Quercetin ameliorates oxidative stress-induced senescence in rat nucleus pulposus-derived mesenchymal stem cells via the miR-34a-5p/SIRT1 axis.

BACKGROUND: Intervertebral disc degeneration (IDD) is a main contributor to low back pain. Oxidative stress, which is highly associated with the progression of IDD, increases senescence of nucleus pulposus-derived mesenchymal stem cells (NPMSCs) and weakens the differentiation ability of NPMSCs in degenerated intervertebral discs (IVDs). Quercetin (Que) has been demonstrated to reduce oxidative stress in diverse degenerative diseases. AIM: To investigate the role of Que in oxidative stress-induced NPMSC damage and to elucidate the underlying mechanism. METHODS: In vitro, NPMSCs were isolated from rat tails. Senescence-associated ß-galactosidase (SA-ß-Gal) staining, cell cycle, reactive oxygen species (ROS), real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, and western blot analyses were used to evaluated the protective effects of Que. Meanwhile the relationship between miR-34a-5p and Sirtuins 1 (SIRT1) was evaluated by dual-luciferase reporter assay. To explore whether Que modulates tert-butyl hydroperoxide (TBHP)-induced senescence of NPMSCs via the miR-34a-5p/SIRT1 pathway, we used adenovirus vectors to overexpress and downregulate the expression of miR-34a-5p and used SIRT1 siRNA to knockdown SIRT1 expression. In vivo, a puncture-induced rat IDD model was constructed, and X rays and histological analysis were used to assess whether Que could alleviate IDD in vivo. RESULTS: We found that TBHP can cause NPMSCs senescence changes, such as reduced cell proliferation ability, increased SA-ß-Gal activity, cell cycle arrest, the accumulation of ROS, and increased expression of senescence-related proteins. While abovementioned senescence indicators were significantly alleviated by Que treatment. Que decreased the expression levels of senescence-related proteins (p16, p21, and p53) and senescence-associated secreted phenotype (SASP), including IL-1ß, IL-6, and MMP-13, and it increased the expression of SIRT1. In addition, the protective effects of Que on cell senescence were partially reversed by miR-34a-5p overexpression and SIRT1 knockdown. In vivo, X-ray, and histological analyses indicated that Que alleviated IDD in a puncture-induced rat model. CONCLUSION: In summary, the present study provides evidence that Que reduces oxidative stress-induced senescence of NPMSCs via the miR-34a/SIRT1 signaling pathway, suggesting that Que may be a potential agent for the treatment of IDD.

Incidence of Spontaneous Resorption of Lumbar Disc Herniation: A Meta-analysis.

STUDY DESIGN: A meta-analysis. OBJECTIVE: This study aimed to analyze the incidence of spontaneous resorption of lumbar disk herniation (LDH) after conservative treatment. SUMMARY OF BACKGROUND DATA: The resorption of intervertebral disks has been more frequently reported, but there is a lack of reference to the probability of resorption. METHODS: We strictly refer to the standard established in the PRISMA (Preferred Reporting Items for a Systematic Review and Meta-analysis) statement, comprehensively searched electronic databases using the terms related to the spontaneous resorption of LDH. Two reviewers independently evaluated the potential studies, extracted, and analyzed the enrolled data. RESULTS: Thirty-one studies with 2233 patients who received conservative treatment were included for this analysis. We found that the pooled overall incidence of disk resorption was 70.39%, 87.77% for disk sequestration, 66.91% for disk extrusion, 37.53% for disk protrusion, and 13.33% for disk bugle, respectively. The resorption incidence in of 25%≤ reduction of disk herniation (RDH) 50%, RDH≥50%, and RDH=100% were 40.19%, 43.62, and 36.89%. The resorption incidence was 66.98% in Japan, 61.66% in the United States, 83.52% in Korea, 60.68% in China, 78.30% in the UK, 56.70% in Italy, and 83.68% in Turkey, respectively. Subgroup analysis showed that there was no significant difference in resorption incidence among prospective, retrospective studies and randomized controlled trials (P=0.77), and there was no significant difference in evaluation method among qualitative and quantitative studies (P=0.05). CONCLUSIONS: The existing evidence shows that the overall resorption incidence of LDH was 70.39%, the resorption incidence of ruptured LDH is higher than that of contained LDH. There are significant differences in the resorption incidence among countries. The resorption process mainly occurred within 6 months of conservative treatment.

Nucleus Pulposus Cells Induce M2 Polarization of RAW264.7 via CX3CL1/CX3CR1 Pathway and M2 Macrophages Promote Proliferation and Anabolism of Nucleus Pulposus Cells.

Background: The mechanisms underlying M2 macrophage polarization induced by nucleus pulposus (NP) cells are unclear. The effects that M2-polarized macrophages have on NP cells are also controversial. Methods: Transcriptome sequencing was performed to detect the gene change profiles between NP cells from ruptured intervertebral disc (IVD) and normal IVD. The main difference on biological activities between the two cell groups were analyzed by GO analysis and KEGG analysis. Virus transduction, flow cytometry, immunofluorescence, RT-PCR, western blot, CCK-8, TUNEL staining, and AO/EB staining were performed to explore the interactions between NP cells and RAW264.7 macrophages. Statistics were performed using SPSS26. Results: 801 upregulated and 276 downregulated genes were identified in NP cells from ruptured IVD in mouse models. According to GO and KEGG analysis, we found that the differentially expressed genes (DEG) were dominantly enriched in inflammatory response, extracellular matrix degradation, blood vessel morphogenesis, immune effector process, ossification, chemokine signaling pathway, macrophage activation, etc. CX3CL1 was one of the top 20% DEG, and we confirmed that both NP tissue and cells expressed remarkably higher level of CX3CL1 in mouse models (p < 0.001∗). Besides, we further revealed that both the recombinant CX3CL1 and NP cells remarkably induced M2 polarization of RAW264.7 (p < 0.001∗), respectively, while this effect was significantly reversed by si-CX3CL1 or JMS-17-2 (p < 0.001∗). Furthermore, we found that M2 macrophages significantly decreased the apoptosis rate (p < 0.001∗) and the catabolic gene levels (p < 0.001∗) of NP cells, while increased the viability, proliferation as well as the anabolic gene levels of NP cells (p < 0.01∗). Conclusions: Via regulating CX3CL1/CX3CR1 pathway, NP cells can induce the M2 macrophage polarization. M2 polarized macrophages can further promote NP cell viability, proliferation, and anabolism, while inhibit NP cell apoptosis and catabolism.

Role of regulatory T cells in spinal cord injury.

Spinal cord injury is an intricate process involving a series of multi-temporal and multi-component pathological events, among which inflammatory response is the core. Thus, it is crucial to find a way to prevent the damaging effects of the inflammatory response. The research has found that Treg cells can suppress the activation, proliferation, and effector functions of many parenchymal cells by multiple mechanisms. This review discusses how Treg cells regulate the inflammatory cells to promote spinal cord recovery. These parenchymal cells include macrophages/microglia, oligodendrocytes, astrocytes, and others. In addition, we discuss the adverse role of Treg cells, the status of treatment, and the prospects of cell-based therapies after spinal cord injury. In conclusion, this review provides an overview of the regulatory role of Treg cells in spinal cord injury. We hope to offer new insights into the treatment of spinal cord injury.

Safety and Efficacy of Local Steroid Application on Dysphagia Following Anterior Cervical Discectomy and Fusion: A Systematic Review and Meta-analysis.

STUDY DESIGN: A systematic review and meta-analysis. OBJECTIVE: To evaluate the safety and efficacy of local steroid application (LSA) on dysphagia after anterior cervical discectomy and fusion (ACDF). SUMMARY OF BACKGROUND DATA: Dysphagia is one of the most common adverse events in the early postoperative period of ACDF. LSA is reported as an effective method to reduce the swelling of soft tissues, thereby decreasing the incidence of dysphagia. However, the safety and efficacy of LSA on dysphagia after ACDF need to be systematically reviewed and analyzed. METHODS: A comprehensive literature search was carried out in the database PubMed, Web of Science, EMBASE, Clinical key, Cochrane library, and Wiley Online Library to screen papers that report LSA in ACDF surgery. The Cochrane Collaboration tool and a methodological index for nonrandomized studies were used for the assessment of study quality. Data were analyzed with the Review Manager 5.3 software. RESULTS: A total of 10 studies were included. The results revealed no significant differences between the steroid group and the control group in ACDF regarding postoperative drainage, estimated blood loss, and neck disability index score ( P > 0.05). LSA significantly alleviates visual analog scale score for neck pain (or odynophagia) ( P < 0.05), reduces the length of hospital stay (weighted mean difference, -1.00 (-1.05 to -0.95); P < 0.001), and mitigates dysphagia rate and prevertebral soft-tissue swelling in the early postoperative period ( P < 0.05). There seemed to be no significant increase in the complication rate and steroid-related adverse events in the steroid group compared with the control group ( P < 0.05). CONCLUSIONS: LSA shows advantages in reducing the length of hospital stay, decreasing dysphagia rate, and mitigating prevertebral soft-tissue swelling in the early postoperative period of ACDF. Further large-scale studies are urgently required for the development of a standard protocol for LSA and further analysis of potential delay complications.

Update on the roles of macrophages in the degeneration and repair process of intervertebral discs.

Intervertebral disc (IVD) degeneration is the common cause of lumbar degenerative diseases, causing severe social and economic burden. The process of IVD degeneration involves a complex of pathologic changes on both extracellular matrix degradation and resident cell apoptosis. In recent years, there is increasing evidence that macrophages play vital roles during the damage and repair process of IVD degeneration. Nevertheless, the interactions between macrophages and IVD are not well understood, even if the IVD has long been regarded as the immune privileged site. Therefore, this review mainly focuses on the progress and obstacles of studies investigating the blood supply, immune response and especially macrophages during the IVD degeneration process.

Influence of the facet joint angle on facet joint degeneration following pedicle screw fixation without fusion in thoracolumbar fractures.

BACKGROUND: Posterior approach pedicle screw fixation without fusion is widely used in the treatment of neurologically intact type A3 thoracolumbar fractures. OBJECTIVE: To analyze the influence of the facet joint (FJ) angle on FJ degeneration following posterior approach pedicle screw fixation without fusion in neurologically intact type A3 thoracolumbar fractures. METHODS: Fifty-eight patients who underwent pedicle screw fixation via the traditional posterior approach (n= 28) or the Wiltse approach (n= 30) were enrolled. A CT scan was performed before fixation and before fixation removal (Within 1.5 to 2 years after fixation) to evaluate the FJs parameters, including FJ inclination (FJI), FJ tropism (FJT), FJ violation, and FJ degeneration grade (FJDG), of three fixed segments and the adjacent segment below the fixed segments. RESULTS: There was no significant difference in FJ violation rate, FJDG deterioration, or FJ angle between the two groups (P> 0.05). FJDG deterioration showed a weak positive correlation with FJI and FJT before fixation, and the angular change in FJI (P< 0.05); and FJT before fixation and the angular change in FJI were risk factors for FJDG deterioration (P< 0.01). CONCLUSION: The Wiltse approach did not increase the rate of FJDG deterioration and FJs angle changes. However, the FJT before fixation and the angular change in FJI were risk factors for FJDG deterioration.

Predictive factors for residual leg numbness after decompression surgery for lumbar degenerative diseases.

BACKGROUND: The purpose of this study is to evaluate the change patterns of leg numbness (LN) after lumbar decompression surgery (LDS), and to find the predictive factors that affect the recovery of numbness. METHODS: Patients who underwent LDS in our institution between August 2020 and July 2021 were prospectively enrolled in this study, and were followed by a 12-month follow-up. The degree of LN, leg pain (LP) and the disability were assessed using the visual analog scale (VAS) and oswestry disability index (ODI). RESULTS: A total of 314 patients finished the 12-month follow-up. The preoperative mean VAS-LN score was 3.49 ± 2.44, which decreased to 1.91 ± 1.30 at 3 months, to 1.29 ± 0.97 at 6 months and to 1.26 ± 0.96 at 12 months after surgery. The preoperative mean VAS-LP score was 6.05 ± 1.30, which decreased to 2.00 ± 0.86 at 3 months, to 1.02 ± 0.80 at 6 months, and to 0.49 ± 0.71 at 12 months after surgery. The preoperative mean ODI score was 27.90 ± 7.08, which decreased to 9.73 ± 3.09 at 3 months, to 6.72 ± 2.98 at 6 months, and to 4.57 ± 2.76 at 12 months after surgery. Via multivariate logistic regression analysis, only preoperative VAS-LN score (p < 0.001*) was identified as a significantly independent predictive factor for residual LN after operation. CONCLUSION: Clinically significant improvement in LN was observed in the majority of patients within 6 months after LDS, and the improvement of VAS-LN was slower than the VAS-LP. High pre-operative VAS-LN score can independently predict the presence of residual LN after surgery at 12-month follow up.

Minimally invasive posterior cervical foraminotomy versus anterior cervical discectomy and fusion for cervical radiculopathy: a meta-analysis.

With the recent development of minimally invasive techniques, minimally invasive posterior cervical foraminotomy (MIS-PCF) has become increasingly popular as a minimally invasive method to treat cervical radiculopathy. However, there are still controversies about whether MIS-PCF is superior to anterior cervical discectomy and fusion (ACDF). The purpose of this study is to evaluate the therapeutic effects of MIS-PCF and ACDF on unilateral cervical radiculopathy without myelopathy. We searched PubMed, Embase, the Cochrane Library, and Scopus comprehensively using the terms related to MIS-PCF. Two reviewers independently evaluated the potential studies, and extracted and analyzed the data of operation time, hospital stay, neck disability index (NDI) score, visual analog scale for neck pain (VAS-neck) and arm pain (VAS-arm) scores, reoperation rate, and complications. Seven studies with 1175 patients were included. The study population was 53.5% male, with a mean age of 48.9. MIS-PCF presented a significantly shorter postoperative hospitalization time compared to ACDF, while the operation time, complication/reoperation rate, and VAS-arm, VAS-neck, and NDI scores were comparable between the two cohorts. In North America, the average cost of MIS-PCF is lower than ACDF. Thus, we suggest that MIS-PCF is an alternative to ACDF for selected patients.

1,25(OH)2D3 Mitigates Oxidative Stress-Induced Damage to Nucleus Pulposus-Derived Mesenchymal Stem Cells through PI3K/Akt Pathway.

Intervertebral disc degeneration (IVDD) is one of the main causes of low back pain. The local environment of the degenerated intervertebral disc (IVD) increases oxidative stress and apoptosis of endogenous nucleus pulposus-derived mesenchymal stem cells (NPMSCs) and weakens its ability of endogenous repair ability in degenerated IVDs. A suitable concentration of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been certified to reduce oxidative stress and cell apoptosis. The current study investigated the protective effect and potential mechanism of 1,25(OH)2D3 against oxidative stress-induced damage to NPMSCs. The present results showed that 1,25(OH)2D3 showed a significant protective effect on NPMSCs at a concentration of 10-10 M for 24 h. Protective effects of 1,25(OH)2D3 were also exhibited against H2O2-induced NPMSC senescence, mitochondrial dysfunction, and reduced mitochondrial membrane potential. The Annexin V/PI apoptosis detection assay, TUNEL assay, immunofluorescence, western blot, and real-time quantitative polymerase chain reaction assay showed that pretreatment with 1,25(OH)2D3 could alleviate H2O2-induced NPMSC apoptosis, including the apoptosis rate and the expression of proapoptotic-related (Caspase-3 and Bax) and antiapoptotic-related (Bcl-2) proteins. The intracellular expression of p-Akt increased after pretreatment with 1,25(OH)2D3. However, these protective effects of 1,25(OH)2D3 were significantly decreased after the PI3K/Akt pathway was inhibited by the LY294002 treatment. In vivo, X-ray, MRI, and histological analyses showed that 1,25(OH)2D3 treatment relieved the degree of IVDD in Sprague-Dawley rat disc puncture models. In summary, 1,25(OH)2D3 efficiently attenuated oxidative stress-induced NPMSC apoptosis and mitochondrial dysfunction via PI3K/Akt pathway and is a promising candidate treatment for the repair of IVDD.

Outcomes of Oblique Lateral Interbody Fusion for Adult Spinal Deformity: A Systematic Review and Meta-Analysis.

STUDY DESIGN: A systematic review and meta-analysis. OBJECTIVES: To evaluate clinical and radiographic outcomes, and perioperative complications of oblique lateral interbody fusion (OLIF) for adult spinal deformity (ASD). METHODS: We performed a systematic review and meta-analysis of related studies reporting outcomes of OLIF for ASD. The clinical outcomes were assessed by visual analogue scale (VAS) and Oswestry Disability Index (ODI). The radiographic parameters were evaluated by sagittal vertical axis (SVA), pelvic tilt (PT), sacral slope (SS), thoracic kyphosis (TK), lumbar lordosis (LL), pelvic incidence-lumbar lordosis (PI-LL), Cobb angle and fusion rate. A random effects model and 95% confidence intervals (CI) were performed to investigate the results. RESULTS: A total of 16 studies involving 519 patients were included in the present study. The mean difference of VAS-back score, VAS-leg score and ODI score before and after surgery was 5.1, 5.0 and 32.3 respectively. The mean correction of LL was 20.6°, with an average of 6.9° per level and the mean correction of Cobb was 16.4°, with an average of 4.7° per level. The mean correction of SVA, PT, SS, TK and PI-LL was 59.3 mm, 11.7°, 6.9°, 9.4° and 20.6° respectively. The mean fusion rate was 94.1%. The incidence of intraoperative and postoperative complications was 4.9% and 29.6% respectively. CONCLUSIONS: OLIF is an effective and safe surgery method in the treatment of mild or moderate ASD and it has advantages in less intraoperative blood loss and lower perioperative complications.

Urolithin a alleviates oxidative stress-induced senescence in nucleus pulposus-derived mesenchymal stem cells through SIRT1/PGC-1α pathway.

BACKGROUND: In degenerative intervertebral disc (IVD), an unfavorable IVD environment leads to increased senescence of nucleus pulposus (NP)-derived mesenchymal stem cells (NPMSCs) and the inability to complete the differentiation from NPMSCs to NP cells, leading to further aggravation of IVD degeneration (IDD). Urolithin A (UA) has been proven to have obvious effects in delaying cell senescence and resisting oxidative stress. AIM: To explore whether UA can alleviate NPMSCs senescence and to elucidate the underlying mechanism. METHODS: In vitro, we harvested NPMSCs from rat tails, and divided NPMSCs into four groups: the control group, H2O2 group, H2O2 + UA group, and H2O2 + UA + SR-18292 group. Senescence-associated ß-Galactosidase (SA-ß-Gal) activity, cell cycle, cell proliferation ability, and the expression of senescence-related and silent information regulator of transcription 1/PPAR gamma coactivator-1α (SIRT1/ PGC-1α) pathway-related proteins and mRNA were used to evaluate the protective effects of UA. In vivo, an animal model of IDD was constructed, and X-rays, magnetic resonance imaging, and histological analysis were used to assess whether UA could alleviate IDD in vivo. RESULTS: We found that H2O2 can cause NPMSCs senescence changes, such as cell cycle arrest, reduced cell proliferation ability, increased SA-ß-Gal activity, and increased expression of senescence-related proteins and mRNA. After UA pretreatment, the abovementioned senescence indicators were significantly alleviated. To further demonstrate the mechanism of UA, we evaluated the mitochondrial membrane potential and the SIRT1/PGC-1α pathway that regulates mitochondrial function. UA protected mitochondrial function and delayed NPMSCs senescence by activating the SIRT1/PGC-1α pathway. In vivo, we found that UA treatment alleviated an animal model of IDD by assessing the disc height index, Pfirrmann grade and the histological score. CONCLUSION: In summary, UA could activate the SIRT1/PGC-1α signaling pathway to protect mitochondrial function and alleviate cell senescence and IDD in vivo and vitro.

Allicin can suppress the activity of vascular endothelial cells probably by regulating JAK2/STAT3 pathway.

Whether allicin can suppress the angiogenesis via inhibiting the activity of vascular endothelial cells (VECs) in preventing epidural hypertrophic scars remains unknown. VECs were treated by allicin at a gradient of concentrations. Cell activity was measured by CCK-8 assay, scratch assay and flow cytometry. Reverse-transcription PCR and Western Blot were used to measure the expression levels of relevant genes and proteins. After treated with allicin at concentrations of 0, 25, 50 and 100 mg/L, the viability of VECs significantly decreased at 24 h (p < 0.001*) and 48 h (p < 0.001*), and migration rate significantly decreased in scratch assay (p = 0.017*) and in Transwell assay (p = 0.021*). As the concentrations of allicin increased, the apoptosis rate of VECs rose up (p = 0.018*). There was no significant difference on cell numbers at S phase (p = 0.25), but cell numbers at G1 phase decreased (p = 0.039*) and at G2 phase increased (p = 0.047*). With the increase of allicin concentrations, the ability of tube formation for VECs significantly decreased (p < 0.001*). Comparing with control group, the expression of PCNA and BCL-2 decreased (p < 0.001*), while the expression of BAX increased significantly (p < 0.001*). Regarding to JAK2/STAT3 pathway, the expression levels of JAK3 and STAT3 decreased significantly with the increase of allicin concentrations (p < 0.001*). Allicin can suppress the activity of VECs probably by regulating JAK2/STAT3 pathway.

Insights of stem cell-based endogenous repair of intervertebral disc degeneration.

Low back pain has become more prevalent in recent years, causing enormous economic burden for society and government. Common therapies used in clinics including conservative treatment and surgery can only relieve pain. Subsequent cell-based treatment such as mesenchymal stem cell transplantation poses problems such as short duration of therapeutic effect and tumorigenesis. Recently, the discovery and identification of stem cell niche and stem/progenitor cells in intervertebral disc bring increased attention to endogenous repair strategy. Therefore, we review the studies involving endogenous repair strategy and present the characteristics and current status of this treatment. Meanwhile, we also discuss the strategy and perspective of endogenous repair strategy in future.

The effect of high glucose on the biological characteristics of nucleus pulposus-derived mesenchymal stem cells.

Diabetes mellitus (DM) is a dependent risk factor in the progression of intervertebral disc degeneration (IVDD). High glucose supply has negative effects on nucleus pulpous (NP) cell and mesenchymal stem cell (MSC) biology. However, the effect of hyperglycaemia on the biological characterization of nucleus pulpous-derived mesenchymal stem cell (NPMSC) has not been investigated previously. Therefore, further exploration of the effects of DM-associated hyperglycaemia on NPMSC biology is important to better understand and develop endogenous repair strategies of DM patient-associated IVDD. Therefore, the cell biological characteristics were compared between NPMSC cultured in media with low glucose concentration (LG-NPMSC) and high glucose concentration (HG-NPMSC). The results demonstrated that HG-NPMSC showed significantly decreased cell proliferation, colony formation ability, migration and wound-healing capability compared with those of LG-NPMSC. HG-NPMSC also showed significantly decreased expressions of stemness genes and mRNA and protein expressions of silent information regulator protein 1 (SIRT1), SIRT6, hypoxia inducible factor-1α (HIF-1α) and glucose transporter 1 (GLUT-1), whereas increased cell apoptosis, cell senescence and caspase-3 expression. These results suggest that high glucose may decrease proliferation and stemness maintenance ability and increase apoptosis and senescence of NPMSC. SIGNIFICANCE OF THE STUDY: We found that high glucose concentration significantly decreased cell proliferation, colony formation ability, migration and wound-healing capability of nucleus pulposus-derived mesenchymal stem cells. Moreover, high glucose cultured nucleus pulposus-derived mesenchymal stem cells showed significantly decreased expression of stemness genes, related mRNA and protein, whereas increased cell apoptosis, cell senescence and expression of caspase-3. The present study indicated that better control of high concentration glucose in the early stage of diabetes mellitus should be recommended to prevent or limit intervertebral disc degeneration.

Allicin Inhibits Proliferation and Promotes Apoptosis of Human Epidural Scar Fibroblasts.

BACKGROUND: Allicin can suppress liver and cardiac fibrosis; thus, we hypothesized that it might prevent scar tissue from extensive epidural fibrosis after laminectomy. METHODS: Human epidural scar fibroblasts were isolated from surgical specimens and treated with allicin at a gradient of concentrations. Morphology, viability, migration rate, cell cycle, and apoptosis rate were measured by fluorescence microscope, Cell Counting Kit-8 assay, scratch assay, and flow cytometry. Western blot was used to measure the expression level of proliferation-related proteins. RESULTS: After treatment by allicin, cell viability (P = 0.042) and migration rate (P = 0.010 in scratch assay and P = 0.025 in transwell assay) decreased significantly in a dose-dependent manner. The percentage of G1 phase cells significantly decreased (P = 0.017), whereas the percentage of S phase cells (P = 0.096) and G2 phase cells (P = 0.038) significantly increased in a dose-dependent manner. Similarly, the percentage of apoptotic cells increased significantly in a dose-dependent manner (P = 0.036). Compared with the control group, the expression level of proliferating cell nuclear antigen protein (P = 0.081) and Bcl-2 protein (P = 0.029) significantly decreased, whereas the BAX protein level significantly increased (P = 0.017). CONCLUSIONS: Allicin can suppress human epidural scar fibroblast migration, induce cell apoptosis, and block cell proliferation at S phase and G2 phase.

6-gingerol protects nucleus pulposus-derived mesenchymal stem cells from oxidative injury by activating autophagy.

BACKGROUND: To date, there has been no effective treatment for intervertebral disc degeneration (IDD). Nucleus pulposus-derived mesenchymal stem cells (NPMSCs) showed encouraging results in IDD treatment, but the overexpression of reactive oxygen species (ROS) impaired the endogenous repair abilities of NPMSCs. 6-gingerol (6-GIN) is an antioxidant and anti-inflammatory reagent that might protect NPMSCs from injury. AIM: To investigate the effect of 6-GIN on NPMSCs under oxidative conditions and the potential mechanism. METHODS: The cholecystokinin-8 assay was used to evaluate the cytotoxicity of hydrogen peroxide and the protective effects of 6-GIN. ROS levels were measured by 2´7´-dichlorofluorescin diacetate analysis. Matrix metalloproteinase (MMP) was detected by the tetraethylbenzimidazolylcarbocyanine iodide assay. TUNEL assay and Annexin V/PI double-staining were used to determine the apoptosis rate. Additionally, autophagy-related proteins (Beclin-1, LC-3, and p62), apoptosis-associated proteins (Bcl-2, Bax, and caspase-3), and PI3K/Akt signaling pathway-related proteins (PI3K and Akt) were evaluated by Western blot analysis. Autophagosomes were detected by transmission electron microscopy in NPMSCs. LC-3 was also detected by immunofluorescence. The mRNA expression of collagen II and aggrecan was evaluated by real-time polymerase chain reaction (RT-PCR), and the changes in collagen II and MMP-13 expression were verified through an immunofluorescence assay. RESULTS: 6-GIN exhibited protective effects against hydrogen peroxide-induced injury in NPMSCs, decreased hydrogen peroxide-induced intracellular ROS levels, and inhibited cell apoptosis. 6-GIN could increase Bcl-2 expression and decrease Bax and caspase-3 expression. The MMP, Annexin V-FITC/PI flow cytometry and TUNEL assay results further confirmed that 6-GIN treatment significantly inhibited NPMSC apoptosis induced by hydrogen peroxide. 6-GIN treatment promoted extracellular matrix (ECM) expression by reducing the oxidative stress injury-induced increase in MMP-13 expression. 6-GIN activated autophagy by increasing the expression of autophagy-related markers (Beclin-1 and LC-3) and decreasing the expression of p62. Autophagosomes were visualized by transmission electron microscopy. Pretreatment with 3-MA and BAF further confirmed that 6-GIN-mediated stimulation of autophagy did not reduce autophagosome turnover but increased autophagic flux. The PI3K/Akt pathway was also found to be activated by 6-GIN. 6-GIN inhibited NPMSC apoptosis and ECM degeneration, in which autophagy and the PI3K/Akt pathway were involved. CONCLUSION: 6-GIN efficiently decreases ROS levels, attenuates hydrogen peroxide-induced NPMSCs apoptosis, and protects the ECM from degeneration. 6-GIN is a promising candidate for treating IDD.

Naringin alleviates H2O2-induced apoptosis via the PI3K/Akt pathway in rat nucleus pulposus-derived mesenchymal stem cells.

Purpose: To investigate the protective effect of naringin (Nar) on H2O2-induced apoptosis of nucleus pulposus-derived mesenchymal stem cells (NPMSC) and the potential mechanism in this process. Methods: Rat NPMSC were cultured in MSC culture medium or culture medium with different concentrations of H2O2. Nar or the combination of Nar and LY294002 was added into the culture medium to investigate the effects of Nar. Cell viability was evaluated by cell counting kit-8 (CCK-8) assay. The apoptosis rate was determined using Annexin V/PI dual staining and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays. Additionally, the levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were analyzed by flow cytometry. ATP level in NPMSC was analyzed via ATP detection kit. Mitochondrial ultrastructure change was observed through transmission electron microscope (TEM). Levels of apoptosis-associated molecules (cleaved caspase-3, Bax and Bcl-2) were evaluated via RT-PCR and western blot, respectively. Results: The cells isolated from NP met the criteria for MSC. H2O2 significantly promoted NPMSC apoptosis in a dose and time-dependent manner. Nar showed no cytotoxicity effect on NPMSC up to a concentration of 100 µM for 24 h. Nar exhibited protective effects against H2O2-induced NPMSC apoptosis including apoptosis rate, expressions of proapoptosis and antiapoptosis related genes and protein. Nar could also alleviate H2O2-induced mitochondrial dysfunction of increased mitochondrial ROS production, reduced MMP, decreased intracellular ATP and mitochondrial ultrastructure change. However, these protected effects were inhibited after LY294002 treatment. Conclusions: Our results demonstrated that Nar efficiently attenuated H2O2-induced NPMSC apoptosis and mitochondrial dysfunction. The activation of ROS-mediated PI3K/Akt pathway may be the potential mechanism in this process.

Injectable Hydrogel Combined with Nucleus Pulposus-Derived Mesenchymal Stem Cells for the Treatment of Degenerative Intervertebral Disc in Rats.

Stem cell-based tissue engineering in treating intervertebral disc (IVD) degeneration is promising. An appropriate cell scaffold can maintain the viability and function of transplanted cells. Injectable hydrogel has the potential to be an appropriate cell scaffold as it can mimic the condition of the natural extracellular matrix (ECM) of nucleus pulposus (NP) and provide binding sites for cells. This study was aimed at investigating the effect of injectable hydrogel-loaded NP-derived mesenchymal stem cells (NPMSC) for the treatment of IVD degeneration (IDD) in rats. In this study, we selected injectable 3D-RGD peptide-modified polysaccharide hydrogel as a cell transplantation scaffold. In vitro, the biocompatibility, microstructure, and induced differentiation effect on NPMSC of the hydrogel were studied. In vivo, the regenerative effect of hydrogel-loaded NPMSC on degenerated NP in a rat model was evaluated. The results showed that NPMSC was biocompatible and able to induce differentiation in hydrogel in vivo. The disc height index (almost 87%) and MRI index (3313.83 ± 227.79) of the hydrogel-loaded NPMSC group were significantly higher than those of other groups at 8 weeks after injection. Histological staining and immunofluorescence showed that the hydrogel-loaded NPMSC also partly restored the structure and ECM content of degenerated NP after 8 weeks. Moreover, the hydrogel could support long-term NPMSC survival and decrease cell apoptosis rate of the rat IVD. In conclusion, injectable hydrogel-loaded NPMSC transplantation can delay the level of IDD and promote the regeneration of the degenerative IVD in the rat model.

The effect of intervertebral disc degenerative change on biological characteristics of nucleus pulposus mesenchymal stem cell: an in vitro study in rats.

Purpose: To evaluate the change on biological characteristics of mesenchymal stem cell (MSC) derived from normal and degenerative intervertebral disc (IVD). Methods: MSC was isolated from normal and degenerative IVD rat model. Immunophenotype detected by flow cytometric analysis, expression of stemness genes determined by reverse-transcription polymerase chain reaction (RT-PCR) and osteogenic, adipogenic and chondrogenic differentiation were compared between MSC derived from normal IVD (N-NPMSC) and degenerative IVD (D-NPMSC). The biological characteristics including cell proliferation, colony formation, apoptosis, caspase-3 activity and mRNA and protein expressions of hypoxia inducible factor-1α (HIF-1α), glucose transporter 1 (GLUT-1), vascular endothelial growth factor (VEGF), silent information regulator protein 1 (SIRT1) and silent information regulator protein 6 (SIRT6) were compared between N-NPMSC and D-NPMSC. Results: Both of N-NPMSC and D-NPMSC highly expressed CD105, CD90 and CD73, and lower expressed CD34 and CD45. There was no significant difference in cell morphology and multipotent differentiation ability between N-NPMSC and D-NPMSC. D-NPMSC showed significantly lower expressions of stemness genes, cell proliferation and colony formation ability. D-NPMSC also exhibited increased cell apoptosis rate and caspase-3 expression, and significantly lower expressions of HIF-1α, GLUT-1, VEGF, SIRT1 and SIRT6 in mRNA and protein levels compared with N-NPMSC. Conclusions: N-NPMSC showed significantly higher proliferation rate, better colony forming and stemness maintenance ability, whereas reduced cell apoptosis rate compared with D-NPMSC. HIF-1α-mediated signal pathway may be involved in the regulation of NPMSC proliferation. These findings indicated that degenerative change of IVD should be taken into account when selecting a source of NPMSC for clinical application.