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BMC Cardiovascular Disorders Nov 2023Induction of chondrogenesis is associated with progressive atherosclerosis. Deficiency of the ADCYAP1 gene encoding pituitary adenylate cyclase-activating peptide...
BACKGROUND
Induction of chondrogenesis is associated with progressive atherosclerosis. Deficiency of the ADCYAP1 gene encoding pituitary adenylate cyclase-activating peptide (PACAP) aggravates atherosclerosis in ApoE deficient (ApoE) mice. PACAP signaling regulates chondrogenesis and osteogenesis during cartilage and bone development. Therefore, this study aimed to decipher whether PACAP signaling is related to atherogenesis-related chondrogenesis in the ApoE mouse model of atherosclerosis and under the influence of a high-fat diet.
METHODS
For this purpose, PACAP/ApoE, PAC1/ApoE, and ApoE mice, as well as wildtype (WT) mice, were studied under standard chow (SC) or cholesterol-enriched diet (CED) for 20 weeks. The amount of cartilage matrix in atherosclerotic lesions of the brachiocephalic trunk (BT) with maximal lumen stenosis was monitored by alcian blue and collagen II staining on deparaffinized cross sections. The chondrogenic RUNX family transcription factor 2 (RUNX2), macrophages [(MΦ), Iba1], and smooth muscle cells (SMC, sm-α-actin) were immunohistochemically analyzed and quantified.
RESULTS
ApoE mice fed either SC or CED revealed an increase of alcian blue-positive areas within the media compared to WT mice. PAC1/ApoE mice under CED showed a reduction in the alcian blue-positive plaque area in the BT compared to ApoE mice. In contrast, PACAP deficiency in ApoE mice did not affect the chondrogenic signature under either diet.
CONCLUSIONS
Our data show that PAC1 deficiency reduces chondrogenesis in atherosclerotic plaques exclusively under conditions of CED-induced hypercholesterolemia. We conclude that CED-related chondrogenesis occurs in atherosclerotic plaques via transdifferentiation of SMCs and MΦ, partly depending on PACAP signaling through PAC1. Thus, PAC1 antagonists or PACAP agonists may offer therapeutic potential against pathological chondrogenesis in atherosclerotic lesions generated under hypercholesterolemic conditions, especially in familial hypercholesterolemia. This discovery opens therapeutic perspectives to be used in the treatment against the progression of atherosclerosis.
Topics: Animals; Mice; Plaque, Atherosclerotic; Pituitary Adenylate Cyclase-Activating Polypeptide; Chondrogenesis; Alcian Blue; Atherosclerosis; Cholesterol; Diet, High-Fat; Apolipoproteins E; Mice, Knockout; Mice, Inbred C57BL
PubMed: 37980508
DOI: 10.1186/s12872-023-03600-5 -
Journal of Ethnopharmacology May 2024Banxia Xiexin decoction (BXD) is a classic traditional Chinese medicine prescription for treating ulcerative colitis (UC). However, its potential mechanism of action is...
ETHNOPHARMACOLOGICAL RELEVANCE
Banxia Xiexin decoction (BXD) is a classic traditional Chinese medicine prescription for treating ulcerative colitis (UC). However, its potential mechanism of action is still unclear.
AIM OF THE STUDY
Reveal the correlation between the beneficial impacts of BXD on UC and the composition of the gut microbiota.
MATERIALS AND METHODS
The major constituents of BXD were identified using the HPLC-DAD technique. An experimental model of UC was induced in male C57BL/6 mice by administering dextran sodium sulfate (DSS). A total of 48 mice were divided into different groups, including control, model, high-dose BXD treatment, medium-dose BXD treatment, low-dose BXD treatment, and a group treated with 5-amino acid salicylic acid (5-ASA). Body weight changes and disease activity index (DAI) scores were documented; colon length, colon index, spleen index, and thymus index scores were determined; myeloperoxidase (MPO) and tumor necrosis factor-α (TNF-α) activities were assessed; and histological staining with hematoxylin-eosin and alcian blue/phosphate Schiff was performed. The immunofluorescence technique was employed to examine the presence of ZO-1 and occludin in the colon tissue. 16S rRNA sequencing was employed to assess the gut microbiota's diversity and metabolomics was utilized to examine alterations in metabolites within the gut microbiota. The impact of BXD on the gut microbiota was confirmed through fecal microbiota transplantation (FMT).
RESULTS
BXD exhibited a positive impact on UC mice, particularly in the high-dose BXD treatment group. The BXD group experienced weight recovery, decreased DAI scores, improved colon length, and restored of spleen and thymus index scores compared to the DSS group. Additionally, BXD alleviated colon damage and the inflammatory response while restoring intestinal barrier function. FMT in BXD-treated mice also showed therapeutic effects in UC mice. At the phylum level, the relative abundance of Desulfobacterota, Deferribacterota and Actinobacteriota increased; at the genus level, g__norank__f__Muribaculaceae, Dubosiella, Akkermansia, and Lactobacillus increased, whereas Faecalibaculum, Alloprevotella, Turicibacter, and g_Paraprevotella decreased. g__norank_f__Muribaculaceae was positively correlated with body weight and colon length and negatively with colon index scores, splenic index scores, and MPO levels; Alloprevotella was positively correlated with splenic index scores, histological scores, and TNF-α levels and negatively with thymus index scores and thymus index scores. Faecalibaculum was positively correlated with colon index scores and MPO levels. Metabolic investigations revealed 58 potential indicators, primarily associated with the metabolism of amino acids, purines, and lipids. Alloprevotella, g_Paraprevotella, and Bifidobacterium were strongly associated with metabolic pathways.
CONCLUSION
BXD showed beneficial therapeutic effects in UC mice. The mechanism may be by promoting the balance and variety of gut microbiota, as well as regulating the metabolism of amino acids, purines, and lipids.
Topics: Male; Animals; Mice; Mice, Inbred C57BL; Colitis, Ulcerative; Gastrointestinal Microbiome; RNA, Ribosomal, 16S; Tumor Necrosis Factor-alpha; Amino Acids; Antifibrinolytic Agents; Purines; Body Weight; Lipids; Dextran Sulfate; Disease Models, Animal; Colon; Colitis; Drugs, Chinese Herbal
PubMed: 38423412
DOI: 10.1016/j.jep.2024.117990 -
Journal of Bone and Mineral Research :... Oct 2023After periprosthetic joint infection (PJI)-dependent revision surgery, a significantly elevated number of patients suffer from prosthesis failure due to aseptic...
After periprosthetic joint infection (PJI)-dependent revision surgery, a significantly elevated number of patients suffer from prosthesis failure due to aseptic loosening and require additional revision surgery despite clearance of the initial infection. The mechanisms underlying this pathology are not well understood, as it has been assumed that the bone stock recovers after revision surgery. Despite clinical evidence suggesting decreased osteogenic potential in PJI, understanding of the underlying biology remains limited. In this study, we investigated the impact of PJI on bone homeostasis in a two-stage exchange approach at explantation and reimplantation. Sixty-four human tibial and femoral specimens (20 control, 20 PJI septic explantation, and 24 PJI prosthesis reimplantation samples) were analyzed for their bone microstructure, cellular composition, and expression of relevant genetic markers. Samples were analyzed using X-ray microtomography, Alcian blue and tartrate-resistant acid phosphatase staining, and RT-qPCR. In patients with PJI, bone volume (BV/TV; 0.173 ± 0.026; p < 0.001), trabecular thickness (164.262 ± 18.841 μm; p < 0.001), and bone mineral density (0.824 ± 0.017 g/cm ; p = 0.049) were reduced; trabecular separation (1833.939 ± 178.501 μm; p = 0.005) was increased. While prevalence of osteoclasts was elevated (N.Oc/BS: 0.663 ± 0.102, p < 0.001), osteoblast cell numbers were lower at explantation (N.Ob/BS: 0.149 ± 0.021; p = 0.047). Mean expression of bone homeostasis markers osteocalcin, osteopontin, Runx2, TSG-6, and FGF-2 was significantly reduced at prosthesis explantation. Despite partial recovery, all analyzed parameters were still significantly impacted at reimplantation. In contrast, mean expression of osteoclastogenesis-stimulating cytokine IL-17a was significantly increased at both explantation and reimplantation. In this study, we found a strong and lasting impact of PJI on the bone homeostasis on a molecular, cellular, and microstructural level. These changes may be responsible for the increased risk of prosthesis failure due to aseptic loosening. Our data suggest there is significant potential in modulating bone homeostasis to improve prosthesis fixation and long-term clinical outcome in affected patients. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
PubMed: 37534610
DOI: 10.1002/jbmr.4892 -
Hua Xi Kou Qiang Yi Xue Za Zhi = Huaxi... Aug 2023This study aims to investigate the effects and mechanisms of chondroitin sulfate (CS), dermatan sulfate (DS), and heparin (HEP) on chondrogenesis of murine chondrogenic...
OBJECTIVES
This study aims to investigate the effects and mechanisms of chondroitin sulfate (CS), dermatan sulfate (DS), and heparin (HEP) on chondrogenesis of murine chondrogenic cell line (ATDC5) cells and the maintenance of murine articular cartilage .
METHODS
ATDC5 and articular cartilage tissue explant were cultured in the medium containing different sulfated glycosaminoglycans. Cell proliferation, differentiation, cartilage formation, and mechanism were observed using cell proliferation assay, Alcian blue staining, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blot, respectively.
RESULTS
Results showed that HEP and DS primarily activated the bone morphogenetic protein (BMP) signal pathway, while CS primarily activated the protein kinase B (AKT) signal pathway, further promoted ATDC5 cell proliferation and matrix production, and increased Sox9, Col2a1, and Aggrecan expression.
CONCLUSIONS
This study investigated the differences and mechanisms of different sulfated glycosaminoglycans in chondrogenesis and cartilage homeostasis maintenance. HEP promotes cartilage formation and maintains the normal state of cartilage tissue , while CS plays a more effective role in the regeneration of damaged cartilage tissue.
Topics: Animals; Mice; Cartilage; Cell Differentiation; Cells, Cultured; Chondrocytes; Chondrogenesis; Glycosaminoglycans
PubMed: 37474471
DOI: 10.7518/hxkq.2023.2023055 -
Journal of Inflammation Research 2023Intervertebral disc degeneration (IDD) is a prevalent degenerative disease and often recognized as the primary cause of lower back pain (LBP). Aucubin (Au) is a natural...
BACKGROUND
Intervertebral disc degeneration (IDD) is a prevalent degenerative disease and often recognized as the primary cause of lower back pain (LBP). Aucubin (Au) is a natural compound with anti-inflammatory properties in various diseases. The present study aimed to confirm the therapeutic effect of Au on IDD and explore its potential mechanism in vivo and in vitro.
METHODS
The process of IDD was simulated using the lumbar spine instability (LSI) model. In vivo, the therapeutic effect of Au on LSI-induced mice was evaluated by micro-CT and histomorphometry. Additionally, immunohistochemistry was applied to detect the cartilage metabolism and inflammasome activation in endplate. In vitro, the cytotoxicity of Au on ATDC5 cells was detected by Cell Counting Kit-8 (CCK-8), and the biological effects of Au were evaluated by Quantitative Real-time PCR (qRT-PCR) and Western blotting.
RESULTS
Micro-CT analysis showed that Au administration significantly alleviated LSI-induced disc volume narrowing and endplate cartilage degeneration, which was further supported by Alcian Blue Hematoxylin/Orange G (ABH/OG) staining. Immunohistochemistry results verified that Au could increase the expression of Col2α1 and Aggrecan, reduce the expression of Mmp-13, and attenuate the degradation of the endplate extracellular matrix (ECM). Mechanistically, we found that Au treatment, both in vivo and in vitro, significantly inhibited NF-κB-NLRP3 inflammasome activation in chondrocytes as determined by the decreased expression of p-P65, NLRP3, and Caspase-1.
DISCUSSION
Taken together, our findings have demonstrated for the first time that Au treatment ameliorated the degeneration of cartilage endplates in IDD may by inhibiting NF-κB-NLRP3 inflammasome activation in chondrocytes and provided a potential candidate for the treatment of IDD.
PubMed: 38084106
DOI: 10.2147/JIR.S439981 -
Coupling imaging mass cytometry with Alcian blue histochemical staining for a single-slide approach.Frontiers in Immunology 2024Imaging mass cytometry (IMC) is a metal mass spectrometry-based method allowing highly multiplex immunophenotyping of cells within tissue samples. However, some...
Imaging mass cytometry (IMC) is a metal mass spectrometry-based method allowing highly multiplex immunophenotyping of cells within tissue samples. However, some limitations of IMC are its 1-µm resolution and its time and costs of analysis limiting respectively the detailed histopathological analysis of IMC-produced images and its application to small selected tissue regions of interest (ROI) of one to few square millimeters. Coupling on a single-tissue section, IMC and histopathological analyses could permit a better selection of the ROI for IMC analysis as well as co-analysis of immunophenotyping and histopathological data until the single-cell level. The development of this method is the aim of the present study in which we point to the feasibility of applying the IMC process to tissue sections previously Alcian blue-stained and digitalized before IMC tissue destructive analyses. This method could help to improve the process of IMC in terms of ROI selection, time of analysis, and the confrontation between histopathological and immunophenotypic data of cells.
Topics: Staining and Labeling; Immunophenotyping; Image Cytometry; Humans; Mass Spectrometry; Animals; Single-Cell Analysis
PubMed: 38742102
DOI: 10.3389/fimmu.2024.1379154 -
Biomedicine & Pharmacotherapy =... Jun 2024Vitamin K2 (VK2) is an effective compound for anti-ferroptosis and anti-osteoporosis, and Semen sojae praeparatum (Dandouchi in Chinese) is the main source of VK2....
Vitamin K2 (VK2) is an effective compound for anti-ferroptosis and anti-osteoporosis, and Semen sojae praeparatum (Dandouchi in Chinese) is the main source of VK2. Chondrocyte ferroptosis and extracellular matrix (ECM) degradation playing a role in the pathogenesis of osteoarthritis (OA). Glutathione peroxidase 4 (GPX4) is the intersection of two mechanisms in regulating OA progression. But no studies have elucidated the therapeutic effects and mechanisms of VK2 on OA. This study utilized an in vivo rat OA model created via anterior cruciate ligament transection (ACLT) and an in vitro chondrocyte oxidative damage model induced by TBHP to investigate the protective effects and mechanisms of action of VK2 in OA. Knee joint pain in mice was evaluated using the Von Frey test. Micro-CT and Safranin O-Fast Green staining were employed to observe the extent of damage to the tibial cartilage and subchondral bone, while immunohistochemistry and PCR were used to examine GPX4 levels in joint cartilage. The effects of VK2 on rat chondrocyte viability were assessed using CCK-8 and flow cytometry assays, and chondrocyte morphology was observed with toluidine blue and alcian blue staining. The impact of VK2 on intracellular ferroptosis-related markers was observed using fluorescent staining and flow cytometry. Protein expression changes were detected by immunofluorescence and Western blot analysis. Furthermore, specific protein inhibitors were applied to confirm the dual-regulatory effects of VK2 on GPX4. VK2 can increase bone mass and cartilage thickness in the subchondral bone of the tibia, and reduce pain and the OARSI score induced by OA. Immunohistochemistry results indicate that VK2 exerts its anti-OA effects by regulating GPX4 to delay ECM degradation. VK2 can inhibit the activation of the MAPK/NFκB signaling pathway caused by reduced expression of intracellular GPX4, thereby decreasing ECM degradation. Additionally, VK2 can reverse the inhibitory effect of RSL3 on GPX4, increase intracellular GSH content and the GSH/GSSG ratio, reduce MDA content, and rescue chondrocyte ferroptosis. The protective mechanism of VK2 may involve its dual-target regulation of GPX4, reducing chondrocyte ferroptosis and inhibiting the MAPK/NFκB signaling pathway to decelerate the degradation of the chondrocyte extracellular matrix.
Topics: Animals; Ferroptosis; Extracellular Matrix; Chondrocytes; Male; Osteoarthritis; Rats, Sprague-Dawley; Rats; Phospholipid Hydroperoxide Glutathione Peroxidase; Mice; Vitamin K 2; Mice, Inbred C57BL; Cartilage, Articular; Disease Models, Animal; Signal Transduction; Cells, Cultured
PubMed: 38759289
DOI: 10.1016/j.biopha.2024.116697 -
Current Protocols Jul 2023Much of the skeletal system develops by endochondral ossification, a process that takes place in early fetal life. This makes the early stages of chondrogenesis, i.e.,...
Much of the skeletal system develops by endochondral ossification, a process that takes place in early fetal life. This makes the early stages of chondrogenesis, i.e., when chondroprogenitor mesenchymal cells differentiate to chondroblasts, challenging to study in vivo. In vitro methods for the study of chondrogenic differentiation have been available for some time. There is currently high interest in developing fine-tuned methodology that would allow chondrogenic cells to rebuild articular cartilage and restore joint functionality. The micromass culture system that relies on embryonic limb bud-derived chondroprogenitor cells is a popular method for the study of the signaling pathways that control the formation and maturation of cartilage. In this protocol, we describe a technique fine-tuned in our laboratory for culturing limb bud-derived mesenchymal cells from early-stage chick embryos in high density (Basic Protocol 1). We also provide a fine-tuned method for high-efficiency transient transfection of cells before plating using electroporation (Basic Protocol 2). In addition, protocols for histochemical detection of cartilage extracellular matrix using dimethyl methylene blue, Alcian blue, and safranin O are also provided (Basic Protocol 3 and Alternate Protocols 1 and 2, respectively). Finally, a step-by-step guide on a cell viability/proliferation assay using MTT reagent is also described (Basic Protocol 4). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Micromass culture of chick embryonic limb bud-derived cells Basic Protocol 2: Transfection of cells with siRNA constructs using electroporation prior to micromass culturing Basic Protocol 3: Qualitative and quantitative assessment of cartilage matrix production using dimethyl methylene blue staining and image analysis Alternate Protocol 1: Qualitative assessment of cartilage matrix production using Alcian blue staining Alternate Protocol 2: Qualitative assessment of cartilage matrix production using safranin O staining Basic Protocol 4: Measurement of mitochondrial activity with the MTT assay.
Topics: Animals; Chick Embryo; Chickens; Methylene Blue; Alcian Blue; Cells, Cultured; Cartilage; Regeneration
PubMed: 37427867
DOI: 10.1002/cpz1.835 -
The Laryngoscope Aug 2023Studies showed that photodynamic therapy (PDT) might be able to prevent vocal fold scar formation when treating laryngeal lesions. We aim to investigate if PDT improves...
OBJECTIVE
Studies showed that photodynamic therapy (PDT) might be able to prevent vocal fold scar formation when treating laryngeal lesions. We aim to investigate if PDT improves vocal wound healing and reduces scar formation in both prophylactic and remodeling procedures performed in vivo.
STUDY DESIGN
In vivo.
METHODS
Vocal fold stripping was performed in Sprague-Dawley rats. PDT was performed with intraperitoneal injection of 100 mg/kg 5-Aminolevulinic Acid (5-ALA) and 635 nm laser irradiation of 20, 40, and 60 J/cm . PDT was performed immediately after surgery to study the prophylactic effect and 4 weeks after surgery to study the remodeling effect. Gene expression was evaluated with real-time PCR at 1 week after PDT. Histologic evaluations were performed 12 weeks after PDT, including hematoxylin-eosin, Masson, Alcian blue staining, and immunohistochemical staining of collagen I and III.
RESULTS
PDT induced similar effects on the vocal fold wound healing outcomes in both prophylactic and remodeling procedures. Expression of MMP8, MMP13, HAS2, and TGFβ1 was significantly elevated. Histologic evaluation revealed significantly increased thickness, decreased density of collagen, and increased deposition of hyaluronic acid in the lamina propria. Immunohistochemistry also revealed better distribution and reduced density of collagen I and III. The most obvious changes were seen in the 60 J/cm PDT group.
CONCLUSION
PDT could significantly improve vocal wound healing by providing both prophylactic effects and remodeling effects. It may be a minimally invasive treatment for vocal fold lesions with slight vocal scarring, and may be used to treat acute or chronic vocal injury to reduce vocal scarring.
LEVEL OF EVIDENCE
N/A Laryngoscope, 133:1943-1951, 2023.
Topics: Rats; Animals; Cicatrix; Vocal Cords; Aminolevulinic Acid; Rats, Sprague-Dawley; Wound Healing; Collagen; Collagen Type I; Photochemotherapy
PubMed: 36278803
DOI: 10.1002/lary.30427 -
Diabetes, Metabolic Syndrome and... 2024Obesity is a growing global problem that causes various complications such as diabetes, cognitive dysfunction, cardiovascular diseases, and hepatobiliary disease....
BACKGROUND
Obesity is a growing global problem that causes various complications such as diabetes, cognitive dysfunction, cardiovascular diseases, and hepatobiliary disease. Alpha-linolenic acid (ALA) has been reported to exhibit multiple pharmaceutical effects. This study aimed to explore the effects of ALA on obesity-induced adipose tissue accumulation, cognitive impairment, inflammation, and colonic mucosal barrier integrity.
METHODS
Mice were fed with high-fat diet (HFD) and were treated with ALA (60 or 100 mg/kg). Body weight, adipose tissue, serum glucose and lipid levels, glucose resistance, and insulin resistance were measured. Cognitive ability was analyzed using the behavior tests. PTP1B and IRS/p-AKT/p-GSK3β/p-Tau signaling were examined to evaluate inflammation and synaptogenesis. Colon mucosal barrier integrity was examined by Alcian blue staining and expression of the tight junction proteins. The production of pro-inflammatory cytokines and liver damages were evaluated. 3T3-L1 cells were used for in vitro experiments. Cell viability, migration and invasion were detected. The levels of ROS, iron, and ferrous ions were measured to assess ferroptosis. Metabolomic analysis of adipose tissues was performed.
RESULTS
ALA treatment prevented HFD-induced adipose tissue accumulation, improved glucose and lipid homeostasis and metabolism. Administration of ALA repressed the HFD-induced increase in insulin levels and insulin resistance index. Serum and colon levels of pro-inflammatory cytokines were decreased after ALA treatment. ALA elevated mitochondrial content in brown adipose tissues. ALA ameliorated obesity-induced cognitive impairment and hippocampal inflammation, enhanced colon mucosa integrity. ALA treatment ameliorated HFD-induced liver damage and lipid accumulation and inhibited differentiation of preadipocyte 3T3-L1 cells into mature adipocytes and induces ferroptosis. Metabolomic analysis suggested that ALA may target the glycerolipid metabolism pathway to ameliorate obesity. Knockdown of AGPAT2 abolished the protective effects of ALA.
CONCLUSION
ALA treatment suppressed adipose accumulation in adipocytes, improved cognitive ability and colon integrity, and alleviated liver damage by modulating the 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2).
PubMed: 38435630
DOI: 10.2147/DMSO.S434671