NEFL、NRN1在二乙酰吗啡致神经元凋亡中的作用

The role of neurofilament light chain(NEFL)and neuritin(NRN1)in neuronal apoptosis induced by diacetylmorphine

目的探究神经丝轻链蛋白(Neurofilament light chain,NEFL)、神经突起素(Neu-ritin,NRN1)在二乙酰吗啡致神经元凋亡中的作用。方法建立二乙酰吗啡成瘾SD大鼠模型,分为Control组、Model组,观察各组大鼠脑组织病理形态学改变、凋亡细胞数及凋亡相关因子蛋白质表达变化;Western blot检测各组大鼠脑组织中NEFL、NRN1、Bax和Bcl-2的蛋白表达;采用NEFL慢病毒转染大鼠原代小脑颗粒细胞,使用Western blot技术检测转染效率,分为NC组、Model组和sh-NEFL+M组,使用二乙酰吗啡干预Model组和sh-NEFL+M组。采用CCK-8法检测细胞活力;流式细胞术检测细胞凋亡率;Western blot检测细胞NEFL、NRN1、Bax和Bcl-2的蛋白表达。结果成瘾SD大鼠脑组织HE染色和Hoechst33342荧光染色显示,与Control组相比,Model组大鼠脑组织出现明显筛网状改变,凋亡细胞数目明显增加;Western blot结果显示,Model组中NEFL、Bax蛋白表达升高,NRN1、Bcl-2蛋白表达降低,差异具有统计学意义(P<0.05);在二乙酰吗啡干预原代小脑颗粒细胞后,与Control组相比,Model组的细胞活力明显降低,凋亡率明显升高,NEFL和Bax蛋白表达显著升高,NRN1和Bcl-2蛋白表达显著降低,差异具有统计学意义(P<0.05);与Model组相比,sh-NEFL+M组细胞活力显著升高,凋亡率显著降低,NEFL、Bax蛋白表达降低,NRN1、Bcl-2蛋白表达升高,差异具有统计学意义(P<0.05)。结论NEFL、NRN1在二乙酰吗啡致小脑颗粒细胞凋亡中发挥重要作用,降低NEFL表达可减少二乙酰吗啡致小脑颗粒细胞凋亡。

Objective To investigate the role of neurofilament light chain(NEFL)and neuritin(NRN1)in neuronal apoptosis caused by diacetylmorphine.Methods A diacetylmorphine addicted SD rat model was established and divided into control group and model group,and the pathological changes in brain tissue,the number of apoptotic cells and the protein expression of apoptosis-related factors were observed in each group;Western blot was used to detect the protein expression of NEFL,NRN1,Bax and Bcl-2 in the brain tissues of the rats in each group.Rat primary cerebellar granule cells were transfected using NEFL lentivirus,and the transfection efficiency was detected using Western blot technique,and divided into NC group,model group and sh-NEFL+M group,and diacetylmorphine was used to intervene in model group and sh-NEFL+M group.Cell viability was detected by CCK-8 assay;apoptosis rate were detected by flow cy-tometry;and protein expression of NEFL,NRN1,Bax and Bcl-2 was detected by Western blot.Results HE staining and Hoechst33342 fluorescence staining of brain tissues of addicted SD rats showed that compared with the control group,the brain tissues of the rats in the model group showed obvious sieve-network al-terations;the number of apoptotic cells were significantly increased;the results of Western blot showed that in the model group,the protein expression of NEFL and Bax was increased,and that of NRN1 and Bcl-2 was decreased,and the results were statistically significant(P<0.05);after diacetylmorphine inter-vened in primary cerebellar granule cells,cell viability was significantly decreased,apoptosis rate was sig-nificantly increased,NEFL and Bax protein expression was significantly increased,and NRN1 and Bcl-2 protein expression was significantly decreased in the model group compared with control group,and the difference was statistically significant(P<0.05);Compared with the model group,the cell viability of sh-NEFL+M group was significantly increased,apoptosis rate was significantly decreased,NEFL and Bax protein expression was decreased,and NRN1 and Bcl-2 protein expression was increased,and the differ-ence was statistically significant(P<0.05).Conclusion NEFL and NRN1 play an important role in cere-bellar granule cell apoptosis caused by diacetylmorphine,and decreasing the expression of NEFL can re-duce cerebellar granule cell apoptosis caused by diacetylmorphine.