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.

Adaptive Neuro-Fuzzy Fusion of Multi-Sensor Data for Monitoring a Pilot's Workload Condition.

To realize an early warning of unbalanced workload in the aircraft cockpit, it is required to monitor the pilot's real-time workload condition. For the purpose of building the mapping relationship from physiological and flight data to workload, a multi-source data fusion model is proposed based on a fuzzy neural network, mainly structured using a principal components extraction layer, fuzzification layer, fuzzy rules matching layer, and normalization layer. Aiming at the high coupling characteristic variables contributing to workload, principal component analysis reconstructs the feature data by reducing its dimension. Considering the uncertainty for a single variable to reflect overall workload, a fuzzy membership function and fuzzy control rules are defined to abstract the inference process. An error feedforward algorithm based on gradient descent is utilized for parameter learning. Convergence speed and accuracy can be adjusted by controlling the gradient descent rate and error tolerance threshold. Combined with takeoff and initial climbing tasks of a Boeing 737-800 aircraft, crucial performance indicators-including pitch angle, heading, and airspeed-as well as physiological indicators-including electrocardiogram (ECG), respiration, and eye movements-were featured. The mapping relationship between multi-source data and the comprehensive workload level synthesized using the NASA task load index was established. Experimental results revealed that the predicted workload corresponding to different flight phases and difficulty levels showed clear distinctions, thereby proving the validity of data fusion.

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.

Anti-proliferative effect of the extract of Guangzao (Fructus Choerospondiatis) on cultured rat cardiac fibroblasts.

OBJECTIVE: To ascertain if total flavonoids of Guangzao (Fructus Choerospondiatis) (TFFC) extracted from Guangzao (Fructus Choerospondiatis) can inhibit angiotensin II-induced proliferation of cardiac fibroblasts (CFs). METHODS: CFs were cultured by the differential attachment method. A model of cell proliferation was established by stimulation with Ang II. Cardiac fibroblasts growth was determined using a hemocytometer. Cell proliferation was detected by methyl thiazole tetrazolium. Lactate dehydrogenase activity was measured by chemical colorimetric method. RESULTS: Proliferation of TFFC-treated (25, 50, 100 mg/L) fibroblasts was significantly less than that of cells in the angiotensin II group (P < 0.01), and TFFC inhibited proliferation in a dose-dependent manner. These inhibitory effects were partly blocked by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME) and 1H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ). CONCLUSION: TFFC inhibited angiotensin II-induced proliferation of cardiac fibroblasts via a mechanism that probably involves activation of the NO-cyclic guanosine monophosphate signaling pathway.

[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.

Interpretable Machine Learning Models Based on SHapley Additive exPlanations for Predicting the Risk of Cerebrospinal Fluid Leakage in Lumbar Fusion Surgery.

STUDY DESIGN: Retrospective study. OBJECTIVES: The objective of this investigation was to formulate and internally verify a customized machine learning (ML) framework for forecasting cerebrospinal fluid leakage (CSFL) in lumbar fusion surgery. This was accomplished by integrating imaging parameters and employing the SHapley Additive exPlanation (SHAP) technique to elucidate the interpretability of the model. SUMMARY OF BACKGROUND DATA: Given the increasing incidence and surgical volume of spinal degeneration worldwide, accurate predictions of postoperative complications are urgently needed. SHAP-based interpretable ML models have not been used for CSFL risk factor analysis in lumbar fusion surgery. METHODS: Clinical and imaging data were retrospectively collected from 3505 patients who underwent lumbar fusion surgery. Six distinct machine learning models were formulated: extreme gradient boosting (XGBoost), decision tree (DT), random forest (RF), support vector machine (SVM), Gaussian naive Bayes (GaussianNB), and K-nearest neighbors (KNN) models. Evaluation of model performance on the test dataset was performed using performance metrics, and the analysis was executed through the SHAP framework. RESULTS: CSFL was detected in 95 out of 3505 patients (2.71%). Notably, the XGBoost model exhibited outstanding accuracy in forecasting CSFLs, with high precision (0.9815), recall (0.6667), accuracy (0.8182), F1 score (0.7347), and AUC (0.7343). Additionally, through SHAP analysis, significant predictors of CSFL were identified, including ligamentum flavum thickness, zygapophysial joint degeneration grade, central spinal stenosis grade, decompression segment count, decompression mode, intervertebral height difference, Cobb angle, intervertebral height index difference, operation mode, lumbar segment lordosis angle difference, Meyerding grade of lumbar spondylolisthesis, and revision surgery. CONCLUSION: The combination of the XGBoost model with the SHAP is an effective tool for predicting the risk of CSFL during lumbar fusion surgery. Its implementation could aid clinicians in making informed decisions, potentially enhancing patient outcomes and lowering healthcare expenses. This study advocates for the adoption of this approach in clinical settings to enhance the evaluation of CSFL risk among patients undergoing lumbar fusion.

ScRNA analysis and ferroptosis-related ceRNA regulatory network investigation in microglia cells at different time points after spinal cord injury.

Spinal cord injuries (SCI) are usually caused by mechanical trauma that leads to serious physical and psychological damage to the patient as well as a huge economic burden to the whole society. The prevention, treatment, and rehabilitation of spinal cord injuries have become a major issue for the medical community today due to the enormous social and economic expenditure induced via spinal cord injuries. Therefore, in-depth research into SCI is necessary. Microglia have been shown to be the key player in the immune inflammatory response after spinal cord injury, but the mechanisms of immune regulation at different time points after spinal cord injury remain unclear. To investigate the inflammatory biomarkers associated with microglia at different time points after SCI, we downloaded single-cell RNA sequencing data from mouse spinal cords 3- and 14-days after the injury and identified subpopulations associated with microglia. Further functional enrichment analysis also confirmed that microglia are associated with immune system regulation at different time points and that both can modulate cytokine production. As ferroptosis is a newly identified non-apoptotic programmed cell death, microglia establish a bridge between ferroptosis and CNS inflammation and may play an important role in spinal cord injury. We then screened for genes differentially expressed in microglia during 3- and 14-days after spinal cord injury and associated with iron death, named Stmn1 and Fgfbr1, respectively, and verified that these pivotal genes are closely related to the immune cells. Finally, we also screened for drug fractions associated with these pivotal genes. Our results predict key genes in the immune inflammatory process associated with microglia at different time points after spinal cord injury at the single-cell level and provide a molecular basis for better treatment of SCI.

Bioinformatics-based diagnosis and evaluation of several pivotal genes and pathways associated with immune infiltration at different time points in spinal cord injury.

Spinal Cord Injury (SCI) is a devastating neurological event. To assess the degree of spinal cord damage and classify the injury, it is recommended to use the 2019 version of the AIS standard. The severity of trauma was evaluated using the Trauma Severity Score, and various classification systems have been proposed for injuries at different parts and segments of the spine. Understanding the regulated signaling pathways and immune processes following SCI can lead to a better understanding of SCI-induced biomarkers and their underlying mechanisms. In this study, two gene expression datasets (GSE464 and GSE45006) from the Gene Expression Omnibus database were utilized. Differential gene expression and co-expression network analysis were performed, revealing 370 shared genes in the 3-day group and 111 shared genes in the 14-day group after SCI. The study used functional enrichment analysis methods such as Gene Set Enrichment Analysis, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes. The ssGSEA method was used to assess the levels and composition of immune infiltration in both the sham (control) and SCI groups. The single-cell transcriptomics dataset GSE182803 was analyzed to identify genes associated with immune marker cells. Four key genes (Ptgs2, Fn1, Ccl2, and Icam1) were identified in the 3-day group, while only one gene (Cyp51) was identified in the 14-day group after SCI. The findings offer significant insights into the immune-related genes and signaling pathways involved in secondary SCI at different time points and hold potential for the development of intervention strategies for acute and chronic post-SCI.

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.

Downregulation of miR-760 Causes Human Intervertebral Disc Degeneration by Targeting the MyD88/Nuclear Factor-Kappa B Signaling Pathway.

Dysregulation of microRNAs (miRNAs) plays a critical role in the development of intervertebral disc degeneration (IDD). In this study, we present evidence from in vitro and in vivo research to elucidate the mechanism underlying the role of miR-760 in IDD. miRNA microarray and quantitative reverse transcription-polymerase chain reaction were used to determine the miRNA profiles in patients with IDD. Functional analysis was performed to evaluate the role of miR-760 in the pathogenesis of IDD. Luciferase reporter and western blotting assays were used to confirm the miRNA targets. The expression of miR-760 was significantly decreased in degenerative nucleus pulposus (NP) cells and negatively correlated with disc degeneration grade. Functional assays demonstrated that miR-760 delivery significantly increased NP cell proliferation and promoted the expression of collagen II and aggrecan. Moreover, MyD88 was identified as a target gene of miR-760. miR-760 effectively suppressed MyD88 expression by interacting with the 3'-untranslated region, which was abolished by miR-760 binding site mutations. An in vivo experiment using an IDD mouse model showed that the upregulation of miR-760 could effectively suspend IDD. Therefore, miR-760 was found to play an important role in IDD and can be used as a promising therapeutic target for the treatment of patients with IDD.

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.

Deficiency of CD93 exacerbates inflammation-induced activation and migration of BV2 microglia by regulating the TAK1/NF-κB pathway.

The role of CD93 in inflammatory response has been reported in multiple previous studies. However, the underlying mechanism of CD93 in microglial activation and migration during neuroinflammation post spinal cord injury (SCI) remains elusive. In the current study, we performed western blot, qRT-PCR, immunofluorescence analyses Transwell assay, and ELISA to determine the expression change and in-depth molecular mechanism of CD93 in microglia post inflammatory initiation. We found that CD93 expression was increased in microglia after SCI in vivo or lipopolysaccharide (LPS) stimuli in vitro. Additionally, CD93 interacted with TAK1 to inhibit NF-κB activation, thus attenuating inflammation and migration of microglia after treatment with LPS. These findings indicate that CD93 might participate in microglia-induced neuroinflammation development post SCI, suggesting that CD93 is a promising target for neuroimmunological regulation.

Omentin-1 promoted proliferation and ameliorated inflammation, apoptosis, and degeneration in human nucleus pulposus cells.

PURPOSE: Intervertebral disc degeneration is an abnormal, cell-mediated process of tissue remodeling, recognized as the principal cause of low back pain affecting 80% of the population worldwide. Inflammatory cytokine, Interleukin-1beta (IL-1ß) is involved in the intervertebral disc degeneration (IDD) process, and it is upregulated in degenerated discs. Omentin-1, also known as intelectin-1, is an adipokine with anti-inflammatory, anti-apoptosis, pro-proliferation, and proangiogenic properties in various types of cells. However, little is known about the effects of omentin-1 on human nucleus pulposus cells (HNPCs). This study aims to investigate the effects of omentin-1 on healthy HNPCs regarding proliferation and further investigate the effects of omentin-1 on IL-1ß-induced inflammation, apoptosis, and degeneration in HNPCs. METHODS: Genes and proteins of interest were measured by qRT-PCR, immunoblotting, and immunofluorescence to conduct related experiments. Cell viability (CCK-8), EdU, and mitochondrial membrane potential (JC-1), flow cytometry assays were used to assess proliferation and apoptosis, respectively. RESULTS: Our study showed that omentin-1 promoted proliferation in normal HNPCs. Furthermore, omentin-1 expression was decreased in IL-1ß-treated HNPCs. Omentin-1 protected against IL-1ß-induced inflammation, apoptosis, and degeneration in HNPCs in vitro via the activation of the PI3K/Akt signaling pathway. CONCLUSION: These findings may contribute to understanding the role of omentin-1 in HNPCs and may be a potential therapeutic candidate for intervertebral disc degeneration.

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.

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.

Activation of glucagon-like peptide-1 receptor in microglia attenuates neuroinflammation-induced glial scarring via rescuing Arf and Rho GAP adapter protein 3 expressions after nerve injury.

Rationale: The neuroinflammation is necessary for glial group initiation and clearance of damaged cell debris after nerve injury. However, the proinflammatory polarization of excessive microglia amplifies secondary injury via enhancing cross-talk with astrocytes and exacerbating neurological destruction after spinal cord injury (SCI). The glucagon-like peptide-1 receptor (GLP-1R) agonist has been previously shown to have a neuroprotective effect in neurodegeneration, whereas its potency in microglial inflammation after SCI is still unknown. Methods: The effect and mechanism of GLP-1R activation by exendin-4 (Ex-4) were investigated in in vitro cultured glial groups and in vivo in SCI mice. Alterations in the gene expression after GLP-1R activation in inflammatory microglia were measured using mRNA sequencing. The microglial polarization, neuroinflammatory level, and astrocyte reaction were detected by using western blotting, flow cytometry, and immunofluorescence. The recoveries of neurological histology and function were also observed using imaging and ethological examinations. Results: GLP-1R activation attenuated microglia-induced neuroinflammation by reversing M1 subtypes to M2 subtypes in vitro and in vivo. In addition, activation of GLP-1R in microglia blocked production of reactive astrocytes. We also found less neuroinflammation, reactive astrocytes, corrected myelin integrity, ameliorated histology, and improved locomotor function in SCI mice treated with Ex-4. Mechanistically, we found that Ex-4 rescued the RNA expression of Arf and Rho GAP adapter protein 3 (ARAP3). Knockdown of ARAP3 in microglia reversed activation of RhoA and the pharmacological effect of Ex-4 on anti-inflammation in vitro. Conclusion: Ex-4 exhibited a previously unidentified role in reducing reactive astrocyte activation by mediation of the PI3K/ARAP3/RhoA signaling pathway, by neuroinflammation targeting microglia, and exerted a neuroprotective effect post-SCI, implying that activation of GLP-1R in microglia was a therapeutical option for treatment of neurological injury.