A20 regulates inflammation through autophagy mediated by NF-κB pathway in human nucleus pulposus cells and ameliorates disc degeneration in vivo.

Intervertebral disc degeneration (IDD) is closely related to loss of the extracellular matrix (ECM), apoptosis and inflammation in nucleus pulposus cells (NPCs). It has been reported that Zinc finger protein A20/TNFAIP3 (A20) can inhibit the activity of the NF-κB pathway and promote autophagy. Therefore, we speculated that A20 can regulate inflammation and ameliorate IDD through autophagy mediated by NF-κB in human NPCs. Our results indicated that the expression of A20 and inflammatory factors in IDD tissues was increased. A20 is an essential negative regulator in the NF-κB pathway. Constructed adenoviral shRNA and overexpression vectors for A20 could effectively regulate the inflammation, autophagy, and activity of NF-κB, which in turn affected the progression of IDD. Inhibition of NF-κB on the basis of knocking down A20 results in increased autophagy, suggesting that A20-regulated autophagy was mediated by NF-κB. In vivo, A20 overexpression could ameliorate the progression of IDD and promote autophagy at the same time, while deletion of A20 leads to low levels of autophagy and severe degeneration. In summary, A20 plays an important role in inhibiting inflammation through autophagy mediated by NF-κB in NPCs and ameliorating IDD.

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

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 1ß (IL-1ß)], 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-1ß, 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.

[Comparative study on effectiveness of modified-transforaminal lumbar interbody fusion and posterior lumbar interbody fusion surgery in treatment of mild to moderate lumbar spondylolisthesis in middle-aged and elderly patients].

OBJECTIVE: To compare the effectiveness of modified transforaminal lumbar interbody fusion (modified-TLIF) and posterior lumbar interbody fusion (PLIF) for mild to moderate lumbar spondylolisthesis in middle-aged and elderly patients. METHODS: The clinical data of 106 patients with mild to moderate lumbar spondylolisthesis (Meyerding classification≤Ⅱ degree) who met the selection criteria between January 2015 and January 2017 were retrospectively analysed. All patients were divided into modified-TLIF group (54 cases) and PLIF group (52 cases) according to the different surgical methods. There was no significant difference in preoperative clinical data of gender, age, disease duration, sliding vertebra, Meyerding grade, and slippage type between the two groups ( P>0.05). The intraoperative blood loss, operation time, postoperative drainage volume, postoperative bed time, hospital stay, and complications of the two groups were recorded and compared. The improvement of pain and function were evaluated by the visual analogue scale (VAS) score and Japanese Orthopedic Association (JOA) score at preoperation, 1 week, and 1, 6, 12 months after operation, and last follow-up, respectively. The effect of slip correction was evaluated by slip angle and intervertebral altitude at preoperation and last follow-up, and the effectiveness of fusion was evaluated according to Suk criteria. RESULTS: All patients were followed up, the modified-TLIF group was followed up 25-36 months (mean, 32.7 months), the PLIF group was followed up 24-38 months (mean, 33.3 months). The intraoperative blood loss, operation time, postoperative drainage volume, postoperative bed time, and hospital stay of the modified-TLIF group were significantly less than those of the PLIF group ( P<0.05). The VAS score and JOA score of both groups were significantly improved at each time point after operation ( P<0.05); the scores of the modified-TLIF group were significantly better than those of the PLIF group at 1 and 6 months after operation ( P<0.05). The slip angle and intervertebral altitude of both groups were obviously improved at last follow-up ( P<0.05), and there was no significant difference between the two groups at preoperation and last follow-up ( P>0.05). At last follow-up, the fusion rate of the modified-TLIF group and the PLIF group was 96.3% (52/54) and 98.1% (51/52), respectively, and no significant difference was found between the two groups ( χ 2=0.000, P=1.000). About complications, there was no significant difference between the two groups in nerve injury on the opposite side within a week, incision infection, and pulmonary infection ( P>0.05). No case of nerve injury on the operation side within a week or dural laceration occurred in the modified-TLIF group, while 8 cases (15.4%, P=0.002) and 4 cases (7.7%, P=0.054) occurred in the PLIF group respectively. CONCLUSION: Modified-TLIF and PLIF are effective in the treatment of mild to moderate lumbar spondylolisthesis in middle-aged and elderly patients. However, modified-TLIF has relatively less trauma, lower blood loss, lower drainage volume, lower incidence of dural laceration and nerve injury, which promotes enhanced recovery after surgery.

TNFAIP3 ameliorates the degeneration of inflammatory human nucleus pulposus cells by inhibiting mTOR signaling and promoting autophagy.

Autophagy is involved in degenerative diseases such as osteoarthritis and disc degeneration. Although, tumor necrosis factor α-induced protein 3 (TNFAIP3) is well-known as a key regulator of inflammation and autophagy, it is still not clear whether TNFAIP3 regulates autophagy to protect from human disc cells degeneration. We hypothesize that TNFAIP3 may also regulate autophagy to inhibit pro-inflammatory cytokines expression in human nucleus pulposus cells (NPCs). In this study, TNFAIP3 expression was increased in degenerative disc tissue as well as LPS-stimulated human NPCs, and the effect of TNFAIP3 in LPS-induced NPCs was further explored. The results demonstrated that pro-inflammatory cytokines expression in TNFAIP3-His cells was decreased, while it was increased in TNFAIP3-siRNA cells. Further molecular mechanism research showed that TNFAIP3-siRNA cells enhanced the phosphorylation of mammalian target of rapamycin (mTOR) and inhibited autophagy. Meanwhile, after treatment of TNFAIP3-siRNA cells with the mTOR inhibitor Torin1, the level of autophagy increased and the decrease of extracellular matrix was reversed. In summary, overexpressed TNFAIP3 can promote autophagy and reduce inflammation in LPS-induced human NPCs. Moreover, autophagy triggered by TNFAIP3 can ameliorate the degeneration of inflammatory human NPCs, providing a potential and an attractive therapeutic strategy for degenerative disease.