Experimental study on the effect of zinc finger protein A20 on lumbar intervertebral disc degeneration in rabbits_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

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;

Corresponding author：

LIU Bo, Email: boliucqmu@qq.com

Keywords：

Intervertebral disc degeneration; gene therapy; zinc finger protein A20; nuclear factor κB pathway; inflammation; rabbit

DOI：

10.7507/1002-1892.202009057

Video：

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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.

Citation： ZHANG Ye, XIA Huiqiang, YI Weiwei, LAN Haiyang, YANG Zhijie, HAN Fei, TANG Pan, LIU Bo. Experimental study on the effect of zinc finger protein A20 on lumbar intervertebral disc degeneration in rabbits. Chinese Journal of Reparative and Reconstructive Surgery, 2021, 35(3): 366-374. doi: 10.7507/1002-1892.202009057 Copy

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5.	Tan Y, Yao X, Dai Z, et al. Bone morphogenetic protein 2 alleviated intervertebral disc degeneration through mediating the degradation of ECM and apoptosis of nucleus pulposus cells via the PI3K/Akt pathway. Int J Mol Med, 2019, 43(1): 583-592.
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7.	Serramito-Gómez I, Boada-Romero E, Slowicka K, et al. The anti-inflammatory protein TNFAIP3/A20 binds the WD40 domain of ATG16L1 to control the autophagic response, NFKB/NF-κB activation and intestinal homeostasis. Autophagy, 2019, 15(9): 1657-1659.
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9.	Kim T, Bae SC, Kang C. Synergistic activation of NF-κB by TNFAIP3 (A20) reduction and UBE2L3 (UBCH7) augment that synergistically elevate lupus risk. Arthritis Res Ther, 2020, 22(1): 93.
10.	Lee SH, Lee HR, Kwon JY, et al. A20 ameliorates inflammatory bowel disease in mice via inhibiting NF-κB and STAT3 activation. Immunol Lett, 2018, 198: 44-51.
11.	蓝海洋, 杨智杰, 夏辉强, 等. 锌指蛋白 A20 对内毒素刺激时人椎间盘髓核细胞炎症及退变的影响. 第三军医大学学报, 2019, 41(6): 543-548.
12.	黑龙, 张佳林, 李宏辉, 等. 经皮纤维环穿刺和纤维环切开法建立兔椎间盘退变模型的比较. 宁夏医科大学学报, 2015, 37(11): 1279-1282.
13.	Vo NV, Hartman RA, Yurube T, et al. Expression and regulation of metalloproteinases and their inhibitors in intervertebral disc aging and degeneration. Spine J, 2013, 13(3): 331-341.
14.	Chen Z, Han Y, Deng C, et al. Inflammation-dependent downregulation of miR-194-5p contributes to human intervertebral disc degeneration by targeting CUL4A and CUL4B. J Cell Physiol, 2019, 234(11): 19977-19989.
15.	Wang S, Li J, Tian J, et al. High amplitude and low frequency cyclic mechanical strain promotes degeneration of human nucleus pulposus cells via the NF-κB p65 pathway. J Cell Physiol, 2018, 233(9): 7206-7216.
16.	Shembade N, Ma A, Harhaj EW. Inhibition of NF-kappaB signaling by A20 through disruption of ubiquitin enzyme complexes. Science, 2010, 327(5969): 1135-1139.
17.	Polykratis A, Martens A, Eren RO, et al. A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis through its ZnF7 ubiquitin-binding domain. Nat Cell Biol, 2019, 21(6): 731-742.
18.	吴丽娟, 陈潇, 冯建男, 等. 锌指蛋白 A20 突变体转基因小鼠脓毒症肺损伤的研究. 第三军医大学学报, 2010, 32(5): 435-438.
19.	Li M, Shi X, Qian T, et al. A20 overexpression alleviates pristine-induced lupus nephritis by inhibiting the NF-κB and NLRP3 inflammasome activation in macrophages of mice. Int J Clin Exp Med, 2015, 8(10): 17430-17440.
20.	Dang Y, Li Z, Wei Q, et al. Protective effect of apigenin on acrylonitrile-induced inflammation and apoptosis in testicular cells via the NF-κB pathway in rats. Inflammation, 2018, 41(4): 1448-1459.
21.	Yi WW, Wen YF, Tan FQ, et al. Impact of NF-κB pathway on the apoptosis-inflammation-autophagy crosstalk in human degenerative nucleus pulposus cells. Aging (Albany NY), 2019, 11(17): 7294-7306.
22.	Jiang W, Zhang X, Hao J, et al. SIRT1 protects against apoptosis by promoting autophagy in degenerative human disc nucleus pulposus cells. Sci Rep, 2014, 4: 7456.

1. Yang S, Zhang F, Ma J, et al. Intervertebral disc ageing and degeneration: The antiapoptotic effect of oestrogen. Ageing Res Rev, 2020, 57: 100978.
2. Chen J, Xie JJ, Jin MY, et al. Sirt6 overexpression suppresses senescence and apoptosis of nucleus pulposus cells by inducing autophagy in a model of intervertebral disc degeneration. Cell Death Dis, 2018, 9(2): 56.
3. Chen ZB, Yu YB, Wa QB, et al. The role of quinazoline in ameliorating intervertebral disc degeneration by inhibiting oxidative stress and anti-inflammation via NF-κB/MAPKs signaling pathway. Eur Rev Med Pharmacol Sci, 2020, 24(4): 2077-2086.
4. Zhong H, Zhou Z, Guo L, et al. The miR-623/CXCL12 axis inhibits LPS-induced nucleus pulposus cell apoptosis and senescence. Mech Ageing Dev, 2021, 194: 111417.
5. Tan Y, Yao X, Dai Z, et al. Bone morphogenetic protein 2 alleviated intervertebral disc degeneration through mediating the degradation of ECM and apoptosis of nucleus pulposus cells via the PI3K/Akt pathway. Int J Mol Med, 2019, 43(1): 583-592.
6. Malynn BA, Ma A. A20: A multifunctional tool for regulating immunity and preventing disease. Cell Immuno, 2019, 340: 103914.
7. Serramito-Gómez I, Boada-Romero E, Slowicka K, et al. The anti-inflammatory protein TNFAIP3/A20 binds the WD40 domain of ATG16L1 to control the autophagic response, NFKB/NF-κB activation and intestinal homeostasis. Autophagy, 2019, 15(9): 1657-1659.
8. Chu Y, Vahl JC, Kumar D, et al. B cells lacking the tumor suppressor TNFAIP3/A20 display impaired differentiation and hyperactivation and cause inflammation and autoimmunity in aged mice. Blood, 2011, 117(7): 2227-2236.
9. Kim T, Bae SC, Kang C. Synergistic activation of NF-κB by TNFAIP3 (A20) reduction and UBE2L3 (UBCH7) augment that synergistically elevate lupus risk. Arthritis Res Ther, 2020, 22(1): 93.
10. Lee SH, Lee HR, Kwon JY, et al. A20 ameliorates inflammatory bowel disease in mice via inhibiting NF-κB and STAT3 activation. Immunol Lett, 2018, 198: 44-51.
11. 蓝海洋, 杨智杰, 夏辉强, 等. 锌指蛋白 A20 对内毒素刺激时人椎间盘髓核细胞炎症及退变的影响. 第三军医大学学报, 2019, 41(6): 543-548.
12. 黑龙, 张佳林, 李宏辉, 等. 经皮纤维环穿刺和纤维环切开法建立兔椎间盘退变模型的比较. 宁夏医科大学学报, 2015, 37(11): 1279-1282.
13. Vo NV, Hartman RA, Yurube T, et al. Expression and regulation of metalloproteinases and their inhibitors in intervertebral disc aging and degeneration. Spine J, 2013, 13(3): 331-341.
14. Chen Z, Han Y, Deng C, et al. Inflammation-dependent downregulation of miR-194-5p contributes to human intervertebral disc degeneration by targeting CUL4A and CUL4B. J Cell Physiol, 2019, 234(11): 19977-19989.
15. Wang S, Li J, Tian J, et al. High amplitude and low frequency cyclic mechanical strain promotes degeneration of human nucleus pulposus cells via the NF-κB p65 pathway. J Cell Physiol, 2018, 233(9): 7206-7216.
16. Shembade N, Ma A, Harhaj EW. Inhibition of NF-kappaB signaling by A20 through disruption of ubiquitin enzyme complexes. Science, 2010, 327(5969): 1135-1139.
17. Polykratis A, Martens A, Eren RO, et al. A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis through its ZnF7 ubiquitin-binding domain. Nat Cell Biol, 2019, 21(6): 731-742.
18. 吴丽娟, 陈潇, 冯建男, 等. 锌指蛋白 A20 突变体转基因小鼠脓毒症肺损伤的研究. 第三军医大学学报, 2010, 32(5): 435-438.
19. Li M, Shi X, Qian T, et al. A20 overexpression alleviates pristine-induced lupus nephritis by inhibiting the NF-κB and NLRP3 inflammasome activation in macrophages of mice. Int J Clin Exp Med, 2015, 8(10): 17430-17440.
20. Dang Y, Li Z, Wei Q, et al. Protective effect of apigenin on acrylonitrile-induced inflammation and apoptosis in testicular cells via the NF-κB pathway in rats. Inflammation, 2018, 41(4): 1448-1459.
21. Yi WW, Wen YF, Tan FQ, et al. Impact of NF-κB pathway on the apoptosis-inflammation-autophagy crosstalk in human degenerative nucleus pulposus cells. Aging (Albany NY), 2019, 11(17): 7294-7306.
22. Jiang W, Zhang X, Hao J, et al. SIRT1 protects against apoptosis by promoting autophagy in degenerative human disc nucleus pulposus cells. Sci Rep, 2014, 4: 7456.

Chinese Journal of Reparative and Reconstructive Surgery

Experimental study on the effect of zinc finger protein A20 on lumbar intervertebral disc degeneration in rabbits

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