Current status and outlook of advances in exosome isolation.

Exosomes are extracellular vesicles with a diameter ranging from 30 to 150 nm, which are an important medium for intercellular communication and are closely related to the progression of certain diseases. Therefore, exosomes are considered promising biomarkers for the diagnosis of specific diseases, and thereby, treatments based on exosomes are being widely examined. For exosome-related research, a rapid, simple, high-purity, and recovery isolation method is the primary prerequisite for exosomal large-scale application in medical practice. Although there are no standardized methods for exosome separation and analysis, various techniques have been established to explore their biochemical and physicochemical properties. In this review, we analyzed the progress in exosomal isolation strategies and proposed our views on the development prospects of various exosomal isolation techniques.

Grape Seed Proanthocyanidins Exert a Neuroprotective Effect by Regulating Microglial M1/M2 Polarisation in Rats with Spinal Cord Injury.

Spinal cord injury (SCI) is a highly disabling disorder for which few effective treatments are available. Grape seed proanthocyanidins (GSPs) are polyphenolic compounds with various biological activities. In our preliminary experiment, GSP promoted functional recovery in rats with SCI, but the mechanism remains unclear. Therefore, we explored the protective effects of GSP on SCI and its possible underlying mechanisms. We found that GSP promoted locomotor recovery, reduced neuronal apoptosis, increased neuronal preservation, and regulated microglial polarisation in vivo. We also performed in vitro studies to verify the effects of GSP on neuronal protection and microglial polarisation and their potential mechanisms. We found that GSP regulated microglial polarisation and inhibited apoptosis in PC12 cells induced by M1-BV2 cells through the Toll-like receptor 4- (TLR4-) mediated nuclear factor kappa B (NF-κB) and phosphatidylinositol 3-kinase/serine threonine kinase (PI3K/AKT) signaling pathways. This suggests that GSP regulates microglial polarisation and prevents neuronal apoptosis, possibly by the TLR4-mediated NF-κB and PI3K/AKT signaling pathways.

Effect of spinal orthosis on clinical outcomes of patients after oblique lumbar interbody fusion: a randomized controlled trial study protocol.

BACKGROUND: Oblique lumbar interbody fusion (OLIF) is an internationally popular minimally invasive technology for the treatment of various lumbar diseases. Since its introduction to China in 2014, OLIF technology has clearly shown its superiority in reconstructing intervertebral stability, restoring intervertebral space height, achieving indirect decompression, and restoring normal lumbar sequence. However, some patients still suffer from persistent symptoms after OLIF, including low back pain and soreness, which indirectly affect the overall surgical efficacy and patient satisfaction. Therefore, some clinicians recommend that patients routinely use spinal orthoses after OLIF to reduce the stress on the lower back muscles and ligaments, thereby relieving or avoiding postoperative residual symptoms or new symptoms. Accordingly, spinal orthosis use after OLIF has emerged as an essential option. However, the role of spinal orthoses in OLIF and their specific impact on postoperative patient clinical outcomes have remained unclear, and there is a lack of strong clinical evidence to indirectly or directly support the role of spinal orthoses in OLIF and demonstrate their impact on patient clinical outcomes. This study aims to investigate the role of spinal orthoses in OLIF by grouping patients based on the use or nonuse of spinal orthosis after OLIF, thus providing a better basis for the majority of patients and physicians. METHODS/DESIGN: We plan to conduct a 1-year randomized controlled trial involving 60 subjects. The subjects will be randomized into two groups: group A (those wearing spinal orthoses after surgery) and group B (those not wearing spinal orthoses after surgery). The clinical outcomes of these patients will be evaluated using the Oswestry disability index, visual analog scale, and Brantigan, Steffee, Fraser 1 day before surgery and 2 weeks and 1, 6, and 12 months after surgery. DISCUSSION: This randomized controlled trial aims to provide a reference for further comprehensive trial design. The findings of this study will provide a better and more scientific basis for the choice of postoperative rehabilitation and treatment for patients undergoing such a procedure. TRIAL REGISTRATION: This study has been registered in the Chinese Clinical Trial Registry (Registration No.: ChiCTR2200059000). Registration date: April 22, 2022. Registration website: http://www.chictr.org.cn/showproj.aspx?proj=166310.

BRD4 Inhibition Suppresses Senescence and Apoptosis of Nucleus Pulposus Cells by Inducing Autophagy during Intervertebral Disc Degeneration: An In Vitro and In Vivo Study.

Intervertebral disc degeneration (IDD) is the most common chronic skeletal muscle degeneration disease. Although the underlying mechanisms remain unclear, nucleus pulposus (NP) autophagy, senescence, and apoptosis are known to play a critical role in this process. Previous studies suggest that bromodomain-containing protein 4 (BRD4) promotes senescent and apoptotic effects in several age-related degenerative diseases. It is not known, however, if BRD4 inhibition is protective in IDD. In this study, we explored whether BRD4 influenced IDD. In human clinical specimens, the BRD4 level was markedly increased with the increasing Pfirrmann grade. At the cellular level, BRD4 inhibition prevented IL-1ß-induced senescence and apoptosis of NP cells and activated autophagy via the AMPK/mTOR/ULK1 signaling pathway. Inhibition of autophagy by 3-methyladenine (3-MA) partially reversed the antisenescence and antiapoptotic effects of BRD4. In vivo, BRD4 inhibition attenuated IDD. Taken together, the results of this study showed that BRD4 inhibition reduced NP cell senescence and apoptosis by induced autophagy, which ultimately alleviated IDD. Therefore, BRD4 may serve as a novel potential therapeutic target for the treatment of IDD.

Mesenchymal stem cell-derived exosomes: therapeutic opportunities and challenges for spinal cord injury.

Spinal cord injury (SCI) often leads to serious motor and sensory dysfunction of the limbs below the injured segment. SCI not only results in physical and psychological harm to patients but can also cause a huge economic burden on their families and society. As there is no effective treatment method, the prevention, treatment, and rehabilitation of patients with SCI have become urgent problems to be solved. In recent years, mesenchymal stem cells (MSCs) have attracted more attention in the treatment of SCI. Although MSC therapy can reduce injured volume and promote axonal regeneration, its application is limited by tumorigenicity, a low survival rate, and immune rejection. Accumulating literature shows that exosomes have great potential in the treatment of SCI. In this review, we summarize the existing MSC-derived exosome studies on SCI and discuss the advantages and challenges of treating SCI based on exosomes derived from MSCs.

NF-κB signalling pathways in nucleus pulposus cell function and intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) is a common clinical degenerative disease of the spine. A series of factors, such as inflammation, oxidative stress and mechanical stress, promote degradation of the extracellular matrix (ECM) of the intervertebral discs (IVD), leading to dysfunction and structural destruction of the IVD. Nuclear factor-κB (NF-κB) transcription factor has long been regarded as a pathogenic factor of IDD. Therefore, NF-κB may be an ideal therapeutic target for IDD. As NF-κB is a multifunctional functional transcription factor with roles in a variety of biological processes, a comprehensive understanding of the function and regulatory mechanism of NF-κB in IDD pathology will be useful for the development of targeted therapeutic strategies for IDD, which can prevent the progression of IDD and reduce potential risks. This review discusses the role of the NF-κB signalling pathway in the nucleus pulposus (NP) in the process of IDD to understand pathological NP degeneration further and provide potential therapeutic targets that may interfere with NF-κB signalling for IDD therapy.

Research progress on the regulatory role of microRNAs in spinal cord injury.

Spinal cord injury (SCI) is a severe CNS injury that results in abnormalities in, or loss of, motor, sensory and autonomic nervous function. miRNAs belong to a new class of noncoding RNA that regulates the production of proteins and biological function of cells by silencing translation or interfering with the expression of target mRNAs. Following SCI, miRNAs related to oxidative stress, inflammation, autophagy, apoptosis and many other secondary injuries are differentially expressed, and these miRNAs play an important role in the progression of secondary injuries after SCI. The purpose of this review is to elucidate the differential expression and functional roles of miRNAs after SCI, thus providing references for further research on miRNAs in SCI.

Different Types of Double-Level Degenerative Lumber Spondylolisthesis: What Is Different in the Sagittal Plane?

BACKGROUND: Degenerative lumber spondylolisthesis (DLS) is a common orthopedic condition, described as a condition that compared with the lower vertebra, the superior vertebra slides forward or backward in the sagittal plane without accompanying isthmic spondylolisthesis. Information pertaining to different types of double-level DLS is scarce. This study aims to analyze parameters of patients with different types of double-level DLS to provide a reference for guiding surgical treatment and restoring sagittal balance of patients with DLS. METHODS: From January 2014 to January 2020, records of patients with double-level DLS were retrospectively reviewed. Patients with double-level DLS were divided into 3 types: anterior, posterior, and combined; the anterior and combined types were studied. The sagittal spinopelvic parameters included C7 tilt, maximal thoracic kyphosis, maximal lumbar lordosis (LLmax), pelvic incidence (PI), pelvic tilt (PT), and sacral slope (SS). After descriptive analysis, demographic and radiographic data were compared. RESULTS: Forty and 18 patients were included in the anterior and combined type groups, respectively. Both groups had different levels of chronic low back pain, but the incidence of radiating leg pain and neurogenic claudication was significantly higher in the anterior type. Oswestry Disability Index and visual analog scale low back scores were also higher in the anterior type. In the anterior type, C7 tilt (7.14 ± 2.15 vs. 5.41 ± 2.28, P = 0.007), LLmax (50.02 ± 14.76 vs. 36.96 ± 14.56, P = 0.003), PI (68.28 ± 9.16 vs. 55.53 ± 14.19, P < 0.001), PT (28.68 ± 7.31 vs. 19.38 ± 4.70, P < 0.001), and PT/PI (42.45 ± 11.22 vs. 36.04 ± 9.87, P = 0.041) were significantly higher. In the anterior type, PI correlated positively with LLmax (r = 0.59) and SS (r = 0.71). LLmax and SS (r = 0.65) had a positive correlation. PT/PI and SS (r = -0.77) had a negative correlation. In the combined type, PI correlated positively with LLmax (r = 0.61) and SS (r = 0.88), and PT/PI correlated negatively with SS (r = -0.81). CONCLUSIONS: In patients with double-level DLS, the sagittal spinopelvic parameters differed between the anterior and combined types. Overall, spinal surgeons should focus on correcting sagittal deformities, relieving postoperative clinical symptoms, and improving quality of life during fusion surgery.

Mesenchymal stem cell-derived exosomes: Toward cell-free therapeutic strategies in regenerative medicine.

Mesenchymal stem cells (MSCs) are multipotent stem cells with marked potential for regenerative medicine because of their strong immunosuppressive and regenerative abilities. The therapeutic effects of MSCs are based in part on their secretion of biologically active factors in extracellular vesicles known as exosomes. Exosomes have a diameter of 30-100 nm and mediate intercellular communication and material exchange. MSC-derived exosomes (MSC-Exos) have potential for cell-free therapy for diseases of, for instance, the kidney, liver, heart, nervous system, and musculoskeletal system. Hence, MSC-Exos are an alternative to MSC-based therapy for regenerative medicine. We review MSC-Exos and their therapeutic potential for a variety of diseases and injuries.

Corrigendum: ß-Sitosterol and Gemcitabine Exhibit Synergistic Anti-Pancreatic Cancer Activity by Modulating Apoptosis and Inhibiting Epithelial-Mesenchymal Transition by Deactivating Akt/GSK-3ß Signaling.

[This corrects the article DOI: 10.3389/fphar.2018.01525.].

Sirtuins and intervertebral disc degeneration: Roles in inflammation, oxidative stress, and mitochondrial function.

Intervertebral disc degeneration (IDD) is one of the main causes of low back pain, which seriously reduces the quality of life of patients and places a heavy economic burden on their families. Cellular senescence is considered to be an important factor leading to IDD, and inflammatory response, oxidative stress, and mitochondrial dysfunction are closely related to intervertebral disc (IVD) senescence. Therefore, inhibition of the inflammatory response and oxidative stress, along with maintaining mitochondrial function, may be useful in treating IDD. The sirtuins are a family of evolutionarily conserved nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylases, which are the major molecules mediating life extension or delay of aging-related diseases. The sirtuin protein family consist of seven members (SIRT1 - 7), which are mainly involved in various aging-related diseases by regulating inflammation, oxidative stress, and mitochondrial function. Among them, SIRT1, SIRT2, SIRT3, and SIRT6 are closely related to IDD. In addition, some activators of sirtuin proteins, such as resveratrol, melatonin, magnolol, 1,4-dihydropyridine (DHP), SRT1720, and nicotinamide mononucleotide (NMN), have been evaluated in preclinical studies for their effects in preventing IDD. This review described the biological functions of sirtuins and the important roles of SIRT1, SIRT2, SIRT3, and SIRT6 in IDD by regulating oxidative stress, inflammatory response, and mitochondrial function. In addition, we introduce the status of some sirtuin activators in IDD preclinical studies. This review will provide a background for further clarification of the molecular mechanism underlying IDD and the development of potential therapeutic drugs.

Proteomics analysis demonstrating rosmarinic acid suppresses cell growth by blocking the glycolytic pathway in human HepG2 cells.

Rosmarinic acid (RA), isolated from herbal balm mint plants, has demonstrated potent anti-tumor properties against liver cancer. However, the precise underlying mechanisms remain unclear. This study aimed to investigate the molecular mechanisms of RA in HepG2 cells. RA anti-tumor activity was assessed using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, and Hoechst 33258 staining. Apoptosis and the cell cycle distribution were evaluated by flow cytometry. A proteomics approach was used to identify differentially expressed proteins following RA treatment in HepG2 cells, and quantitative reverse transcription-quantitative polymerase chain reaction was used to validate the results. Bioinformatics analysis was also implemented to further understand the identified proteins, and western blotting was used to analyze the associated proteins. Our results suggested that RA treatment significantly inhibits the viability of HepG2 cells. The MTT and LDH assays indicated dose-dependent decreases in cell proliferation following RA treatment. Hoechst 33258 staining and flow cytometry analysis showed that RA exhibits an apoptosis-inducing effect and induces cell cycle arrest in G1. The proteomics analysis successfully identified 16 differentially expressed proteins. Bioinformatics analysis indicated that the identified proteins participated in several biological processes and exhibited various molecular functions, mainly related to inactivation of the glycolytic pathway. Further western blotting analysis showed that RA could downregulate the expression of glucose transporter-1 and hexokinase-2, leading to the suppression of glucose consumption and generation of lactate and ATP. Taken together, our study found that RA exhibits significant cytotoxic effects by inhibiting cell proliferation and inducing apoptosis and cell cycle arrest, possibly by blocking the glycolytic pathway in human HepG2 cells.

ß-Sitosterol and Gemcitabine Exhibit Synergistic Anti-pancreatic Cancer Activity by Modulating Apoptosis and Inhibiting Epithelial-Mesenchymal Transition by Deactivating Akt/GSK-3ß Signaling.

ß-sitosterol (BS), a major bioactive constituent present in plants, has shown potent anti-cancer activity against many human cancer cells, but its activity in pancreatic cancer (PC) cells has rarely been reported. Gemcitabine (GEM) is one of the first-line drugs for PC therapy, however, the treatment effect is not sustained due to prolonged drug resistance. In this study, we firstly studied the anti-PC activity and the mechanism of BS alone and in combination with GEM in vitro and in vivo. BS effectively inhibited the growth of PC cell lines by inhibiting proliferation, inducing G0/G1 phase arrest and apoptosis, suppressed the NF- kB activity, and increased expression of the protein Bax but decreased expression of the protein Bcl-2. Moreover, BS inhibited migration and invasion and downregulated epithelial-mesenchymal transition (EMT) markers and AKT/GSK-3ß signaling pathways. Furthermore, the combination of BS and GEM exhibited a significant synergistic effect in MIAPaCa-2 and BXPC-3 cells. More importantly, the combined treatment with BS and GEM lead to significant growth inhibition of PC xenografts. Overall, our data revealed a promising treatment option for PC by the combination therapy of BS and GEM.

Lariciresinol induces apoptosis in HepG2 cells via mitochondrial-mediated apoptosis pathway.

Lariciresinol (LA) is one of the main active ingredients in many traditional medicinal plants such as Patrinia, and has the role of anti-liver cancer. However, the precise mechanisms are unclear. This study investigated the molecular mechanisms of LA against HepG2 cells. LA anti-tumor activity was assessed with the CCK-8, Ki-67, and immunofluorescence staining. Cells apoptotic ratio was evaluated by Annexin V/PI double-staining assay. A proteomic approach was used to identify differentially expressed proteins after LA treatment. JC-1 staining was carried out to detect the mitochondrial membrane potential (ΔΨm), and the Western blot analysis was used to analyse the apoptosis-associated proteins. Our results suggested that LA significantly suppressed the viability of HepG2 cells. The CCK-8 and Ki-67 expression indicated dose-dependent decreases in cell proliferation. Flow cytometry analysis showed that LA exhibited a apoptosis-inducing effect. The proteomic study observed the presence of apoptosis-associated proteins and mitochondrial dysfunction in HepG2 cells after LA-treatment. Further analysis showed that LA could trigger the mitochondrial-mediated apoptosis pathway, based on a decrease in ΔΨm; deliver of cytochrome c; activation of caspase-9/-3 and poly(ADP-ribose) polymerase; and decrease of the proportion of Bcl-2/Bax. Collectively, our studies found that LA exhibits significant cytotoxic effects by inhibiting cell proliferation, inducing apoptosis, possibly via activation of the mitochondrial-mediated apoptosis pathway.

Proteomic analysis of apoptosis induction by lariciresinol in human HepG2 cells.

Lariciresinol (LA) is a traditional Chinese medicine possessing anticancer activity, but its mechanism of action remains unclear. The present study explored the effects of LA on human HepG2 cells and the underlying mechanism. Our data indicated that LA inhibited cell proliferation and induced cell cycle arrest in S phase, subsequently resulting in apoptosis in HepG2 cells. Using a proteomics approach, eight differentially expressed proteins were identified. Among them, three proteins, glyceraldehyde-3-phosphate, UDP-glucose 4-epimerase, and annexin A1, were upregulated, while the other five proteins, heat shock protein 27, haptoglobin, tropomodulin-2, tubulin alpha-1A chain, and brain acid soluble protein 1, were downregulated; all of these proteins are involved in cell proliferation, metabolism, cytoskeletal organization, and movement. Network analysis of these proteins suggested that the ubiquitin-conjugating enzyme (UBC) plays an important role in the mechanism of LA. Western blotting confirmed downregulation of heat shock protein 27 and upregulation of ubiquitin and UBC expression levels in LA-treated cells, consistent with the results of two-dimensional electrophoresis and a STRING software-based analysis. Overall, LA is a multi-target compound with anti-cancer effects potentially related to the ubiquitin-proteasome pathway. This study will increase our understanding of the anticancer mechanisms of LA.