miR-186-5p通过抑...通路改善脊髓损伤后神经修复_蔡军.pdf

基金项目:新疆维吾尔自治区自然科学基金项目(2018D01C011)作者简介:蔡军，男，1985 09 生，硕士，主治医师，E-mail:181106153 qq com收稿日期:2022 11 18mi-186-5p 通过抑制 HMGB1 信号通路改善脊髓损伤后神经修复蔡军，艾孜孜江买买塔吾拉，张玉新*(新疆喀什地区第一人民医院脊柱外科，喀什844000;*通讯作者，E-mail:zhang_yx1992163 com)摘要:目的探究 mi-186-5p 在脊髓损伤(SCI)中的变化及对神经修复的影响。方法将小胶质细胞分为对照组(正常培养)、脂多糖(LPS)组(1 g/ml LPS 刺激 24 h)、LPS+mi-186-5p-mimic 组(转染 mi-186-5p-mimic 后 1 g/ml LPS 刺激24 h)和 LPS+mi-186-5p-inhibitor 组(转染 mi-186-5p-inhibitor 后 1 g/ml LPS 刺激 24 h)。通过 T-PC 检测细胞中 mi-186-5p 水平，通过 T-PC 和 Western blot 检测细胞中高迁移率族蛋白 1(HMGB1)mNA 和蛋白表达水平，通过 ELISA 检测细胞中肿瘤坏死因子 (TNF-)和白细胞介素 6(IL-6)的水平。将大鼠分为假手术组、SCI+LV-NC 组和 SCI+LV-mi-186-5p 组，每组 10 只大鼠，假手术组大鼠仅行椎板切除术，其余组采用脊髓打击器建立 SCI 模型。建模后，假手术组大鼠正常饲养，SCI+LV-NC 组和 SCI+LV-mi-186-5 组大鼠分别脊髓注射空载体慢病毒和过表达 mi-186-5p 的重组慢病毒。通过T-PC 检测脊髓组织中 mi-186-5p 水平。通过 Western blot 检测脊髓组织中 HMGB1、Toll 样受体 4(TL4)、p-p65、total p65、诱导型一氧化氮合酶(iNOS)、血红素加氧酶 1(HO-1)、超氧化物歧化酶 1(SOD-1)、Bax 和 Bcl-2 的蛋白水平。ELISA 检测脊髓组织中的 TNF-和 IL-6 的水平;免疫荧光检测脊髓组织中的离子钙结合接头蛋白分子 1(IBA-1)、iNOS、胶质纤维酸性蛋白(GFAP)和神经丝蛋白 200(NF-200)的表达;通过 BBB 评分评价大鼠的运动功能;通过尼氏染色测定脊髓前角运动神经元数量。结果与对照组相比，LPS 组 mi-186-5p 表达降低(P 0 05)，而 HMGB1、iNOS、TNF-和 IL-6 水平升高(P0 05)。与 LPS 组相比，LPS+mi-186-5p-mimic 组 mi-186-5p 表达升高(P 0 05)，而 HMGB1、iNOS、TNF-和 IL-6 水平降低(P 005)。与 LPS 组相比，LPS+mi-186-5p-inhibitor 组 mi-186-5p 水平降低(P 0 05)，而 HMGB1、iNOS、TNF-和 IL-6 水平升高(P0 05)。与假手术组相比，SCI+LV-NC 组大鼠脊髓出现明显损伤，iNOS/IBA-1 比例升高(P 0 05)，TNF-和 IL-6水平升高(P0 05)，Bax 蛋白表达上调(P0 05)，Bcl-2、HO-1 和 SOD-1 蛋白表达下调(P 0 05)，GFAP 和 IBA-1 的阳性染色比例增加(P0 05)，HMGB1 和 TL4 的蛋白水平及 NF-B p65 的磷酸化水平增加(P 0 05)，BBB 评分降低(P 0 05)，脊髓前角运动神经元数量减少(P 0 05)。与 SCI+LV-NC 组相比，SCI+LV-mi-186-5p 组脊髓损伤区域减少，iNOS/IBA-1比例降低(P0 05)，TNF-和 IL-6 水平降低(P0 05)，Bax 蛋白表达下调(P 0 05)，Bcl-2、HO-1 和 SOD-1 蛋白表达上调(P0 05)，GFAP 和 IBA-1 的阳性染色比例降低(P 0 05)，HMGB1 和 TL4 的蛋白水平及 NF-B p65 的磷酸化水平降低(P0 05)，BBB 评分升高(P 0 05)，脊髓前角运动神经元数量增多(P 0 05)。结论上调 mi-186-5p 通过抑制HMGB1/TL4/NF-B 通路提高了脊髓损伤大鼠的运动功能和神经修复。关键词:脊髓损伤;mi-186-5p;高迁移率族蛋白 1;HMGB1/TL4/NF-B 通路;神经修复中图分类号:363文献标志码:A文章编号:1007 6611(2023)04 0470 10DOI:1013753/j issn1007 6611202304009Mi-186-5p improves nerve repair after spinal cord injury by inhibiting HMGB1 signaling pathwayCAI Jun，Aizizijiang Maimaitawula，ZHANG Yuxin*(Department of Spinal Orthopaedics，First People s Hospital of Kashi，Kashi844000，China;*Corresponding author，E-mail:zhang_yx1992163 com)Abstract:ObjectiveTo investigate the expression of mi-186-5p in spinal cord injury(SCI)and its effect on nerve repairMethodsThe microglia were divided into control group(normal culture)，lipopolysaccharide(LPS)group(1 g/ml LPS for 24 h)，LPS+mi-186-5p-mimic group(1 g/ml LPS for 24 h after transfection with mi-186-5p-mimic)and LPS+mi-186-5p-inhibitor group(1 g/ml LPS for 24 h after transfection with LPS+mi-186-5p-inhibitor)The level of mi-186-5p in cells was detected by T-PC，the expression of high mobility group protein 1(HMGB1)mNA and protein was detected by T-PC and Western blot，and thelevels of tumor necrosis factor-(TNF-)and interleukin-6(IL-6)were detected by ELISA The rats were divided into shamgroup，SCI+LV-NC group and SCI+LV-mi-186-5p group，with 10 rats in each group The rats in sham group only received lami-nectomy，and SCI model was established using impactor in the other groups After modeling，the rats were fed normally in sham group，the rats were injected with empty vector lentivirus and recombinant lentivirus overexpressing mi-186-5p in SCI+LV-NC group and SCI+LV-mi-186-5 group，respectively The level of mi-186-5p in spinal cord tissue was detected by T-PC The protein levels ofHMGB1，Toll-like receptor 4(TL4)，p-p65，total p65，induction nitric oxide synthase(iNOS)，heme oxygenase-1(HO-1)，super-074J Shanxi Med Univ，Apr 2023，Vol 54 No 4oxide dismutase-1(SOD-1)，Bax and Bcl-2 in spinal cord tissue were detected by Western blot The levels of TNF-and IL-6 in spinalcord tissue were detected by ELISA The expression levels of ionized calcium-binding adapter molecule 1(IBA-1)，iNOS，glialfibrillary acidic protein(GFAP)and neurofilament-200(NF-200)in spinal cord tissue were detected by immunofluorescence The motorfunction of rats was evaluated by BBB score The number of motoneurons in the anterior horn of spinal cord was measured by NisslstainingesultsCompared with control group，the expression of mi-186-5p was decreased in LPS group(P0 05)，while thelevels of HMGB1，iNOS，TNF-and IL-6 were increased(P 0 05)Compared with LPS group，the expression of mi-186-5p inLPS+mi-186-5p-mimic group was increased(P0 05)，while the levels of HMGB1，iNOS，TNF-and IL-6 were decreased(P 0 05)Compared with LPS group，the level of mi-186-5p was decreased in LPS+mi-186-5p-inhibitor group(P0 05)，while thelevels of HMGB1，iNOS，TNF-and IL-6 were increased(P 0 05)Compared with sham group，there was obvious spinal cordinjury in SCI+LV-NC group Compared with sham group，the proportion of iNOS/IBA-1 in SCI+LV-NC group was increased(P 005)，the levels of TNF-and IL-6 were increased(P0 05)，the expression of Bax protein was increased(P0 05)，the expres-sion levels of Bcl-2，HO-1 and SOD-1 protein were decreased(P0 05)，the positive staining proportions o