摘要
目的探讨微RNA(miR)-708-5p在结核性脑膜炎(TBM)小鼠脑损伤中的作用及机制。方法将48只BALB/c小鼠随机分为假手术组、TBM组、TBM+miR-708-5pinhibitor组及TBM+NC-inhibitor组,每组12只。TBM组、TBM+miR-708-5pinhibitor组及TBM+NC-inhibitor组小鼠经尾静脉注射0.2mL结核分枝杆菌H37Rv菌悬液(2.5×10^9CFU·L^-1)制备TBM模型,假手术组小鼠给予等体积磷酸盐缓冲液。造模成功后,TBM+miR-708-5pinhibitor组、TBM+NC-inhibitor组小鼠分别经尾静脉注射0.2mLmiR-708-5pinhibitor(2mg·kg^-1)和NC-inhibitor(2mg·kg^-1),每2d注射1次,连续1周。将对数生长期小鼠脑膜细胞GIBH001根据转染载体的不同分为对照组、miR-708-5p过表达组、补体C1q肿瘤坏死因子相关蛋白1(C1qtnf1)过表达组及miR-708-5p+C1qtnf1过表达组,分别转染NC-mimic和Vector、miR-708-5pmimic和Vector、NC-mimic和pcDNA-C1qtnf1、miR-708-5pmimic和pcDNA-C1qtnf1;另根据转染载体的不同,将GIBH001细胞分为空白组、NC-inhibitor组及miR-708-5pinhibitor组,空白组细胞不做处理,NC-inhibitor组、miR-708-5pinhibitor组细胞分别转染NC-inhibitor、miR-708-5pinhibitor。采用干湿法检测各组小鼠脑组织含水量;采用伊文思蓝实验检测各组小鼠血脑屏障通透性;采用酶联免疫吸附法检测各组小鼠脑组织中肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)水平;采用实时荧光定量聚合酶链反应法检测小鼠脑组织中miR-708-5p和C1qtnf1mRNA相对表达量以及各组GIBH001细胞中C1qtnf1mRNA相对表达量;采用Westernblot法检测各组小鼠脑组织中和对照组、miR-708-5p过表达组、C1qtnf1过表达组、miR-708-5p+C1qtnf1过表达组GIBH001细胞中磷脂酰肌醇3激酶(PI3K)、磷酸化磷脂酰肌醇3激酶(p-PI3K)、蛋白激酶B(Akt)、磷酸化蛋白激酶B(p-Akt)蛋白相对表达量。结果TBM组小鼠脑组织同侧基底神经节含水量显著高于假手术组(P<0.05);TBM+miR-708-5pinhibitor组小鼠脑组织同侧基底神经节含水量显著低于TBM+NC-inhibitor组(P<0.05);4组小鼠对侧皮质、对侧基底神经节、同侧皮质、小脑含水量比较差异均无统计学意义(P>0.05)。TBM组小鼠血脑屏通透性显著高于假手术组(P<0.05);TBM+miR-708-5pinhibitor组小鼠血脑屏障通透性显著低于TBM+NC-inhibitor组(P<0.05)。TBM组小鼠脑组织中TNF-α、IL-1β水平均高于假手术组(P<0.05);TBM+miR-708-5pinhibitor组小鼠脑组织中TNF-α、IL-1β水平显著低于TBM+NC-inhibitor组(P<0.05)。与假手术组比较,TBM组小鼠脑组织中miR-708-5p的相对表达量显著升高(P<0.05),C1qtnf1mRNA相对表达量显著降低(P<0.05);与TBM+NC-inhibitor组比较,TBM+miR-708-5pinhibitor组小鼠脑组织中miR-708-5p的相对表达量显著降低(P<0.05),C1qtnf1mRNA相对表达量显著升高(P<0.05)。miR-708-5p过表达组细胞中C1qtnf1mRNA相对表达量低于对照组(P<0.05);C1qtnf1过表达组细胞中C1qtnf1 mRNA相对表达量高于对照组(P<0.05);miR-708-5p+C1qtnf1过表达组细胞中C1qtnf1mRNA相对表达量显著低于C1qtnf1过表达组(P<0.05);miR-708-5p+C1qtnf1过表达组细胞中C1qtnf1mRNA的相对表达量与对照组比较差异无统计学意义(P>0.05)。NC-inhibitor组细胞中C1qtnf1mRNA相对表达量与空白组比较差异无统计学意义(P>0.05);miR-708-5pinhibitor组细胞中C1qtnf1mRNA相对表达量显著低于空白组(P<0.05);miR-708-5pinhibitor组细胞中C1qtnf1mRNA相对表达量显著低于NC-inhibitor组(P<0.05)。TBM组小鼠脑组织中p-PI3K/PI3K、p-Akt/Akt显著高于假手术组(P<0.05);TBM+miR-708-5pinhibitor组小鼠脑组织中p-PI3K/PI3K、p-Akt/Akt显著低于TBM+NC-inhibitor组(P<0.05)。miR-708-5p过表达组细胞中p-PI3K/PI3K、p-Akt/Akt均显著低于对照组(P<0.05);C1qtnf1过表达组细胞中p-PI3K/PI3K、p-Akt/Akt均显著高于对照组(P<0.05);miR-708-5p+C1qtnf1过表达组细胞中p-PI3K/PI3K、p-Akt/Akt均显著低于C1qtnf1过表达组(P<0.05);miR-708-5p+C1qtnf1过表达组细胞中p-PI3K/PI3K、p-Akt/Akt与对照组比较差异无统计学意义(P>0.05)。结论miR-708-5p可促进TBM小鼠脑损伤的发生,其机制可能与下调C1qtnf1的表达、抑制PI3K/Akt信号通路有关。
Objective To investigate the role and mechanism of microRNA(miR)-708-5 p in brain injury of mice with tuberculous meningitis(TBM).Methods Forty-eight BALB/c mice were randomly divided into sham operation group,TBM group,TBM+miR-708-5 p inhibitor group and TBM+NC-inhibitor group,with 12 mice in each group.Mice in the TBM group,TBM+miR-708-5 p inhibitor group and TBM+NC-inhibitor group were injected with 0.2 m L of Mycobacterium tuberculosis H37 Rv suspension(2.5×10^9 CFU·L^-1)via tail vein to establish the TBM model,and the mice in the sham operation group were given equal volume of phosphate buffered solution.After successful modeling,mice in TBM+miR-708-5 p inhibitor group and TBM+NC-inhibitor group were respectively injected with 0.2 m L of miR-708-5 p inhibitor(2 mg·kg^-1)and NC-inhibitor(2 mg·kg^-1)via tail vein once every 2 days for 1 week.Mice meningeal cells GIBH001 at logarithmic growth stage were divided into control group,miR-708-5 p overexpression group,complement C1 q tumor necrosis factor-related protein 1(C1 qtnf1)overexpression group and miR-708-5 p+C1 qtnf1 overexpression group according to different transfection vectors.The cells in above four groups were transfected with NC-mimics and Vector,miR-708-5 p mimic and Vector,NC-mimic and pc DNA-C1 qtnf1,miR-708-5 p mimic and pc DNA-C1 qtnf1,respectively.Additionally,the GIBH001 cells were divided into blank group,NC-inhibitor group and miR-708-5 p inhibitor group according to different transfection vectors.The cells in the blank group were not treated;the cells in the NC-inhibitor group and miR-708-5 p inhibitor group were transfected with NC-inhibitor and miR-708-5 p inhibitor,respectively.The water content of the brain tissue of mice in each group was detected by dry and wet method;the permeability of the blood-brain barrier of mice in each group was detected by the Evans blue test;the levels of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in the brain tissue of mice in each group were detected by enzyme-linked immunosorbent assay;the relative expression levels of miR-708-5 p and C1 qtnf1 mRNA in the brain tissue of mice and C1 qtnf1 mRNA in the GIBH001 cells in each group were detected by real-time fluorescent quantitative polymerase chain reaction method;the relative expression levels of phosphatidylinositol 3 kinase(PI3 K),phosphorylated phosphatidylinositol 3 kinase(p-PI3 K),protein kinase B(Akt)and phosphorylated protein kinase B(p-Akt)in the brain tissue of mice and the GIBH001 cells in the control group,miR-708-5 p overexpression group,C1 qtnf1 overexpression group,miR-708-5 p+C1 qtnf1 overexpression group were detected by Western blot.Results The water content of the ipsilateral basal ganglia in the brain tissue of mice in the TBM group was significantly higher than that in the sham operation group(P<0.05);the water content of the ipsilateral basal ganglia in the brain tissue of mice in the TBM+miR-708-5 p inhibitor group was significantly lower than that in the TBM+NC-inhibitor group(P<0.05);there was no significant difference in the water content of contralateral cortex,contralateral basal ganglia,ipsilateral cortex and cerebellum of mice among the four groups(P>0.05).The blood-brain barrier permeability of mice in the TBM group was significantly higher than that in the sham operation group(P<0.05);the blood-brain barrier permeability of mice in the TBM+miR-708-5 p inhibitor group was significantly lower than that in the TBM+NC-inhibitor group(P<0.05).The levels of TNF-αand IL-1βin the brain tissue of mice in the TBM group were higher than those in the sham operation group(P<0.05);the levels of TNF-αand IL-1βin the brain tissue of mice in the TBM+miR-708-5 p inhibitor group were significantly lower than those in the TBM+NC-inhibitor group(P<0.05).Compared with the sham operation group,the relative expression of miR-708-5 p in the brain tissue of mice in the TBM group was significantly increased(P<0.05),and the relative expression of C1 qtnf1 mRNA was significantly reduced(P<0.05);compared with the TBM+NC-inhibitor group,the relative expression of miR-708-5 p in the brain tissue of mice in the TBM+miR-708-5 p inhibitor group was significantly reduced(P<0.05),and the relative expression of C1 qtnf1 mRNA was significantly increased(P<0.05).The relative expression of C1 qtnf1 mRNA of the cells in the miR-708-5 p overexpression group was lower than that in the control group(P<0.05);the relative expression of C1 qtnf1 mRNA of the cells in the C1 qtnf1 overexpression group was higher than that in the control group(P<0.05);the relative expression of C1 qtnf1 mRNA of the cells in the miR-708-5 p+C1 qtnf1 overexpression group was significantly lower than that in the C1 qtnf1 overexpression group(P<0.05).There was no significant difference in the relative expression of C1 qtnf1 mRNA of the cells between the miR-708-5 p+C1 qtnf1 overexpression group and the control group(P>0.05).There was no significant difference in the relative expression of C1 qtnf1 mRNA of the cells between the NC-inhibitor group and the blank group(P>0.05);the relative expression of C1 qtnf1 mRNA of the cells in the miR-708-5 p inhibitor group was significantly lower than that in the blank group(P<0.05);the relative expression of C1 qtnf1 mRNA of the cells in the miR-708-5 p inhibitor group was significantly lower than that in the NC-inhibitor group(P<0.05).The p-PI3 K/PI3 K and p-Akt/Akt in the brain tissue of mice in the TBM group were significantly higher than those in the sham operation group(P<0.05);the p-PI3 K/PI3 K and p-Akt/Akt in the brain tissue of mice in the TBM+miR-708-5 p inhibitor group were significantly lower than those in the TBM+NC-inhibitor group(P<0.05).The p-PI3 K/PI3 K and p-Akt/Akt of the cells in the miR-708-5 p overexpression group were significantly lower than those in the control group(P<0.05);the p-PI3 K/PI3 K,p-Akt/Akt of the cells in the C1 qtnf1 overexpression group were significantly higher than those in the control group(P<0.05);the p-PI3 K/PI3 K,p-Akt/Akt of the cells in the miR-708-5 p+C1 qtnf1 overexpression group were significantly lower than those in the C1 qtnf1 overexpression group(P<0.05);there was no significant difference in the p-PI3 K/PI3 K,p-Akt/Akt of the cells between the MiR-708-5 p+C1 qtnf1 overexpression group and the control group(P>0.05).Conclusion MiR-708-5 p can promote the occurrence of brain injury in TBM mice,and its mechanism may be related to down-regulating the expression of C1 qtnf1 and inhibiting the PI3 K/Akt signaling pathway.
作者
吴芳
郭俊
李蓓
齐红艳
WU Fang;GUO Jun;LI Bei;QI Hongyan(Department of Neurology,Xi'an Children's Hospital,,Xi'an 710000,Shaanxi Province,China;Department of Neurology,Tangdu Hospital,,Xi'an 710000,Shaanxi Province,China)
出处
《新乡医学院学报》
CAS
2021年第1期11-17,共7页
Journal of Xinxiang Medical University
基金
陕西省自然科学基础研究计划面上项目(编号:2016JM8033)。
