紫红獐牙菜酮提取物抑制脊髓损伤后炎性反应及凋亡的机制研究  

Mechanism of inhibition of inflammatory response and apoptosis after spinal cord injury by Swertia punicea hemsl xanthone extract

作　　者：刘振刚 施建东 洪剑鑫 刘宇鹏[2] 卢一生 Liu Zhengang;Shi Jiandong;Hong Jianxin;Liu Yupeng;Lu Yisheng(Department of Orthopedics,No.903 Hospital,Joint Logistics Support Force of PLA,Hangzhou 310002,China;Department of Orthopedics,Zhongshan Hospital Affiliated to Dalian University,Dalian 116085,China)

机构地区：[1]解放军联勤保障部队第九〇三医院骨科,杭州310002 [2]大连大学附属中山医院骨科,116085

出　　处：《中华实验外科杂志》2022年第5期920-923,共4页Chinese Journal of Experimental Surgery

摘　　要：目的探讨紫红獐牙菜酮提取物抑制脊髓损伤后炎性反应及凋亡机制。方法PC-12细胞购自美国典型培养物保藏中心。采用脂多糖(LPS)处理PC-12细胞建立脊髓损伤细胞模型(LPS组),未经处理细胞为NC组;不同浓度紫红獐牙菜酮提取物处理LPS组细胞;将抗微小核糖核酸(miR)-NC和抗miR-136-5p转染至LPS细胞(标记为抗miR-NC+LPS组、抗miR-136-5p+LPS组);将miR-NC、miR-136-5p转染至60μg/ml紫红獐牙菜酮提取物处理的LPS组细胞(标记为60μg/ml紫红獐牙菜酮提取物+miR-NC+LPS组、60μg/ml紫红獐牙菜酮提取物+miR-136-5p+LPS组)。溴化-3-(4,5-二甲基-2-噻唑)-2,5-二苯基四氮唑(MTT)检测细胞活性;流式细胞仪检测细胞凋亡;蛋白质印迹法(Western blot)检测剪切的半胱天冬酶-3(cleaved Caspase-3)、磷酸化p65(p-p65)及磷酸化IκBα(p-IκBα)蛋白表达;酶联免疫吸附法法检测白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)及肿瘤坏死因子α(TNF-α)表达;实时荧光定量聚合酶链式反应(RT-qPCR)检测miR-136-5p表达。两组间比较行t检验,多组间比较采用单因素方差分析,组间两两比较采用SNK检验。结果15 mg/ml紫红獐牙菜酮提取物+LPS组、30 mg/ml紫红獐牙菜酮提取物+LPS组及60 mg/ml紫红獐牙菜酮提取物+LPS组细胞活性显著高于LPS组(0.67±0.06、0.96±0.09、1.05±0.10比0.54±0.05),凋亡率[(16.13±1.25)%、(10.23±0.91)%、(6.89±0.63)%比(19.43±1.61)%]、cleaved Caspase-3蛋白表达水平(0.71±0.06、0.56±0.05、0.43±0.04比0.87±0.07)、IL-1β[(131.02±11.63)、(95.36±9.63)、(72.69±6.94)pg/ml比(169.32±13.52)pg/ml]、IL-6[(203.81±18.51)、(151.87±13.21)、(115.93±10.06)pg/ml比(243.61±22.25)pg/ml]、TNF-α[(1.82±0.04)、(1.43±0.12)、(1.14±0.09)pg/ml比(2.13±0.19)pg/ml]及miR-136-5p(2.21±0.17、1.61±0.14、1.23±0.11比2.67±0.24)表达显著低于LPS组,差异有统计学意义(F=83.393、288.901、125.546、173.231、131.432、116.412、167.637,P<0.05)。抗miR-136-5p+LPS组细胞�Objective To study the mechanism of inhibition of inflammatory response and apoptosis after spinal cord injury by Swertia punicea hemsl xanthone extract.Methods The PC-12 cells were treated with lipopolysaccharide(LPS)to establish a spinal cord injury cell model(LPS group),and the untreated cells served as the normal control(NC)group;the LPS group cells were treated with different concentrations of Swertia punicea hemsl xanthone extract;the anti-micro ribonucleic acid(miR)-NC and anti-miR-136-5p were transfected into cells in LPS group(labeled as anti-miR-NC+LPS group,anti-miR-136-5p+LPS group);miR-NC and miR-136-5p were transfected into the cells in LPS group treated with 60μg/ml Swertia punicea hemsl xanthone extract(labeled as 60μg/ml Swertia punicea hemsl xanthone extract+miR-NC+LPS group,and 60μg/ml Swertia punicea hemsl xanthone extract+miR-136-5p+LPS group,respectively).The MTT assay was used to measure cell viability.The flow cytometry was used to examine cell apoptosis.Western blotting was used to detect cleaved Caspase-3(cleaved Caspase-3),phosphorylated p65(Phospho-p65,p-p65)and phosphorylated IκBα(p-IκBα)protein expression.The enzyme-linked immunosorbent assay was used to detect interleukin-1β(IL-1β),IL-6 and tumor necrosis factor-α(TNF-α)expression.The real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect microribonucleic acid-136-5p(miR-136-5p)expression.The t test was used for comparison between two groups,one-way analysis of variance was used for comparison between multiple groups,and SNK test was used for pairwise comparison between groups.Results The cell viability in anti-miR-136-5p+LPS group was significantly higher than in the anti-miR-NC+LPS group(1.02±0.10 vs.0.56±0.03),and apoptosis rate[(6.35±0.59)%vs.(18.46±1.57)%],Cleaved-caspase-3(0.43±0.04 vs.0.88±0.07),IL-1β[(65.39±6.11)vs.(170.02±14.15)pg/ml],IL-6[(98.61±9.24)vs.(241.02±19.47)pg/ml],TNF-α[(1.01±0.09)vs.(2.18±0.17)pg/ml]and miR-136-5p(0.46±0.04 vs.1.00±0.11)were significan

关 键 词：紫红獐牙菜酮提取物 微小RNA 脊髓损伤 炎性反应 凋亡 

分 类 号：R285.5[医药卫生—中药学]