Zhang Ye, Xia Huiqiang, Yi Weiwei, Lan Haiyang, Yang Zhijie, Han Fei, Tang Pan, Liu Bo
Department of Orthopedics, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, P.R.China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2021 Mar 15;35(3):366-374. doi: 10.7507/1002-1892.202009057.
To investigate the effect of zinc finger protein A20 on lumbar intervertebral disc degeneration in rabbits.
Twenty-six 3-month-old New Zealand rabbits, 2.0-2.5 kg in weight, were used to establish the model of intervertebral disc degeneration at L , L , and L by transabdominal needle puncture. At 4 weeks after operation, the 24 rabbits were randomly divided into 4 groups after successful modeling, which checked by MRI. The target intervertebral discs of each group were injected with zinc finger protein A20 overexpressed adenovirus (Ov-A20 group), empty carrier adenovirus (NC group), phosphate buffer saline (control group), and shRNA-A20 adenovirus (Sh-A20 group). The biological responses of animals in each group were comprehensive scored before 1 day of injection and after 1, 2, 3, and 6 days of injection. At 2, 4, and 8 weeks after injection, the animals in each group were observed by MRI to obtain the exact T2 relaxation time (T2 signal value). After MRI examination, the animals were killed to take the degenerative intervertebral disc tissue; and the tissue was detected by Alcian blue staining to observed the intervertebral disc degeneration. The expressions of zinc finger protein A20, collagen Ⅱ, and aggrecan were detected by immunohistochemistry staining. The expressions of zinc finger protein A20, nuclear factor κB binding protein [P65, phosphate P65 (P-P65), collagen Ⅱ, aggrecan], inflammatory factors [tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β)], autophagy-related protein [LC3 (LC3Ⅱ/LC3Ⅰ) and P62] were detected by Western blot.
The comprehensive score of biological response in each group after injection was significantly lower than that before injection ( <0.05). At 6 days after injection, the comprehensive score of biological response in the Sh-A20 group was significantly lower than that in other groups ( <0.05), and there was no significant difference among other groups ( >0.05). The detection of MRI showed that the T2 signal value in the Ov-A20 group was the highest at 2, 4, and 8 weeks after injection ( <0.05), and the T2 signal value in the Sh-A20 group was the lowest at 2 and 4 weeks after injection ( <0.05). There was no significant difference between other groups ( >0.05). Alcian blue staining showed that the expression of aggrecan was the highest in Ov-A20 group and the lowest in Sh-A20 group at 4 weeks ( <0.05); the expression of aggrecan in Ov-A20 group was the highest at 8 weeks ( <0.05), and there was no significant difference between other groups ( >0.05). Immunohistochemical staining showed that the expressions of zinc finger protein A20, collagen Ⅱ, and aggrecan were the highest in Ov-A20 group and lowest in Sh-A20 group ( <0.05). Western blot showed that the expressions of zinc finger protein A20, collagen Ⅱ, aggrecan, and LC3 (LC3Ⅱ/LC3Ⅰ) proteins were the highest in the Ov-A20 group and the lowest in Sh-A20 group ( <0.05), while the expressions of P-P65, TNF-α, IL-1β, and P62 proteins were the lowest in Ov-A20 group and the highest in Sh-A20 group ( <0.05). There was no significant difference in the expression of p65 protein between groups ( >0.05).
Zinc finger protein A20 can effectively regulate the process of lumbar intervertebral disc degeneration in rabbits by inhibiting inflammation.
探讨锌指蛋白A20对兔腰椎间盘退变的影响。
选取26只3月龄、体重2.0 - 2.5 kg的新西兰兔，经腹针刺法建立L、L、L节段椎间盘退变模型。术后4周，经磁共振成像（MRI）检查建模成功后，将24只兔随机分为4组。对每组目标椎间盘分别注射过表达锌指蛋白A20的腺病毒（Ov - A20组）、空载体腺病毒（NC组）、磷酸盐缓冲液（对照组）和shRNA - A20腺病毒（Sh - A20组）。在注射前1天及注射后1、2、3和6天对每组动物的生物学反应进行综合评分。注射后2、4和8周，通过MRI观察每组动物，获取确切的T2弛豫时间（T2信号值）。MRI检查后，处死动物获取退变椎间盘组织；采用阿尔新蓝染色检测组织，观察椎间盘退变情况。通过免疫组织化学染色检测锌指蛋白A20、Ⅱ型胶原和聚集蛋白聚糖的表达。采用蛋白质免疫印迹法检测锌指蛋白A20、核因子κB结合蛋白[P65、磷酸化P65（P - P65）、Ⅱ型胶原、聚集蛋白聚糖]、炎症因子[肿瘤坏死因子α（TNF - α）、白细胞介素1β（IL - 1β）]、自噬相关蛋白[微管相关蛋白1轻链3（LC3，LC3Ⅱ/LC3Ⅰ）和P62]的表达。
注射后每组生物学反应综合评分均显著低于注射前（<0.05）。注射后6天，Sh - A20组生物学反应综合评分显著低于其他组（<0.05），其他组间差异无统计学意义（>0.05）。MRI检测显示，注射后2、4和8周，Ov - A20组T2信号值最高（<0.05），注射后2和4周，Sh - A20组T2信号值最低（<0.05）。其他组间差异无统计学意义（>0.05）。阿尔新蓝染色显示，4周时Ov - A20组聚集蛋白聚糖表达最高，Sh - A20组最低（<0.05）；8周时Ov - A20组聚集蛋白聚糖表达最高（<0.05），其他组间差异无统计学意义（>0.05）。免疫组织化学染色显示，Ov - A20组锌指蛋白A20、Ⅱ型胶原和聚集蛋白聚糖表达最高，Sh - A20组最低（<0.05）。蛋白质免疫印迹法显示，Ov - A20组锌指蛋白A20、Ⅱ型胶原、聚集蛋白聚糖和LC3（LC3Ⅱ/LC3Ⅰ）蛋白表达最高，Sh - A20组最低（<0.05），而Ov - A20组P - P65、TNF - α、IL - 1β和P62蛋白表达最低，Sh - A20组最高（<0.05）。各组间p65蛋白表达差异无统计学意义（>0.05）。
锌指蛋白A20可通过抑制炎症有效调控兔腰椎间盘退变进程。
