승고,△Ψm강저,p-ERKs화p-Akt표체하조,Dex+Bup조세포핵고축솔승고,△Ψm강저,p-ERK표체하조,p-AKt표체상조,Dex조p-ERKs화p-Akt표체상조(P<0.01).여Bup조비교,Dex+Bup조LDH석방솔화세포핵고축솔강저,△Ψm증가,p-ERKs화p-Akt표체상조(P<0.01).결론 지새미송가감경포비잡인유도N2a세포독성,기궤제가능여회복선립체막전위、억제Akt화ERKs탈린산화유관.
Objective To investigate the effect of dexamethasone on the toxicity of bupivacaine in murine neurons.Methods Murine neuroblastoma cell line N2a was obtained from ATCC cell bank (USA). The cells were cultured in 10% fetal cow serum/MEM culture medium and divided into 4 groups voup I control (Con); group II bupivacaine ( Bup); group Ⅲ dexamethasone (Dex) and group IV Dex + Bup. The culture medium contained bupivacaine 900 μmol/L in group Bup and dexamethasone 1 μmol/L in group Dex respectively. In group Dex + Bup ( IV ) Bup was added to the culture medium with a final concentration of 900 μmol/L at 12 h after pretreatment with Dex 1 μmol/L. The cells were inoculated in 24 well plates (0.5 ml in each well, 24 wells in each group) and 10 cm culture dishes (7 ml in each dish, 4 dishes in each group). The release rate of LDH was calculated and the morphology of the cells and nucleus condensation (by Hoechst 3334224 fluorescent staining) was detected at 9 h of incubation in 24 well plates. The mitochondrial transmembrane potential (by JC-1 assay) and phosphorylation of Akt and ERKs (by Western blot) were measured at 5 h of incubation in 24 well plates and in culture dishes respectively. ResultsBupivacaine caused severe damage to the N2a cells as evidenced by increase in LDH release and nucleus condensation (apoptosis), dephosphorylation of Akt and ERKs, decrease in mitochondrial transmembrane potential and severe morphological changes. Dexamethasone pretreatment significantly attenuated bupivacaine-induced neurotoxicity. Conclusion Dexamethasone can protect N2a cells from bupivacaine-induced neurotoxicity through stabilization of mitochondrial transmembrane potential and inhibition of dephosphorylation of Akt and ERKs.