BDNF通过Akt信号通路减轻罗哌卡因诱导的神经细胞损伤

Brain derived neurotrophic factor alleviates neuronal injury induced by ropivacaine through threonine protein kinase signaling pathway

目的探讨脑源性神经营养因子(BDNF)对罗哌卡因诱导的神经细胞损伤的保护作用及其机制。方法(1)实验一:分别用0、1、2、3、4、5 mmol/L罗哌卡因刺激人神经母细胞瘤细胞SH-SY5Y 48 h,于显微镜下观察各组细胞的形态变化,采用噻唑蓝(MTT)法检测各组细胞的活性情况,采用流式细胞术检测各组细胞的凋亡情况,采用免疫组化染色检测各组细胞中蛋白激酶B(Akt)、增殖细胞核抗原(PCNA)阳性细胞的表达。(2)实验二:分别用0、1、3、5、7 mmol/L罗哌卡因刺激SH-SY5Y细胞48 h,采用MTT法检测各组细胞的活性情况并计算出罗哌卡因的半抑制浓度(IC50)。将SH-SY5Y细胞分为对照组(磷酸盐缓冲液刺激48 h)、罗哌卡因组(IC50浓度罗哌卡因刺激48 h)、BDNF+罗哌卡因组(20μg/L BDNF刺激2 h+IC50浓度罗哌卡因刺激48 h)、Akt信号通路激活剂SC79+罗哌卡因组(5 mg/L SC79刺激2 h+IC50浓度罗哌卡因刺激48 h)、BDNF+Akt信号通路抑制剂API-2+罗哌卡因组(20μg/L BDNF及10μmol/L API-2刺激2 h+IC50浓度罗哌卡因刺激48 h),于显微镜下观察各组细胞的形态变化,采用MTT法检测各组细胞的活性情况,采用流式细胞术检测各组细胞的凋亡情况,采用免疫组化染色检测各组细胞中Akt、PCNA阳性细胞的表达,采用逆转录-聚合酶链反应(RT-PCR)及Western blotting检测各组细胞中B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸蛋白酶-3(Caspase-3)mRNA及蛋白的表达,采用Western blotting检测各组细胞中Akt、磷脂酰肌醇3-激酶(PI3K)蛋白的表达。结果(1)实验一:与0 mmol/L罗哌卡因组比较,1、2、3、4、5 mmol/L罗哌卡因组的细胞活性明显降低,细胞凋亡率明显升高,Akt、PCNA阳性细胞数明显降低,差异均有统计学意义(P<0.05)。(2)实验二:与对照组比较,罗哌卡因组的细胞活性明显降低,细胞凋亡率明显升高,Akt、PCNA阳性细胞数明显降低,Bcl-2 mRNA及蛋白的表达均明显降低,Bax、Caspase-3 mRNA及蛋白的表达均明显升高,p-Akt、p-PI3K蛋白的表达均明显降低,差异均有统计学意义(P<0.05)。与罗哌卡因组比较,BDNF+罗哌卡因组、SC79+罗哌卡因组的细胞活性明显升高,细胞凋亡率明显降低,Akt、PCNA阳性细胞数明显升高,Bcl-2 mRNA及蛋白的表达明显升高,Bax、Caspase-3 mRNA及蛋白的表达明显降低,p-Akt、p-PI3K蛋白的表达明显升高,差异均有统计学意义(P<0.05)。与BDNF+罗哌卡因组、SC79+罗哌卡因组比较,BDNF+API-2+罗哌卡因组的细胞活性明显降低,细胞凋亡率明显升高,Akt、PCNA阳性细胞数明显降低,Bcl-2 mRNA及蛋白的表达明显降低,Bax、Caspase-3 mRNA及蛋白的表达明显升高,p-Akt、p-PI3K蛋白的表达明显降低,差异均有统计学意义(P<0.05)。结论BDNF可通过激活Akt信号通路减轻罗哌卡因引起的神经细胞损伤,从而调节神经细胞的增殖和凋亡。

Objective To investigate the protective effect of brain-derived neurotrophic factor(BDNF)on neuronal injury induced by ropivacaine(Rop)and its mechanism.Methods(1)Experiment one:0,1,2,3,4 and 5 mmol/L Rop was used to stimulate SH-SY5Y cells for 48 h to induce neuronal injury;the morphological changes of the cells were observed under microscope;MTT assay was used to detect the cell activity;flow cytometry was used to detect the cell apoptosis,and immunohistochemistry was used to detect the expressions of protein kinase B(Akt)and proliferating cell nuclear antigen(PCNA).(2)Experiment two:SH-SY5Y cells were treated with 0,1,3,5,7 mmol/L Rop,respectively;the cell activity was measured 48 h after Rop treatment;the semi inhibitory concentration(IC50)of Rop was calculated by MTT assay;the SH-SY5Y cells were divided into control group(PBS for 48 h),Rop group(Rop at IC50 for 48 h),BDNF+Rop group(20μg/L BDNF for 2 h,and Rop at IC50 for 48 h),Akt pathway activator SC79+Rop group(5 mg/L SC79 for 2 h,and Rop at IC50 for 48 h),and BDNF+Akt pathway inhibitor API-2+Rop group(20μg/L BDNF+10μmol/L API-2 for 2 h,Rop at IC50 for 48 h);the morphological changes of the cells were observed under microscope;MTT assay was used to detect the cell activity;flow cytometry was used to detect the cell apoptosis,and immunohistochemistry was used to detect the expressions of Akt and PCNA;the expressions of B lymphoma-2(Bcl-2),Bcl-2-associated X(Bax)and cysteine protease-3(Caspase-3)were detected by reverse transcription(RT)-PCR and Western blotting.Western blotting was used to detect the expressions of Akt and phosphatidylinositol 3-kinase(PI3K).Results(1)As compared with the 0 mmol/L Rop group,the 1,2,3,4,and 5 mmol/L Rop group had significantly decreased cell activity,significantly increased apoptosis rate,and statistically smaller number of Akt and PCNA positive cells(P<0.05).(2)As compared with the control group,the Rop group had significantly decreased cell activity,statistically increased apoptosis rate,significantly smaller number of Akt and PCNA positive cells,significantly decreased Bcl-2 mRNA and protein expressions,significantly increased Bax and caspase-3 mRNA and protein expressions,and significantly decreased phosphorylated-(p-)Akt and p-PI3K protein expressions;as compared with the Rop group,the BDNF+Rop group and SC79+Rop group had significantly higher cell activity,significantly decreased apoptosis rate,significantly larger number of Akt and PCNA positive cells,significantly increased Bcl-2 mRNA and protein expressions,statistically decreased mRNA and protein expressions of Bax and Caspase-3,and significantly increased p-Akt and p-PI3K protein expressions(P<0.05);as compared with the BDNF+Rop group and SC79+Rop group,the BDNF+API-2+Rop group had significantly lower cell activity,significantly increased apoptosis rate,significantly smaller number of Akt and PCNA positive cells,significantly decreased Bcl-2 mRNA and protein expressions,statistically increased mRNA and protein expressions of Bax and Caspase-3,and significantly decreased p-Akt and p-PI3K protein expressions(P<0.05).Conclusion BDNF can alleviate ropivacaine-induced neuronal injury by activating Akt signaling pathway,consequently modulating the proliferation and apoptosis of neurons.