Cross-sectional studies of the causal link between asthma and osteoporosis: insights from Mendelian randomization and bioinformatics analysis.

The study, using data from Chongqing, China, and employing Mendelian randomization along with bioinformatics, establishes a causal link between asthma and osteoporosis, beyond glucocorticoid effects. Asthma may contribute to osteoporosis by accelerating bone turnover through inflammatory factors, disrupting the coupling between osteoblasts and osteoclasts, ultimately leading to osteoporosis. INTRODUCTION: Asthma and osteoporosis are prevalent health conditions with substantial public health implications. However, their potential interplay and the underlying mechanisms have not been fully elucidated. Previous research has primarily focused on the impact of glucocorticoids on osteoporosis, often overlooking the role of asthma itself. METHODS: We conducted a multi-stage stratified random sampling in Chongqing, China and excluded individuals with a history of glucocorticoid use. Participants underwent comprehensive health examinations, and their clinical data, including asthma status, were recorded. Logistic regression and Mendelian randomization were employed to investigate the causal link between asthma and osteoporosis. Furthermore, bioinformatics analyses and serum biomarker assessments were conducted to explore potential mechanistic pathways. RESULTS: We found a significant association between asthma and osteoporosis, suggesting a potential causal link. Mendelian Randomization analysis provided further support for this causal link. Bioinformatics analyses revealed that several molecular pathways might mediate the impact of asthma on bone health. Serum alkaline phosphatase levels were significantly elevated in the asthma group, suggesting potential involvement in bone turnover. CONCLUSION: Our study confirms a causal link between asthma and osteoporosis and highlights the importance of considering asthma in osteoporosis prediction models. It also suggests that asthma may accelerate osteoporosis by increasing bone turnover through inflammatory factors, disrupting the coupling between osteoblasts and osteoclasts, ultimately leading to bone loss.

Loss of Fgfr1 in chondrocytes inhibits osteoarthritis by promoting autophagic activity in temporomandibular joint.

Temporomandibular joint osteoarthritis (TMJ OA) is a common degenerative disease with few effective disease-modifying treatments in the clinic. Fibroblast growth factor (FGF) signaling is implicated in articular cartilage homeostasis, but the functional roles of FGFR1 in TMJ OA remain largely unknown. In this study, we report that deletion of Fgfr1 in TMJ chondrocytes delayed TMJ OA progression in the age-associated spontaneous OA model and the abnormal dental occlusion OA model. Immunohistochemical staining revealed that Fgfr1 deficiency decreased the expressions of MMP13 (matrix metalloproteinase-13), ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5), and COL10A1 but increased aggrecan expression level in two TMJ OA models. Furthermore, our data show that inactivation of FGFR1 signaling may promote autophagic activity in TMJ. FGFR1 inhibitor decreased the expressions of Mmp13, Adamts5, and Runx2 in IL-1ß-stimulated condylar chondrocytes, whereas autophagy inhibitors abrogated the protective effects of the FGFR1 inhibitor. Thus, our study indicates inactivated FGFR1 signaling ameliorates TMJ OA progression partially by promoting autophagic activity. Manipulation of this signaling may be a potential therapeutic approach to modify TMJ OA.

Postnatal deletion of Alk5 gene in meniscal cartilage accelerates age-dependent meniscal degeneration in mice.

Activation of transforming growth factor-ß (TGF-ß) signaling has been used to enhance healing of meniscal degeneration in several models. However, the exact role and molecular mechanism of TGF-ß signaling in meniscus maintenance and degeneration are still not understood due to the absence of in vivo evidence. In this study, we found that the expression of activin receptor-like kinases 5 (ALK5) in the meniscus was decreased with the progression of age and/or osteoarthritis induced meniscal degeneration. Col2α1 positive cells were found to be specifically distributed in the superficial and inner zones of the anterior horn, as well as the inner zone of the posterior horn in mice, indicating that Col2α1-CreERT2 mice can be a used for studying gene function in menisci. Furthermore, we deleted Alk5 in Col2α1 positive cells in meniscus by administering tamoxifen. Alterations in the menisci structure were evaluated histologically. The expression levels of genes and proteins associated with meniscus homeostasis and TGF-ß signaling were analyzed by quantitative real-time PCR analysis (qRT-PCR) and immunohistochemistry (IHC). Our results revealed severe and progressive meniscal degeneration phenotype in 3- and 6-month-old Alk5 cKO mice compared with Cre-negative control, including aberrantly increased hypertrophic meniscal cells, severe fibrillation, and structure disruption of meniscus. qRT-PCR and IHC results showed that disruption of anabolic and catabolic homeostasis of chondrocytes may contribute to the meniscal degeneration phenotype observed in Alk5 cKO mice. Thus, TGF-ß/ALK5 signaling plays a chondro-protective role in menisci homeostasis, in part, by inhibiting matrix degradation and maintaining extracellular matrix proteins levels in meniscal tissues.

FGFR3 Deficiency Causes Multiple Chondroma-like Lesions by Upregulating Hedgehog Signaling.

Most cartilaginous tumors are formed during skeletal development in locations adjacent to growth plates, suggesting that they arise from disordered endochondral bone growth. Fibroblast growth factor receptor (FGFR)3 signaling plays essential roles in this process; however, the role of FGFR3 in cartilaginous tumorigenesis is not known. In this study, we found that postnatal chondrocyte-specific Fgfr3 deletion induced multiple chondroma-like lesions, including enchondromas and osteochondromas, adjacent to disordered growth plates. The lesions showed decreased extracellular signal-regulated kinase (ERK) activity and increased Indian hedgehog (IHH) expression. The same was observed in Fgfr3-deficient primary chondrocytes, in which treatment with a mitogen-activated protein kinase (MEK) inhibitor increased Ihh expression. Importantly, treatment with an inhibitor of IHH signaling reduced the occurrence of chondroma-like lesions in Fgfr3-deficient mice. This is the first study reporting that the loss of Fgfr3 function leads to the formation of chondroma-like lesions via downregulation of MEK/ERK signaling and upregulation of IHH, suggesting that FGFR3 has a tumor suppressor-like function in chondrogenesis.

Anemonin attenuates osteoarthritis progression through inhibiting the activation of IL-1ß/NF-κB pathway.

The osteoarthritis (OA) progression is now considered to be related to inflammation. Anemonin (ANE) is a small natural molecule extracted from various kinds of Chinese traditional herbs and has been shown to inhibiting inflammation response. In this study, we examined whether ANE could attenuate the progression of OA via suppression of IL-1ß/NF-κB pathway activation. Destabilization of the medial meniscus (DMM) was performed in 10-week-old male C57BL/6J mice. ANE was then intra-articularly injected into joint capsule for 8 and 12 weeks. Human articular chondrocytes and cartilage explants challenged with interleukin-1ß (IL-1ß) were treated with ANE. We found that ANE delayed articular cartilage degeneration in vitro and in vivo. In particular, proteoglycan loss and chondrocyte hypertrophy were significantly decreased in ANE -treated mice compared with vehicle-treated mice. ANE decreased the expressions of matrix metalloproteinase-13 (MMP13), A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), collagen X (Col X) while increasing Aggrecan level in murine with DMM surgery. ANE treatment also attenuated proteoglycan loss in human cartilage explants treated with IL-1ß ex vivo. ANE is a potent protective molecule for OA; it delays OA progression by suppressing ECM loss and chondrocyte hypertrophy partially by suppressing IL-1ß/NF-κB pathway activation.

mTOR mutation disrupts larval zebrafish tail fin regeneration via regulating proliferation of blastema cells and mitochondrial functions.

BACKGROUND: The larval zebrafish tail fin can completely regenerate in 3 days post amputation. mTOR, the main regulator of cell growth and metabolism, plays an essential role in regeneration. Lots of studies have documented the role of mTOR in regeneration. However, the mechanisms involved are still not fully elucidated. MATERIALS AND RESULTS: This study aimed to explore the role and mechanism of mTOR in the regeneration of larval zebrafish tail fins. Initially, the spatial and temporal expression of mTOR signaling in the larval fin was examined, revealing its activation following tail fin amputation. Subsequently, a mTOR knockout (mTOR-KO) zebrafish line was created using CRISPR/Cas9 gene editing technology. The investigation demonstrated that mTOR depletion diminished the proliferative capacity of epithelial and mesenchymal cells during fin regeneration, with no discernible impact on cell apoptosis. Insight from SMART-seq analysis uncovered alterations in the cell cycle, mitochondrial functions and metabolic pathways when mTOR signaling was suppressed during fin regeneration. Furthermore, mTOR was confirmed to enhance mitochondrial functions and Ca2 + activation following fin amputation. These findings suggest a potential role for mTOR in promoting mitochondrial fission to facilitate tail fin regeneration. CONCLUSION: In summary, our results demonstrated that mTOR played a key role in larval zebrafish tail fin regeneration, via promoting mitochondrial fission and proliferation of blastema cells.

Lymphatic vessels: roles and potential therapeutic intervention in rheumatoid arthritis and osteoarthritis.

Lymphatic vessel networks are a main part of the vertebrate cardiovascular system, which participate in various physiological and pathological processes via regulation of fluid transport and immunosurveillance. Targeting lymphatic vessels has become a potent strategy for treating various human diseases. The presence of varying degrees of inflammation in joints of rheumatoid arthritis (RA) and osteoarthritis (OA), characterized by heightened infiltration of inflammatory cells, increased levels of inflammatory factors, and activation of inflammatory signaling pathways, significantly contributes to the disruption of cartilage and bone homeostasis in arthritic conditions. Increasing evidence has demonstrated the pivotal role of lymphatic vessels in maintaining joint homeostasis, with their pathological alterations closely associated with the initiation and progression of inflammatory joint diseases. In this review, we provide a comprehensive overview of the evolving knowledge regarding the structural and functional aspects of lymphatic vessels in the pathogenesis of RA and OA. In addition, we summarized the potential regulatory mechanisms underlying the modulation of lymphatic function in maintaining joint homeostasis during inflammatory conditions, and further discuss the distinctions between RA and OA. Moreover, we describe therapeutic strategies for inflammatory arthritis based on lymphatic vessels, including the promotion of lymphangiogenesis, restoration of proper lymphatic vessel function through anti-inflammatory approaches, enhancement of lymphatic contractility and drainage, and alleviation of congestion within the lymphatic system through the elimination of inflammatory cells. At last, we envisage potential research perspectives and strategies to target lymphatic vessels in treating these inflammatory joint diseases.

An Hsp70 promoter-based mouse for heat shock-induced gene modulation.

Physical therapy is extensively employed in clinical settings. Nevertheless, the absence of suitable animal models has resulted in an incomplete understanding of the in vivo mechanisms and cellular distribution that respond to physical stimuli. The objective of this research was to create a mouse model capable of indicating the cells affected by physical stimuli. In this study, we successfully established a mouse line based on the heat shock protein 70 (Hsp70) promoter, wherein the expression of CreERT2 can be induced by physical stimuli. Following stimulation of the mouse tail, ear, or cultured calvarias with heat shock (generated by heating, ultrasound, or laser), a distinct Cre-mediated excision was observed in cells stimulated by these physical factors with minimal occurrence of leaky reporter expression. The application of heat shock to Hsp70-CreERT2; FGFR2-P253R double transgenic mice or Hsp70-CreERT2 mice infected with AAV-BMP4 at calvarias induced the activation of Cre-dependent mutant FGFR2-P253R or BMP4 respectively, thereby facilitating the premature closure of cranial sutures or the repair of calvarial defects. This novel mouse line holds significant potential for investigating the underlying mechanisms of physical therapy, tissue repair and regeneration, lineage tracing, and targeted modulation of gene expression of cells in local tissue stimulated by physical factor at the interested time points. KEY MESSAGES: In the study, an Hsp70-CreERT2 transgenic mouse was generated for heat shock-induced gene modulation. Heat shock, ultrasound, and laser stimulation effectively activated Cre expression in Hsp70-CreERT2; reporter mice, which leads to deletion of floxed DNA sequence in the tail, ear, and cultured calvaria tissues of mice. Local laser stimuli on cultured calvarias effectively induce Fgfr2-P253R expression in Hsp70-mTmG-Fgfr2-P253R mice and result in accelerated premature closure of cranial suture. Heat shock activated AAV9-FLEX-BMP4 expression and subsequently promoted the repair of calvarial defect of Hsp70-CreERT2; Rosa26-mTmG mice.

A Novel Cargo Delivery System-AnCar-ExoLaIMTS Ameliorates Arthritis via Specifically Targeting Pro-Inflammatory Macrophages.

Macrophages are heterogenic phagocytic cells that play distinct roles in physiological and pathological processes. Targeting different types of macrophages has shown potent therapeutic effects in many diseases. Although many approaches are developed to target anti-inflammatory macrophages, there are few researches on targeting pro-inflammatory macrophages, which is partially attributed to their non-s pecificity phagocytosis of extracellular substances. In this study, a novel recombinant protein is constructed that can be anchored on an exosome membrane with the purpose of targeting pro-inflammatory macrophages via antigen recognition, which is named AnCar-ExoLaIMTS . The data indicate that the phagocytosis efficiencies of pro-inflammatory macrophages for different AnCar-ExoLaIMTS show obvious differences. The AnCar-ExoLaIMTS3 has the best targeting ability for pro-inflammatory macrophages in vitro and in vivo. Mechanically, AnCar-ExoLaIMTS3 can specifically recognize the leucine-rich repeat domain of the TLR4 receptor, and then enter into pro-inflammatory macrophages via the TLR4-mediated receptor endocytosis pathway. Moreover, AnCar-ExoLaIMTS3 can efficiently deliver therapeutic cargo to pro-inflammatory macrophages and inhibit the synovial inflammatory response via downregulation of HIF-1α level, thus ameliorating the severity of arthritis in vivo. Collectively, the work established a novel gene/drug delivery system that can specifically target pro-inflammatory macrophages, which may be beneficial for the treatments of arthritis and other inflammatory diseases.

Genetic inhibition of fibroblast growth factor receptor 1 in knee cartilage attenuates the degeneration of articular cartilage in adult mice.

OBJECTIVE: Fibroblast growth factor (FGF) family members are involved in the regulation of articular cartilage homeostasis. The aim of this study was to investigate the function of FGF receptor 1 (FGFR-1) in the development of osteoarthritis (OA) and its underlying mechanisms. METHODS: FGFR-1 was deleted from the articular chondrocytes of adult mice in a cartilage-specific and tamoxifen-inducible manner. Two OA models (aging-associated spontaneous OA, and destabilization-induced OA), as well as an antigen-induced arthritis (AIA) model, were established and tested in Fgfr1-deficient and wild-type (WT) mice. Alterations in cartilage structure and the loss of proteoglycan were assessed in the knee joints of mice of either genotype, using these 3 arthritis models. Primary chondrocytes were isolated and the expression of key regulatory molecules was assessed quantitatively. In addition, the effect of an FGFR-1 inhibitor on human articular chondrocytes was examined. RESULTS: The gross morphologic features of Fgfr1-deficient mice were comparable with those of WT mice at both the postnatal and adult stages. The articular cartilage of 12-month-old Fgfr1-deficient mice displayed greater aggrecan staining compared to 12-month-old WT mice. Fgfr1 deficiency conferred resistance to the proteoglycan loss induced by AIA and attenuated the development of cartilage destruction after surgically induced destabilization of the knee joint. The chondroprotective effect of FGFR-1 inhibition was largely associated with decreased expression of matrix metalloproteinase 13 (MMP-13) and up-regulation of FGFR-3 in mouse and human articular chondrocytes. CONCLUSION: Disruption of FGFR-1 in adult mouse articular chondrocytes inhibits the progression of cartilage degeneration. Down-regulation of MMP-13 expression and up-regulation of FGFR-3 levels may contribute to the phenotypic changes observed in Fgfr1-deficient mice.

Dexmedetomidine Suppresses Glutamate-Stimulated Neuronal Injury Via MicroRNA-433/Janus Kinase 2/Signal Transducer and Activator of Transcription 3 Pathway.

Background: Cerebral ischemia has been a hotpot in the prevention and treatment of cerebral ischemia. Dexmedetomidine (Dex) is a new type of highly selective α2 adrenergic receptor agonist with pharmacological properties. Objective: Quantitative studies have shown that Dex has a protective effect on glutamate (Glu)-induced neuronal damage. however, its mechanism has not been fully elucidated. The purpose of this study was to explore the underlying molecular mechanism by which Dex ameliorates Glu-induced neuronal injury by regulating miR-433/JAK2/STAT3 axis. Materials and Methods: A model of neuronal injury was constructed by Glu treatment and intervened with Dex. miRNA expression profiling assay was conducted to screen potential miRNAs affected by Dex. Cell viability, lactate dehydrogenase (LDH) release and apoptosis were detected by MTT assay, LDH kit, and TUNEL staining, respectively. Oxidative stress indicators were assessed by ELISA whereas mitochondrial membrane potential (MMP) was assessed by C11-BODIPY581/591 staining. The targeting relationship between the miR-433 and JAK2 was verified by dual-luciferase reporter assay and gene expression was analyzed by quantitative PCR and Western blot. Results: Glu treatment decreased cell viability and MMP and promoted LDH release, apoptosis and oxidative damage. Glu-induced changes in neurons were reversed after Dex treatment through upregulating the miR-433 expression to block the activation of JAK2/STAT3 pathway. Conclusions: Dex protects against Glu-induced neuronal injury by regulating miR-433/JAK2/STAT3 pathway, which provides new insights into the treatment of neuronal injury.

Effects of different over-fired drying methods on the aroma of Lu'an Guapian tea.

Over-fired drying, a crucial process in the production of Lu'an Guapian (LAGP) tea, greatly enriches the tea's aroma. In this study, the aroma compounds of LAGP tea processed through pulley charcoal drying (PCD), roller drying (RD), roller-conveyor drying (RCD), and hot air drying (HD) were analyzed using gas chromatography-mass spectrometry. A subsequent analysis of aroma extraction dilution analysis and odor activity values revealed that (E)-ß-ionone, dimethyl sulfide, (E,E)-2,4-heptadienal, geraniol, linalool, benzeneacetaldehyde, coumarin, 2-ethyl-3,5-dimethyl-pyrazine, indole, hexanal, (Z)-jasmone, and (Z)-3-hexen-1-ol were the key contributors to the samples' aroma variation. Moreover, a quantitative descriptive analysis and aroma recombination and omission experiments analysis revealed that (E)-ß-ionone is the most critical contributor to the formation of floral aroma in tea processed using PCD, whereas (E,E)-2,4-heptadienal is responsible for the more pronounced fresh aroma in tea processed using HD. In addition, 2-ethyl-3,5-dimethyl-pyrazine contributes to the formation of a roasted aroma in tea processed using RD and RCD. The study results provide a theoretical basis for choosing the processing method, especially for drying, to obtain high-quality LAGP tea.

Sevoflurane Suppresses the Growth, Metastasis, and Invasion of Endometrial Carcinoma Cells via miR-195-5p/JAK2 Axis.

BACKGROUND: Highly invasive and destructive endometrioma is one of the most familiar primary malignant tumors among women. It has been studied that sevoflurane can influence the development of various malignancies. But whatever sevoflurane could influence endometrial tumors is unknown. MATERIALS AND METHODS: Through CCK8 and transwell analysis, we investigated the influence of sevoflurane on the development of endometrial tumors in vitro. Then, we studied the function of miRNA-195-5p to promote sevoflurane to inhibit the development of endometrial tumors. Then, we predicted the target genes of miRNA-195-5p by online software and focused on JAK2. Through luciferase assay, we proved the direct binding and regulation of miRNA-195-5p to JAK2. RESULTS: We showed that sevoflurane could inhibit the growth, metastasis, and invasion of endometrial tumors via miRNA-195-5p/JAK2 axis. CONCLUSIONS: Our research shows the function of sevoflurane in inhibiting the development of endometrial tumors via miRNA-195-5p/JAK2 axis. Our findings proved that sevoflurane is potentially beneficial for endometrial carcinoma patients with surgery and may be helpful for the choice of anesthetics in endometrial carcinoma operations.

Improving the flavor of summer green tea (Camellia sinensis L.) using the yellowing process.

Summer green tea (SGT) has poor flavor due to its high levels of bitterness and astringency. The present study aimed to improve the flavor of SGT using the yellowing process. The results showed that after the yellowing process, the sweetness and overall acceptability increased, and the content of gallated catechins and flavonol glycosides decreased by 30.2% and 27.4%, respectively, as did the bitterness and astringency of SGT. Yellowing caused a decrease in the concentration of some aroma compounds, such as (z)-3-hexen-1-ol, 1-hexanol, pentanal, heptanal and 1-octanol, which caused grassy, floral and fruity aromas. In contrast, the concentrations of 1-octen-3-ol, benzene acetaldehyde and ß-ionone increased, which have mushroom and sweet aromas. Meanwhile, the sweetness and umami of SGT were enhanced by the addition of selected aroma compounds (1-octen-3-ol, benzene acetaldehyde and ß-ionone), demonstrating that the yellowing process improves the flavor of SGT through odor-taste interactions.

Association rules analysis on patterns of multimorbidity in adults: based on the National Health and Nutrition Examination Surveys database.

OBJECTIVE: To explore the prevalence and patterns of multimorbidity in population with different genders and age ranges. DESIGN: A cross-sectional study. SETTING: National Health and Nutrition Examination Surveys database. PARTICIPANTS: 12 576 patients. PRIMARY AND SECONDARY OUTCOME MEASURES: The prevalence and patterns of multimorbidity. RESULTS: High cholesterol had the highest prevalence in all population (33.4 (95% CI: 32.0 to 34.9)) and males. In females <65 years, the most prevalent disease was sleep disorder (32.1 (95% CI: 29.6 to 34.5)) while in females ≥65 years, hypertension was the most prevalent disease (63.9 (95% CI: 59.9 to 67.9)). Hypertension and high cholesterol were associated with the highest support (occur together most frequently) in all population regardless of genders. Hypertension displayed the highest betweenness centrality (mediating role in the network) followed by high cholesterol and arthritis in all population. For males aged <65 years, hypertension and high cholesterol presented the highest betweenness centrality. In males ≥65 years, hypertension, high cholesterol and arthritis were the top three diseases of degree centrality (direct association with other conditions). As for females ≥65 years, hypertension showed the highest betweenness centrality followed by high cholesterol and arthritis. The associations of hypertension, arthritis and one other item with high cholesterol presented the highest support in all population. In males, the associations of depression, hypertension with sleep disorders had the highest lift (the chance of co-occurrence of the conditions and significant association). Among females, the associations of depression, arthritis with sleep disorders had the highest lift. CONCLUSION: Hypertension and high cholesterol were prevalent in all population, regardless of females and males. Hypertension and high cholesterol, arthritis and hypertension, and diabetes and hypertension were more likely to coexist. The findings of this study might help make plans for the management and primary care of people with one or more diseases.

Rapid identification of the green tea geographical origin and processing month based on near-infrared hyperspectral imaging combined with chemometrics.

The geographical origin and processing month of green tea greatly affect its economic value and consumer acceptance. This study investigated the feasibility of combining near-infrared hyperspectral imaging (NIR-HSI) with chemometrics for the identification of green tea. Tea samples produced in three regions of Chongqing (southeastern Chongqing, northeastern Chongqing, and western Chongqing) for four months (from May to August 2020) were collected. Principal component analysis (PCA) was used to reduce data dimensionality and visualize the clustering of samples in different categories. Linear partial least squares-discriminant analysis (PLS-DA) and nonlinear support vector machine (SVM) algorithms were used to develop discriminant models. The PCA-SVM models based on the first four and first five principal components (PCs) achieved the best accuracies of 97.5% and 95% in the prediction set for geographical origin and processing month of green tea, respectively. This study demonstrated the feasibility of HSI in the identification of green tea species, providing a rapid and nondestructive method for the evaluation and control of green tea quality.

Exogenous Indian hedgehog antagonist damages intervertebral discs homeostasis in adult mice.

Background: Vismodegib, as an exogenous Indian hedgehog (Ihh) antagonist, has been approved by the Food and Drug Administration (FDA) for the clinical treatment of patients with basal cell carcinoma, and previous observations implicate the potential therapeutic of vismodegib in osteoarthritis treatment. However, there is no direct evidence for the role of Ihh signaling in intervertebral discs (IVDs) homeostasis of adult mice. The aim of the present study is to assess the effect of systemic administration of Smoothened inhibitor (SMOi) - vismodegib on IVDs homeostasis during the adult stage. Methods: The expression of glioma-associated oncogene homolog 1 (Gli1), the downstream targeting gene of Ihh signaling, in IVDs of adult mice after receiving systemic administration of SMOi was examined by immunohistochemistry. The pathological changes of vertebral bodies after SMOi treatment were evaluated by X-ray and micro-CT. The effects of SMOi on homeostasis of IVDs including cartilaginous endplates (CEP), growth plates (GP) and annulus fibrous (AF) were evaluated by histological analysis. The expressions of Aggrecan, Matrix metalloproteinase 13 (MMP13) and Runt-related transcription factor 2 (Runx2), in IVDs were also investigated by immunohistochemistry. Changes in chondrocyte apoptosis and proliferation in IVDs were evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and analyzing the expression of the cell proliferation antigen Ki-67. Results: Systemic administration of SMOi significantly decreased the expression of Gli1 in IVDs that indicating effective inhibition of Ihh signaling. Bone mass of vertebral bodies was diminished after SMOi treatment. Moreover, IVDs degeneration (IDD) like defects including CEP sclerosis, degenerative nucleus pulposus (NP) and fissure within AF, as well as narrowed or fused GP and loss bone mass of vertebral bodies was observed in SMOi-treated mice. The severity of IDD was time-dependent with the administration of SMOi treatment after 2-8 weeks. The expressions of Aggrecan, MMP13 and Runx2 in IVDs of mice receiving SMOi treatment were significantly decreased. In addition, chondrocyte apoptosis was significantly enhanced, while chondrocyte proliferation was significantly inhibited. Conclusions: Our study propose that systemic administration of vismodegib damages IVDs homeostasis via inhibition of Ihh signaling in adult mice. The clinical application of Ihh signaling antagonists such as vismodegib should be careful considering these side adverse. The Translational Potential of this Article: Vismodegib as an exogenous antagonist of Ihh signaling has been approved by the FDA for the clinical treatment of patients with basal cell carcinoma. However, it is still unknown whether vismodegib will has adverse effects on the patient or animal model of IVDs cartilage homeostasis. Based on our study, systemic administration of vismodegib damages IVDs homeostasis via inhibition of Ihh signaling in adult mice and special attention should be paid to the clinical application of vismodegib.

Expansion of FGFR3-positive nucleus pulposus cells plays important roles in postnatal nucleus pulposus growth and regeneration.

BACKGROUND: Intervertebral disc degeneration (IVDD) can cause low back pain, a major public health concern. IVDD is characterized with loss of cells especially those in nucleus pulposus (NP), due to the limited proliferative potential and regenerative ability. Few studies, however, have been carried out to investigate the in vivo proliferation events of NP cells and the cellular contribution of a specific subpopulation of NP during postnatal growth or regeneration. METHODS: We generated FGFR3-3*Flag-IRES-GFP mice and crossed FGFR3-CreERT2 mice with Rosa26-mTmG, Rosa26-DTA and Rosa26-Confetti mice, respectively, to perform inducible genetic tracing studies. RESULTS: Expression of FGFR3 was found in the outer region of NP with co-localized expressions of proliferating markers. By fate mapping studies, FGFR3-positive (FGFR3+) NP cells were found proliferate from outer region to inner region of NP during postnatal growth. Clonal lineage tracing by Confetti mice and ablation of FGFR3·+ NP cells by DTA mice further revealed that the expansion of the FGFR3+ cells was required for the morphogenesis and homeostasis of postnatal NP. Moreover, in degeneration and regeneration model of mouse intervertebral disc, FGFR3+ NP cells underwent extensive expansion during the recovery stage. CONCLUSION: Our present work demonstrates that FGFR3+ NP cells are novel subpopulation of postnatal NP with long-existing proliferative capacity shaping the adult NP structure and participating in the homeostasis maintenance and intrinsic repair of NP. These findings may facilitate the development of new therapeutic approaches for IVD regeneration.

Inhibition of aberrant Hif1α activation delays intervertebral disc degeneration in adult mice.

The intervertebral disc (IVD) is the largest avascular tissue. Hypoxia-inducible factors (HIFs) play essential roles in regulating cellular adaptation in the IVD under physiological conditions. Disc degeneration disease (DDD) is one of the leading causes of disability, and current therapies are ineffective. This study sought to explore the role of HIFs in DDD pathogenesis in mice. The findings of this study showed that among HIF family members, Hif1α was significantly upregulated in cartilaginous endplate (EP) and annulus fibrosus (AF) tissues from human DDD patients and two mouse models of DDD compared with controls. Conditional deletion of the E3 ubiquitin ligase Vhl in EP and AF tissues of adult mice resulted in upregulated Hif1α expression and age-dependent IVD degeneration. Aberrant Hif1α activation enhanced glycolytic metabolism and suppressed mitochondrial function. On the other hand, genetic ablation of the Hif1α gene delayed DDD pathogenesis in Vhl-deficient mice. Administration of 2-methoxyestradiol (2ME2), a selective Hif1α inhibitor, attenuated experimental IVD degeneration in mice. The findings of this study show that aberrant Hif1α activation in EP and AF tissues induces pathological changes in DDD, implying that inhibition of aberrant Hif1α activity is a potential therapeutic strategy for DDD.

Low-intensity pulsed ultrasound inhibits fibroblast-like synoviocyte proliferation and reduces synovial fibrosis by regulating Wnt/ß-catenin signaling.

OBJECTIVE: Synovial fibrosis is a characteristic symptom of osteoarthritis (OA), which is closely associated with joint pain and stiffness. Previous studies have reported that low-intensity pulsed ultrasound (LIPUS) can alleviate cartilage degradation in OA. However, the functions and mechanisms of LIPUS in OA synovial fibrosis are still unknown. METHODS: The destabilization of the medial meniscus (DMM) mouse model of OA was established in C57 male mice and fibroblast-like synoviocytes (FLS) were isolated from synovial tissue of OA patients. The knee joint diameter, Masson's trichrome (MT) and Hematoxylin-eosin (HE) staining were used to evaluate synovial fibrosis and hyperplasia. The Immunohistochemistry (IHC) staining was performed to detected the expression of synovial fibrosis makers and the activation of Wnt/ß-catenin signaling in vivo. FLS were treated with TGF-ß1 to serve as an in vitro model of synovial fibrosis, Wnt3a was used to activate the Wnt/ß-catenin signaling in cells. Cell proliferation was detected by using EdU assay, cell viability was performed by CCK8 assay. The protein levels of α-SMA, CTGF, Col Ⅰ, ß-catenin, active ß-catenin, c-Myc and cyclin D1 were examined by western blot and immunofluorescence staining. RESULTS: Two weeks after the LIPUS treatment, the synovial fibrosis, synovial hyperplasia and synoviocyte proliferation in the DMM model were significantly decreased. In vitro, LIPUS directly inhibited the TGF-ß1-induced fibrotic response and proliferation of FLS. Meanwhile, LIPUS suppressed Wnt/ß-catenin signaling in the synovium of DMM mice and cultured FLS. More importantly, we found that the synovial fibrosis makers, Wnt/ß-catenin pathway downstream proteins and FLS proliferation were significantly decreased in Wnt3a-stimulated FLS following LIPUS treatment. CONCLUSIONS: Our results present a novel role of LIPUS in OA-related synovial fibrosis, which is associated with its ability to repress Wnt/ß-catenin signaling in FLS. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This study provides new insight into the clinical application of LIPUS as a therapeutic option to manage synovial fibrosis in OA.