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

Beta-ecdysone (βEcd) is a phytoecdysteroid found in the dry roots and seeds of the asteraceae and achyranthes plants, and is reported to increase osteogenesis in vitro. Since glucocorticoid (GC) excess is associated with a decrease in bone formation, the purpose of this study was to determine if treatment with βEcd could prevent GC-induced osteoporosis. Two-month-old male Swiss-Webster mice (n=8-10/group) were randomized to either placebo or slow release prednisolone pellets (3.3mg/kg/day) and treated with vehicle control or βEcd (0.5mg/kg/day) for 21days. GC treatment inhibited age-dependent trabecular gain and cortical bone expansion and this was accompanied by a 30-50% lower bone formation rate (BFR) at both the endosteal and periosteal surfaces. Mice treated with only βEcd significantly increased bone formation on the endosteal and periosteal bone surfaces, and increased cortical bone mass were their controls to compare to GC alone. Concurrent treatment of βEcd and GC completely prevented the GC-induced reduction in BFR, trabecular bone volume and partially prevented cortical bone loss. In vitro studies determined that βEcd prevented the GC increase in autophagy of the bone marrow stromal cells as well as in whole bone. In summary, βEcd prevented GC induced changes in bone formation, bone cell viability and bone mass. Additional studies are warranted of βEcd for the treatment of GC induced bone loss.

Prevention of glucocorticoid induced bone changes with beta-ecdysone.

One of the strongest predictors for osteoporosis is peak bone mass. Interventions to augment peak bone mass have yet to be developed. β-Ecdysone (βEcd), a natural steroid-like compound produced by arthropods to initiate metamorphosis, is believed to have androgenic effects and so may be used to augment bone mass.

β-Ecdysone Augments Peak Bone Mass in Mice of Both Sexes.

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.

Reducing the south and reinforcing the north method (RSRN) has a positive effect on atherosclerosis disease. However, there is a lack of objective standards based on the quantification of 4 diagnostic methods in evaluating the improvement or effectiveness of the treatment. This study aimed to explore the quantitative evaluation of the therapeutic effect of RSRN on postmenopausal atherosclerosis based on the 4 diagnostic methods. The observational prospective cohort study was conducted at Longhua hospital Shanghai University of traditional Chinese medicine. According to the inclusion criteria, 96 patients (disease group) and 38 healthy cases (control group) were selected, the pulse parameters were compared between the 2 groups to demonstrate the reliability and success of the disease model. Then 4 diagnostic information before and after RSRN treatment were collected and statistical analyzed by 1-way analysis of variance (ANOVA) (with Bonferroni correction). Furthermore, social network analysis was used to analyze the changes of symptoms, tongue, pulse, and complexion characteristics before and after treatment. There was a significant difference in pulse parameters between the disease group and the control group. The pulse parameters t1, h3, h3/h1, h4/h1, S, As, and w values in disease group were higher than those in control group, while the h5, h5/h1, and Ad values were lower than those in control group (P < .05). After the treatment of RSRN, the clinical symptoms of patients were greatly improved. The facial color indexes L, a, b values of the disease group at week 6 were different from those at week 0 (P < .05). The overall brightness and chroma of the patient's facial color were significantly improved. The patients had virtual string pulse at week 0, and mainly string I and string II at week 7. The pulse parameters t1, t5, w, w/t, h1, h5, h3/h1, and h5/h1 values at week 7 were different from those at weeks 0, 1, 2 (P < .05); the tongue image was mainly red and crimson, peeling or greasy fur at week 0, while at weeks 6, 7, mainly light red, or thin white tongue. The RSRN method can regulate the complexion, tongue and pulse condition, clinical symptoms of postmenopausal atherosclerosis.

Evaluation of reducing the south and reinforcing the north method on postmenopausal atherosclerosis disease based on 4 diagnostic objectifications: A prospective observational study.

Aging refers to a progressive decline in biological functions, leading to age-related diseases and mortality. The transition metals, including iron, copper, and manganese, play important roles in human physiological and pathological processes. Substantial research has demonstrated that senescent cells accumulate higher levels of transition metals, which in turn accelerates the process of cellular senescence and related diseases through mechanisms such as production of excessive reactive oxygen species (ROS), induction of oxidative stress, DNA damage, and mitochondrial dysfunction. This review article provides a comprehensive overview of the causes of transition metal accumulation in senescent cells, as well as the mechanisms by which it further promotes cellular senescence and related diseases. The aim is to provide insights into anti-aging and treatment of aging-related diseases caused by transition metal accumulation.

[Transition metal accumulation and cellular senescence].

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
Prevention of glucocorticoid induced bone changes with beta-ecdysone. Bone\| PubMed ID: 25585248	2015
β-Ecdysone Augments Peak Bone Mass in Mice of Both Sexes. Clinical orthopaedics and related research\| PubMed ID: 25822452	2015
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
Evaluation of reducing the south and reinforcing the north method on postmenopausal atherosclerosis disease based on 4 diagnostic objectifications: A prospective observational study. Medicine\| PubMed ID: 38579101	2024
[Transition metal accumulation and cellular senescence]. Sheng li xue bao : [Acta physiologica Sinica]\| PubMed ID: 38939936	2024
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

Weiwei Dai

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