葛根素通过调控miR-214抑制脊髓损伤后神经元炎症反应及凋亡的实验研究  

The experimental study of puerarin by regulating miR-214 to inhibit neuronal inflammation and apoptosis after spinal cord injury

作　　者：王光健 王德华 谭响 谢继勇 WANG Guang-jian;WANG De-hua;TAN Xiang;XIE Ji-yong(Department of Orthopedics,Chongqing Rongchang District People’s Hospital,Chongqing,402460,China)

机构地区：[1]重庆市荣昌区人民医院骨科,重庆402460

出　　处：《颈腰痛杂志》2023年第4期508-512,516,共6页The Journal of Cervicodynia and Lumbodynia

摘　　要：目的探讨葛根素是否通过介导miR-214抑制过氧化氢(hydrogen peroxide,H_(2)O_(2))诱导的脊髓神经元炎症反应及凋亡。方法体外培养大鼠脊髓神经元细胞,以H_(2)O_(2)刺激构建脊髓神经元损伤模型,实验分为:Con组、H_(2)O_(2)组、H_(2)O_(2)+Pue组、H_(2)O_(2)+Pue+anti-miR-NC组和H_(2)O_(2)+Pue+anti-miR-214组。实时荧光定量PCR(real time fluorescence quantitative PCR,RT-qPCR)检测各组miR-214的表达,CCK-8法检测细胞活性,DCFH-DA探针法检测细胞内活性氧(reactive oxygen species,ROS)水平,试剂盒检测超氧化物歧化酶(superoxide dismutase,SOD)活性,酶联免疫吸附测定法分析肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)和白细胞介素-6(interleukin-6,IL-6)含量,流式细胞术检测细胞凋亡,蛋白印迹法(Western blot)检测裂解半胱氨酸天冬氨酸蛋白酶3(Cleaved caspase-3)表达水平。结果与Con组相比,H_(2)O_(2)组细胞活性、SOD活性和miR-214的表达显著降低,细胞内ROS水平、TNF-α、IL-1β和IL-6的含量、凋亡率、Cleaved caspase-3的表达水平显著升高,差异均有统计学意义(P<0.05);与H_(2)O_(2)组相比,H_(2)O_(2)+Pue组细胞活性、SOD活性和miR-214的表达明显升高,细胞内ROS水平、TNF-α、IL-1β和IL-6的含量、凋亡率、Cleaved caspase-3的表达水平明显降低,差异均有统计学意义(P<0.05);抑制miR-214能够逆转葛根素对H_(2)O_(2)诱导的脊髓神经元细胞的损伤保护作用。结论葛根素能够通过上调miR-214的表达抑制H_(2)O_(2)诱导的脊髓神经元细胞凋亡和炎症反应,对脊髓神经元细胞损伤起保护作用。Objective To investigate whether puerarin can inhibit the inflammatory response and apoptosis of spinal cord neurons induced by hydrogen peroxide(H_(2)O_(2))by mediating miR-214.Methods Rat spinal cord neuronal cells were cultured in vitro,and H_(2)O_(2)stimulation was used to construct spinal cord neuronal injury models.The experiment was divided into:Con group,H_(2)O_(2)group,H_(2)O_(2)+Pue group,H_(2)O_(2)+Pue+anti-miR-NC group and H_(2)O_(2)+Pue+anti-miR-214 group.Real-time fluorescent quantitative PCR(RT-qPCR)was used to detect the expression of miR-214 in each group,CCK-8 method to detect cell viability,DCFH-DA probe to detect intracellular reactive oxygen species(ROS)levels,the kit to detect superoxide dismutase(SOD)activity,ELISA to analyse tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)content,flow cytometry to detect cell apoptosis,and Western blot to detect the expression level of cleaved caspase-3.Results Compared with the Con group,the cell activity,SOD activity and miR-214 expression in the H_(2)O_(2)group were significantly reduced,while the intracellular ROS level,TNF-α,IL-1βand IL-6 content,apoptosis rate,cleaved caspase-3 were markedly raised,all with statistically significant difference(P<0.05).Compared with the H_(2)O_(2)group,the cell activity,SOD activity and miR-214 expression in the H_(2)O_(2)+Pue group significantly increased,and the intracellular ROS level,TNF-α,IL-1βand IL-6 content,apoptosis rate,and cleaved caspase-3 expression levels significantly decreased,and the difference was statistically significant(P<0.05).The inhibition of miR-214 could reverse the protective effect of puerarin on H_(2)O_(2)-induced spinal cord neuronal cell damage.Conclusion Puerarin can inhibit H_(2)O_(2)-induced spinal cord neuronal cell apoptosis and inflammation by up-regulating the expression of miR-214,and thereby protecting spinal cord neuronal cell injury.

关 键 词：葛根素 miR-214 脊髓神经元细胞 凋亡 炎症反应 

分 类 号：R744[医药卫生—神经病学与精神病学]