circLphn3调节miR-882/NLRX1轴对创伤性脑损伤小鼠神经元损伤的影响

Effect of circLphn3 on neuronal damage inmicewith traumatic brain injury by regulating the miR-882/NLRX1 axis

目的探讨circLphn3对创伤性脑损伤(Traumatic brain injury,TBI)小鼠神经元损伤的改善作用及对微小RNA(microRNA,miR)-882/NLR家族成员X1(NLR family member X1,NLRX1)轴的调节作用。方法撞击法构建TBI小鼠模型,根据注射腺病毒的类型将小鼠随机分为TBI组、过表达对照(over-NC)组、circLphn3过表达(over-circLphn3)组、circLphn3过表达+miR-882激动剂对照(over-circLphn3+agomir-NC组)组、circLphn3过表达+miR-882激动剂(over-circLphn3+miR-882 agomir)组,每组各12只;随机取12只C57BL/6小鼠只钻孔不撞击作为假手术(Sham)组;采用Longa 5级评分法评估小鼠神经功能损伤情况;Morris水迷宫实验评估小鼠学习记忆能力;酶联免疫吸附法(Enzyme linked immunosorbent assay,ELISA)试剂盒检测血清白细胞介素-1β(Interleukin-1β,IL-1β)和肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)水平;伊红(Hematoxylin-eosin,HE)染色法和脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(Deoxyribonucleotide terminal transferase mediated nick end labeling,TUNEL)观察伤侧皮层组织形态及细胞凋亡情况;逆转录定量聚合酶链反应(Reverse transcription quantitative polymerase chain reaction,RT-qPCR)检测伤侧皮层组织miR-882,circLphn3,NLRX1 mRNA表达水平;Western blot检测伤侧皮层组织NLRX1,B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关蛋白(Bcl-2 associated protein,Bax)蛋白表达水平;双荧光素酶报告基因实验验证circLphn3与miR-882的靶向关系。结果与Sham组比较,TBI组小鼠伤侧皮层组织有出血、炎性细胞浸润等损伤,神经功能评分、在原木板处停留时间、circLphn3水平、NLRX1 mRNA和蛋白水平、Bcl-2蛋白水平显著下降(P<0.05),寻找木板所耗时间、IL-1β,TNF-α、细胞凋亡率、miR-882水平、Bax蛋白水平显著增高(P<0.05);与TBI组、over-NC组比较,over-circLphn3组小鼠伤侧皮层组织损伤减轻,神经功能评分、在原木板处停留时间、circLphn3水平、NLRX1 mRNA和蛋白水平、Bcl-2蛋白水平显著上升(P<0.05),寻找木板所耗时间、IL-1β,TNF-α水平、细胞凋亡率、miR-882水平、Bax蛋白水平显著下降(P<0.05);与over-circLphn3组、over-circLphn3+agomir-NC组比较,over-circLphn3+miR-882 agomir组小鼠伤侧皮层组织损伤加重,神经功能评分、在原木板处停留时间、circLphn3水平、NLRX1 mRNA和蛋白水平、Bcl-2蛋白水平显著下降(P<0.05),寻找木板所耗时间、IL-1β,TNF-α水平、细胞凋亡率、miR-882水平、Bax蛋白水平显著增高(P<0.05)。circLphn3与miR-882存在结合位点。结论过表达circLphn3可能通过调节miR-882/NLRX1轴来减轻TBI小鼠神经元损伤。

Objective To investigate the improved effect of circLphn3 on neuronal damage in traumatic brain injury(TBI)mice and its regulatory effect on the microRNA(miR)-882/NLR family member X1(NLRX1)axis.Methods Mouse model of TBI was established with a controlled cortical impact method.According to the difference of injected adenovirus,the mice were randomly divided into TBI group,overexpression control(over-NC)group,circLphn3 overexpression(over-circLphn3)group,and circLphn3 overexpression+miR-882 agonist control(over-circLphn3+ago-NC group)group and circLphn3 overexpression+miR-882 agonist(over-circLphn3+miR-882 agonist)group,for 12 mice in each group.Twelve C57BL/6 mice were randomly selected to drill without impact as the sham operation(Sham)group.The neurological deficits were assessed by Longa 5-grade scale.Morris water maze test was applied to evaluate the learning and memory ability of mice.Serum interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)were detected by enzyme linked immunosorbent assay(ELISA)kits.Hematoxylin eosin(HE)and deoxyribonucleotide terminal transferase mediated nick end labeling(TUNEL)staining were applied to observe the morphology of brain tissue and cell apoptosis of the injured cortex.Reverse transcription quantitative polymerase chain reaction(RT-qPCR)was used to detect the mRNA expression of miR-882,circLphn3 and NLRX1 in the injured cortex.Western blot was applied to detect the proteins expression of NLRX1,B-cell lymphoma-2(Bcl-2)and Bcl-2 associated X protein(Bax)in the injured cortex.Dual-luciferase reporter gene assay was applied to verify the targeting relationship between circLphn3 and miR-882.Results Compared with the Sham group,the mice in TBI group suffered from hemorrhage,inflammatory cell infiltration and other damages in the injured cortex,while the neurological deficits score,residence time at the original board,circLphn3level,mRNA and protein expression of NLRX1,and the protein expression of Bcl-2 were significantly decreased(P<0.05),and the time taken to find the board,serum IL-1β,TNF-α,cell apoptosis rate,miR-882 level,and the protein expression of Bax were significantly increased(P<0.05).Compared with the TBI group and the over-NC group,the mice in the over-circLphn3 group had reduced damage of the injured cortex,and the neurological deficits score,residence time at the original board,circLphn3 level,mRNA and protein expression of NLRX1,and the protein expression of Bcl-2 were significantly increased(P<0.05),while the time taken to find the board,serum IL-1β,TNF-α,cell apoptosis rate,miR-882 level,and the protein expression of Bax were significantly decreased(P<0.05).Compared with the over-circLphn3 group and the over-circLphn3+ago-NC group,the mice in the over-circLphn3+miR-882 agonist group had more severe damage of the injured cortex,and the neurological deficits score,residence time at the original board,circLphn3 level,mRNA and protein expression of NLRX1,and the protein expression of Bcl-2 were significantly decreased(P<0.05),while the time taken to find the board,serum IL-1β,TNF-α,cell apoptosis rate,miR-882 level,and the protein expression of Bax were significantly increased(P<0.05).There was a binding site between circLphn3 and miR-882.Conclusion Overexpression of circLphn3 may reduce neuronal damage in TBI mice by regulating the miR-882/NLRX1 axis.