Single-cell and spatial transcriptomics reveal metastasis mechanism and microenvironment remodeling of lymph node in osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumor and is highly prone to metastasis. OS can metastasize to the lymph node (LN) through the lymphatics, and the metastasis of tumor cells reestablishes the immune landscape of the LN, which is conducive to the growth of tumor cells. However, the mechanism of LN metastasis of osteosarcoma and remodeling of the metastatic lymph node (MLN) microenvironment is not clear. METHODS: Single-cell RNA sequencing of 18 samples from paracancerous, primary tumor, and lymph nodes was performed. Then, new signaling axes closely related to metastasis were identified using bioinformatics, in vitro experiments, and immunohistochemistry. The mechanism of remodeling of the LN microenvironment in tumor cells was investigated by integrating single-cell and spatial transcriptomics. RESULTS: From 18 single-cell sequencing samples, we obtained 117,964 cells. The pseudotime analysis revealed that osteoblast(OB) cells may follow a differentiation path from paracancerous tissue (PC) → primary tumor (PT) → MLN or from PC → PT, during the process of LN metastasis. Next, in combination of bioinformatics, in vitro and in vivo experiments, and immunohistochemistry, we determined that ETS2/IBSP, a new signal axis, might promote LN metastasis. Finally, single-cell and spatial dissection uncovered that OS cells could reshape the microenvironment of LN by interacting with various cell components, such as myeloid, cancer-associated fibroblasts (CAFs), and NK/T cells. CONCLUSIONS: Collectively, our research revealed a new molecular mechanism of LN metastasis and clarified how OS cells influenced the LN microenvironment, which might provide new insight for blocking LN metastasis.

UPLC/Q-TOF-MS-based Metabolomics Study of the Antiosteoporosis Effects of Vaccarin in Ovariectomized Mice.

Osteoporosis is a systemic and metabolic bone disease that usually occurs in postmenopausal women, which mainly manifests as bone loss and increased bone fragility that both facilitate fracture. However, few drugs for osteoporosis have shown good efficacy and limited side effects. Vaccarin has demonstrated its antiosteoporosis effects by inhibiting the formation and osteolytic activities of osteoclasts in our previous investigation. In this study, multivariate statistical analysis and ultrahigh-performance liquid chromatography and quadrupole time-of-flight tandem mass spectrometry were used to analyze the serum metabolites of ovariectomized mice treated with or without vaccarin. As a result, 9 serum metabolites were identified as biomarkers. The metabolic levels of 3 crucial biomarkers, namely, lysophosphatidylcholine [22 : 6, (4Z, 7Z, 10Z, 13Z, 16Z, 19Z)], 1-linoleoylglycerophosphocholine and 1-palmitoyl-Sn-glycero-3-phosphocholine, that were correlated with glycerophospholipid metabolism increased and then decreased significantly after vaccarin treatment. Molecular docking analysis and osteoclasts differentiation experiment further revealed that vaccarin may bind with phospholipase A2 and downregulated its activity to reduce the osteoclastogenesis. Therefore, the occurrence of osteoporosis is closely related with glycerophospholipid metabolism disorders, and vaccarin exerts antiosteoporosis effects by reducing the levels of glycerophospholipid metabolites.

P14ARF inhibits regional inflammation and vascularization in intervertebral disc degeneration by upregulating TIMP3.

In this study, we identified P14 alternate reading frame (P14ARF) as a novel regulator of inflammation and vascularization in intervertebral disk degeneration (IVDD). We collected IVD tissues from IVDD patients and normal individuals for analysis of P14ARF expression. We also induced experimental IVDD by needle puncture injuries in the caudal intervertebral disks of Sprague-Dawley (SD) rats and achieved recombinant adenovirus-mediated P14ARF overexpression in experimental IVDD rats. Regulation relationships between P14ARF and tissue inhibitors of metalloproteinases-3 (TIMP3) were confirmed in P14ARF-overexpressed and TIMP3-depleted nucleus pulposus (NP) cells. Tube formation in vitro was evaluated in coculture systems of human umbilical vein endothelial cells (HUVECs) and rat degenerated NP cells (DNPCs). Inflammatory response was assessed from levels of TNF-α, IL-1ß, and IL-6 and neovascularization from expression of endothelial growth factor (VEGF). The P14ARF and TIMP3 were downregulated in degenerated IVD tissue derived from patients and experimental IVDD rats. Overexpressed P14ARF suppressed inflammatory cytokine levels and vascularization. There was decreased in vitro tube formation in response to P14ARF overexpression and TIMP3 elevation. Finally, attenuated inflammatory responses and suppression of VEGF were achieved by P14ARF-mediated promotion of TIMP3 in rat DNPCs. Taken together, the present study reveals that P14ARF/TIMP3 modulation of inflammatory response and vascularization in the context of IVDD highlights a potential target for future therapeutic strategies.

Articular chondrocyte-derived extracellular vesicles promote cartilage differentiation of human umbilical cord mesenchymal stem cells by activation of autophagy.

BACKGROUND: Umbilical cord mesenchymal stem cell (HUCMSC)-based therapies were previously utilised for cartilage regeneration because of the chondrogenic potential of MSCs. However, chondrogenic differentiation of HUCMSCs is limited by the administration of growth factors like TGF-ß that may cause cartilage hypertrophy. It has been reported that extracellular vesicles (EVs) could modulate the phenotypic expression of stem cells. However, the role of human chondrogenic-derived EVs (C-EVs) in chondrogenic differentiation of HUCMSCs has not been reported. RESULTS: We successfully isolated C-EVs from human multi-finger cartilage and found that C-EVs efficiently promoted the proliferation and chondrogenic differentiation of HUCMSCs, evidenced by highly expressed aggrecan (ACAN), COL2A, and SOX-9. Moreover, the expression of the fibrotic marker COL1A and hypertrophic marker COL10 was significantly lower than that induced by TGF-ß. In vivo, C-EVs induced HUCMSCs accelerated the repair of the rabbit model of knee cartilage defect. Furthermore, C-EVs led to an increase in autophagosomes during the process of chondrogenic differentiation, indicating that C-EVs promote cartilage regeneration through the activation of autophagy. CONCLUSIONS: C-EVs play an essential role in fostering chondrogenic differentiation and proliferation of HUCMSCs, which may be beneficial for articular cartilage repair.

Rational engineering of ferritin nanocages for targeted therapy of osteoarthritis.

Intra-articular (IA) drug delivery to treat osteoarthritis (OA) is limited by the short retention time of drugs in the joints due to poor specific targeting and non-responsiveness under acidic environment. A cartilage-targeting peptide was engineered to the surface of ferritin nanocages (CT-Fn) and loaded with an anti-inflammatory drug, metformin (Met), via the self-assembling nature of Fn nanocages. It demonstrated that the CT-Fn/Met could specifically bind to type II collagen, leading to the downregulation of catabolic markers of OA and promotion of cartilage-specific makers in IL-1ß-induced chondrocytes. IA delivery of CT-Fn/Met prolonged the retention time for 3 weeks and remarkably reduced inflammation. Moreover, better release under acidic conditions which enabling longer retention time of Met after IA delivery in OA joints for one more week. CT-Fn/Met could target and efficiently enter chondrocytes, further inducing prolonged IA accumulation and achieving enhanced therapeutic efficacy for OA treatment.

Andrographolide prevents human nucleus pulposus cells against degeneration by inhibiting the NF-κB pathway.

Intervertebral disc degeneration (IDD) is among the most common spinal disorders, pathologically characterized by excessive cell apoptosis and production of proinflammatory factors. Pharmacological targeting of nucleus pulposus (NP) degeneration may hold promise in IDD therapy, but it is limited by adverse side effects and nonspecificity of drugs. In this study, we used a natural compound, andrographolide (ANDRO), which has been widely used to intervene inflammatory and apoptotic diseases in the investigation of NP degeneration based on IDD-patients-derived NP cells by lipopolysaccharide (LPS) treatment for the preservation of degeneration. The results showed that LPS maintained the degeneration status of NP cells as evidenced by a high apoptosis rate and the expression of degenerative and inflammatory mediators after LPS treatment. ANDRO reversed the effects of LPS-caused degeneration of NP cells and maintained the phenotype of NP cells, as demonstrated by flow cytometry, degenerative mediators (ADAMTS4 and ADAMTS5), inflammatory factors (COX2, PGE2, MMP-13, and MMP-3), biomarkers of NP cells (SOX9, ACAN, and COL2A1) expressions, and glycosaminoglycan secretion. We also found the involvement of the nuclear factor kappa-light-chain-enhancer of the activated B cells (NF-κB) pathway in ANDRO treatment, indicating that ANDRO prevented the LPS-preserved degeneration of NP cells by inhibiting the NF-κB pathway. This study may provide a reference for clinic medication of IDD therapy.

Propofol alleviates hypoxia-induced nerve injury in PC-12 cells by up-regulation of microRNA-153.

BACKGROUND: Although the neuroprotective role of propofol has been identified recently, the regulatory mechanism associated with microRNAs (miRNAs/miRs) in neuronal cells remains to be poorly understood. We aimed to explore the regulatory mechanism of propofol in hypoxia-injured rat pheochromocytoma (PC-12) cells. METHODS: PC-12 cells were exposed to hypoxia, and cell viability and apoptosis were assessed by CCK-8 assay and flow cytometry assay/Western blot analysis, respectively. Effects of propofol on hypoxia-injured cells were measured, and the expression of miR-153 was determined by stem-loop RT-PCR. After that, whether propofol affected PC-12 cells under hypoxia via miR-153 was verified, and the downstream protein of miR-153 as well as the involved signaling cascade was finally explored. RESULTS: Hypoxia-induced decrease of cell viability and increase of apoptosis were attenuated by propofol. Then, we found hypoxia exposure up-regulated miR-153 expression, and the level of miR-153 was further elevated by propofol in hypoxia-injured PC-12 cells. Following experiments showed miR-153 inhibition reversed the effects of propofol on hypoxia-treated PC-12 cells. Afterwards, we found BTG3 expression was negatively regulated by miR-153 expression, and BTG3 overexpression inhibited the mTOR pathway and AMPK activation. Besides, hypoxia inhibited the mTOR pathway and AMPK, and these inhibitory effects could be attenuated by propofol. CONCLUSION: Propofol protected hypoxia-injured PC-12 cells through miR-153-mediataed down-regulation of BTG3. BTG3 could inhibit the mTOR pathway and AMPK activation.

Masticatory Muscles Dysfunction after CT-guided Percutaneous Trigeminal Radiofrequency Thermocoagulation for Trigeminal Neuralgia: A Detailed Analysis.

OBJECTIVE: The aim of this study was to investigate the severity and the natural course of masticatory muscles weakness that developed after CT-guided percutaneous trigeminal radiofrequency thermocoagulation (PT-RFT) for the treatment of idiopathic trigeminal neuralgia (ITN). METHODS: Twenty-seven patients with ITN were treated by CT-guided percutaneous trigeminal radiofrequency thermocoagulation. Each patients' occlusal function and surface electromyographic (sEMG) activity of the ipsilateral anterior temporalis (TA) and masseter muscles (MM) at mandibular postural position (MPP), and during a fast maximum voluntary clenching (MVC) from MPP to intercuspal position (ICP), were simultaneously recorded by the T-Scan III system and Bio-pak sEMG III system before (baseline), 3 days, 3 months, and 12 months after procedure. The incidence, degree, and prognosis of masticatory muscles dysfunction related to trigeminal nerve motor-branch injury were analyzed. RESULTS: Three days and 3 months after procedure, both the occlusal symmetry and the sEMG activity of ipsilateral TA and MM became significantly decreased compared to the baseline (P < 0.05). However, they demonstrated a gradual improvement toward preoperative values in follow-up, returning to complete in 23 patients at 12 months after procedure. None reported permanent masticatory paralysis. Pain relief was most significant on the third day after procedure. At the final clinical visit, a pain-free status was observed in 25 patients (92.6%). Meanwhile, the intensity of facial dysesthesia was mildest, whereas there were statistic differences compared with baseline. CONCLUSION: CT-guided PT-RFT for ITN remains an effective and safe surgical procedure, but there is a high rate of temporary masticatory dysfunction during a short time after procedure, appearing to be reversible in a period of 12 months.

Global trends in the research on Legg-Calve-Perthes disease in Web of Science.

Background: Legg-Calve-Perthes disease (LCPD) is a form of idiopathic femoral head necrosis that can lead to permanent femoral head deformities and premature osteoarthritis in children under the age of 15. Its pathogenesis is utterly and remains to be clarified. Although many research publications on LCPD have emerged during the last few decades, few systematic bibliometric analyses of these articles have been reported. Methods: A bibliometric analysis was performed to investigate the development processes and hotspots, as well as the collaboration and influence among countries, institutions, authors, journals, and keywords of papers relevant to LCPD from the Web of Science Core Collection (WoSCC) during the period from 1 January 2000 to 30 June 2023. Results: A total of 2,205 researchers from 916 institutions across 53 countries/regions have contributed to 673 papers published in 199 academic journals. The research on LCPD has shown significant fluctuations but a gradual increase in the number of articles published over the last two decades. The United States leads in the number of publications of LCPD, with the Texas Scottish Rite Hospital for Children being the most productive institution. English, as the most widely used language in the world, was undoubtedly the most popular language. Herring JA, who acted as both the corresponding and first author, has contributed to the most co-cited papers published. The most number of LCPD papers are published in the Journal of Pediatric Orthopaedics, whereas the Journal of Bone and Joint Surgery American Volume garnered the highest total citations, indicating the great importance of these two journals in the field of orthopedics. The most frequently used keywords in published articles were related to the symptoms, mechanisms, and prognosis, revealing the research focus of most scholars. Conclusion: Our research described the development trends and hotspots in the research field of LCPD and will help researchers make better decisions.

Analysis of the Genetic Model of the Extremely Narrow Channel Shallow Water Delta in DL-A Oilfield, Bohai Bay Basin.

In recent years, the oil and gas reserves discovered in shallow water deltas in China have continued to grow. The research on shallow water delta deposition models and depositional genesis is becoming more and more mature. In this latest discovery, a unique type of extremely narrow channel shallow water delta deposit was found at the top of the V oil group in the lower part of the Minghuazhen Formation during the Neogene period at DL-A Oilfield, located in the Bohai Bay Basin. The width of most single channels in this deposit measures between 100 and 200m, which is relatively rare and differs from existing research. To better understand this unique narrow channel shallow water delta deposit, a range of analysis methods were conducted including trace element analysis, major element analysis, grain size analysis, core observation, casting thin section observation, 3D seismic analysis, and other methods. These analyses were used to determine the sedimentary environment and sedimentary genesis of the deposit in the study area. The results show the following: (1) The top of the V oil group in the lower part of Minghuazhen Formation was deposited with a strong oxidizing environment. In the early stage, the climate was dry and cold, and gradually changed to warm and humid in the late stage. (2) Due to the frequent exposure to the surface, obvious weathered surfaces and sedimentary discontinuities were observed on the cores; the particle size analysis shows that the lamina types developed in the study area are clastic-clay laminae and clay-clastic laminae, which are mostly developed in shallow lakes area. (3) Observations of cores and thin sections also indicated that the hydrodynamic conditions frequently changed in the study area, alternating between strong and weak hydrodynamic conditions in a short period due to the alternating occurrence of flood and dry periods during the rainy season. Weak hydrodynamic conditions and slow water flow result in insufficient undercutting and sidecutting of rivers. The alternating occurrence of flood periods and dry periods has led to the development of crevasse splays and frequent river channel diversions, resulting in the inability of long-term stable development of the river channel. Besides, the change of water level has also led to the rebuilding of the river. Therefore, the multiple effects led to the formation of an extremely narrow channel shallow water delta. The accuracy of the sedimentary model is verified by a comparative study of the Shaliu River and Buha River in the modern Qinghai Lake. The new extremely narrow channels deposition model proposed this time further improves the deposition theory. At the same time, the modern depositional characteristics of the Shaliu River and Buha River also reveal the reservoir deposition between channels that cannot be distinguished by seismic data, providing guidance for the development of oil and gas in the study area.