purification-expansion-flow-cytometry-based-phenotyping-mouse-derived

Purification, Expansion, and Flow Cytometry-Based Phenotyping of Mouse Derived Bone Marrow Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of stem cells that can self-renew and differentiate into multiple cell lineages. Among them, MSCs found in ...

Longhua Hospital, Shanghai University of Traditional Chinese Medicine

Shanghai University of Traditional Chinese Medicine

Inflammation is the pathophysiological basis of hyperlipidemia-related disease (HRD). Reducing the south and reinforcing the north method (RSRN) has a positive effect on HRD. However, the pharmacological mechanisms of RSRN are still unclear in the treatment of HRD. We obtained RSRN compounds from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) and identified potential targets of these compounds through target fishing based on the TCMSP databases. Next, we identified the HRD targets by using multiple databases. Then, the overlapping genes between the RSRN potential targets and the HRD targets were used to establish a protein-protein interaction (PPI) network, and we further analyzed their interactions and identified the major hub genes in this network. Subsequently, the Metascape database was utilized to conduct the enrichment of Gene Ontology biological processes (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. A total of 187 potential active components and 106 related core targets were obtained and identified overall. Then after the Metascape enrichment analysis, a total of 148 KEGG pathways were screened, which were mainly associated with AGE-RAGE signaling pathway, PI3K-Akt signaling pathway, TNF signaling pathway, and NF-kappa B signaling pathway. Furthermore, 34 hub genes, such as AKT1, NF-Bp65(RELA), IB(CHUK), MAPK8, and MAPK14, CCND1, were considered potential therapeutic targets. Furthermore, evaluations of protein levels of NF-Bp65, IB, TNF-, IL-1 , and IL-6 were performed for experimental validation. RSRN can reduce the expression of NF-Bp65 protein, increase the level of IB protein, and reduce the protein levels of TNF-, IL-1, and IL-6 in ovariectomized rats. The results indicate that the mechanism of RSRN against inflammation may be related to AKT1, NF-Bp65, IB, MAPK8, and MAPK14, as well as TNF, NF-kappa B, PI3K-Akt signaling pathways.

Effects of Reducing the South and Reinforcing the North Method on Inflammatory Injury Induced by Hyperlipidemia.

The osteocyte lacuno-canalicular system (LCS) plays a crucial role in maintaining bone homeostasis and mediating cellular mechanotransduction. Current histological techniques, particularly the Ploton silver nitrate staining method, face challenges such as variations in solution concentrations and types as well as a lack of standardization, which limits their broader application in osteocyte research. In this study, we present a simplified and more effective silver nitrate staining protocol designed to address these issues. Our method utilizes a 1 mol/L silver nitrate solution combined with optimized gelatin-formic acid solutions at varying concentrations (0.05%-0.5% type-B gelatin and 0.05%-5% formic acid, or 1%-2% type-B gelatin and 0.1%-2% formic acid). Staining is performed for 1 h under 254 nm ultraviolet light or 90 min under room light, followed by washing with Milli-Q water to terminate staining. This novel optimized method yields consistent and distinct staining of the osteocyte LCS across multiple species, demonstrating superior efficiency and reliability compared to the Ploton method. It will significantly advance research in osteocyte biology and provide a valuable tool for exploring the adaptive evolution of osteocyte LCS morphology and function across various taxa. Key features • A novel optimized silver nitrate method using a 1 mol/L silver nitrate solution with type-B gelatin-formic acid solution effectively stains the osteocyte LCS. • The novel optimized method is more efficient for staining the osteocyte LCS across different species. • The novel optimized method is simpler to perform and more cost-effective than conventional methods. Graphical overview  Created in BioRender. Libo, W. (2024) https://BioRender.com/c10z002.

A Novel Optimized Silver Nitrate Staining Method for Visualizing and Quantifying the Osteocyte Lacuno-Canalicular System (LCS).

A 50% (w/v, equivalent to 2.943 mol/L) silver nitrate solution is commonly used to stain and characterize the osteocyte lacuno-canalicular system (LCS) in bone biology research. However, variability in reagent concentrations and types, along with inconsistent staining procedures, have limited the broader application of this method in osteocyte research. In this study, we present a novel optimized silver nitrate staining technique aimed at addressing these limitations. This new method utilizes a 1 mol/L (equivalent to 16.987%, w/v) silver nitrate solution in combination with a type-B gelatin-formic acid solution at various concentrations (0.05%-0.5% gelatin and 0.05%-5% formic acid, or 1%-2% gelatin and 0.1%-2% formic acid) in volume ratios of 4:1, 2:1, or 1:1, or a 0.5 mol/L silver nitrate solution at a 4:1 ratio. The staining process is carried out for 1 hour under ultraviolet light or 90 minutes under regular room light (or dark), followed by washing with Milli-Q water to terminate the reaction. We applied this new method to stain the osteocyte LCS in bone samples from different species and pathological bone models. The technique consistently produced clear, distinct staining patterns across all samples. Moreover, our novel method revealed a greater number of LCS compared to the traditional 50% silver nitrate solution. This suggests that the commonly used 50% silver nitrate method may disrupt or inadequately reveal the LCS in bones, potentially leading to an underestimation of LCS density and number. In conclusion, our novel silver nitrate staining method provides a simpler and more cost-effective alternative to the traditional technique. By offering a more accurate and comprehensive analysis of the LCS across species, this approach has the potential to advance research on osteocyte morphogenesis, as well as the functional and evolutionary adaptations of the osteocyte LCS across different taxa.

Article	Year
Effects of Reducing the South and Reinforcing the North Method on Inflammatory Injury Induced by Hyperlipidemia. Evidence-based complementary and alternative medicine : eCAM\| PubMed ID: 34594388	2021
A Novel Optimized Silver Nitrate Staining Method for Visualizing and Quantifying the Osteocyte Lacuno-Canalicular System (LCS). Bio-protocol\| PubMed ID: 40291414	2025
A novel optimized silver nitrate staining method for visualizing the osteocyte lacuno-canalicular system. Frontiers in endocrinology\| PubMed ID: 40444242	2025

Chenglong Wang

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