[1]吴　芳,郭　俊,李　蓓,等.微RNA-708-5p对结核性脑膜炎小鼠脑损伤的影响[J].新乡医学院学报,2021,38(1):011-17.[doi:10.7683/xxyxyxb.2021.01.003]
　WU Fang,GUO Jun,LI Bei,et al.Effect of microRNA-708-5p on brain damage of mice with tuberculous meningitis[J].Journal of Xinxiang Medical University,2021,38(1):011-17.[doi:10.7683/xxyxyxb.2021.01.003]
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微RNA-708-5p对结核性脑膜炎小鼠脑损伤的影响
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
38
期数:
2021年1
页码:
011-17
栏目:
基础研究
出版日期:
2021-01-05

文章信息/Info

Title:
Effect of microRNA-708-5p on brain damage of mice with tuberculous meningitis
作者:
吴　芳1; 郭　俊2; 李　蓓1; 齐红艳1
（1.西安市儿童医院神经内科，陕西　西安　710000；2.唐都医院神经内科，陕西　西安　710000）
Author(s):
WU Fang1; GUO Jun2; LI Bei1; QI Hongyan1
（1.Department of Neurology,Xi′an Children′s Hospital,Xi′an 710000,Shaanxi Province,China2.Department of Neurology,Tangdu Hospital,Xi′an 710000,Shaanxi Province,China）
关键词:
结核性脑膜炎; 微RNA-708-5p; 补体C1q肿瘤坏死因子相关蛋白１; 磷脂酰肌醇3激酶/蛋白激酶B信号通路
Keywords:
tuberculous meningitismicroRNA-708-5pcomplement C1q tumor necrosis factor-related protein 1phosphatidylinositol 3 kinase /protein kinase B signaling pathway
分类号:
R363
DOI:
10.7683/xxyxyxb.2021.01.003
文献标志码:
A
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-708-5p inhibitor组及TBM+NC-inhibitor组，每组12只。TBM组、TBM+miR-708-5p inhibitor组及TBM+NC-inhibitor组小鼠经尾静脉注射0.2 mL结核分枝杆菌H37Rv菌悬液（2.5×109 CFU·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-708-5p inhibitor组、TBM+NC-inhibitor组小鼠分别经尾静脉注射0.2 mL miR-708-5p inhibitor（2 mg·kg-1）和NC-inhibitor（2mg·kg-1），每2 d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-708-5p mimic和 Vector、NC-mimic和 pcDNA-C1qtnf1、miR-708-5p mimic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-708-5p inhibitor组，空白组细胞不做处理，NC-inhibitor组、miR-708-5p inhibitor组细胞分别转染 NC-inhibitor、miR-708-5p inhibitor。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C1qtnf1 mRNA相对表达量以及各组GIBH001细胞中C1qtnf1 mRNA相对表达量；采用Western blot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-708-5p inhibitor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-708-5p inhibitor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-708-5p inhibitor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），c1qtnf1 mrna相对表达量显著降低（P<0.05）；与tbm+nc-inhibitor组比较，tbm+mir-708-5p inhibitor组小鼠脑组织中mir-708-5p的相对表达量显著降低（P<0.05），c1qtnf1 mrna相对表达量显著升高（P<0.05）。mir-708-5p过表达组细胞中c1qtnf1 mrna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c1qtnf1 mrna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c1qtnf1 mrna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C1qtnf1 mRNA相对表达量与空白组比较差异无统计学意义（P>0.05）；miR-708-5p inhibitor组细胞中C1qtnf1 mRNA相对表达量显著低于空白组（P<0.05）；mir-708-5p inhibitor组细胞中c1qtnf1="" mrna相对表达量显著低于nc-inhibitor组（PPPPPPP>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。undefinedundefinedundefinedundefinedundefinedundefinedundefinedundefinedundefinedundefinedundefinedundefinedundefinedundefined/i="">/i="">/i="">/i="">/i="">/html>
Abstract:
Objective　To investigate the role and mechanism of microRNA (miR) -708-5p 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-708-5p inhibitor group and TBM+NC-inhibitor group,with 12 mice in each group.Mice in the TBM group,TBM+miR-708-5p inhibitor group and TBM+NC-inhibitor group were injected with 0.2 mL of Mycobacterium tuberculosis H37Rv suspension (2.5×109 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-708-5p inhibitor group and TBM+NC-inhibitor group were respectively injected with 0.2 mL of miR-708-5p 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-708-5p overexpression group,complement C1q tumor necrosis factor-related protein 1 (C1qtnf1) overexpression group and miR-708-5p+C1qtnf1 overexpression group according to different transfection vectors.The cells in above four groups were transfected with NC-mimics and Vector,miR-708-5p mimic and Vector,NC-mimic and pcDNA-C1qtnf1,miR-708-5p mimic and pcDNA-C1qtnf1,respectively.Additionally,the GIBH001 cells were divided into blank group,NC-inhibitor group and miR-708-5p 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-708-5p inhibitor group were transfected with NC-inhibitor and miR-708-5p 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-708-5p and C1qtnf1 mRNA in the brain tissue of mice and C1qtnf1 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 (PI3K),Phosphorylated phosphatidylinositol 3 kinase (P-PI3K),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-708-5p overexpression group,C1qtnf1 overexpression group,miR-708-5p+C1qtnf1 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-708-5p 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,contrala-teral 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-708-5p inhibitor group was significantly lower than that in the TBM+NC-inhibitor group (P<0.05).The levels of TNF-α and IL-1β in 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-α and IL-1β in the brain tissue of mice in the TBM+miR-708-5p 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-708-5p in the brain tissue of mice in the TBM group was significantly increased (P<0.05),and the relative expression of C1qtnf1 mRNA was significantly reduced (P<0.05);compared with the TBM+NC-inhibitor group,the relative expression of miR-708-5p in the brain tissue of mice in the TBM+miR-708-5p inhibitor group was significantly reduced (P<0.05),and the relative expression of C1qtnf1 mRNA was significantly increased (P<0.05).The relative expression of C1qtnf1 mRNA of the cells in the miR-708-5p overexpression group was lower than that in the control group (P<0.05);the relative expression of C1qtnf1 mRNA of the cells in the C1qtnf1 overexpression group was higher than that in the control group (P<0.05);the relative expression of C1qtnf1 mRNA of the cells in the miR-708-5p+C1qtnf1 overexpression group was significantly lower than that in the C1qtnf1 overexpression group (P<0.05).There was no significant difference in the relative expression of C1qtnf1 mRNA of the cells between the miR-708-5p+C1qtnf1 overexpression group and the control group (P>0.05).There was no significant difference in the relative expression of C1qtnf1 mRNA of the cells between the NC-inhibitor group and the blank group (P>0.05);the relative expression of C1qtnf1 mRNA of the cells in the miR-708-5p inhibitor group was significantly lower than that in the blank group (P<0.05);the relative expression of C1qtnf1 mRNA of the cells in the miR-708-5p inhibitor group was significantly lower than that in the NC-inhibitor group (P<0.05).The p-PI3K/PI3K 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-PI3K/PI3K and p-Akt/Akt in the brain tissue of mice in the TBM+miR-708-5p inhibitor group were significantly lower than those in the TBM+NC-inhibitor group (P<0.05).The p-PI3K/PI3K and p-Akt/Akt of the cells in the miR-708-5p overexpression group were significantly lower than those in the control group (P<0.05);the p-PI3K/PI3K,P-Akt/Akt of the cells in the C1qtnf1 overexpression group were significantly higher than those in the control group (P<0.05);the p-PI3K/PI3K,P-Akt/Akt of the cells in the miR-708-5p+C1qtnf1 overexpression group were significantly lower than those in the C1qtnf1 overexpression group (P<0.05);there was no significant difference in the p-PI3K/PI3K,P-Akt/Akt of the cells between the MiR-708-5p+C1qtnf1 overexpression group and the control group (P>0.05).Conclusion　MiR-708-5p can promote the occurrence of brain injury in TBM mice,and its mechanism may be related to down-regulating the expression of C1qtnf1 and inhibiting the PI3K/Akt signaling pathway.

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更新日期/Last Update: 2021-01-05