Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae

Background: Previous studies reported that melatonin exerts its effect on mesenchymal stem cell (MSC) survival and differentiation into osteogenic and adipogenic lineage. In the current study we aimed to explore the effect of melatonin on osteoporosis and relevant mechanisms. Methods: Real-time qualitative polymerase chain reaction (RT-qPCR) and Western blot analysis were conducted to determine expression of HGF, PTEN, and osteoblast differentiation-related genes in ovariectomy (OVX)-induced osteoporosis mice and the isolated bone marrow MSCs (BMSCs). Pre-conditioning with melatonin (1 μmol/l, 10 μmol/l and 100 μmol/l) was carried out in OVX mice BMSCs. Bone microstructure was analyzed using micro-computed tomography and the contents of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase 5b (TRAP5b) were detected by enzyme-linked immunosorbent assay in serum. BMSC proliferation was measured by cell-counting kit (CCK)-8 assay. Alizarin red S (ARS) staining and ALP activity assay were performed to assess BMSC mineralization and calcification. The activity of the Wnt/β-catenin pathway was evaluated by dual-luciferase reporter assay. Results: Melatonin prevented bone loss in OVX mice. Melatonin increased ALP expression and reduced TRAP5b expression. HGF and β-catenin were downregulated, while PTEN was upregulated in the femur of OVX mice. Melatonin elevated HGF expression and then stimulated BMSC proliferation and osteogenic differentiation. Additionally, HGF diminished the expression of PTEN, resulting in activated Wnt/β-catenin pathway both in vitro and in vivo. Furthermore, melatonin was shown to ameliorate osteoporosis in OVX mice via the HGF/ PTEN/ Wnt/β-catenin axis. Conclusion: Melatonin could potentially enhance osteogenic differentiation of BMSCs and retard bone loss through the HGF/ PTEN/ Wnt/β-catenin axis.

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Osteoporosis Recovery byAntrodia camphorataAlcohol Extracts through Bone Regeneration in SAMP8 Mice

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2016/2617868 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

Hen-Yu Liu ◽

Chiung-Fang Huang ◽

Chun-Hao Li ◽

Ching-Yu Tsai ◽

Wei-Hong Chen ◽

...

Keyword(s):

Bone Loss ◽

Bone Matrix ◽

Mineral Density ◽

Antrodia Camphorata ◽

Osteoporosis Therapy ◽

Alizarin Red ◽

Alcohol Extract ◽

Samp8 Mice

Antrodia camphoratahas previously demonstrated the efficacy in treating cancer and anti-inflammation. In this study, we are the first to evaluateAntrodia camphorataalcohol extract (ACAE) for osteoporosis recoveryin vitrowith preosteoblast cells (MC3T3-E1) andin vivowith an osteoporosis mouse model established in our previous studies, ovariectomized senescence accelerated mice (OVX-SAMP8). Our results demonstrated that ACAE treatment was slightly cytotoxic to preosteoblast at 25 μg/mL, by which the osteogenic gene expression (RUNX2, OPN, and OCN) was significantly upregulated with an increased ratio of OPG to RANKL, indicating maintenance of the bone matrix through inhibition of osteoclastic pathway. Additionally, evaluation by Alizarin Red S staining showed increased mineralization in ACAE-treated preosteoblasts. Forin vivostudy, our results indicated that ACAE inhibits bone loss and significantly increases percentage bone volume, trabecular bone number, and bone mineral density in OVX-SAMP8 mice treated with ACAE. Collectively,in vitroandin vivoresults showed that ACAE could promote osteogenesis and prevent bone loss and should be considered an evidence-based complementary and alternative medicine for osteoporosis therapy through the maintenance of bone health.

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Effects of raloxifene against letrozole-induced bone loss in chemically-induced model of menopause in mice

Molecular and Cellular Endocrinology ◽

10.1016/j.mce.2016.11.005 ◽

2017 ◽

Vol 440 ◽

pp. 34-43 ◽

Cited By ~ 5

Author(s):

Abul Kalam ◽

Sushama Talegaonkar ◽

Divya Vohora

Keyword(s):

Bone Loss ◽

Chemically Induced

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Exploration of Anti-inflammatory Mechanism of Forsythiaside A and Forsythiaside B in CuSO4-Induced Inflammation in Zebrafish by Metabolomic and Proteomic Analyses

10.21203/rs.2.19758/v1 ◽

2019 ◽

Author(s):

Lihong Gong ◽

Linyuan Yu ◽

Xiaohong Gong ◽

Cheng Wang ◽

Naihua Hu ◽

...

Keyword(s):

Sugar Metabolism ◽

Amino Sugar ◽

Zebrafish Model ◽

Zebrafish Larvae ◽

Chemically Induced ◽

Inflammatory Mechanism ◽

Forsythia Suspensa ◽

Proteomic Analyses ◽

Anti Inflammatory ◽

Inflammatory Effect

Abstract Background: Inflammation is a general pathological phenomenon that operates during severe disturbance of homeostasis. Forsythiaside A (FA) and Forsythiaside B (FB) are isolated from air-dried fruits of Forsythia suspensa, which show a significant effect against inflammation. However, the anti-inflammatory effect and therapeutic mechanism have not yet been clarified in zebrafish. Methods: In this study, the anti-inflammatory effects of FA and FB were investigated in CuSO4-induced zebrafish model. Intracellular ROS and NO generation in zebrafish were performed using fluorescent probe dyes. Metabolomics and proteomics using liquid chromatography mass spectrometry were carried out to identify the expression of metabolites and proteins associated with chemically induced inflammation in zebrafish larvae. Quantitative PCR was performed to detect the progressive changes in the genes.Results: FA and FB inhibited neutrophils migration to the damaged neuromasts and remarkably reduced CuSO4-induced ROS and NO generation in zebrafish. Metabolomics analyses indicated that the Nicotinate and nicotinamide metabolism, Amino sugar and nucleotide sugar metabolism, Pyrimidine metabolism and Purine metabolism were mainly involved. The proteomic analyses identified 146 differentially expressed proteins, among which, the expression of collagen (col2a1b, col9a2, col9a1b), nme3, wdr3, mrps7, srpx, gch2, ptpn11a, rcvrn2, nit2, scaf4a and zgc:162509 were all reversed both in FA and FB groups. FA and FB could reverse the abnormal expression of these metabolites and proteins to alleviate chemical damage to the neuromasts in the lateral line of zebrafish.Conclusions: FA and FB possess remarkable anti-inflammatory effect and could be used to protect chemically induced neuromasts damage in zebrafish larvae.

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Multivesicular Liposomes for Glucose-Responsive Insulin Delivery

Pharmaceutics ◽

10.3390/pharmaceutics14010021 ◽

2021 ◽

Vol 14 (1) ◽

pp. 21

Author(s):

Guangqu Liu ◽

Suping He ◽

Yu Ding ◽

Cai Chen ◽

Qingchun Cai ◽

...

Keyword(s):

Glucose Oxidase ◽

Diabetes Management ◽

Double Emulsion ◽

Insulin Delivery ◽

Diabetic Rats ◽

Alizarin Red ◽

Glucose Levels ◽

Chemically Induced

An intelligent insulin delivery system is highly desirable for diabetes management. Herein, we developed a novel glucose-responsive multivesicular liposome (MVL) for self-regulated insulin delivery using the double emulsion method. Glucose-responsive MVLs could effectively regulate insulin release in response to fluctuating glucose concentrations in vitro. Notably, in situ released glucose oxidase catalyzed glucose enrichment on the MVL surface, based on the combination of (3-fluoro-4-((octyloxy)carbonyl)phenyl)boronic acid and glucose. The outer MVL membrane was destroyed when triggered by the local acidic and H2O2-enriched microenvironment induced by glucose oxidase catalysis in situ, followed by the further release of entrapped insulin. Moreover, the Alizarin red probe and molecular docking were used to clarify the glucose-responsive mechanism of MVLs. Utilizing chemically induced type 1 diabetic rats, we demonstrated that the glucose-responsive MVLs could effectively regulate blood glucose levels within a normal range. Our findings suggest that glucose-responsive MVLs with good biocompatibility may have promising applications in diabetes treatment.

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Using Acoustic Waves to Modulate Stem Cell Growth and Differentiation

Volume 3: Biomedical and Biotechnology Engineering ◽

10.1115/imece2017-71341 ◽

2017 ◽

Author(s):

Jacqueline A. Maynard ◽

Ahmad S. Arabiyat ◽

Anna Elefante ◽

Lucas Shearer ◽

Eoin King ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Bone Loss ◽

Acoustic Waves ◽

Stem Cell Differentiation ◽

Cell Number ◽

Sound Waves ◽

Alizarin Red ◽

Skeletal Mass

During spaceflight, the loss of mechanical loads due to microgravity leads to rapid bone loss, where bone deteriorates at a rate of 1–2% per month, where some astronauts can lose as much as 20% of their skeletal mass in a single expedition [NASA, 2001]. In order to prevent muscle and bone loss, long-term space flight exercise regimes are strictly implemented [Shackleford, 2004]. Current research has demonstrated that mechanical vibrations can help to maintain or improve bone mass [Chan, 2013] and reduce adiposity [Chen, 2015, Sen, 2011] when signals are applied at the appropriate frequency and amplitude. We have developed an acoustic sound chamber that can apply sound waves to stem cells grown in vitro. Characterization of the culture conditions inside the vibration chamber showed considerable variance across the culture plates where an applied acceleration of 0.6g varied at different spots in a 12-well tissue culture plate from as low as 0.47g to 0.78g. We believe the variance is caused by differences in the rigidity of the culture plates that makes the waves transmit inconsistently through the plastic. We hypothesized acoustic waves would induce osteogenic differentiation when applied to stem cells. We utilized pre-osteoblastic stem cells (MC3T3-E1-Subclone 4) to observe the effects of acoustic waves when applied at 0.3g and 0.6g, compared to non-vibrated controls. Cells were vibrated for 30 minutes a day for either 6 days (n = 24/group) or 12 days (n = 12/group). Cellular changes were characterized by assessing well-by-well cell number by a manual cell count and mineral content by Alizarin Red S staining. Differences between groups were determined using One-Way ANOVA with a post hoc test: Student’s t-test. To assess the effects of the variance across the culture plates, correlative analysis was conducted for well-by-well variation using Regression Analysis. Acoustically vibrated wells had 10x more cells after 6 days and showed more mineralization than non-vibrated wells at both 6 and 12 days. Acoustic waves have the ability to increase cell proliferation and can drive stem cell differentiation towards an osteoblastic lineage, this could lead to therapies that prevent bone loss during spaceflight.

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Curculigoside promotes osteogenic differentiation of ADSCs to prevent ovariectomized-induced osteoporosis

Journal of Orthopaedic Surgery and Research ◽

10.1186/s13018-021-02389-3 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Wei-Li You ◽

Zheng-Long Xu

Keyword(s):

Bone Loss ◽

Western Blot ◽

Osteogenic Differentiation ◽

Signaling Pathway ◽

Akt Signaling ◽

Calcium Deposition ◽

Phenolic Glycoside ◽

Akt Signaling Pathway ◽

Alizarin Red ◽

Alp Activity

Abstract Background Curculigoside is a natural phenolic glycoside compound produced by Curculigo orchioides Gaertn. This study aimed to explore the effects of curculigoside in promoting the osteogenic differentiation of adipose-derived stem cells (ADSCs) as well as the underlying mechanism. Methods ADSCs were treated with curculigoside at different concentrations (0 μmol/L, 1 μmol/L, 2.5 μmol/L, 5 μmol/L, 10 μmol/L, and 20 μmol/L), and cell viability was assessed by CCK-8 assay. Then, the alkaline phosphatase (ALP) activity was determined, and alizarin red S (ARS) staining was performed to measure the extracellular mineralization of curculigoside. Information about protein-chemical interactions is provided by the search tool for interactions of chemicals (STITCH) database. Then, LY294002 was administered to explore the mechanism by which curculigoside promotes the osteogenic differentiation of ADSCs. Western blot assays were performed to assess changes in the expression of osteogenic-related markers and the phosphorylation of PI3K and AKT. Finally, we established an ovariectomized (OVX)-induced osteoporosis mouse model and administered curculigoside to explore the effects of curculigoside in preventing bone loss in vivo. Results The CCK-8 assay indicated that curculigoside did not induce cytotoxicity at a concentration of 5 μmol/L after 48 h. The ALP and ARS results revealed that the induced group had higher ALP activity and calcium deposition than the control group. Moreover, the curculigoside group exhibited increased biomineralization, ALP activity, and ARS staining compared to the induced and control groups, and these effects were partially inhibited by LY294002. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the target genes of curculigoside were mainly involved in the PI3K-Akt signaling pathway. PCR and western blot analysis showed that the expression of RUNX2, ALP, and Osterix was upregulated in curculigoside-treated ADSCs, but this effect was partially reversed by the PI3K inhibitor LY294002. Moreover, the curculigoside-treated group exhibited significantly increased phosphorylation of AKT to P-AKT compared with the osteogenic induction group. After treatment with curculigoside, the mice had a higher bone volume than the OVX mice, suggesting partial protection from cancellous bone loss. In addition, when LY294002 was added, the protective effects of curculigoside could be neutralized. Conclusions Curculigoside could induce the osteogenic differentiation of ADSCs and prevent bone loss in an OVX model through the PI3K/Akt signaling pathway.

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Protective Role of Galanin during Chemically Induced Inflammation in Zebrafish Larvae

Biology ◽

10.3390/biology10020099 ◽

2021 ◽

Vol 10 (2) ◽

pp. 99

Author(s):

Natalia Nowik ◽

Anna Przyborowska ◽

Waldemar Sienkiewicz ◽

Piotr Podlasz

Keyword(s):

Pathological Condition ◽

Protective Role ◽

Neuroendocrine System ◽

Biological Processes ◽

Zebrafish Larvae ◽

Chemically Induced ◽

Enhanced Expression ◽

The Central Nervous System ◽

Endocrine Axis

During a pathological condition, many different systems are involved in the response of an affected organism. Galanin is considered to be a neuropeptide that plays an important role in the central nervous system; however, it is involved in many other biological processes, including the immune response. During our studies, we showed that galanin became upregulated in zebrafish larvae when exposed to copper sulfate. Moreover, the presence of normal levels of galanin, administration of a galanin analog NAX 5055 or galanin overexpression led to lowered lateral line damage and enhanced expression of inflammatory markers compared to the knockout larvae. The results showed that the neuroendocrine system acts multifunctionally and should be considered as a part of the complex neuro–immune–endocrine axis.

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Catalpol Protects Against High Glucose-Induced Bone Loss by Regulating Osteoblast Function

Frontiers in Pharmacology ◽

10.3389/fphar.2021.626621 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lu Zhao ◽

Wei Du ◽

Dandan Zhao ◽

Xueyan Ji ◽

Yanfei Huang ◽

...

Keyword(s):

Bone Loss ◽

Trabecular Bone ◽

High Glucose ◽

Type I Collagen ◽

Type I ◽

Protective Effects ◽

Gap Formation ◽

Alizarin Red ◽

Diabetic Osteoporosis ◽

And Migration

Objective: The overall objective of this study was to investigate the effects of catalpol on bone remodeling of diabetic osteoporosis by regulating osteoblast differentiation and migration.Method: Using a murine model of diabetic osteoporosis, to detect the protective effects of catalpol on bone loss, architectural deterioration of trabecular bone and bone metabolism biomarkers were tested. A model of MC3T3-E1 cells was established by treatment with high glucose; the regulatory role of catalpol in the differentiation and migration was tested by Western blot, ALP staining, and Alizarin Red staining.Results: Catalpol treatment markedly ameliorated trabecular bone deterioration by reducing degenerative changes of the trabecular structure by improving the bone formation marker levels of ALP, osteopontin, type I collagen, and osteocalcin, as well as the level of OPG/RANKL. Catalpol enhanced cell motility and scattering following gap formation of MC3T3-E1 cells.Conclusion: The results indicated that catalpol exhibits a protective effect against diabetic osteoporosis by regulating the differentiation and migration of osteoblast.

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