A20 attenuates pyroptosis and apoptosis in nucleus pulposus cells via promoting mitophagy and stabilizing mitochondrial dynamics.

BACKGROUND: A20 is an anti-inflammatory molecule in nucleus pulposus (NP) cells. The anti-inflammatory properties of A20 are mainly attributed to its ability to suppress the NF-κB pathway. However, A20 can protect cells from death independently of NF-κB regulation. This study aimed to investigate the effects of A20 on pyroptosis and apoptosis of NP cells induced by lipopolysaccharide (LPS). METHODS: NP cells induced by LPS were used as an in vitro model of the inflammatory environment of the intervertebral disc. Pyroptosis, apoptosis, and mitophagy marker proteins were detected. Then, NP cells were transfected with A20 overexpressed lentivirus or A20-siRNA. Annexin V FITC/PI, Western blotting, and immunofluorescence assays were used to detect the apoptosis, pyroptosis, and mitophagy of NP cells. Furthermore, the expressions of A20, related proteins, and related inflammatory cytokines were detected by western blotting, and ELISA. RESULTS: Apoptosis and pyroptosis of NP cells increased gradually treated with LPS for 12 h, 24 h, and 48 h. Differently, the level of mitophagy increased first and then decreased, and was the highest at LPS treatment for 12 h. Overexpression or knockdown of A20 in NP cells revealed that A20 attenuated the pyroptosis, apoptosis, and production of inflammatory cytokines of NP cells induced by LPS, while A20 sponsored mitophagy, reduced ROS production and collapse of mitochondrial membrane potential (ΔΨm). Moreover, A20 also promoted mitochondrial dynamic homeostasis and attenuated LPS-induced excessive mitochondrial fission. Excitingly, inhibition of mitophagy attenuated the effect of A20 on the negative regulation of pyroptosis of NP cells induced by LPS. Pyroptosis was accompanied by a large release of inflammatory cytokines. Inhibition of pyroptosis also significantly reduced apoptosis of NP cells. Finally, The mitochondria-targeted active peptide SS-31 inhibited LPS-induced pyroptosis and ROS production in NP cells. CONCLUSIONS: To sum up, A20 attenuates pyroptosis and apoptosis of NP cells via promoting mitophagy and stabilizing mitochondrial dynamics. Besides, A20 reduces LPS-induced NP cell apoptosis by inhibiting NLRP3 inflammasome-mediated pyroptosis. It provides theoretical support for the reduction of functional NP cell loss in the intervertebral disc through the gene-targeted intervention of A20.

Comparison of percutaneous endoscopic lumbar discectomy versus microendoscopic discectomy for the treatment of lumbar disc herniation: a meta-analysis.

PURPOSE: We conducted a systematic review and meta-analysis to compare the clinical outcomes of percutaneous endoscopic lumbar discectomy (PELD) and microendoscopic discectomy (MED) for the treatment of lumbar disc herniation (LDH), and to clarify whether PELD is more superior to MED. METHODS: We performed a comprehensive search in the databases of MEDLINE, EMBASE, PubMed, Web of Science, Cochrane database, CNKI, and Wanfang Data to acquire all relevant studies up to July 2018. The searched literatures were then screened according to the strict inclusion and exclusion criteria. The critical data were extracted and analyzed utilizing Review Manager software. The pooled effects were calculated by mean difference (MD) or odds ratio (OR) with 95% confidence intervals (CI) on the basis of data attributes. RESULTS: A total of 18 studies (2161 patients, 1093 in the PELD group and 1068 in the MED group) were included in this systematic review and meta-analysis. At last follow-up, the results revealed that no significant difference was found between PELD group and MED group with respect to ODI (MD - 0.30; 95% CI - 1.02 to 0.42; P = 0.41), VAS-leg pain (MD - 0.18; 95% CI - 0.45 to 0.09; P = 0.19), VAS-unspecified (MD - 0.00; 95% CI - 0.05 to 0.04; P = 0.94), excellent & good rate (OR, 1.04; 95% CI 0.68 to 1.59; P = 0.86), total complication rate (OR, 0.96; 95% CI 0.65 to 1.43; P = 0.85), dural tear rate (OR, 0.39; 95% CI 0.10 to 1.55; P = 0.18), and residue or recurrence rate (OR, 2.22; 95% CI 1.02 to 4.83; P = 0.05). When compared to MED group, the PELD group showed significantly better results with regard to shorter length of incision (MD - 1.18; 95% CI - 1.39 to - 0.97; P < 0.00001), less blood loss (MD - 45.17; 95% CI - 64.74 to - 25.60; P < 0.00001), shorter post-operative in-bed time (MD - 59.11; 95% CI - 71.19 to - 47.04; P < 0.00001), shorter post-operative hospital stay (MD - 3.07; 95% CI - 4.81 to - 1.33; P < 0.00001), shorter total hospital stay (MD - 2.29; 95% CI - 3.03 to - 1.55; P < 0.00001), and lower VAS-back pain at last follow-up (MD - 0.77; 95% CI - 1.31 to - 0.24; P = 0.005), but with significantly worse results such as more fluoroscopy (MD 7.63; 95% CI 5.25 to 10.01; P < 0.00001) and higher re-operation rate (OR, 2.67; 95% CI 1.07 to 6.67; P = 0.04). Although no significant difference was found between the two groups in terms of duration of operation (MD 6.27; 95% CI - 2.44 to 14.98; P = 0.16) and total hospital cost (MD - 0.69; 95% CI - 12.60 to 11.23; P = 0.91), further subgroup analysis revealed that the duration of operation was significantly longer in the PELD group compared with the MED group in "Years before 2016" (MD 24.97; 95% CI 7.07 to 42.87; P = 0.006) and "Year 2016 to 2017" (MD 6.57; 95% CI 0.58 to 12.55; P = 0.03) subgroups but not in the subgroup "Year 2018" (MD - 5.66; 95% CI - 18.84 to 7.53; P = 0.40), and that the total hospital cost was significantly more in the PELD group compared with the MED group in the subgroup "Southeast of China" (MD 6.67; 95% CI 3.23 to 10.28; P = 0.0002) but not in the subgroup "Midwest of China" (MD - 8.09; 95% CI - 17.99 to 1.80; P = 0.11). CONCLUSIONS: For the treatment of LDH, both of PELD and MED can reach excellent results and no superiority was found between the two minimally invasive procedures with regard to duration of operation, ODI, VAS-leg pain, VAS-unspecified, excellent & good rate, total complication rate, dural tear rate, and residue or recurrence rate. While PELD can achieve better outcomes with respect to the length of incision, blood loss, post-operative in-bed time, post-operative hospital stay, total hospital stay, and VAS-back pain at last follow-up, however, MED showed certain advantages of less fluoroscopic times and lower re-operation rate. More practice and development are needed to make up for the deficiencies of PELD. Besides, the economic factor should also be considered according to different regions before making the treatment strategies. Well-defined randomized controlled trials with large samples are needed to further confirm these results.

Lumbar disc herniation treated by microendoscopic discectomy : Prognostic predictors of long-term postoperative outcome.

PURPOSE: Microendoscopic discectomy (MED) is becoming an established and effective minimally invasive surgical method for the treatment of lumbar disc herniation (LDH); however, the absence of prognostic factors for long-term outcomes after MED results in a lack of specific criteria for appropriate patient selection. Therefore, we evaluated the long-term outcomes and associated predictors in patients who underwent MED for LDH. MATERIAL AND METHODS: Baseline and follow-up data for 664 LDH patients who suffered from sciatica and underwent primary MED were reviewed retrospectively. Variables at baseline that were analyzed as potential prognostic factors included sociodemographic characteristics, clinical findings, and imaging features. Follow-up data including improvements in the Visual Analog Scale (VAS) score and Oswestry Disability Index (ODI), postoperative low back pain (LBP), reoperation, and excellent/good results according to the modified MacNab criteria were set as outcome variables for univariate and further multivariate logistic regression analyses. RESULTS: The mean follow-up period was 63.8 ± 20.0 months (range 24-96 months). On average, sufficient improvements in both the VAS score (72.8%) and ODI (63.4%) were observed. In addition, a low postoperative LBP rate (23.9%), low reoperation rate (7.1%), and high rate of excellent/good clinical outcomes (89.9%) were achieved. A multivariate analysis indicated that older age, shorter disease duration, higher preoperative VAS score, lower preoperative ODI, shorter surgical time, lower severity of disc and adjacent disc degeneration, and lower severity of lumbar multifidus atrophy contributed to superior clinical outcomes. CONCLUSION: Excellent long-term outcomes after primary MED were achieved and specific sociodemographic, clinical, and imaging variables were identified as prognostic factors that can be used to guide patient selection and clinical decision making.

A cohort study comparing the serum levels of pro- or anti-inflammatory cytokines in patients with lumbar radicular pain and healthy subjects.

PURPOSE: The factors influencing the presence or absence of pain in sciatica secondary to disc herniation remain incompletely understood. We hypothesized that the imbalance in inflammatory cytokines is implicated in the generation of pain. In our study, serum levels of pro-inflammatory and anti-inflammatory cytokines were investigated among patients with severe sciatica; the serum levels were compared with those of patients with mild sciatica and healthy subjects. METHODS: In this prospective study, blood protein levels of the pro-inflammatory cytokines, namely, interleukin-6 (IL-6), interleukin-8 (IL-8),and tumor necrosis factor-α (TNF-α), and the anti-inflammatory cytokines, namely, interleukin-4 (IL-4) and interleukin-10 (IL-10), of 58 patients with severe sciatica, 50 patients with mild sciatica, and 30 healthy control subjects were analyzed through ELISA. Physical and mental health symptoms were determined using the Oswestry Disability Index (ODI) and short form-36 (SF-36) questionnaire. Spearman rank correlation coefficient was also determined to calculate the correlation between the scores obtained from the questionnaires and the serum levels of cytokines. RESULTS: IL-6 protein was detected in the three groups and median levels were about 1.5 times higher in patients with severe sciatica than the mild sciatica group (p = 0.02) and the controls (p = 0.03). Median levels of IL-8 in sciatica patients were higher than those of the healthy controls (p = 0.001 for severe sciatica, p = 0.02 for mild sciatica). The TNF-α protein values were approximately twofold higher in the severe sciatica group than in the mild sciatica group (p < 0.01) and in the healthy control group (p < 0.01). Median levels of IL-4 were about 2.5-fold higher in mild sciatica (p < 0.01) and about twofold higher in patients with severe sciatica (p = 0.012) when compared with controls. Median protein levels of IL-10 showed a trend to be higher in patients with mild sciatica compared with severe sciatica (p < 0.01) and with healthy controls (p < 0.01). ODI was significantly correlated with IL-6 (r = 0.394, p = 0.013), TNF-α (r = 0.629, p < 0.001), and IL-10 (r = -0.415, p = 0.009). ODI was not significantly correlated with IL-4 (r = -0.174, p = 0.29) and IL-8 (r = -0.133, p = 0.418). CONCLUSIONS: These findings support our hypothesis that sciatica pain is accompanied by the imbalance in inflammatory cytokines.

The presence of stem cells in potential stem cell niches of the intervertebral disc region: an in vitro study on rats.

PURPOSE: The potential of stem cell niches (SCNs) in the intervertebral disc (IVD) region, which may be of great significance in the regeneration process, was recently proposed. To the best of our knowledge, no previous in vitro study has examined the characteristics of stem cells derived from the potential SCN of IVD (ISN). Therefore, increasing knowledge on ISN-derived stem cells (ISN-SCs) may provide a greater understanding of IVD degeneration and regeneration processes. We aimed to demonstrate the existence of ISN-SCs and to compare their characteristics with bone marrow mesenchymal stem cells (BMSCs) in vitro. METHODS: Sprague-Dawley rats (male, 10-week-old) were used in this study. ISN tissues were separated by ophthalmic surgical instruments under a dissecting microscope according to the anatomical areas. BMSCs and cells isolated from the ISN tissues were cultured and expanded in vitro. Passage 4 populations were used for further analysis with respect to colony-forming ability, cellular immunophenotype, cell cycle, stem cell-related gene expression, and proliferation and multipotential differentiation capacities. RESULTS: In general, both of ISN-SCs and mesenchymal stromal cells (MSCs) met the minimal criteria for the definition of multipotent mesenchymal stromal cells, including adherence to plastic, specific surface antigen expression, and multipotent differentiation potential. Especially, ISN-SCs even showed greater potential of osteogenesis and chondrogenesis. The ISN-SCs also expressed stem cell-related genes that were comparable to those of BMSCs, and had colony-forming and self-renewal abilities. CONCLUSIONS: To the best of our knowledge, this is the first in vitro study aimed towards determining the existence and characteristics of ISN-SCs, which belong to the MSC family and with greater osteogenic and chondrogenic abilities than BMSCs according to our data. This finding may be of great significance for additional studies that investigate the migration of ISN-SCs into the IVD, and may provide a new perspective on different biological approaches for IVD self-regeneration.

Evaluation of transforaminal endoscopic lumbar discectomy in the treatment of lumbar disc herniation.

PURPOSE: The purpose of this study was to evaluate the efficacy of transforaminal endoscopic lumbar discectomy (TELD) in the treatment of lumbar disc herniation (LDH) and to identify the relationship between TELD efficacy and age. METHODS: A total of 207 consecutive LDH patients who had undergone TELD with the THESSYS system from January 2013 to September 2014 were divided into two groups on the basis of their age, with 108 cases in the ≤ 45-year-old age group and 99 cases in the >45-year-old group. The Oswestry Disability Index (ODI) was used to quantify the pain relief. The degree of pain and disability were measured on the basis of the visual analog scale (VAS) and the modified MacNab criteria. Complications, duration of hospital stay, surgical costs, and operation time were recorded and compared between the two groups. Spearman's coefficient of rank correlation was used to assess the learning curves for TELD. RESULTS: The mean pre-operative and postoperative VAS and ODI scores significantly improved in both age ≤ 45 group and age >45 group, with no significant differences between them. In age ≤45 group, 56 % had excellent outcomes, 28 % good, 14 % fair, and 3 % poor. In the age >45 group, 51 % had excellent outcomes, 20 % good, 25 % fair, and 4 % poor. The average lengths of hospital stay for the age ≤ 45 group and age >45 group were 6.8 and 8.4 days, respectively. The mean time to return to work or normal activities was ten days for the age ≤ 45 group and 15 days for the age >45 group. The mean operative time for the age ≤ 45 group was 94 minutes and that for age >45 group was 97 minutes. The surgical cost of age ≤ 45 group was 15,480 RMB, which was lower than the 16,381 RMB of age >45 group. A total of 14 patients in the age ≤ 45 group and 13 patients in age >45 group used analgesic medications. Three and five recurrences were reported in the age ≤ 45 group and age >45, respectively. The steep learning curves of operative time plotted against the number of surgeries conducted suggest that the TELD technique can be mastered quickly in terms of reducing the duration of operation. CONCLUSIONS: The efficacy of TELD is relatively good for the selected young and elderly patients in this study. Therefore, age is not a predictor of TELD surgery-related outcomes.

[In vitro effect of total flavones of Fructus Chorspondiatis on expression of collagen type I and type III mRNA and protein of cultured rat cardiac fibroblasts].

This study aims to investigate the effect of total flavones of Fructus Chorspondiatis (TFFC) on the mRNA and protein expression of collagen type I and III of rat cardiac fibroblasts (CFs) induced by angiotensin II (Ang II), and explore its anti-myocardial fibrosis molecular mechanism. Neonatal rat CFs were prepared from Sprague-Dawley rats (1-3 d after birth). The expression of collagen type I and III mRNA and protein were measured by RT-PCR and Western blotting, respectively. The study showed that stimulation of neonatal rat CFs with 100 nmol.L-1 of Ang II for 72 h resulted in a significant increase of the expression of collagen type I and III mRNA and protein. The changes on the expression level were blocked by TFFC. The results demonstrated that TFFC can inhibit myocardial fibrosis induced by Ang II in rats, which is probably associated with the collagen type I and III mRNA and protein levels up-regulated by Ang II, and TFFC was shown to decrease the expression levels of collagen type I and III mRNA and protein.

A20 ameliorates disc degeneration by suppressing mTOR/BNIP3 axis-mediated mitophagy.

BACKGROUND: The pathological mechanism of intervertebral disc degeneration (IDD) is an unanswered question that we are committed to exploring. A20 is an anti-inflammatory protein of nucleus pulposus (NP) cells and plays a protective role in intervertebral disc degeneration. OBJECTIVE: This study aims to investigate the molecular mechanism by which A20 attenuates disc degeneration. METHODS: The proteins of interest were measured by immunoblotting, immunofluorescence, ELISA assay, and immunohistochemical technique to conduct related experiments. Immunofluorescence assays and mitochondrial membrane potential (JC-1) were used to assess mitophagy and mitochondrial fitness, respectively. RESULTS: Here, we demonstrated that A20 promoted mitophagy, attenuated pyroptosis, and inhibited the degradation of the extracellular matrix, consequently significantly ameliorating disc degeneration. Mechanistically, A20 reduces pyroptosis and further suppresses cellular mTOR activity. On the one hand, A20-induced mTOR inhibition triggers BNIP3-mediated mitophagy to ensure mitochondrial fitness under LPS stimulation, as a result of mitigating mitochondrial dysfunction induced by LPS. On the other hand, A20-induced mTOR inhibition reduces the loss of mitochondrial membrane potential and the generation of Mitochondrial ROS. CONCLUSION: The study revealed that A20 promotes BNIP3-mediated mitophagy by suppressing mTOR pathway activation against LPS-induced pyroptosis.

CircRNA-Associated ceRNA Network Reveals Focal Adhesion and Metabolism Pathways in Neuropathic Pain.

Background: Increasing evidence has shown that noncoding RNAs perform a remarkable function in neuropathic pain (NP); nonetheless, the mechanisms underlying the modulation of competitive endogenous RNA in NP remain uncertain. The goal of this research was to investigate the molecular processes underlying NP. Methods: We utilized the Gene Expression Omnibus (GEO) to obtain NP-related microarray datasets that included the expression patterns of circular RNAs (circRNAs) and messenger RNAs (mRNAs). Following that, bioinformatics analyses and a molecular biology experiment were carried out. Results: According to the findings, carrying out enrichment studies of the targeted genes had an impact on a variety of NP-related pathways. Notably, we isolated a ceRNA subnetwork incorporating two upregulated circRNAs (Esrrg and Map3k3) which primarily participate in the focal adhesion pathway by regulating Integrin Subunit Beta 4 (ITGB4) and two downregulated circRNAs (Dgkb and Atp2a2), which potentially regulate metabolism-related molecule Lipase A (LIPA). Conclusions: According to our findings, the focal adhesion and metabolic signaling pathways could be critical in the advancement of NP, and some circRNA might regulate this biological process through the ceRNA network, which might offer pertinent insights into the underlying mechanisms.

Small extracellular vesicles from hypoxic mesenchymal stem cells alleviate intervertebral disc degeneration by delivering miR-17-5p.

Minimally invasive repair strategies are a very promising approach for the treatment of intervertebral disc degeneration (IDD). In recent years, small extracellular vesicles (sEVs) secreted from mesenchymal stem cells (MSCs) have been shown great potential in alleviating IDD. However, in vitro experiments, MSCs are usually exposed to a normoxic micro-environment, which differs greatly from the hypoxic micro-environment in vivo. The primary purpose of our research was to determine whether sEVs isolated from MSCs under hypoxic status (H-sEVs) exhibit a more beneficial effect on protecting IDD compared with sEVs derived from MSCs under normoxic status (N-sEVs). A tail IDD rat model and a series of experiments in vitro were conducted to compare the beneficial effects of PBS, N-sEVs, and H-sEVs treatment. Then, to validate the role of sEVs miRNAs in IDD, a miRNA microarray sequencing analysis and a series of rescue experiments were conducted. Luciferase activity, RNA-ChIP and western blot were performed to explore the potential mechanisms. The results indicate that sEVs alleviate IDD by ameliorating the homeostatic imbalance between anabolism and catabolism in vivo and in vitro. Microarray sequencing result shows that miR-17-5p is maximally enriched in H-sEVs. Toll-like receptor 4 (TLR4) was determined to be a target downstream gene of miR-17-5p. Finally, it was found that H-sEVs miR-17-5p may modulate proliferation and synthesis of human nucleus pulposus cells (HNPCs) matrix via TLR4 pathway. In conclusion, H-sEVs miR-17-5p alleviate IDD via promoting HNPCs matrix proliferation and synthesis, providing new therapeutic targets for IDD. STATEMENT OF SIGNIFICANCE: Intervertebral disc degeneration (IDD) is the primary cause of low back pain (LBP), which is a huge burden to society. Our research demonstrates for the first time that hypoxic pretreatment of small extracellular vesicles (H-sEVs) effectively alleviated the progress of IDD. In short, in the present research, we found that H-sEVs miR-17-5p could modulate proliferation and synthesis of nucleus pulposus cells (NPCs) matrix via TLR4/PI3K/AKT pathway. Therefore, hypoxic pre-treatment is a prospective and efficient method to optimize the therapeutic effect of MSCs-derived sEVs. miRNA and MSCs-derived sEVs combination may be a promising therapeutic approach for IDD.

GATA4 promotes the senescence of nucleus pulposus cells via NF-κB pathway.

PURPOSE: Cell senescence plays a vital role in intervertebral disc degeneration. The regulatory mechanism of the cellular senescence of nucleus pulposus cells has not been fully elucidated. A recent study identified GATA4 as an emerging regulator of IMR90 cellular senescence. However, whether GATA4 controls senescence in nucleus pulposus cells still needs to be explored. METHODS: Nucleus pulposus cells were exposed to acidified medium mimic the acid environment of intervertebral disc degeneration. RESULTS: We found that GATA4 protein expression was significantly upregulated in older rats and nucleus pulposus cells undergoing stress-induced aging. Moreover, the data indicated that inhibition of GATA4 significantly inhibited the senescence of nucleus pulposus cells cultured under acidic conditions and that over expression of GATA4 promoted a senescence phenotype. The NF-κB pathway has been confirmed in this study to play a role in the regulation of nucleus pulposus cell senescence by GATA4. By using the NF-κB pathway inhibitor, PDTC (100 µmol/L), significantly decreased the IL-6, matrix metallopeptidase (MMP)-2, MMP-3, MMP-9, MMP-13, ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS)-4, ADAMTS-5 expression level, and increased Aggrecan and typeⅡcollagen expression level in GATA4 transfected nucleus pulposus cells compared with the group in the absence of PDTC. CONCLUSION: This outcome suggested that GATA4 might play a significant role in nucleus pulposus cell senescence through the NF-κB signaling pathway, and GATA4 is a promising target for intervertebral disc degeneration treatment in the future.

The mitochondrial antioxidant SS-31 attenuated lipopolysaccharide-induced apoptosis and pyroptosis of nucleus pulposus cells via scavenging mitochondrial ROS and maintaining the stability of mitochondrial dynamics.

Evidence has shown that effects from inflammation and mitochondrial dysfunction lead to pyroptosis and apoptosis of nucleus pulposus (NP) cells. Damaged mitochondria release dangerous molecules such as reactive oxygen species (ROS), activating the NLRP3 inflammasome. SS-31 is a mitochondria-targeting peptide that has been used in the treatment of many diseases by scavenging ROS and ameliorating mitochondrial function. This study found that SS-31 ameliorated lipopolysaccharide (LPS)-induced loss of cell viability, ROS production, and apoptosis in NP cells. Moreover, mitochondrial dynamics and ATP synthesis were restored on pretreatment with SS-31 compared with the LPS group. For the molecular mechanism research, SS-31 stabilized mitochondrial morphology and inhibited the activation of the NF-κB pathway and the activation of the NLRP3 inflammasome. To evaluate whether the inhibition of NLRP3 inflammasome activation by SS-31 is dependent on the clearance of mitochondrial ROS, we comparatively analyzed the activation of NLRP3 inflammasome in NP cells pretreated with SS-31 and the ROS scavenger N-acetyl-L-cysteine (NAC). The results indicate that SS-31 could inhibit NLRP3 inflammasome activation by limiting the production of mitochondrial ROS. To sum up, our results revealed that SS-31 inhibits LPS-induced apoptosis, pyroptosis, and inflammation in NP cells via scavenging ROS and maintaining the stability of mitochondrial dynamics, which could be considered a promising therapeutic intervention for disk degeneration.

A20 of nucleus pulposus cells plays a self-protection role via the nuclear factor-kappa B pathway in the inflammatory microenvironment.

AIMS: Inflammatory response plays a pivotal role in the pathophysiological process of intervertebral disc degeneration (IDD). A20 (also known as tumour necrosis factor alpha-induced protein 3 (TNFAIP3)) is a ubiquitin-editing enzyme that restricts nuclear factor-kappa B (NF-κB) signalling. A20 prevents the occurrence of multiple inflammatory diseases. However, the role of A20 in the initiation of IDD has not been elucidated. The aim of the study was to investigate the effect of A20 in senescence of TNF alpha (TNF-α)-induced nucleus pulposus cells (NPCs). METHODS: Immunohistochemical staining was performed to observe the expression of A20 in normal and degenerated human intervertebral discs. The NPCs were dissected from the tail vertebrae of healthy male Sprague-Dawley rats and were cultured in the incubator. In the experiment, TNF-α was used to mimic the inflammatory environment of IDD. The cell viability and senescence were examined to investigate the effect of A20 on TNF-α-treated NPCs. The expression of messenger RNA (mRNA)-encoding proteins related to matrix macromolecules (collagen II, aggrecan) and senescence markers (p53, p16). Additionally, NF-κB/p65 activity of NPCs was detected within different test compounds. RESULTS: The expression of A20 was upregulated in degenerate human intervertebral discs. The A20 levels of NPCs in TNF-α inflammatory microenvironments were dramatically higher than those of the control group. TNF-α significantly decreased cell proliferation potency but increased senescence-associated beta-galactosidase (SA-ß-Gal) activity, the expression of senescence-associated proteins, the synthesis of extracellular matrix, and G1 cycle arrest. The senescence indicators and NF-κB/p65 expression of A20 downregulated group treated with TNF-α were significantly upregulated compared to TNF-α-treated normal NPCs. CONCLUSION: A20 has a self-protective effect on the senescence of NPCs induced by TNF-α. The downregulation of A20 in NPCs exacerbated the senescence of NPCs induced by TNF-α.Cite this article: Bone Joint Res. 2020;9(5):225-235.

Tumor necrosis factor α modulates sodium-activated potassium channel SLICK in rat dorsal horn neurons via p38 MAPK activation pathway.

The dorsal horn (DH) of the spinal cord is the integrative center that processes and transmits pain sensation. Abnormal changes in ion channel expression can enhance the excitability of pain-related DH neurons. Sodium-activated potassium (KNa) channels are highly expressed particularly in the central nervous system; however, information about whether rat DH neurons express the SLICK channel protein is lacking, and the direct effects on SLICK in response to inflammation and the potential signaling pathway mediating such effects are yet to be elucidated. Here, using cultured DH neurons, we have shown that tumor necrosis factor-α inhibits the total outward potassium current IK and the KNa current predominantly as well as induces a progressive loss of firing accommodation. However, we found that this change in channel activity is offset by the p38 inhibitor SB202190, thereby suggesting the modulation of SLICK channel activity via the p38 MAPK pathway. Furthermore, we have demonstrated that the tumor necrosis factor-α modulation of KNa channels does not occur at the level of SLICK channel gating but arises from possible posttranslational modification.

Resveratrol attenuated TNF-α-induced MMP-3 expression in human nucleus pulposus cells by activating autophagy via AMPK/SIRT1 signaling pathway.

Resveratrol (RSV) is known to play a role of anti-TNF-α in a number of cell types. However, whether RSV modulates the effects of TNF-α on human nucleus pulposus (NP) cells is unknown. The purpose of this study is to investigate whether RSV regulates TNF-α-induced matrix metalloproteinase-3 (MMP-3) expression. Via quantitative real-time polymerase chain reaction (qRT-PCR) analysis, we found that MMP-3 expression induced by TNF-α was inhibited by RSV treatment. Depending on Western blot and qRT-PCR assay, we found that RSV induced autophagy in human NP cells, whereas inhibition of autophagy remarkably abolished the restraining role of RSV in the TNF-α-mediated up-regulation of MMP-3. Furthermore, RSV increased SIRT1 expression and SIRT1 knockdown significantly suppressed RSV-induced autophagy in NP cells. RSV also activated AMP-activated protein kinase (AMPK), while inhibition of AMPK notably abolished RSV-induced SIRT1 expression. Our data showed that RSV attenuated TNF-α-induced MMP-3 expression in human NP cells by activating autophagy via AMPK/SIRT1 signaling pathway. This new finding suggested that RSV might act as a novel preventive and therapeutic role in intervertebral disc degeneration.

Tumor necrosis factor alpha promotes the proliferation of human nucleus pulposus cells via nuclear factor-κB, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase.

Although tumor necrosis factor alpha (TNF-α) is known to play a critical role in intervertebral disc (IVD) degeneration, the effect of TNF-α on nucleus pulposus (NP) cells has not yet been elucidated. The aim of this study was to explore the effect of TNF-α on proliferation of human NP cells. NP cells were treated with different concentrations of TNF-α. Cell proliferation was determined by cell counting kit-8 (CCK-8) analysis and Ki67 immunofluorescence staining, and expression of cyclin B1 was studied by quantitative real-time RT-PCR. Cell cycle was measured by flow cytometry and cell apoptosis was analyzed using an Annexin V-fluorescein isothiocyanate (FITC) & propidium iodide (PI) apoptosis detection kit. To identify the mechanism by which TNF-α induced proliferation of NP cells, selective inhibitors of major signaling pathways were used and Western blotting was carried out. Treatment with TNF-α increased cell viability (as determined by CCK-8 analysis) and expression of cyclin B1 and the number of Ki67-positive and S-phase NP cells, indicating enhancement of proliferation. Consistent with this, NP cell apoptosis was suppressed by TNF-α treatment. Moreover, inhibition of NF-κB, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) blocked TNF-α-stimulated proliferation of NP cells. In conclusion, the current findings suggest that the effect of TNF-α on IVD degeneration involves promotion of the proliferation of human NP cells via the NF-κB, JNK, and p38 MAPK pathways.

Outcomes of Microendoscopic Discectomy and Percutaneous Transforaminal Endoscopic Discectomy for the Treatment of Lumbar Disc Herniation: A Comparative Retrospective Study.

STUDY DESIGN: Retrospective, case control evaluation of 86 patients who underwent microendoscopic discectomy (MED) and percutaneous transforaminal endoscopic discectomy (PTED) for the treatment of lumbar disc herniation (LDH). PURPOSE: To evaluate the safety and the outcomes of MED and PTED for the treatment of LDH. OVERVIEW OF LITERATURE: MED and PTED are minimally invasive surgical techniques for lower back pain. Studies to date have shown that MED and PTED are safe and effective treatment modalities for LDH. METHODS: A retrospective study was performed in patients with LDH treated with MED (n=50) and transforaminal endoscopic discectomy (PTED; n=36) in our hospital. All patients were followed-up with self-evaluation questionnaires, Oswestry disability index (ODI), medical outcomes study 36-item short form health survey and MacNab criteria. All the patients in both groups were followed up to 12 months after the operation. RESULTS: ODI questionnaire responses were not statistically different between the MED and PTED groups (53.00 vs. 48.72) before treatment. Average scores and minimal disability after 5 days to 12 months of follow-up were 4.96 in the MED group and 3.61 in the PTED group. According to MacNab criteria, 92.0% of the MED group and 94.4% of the PTED group had excellent or good results with no significant difference. CONCLUSIONS: There was no significant difference between MED and PTED outcomes. Further large-scale, randomized studies with long-term follow-up are needed.

New cage for posterior minimally invasive lumbar interbody fusion: a study in vitro and in vivo.

OBJECTIVE: To design a new type of interbody fusion device made of nickel titanium NiTi shape memory alloy and to compare segmental stiffness after various posterior lumbar interbody fusion (PLIF) procedures in vitro and in vivo. METHODS: Twelve sheep lumbar functional spinal units were randomly allocated to four groups. One acted as controls (N); the other three were treated with autogenous iliac crest bone dowel graft (L), a threaded cylindrical titanium (KC) interbody fusion device (TFC) or a new type of interbody fusion device made of NiTi shape memory alloy (NT) containing autogenous iliac crest graft. In addition, 15 sheep were allocated to three groups; one served as controls and the other two underwent TFC (KC) or NiTi-FC (NT). Nondestructive mechanical tests were performed in pure compression, extension, lateral bending and torsion. The operated spines were photographed regularly to assess changes in interbody height and degree of fusion. The animals were killed at 6 months for histologic testing. RESULTS: Biomechanical tests showed both the strength and axial stiffness of the NT and KC groups were significantly higher than those of the control group and L group (P < 0.05). When the mechanical performance in torque and torsion of each group were compared, the same results could be obtained. The maximal destructive load of the NiTi-TFC was 11 200 N and the safety coefficient was above 1.2. Radiological observations revealed that the bone callus around the interbody fusion device were gradually increased postoperatively (2 months, no obvious; 4 months, poorly define; 6 months, dense). The KC and NT group had lost 16% and 16.5% of their postoperative height but remained well above normal disc height (P < 0.05). Histologic examination showed new trabeculation connected with that of the host. CONCLUSION: The mechanical characteristics of the NiTi-TFC are excellent and it is safe and reliable.

Ex vivo observation of human nucleus pulposus chondrocytes isolated from degenerated intervertebral discs.

STUDY DESIGN: We performed an ex vivo study to observe cell morphology and viability of human nucleus pulposus (NP) chondrocytes isolated from degenerated intervertebral discs (IVD). PURPOSE: To better understand the biological behavior of NP chondrocytes in monolayer cultures. OVERVIEW OF LITERATURE: Biological repair of IVDs by cell-based therapy has been shown to be feasible in clinical trials. As one of the most promising transplanting seeds, how the isolated NP chondrocytes behavior ex vivo has not been fully understood. METHODS: Human NP chondrocytes were harvested from 20 degenerated IVDs and cultured in monolayers. Histological and immunochemistry staining was used to detect cell morphology change. Cell viability was studied by analyzing cell cycle distribution and apoptotic rate in the primary and subculuted cells. RESULTS: The round or polygonal primary NP chondrocytes had an average adherence time of 7 days and took nearly 31 days to reach 95% confluence. The spindle-shaped P1 NP chondrocytes increased growth kinetics and took about 12 hours to adhere and 6.6 days to get 95% confluent. Immunochemistry staining of collagen II was positive in the cell cytoplasm. Nearly 90% of the confluent NP chondrocytes stayed in G1 phase while 16% underwent apoptosis. No significant difference of the collagen II expression, cell cycle distribution or the apoptosis indices were detected between the primary and subcultured NP chondrocytes. CONCLUSIONS: Human NP chondrocytes undergo significant morphological change in monolayer cultures. Cell cycle distribution pattern and apoptosis index of the cutured NP chondrocytes potentially influence their clinical transplantation or laboratory use.