Irisin alleviates obesity-induced bone loss by inhibiting interleukin 6 expression via TLR4/MyD88/NF-κB axis in adipocytes.

INTRODUCTION: Obesity-induced bone loss affects the life quality of patients all over the world. Irisin, one of the myokines, plays an essential role in bone and fat metabolism. OBJECTIVE: Investigate the effects of irisin on bone metabolism via adipocytes in the bone marrow microenvironment. METHODS: In this study, we fed fibronectin type III domain-containing protein 5 (FNDC5, the precursor protein of irisin) knockout mice (FNDC5-/-) with a high-fat diet (HFD) for 10 weeks. The quality of bone mass was assessed by micro-CT analysis, histological staining, and dynamic bone formation. In vitro, the lipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) was assayed by Oil Red O staining, and the osteogenic differentiation was assayed by alkaline phosphatase staining. Meanwhile, the gene expression in the BMSC-differentiated adipocytes by RNA sequence and the involved pathway of irisin were determined by western blot and qRT-PCR were performed. RESULTS: The FNDC5-/- mice fed with a HFD showed an increased body weight, fat content of the bone marrow and bone, and a decreased bone formation compared with those with a standard diet (SD). In vitro, irisin inhibited the differentiation of BMSCs into adipocytes and alleviated the inhibition of osteogenesis derived from BMSCs by the adipocyte supernatant. RNA sequence and blocking experiment showed that irisin reduced the production of interleukin 6 (IL-6) in adipocytes through downregulating the TLR4/MyD88/NF-κB pathway. Immunofluorescence staining of bone marrow further confirmed an increased IL-6 expression in the FNDC5-/- mice fed with HFD compared with those fed with SD, which suffered serious bone loss. CONCLUSION: Irisin downregulates activation of the TLR4/MyD88/NF-κB pathway, thereby reducing IL-6 production in adipocytes to enhance the osteogenesis of BMSCs. Thus, the rescue of osteogenesis of BMSCs, initially inhibited by IL-6, is a potential therapeutic target to mitigate obesity-induced osteoporosis.

Fibromodulin facilitates the osteogenic effect of Masquelet's induced membrane by inhibiting the TGF-ß/SMAD signaling pathway.

Masquelet's induced membrane (IM) technique is a promising treatment strategy for the repair of substantial bone defects. The formation of an IM around polymethylmethacrylate bone cement plays a crucial role in this technique. Several studies have indicated that IMs have bioactivity because they contain abundant blood vessels, a variety of cells, and biological factors. The bioactivity of an IM increases during the initial stages of formation, thereby facilitating bone regeneration and remodeling. Nevertheless, the precise mechanisms underlying the enhancement of IM bioactivity and the promotion of bone regeneration necessitate further investigation. In this study, we successfully developed a Masquelet IM model of critical femur defects in rats. By employing proteomics analysis and biological detection techniques, we identified fibromodulin (FMOD) as a pivotal factor contributing to angiogenesis and the enhanced bioactivity of the IM. A significant increase in angiogenesis and the expression of bioactive factors in the IM was also observed with the upregulation of FMOD expression. Furthermore, this effect is mediated through the inhibition of the transforming growth factor beta (TGF-ß)/SMAD signaling pathway. We also demonstrated that administering recombinant human FMOD enhanced osteogenesis in rat bone marrow mesenchymal stem cells and angiogenesis in human umbilical vein endothelial cells in vitro. Furthermore, the negative regulatory effect of the TGF-ß signaling pathway was verified. In conclusion, this study provides a novel theoretical basis for the application of IMs in bone-defect reconstruction and explores possible new mechanisms that may play an important role in promoting the bioactivity and osteogenic potential of IMs.

Overexpression of ETV2 in BMSCs promoted wound healing in cutaneous wound mice by triggering the differentiation of BMSCs into endothelial cells and modulating the transformation of M1 phenotype macrophages to M2 phenotype macrophages.

This study aimed to investigate the effects of E26-transformation-specific variant-2 (ETV2) overexpression on wound healing in a cutaneous wound (CW) model and clarify associated mechanisms. pLVX-ETV2 lentivirus expressing ETV2 was constructed and infected into BMSCs to generate ETV2-overexpressed BMSCs (BMSCs+pLVX+ETV2). The RT-PCR assay was applied to amplify ETV2, VE-cadherin, vWF, ARG-1, IL-6, iNOS, TGF-ß, IL-10, TNF-α. Western blot was used to determine expression of VE-cadherin and vWF. ETV2 induced differentiation of BMSCs into ECs by increasing CDH5/CD31, triggering tube-like structures, inducing Dil-Ac-LDL positive BMSCs. ETV2 overexpression increased the gene transcription and expression of VE-cadherin and vWF (P<0.01). Transcription of M1 phenotype specific iNOS gene was lower and transcription of M2 phenotype specific ARG-1 gene was higher in the RAW264.7+BMSCs+ETV2 group compared to the RAW264.7+BMSCs+pLVX group (P<0.01). ETV2 overexpression (RAW264.7+BMSCs+ETV2) downregulated IL-6 and TNF-α, and upregulated IL-10 and TGF-ß gene transcription compared to RAW264.7+BMSCs+pLVX group (P<0.01). ETV2-overexpressed BMSCs promoted wound healing in CW mice and triggered the migration of BMSCs to the wound region and macrophage activation. ETV2-overexpressed BMSCs promoted collagen fibers and blood vessel formation in the wound region of CW mice. In conclusion, this study revealed a novel biofunction of ETV2 molecule in the wound healing process. ETV2 overexpression in BMSCs promoted wound healing in CW mice by triggering BMSCs differentiation into endothelial cells and modulating the transformation of M1 pro-inflammatory and M2 anti-inflammatory macrophages in vitro and in vivo.

Explore novel molecular mechanisms of FNDC5 in ischemia-reperfusion (I/R) injury by analyzing transcriptome changes in mouse model of skeletal muscle I/R injury with FNDC5 knockout.

BACKGROUND: Irisin, a myokine derived from proteolytic cleavage of the fibronectin type III domain-containing protein 5 (FNDC5) protein, is crucial in protecting tissues and organs from ischemia-reperfusion (I/R) injury. However, the underlying mechanism of its action remains elusive. In this study, we investigated the expression patterns of genes associated with FNDC5 knockout to gain insights into its molecular functions. METHODS: We employed a mouse model of skeletal muscle I/R injury with FNDC5 knockout to examine the transcriptional profiles using RNA sequencing. Differentially expressed genes (DEGs) were identified and subjected to further analyses, including gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, protein-protein interaction (PPI) network analysis, and miRNA-transcription factor network analysis. The bioinformatics findings were validated using qRT-PCR and Western blotting. RESULTS: Comparative analysis of skeletal muscle transcriptomes between wild-type (WT; C57BL/6), WT-I/R, FNDC5 knockout (KO), and KO-I/R mice highlighted the significance of FNDC5 in both physiological conditions and I/R injury. Through PPI network analysis, we identified seven key genes (Col6a2, Acta2, Col4a5, Fap, Enpep, Mmp11, and Fosl1), which facilitated the construction of a TF-hub genes-miRNA regulatory network. Additionally, our results suggested that the PI3K-Akt pathway is predominantly involved in FNDC5 deletion-mediated I/R injury in skeletal muscle. Animal studies revealed reduced FNDC5 expression in skeletal muscle following I/R injury, and the gastrocnemius muscle with FNDC5 knockout exhibited larger infarct size and more severe tissue damage after I/R. Moreover, Western blot analysis confirmed the upregulation of Col6a2, Enpep, and Mmp11 protein levels following I/R, particularly in the KO-I/R group. Furthermore, FNDC5 deletion inhibited the PI3K-Akt signaling pathway. CONCLUSION: This study demonstrates that FNDC5 deletion exacerbates skeletal muscle I/R injury, potentially involving the upregulation of Col6a2, Enpep, and Mmp11. Additionally, the findings suggest the involvement of the PI3K-Akt pathway in FNDC5 deletion-mediated skeletal muscle I/R injury, providing novel insights into the molecular mechanisms underlying FNDC5's role in this pathological process.

Oxymatrine ameliorates osteoarthritis via the Nrf2/NF-κB axis in vitro and in vivo.

PURPOSE: Osteoarthritis (OA) is a common degenerative joint disorder. Currently, the underlying etiology of OA is still far from fully elucidated and there is no cure for OA progression. Previous studies have demonstrated that oxymatrine (OMT) could inhibit inflammation and oxidative stress in several animal models. However, the potential effects of OMT on OA remain largely elusive. The aim of the study is to investigate the anti-inflammatory and chondrocyte protective effect of OMT, and delineate the potential mechanism in vitro and in vivo. METHODS: Western blotting, RT-PCR, ELISA and tissue staining were employed to explore the mechanisms by which OMT exerted a protective effect on IL-1ß-induced production of pro-inflammation cytokines and extracellular matrix (ECM) degradation in primary murine chondrocytes and DMM mouse models. RESULTS: The results showed that OMT reduced the IL-1ß-induced over-production of pro-inflammation cytokines and ECM degradation. Mechanistically, OMT inhibited the NF-κB pathway via activating Nrf2. In vivo studies also demonstrated that OMT ameliorated OA progression. CONCLUSIONS: OMT reduced pro-inflammation cytokines, ECM degradation and OA progression via activating Nrf2 and inhibiting NF-κB pathway.

Phillygenin inhibits inflammation in chondrocytes via the Nrf2/NF-κB axis and ameliorates osteoarthritis in mice.

Objective: Osteoarthritis (OA), widely seen in the elderly, is featured by cartilage degradation, subchondral bone remolding, and synovium inflammation. Currently, there is no cure for OA development. Phillygenin (PHI), an active ingredient from the Forsythiae Fructus, possesses many biological properties, such as anti-inflammation and anti-oxidative stress in several diseases. However, the potential effects and underlying mechanisms of PHI on OA remain unclear. Methods: Western blotting, RT-PCR, ELISA and tissue staining were employed to explore the mechanisms by which PHI exerted a protective effect on IL-1ß-induced production of pro-inflammation cytokines and extracellular matrix (ECM) degradation in primary murine chondrocytes and destabilization of the medial meniscus (DMM) mouse models. Results: In this study, we found that PHI inhibited the production of pro-inflammation cytokines and ECM degradation induced by IL-1ß in primary murine chondrocytes. Mechanically, PHI inhibited the NF-κB pathway via activating nuclear factor (erythrluteolind-derived 2)-like 2 (Nrf2). In vivo experiments also confirmed the chondroprotection of PHI in DMM mouse models. Conclusion: PHI alleviated IL-1ß-induced inflammation cytokines and ECM degradation via activating Nrf2 and inhibiting NF-κB pathway. The translational potential of this article: This study provides a biological rationale for the use of PHI as a potential candidate for OA treatment.

A novel modified Winograd surgical tricks and tips with a "bird flap" for the treatment of ingrown toenails.

BACKGROUND: The Winograd technique is the most commonly used surgical treatment for ingrown toenails. We describe a novel modified approach, more effective and simpler to perform with a better cosmetic outcome. METHODS: We retrospectively included 45 and 39 patients with 67 and 58 ingrown toenails who underwent our modified Winograd technique and the Winograd technique, respectively, from July 2017 to June 2020, and obtained data after 3, 6, and 12 months of follow-up. RESULTS: No significant differences in the postoperative time taken to return to regular activities in the modified Winograd and traditional Winograd groups (p = 0.103) and regarding the recurrence in both groups (p = 0.055) were found. The extent of proximal germinal matrix exposure with the modified Winograd technique was significantly more clearly revealed than in the traditional Winograd method contextually (p < 0.05). The postoperative appearance satisfaction rate was significantly higher in the modified Winograd group than in the traditional Winograd group (p = 0.029). CONCLUSION: The modified Winograd technique is effective in treating ingrown toenails.

Four-Octyl Itaconate Protects Chondrocytes against H2O2-Induced Oxidative Injury and Attenuates Osteoarthritis Progression by Activating Nrf2 Signaling.

Nrf2 is a critical regulator of the antioxidant defense systems in cellular protection. Emerging evidence has shown that four-octyl itaconate (OI) activates Nrf2 cascade. In this study, the chondroprotective effects of OI on H2O2-stimulated chondrocytes and DMM-induced osteoarthritis (OA) progression were investigated. In primary murine chondrocytes, OI interrupted the binding of Keap1 and Nrf2, leading to accumulation and nuclear translocation of Nrf2 protein, as well as transcription and expression of Nrf2-dependent genes, such as HO-1, NQO1, and GCLC. Furthermore, OI inhibited cell death and apoptosis, as well as H2O2-stimulated ROS generation, lipid peroxidation, superoxide accumulation, and mitochondrial depolarization in chondrocytes, which were abolished by the silence or depletion of Nrf2. In addition, in vivo experiments revealed the therapeutic effects of OI on OA progression in a DMM mouse model. Collectively, these results suggested that OI might serve as a potential treatment for OA progression.

Myokine Irisin promotes osteogenesis by activating BMP/SMAD signaling via αV integrin and regulates bone mass in mice.

Irisin is well-known to contribute to bone homeostasis due to its bidirectional regulation on osteogenesis and osteoclastogenesis. However, the mechanisms of irisin involved in mesenchymal stem/stromal cells (MSCs)-derived osteogenesis are still under investigated. Fibronectin type III domain-containing protein 5 (FNDC5) is the precursor protein of irisin, compare with wild type (WT) littermates, FNDC5-/- mice lost bone mass significantly, collectively evidenced by the decrease of bone mineral density (BMD), impaired bone formation and reduced N-terminal propertied of type I procollagen (P1NP) in sera. Meanwhile, the bone resorbing of FNDC5-/- mice has enhanced accompanied by increased tartrate phosphatase (TRAP) staining cells morphologically and cross-Linked C-telopeptide of type 1 collagen (CTX) level in sera. In vitro study showed that lack of irisin impeded the MSC-derived osteogenesis of FNDC5-/- mice. The addition of irisin promote the osteogenesis of WT and irisin-deficient MSCs, by activating αV integrin-induced ERK/STAT pathway, subsequently enhancing bone morphogenetic protein 2 (BMP2) expression and BMP/SMAD signaling activation. Taken together, these findings further indicate that irisin regulates bone homeostasis. Moreover, irisin promotes MSC-derived osteogenesis by binding to αV integrin and activating BMP/SMAD signaling consequently. Thus, irisin may be a promising therapeutic target for osteoporosis and bone defects.

Hyaluronic acid hydrogel encapsulated BMP-14-modified ADSCs accelerate cartilage defect repair in rabbits.

BACKGROUND: Cartilage defect has a limited capacity to heal. In this context, we hypothesized that hyaluronic acid (HA) hydrogel encapsulated BMP-14-modified adipose-derived mesenchymal stem cells (ADSCs) could accelerate cartilage defect repair in rabbits. METHODS: ADSCs were isolated and identified by flow cytometry. ADSCs were treated with adenovirus vector encoding BMP-14 (Ad-BMP-14) or adenovirus vector encoding control (Ad-ctrl). Real-time PCR (RT-PCR) and western blot assay was performed to verify the transfection efficacy and chondrogenic differentiation markers (ACAN, Collagen II and SOX9). Rabbit cartilage defect model was performed and randomly divided into following groups: control group, HA hydrogel + ADSCs, ADSCs, HA hydrogel + BMP-14 transfected ADSCs, HA hydrogel + BMP-14 transfected ADSCs. At 6, 9 and 12 weeks after surgery, scanning electron microscopy, hematoxylin-eosin, Safranin-O/Fast Green and immunohistochemical staining for Collagen II were performed to determine the role of HA hydrogel encapsulated BMP-14-modified ADSCs in cartilage repair in vivo. RESULTS: ADSCs were successfully isolated and positively expressed CD29, CD44 and CD90. Transfection efficacy of Ad-BMP-14 was verified by RT-PCR and western blot assay. Moreover, Ad-BMP-14 could significantly increased chondrogenic differentiation markers (ACAN, Collagen II and SOX9). The LV-BMP-14-ADSCs and HA hydrogel + LV-BMP-14-ADSCs groups revealed smoother surface cartilage repair that was level with the surrounding cartilage and almost complete border integration. CONCLUSIONS: HA hydrogel encapsulated BMP-14-modified ADSCs accelerate cartilage defect repair in rabbits. We need to further validate the specific mechanism of action of HA hydrogel encapsulated LV-BMP-14-ADSCs involved in the repairing cartilage damage in vivo.

Irisin recouples osteogenesis and osteoclastogenesis to protect wear-particle-induced osteolysis by suppressing oxidative stress and RANKL production.

The disruption of bone homeostasis with the decrease in osteoblastic bone formation and facilitated osteoclastic bone resorption is the leading cause of periprosthetic osteolysis. Accumulative studies have indicated that irisin has the function of maintaining and rebalancing bone homeostasis. In this study, we explored the protective effect of irisin on wear-particle-induced osteolysis in mice. The results showed that irisin effectively inhibited titanium (Ti) particle-induced calvarial osteolysis, supported by a lower bone loss and existence of more collagen, compared with the ones stressed by Ti particles. Further analysis demonstrated that irisin not only rescued Ti-particle-impaired osteogenesis derived from bone mesenchymal stem cells (BMSCs) but also alleviated the increase in wear-particle-induced nuclear factor-κB ligand (RANKL) secreted by BMSCs-derived osteoblasts, which consequently restrained the activation of osteoclasts. Meanwhile, irisin inhibited osteoclastogenesis by the direct inactivation of reactive oxygen species (ROS) signaling. These results revealed that irisin functions to fight against osteolysis caused by wear particles through rebalancing the periprosthetic bone homeostasis microenvironment, which may provide a potential therapeutic strategy for the management of osteolysis and induced prosthetic loosening.