Chicoric acid ameliorates sepsis-induced cardiomyopathy via regulating macrophage metabolism reprogramming.

BACKGROUND: Sepsis-related cardiac dysfunction is believed to be a primary cause of high morbidity and mortality. Metabolic reprogramming is closely linked to NLRP3 inflammasome activation and dysregulated glycolysis in activated macrophages, leading to inflammatory responses in septic cardiomyopathy. Succinate dehydrogenase (SDH) and succinate play critical roles in the progression of metabolic reprogramming in macrophages. Inhibition of SDH may be postulated as an effective strategy to attenuate macrophage activation and sepsis-induced cardiac injury. PURPOSE: This investigation was designed to examine the role of potential compounds that target SDH in septic cardiomyopathy and the underlying mechanisms involved. METHODS/RESULTS: From a small molecule pool containing about 179 phenolic compounds, we found that chicoric acid (CA) had the strongest ability to inhibit SDH activity in macrophages. Lipopolysaccharide (LPS) exposure stimulated SDH activity, succinate accumulation and superoxide anion production, promoted mitochondrial dysfunction, and induced the expression of hypoxia-inducible factor-1α (HIF-1α) in macrophages, while CA ameliorated these changes. CA pretreatment reduced glycolysis by elevating the NAD+/NADH ratio in activated macrophages. In addition, CA promoted the dissociation of K(lysine) acetyltransferase 2A (KAT2A) from α-tubulin, and thus reducing α-tubulin acetylation, a critical event in the assembly and activation of NLRP3 inflammasome. Overexpression of KAT2A neutralized the effects of CA, indicating that CA inactivated NLRP3 inflammasome in a specific manner that depended on KAT2A inhibition. Importantly, CA protected the heart against endotoxin insult and improved sepsis-induced cardiac mitochondrial structure and function disruption. Collectively, CA downregulated HIF-1α expression via SDH inactivation and glycolysis downregulation in macrophages, leading to NLRP3 inflammasome inactivation and the improvement of sepsis-induced myocardial injury. CONCLUSION: These results highlight the therapeutic role of CA in the resolution of sepsis-induced cardiac inflammation.

TNF-α-Induced KAT2A Impedes BMMSC Quiescence by Mediating Succinylation of the Mitophagy-Related Protein VCP.

Regular quiescence and activation are important for the function of bone marrow mesenchymal stem cells (BMMSC), multipotent stem cells that are widely used in the clinic due to their capabilities in tissue repair and inflammatory disease treatment. TNF-α is previously reported to regulate BMMSC functions, including multilineage differentiation and immunoregulation. The present study demonstrates that TNF-α impedes quiescence and promotes the activation of BMMSC in vitro and in vivo. Mechanistically, the TNF-α-induced expression of KAT2A promotes the succinylation of VCP at K658, which inhibits the interaction between VCP and MFN1 and thus inhibits mitophagy. Furthermore, activated BMMSC exhibits stronger fracture repair and immunoregulation functions in vivo. This study contributes to a better understanding of the mechanisms of BMMSC quiescence and activation and to improving the effectiveness of BMMSC in clinical applications.

Targeting macrophage M1 polarization suppression through PCAF inhibition alleviates autoimmune arthritis via synergistic NF-κB and H3K9Ac blockade.

Sustained inflammatory invasion leads to joint damage and progressive disability in several autoimmune rheumatic diseases. In recent decades, targeting M1 macrophage polarization has been suggested as a promising therapeutic strategy for autoimmune arthritis. P300/CBP-associated factor (PCAF) is a histone acetyltransferase (HAT) that exhibits a strong positive relationship with the proinflammatory microenvironment. However, whether PCAF mediates M1 macrophage polarization remains poorly studied, and whether targeting PCAF can protect against autoimmune arthritis in vivo remains unclear. Commonly used drugs can cause serious side effects in patients because of their extensive and nonspecific distribution in the human body. One strategy for overcoming this challenge is to develop drug nanocarriers that target the drug to desirable regions and reduce the fraction of drug that reaches undesirable targets. In this study, we demonstrated that PCAF inhibition could effectively inhibit M1 polarization and alleviate arthritis in mice with collagen-induced arthritis (CIA) via synergistic NF-κB and H3K9Ac blockade. We further designed dextran sulfate (DS)-based nanoparticles (DSNPs) carrying garcinol (a PCAF inhibitor) to specifically target M1 macrophages in inflamed joints of the CIA mouse model via SR-A-SR-A ligand interactions. Compared to free garcinol, garcinol-loaded DSNPs selectively targeted M1 macrophages in inflamed joints and significantly improved therapeutic efficacy in vivo. In summary, our study indicates that targeted PCAF inhibition with nanoparticles might be a promising strategy for treating autoimmune arthritis via M1 macrophage polarization inhibition.

ALKBH5 facilitates CYP1B1 mRNA degradation via m6A demethylation to alleviate MSC senescence and osteoarthritis progression.

Improving health and delaying aging is the focus of medical research. Previous studies have shown that mesenchymal stem cell (MSC) senescence is closely related to organic aging and the development of aging-related diseases such as osteoarthritis (OA). m6A is a common RNA modification that plays an important role in regulating cell biological functions, and ALKBH5 is one of the key m6A demethylases. However, the role of m6A and ALKBH5 in MSC senescence is still unclear. Here, we found that the m6A level was enhanced and ALKBH5 expression was decreased in aging MSCs induced by multiple replications, H2O2 stimulation or UV irradiation. Downregulation of ALKBH5 expression facilitated MSC senescence by enhancing the stability of CYP1B1 mRNA and inducing mitochondrial dysfunction. In addition, IGF2BP1 was identified as the m6A reader restraining the degradation of m6A-modified CYP1B1 mRNA. Furthermore, Alkbh5 knockout in MSCs aggravated spontaneous OA in mice, and overexpression of Alkbh5 improved the efficacy of MSCs in OA. Overall, this study revealed a novel mechanism of m6A in MSC senescence and identified promising targets to protect against aging and OA.

Formation mechanism and environmental drivers of Alexandrium catenella bloom events in the coastal waters of Qinhuangdao, China.

In the last 5 years, paralytic shellfish toxins (PSTs) have been recurrently detected in mollusks farmed in the mussel culture area of Qinhuangdao city, along with the occurrence of toxic outbreaks linked to dinoflagellate species of the Alexandrium genus. To understand the formation mechanism and variation of these events, continuous and comprehensive PSTs monitoring was carried out between 2017 and 2020. Through the analysis of both phytoplankton and cysts via light microscopy and quantitative polymerase chain reaction, it was shown that Alexandrium catenella was responsible for the production of PSTs, which consisted mainly of gonyautoxins 1,4 (GTX1/4, 87%) and GTX2/3 (13%). During bloom events in 2019, mussels accumulated the highest PSTs value (929 µg STX di-HCl eq·kg-1) in conjunction with the peak of cell abundances, and toxin profiles were consistent with high distributions of GTX1/4, GTX2/3, and Neosaxitoxin. Toxin metabolites vary in different substances and mainly transferred to a stable proportion of α-epimer: ß-epimers 3:1. The environmental drivers of Alexandrium blooms included the continuous rise of water temperature (>4 °C) and calm weather with low wind speed and no significant precipitation. By comparing toxin profiles and method sensitivity, it was found that dissolved toxins in seawater are more useful for early warning. These results have important implications for the effective monitoring and management of paralytic shellfish poisoning outbreaks.

Impairment of APPL1/Myoferlin facilitates adipogenic differentiation of mesenchymal stem cells by blocking autophagy flux in osteoporosis.

An imbalance of human mesenchymal stem cells (hMSCs) adipogenic and osteogenic differentiation is crucial in the pathogenesis of osteoporosis, and elucidation of the underlying mechanism is urgently needed. APPL1, an adaptor protein of the adiponectin receptor, was recently shown to be closely related to bone mass. However, the role of APPL1 in the imbalance of hMSC differentiation in osteoporosis is unclear. Therefore, we aimed to explore the mechanisms by which APPL1 alters hMSCs adipogenic differentiation in osteoporosis. Here, we found that APPL1 expression was downregulated in elderly patients with osteoporosis and in mouse osteoporosis model. APPL1 negatively regulated hMSC adipogenic differentiation in vivo and in vitro. Mechanistically, by enhancing ubiquitination-mediated Myoferlin degradation, downregulated APPL1 expression increased the risk of lysosome dysfunction during hMSCs adipogenic differentiation. Lysosomal dysfunction inhibited autophagy flux by suppressing autophagosome degradation and promoted hMSC differentiation towards the adipocyte lineage. Our findings suggest that APPL1/Myoferlin downregulation promoted hMSCs adipogenic differentiation by inhibiting autophagy flux, further impairing the balance of hMSCs adipogenic and osteogenic differentiation in osteoporosis; the APPL1/ Myoferlin axis may be a promising diagnostic and therapeutic target for osteoporosis.

Single-cell RNA sequencing analysis of human bone-marrow-derived mesenchymal stem cells and functional subpopulation identification.

Mesenchymal stem cells (MSCs) are a common kind of multipotent cell in vivo, but their heterogeneity limits their further applications. To identify MSC subpopulations and clarify their relationships, we performed cell mapping of bone-marrow-derived MSCs through single-cell RNA (scRNA) sequencing. In our study, three main subpopulations, namely, the stemness subpopulation, functional subpopulation, and proliferative subpopulation, were identified using marker genes and further bioinformatic analyses. Developmental trajectory analysis showed that the stemness subpopulation was the root and then became either the functional subpopulation or the proliferative subpopulation. The functional subpopulation showed stronger immunoregulatory and osteogenic differentiation abilities but lower proliferation and adipogenic differentiation. MSCs at different passages or isolated from different donors exhibited distinct cell mapping profiles, which accounted for their corresponding different functions. This study provides new insight into the biological features and clinical use of MSCs at the single-cell level, which may contribute to expanding their application in the clinic.

Mitochondrion-encoded circular RNAs are widespread and translatable in plants.

Nucleus-encoded circular RNAs (ncircRNAs) have been widely detected in eukaryotes, and most circRNA identification algorithms are designed to identify them. However, using these algorithms, few mitochondrion-encoded circRNAs (mcircRNAs) have been identified in plants, and the role of plant mcircRNAs has not yet been addressed. Here, we developed a circRNA identification algorithm, mitochondrion-encoded circRNA identifier, based on common features of plant mitochondrial genomes. We identified 7,524, 9,819, 1,699, 1,821, 1,809, and 5,133 mcircRNAs in maize (Zea mays), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), tomato (Solanum lycopersicum), cucumber (Cucumis sativus), and grape (Vitis vinifera), respectively. These mcircRNAs were experimentally validated. Plant mcircRNAs had distinct characteristics from ncircRNAs, and they were more likely to be derived from RNA degradation but not intron backsplicing. Alternative circularization was prevalent in plant mitochondria, and most parental genomic regions hosted multiple mcircRNA isoforms, which have homogenous 5' termini but heterogeneous 3' ends. By analysis of mitopolysome and mitoribosome profiling data, 1,463 mcircRNAs bound to ribosomes were detected in maize and Arabidopsis. Further analysis of mass spectrometry-based proteomics data identified 358 mcircRNA-derived polypeptides. Overall, we developed a computational pipeline that efficiently identifies plant mcircRNAs, and we demonstrated mcircRNAs are widespread and translated in plants.

Osteogenic differentiation of mesenchymal stem cells promotes c-Jun-dependent secretion of interleukin 8 and mediates the migration and differentiation of CD4+ T cells.

BACKGROUND: The immune system and the skeletal system have complex interactions in the bone marrow and even in the joints, which has promoted the development of the concept of osteoimmunology. Some evidence has indicated that T cells and B cells contribute to the balance between the resorption and formation of bone. However, there has been little discussion on the regulation of CD4+ T lymphocytes by cells involved in bone metabolism. Mesenchymal stem cells (MSCs), which exert core functions related to immunoregulation and osteogenic differentiation, are crucial cells linked to both bone metabolism and the immune system. Previous studies have shown that the immunoregulatory capacity of MSCs changes following differentiation. However, it is still unclear whether the osteogenic differentiation of MSCs affects the migration and differentiation of CD4+ T cells. METHODS: MSCs were cultured in growth medium or osteogenic medium for 10 days and then cocultured with CD4+ T cells. CD4+ T cell migration and differentiation were detected by flow cytometry. Further, gene expression levels of specific cytokines were analyzed by quantitative real-time PCR and enzyme-linked immunosorbent assays. A Proteome Profiler Human XL Cytokine Array Kit was used to analyze supernatants collected from MSCs. Alizarin red S staining and Alkaline phosphatase assay were used to detect the osteogenic differentiation of MSCs. RESULTS: Here, we found that the migration of CD4+ T cells was elevated, and the capacity to induce the differentiation of regulatory T (Treg) cells was weakened during MSC osteogenic differentiation, while the differentiation of T helper 1 (Th1), T helper 2 (Th2) and T helper 17 (Th17) cells was not affected. Further studies revealed that interleukin (IL)-8 was significantly upregulated during MSC osteogenic differentiation. Both a neutralizing antibody and IL-8-specific siRNA significantly inhibited the migration of CD4+ T cells and promoted the differentiation of Treg cells. Finally, we found that the transcription factor c-Jun was involved in regulating the expression of IL-8 and affected the osteogenic differentiation of MSCs, thereby mediating the migration and differentiation of CD4+ T cells. CONCLUSION: This study demonstrated that MSC osteogenic differentiation promoted c-Jun-dependent secretion of IL-8 and mediated the migration and differentiation of CD4+ T cells. These results provide a further understanding of the crosstalk between bone and the immune system and reveal information about the relationship between osteogenesis and inflammation in the field of osteoimmunology.

Full-endoscopic uniportal retropharyngeal odontoidectomy: A preliminary case report.

Summary of background data: Odontoidectomy aims to decompress the medulla oblongata and is usually performed through the classical transoral approach, which affects oropharynx and accompanied with high rate of complications comprising swallowing and respiratory tract. We have developed a minimal invasive method via a standard cervical anterior approach: full-endoscopic trans-cervical odontoidectomy, which provides an alternative access for the resection of odontoid process and medulla oblongata decompression without traversing potentially contaminated cavities. Methods: From 2018 to 2020, three patients with either odontoid process lesion or basilar invagination underwent full-endoscopic uniportal trans-cervical odontoidectomy with/without combining the posterior instrumentation. With fluoroscopic guidance, a uniportal endoscope sleeve was placed inside of the odontoid process; then odontoid process was gradually resected from the inside to outside under endoscopic monitoring. Postoperative images and clinical data were collected during post-op follow-up. Result: Patients were soon extubated after surgery when patients wake up from general anesthesia. There were no severely perioperative complications, especially dysphagia and airway obstruction, and the symptoms and neurological function was improved immediately after surgery. The final pathology of one patient with odontoid osteolytic lesion was confirmed as plasmacytoma. The postoperative CT scans proved that the range of odontoid process resection was consistent with the preoperative expectation. Conclusion: In summary, our proposed endoscopic trans-cervical odontoidectomy provides a valid choice for non-oral approach, which would reduce postoperative approach related complications and accelerate postoperative recovery.

TNF-α-mediated m6A modification of ELMO1 triggers directional migration of mesenchymal stem cell in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a type of rheumatic disease characterized by chronic inflammation and pathological osteogenesis in the entheses. Previously, we demonstrated that enhanced osteogenic differentiation of MSC from AS patients (AS-MSC) resulted in pathological osteogenesis, and that during the enhanced osteogenic differentiation course, AS-MSC induced TNF-α-mediated local inflammation. However, whether TNF-α in turn affects AS-MSC remains unknown. Herein, we further demonstrate that a high-concentration TNF-α treatment triggers enhanced directional migration of AS-MSC in vitro and in vivo, which enforces AS pathogenesis. Mechanistically, TNF-α leads to increased expression of ELMO1 in AS-MSC, which is mediated by a METTL14 dependent m6A modification in ELMO1 3'UTR. Higher ELMO1 expression of AS-MSC is found in vivo in AS patients, and inhibiting ELMO1 in SKG mice produces therapeutic effects in this spondyloarthritis model. This study may provide insight into not only the pathogenesis but also clinical therapy for AS.

Four new dammarane triterpenoid glycosides from the leaves of Cyclocarya paliurus and their SIRT1 activation activities.

Four new C-11 monosaccharide attached dammarane triterpenoid glycosides cypaliurusides SV (1-4), along with nine known dammarane triterpenoid glycosides (5-13) were isolated from a CHCl3-soluble extract of the leaves of Cyclocarya paliurus. All characterized compounds were assayed for their cytotoxicities against HepG2 cells and 10 compounds were evaluated for the agonistic effects on sirtuin1 (SIRT1). The results showed that compounds 1, 5 and 6 were strongly cytotoxic in HepG2 cell line. Two dammarane triterpenoid glycosides 3 and 10 exhibited agonistic activities on SIRT1 with IC50 of 10 µM and 20 µM, respectively.

IRF2-mediated upregulation of lncRNA HHAS1 facilitates the osteogenic differentiation of bone marrow-derived mesenchymal stem cells by acting as a competing endogenous RNA.

BACKGROUND: Mesenchymal stem cells (MSCs) are the major source of osteoblasts. Long noncoding RNAs (lncRNAs) are abundantly expressed RNAs that lack protein-coding potential and play an extensive regulatory role in cellular biological activities. However, the regulatory network of lncRNAs in MSC osteogenesis needs further investigation. METHODS: QRT-PCR, western blot, immunofluorescence, and immunohistochemistry assays were used to determine the levels of relevant genes. The osteogenic differentiation capability was evaluated by using Alizarin Red S (ARS) staining, alkaline phosphatase activity assays, hematoxylin & eosin staining or micro-CT. RNA fluorescence in situ hybridization (FISH) and RNAscope were used to detect HHAS1 expression in cells and bone tissue. A microarray assay was performed to identify differentially expressed microRNAs. RNA immunoprecipitation and RNA pull-down were used to explore the interactions between related proteins and nucleic acids. RESULTS: The level of lncRNA HHAS1 increased during bone marrow-derived MSC (BMSC) osteogenesis and was positively related to the levels of osteogenic genes and ARS intensity. HHAS1 was located in both the cytoplasm and the nucleus and was expressed in human bone tissue. HHAS1 facilitated BMSC osteogenic differentiation by downregulating miR-204-5p expression and enhancing the level of RUNX family transcription factor 2 (RUNX2). In addition, interferon regulatory factor 2 (IRF2) was increased during BMSC osteogenic differentiation and interacted with the promoter of HHAS1, which resulted in the transcriptional activation of HHAS1. Furthermore, IRF2 and HHAS1 helped improve bone defect repair in vivo. CONCLUSIONS: Our study identified a novel lncRNA, HHAS1, that facilitates BMSC osteogenic differentiation and proposed a role for the IRF2/HHAS1/miR-204-5p/RUNX2 axis in BMSC osteogenesis regulation. These findings help elucidate the regulatory network of BMSC osteogenesis and provide potential targets for clinical application.

The N6-methyladenosine demethylase ALKBH5 negatively regulates the osteogenic differentiation of mesenchymal stem cells through PRMT6.

N6-methyladenosine (m6A) modification is widespread in messenger RNAs and increasing evidence suggests the crucial roles of m6A in cell differentiation and tissue development. However, whether m6A modulates the osteogenic differentiation of mesenchymal stem cells (MSCs) has not been fully elucidated. Here we show that conditional knockout of the demethylase Alkbh5 in bone marrow MSCs strengthened bone mass in mice. Loss- and gain-of-function studies demonstrated that ALKBH5 negatively regulates the osteogenic differentiation of MSCs in vitro. At a mechanistic level, meRIP-seq and RNA-seq in MSCs following knockdown of ALKBH5 revealed changes in transcripts of PRMT6 containing consensus m6A motifs required for demethylation by ALKBH5. Furthermore, we found that ALKBH5 accelerates the degradation rate of PRMT6 mRNA in an m6A-dependent manner, and that the ALKBH5-PRMT6 axis regulates the osteogenesis of MSCs, mainly through activation of the PI3K/AKT pathway. Thus, our work reveals a different facet of the novel ALKBH5-PRMT6 axis that modulates the osteogenic differentiation of MSCs, which can serve as a target to improve the clinical use of MSCs.

A multicenter survey of perioperative anxiety in China: Pre- and postoperative associations.

OBJECTIVES: To describe patient characteristics associated with preoperative anxiety and subsequently assess the relationship between preoperative anxiety and postoperative anxiety, pain, sleep quality, nausea and vomiting. METHODS: The study collected data from patients undergoing elective operation from 12 hospitals in China. The State-Trait Anxiety Inventory (STAI) and the Athens Insomnia Scale (AIS) were used to assess anxiety and sleep quality before surgery. Evaluations of anxiety, pain, sleep quality, nausea and vomiting were quantified using the Visual Analogue Scale on postoperative days 1 and 2. RESULTS: Data from 997 patients were analyzed. Preoperatively, 258 (25.9%) patients had high anxiety (STAI-State>44). Multivariate analyses showed a significant relationship between high anxiety and female gender (OR: 1.66, 95% CI: 1.08-2.57, p = 0.02), highly invasive surgery (OR: 2.29, 95% CI: 1.29-4.06, p = 0.005), higher trait anxiety (OR: 1.24, 95% CI: 1.20-1.28, p < 0.001) and insomnia (AIS ≥ 6, OR: 1.79, 95% CI: 1.17-2.76, p = 0.008). Preoperative anxiety demonstrated a negative correlation with postoperative anxiety following highly invasive surgery; this became a positive relationship following less invasive surgery. Preoperative anxiety was also positively related to postoperative pain and poor sleep quality. The correlation between preoperative anxiety and postoperative nausea and vomiting was not statistically significant. CONCLUSION: Female gender, highly invasive surgery, higher trait anxiety and insomnia are independent risk factors for high preoperative anxiety. Surgical invasiveness influences association between pre- and postoperative anxiety. Higher preoperative anxiety is related to poorer sleep quality and more severe pain postoperatively.

Autophagy-Mediated Activation of Mucosal-Associated Invariant T Cells Driven by Mesenchymal Stem Cell-Derived IL-15.

Mucosal-associated invariant T (MAIT) cells are innate-like unconventional T cells that are abundant in humans and have attracted increasing attention in recent years. Mesenchymal stem cells (MSCs) are crucial regulators of immune cells. However, whether MAIT cells are regulated by MSCs is unclear. Here, we explored the effect of MSCs on MAIT cells and revealed the underlying mechanism. We found that MSCs did not influence the proliferation of MAIT cells but strikingly induced an activated phenotype with an increased expression of CD69, TNF-α, IFN-γ, and granzyme B. Moreover, MSCs activated MAIT cells in a TCR-MR1-independent mechanism through MSC-secreted IL-15. We revealed that MSC-derived IL-15 activated MAIT cells by enhancing autophagy activity, which was abolished by the autophagy inhibitor 3-methyladenine. Based on our findings, MAIT cells are activated by MSCs through IL-15-induced autophagy, which may help elucidate the mechanisms underlying some immune responses and diseases and provide guidance for future research.

SNP-adjacent super enhancer network mediates enhanced osteogenic differentiation of MSCs in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a rheumatic disease with pathological osteogenesis that causes bony ankylosis and even deformity over time. Mesenchymal stem cells (MSCs) are multipotent stem cells that are the main source of osteoblasts. We previously demonstrated that enhanced osteogenic differentiation of MSCs from AS patients (ASMSCs) is related to pathological osteogenesis in AS. However, the more concrete mechanism needs further exploration. Super enhancers (SEs) are dense clusters of stitched enhancers that control cell identity determination and disease development. Single-nucleotide polymorphisms (SNPs) regulate the formation and interaction of SEs and denote genes accounting for AS susceptibility. Via integrative analysis of multiomic data, including histone 3 lysine 27 acetylation (H3K27ac), chromatin immunoprecipitation sequencing (ChIP-seq), SNPs and RNA sequencing (RNA-seq) data, we discovered a transcription network mediated by AS SNP-adjacent SEs (SASEs) in ASMSCs and identified key genes, such as Toll-like receptor 4 (TLR4), interleukin 18 receptor 1 (IL18R1), insulin-like growth factor binding protein 4 (IGFBP4), transportin 1 (TNPO1) and proprotein convertase subtilisin/kexin type 5 (PCSK5), which are pivotal in osteogenesis and AS pathogenesis. The SASE-regulated network modulates the enhanced osteogenic differentiation of ASMSCs by synergistically activating the PI3K-Akt, NF-kappaB and Hippo signaling pathways. Our results emphasize the crucial role of the SASE-regulated network in pathological osteogenesis in AS, and the preferential inhibition of ASMSC osteogenic differentiation by JQ1 indicates that SEs may be attractive targets in future treatment for new bone formation in AS.

GAS5 protects against osteoporosis by targeting UPF1/SMAD7 axis in osteoblast differentiation.

Osteoporosis is a common systemic skeletal disorder resulting in bone fragility and increased fracture risk. It is still necessary to explore its detailed mechanisms and identify novel targets for the treatment of osteoporosis. Previously, we found that a lncRNA named GAS5 in human could negatively regulate the lipoblast/adipocyte differentiation. However, it is still unclear whether GAS5 affects osteoblast differentiation and whether GAS5 is associated with osteoporosis. Our current research found that GAS5 was decreased in the bones and BMSCs, a major origin of osteoblast, of osteoporosis patients. Mechanistically, GAS5 promotes the osteoblast differentiation by interacting with UPF1 to degrade SMAD7 mRNA. Moreover, a decreased bone mass and impaired bone repair ability were observed in Gas5 heterozygous mice, manifesting in osteoporosis. The systemic supplement of Gas5-overexpressing adenoviruses significantly ameliorated bone loss in an osteoporosis mouse model. In conclusion, GAS5 promotes osteoblast differentiation by targeting the UPF1/SMAD7 axis and protects against osteoporosis.

Enhanced Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells in Ossification of the Posterior Longitudinal Ligament Through Activation of the BMP2-Smad1/5/8 Pathway.

Ossification of the posterior longitudinal ligament (OPLL) is characterized by ectopic OPLL. To date, the specific molecular pathogenesis of OPLL has not been clearly elucidated. In this study, bone marrow-derived mesenchymal stem cells obtained from healthy donors (HD-MSCs) and patients with OPLL (OPLL-MSCs) were cultured in osteogenic differentiation medium for 21 days. The osteogenic differentiation capacity was determined by alizarin red S (ARS) and alkaline phosphatase (ALP) assays. Gene expression levels of osteoblastic markers were measured by quantitative reverse transcription-polymerase chain reaction. Protein levels of related genes and the activation of related signaling pathways were measured by western blotting. LDN193189 was used to inhibit the Smad1/5/8 pathway, and small interfering RNA was used to regulate BMP2 expression. Our results showed that the OPLL-MSCs had stronger ARS staining and ALP activity and higher expression of RUNX2, Osterix, and OCN than the HD-MSCs. During osteogenic differentiation, the Smad1/5/8 pathway was overactivated in the OPLL-MSCs, and LDN193189 inhibition reversed the enhanced osteogenic ability of these cells. Besides, BMP2 was upregulated in the OPLL-MSCs. After BMP2 knockdown, the abnormal osteogenic differentiation of OPLL-MSCs was rescued. Thus, abnormal activation of the BMP2-Smad1/5/8 pathway induces enhanced osteogenic differentiation of OPLL-MSCs compared with HD-MSCs. These findings reveal a mechanism of pathological osteogenesis in OPLL and provide a new perspective on inhibiting pathological osteogenesis by regulating BMP2.