Objective: To investigate the effect of zinc finger protein A20 on lumbar intervertebral disc degeneration in rabbits.
Methods: 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 3, 4, L 4, 5, and L 5, 6 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.
Results: The comprehensive score of biological response in each group after injection was significantly lower than that before injection ( P<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 ( P<0.05), and there was no significant difference among other groups ( P>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 ( P<0.05), and the T2 signal value in the Sh-A20 group was the lowest at 2 and 4 weeks after injection ( P<0.05). There was no significant difference between other groups ( P>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 ( P<0.05); the expression of aggrecan in Ov-A20 group was the highest at 8 weeks ( P<0.05), and there was no significant difference between other groups ( P>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 ( P<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 ( P<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 ( P<0.05). There was no significant difference in the expression of p65 protein between groups ( P>0.05).
Conclusion: Zinc finger protein A20 can effectively regulate the process of lumbar intervertebral disc degeneration in rabbits by inhibiting inflammation.
目的: 探讨锌指蛋白 A20 对兔腰椎间盘退变的影响。.
方法: 取 3 月龄新西兰大白兔 26 只，体质量 2.0～2.5 kg，经腹细针穿刺法制备 L 3、4、L 4、5、L 5、6 椎间盘退变模型，其中 24 只术后 4 周 MRI 检查明确造模成功，随机分为 4 组（ n=6），于目标椎间盘中分别注射锌指蛋白 A20 过表达腺病毒（过表达 A20 组）、空载体腺病毒（空载体组）、PBS 液（对照组）、锌指蛋白 A20干扰腺病毒（干扰 A20 组）。于注射前 1 d 及注射后 1、2、3、6 d 行生物反应综合评分；注射后 2、4、8 周，各组行 MRI 检查并测量 T2 弛豫时间（T2 信号值）后，取材行阿利辛蓝染色观察椎间盘髓核细胞退变情况，免疫组织化学染色检测锌指蛋白 A20 以及椎间盘退变相关指标（Ⅱ型胶原、蛋白聚糖）的表达，Western blot 检测锌指蛋白 A20、NF-κB 结合蛋白［P65、磷酸化 P65（phosphate P65，P-P65）、Ⅱ型胶原、蛋白聚糖］、自噬相关蛋白［LC3 （LC3Ⅱ/LC3Ⅰ）、P62］以及炎症因子（TNF-α、IL-1β）的表达。.
结果: 各组注射后各时间点生物反应综合评分均明显低于注射前 1 d（ P<0.05）；注射后 6 d 干扰 A20 组评分明显低于其他组（ P<0.05），其他组间比较差异均无统计学意义（ P>0.05）。MRI 检测提示，注射后 2、4、8 周过表达 A20 组 T2 信号值均最高（ P<0.05），2、4 周时干扰 A20 组最低（ P<0.05），其余组间差异均无统计学意义（ P>0.05）。阿利辛蓝染色显示，注射后 4 周过表达 A20 组蛋白聚糖含量最高（ P<0.05）、干扰 A20 组最低（ P<0.05）；8 周时过表达 A20 组蛋白聚糖含量显著高于其他组（ P<0.05），其他组间比较差异无统计学意义（ P>0.05）。免疫组织化学染色示，锌指蛋白 A20、Ⅱ型胶原、蛋白聚糖表达过表达 A20 组最高（ P<0.05），干扰 A20 组上述蛋白表达最低（ P<0.05）。Western blot 检测示锌指蛋白 A20、蛋白聚糖、Ⅱ型胶原、LC3 （LC3Ⅱ/LC3Ⅰ）蛋白相对表达量过表达 A20 组最高、干扰 A20 组最低，而 P-P65、TNF-α、IL-1β、P62 蛋白相对表达量过表达 A20 组最低、干扰 A20 组最高，与其他组比较差异均有统计学意义（ P<0.05）；各组 P65 蛋白相对表达量差异均无统计学意义（ P>0.05）。.
结论: 锌指蛋白 A20 能通过抑制炎症反应，有效延缓兔腰椎间盘退变的进程。.
Keywords: Intervertebral disc degeneration; gene therapy; inflammation; nuclear factor κB pathway; rabbit; zinc finger protein A20.