Fibroblasts inhibit osteogenesis by regulating nuclear-cytoplasmic shuttling of YAP in mesenchymal stem cells and secreting DKK1.

BACKGROUND: Fibrous scars frequently form at the sites of bone nonunion when attempts to repair bone fractures have failed. However, the detailed mechanism by which fibroblasts, which are the main components of fibrous scars, impede osteogenesis remains largely unknown. RESULTS: In this study, we found that fibroblasts compete with osteogenesis in both human bone nonunion tissues and BMP2-induced ectopic osteogenesis in a mouse model. Fibroblasts could inhibit the osteoblastic differentiation of mesenchymal stem cells (MSCs) via direct and indirect cell competition. During this process, fibroblasts modulated the nuclear-cytoplasmic shuttling of YAP in MSCs. Knocking down YAP could inhibit osteoblast differentiation of MSCs, while overexpression of nuclear-localized YAP-5SA could reverse the inhibition of osteoblast differentiation of MSCs caused by fibroblasts. Furthermore, fibroblasts secreted DKK1, which further inhibited the formation of calcium nodules during the late stage of osteogenesis but did not affect the early stage of osteogenesis. Thus, fibroblasts could inhibit osteogenesis by regulating YAP localization in MSCs and secreting DKK1. CONCLUSIONS: Our research revealed that fibroblasts could modulate the nuclear-cytoplasmic shuttling of YAP in MSCs, thereby inhibiting their osteoblast differentiation. Fibroblasts could also secrete DKK1, which inhibited calcium nodule formation at the late stage of osteogenesis.

Fibroblasts inhibit osteogenesis by regulating nuclear-cytoplasmic shuttling of YAP in mesenchymal stem cells and secreting DKK1

BACKGROUND: Fibrous scars frequently form at the sites of bone nonunion when attempts to repair bone fractures have failed. However, the detailed mechanism by which fibroblasts, which are the main components of fibrous scars, impede osteogenesis remains largely unknown. RESULTS: In this study, we found that fibroblasts compete with osteogenesis in both human bone nonunion tissues and BMP2-induced ectopic osteogenesis in a mouse model. Fibroblasts could inhibit the osteoblastic differentiation of mesenchymal stem cells (MSCs) via direct and indirect cell competition. During this process, fibroblasts modulated the nuclear-cytoplasmic shuttling of YAP in MSCs. Knocking down YAP could inhibit osteoblast differentiation of MSCs, while overexpression of nuclear-localized YAP-5SA could reverse the inhibition of osteoblast differentiation of MSCs caused by fibroblasts. Furthermore, fibroblasts secreted DKK1, which further inhibited the formation of calcium nodules during the late stage of osteogenesis but did not affect the early stage of osteogenesis. Thus, fibroblasts could inhibit osteogenesis by regulating YAP localization in MSCs and secreting DKK1. CONCLUSIONS: Our research revealed that fibroblasts could modulate the nuclear-cytoplasmic shuttling of YAP in MSCs, thereby inhibiting their osteoblast differentiation. Fibroblasts could also secrete DKK1, which inhibited calcium nodule formation at the late stage of osteogenesis.

Contrast-enhanced magnetic resonance image segmentation based on improved U-Net and Inception-ResNet in the diagnosis of spinal metastases.

Objective: The objective of this study was to investigate the use of contrast-enhanced magnetic resonance imaging (CE-MRI) combined with radiomics and deep learning technology for the identification of spinal metastases and primary malignant spinal bone tumor. Methods: The region growing algorithm was utilized to segment the lesions, and two parameters were defined based on the region of interest (ROI). Deep learning algorithms were employed: improved U-Net, which utilized CE-MRI parameter maps as input, and used 10 layers of CE images as input. Inception-ResNet model was used to extract relevant features for disease identification and construct a diagnosis classifier. Results: The diagnostic accuracy of radiomics was 0.74, while the average diagnostic accuracy of improved U-Net was 0.98, respectively. the PA of our model is as high as 98.001%. The findings indicate that CE-MRI based radiomics and deep learning have the potential to assist in the differential diagnosis of spinal metastases and primary malignant spinal bone tumor. Conclusion: CE-MRI combined with radiomics and deep learning technology can potentially assist in the differential diagnosis of spinal metastases and primary malignant spinal bone tumor, providing a promising approach for clinical diagnosis.

High-concentrate diet elevates histone lactylation mediated by p300/CBP through the upregulation of lactic acid and induces an inflammatory response in mammary gland of dairy cows.

High-concentrate diet can cause metabolic diseases, such as subacute ruminal acidosis (SARA), and secondary mastitis. To investigate the effect of SARA induced by high-concentrate diet on the lysine lactylation (Kla) and inflammatory responses in the mammary gland of dairy cows and the mechanism between them, we selected twelve mid-lactation Holstein cows with similar body conditions for modelling. They were randomly divided into two groups, fed a low-concentrate diet (LC) and a high-concentrate diet (HC) for 21 days. Our results showed that high-concentrate diet feeding significantly reduced ruminal pH, and the pH was below 5.6 for more than 3 h per day, indicating successful induction of the SARA model. Lactic acid concentrations in mammary gland and plasma were higher in the HC group than that in the LC group. HC diet feeding significantly up-regulated the expression levels of the Pan Kla, H3K18la, p300/CBP and monocarboxylate transporter 1 (MCT1) in the mammary gland. In addition, the mRNA expression levels of inflammatory factors were significantly regulated, including IL-1ß, IL-1α, IL-6, IL-8, SAA3, and TNF-α, while the anti-inflammatory factor IL-10 was down-regulated. The mammary gland of HC group was structurally disorganized with incomplete glandular vesicles, with a large number of detached mammary epithelial cells and inflammatory cells infiltration. The up-regulation of TLR4, TNF-α, p-p65, and p-IκBα indicated that the TLR4/NF-κB signaling pathway was activated. In conclusion, this study found that HC diet feeding can induce SARA and increase the concentration of lactic acid in mammary gland and plasma. Then, lactic acid could be transported into cells by MCT1 and up-regulate the expression level of histone lactylation mediated by p300/CBP, and subsequently promote the activation of TLR4/NF-κB signaling pathway, ultimately causing inflammatory responses in the mammary gland.

Synergistic Effect of QNZ, an Inhibitor of NF-κB Signaling, and Bone Morphogenetic Protein 2 on Osteogenic Differentiation in Mesenchymal Stem Cells through Fibroblast-Induced Yes-Associated Protein Activation.

Biomaterials carrying recombinant human bone morphogenetic protein 2 (BMP2) have been developed to enhance bone regeneration in the treatment of bone defects. However, various reports have shown that in the bone repair microenvironment, fibroblasts can inhibit BMP2-induced osteogenic differentiation in mesenchymal stem cells (MSCs). Thus, factors that can target fibroblasts and improve BMP2-mediated osteogenesis should be explored. In this project, we focused on whether or not an inhibitor of the NF-κB signaling pathway, QNZ (EVP4593), could play a synergistic role with BMP2 in osteogenesis by regulating the activity of fibroblasts. The roles of QNZ in regulating the proliferation and migration of fibroblasts were examined. In addition, the effect of QNZ combined with BMP2 on the osteogenic differentiation of MSCs was evaluated both in vitro and in vivo. Furthermore, the detailed mechanisms by which QNZ improved BMP2-mediated osteogenesis through the modulation of fibroblasts were analyzed and revealed. Interestingly, we found that QNZ inhibited the proliferation and migration of fibroblasts. Thus, QNZ could relieve the inhibitory effects of fibroblasts on the homing and osteogenic differentiation of mesenchymal stem cells. Furthermore, biomaterials carrying both QNZ and BMP2 showed better osteoinductivity than did those carrying BMP2 alone both in vitro and in vivo. It was found that the mechanism of QNZ involved reactivating YAP activity in mesenchymal stem cells, which was inhibited by fibroblasts. Taken together, our results suggest that QNZ may be a candidate factor for assisting BMP2 in inducing osteogenesis. The combined application of QNZ and BMP2 in biomaterials may be promising for the treatment of bone defects in the future.

Protective Effects of N-Acetylcysteine on Lipopolysaccharide-Induced Respiratory Inflammation and Oxidative Stress.

As the leading cause of bovine respiratory disease (BRD), bacterial pneumonia can result in tremendous losses in the herd farming industry worldwide. N-acetylcysteine (NAC), an acetylated precursor of the amino acid L-cysteine, has been reported to have anti-inflammatory and antioxidant properties. To explore the protective effect and underlying mechanisms of NAC in ALI, we investigated its role in lipopolysaccharide (LPS)-induced bovine embryo tracheal cells (EBTr) and mouse lung injury models. We found that NAC pretreatment attenuated LPS-induced inflammation in EBTr and mouse models. Moreover, LPS suppressed the expression of oxidative-related factors in EBTr and promoted gene expression and the secretion of inflammatory cytokines. Conversely, the pretreatment of NAC alleviated the secretion of inflammatory cytokines and decreased their mRNA levels, maintaining stable levels of antioxidative gene expression. In vivo, NAC helped LPS-induced inflammatory responses and lung injury in ALI mice. The relative protein concentration, total cells, and percentage of neutrophils in BALF; the level of secretion of IL-6, IL-8, TNF-α, and IL-1ß; MPO activity; lung injury score; and the expression level of inflammatory-related genes were decreased significantly in the NAC group compared with the LPS group. NAC also ameliorated LPS-induced mRNA level changes in antioxidative genes. In conclusion, our findings suggest that NAC affects the inflammatory and oxidative response, alleviating LPS-induced EBTr inflammation and mouse lung injury, which offers a natural therapeutic strategy for BRD.

Sodium butyrate promotes lipopolysaccharide-induced innate immune responses by enhancing mitogen-activated protein kinase activation and histone acetylation in bovine mammary epithelial cells.

The innate immune response plays a crucial role in recovery from infectious diseases by promoting the clearance of pathogens. Sodium butyrate (NaB) is an energy source for cellular processes with the potential to regulate the innate immune response. The present study aimed to evaluate the effect of NaB on the innate immune response in a bovine mammary alveolar cell line (MAC-T) initiated by lipopolysaccharides (LPS). Thus, treatments were conducted as follows: treated with 1× PBS for 24 h (control), pretreated with 1 mM NaB (optimized by cell viability assays and dose-dependent experiment) for 18 h followed by treatment of 1× PBS for 6 h (NaB), pretreated with 1× PBS for 18 h followed by stimulation with LPS (1 µg/mL) for 6 h (LPS), and pretreated with 1 mM NaB for 18 h followed by stimulation with LPS (1 µg/mL) for 6 h (NaB + LPS). Different inhibitors were also used to elucidate the underlying mechanism. Furthermore, cells were treated with NaB and heat-inactivated Escherichia coli to test the effect of NaB on transcription of genes related to the innate immune response triggered by the major causative pathogen of mastitis. Each treatment had 3 replicates and was repeated 3 times. Proinflammatory cytokines, chemokines, and ß-defensins are crucial secretion factors in innate immunity, and transcription of these factors was increased by NaB during challenge with LPS or heat-inactivated E. coli in MAC-T cells. Acetylation of histone H3 protein, which promotes gene expression by affecting the structure of chromatin, was also upregulated by NaB in response to LPS stimulation. P38 mitogen-activated protein kinases (MAPK), JNK, and Erk 1 and 2 are key upstream regulators of the expression of proinflammatory cytokines, chemokines, and ß-defensins, and their activity was enhanced by NaB during LPS stimulation. Furthermore, inhibitors were used to assess the role of MAPK signaling in the effects of NaB. The results showed that inhibitors of p38 MAPK, Erk, and JNK attenuated the NaB-induced upregulation of TNF and ß-defensin 5 (DEFB5) transcription, and that the inhibitor of Erk attenuated the NaB-induced upregulation of IL1B transcription during LPS challenge. Enhanced transcription of CXCL8 by NaB was blocked by the inhibitor of Erk and p38 MAPK during LPS stimulation. Overall, NaB boosted the LPS-induced innate immune response by promoting the expression of proinflammatory cytokines, chemokines, and ß-defensins, which was associated with enhanced MAPK signaling activation and histone H3 acetylation.

Dihydromyricetin protects against lipopolysaccharide­induced cardiomyocyte injury through the toll­like receptor­4/nuclear factor­κB pathway.

Dihydromyricetin (DHM) is a bioactive flavonoid compound extracted from the stems and leaves of Ampelopsis grossedentata. Previous studies have indicated that DHM has antioxidation and antitumor capabilities, while the effect of DHM on lipopolysaccharide (LPS)­induced cardiomyocyte injury has not been clarified. Therefore, the aim of the present study was to investigate the effect of DHM on LPS­induced cardiomyocyte injury. In the present study, cardiomyocytes were randomized to the control (PBS), LPS and DHM + LPS groups. The LPS group was treated with 10 µg/ml LPS for 12 h and the DHM + LPS group was treated with 100 or 25 µM DHM 12 h prior to treatment with LPS. The protective effect of DHM on LPS­induced cardiomyocytes injury was evaluated by Cell Counting kit­8 assay, TUNEL staining, reverse transcription­quantitative polymerase chain reaction and western blot analysis. The results demonstrated that LPS treatment led to cardiomyocyte apoptosis, and these effects were significantly attenuated by DHM. Furthermore, LPS treatment downregulated the expression of B­cell lymphoma 2 apoptosis regulator (Bcl­2) and induced increased expression of Bcl­2­associated X apoptosis regulator (Bax). Additionally, DHM treatment reversed LPS­induced changes in Bcl­2 expression and Bax expression in cardiomyocytes, and rescued cells from apoptosis. In addition, increased mRNA expression levels of tumor necrosis factor­α and interleukin­6 induced by LPS were attenuated following treatment with DHM. Further investigation demonstrated that DHM suppressed the activation of toll­like receptor­4 (TLR4), which is involved in regulating the downstream signaling pathway of nuclear factor­κB (NF­κB). DHM attenuated LPS­induced cardiomyocyte injury, the potential mechanism responsible for this effect may involve inhibition of TLR4 activation and subsequent regulation of the associated downstream signaling pathway of NF­κB. The current study indicates that DHM may protect cardiomyocytes against LPS­induced injury by inhibition of the TLR4/NF­κB signaling pathway. The results of the present study may provide support for the development DHM as a strategy for the treatment of heart failure in septic shock.

Grape seed proanthocyanidin extract attenuates airway inflammation and hyperresponsiveness in a murine model of asthma by downregulating inducible nitric oxide synthase.

Allergic asthma is characterized by hyperresponsiveness and inflammation of the airway with increased expression of inducible nitric oxide synthase (iNOS) and overproduction of nitric oxide (NO). Grape seed proanthocyanidin extract (GSPE) has been proved to have antioxidant, antitumor, anti-inflammatory, and other pharmacological effects. The purpose of this study was to examine the role of GSPE on airway inflammation and hyperresponsiveness in a mouse model of allergic asthma. BALB/c mice, sensitized and challenged with ovalbumin (OVA), were intraperitoneally injected with GSPE. Administration of GSPE remarkably suppressed airway resistance and reduced the total inflammatory cell and eosinophil counts in BALF. Treatment with GSPE significantly enhanced the interferon (IFN)- γ level and decreased interleukin (IL)-4 and IL-13 levels in BALF and total IgE levels in serum. GSPE also attenuated allergen-induced lung eosinophilic inflammation and mucus-producing goblet cells in the airway. The elevated iNOS expression observed in the OVA mice was significantly inhibited by GSPE. In conclusion, GSPE decreases the progression of airway inflammation and hyperresponsiveness by downregulating the iNOS expression, promising to have a potential in the treatment of allergic asthma.