Idiopathic pulmonary fibrosis(IPF) is a chronic progressive interstitial lung disease characterized by a complex pathogenesis and limited treatment options. Although studies have indicated that lipid metabolism dysregulation is associated with the progression of IPF, the core regulatory mechanisms remain unclear. By integrating RNA sequencing data from the GEO database, we identified four key genes related to lipid metabolism: peroxisome proliferator-activated receptor gamma(PPARG), secreted phosphoprotein 1(SPP1), caspase 3(CASP3), and platelet endothelial cell adhesion molecule 1(PECAM1). Further validation using single-cell RNA sequencing revealed the cell-specific expression patterns of these genes. The results found that PPARG was significantly downregulated in alveolar macrophages while SPP1 was significantly upregulated. Mechanistic studies indicated that PPARG negatively regulated SPP1 expression, and the interaction between SPP1 and cluster of differentiation 44(CD44) activated intercellular signaling pathways that promoted fibrosis. Through network pharmacology and molecular docking, it was predicted that the bioactive components of the traditional Chinese medicine formula, namely Buyang Huanwu Decoction may target PPARG to modulate lipid metabolism pathways. In a bleomycin-induced rat model with IPF, this paper randomly divided the rats into six groups(control, group, model group, pirfenidone group, and low, middle, and high-dose groups of Buyang Huanwu Decoction). The results demonstrated that Buyang Huanwu Decoction treatment significantly improved tissue pathological damage, reduced collagen deposition, and alleviated lipid metabolism dysregulation. Western blot analysis confirmed that Buyang Huanwu Decoction mediated the upregulation of PPARG and inhibited the activation of the SPP1/CD44 pathway. The multi-omics study elucidated the role of the PPARG/SPP1/CD44 pathway as a key regulatory factor in lipid metabolism in IPF, providing evidence that Buyang Huanwu Decoction exerted its antifibrotic effects through this novel mechanism and thus offering new insights into the therapeutic prospects for IPF.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Signal Transduction/drug effects* ; PPAR gamma/genetics* ; Humans ; Osteopontin/genetics* ; Lipid Metabolism/drug effects* ; Idiopathic Pulmonary Fibrosis/genetics* ; Hyaluronan Receptors/genetics* ; Rats ; Male ; Rats, Sprague-Dawley ; Molecular Docking Simulation

OBJECTIVE: To investigate the effect of stretch on long non-coding RNA taurine upregulated gene 1 (TUG1)-mediated miR-545-3p/cannbinoida receptor 2 (CNR2) pathway regulating bone regeneration in the distraction area of rats during distraction osteogenesis. METHODS: Thirty-six 10-week-old male Sprague Dawley rats were randomly divided into 3 groups ( n=12 in each group): group A (femoral fracture+injection of interfering RNA), group B (distraction osteogenesis+injection of interfering RNA), and group C (distraction osteogenesis+injection of TUG1). Groups A and B were injected with 60 μg of interfering RNA at the beginning of incubation period (immediate after operation), the beginning of distraction phase (7 days after operation), and the end of distraction phase (21 days after operation), and group C was injected with 60 μg of synthetic TUG1 in vivo interfering sequence at the same time. The general situation of rats in each group was observed during the experiment. The mineralization of fracture space or distraction area was observed by X-ray films at 21, 35, and 49 days after operation. At 49 days after operation, the samples of the distraction area were taken for HE staining to observe the mineralization, and real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expressions of osteoblast-related genes such as TUG1, miR-545-3p, CNR2, alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Blood samples were collected from the abdominal aorta of the rats, and the expressions of ALP and C terminal telopeptide of type Ⅰ (CTX-Ⅰ) protein were detected by ELISA assay. RESULTS: The results of X-ray film and HE staining observations showed that osteogenesis in group C was superior to groups A and B at the same time point. The results of qRT-PCR showed that the relative mRNA expressions of TUG1, CNR2, ALP, OCN, and OPN in group C were significantly higher than those in group A and group B, and the relative mRNA expression of miR-545-3p in group C was significantly lower than that in group A and group B ( P<0.05). The relative mRNA expressions of TUG1 and ALP in group B were significantly higher than those in group A, and the relative mRNA expression of miR-545-3p in group B was significantly lower than that in group A ( P<0.05). There was no significant difference in the relative mRNA expressions of CNR2, OCN, and OPN between group A and group B ( P>0.05). The results of ELISA showed that the expressions of ALP and CTX-Ⅰ protein were significantly higher in group C than in group A and group B, and in group B than in group A ( P<0.05). CONCLUSION Under the action of stretch, the expression of TUG1 in the femoral distraction area of rats increases, which promotes the expression of CNR2 by inhibiting the expression of miR-545-3P, which is helpful to the mineralization of the extension area and osteogenesis.
Animals ; MicroRNAs/genetics* ; Rats, Sprague-Dawley ; Male ; Osteogenesis, Distraction/methods* ; Rats ; RNA, Long Noncoding/metabolism* ; Osteopontin/genetics* ; Osteogenesis ; Bone Regeneration ; RNA, Small Interfering/genetics* ; Osteocalcin/genetics* ; Alkaline Phosphatase/metabolism* ; Osteoblasts/cytology* ; Signal Transduction ; Femoral Fractures/surgery*

BACKGROUND: Congenital scoliosis (CS) is a complex spinal malformation of unknown etiology with abnormal bone metabolism. Fibroblast growth factor 23 (FGF23), secreted by osteoblasts and osteocytes, can inhibit bone formation and mineralization. This research aims to investigate the relationship between CS and FGF23. METHODS: We collected peripheral blood from two pairs of identical twins for methylation sequencing of the target region. FGF23 mRNA levels in the peripheral blood of CS patients and age-matched controls were measured. Receiver operator characteristic (ROC) curve analyses were conducted to evaluate the specificity and sensitivity of FGF23. The expression levels of FGF23 and its downstream factors fibroblast growth factor receptor 3 (FGFr3)/tissue non-specific alkaline phosphatase (TNAP)/osteopontin (OPN) in primary osteoblasts from CS patients (CS-Ob) and controls (CT-Ob) were detected. In addition, the osteogenic abilities of FGF23-knockdown or FGF23-overexpressing Ob were examined. RESULTS: DNA methylation of the FGF23 gene in CS patients was decreased compared to that of their identical twins, accompanied by increased mRNA levels. CS patients had increased peripheral blood FGF23 mRNA levels and decreased computed tomography (CT) values compared with controls. The FGF23 mRNA levels were negatively correlated with the CT value of the spine, and ROCs of FGF23 mRNA levels showed high sensitivity and specificity for CS. Additionally, significantly increased levels of FGF23, FGFr3, OPN, impaired osteogenic mineralization and lower TNAP levels were observed in CS-Ob. Moreover, FGF23 overexpression in CT-Ob increased FGFr3 and OPN levels and decreased TNAP levels, while FGF23 knockdown induced downregulation of FGFr3 and OPN but upregulation of TNAP in CS-Ob. Mineralization of CS-Ob was rescued after FGF23 knockdown. CONCLUSIONS Our results suggested increased peripheral blood FGF23 levels, decreased bone mineral density in CS patients, and a good predictive ability of CS by peripheral blood FGF23 levels. FGF23 may contribute to osteopenia in CS patients through FGFr3/TNAP / OPN pathway.
Humans ; Osteopontin/genetics* ; Alkaline Phosphatase/metabolism* ; Receptor, Fibroblast Growth Factor, Type 3/metabolism* ; Scoliosis/genetics* ; Osteoblasts/metabolism* ; Calcinosis ; RNA, Messenger/metabolism* ; Bone Diseases, Metabolic/metabolism* ; Fibroblast Growth Factors/genetics*

BACKGROUND: Although osteopontin (OPN) is expressed in the liver and pigment gallstones of patients with hepatolithiasis, its role in pigment gallstone formation remains unclear. This study aimed to explore the function of OPN in pigment gallstone formation. METHODS: Rats were fed a chow diet (CD) or lithogenic diet (LD) for 10 consecutive weeks; blocking tests were then performed using an OPN antibody (OPN-Ab). Incidence of gallstones and levels of several bile components, OPN, tumor necrosis factor alpha (TNF-α), and cholesterol 7 alpha-hydroxylase (CYP7A1) were analyzed. To determine TNF-α expression in hepatic macrophages and both CYP7A1 and bile acid (BA) expression in liver cells, recombinant rat OPN and recombinant rat TNF-α were used to treat rat hepatic macrophages and rat liver cells, respectively. Chi-square or Fisher exact tests were used to analyze qualitative data, Student t-test or one-way analysis of variance were used to analyze qualitative data. RESULTS: Incidence of gallstones was higher in LD-fed rats than in CD-fed rats (80% vs. 10%, P < 0.05). BA content significantly decreased in bile (t = -36.08, P < 0.01) and liver tissue (t = -16.16, P < 0.01) of LD-fed rats. Both hepatic OPN protein expression (t = 9.78, P < 0.01) and TNF-α level (t = 8.83, P < 0.01) distinctly increased in the LD group; what's more, CYP7A1 mRNA and protein levels (t = -12.35, P < 0.01) were markedly down-regulated in the LD group. Following OPN-Ab pretreatment, gallstone formation decreased (85% vs. 25%, χ2 = 14.55, P < 0.01), liver TNF-α expression (F = 20.36, P < 0.01) was down-regulated in the LD group, and CYP7A1 expression (F = 17.51, P < 0.01) was up-regulated. Through CD44 and integrin receptors, OPN promoted TNF-α production in macrophage (F = 1041, P < 0.01), which suppressed CYP7A1 expression (F = 48.08, P < 0.01) and reduced liver BA synthesis (F = 119.4, P < 0.01). CONCLUSIONS We provide novel evidence of OPN involvement in pigmented gallstone pathogenesis in rats.
Animals ; Diet/adverse effects* ; Gallstones/etiology* ; Lithiasis ; Liver ; Liver Diseases ; Osteopontin/genetics* ; Rats

Objective: To explore the association between serum levels of osteopontin (OPN) and systolic pulmonary artery pressure (sPAP) in healthy men following acute high altitude exposure. Methods: According to the inclusion and exclusion criteria, this observational study included 94 male subjects (aged from 18 to 30 years, dwelling in lowland<500 m) who ascended to Litang (4 100 m) from Chongqing (400 m) by bus with a stair-like journey within 7 days in June 2013. Data including basic information, OPN, superoxide dismutase (SOD), and malondialdehyde (MDA) and echocardiographic derived sPAP were collected within 48 hours before ascent and within 2-7 hours after arrival. Accordingly, subjects were divided into 3 groups based on the tertiles of sPAP after acute high altitude exposure: low sPAP group (26.8-32.3 mmHg (1 mmHg=0.133 kPa)) (n=31), middle sPAP group (32.4-37.4 mmHg) (n=32) and high sPAP group (37.5-55.6 mmHg) (n=31). Associations of serum OPN and SOD levels with sPAP were analysed by univariate and multivariate linear regression analysis. Results: After acute high altitude exposure, the levels of sPAP were significantly increased (P<0.001). There were no differences in age, height, weight, body mass index, percent of Han nationality and smoking among 3 subgroups. However, following acute high altitude exposure, the levels of heart rate, systolic and diastolic blood pressure elevated (all P<0.05), whereas the levels of oxygen saturation were reduced in the total subjects and all subgroups (all P<0.05). Moreover, systolic blood pressure of subjects in the high sPAP group was higher than that in low and middle sPAP groups (both P<0.05), and diastolic blood pressure of subjects in high sPAP group was higher than that in low sPAP group (P<0.05). The serum levels of OPN were increased in total cohort(27.9 (22.5,34.0) μg/L vs. 25.6 (18.4, 33.1) μg/L, P<0.05)， and high sPAP group (P<0.05), whereas no differences were found in serum SOD and MDA levels among groups. Furthermore, the serum level of OPN in high sPAP group was higher than that in low sPAP group at high altitude (P<0.05), and there was a trend for decline in SOD level with increasing sPAP (P>0.05). Results from univariable linear regression analysis showed that the serum levels of OPN (r=0.32, P=0.002) and SOD (r=-0.22，P=0.032) were linearly correlated with sPAP in total cohort after high altitude exposure. Multivariate regression analysis showed that the serum levels of OPN(β=0.310，P=0.002) and SOD (β=-0.199，P=0.043) were independently associated with the levels of sPAP at high altitude. Conclusion: After acute high altitude exposure, the serum level of OPN is positively associated with sPAP, suggesting that OPN may be a novel bio-marker for predicting the increase of pulmonary pressure in response to acute high altitude exposure.
Adolescent ; Adult ; Altitude ; Blood Pressure Determination ; Humans ; Male ; Osteopontin ; Pulmonary Artery ; Systole ; Young Adult

Biomarkers for hepatocellular carcinoma (HCC) following curative resection are not currently sufficient for prognostic indication of overall survival (OS) and disease-free survival (DFS). The aim of this study was to investigate the prognostic performance of osteopontin (OPN), matrix metalloproteinase 7 (MMP7), and pregnancy specific glycoprotein 9 (PSG9) in patients with HCC. A total of 179 prospective patients with HCC provided plasma before hepatectomy. Plasma OPN, MMP7, and PSG9 levels were determined by enzyme-linked immunosorbent assay. Correlations between plasma levels, clinical parameters, and outcomes (OS and DFS) were overall analyzed. High OPN ( ⩾ 149.97 ng/mL), MMP7 ( ⩾ 2.28 ng/mL), and PSG9 ( ⩾ 45.59 ng/mL) were prognostic indicators of reduced OS (P < 0.001, P < 0.001, and P = 0.007, respectively). Plasma PSG9 protein level was an independent factor in predicting OS (P = 0.008) and DFS (P = 0.038). Plasma OPN + MMP7 + PSG9 elevation in combination was a prognostic factor for OS (P < 0.001). OPN was demonstrated to be a risk factorassociated OS in stage I patients with HCC and patients with low α-fetoprotein levels ( < 20 ng/mL). These findings suggested that OPN, MMP7, PSG9 and their combined panels may be useful for aiding in tumor recurrence and mortality risk prediction of patients with HCC, particularly in the early stage of HCC carcinogenesis.
Adult ; Aged ; Biomarkers, Tumor ; blood ; Carcinoma, Hepatocellular ; blood ; mortality ; Enzyme-Linked Immunosorbent Assay ; Female ; Hepatectomy ; Humans ; Liver Neoplasms ; blood ; mortality ; Male ; Matrix Metalloproteinase 7 ; blood ; Middle Aged ; Osteopontin ; blood ; Pregnancy-Specific beta 1-Glycoproteins ; analysis ; Prognosis ; Prospective Studies ; Risk Assessment ; Survival Analysis

BACKGROUND: Bioprinting has recently appeared as a powerful tool for building complex tissue and organ structures. However, the application of bioprinting to regenerative medicine has limitations, due to the restricted choices of bio-ink for cytocompatible cell encapsulation and the integrity of the fabricated structures. METHODS: In this study, we developed hybrid bio-inks based on acrylated hyaluronic acid (HA) for immobilizing bioactive peptides and tyramine-conjugated hyaluronic acids for fast gelation. RESULTS: Conventional acrylated HA-based hydrogels have a gelation time of more than 30 min, whereas hybrid bioink has been rapidly gelated within 200 s. Fibroblast cells cultured in this hybrid bio-ink up to 7 days showed < 90% viability. As a guidance cue for stem cell differentiation, we immobilized four different bio-active peptides: BMP-7-derived peptides (BMP-7D) and osteopontin for osteogenesis, and substance-P (SP) and Ac-SDKP (SDKP) for angiogenesis. Mesenchymal stem cells cultured in these hybrid bio-inks showed the highest angiogenic and osteogenic activity cultured in bio-ink immobilized with a SP or BMP-7D peptide. This bio-ink was loaded in a three-dimensional (3D) bioprinting device showing reproducible printing features. CONCLUSION: We have developed bio-inks that combine biochemical and mechanical cues. Biochemical cues were able to regulate differentiation of cells, and mechanical cues enabled printing structuring. This multi-functional bio-ink can be used for complex tissue engineering and regenerative medicine.
Bioprinting ; Cues ; Fibroblasts ; Hyaluronic Acid ; Hydrogel ; Hydrogels ; Mesenchymal Stromal Cells ; Osteogenesis ; Osteopontin ; Peptides ; Regeneration* ; Regenerative Medicine ; Stem Cells ; Tissue Engineering

PURPOSE: The purpose of this paper was to determine the ability of a mixture consisting of mesenchymal stem cells, beta-tricalcium phosphate β-TCP), and hydrogel, to support cells and form new tissue. MATERIALS AND METHODS: A composite was produced by adding β-TCP to hydrogel, and mesenchymal stem cells were cultivated in the composite. Then, reverse transcription polymerase chain reaction (RT-PCR) was conducted to measure the level of gene expression for the new bone formation in the cells. Moreover, a composite in which the mesenchymal stem cells were added was injected into the subcutaneous fat of sprague-dawley rats. After four weeks, H&E, Masson trichrome, silver nitrate staining, and osterix immunohistochemical staining were conducted by taking the tissue to evaluate whether the composite supported mesenchymal stem cells and formed new tissue. RESULTS: By using RT-PCR, we found that the level of gene expression became significantly higher in 3-dimensional gel culture with RUNX2 by 1.26 times, with osteopontin by 1.23 times, transforming growth factor-β by 2.12 times, osterix by 1.07 times, type I collagen by 1.3 times, and fibronectin by 1.3 times. In the animal experiment in which a composite was transplanted into the subcutaneous fat, newly formed tissue was observed. Also, it was found that the composite prevented mesenchymal stem cells from leaving and formed new tissue. Osteogenic differentiation cells in the tissue was observed through osterix immunostaining. CONCLUSION: It was identified that the composite prevented mesenchymal stem cells dispersal and contributed to the formation of neogenic tissue. Therefore we conclude that the composite plays a role of a scaffold to support the implanted cells and form neogenic tissue more effectively.
Animal Experimentation ; Collagen Type I ; Fibronectins ; Gene Expression ; Hydrogel ; Mesenchymal Stromal Cells* ; Osteogenesis* ; Osteopontin ; Polymerase Chain Reaction ; Rats, Sprague-Dawley ; Reverse Transcription ; Silver Staining ; Subcutaneous Fat

Osteopontin (OPN) is a phosphorylated glycoprotein secreted into body fluids by various cell types. OPN contains arginine-glycine-aspartate (RGD) and serine-leucine-alanine-tyrosine (SLAY) motifs that bind to several integrins and mediate a wide range of cellular processes. In the present study, the proangiogenic effects of a 20-amino-acid OPN peptide (OPNpt20) containing RGD and SLAY motifs were examined in human umbilical vein endothelial cells (HUVECs) and in a rat focal cerebral ischemia model. OPNpt20 exerted robust proangiogenic effects in HUVECs by promoting proliferation, migration and tube formation. These effects were significantly reduced in OPNpt20-RAA (RGD->RAA)-treated cells, but only slightly reduced in OPNpt20-SLAA (SLAY->SLAA)-treated cells. Interestingly, a mutant peptide without both motifs failed to induce these proangiogenic processes, indicating that the RGD motif is crucial and that SLAY also has a role. In OPNpt20-treated HUVEC cultures, AKT and ERK signaling pathways were activated, but activation of these pathways and tube formation were suppressed by anti-αvβ3 antibody, indicating that OPNpt20 stimulates angiogenesis via the αvβ3-integrin/AKT and ERK pathways. The proangiogenic function of OPNpt20 was further confirmed in a rat middle cerebral artery occlusion model. Total vessel length and vessel densities were markedly greater in OPNpt20-treated ischemic brains, accompanied by induction of proangiogenic markers. Together, these results demonstrate that the 20-amino-acid OPN peptide containing RGD and SLAY motifs exerts proangiogenic effects, wherein both motifs have important roles, and these effects appear to contribute to the neuroprotective effects of this peptide in the postischemic brain.
Animals ; Body Fluids ; Brain Ischemia ; Brain ; Glycoproteins ; Human Umbilical Vein Endothelial Cells ; Infarction, Middle Cerebral Artery ; Integrins ; MAP Kinase Signaling System ; Neuroprotective Agents ; Osteopontin ; Rats

Bone defect occurs as a consequence of many conditions. Diseased bones don’t heal properly and defects in face area need proper bone reconstruction to avoid psychological and social problems. Tissue engineering is an emerging new modality of treatment. We thought to study different methods to fill skull bone defect in rats in order to find the most safe and effective method. So, this study was designed to evaluate the efficacy of acellular dermal graft (ADM) versus propylene mesh both either loaded or unloaded with bone marrow derived mesenchymal stem cells (BM-MSCs) in healing of skull bone defect of a 5 mm diameter. The study included 36 adult male Wistar albino rats that were divided into three groups according to the way of filling skull bone defect. Group I: Ia (sham control), Ib (negative control). Group II: IIa (unseeded propylene), IIb (seeded propylene) and Group III: IIIa (unseeded ADM), IIIb (seeded ADM). The trephine operation was done on the left parietal bone. Specimens were collected four weeks postoperative and processed for H&E, osteopontin immunohistochemistry and scanning electron microscope. Morphometric and statistical analysis were also performed. After studying the results of the experiment, we found that propylene mesh and ADM were suitable scaffolds that could support new bone formation in clavarial bone defect. Healing of skull bone defect was better in rats that received seeded scaffolds more than rats with unseeded scaffolds. The seeded ADM showed significant increase in bone forming activity as confirmed by histomorphometric and statistical results.
Adult ; Animals ; Bone Marrow ; Humans ; Immunohistochemistry ; Male ; Mesenchymal Stromal Cells ; Methods ; Osteogenesis ; Osteopontin ; Parietal Bone ; Rats ; Skull ; Social Problems ; Tissue Engineering ; Transplants