Resveratrol-enhanced SIRT1-mediated osteogenesis in porous endplates attenuates low back pain and anxiety behaviors.

Low back pain (LBP) is a major clinical problem that lacks effective treatments. The sensory innervation in porous vertebral endplates and anxiety contributes to spinal hyperalgesia. We hypothesized that SIRT1 activator resveratrol alleviates LBP and anxiety via promotion of osteogenesis in the porous endplates. The hyperalgesia and anxiety-related behaviors; sensory innervation, inflammation and porosity of endplates; and osteogenic/osteoclastic factors expression were measured following resveratrol treatment after lumbar spine instability (LSI) surgery. To explore whether resveratrol promotes endplates osteogenesis and thus alleviates LBP through activation of SIRT1 in the osteoprogenitor cells of endplates, SIRT1OSX-/- mice were employed. Additionally, the levels of inflammation markers, phosphorylation of cAMP response element-binding protein (pCREB), and brain-derived neurotrophic factor (BDNF) in hippocampus were evaluated. After 4 or 8 weeks LSI surgery, the mice suffered from hyperalgesia and anxiety, which were efficiently attenuated by resveratrol at 8 weeks. Resveratrol treatment-enhanced osteogenesis and decreased endplates porosities accompanied with the reduction of TNFα, IL-1ß, and COX2 levels and CGRP+ nerve fibers innervation in porous endplates. Resveratrol-mediated endplates osteogenesis, decreased endplates porosities, and analgesic and antianxiety effects were abrogated in SIRT1OSX-/- mice. Furthermore, resveratrol relieved inflammation and increased pCREB and BDNF expression in the hippocampus after 8 weeks, which alleviate anxiety-related behaviors. This study provides that resveratrol-mediated porous endplates osteogenesis via the activation of SIRT1 markedly blocked sensory innervation and inflammation in endplates, therefore, alleviating LSI surgery-induced LBP and hippocampus-related anxiety.

Novel Insights into the Stemness and Immune Privilege of Mesenchymal Stem Cells from Human Wharton Jelly by Single-Cell RNA Sequencing.

BACKGROUND Fundamental and clinical interest in mesenchymal stem cells (MSCs) has risen dramatically over the past 3 decades. The immunomodulatory and differentiation abilities are the main mechanisms in vitro and in vivo. However, increasing evidence casts doubt on the stemness and immunogenicity of MSCs. MATERIAL AND METHODS We conducted a high-throughput 10x RNA sequencing and Smart-seq2 scRNA-seq analysis to reveal gene expression of Wharton jelly MSCs (WJ-MSCs) at a single-cell level. Multipotent differentiation, subpopulations, marker genes, human leucocyte antigen (HLA) gene expression, and cell cluster trajectory analysis were evaluated. RESULTS The WJ-MSCs had considerable heterogeneity between cells in terms of gene expression. They highly, partially, and hardly expressed genes related to mesodermal differentiation, endodermal differentiation, and ectodermal differentiation, respectively. Some cells seem to be bipotent or unipotent stem cells. Further, Monocle and cell cluster trajectory analysis demonstrated that 1 of the 3 divided clusters performed as stem cells, accounting for 12.6% of the population. The marker genes for a stem cell cluster were CRIM1, GLS, PLOD2, NEXN, ACTR2, FN1, MBNL1, LMOD1, COL3A1, NCL, SEC62, EPRS, COL5A2, COL8A1, and VCAN. In addition, the MSCs also highly, partially, and hardly expressed HLA-I antigen genes, HLA-II genes, and the HLA-G gene, respectively, indicating that MSCs probably have immunogenicity. A Kyoto Encyclopedia of Genes and Genomes pathway analysis of the 3 clusters demonstrated that they were mainly connected with viral infectious diseases, cancer, and endocrine and metabolic disorders. The most expressed transcription factors were zf-C2H2, HMG/HMGY, and Homeobox. CONCLUSIONS We found that only a subpopulation of WJ-MSCs are real stem cells and WJ-MSCs probably do not have immune privilege.

Melatonin exerts neuroprotective effects in mice with spinal cord injury by activating the Nrf2/Keap1 signaling pathway via the MT2 receptor.

Spinal cord injury (SCI) is a devastating event that often leads to severe disability, and effective treatments for SCI are currently limited. The present study investigated the potential effects and specific mechanisms of melatonin treatment in SCI. Mice were divided into Sham (Sham), Vehicle (Veh), Melatonin (Mel), and Melatonin + 4-phenyl-2-propionamidotetralin (4P-PDOT) (Mel + 4PP) groups based on randomized allocation. The expression of MT2 and the nuclear factor-erythroid 2-related factor 2 (Nrf2)/Keap1 signaling pathways were examined, along with oxidative stress indicators, inflammatory factors and GFAP-positive cells near the injury site. The polarization of microglial cells in different inflammatory microenvironments was also observed. Cell survival, motor function recovery and spinal cord tissue morphology were assessed using staining and Basso Mouse Scale scores. On day 7 after SCI, the results revealed that melatonin treatment increased MT2 protein expression and activated the Nrf2/Keap1 signaling pathway. It also reduced GFAP-positive cells, mitigated oxidative stress, and suppressed inflammatory responses around the injury site. Furthermore, melatonin treatment promoted the polarization of microglia toward the M2 type, increased the number of neutrophil-positive cells, and modulated the transcription of Bax and Bcl2 in the injured spinal cord. Melatonin treatment alleviated the severity of spinal injuries and facilitated functional recovery in mice with SCI. Notably, blocking MT2 with 4P-PDOT partially reversed the neuroprotective effects of melatonin in SCI, indicating that the activation of the MT2/Nrf2/Keap1 signaling pathway contributes to the neuroprotective properties of melatonin in SCI. The therapeutic and translational potentials of melatonin in SCI warrant further investigation.

Risk Factors for New Adjacent and Remote Vertebral Fracture After Percutaneous Vertebroplasty.

OBJECTIVE: To analyze the risk factors of new adjacent vertebral fractures (AVF) and remote vertebral fractures (RVF) after percutaneous vertebroplasty (PVP) for osteoporotic vertebral compression fractures (OVCFs). METHODS: Patients who underwent additional PVP for new OVCFs were enrolled. In addition, we set a 1:1 age-, sex-, surgical segment-, and surgical date-matched control group, in which patients underwent PVP without new OVCFs. Data on body mass index, occurrence time of second PVP, vertebral computed tomography (CT) Hounsfield Unit (HU) at the fracture adjacent segment, and RVF segment were collected. RESULTS: A total of 44 patients who underwent additional PVP for new OVCFs at our hospital were included. AVF occurred significantly earlier than RVF (13.5 ± 14.1 vs. 30.4 ± 20.1 months, P = 0.007). Compared to the control group, the AVF segment CT HU was significantly lower in patients with AVF (28.7 ± 16.7 vs. 61.3 ± 14.7, P = 0.000), while there was no significant difference between patients with RVF and control group including both adjacent and RVF segment CT HU. Receiver operating characteristic curves identified a cutoff value of 43 for using adjacent segment CT HU to differentiate patients with AVF from controls, with a sensitivity of 80% and a specificity of 88.9%. CONCLUSIONS: Our study showed that the risk factors for AVF and RVF after PVP surgery were different. The occurrence of AVF was earlier and associated with low adjacent segment CT HU values, whereas the preoperative CT HU in both adjacent and RVF segments was not found to be associated with RVF.

Inflammatory response in traumatic brain and spinal cord injury: The role of XCL1-XCR1 axis and T cells.

BACKGROUND: Traumatic brain injury (TBI) and spinal cord injury (SCI) are acquired injuries to the central nervous system (CNS) caused by external forces that cause temporary or permanent sensory and motor impairments and the potential for long-term disability or even death. These conditions currently lack effective treatments and impose substantial physical, social, and economic burdens on millions of people and families worldwide. TBI and SCI involve intricate pathological mechanisms, and the inflammatory response contributes significantly to secondary injury in TBI and SCI. It plays a crucial role in prolonging the post-CNS trauma period and becomes a focal point for a potential therapeutic intervention. Previous research on the inflammatory response has traditionally concentrated on glial cells, such as astrocytes and microglia. However, increasing evidence highlights the crucial involvement of lymphocytes in the inflammatory response to CNS injury, particularly CD8+ T cells and NK cells, along with their downstream XCL1-XCR1 axis. OBJECTIVE: This review aims to provide an overview of the role of the XCL1-XCR1 axis and the T-cell response in inflammation caused by TBI and SCI and identify potential targets for therapy. METHODS: We conducted a comprehensive search of PubMed and Web of Science using relevant keywords related to the XCL1-XCR1 axis, T-cell response, TBI, and SCI. RESULTS: This study examines the upstream and downstream pathways involved in inflammation caused by TBI and SCI, including interleukin-15 (IL-15), interleukin-12 (IL-12), CD8+ T cells, CD4+ T cells, NK cells, XCL1, XCR1+ dendritic cells, interferon-gamma (IFN-γ), helper T0 cells (Th0 cells), helper T1 cells (Th1 cells), and helper T17 cells (Th17 cells). We describe their proinflammatory effect in TBI and SCI. CONCLUSIONS: The findings suggest that the XCL1-XCR1 axis and the T-cell response have great potential for preclinical investigations and treatments for TBI and SCI.

Role of dendritic cells in spinal cord injury.

BACKGROUND: Inflammation can worsen spinal cord injury (SCI), with dendritic cells (DCs) playing a crucial role in the inflammatory response. They mediate T lymphocyte differentiation, activate microglia, and release cytokines like NT-3. Moreover, DCs can promote neural stem cell survival and guide them toward neuron differentiation, positively impacting SCI outcomes. OBJECTIVE: This review aims to summarize the role of DCs in SCI-related inflammation and identify potential therapeutic targets for treating SCI. METHODS: Literature in PubMed and Web of Science was reviewed using critical terms related to DCs and SCI. RESULTS: The study indicates that DCs can activate microglia and astrocytes, promote T-cell differentiation, increase neurotrophin release at the injury site, and subsequently reduce secondary brain injury and enhance functional recovery in the spinal cord. CONCLUSIONS: This review highlights the repair mechanisms of DCs and their potential therapeutic potential for SCI.

Recent advances in senescence-associated secretory phenotype and osteoporosis.

The worldwide elderly population is on the rise, and aging is a major osteoporosis risk factor. Senescent cells accumulation can have a detrimental effect the body as we age. The senescence-associated secretory phenotype (SASP), an essential cellular senescence hallmark, is an important mechanism connecting cellular senescence to osteoporosis. This review describes in detail the characteristics of SASPs and their regulatory agencies, and shed fresh light on how SASPs from different senescent cells contribute to osteoporosis development. Furthermore, we summarized various innovative therapy techniques that target SASPs to lower the burden of osteoporosis in the elderly and discussed the potential challenges of SASPs-based therapy for osteoporosis as a new clinical trial.

Cell Reprogramming Strategies for Treating Osteoarthritis and Intervertebral Disc Degeneration.

Osteoarthritis (OA) and intervertebral disc degeneration (IVDD) are the most common degenerative bone and joint diseases, posing a major threat to patients' physical and mental health due to the occurrence of chronic pain and disability. Within this context, the absence of efficacious therapies has led to a growing interest in regenerative medicine. In particular, as a method that can erase the memory of differentiation and re-endow cells with pluripotency, cell reprogramming technologies have ushered in a new era of personalized therapy, which not only show great potential for the treatment of degenerative osteoarthropathies but also promise to achieve tissue regenerative and repair. However, compared to other areas of research, reprogramming technologies to treat OA and IVDD are still in the preliminary stages and require further investigation. This paper briefly introduces the characteristics of cell reprogramming; summarizes the pathological mechanisms of reprogramming to improves energy metabolism, aging, inflammation, oxidative stress, and immune imbalance in OA and IVDD under the background of microenvironment and immunity; highlights the significant advantages of reprogramming-derived cells compared to embryonic stem cells and mesenchymal stem cells, based on these advances, providing important strategies for its development and clinical application in OA and IVDD.

Implant failure and revision strategies after total spondylectomy for spinal tumors.

Background: Although there have been several risk factors reported for implant failure (IF), little consensus exists. Potential applicable measures to protect patients from IF are relatively few. This study aimed to discover new risk factors for IF and explore potential protective measures from IF after total spondylectomy for spinal tumors. Methods: A total of 145 patients undergoing total spondylectomy for thoracic and lumbar spinal tumors between 2010 and 2021 were included from three tertiary university hospitals. Patient demographic and surgical characteristics and follow-up outcomes were collected. Results: During a mean follow-up of 53.77 months (range, 12 to 149 months), 22 of 145 patients (15.17%) developed IF. Patients undergoing thoracolumbar junctional region (T12/L1) resection were more likely to develop IF compared to those undergoing surgery at other vertebral levels (HR = 21.622, 95% CI = 3.567-131.084, P = 0.001). Patients undergoing titanium mesh cage reconstruction were more likely to develop IF compared to patients undergoing expandable titanium cage reconstruction (HR = 8.315, 95% CI = 1.482-46.645, P = 0.016). Patients with bone cement augmentation around the cage were less likely to develop IF compared to those not receiving bone cement augmentation (HR = 0.015, 95% CI = 0.002-0.107, P < 0.001). Of the 22 patients with IF, 14 (63.63%) accepted personalized revision surgery. Conclusion: The use of an expandable cage and the use of bone cement augmentation around the anterior column support cage are protective measures against IF after total spondylectomy.

New Progress in Basic Research of Macrophages in the Pathogenesis and Treatment of Low Back Pain.

Low back pain (LBP) is quite common in clinical practice, which can lead to long-term bed rest or even disability. It is a worldwide health problem remains to be solved. LBP can be induced or exacerbated by abnormal structure and function of spinal tissue such as intervertebral disc (IVD), dorsal root ganglion (DRG) and muscle; IVD degeneration (IVDD) is considered as the most important among all the pathogenic factors. Inflammation, immune response, mechanical load, and hypoxia etc., can induce LBP by affecting the spinal tissue, among which inflammation and immune response are the key link. Inflammation and immune response play a double-edged sword role in LBP. As the main phagocytic cells in the body, macrophages are closely related to body homeostasis and various diseases. Recent studies have shown that macrophages are the only inflammatory cells that can penetrate the closed nucleus pulposus, expressed in various structures of the IVD, and the number is positively correlated with the degree of IVDD. Moreover, macrophages play a phagocytosis role or regulate the metabolism of DRG and muscle tissues through neuro-immune mechanism, while the imbalance of macrophages polarization will lead to more inflammatory factors to chemotaxis and aggregation, forming an "inflammatory waterfall" effect similar to "positive feedback," which greatly aggravates LBP. Regulation of macrophages migration and polarization, inhibition of inflammation and continuous activation of immune response by molecular biological technology can markedly improve the inflammatory microenvironment, and thus effectively prevent and treat LBP. Studies on macrophages and LBP were mainly focused in the last 3-5 years, attracting more and more scholars' attention. This paper summarizes the new research progress of macrophages in the pathogenesis and treatment of LBP, aiming to provide an important clinical prevention and treatment strategy for LBP.

Short-term Efficacy of the Percutaneous Vertebroplasty Using a Curved Versus Straight Vertebroplasty Needle in Osteoporotic Vertebral Compression Fractures.

The purpose of this study was to compare the efficacy of the curved puncture approach with 2 conventional approaches in percutaneous vertebroplasty (PVP) for the treatment of single-level osteoporotic vertebral compression fractures. Ninety-six patients with a single-level thoracolumbar vertebral fracture were surgically treated in the authors' department from February 2016 to February 2018. Patients were randomly divided into 3 groups, including 25 patients who had PVP punctured with a curved vertebroplasty needle (group C), 40 patients with unipedicular PVP with a straight vertebroplasty needle (group U), and 31 patients with bipedicular PVP with a straight vertebroplasty needle (group B). The short-term efficacies of PVP using different vertebroplasty needles were compared. Significant differences were tested preoperatively and postoperatively in vertebral body height variation, visual analog scale score, and Oswestry Disability Index in each of the 3 groups (P<.05). There was no significant difference among the groups in terms of Cobb angle correction and bone cement leakage. Group C and group U were superior to group B in terms of operative time and injected cement volume (P<.05). Twenty-four (96.0%) patients in group C and 29 (93.5%) patients in group B had centered cement distribution without significant differences (P>.05), which was superior to group U (P<.05). Curved puncture PVP achieved a satisfactory clinical outcome for osteoporotic vertebral compression fractures, with the advantages of less operative time, less injected cement volume, and more reasonable cement distribution for stabilization of the affected vertebrae. [Orthopedics. 2021;44(1):e131-e138.].

Hsa-miR-557 Inhibits Osteosarcoma Growth Through Targeting KRAS.

Objective: Osteosarcoma is the most common malignancy in the skeletal system; studies showed an important role of miRNAs in tumorigenesis, indicating miRNAs as possible therapeutic molecules. This study found abnormal hsa-miR-557 expression levels in osteosarcoma and tried to explore the potential function and the mechanism. Methods: Differential expression genes of osteosarcoma were analyzed using GSE28423 from the GEO database. Survival analysis of miRNAs was conducted with data obtained from the TARGET-OS database. STRING and miRDIP were used to predict target genes of hsa-miR-557; KRAS was then verified using dual-luciferase reporter assay. Expression of genes was detected by qPCR, and levels of proteins were detected by Western blot. The proliferation ability of cells was detected by CCK-8 and cell cycle analysis. Tumor formation assay in nude mice was used to detect the influence of osteosarcoma by hsa-miR-557 in vivo. Results: Analysis from the GEO and TARGET databases found 12 miRNAs that are significantly related to the osteosarcoma prognosis, 7 downregulated (hsa-miR-140-3p, hsa-miR-564, hsa-miR-765, hsa-miR-1224-5p, hsa-miR-95, hsa-miR-940, and hsa-miR-557) and 5 upregulated (hsa-miR-362-3p, hsa-miR-149, hsa-miR-96, hsa-miR-744, and hsa-miR-769-5p). CCK-8 analysis and cell cycle analysis found that hsa-miR-557 could significantly inhibit the proliferation of osteosarcoma cells. The tumor formation assay in nude mice showed that tumor sizes and weights were inhibited by hsa-miR-557 transfection. Further studies also proved that hsa-miR-557 could target the 3'UTR of KRAS and modulate phosphorylation of downstream proteins. Conclusion: This study showed that hsa-miR-557 could inhibit osteosarcoma growth both in vivo and in vitro, by modulating KRAS expression.

Sprifermin: Effects on Cartilage Homeostasis and Therapeutic Prospects in Cartilage-Related Diseases.

When suffering from osteoarthritis (OA), articular cartilage homeostasis is out of balance and the living quality declines. The treatment of knee OA has always been an unsolved problem in the world. At present, symptomatic treatment is mainly adopted for OA. Drug therapy is mainly used to relieve pain symptoms, but often accompanied with adverse reactions; surgical treatment involves the problem of poor integration between the repaired or transplanted tissues and the natural cartilage, leading to the failure of repair. Biotherapy which aims to promote cartilage in situ regeneration and to restore endochondral homeostasis is expected to be an effective method for the prevention and treatment of OA. Disease-modifying osteoarthritis drugs (DMOADs) are intended for targeted treatment of OA. The DMOADs prevent excessive destruction of articular cartilage through anti-catabolism and stimulate tissue regeneration via excitoanabolic effects. Sprifermin (recombinant human FGF18, rhFGF18) is an effective DMOAD, which can not only promote the proliferation of articular chondrocyte and the synthesis of extracellular matrix, increase the thickness of cartilage in a dose-dependent manner, but also inhibit the activity of proteolytic enzymes and remarkedly slow down the degeneration of cartilage. This paper reviews the unique advantages of Sprifermin in repairing cartilage injury and improving cartilage homeostasis, aiming to provide an important strategy for the effective prevention and treatment of cartilage injury-related diseases.

A patient with Turner syndrome received the percutaneous vertebroplasty seven times: a case report and literature review.

BACKGROUND: Turner syndrome (TS) is characterized as the complete or partial absence of one X chromosome and is an extremely rare disease affecting approximately 1:2500 live female births. Though the prevalence of osteoporosis among women with TS is estimated to be around 55-64% and they suffer more frequently from fractures than normal, few reports concerning TS patients with osteoporosis are able to be seen due to tiny number of patients. CASE PRESENTATION: Here, we report a rare case of TS with osteoporosis, who has undergone percutaneous vertebroplasty (PVP) seven times because of several vertebral compression fractures (VCFs). G-banded karyotype analysis was performed and the result was 45,X[43]/47,XXX[17], indicating that the patient was a mosaicism of TS karyotype and Trisomy X syndrome karyotype. TS is the underlying cause of low level of estrogen for this patient. The interaction of aging, estrogen deficiency and intestinal dysbacteriosis leads to her severe osteoporosis and multi-segmental VCFs. The aim of this report is to provide recommendations regarding the management of TS patients with osteoporosis by reviewing the clinical presentation of TS, the influence of estrogen deficiency in osteoporosis, etc. CONCLUSIONS: Early diagnosis and hormone replacement treatment are essential for TS patients to prevent osteoporosis and reduce the risk of fractures. This is a rare case report describing TS patient with severe osteoporosis and VCFs.

Curcumin Modulates the Crosstalk Between Macrophages and Bone Mesenchymal Stem Cells to Ameliorate Osteogenesis.

Bone healing is thought to be influenced by the cross-talk between bone forming and immune cells. In particular, macrophages play a crucial role in the regulation of osteogenesis. Curcumin, the major bioactive polyphenolic ingredient of turmeric, has been shown to regulate inflammatory response and osteogenic activities. However, whether curcumin could regulate macrophage polarization and subsequently influence osteogenesis remain to be elucidated. In this study, the potential immunomodulatory capability of curcumin on inflammatory response and phenotype switch of macrophages and the subsequent impact on osteogenic differentiation of MSCs are investigated. We demonstrated that curcumin exhibited significant anti-inflammatory effect by polarizing the macrophages toward anti-inflammatory phenotype, with increased expression of IL-4, IL-10, and CD206, and decreased expression of IL-1ß, TNF-α, CCR7, and iNOS. In addition, curcumin could improve the osteo-immune microenvironment via promoting osteogenesis-related regenerative cytokine BMP-2 and TGF-ß production. Moreover, the co-cultured test of macrophages and BMSCs showed that curcumin-modulated macrophages conditioned medium could promote osteogenic differentiation of BMSCs with increased gene (ALP, Runx-2, OCN, and OPN) and protein (Runx-2 and OCN) expression levels, enhanced ALP activity, and obvious formation of mineralized nodules. Taken together, with the interaction between curcumin-conditioned macrophage and curcumin-stimulated BMSCs, curcumin could remarkably enhance the osteogenic differentiation of BMSCs in LPS-activated inflammatory macrophage-BMSCs coculture system.

A Review of Gene Therapy Delivery Systems for Intervertebral Disc Degeneration.

BACKGROUND: Intervertebral Disc (IVD) degeneration is a major public health concern, and gene therapy seems a promising approach to delay or even reverse IVD degeneration. However, the delivery system used to transfer exogenous genes into intervertebral disc cells remains a challenge. METHODS: The MEDLINE, Web of Science, and Scopus databases were searched for English-language articles related to gene therapy for IVD degeneration articles from 1999 to May 2019. The keywords included "gene therapy" AND "intervertebral disc". The history of the development of different delivery systems was analysed, and the latest developments in viral and non-viral vectors for IVD degeneration treatment were reviewed. RESULTS: Gene therapy delivery systems for IVD degeneration are divided into two broad categories: viral and non-viral vectors. The most commonly used viral vectors are adenovirus, adeno-associated virus (AAV), and lentivirus. Enthusiasm for the use of adenovirus vectors has gradually declined and has been replaced by a preference for lentivirus and AAV vectors. New technologies, such as RNAi and CRISPR, have further enhanced the advantage of viral vectors. Liposomes are the classic non-viral vector, and their successors, polyplex micelles and exosomes, have more potential for use in gene therapy for IVD degeneration. CONCLUSION: Lentivirus and AAV are the conventional viral vectors used in gene therapy for IVD degeneration, and the new technologies RNAi and CRISPR have further enhanced their advantages. Nonviral vectors, such as polyplex micelles and exosomes, are promising gene therapy vectors for IVD degeneration.

Synergistic Utilization of Necrostatin-1 and Z-VAD-FMK Efficiently Promotes the Survival of Compression-Induced Nucleus Pulposus Cells via Alleviating Mitochondrial Dysfunction.

We recently reported that necroptosis contributed to compression-induced nucleus pulposus (NP) cells death. In the current study, we investigated the regulative effect of necroptosis inhibitor Necrostatin-1 on NP cells apoptosis and autophagy. Necrostatin-1, autophagy inhibitor 3-Methyladenine and apoptosis inhibitor Z-VAD-FMK were employed, and NP cells were exposed to 1.0 MPa compression for 0, 24 and 36 h. Necroptosis-associated molecules were measured by Western blot and RT-PCR. Autophagy and apoptosis levels were evaluated by Western blot and quantified by flow cytometry after monodansylcadaverine and Annexin V-FITC/propidium iodide staining, respectively. The cell viability and cell death were also examined. Furthermore, we measured mitochondrial membrane potential (MMP), mitochondrial permeability transition pore (MPTP) and indices of oxidative stress to assess mitochondrial dysfunction. The results established that Necrostatin-1 blocked NP cells autophagy, and 3-Methyladenine had little influence on NP cells necroptosis. The Necrostatin-1+3-Methyladenine treatment exerted almost the same role as Necrostatin-1 in reducing NP cells death. Necrostatin-1 restrained NP cells apoptosis, while Z-VAD-FMK enhanced NP cells necroptosis. The Necrostatin-1+Z-VAD-FMK treatment provided more prominent role in blocking NP cells death compared with Necrostatin-1, consistent with increased MMP, reduced opening of MPTP and oxidative stress. In summary, the synergistic utilization of Necrostatin-1 and Z-VAD-FMK is a very worthwhile solution in preventing compression-mediated NP cells death, which might be largely attributed to restored mitochondrial function.