COL1A1 Expression Research Articles

A combined analysis of bulk RNA-seq and scRNA-seq was performed to investigate the molecular mechanisms associated with the occurrence of myocardial infarction

BackgroundMyocardial infarction (MI) induces complex transcriptional changes across diverse cardiac cell types. Single-cell RNA sequencing (scRNA-seq) provides an unparalleled ability to discern cellular diversity during infarction, yet the veracity of these discoveries necessitates confirmation. This investigation sought to elucidate MI mechanisms by integrating scRNA-seq and bulk RNA-seq data.MethodsPublicly available scRNA-seq (GSE136088) and bulk RNA-seq (GSE153485) data from mice MI models were analyzed. Cell types were annotated, and differential expression analysis conducted. Bulk RNA-seq underwent quality control, principal component analysis, and differential expression analysis.ResultsIn scRNA-seq data, the comparison between MI and sham groups unveiled a reduction in endothelial cell populations, but macrophages and monocytes increased. Within fibroblast subgroups, three distinct categories were discerned, with two exhibiting upregulation in MI. Notably, endothelial cells exhibited an elevated expression of genes associated with apoptosis and ferroptosis. In bulk RNA-seq analysis, distinct patterns emerged when comparing MI and sham groups. Specifically, six genes linked to endothelial ferroptosis exhibited heightened expression in MI group, thereby corroborating the scRNA-seq findings. Moreover, the examination of isolated cardiac macrophages from mice MI model revealed increased expression of Spp1, Col1a2, Col3a1, Ctsd, and Lgals3 compared to sham group, thus substantiating the dysregulation of macrophage apoptosis-related proteins following MI.ConclusionMI altered the transcriptomic landscapes of cardiac cells with increased expression of apoptotic genes. Moreover, the upregulation of macrophage apoptosis marker was confirmed within MI models. The presence of endothelial cell depletion and ferroptosis in MI has been demonstrated.

Scopolamine mitigates oophorectomy-induced osteoporosis: potential mechanisms and direct effects on bone structure

The aim of our research is to investigate the therapeutic effects of scopolamine (SCO) on osteoporosis and to explore the underlying mechanism. To study osteoporosis, we established an ovariectomy (OVX) model. The rats were divided into four groups: sham operation, OVX, OVX+SCO, and OVX+SCO+ML385 (Nrf2 inhibitor). ELISA, Realtime PCR, Western blot, and kits were utilised to assess the expression of related proteins and substances. The OVX rats exhibited significant weight gain, reduced bone volume, destruction of trabecular and cortical bone microstructure, decreased expression of ALP, OCN, OPN, COL1A1, Runx2, Nrf2 proteins, and CAT, SOD, GST, GPX levels while increased expression of TRAP protein and ROS levels. SCO was able to restore these indices in OVX rats, while ML385 blocked the effects of SCO. In conclusion, SCO inhibits oxidative stress response to exert therapeutic effects on osteoporosis by activating the Nrf2 signalling pathway.

Suramin enhances proliferation, migration, and tendon gene expression of human supraspinatus tenocytes.

Rotator cuff tendinopathy is a common musculoskeletal disorder with limited pharmacological treatment strategies. This study aimed to investigate tenocytes' functional in vitro response from a ruptured supraspinatus tendon to suramin administration and to elucidate whether suramin can enhance tendon repair and modulate the inflammatory response to injury. Tenocytes were obtained from human supraspinatus tendons (n = 6). We investigated the effect of suramin on LPS-induced inflammatory responses and the underlying molecular mechanisms in THP-1 macrophages. Suramin enhanced the proliferation, cell viability, and migration of tenocytes. It also increased the protein expression of PCNA and Ki-67. Suramin-treated tenocytes exhibited increased expression of COL1A1, COL3A1, TNC, SCX, and VEGF. Suramin significantly reduced LPS-induced iNOS, COX2 synthesis, inflammatory cytokine TNF-α production, and inflammatory signaling by influencing the NF-κB pathways in THP-1 cells. Our results suggest that suramin holds great promise as a therapeutic option for treating rotator cuff tendinopathy.

High expression of COL8A1 predicts poor prognosis and promotes EMT in papillary thyroid cancer.

Collagen type VIII α 1 chain (COL8A1), a collagen type VIII protein, has been suggested to exert various functions in progression of multiple cancers. However, the effect of COL8A1 in papillary thyroid cancer (PTC) has not been elucidated. The Cancer Genome Atlas (TCGA) databases were applied to investigate the COL8A1 expression and its clinical significance in PTC. The COL8A1 expression level was further validated using Gene Expression Omnibus (GEO) data and clinical paired PTC tissues. Additionally, Kaplan-Meier curve was used to analyze the prognosis. The cell migrative and invasive abilities were evaluated by wound healing assay and Transwell assay. CCK8 assays were used to evaluate proliferation of PTC cells. Western blotting was conducted to explore the potential mechanisms involved in the pro-tumor role of COL8A1. The correlation between immune cell infiltration and COL8A1 was analyzed using Tumor Immune Estimation Resource (TIMER) database and the single-sample GSEA (ssGSEA) method. We found that COL8A1 was upregulated in PTC (P<0.05). High COL8A1 expression level was significantly associated with advanced T stage (P<0.01), N stage (P<0.001) and poor prognosis (P=0.0142) in PTC. Furthermore, cell migration and invasion were significantly reduced following COL8A1 knockdown (P<0.001). Mechanistic studies demonstrated that the epithelial-to-mesenchymal transition (EMT) related proteins (FN1, MMP9, MMP7, ZEB2 and Twist1) and phosphorylation of AKT and ERK were obviously down-regulated after COL8A1 knockdown (P<0.01). Moreover, COL8A1 expression was correlated with immune cell infiltration. Our study demonstrates that COL8A1 may function as an oncogene and a potential prognostic biomarker for PTC patients.

Upregulation of MFAP5 Enhances COL1A1 Expression, Promoting Epithelial-Mesenchymal Transition in Gastric Cancer Cells.

Gastric cancer (GC) is one of the most common types of cancer. Earlier research has suggested an association of microfibril-associated protein 5 (MFAP5) and collagen type I alpha 1 (COL1A1) with the progression of various tumors. However, the specific roles and mechanisms of action of MFAP5 and COL1A1 in the context of GC are yet to be fully elucidated. Thus, the objective of this study is to investigate the functions of MFAP5 and COL1A1 in the epithelial-mesenchymal transition (EMT) of GC and to unravel the associated molecular mechanisms. We examined the MFAP5 expression level in GC through real-time polymerase chain reaction (RT-PCR), western blotting, and immunohistochemistry. Subsequently, shRNA interference was employed to knockdown the expression of MFAP5 or COL1A1 in GC cells. Cell viability assay, Transwell assay, RT-PCR, and western blotting were then used to explore the impact of MFAP5 and COL1A1 on GC progression and metastasis, along with GC cell proliferation, migration, and EMT. Increased MFAP5 levels were observed in both GC tissues and cells (p < 0.05), with decreased MFAP5 levels significantly impeding GC cell activity and GC progression and metastasis (p < 0.05). Additionally, the pronounced reduction in the COL1A1 expression level effectively alleviated the migration and EMT processes induced by MFAP5 overexpression in GC cells (p < 0.05). These results indicate that MFAP5 plays a role in initiating the process of EMT in GC cells through the upregulation of COL1A1 expression.

Rosmarinic acid promotes cartilage regeneration through Sox9 induction via NF-κB pathway inhibition in mouse osteoarthritis progression

BackgroundThe natural polyphenolic compound known as Rosmarinic acid (RosA) can be found in various plants. Although its potential health benefits have been extensively studied, its effect on osteoarthritis (OA) progression and cartilage regeneration function still needs to be fully elucidated in OA animal models. This study elucidated the effect of RosA on OA progression and cartilage regeneration. MethodsIn vitro assessments were conducted using RT-PCR, qRT-PCR, Western blotting, and ELISA to measure the effects of RosA. The molecular mechanisms of RosA were determined by analyzing the translocation of p65 into the nucleus using immunocytochemistry (ICC). Histological analysis of cartilage explant was performed using alcian blue staining and immunohistochemistry (IHC). For in vivo analysis, the destabilization of the medial meniscus (DMM)-induced OA mouse model was utilized to evaluate cartilage destruction through Safranin-O staining. The expression of catabolic and anabolic factors in mice knee joints was quantified by immunohistochemistry. ResultsThe expression of catabolic factors in chondrocytes was significantly impeded by RosA. It also suppressed the NF-κB signaling pathway by decreasing phosphorylation of p65 and reducing degradation of IκB protein. In ex vivo experiments, RosA protected sulfated proteoglycan erosion triggered by IL-1β and suppressed the catabolic factors in cartilage explant. RosA treatment in animal models resulted in preventing cartilage destruction and reducing catabolic factors in the cartilage. RosA was also found to promote the expression of Sox9, Col2a1, and Acan in vitro, ex vivo, and in vivo analyses. ConclusionsRosA attenuated the OA progression by suppressing the catabolic factors expression. These effects were facilitated through the suppression of the NF-κB signaling pathway. Additionally, it promotes cartilage regeneration by inducing anabolic factors. Therefore, RosA shows potential as an effective therapeutic agent for treating OA.

Impact of Polydeoxyribonucleotides on the Morphology, Viability, and Osteogenic Differentiation of Gingiva-Derived Stem Cell Spheroids

Background and Objectives: Polydeoxyribonucleotides (PDRN), composed of DNA fragments derived from salmon DNA, is widely recognized for its regenerative properties. It has been extensively used in medical applications, such as dermatology and wound healing, due to its ability to enhance cellular metabolic activity, stimulate angiogenesis, and promote tissue regeneration. In the field of dentistry, PDRN has shown potential in promoting periodontal healing and bone regeneration. This study aims to investigate the effects of PDRN on the morphology, survival, and osteogenic differentiation of gingiva-derived stem cell spheroids, with a focus on its potential applications in tissue engineering and regenerative dentistry. Materials and Methods: Gingiva-derived mesenchymal stem cells were cultured and formed into spheroids using microwells. The cells were treated with varying concentrations of PDRN (0, 25, 50, 75, and 100 μg/mL) and cultivated in osteogenic media. Cell morphology was observed over seven days using an inverted microscope, and viability was assessed with Live/Dead Kit assays and Cell Counting Kit-8. Osteogenic differentiation was evaluated by measuring alkaline phosphatase activity and calcium deposition. The expression levels of osteogenic markers RUNX2 and COL1A1 were quantified using real-time polymerase chain reaction. RNA sequencing was performed to assess the gene expression profiles related to osteogenesis. Results: The results demonstrated that PDRN treatment had no significant effect on spheroid diameter or cellular viability during the observation period. However, a PDRN concentration of 75 μg/mL significantly enhanced calcium deposition by Day 14, suggesting increased mineralization. RUNX2 and COL1A1 mRNA expression levels varied with PDRN concentration, with the highest RUNX2 expression observed at 25 μg/mL and the highest COL1A1 expression at 75 μg/mL. RNA sequencing further confirmed the upregulation of genes involved in osteogenic differentiation, with enhanced expression of RUNX2 and COL1A1 in PDRN-treated gingiva-derived stem cell spheroids. Conclusions: In summary, PDRN did not significantly affect the viability or morphology of gingiva-derived stem cell spheroids but influenced their osteogenic differentiation and mineralization in a concentration-dependent manner. These findings suggest that PDRN may play a role in promoting osteogenic processes in tissue engineering and regenerative dentistry applications, with specific effects observed at different concentrations.

Effects of Shaofu Zhuyu decoction on intestinal flora and fibrosis in a mouse model of endometriosis

Shaofu Zhuyu decoction has been widely used to treat gynecological diseases; however, its mechanism of action in endometriosis remains unclear. We analyzed Shaofu Zhuyu decoction’s chemical composition using ultra-high performance liquid chromatography-mass spectrometry. In an endometriosis mouse model, ectopic lesions weight measurements and hematoxylin and eosin staining were used to assess the therapeutic efficacy of Shaofu Zhuyu decoction. Effects on intestinal microflora were analyzed using 16S ribosomal ribonucleic acid sequencing, and impacts on focal fibrosis were analyzed using Masson’s trichrome staining. Moreover, fibrosis- and metabolism-related proteins were assessed using immunohistochemistry and enzyme-linked immunosorbent assay. The study identified 157 chemical constituents within Shaofu Zhuyu decoction. Shaofu Zhuyu decoction treatment in mice with endometriosis resulted in a reduction in ectopic lesions weight (P<0.05) and delayed disease progression. Moreover, it improved the diversity and abundance of intestinal flora, and decreased the expression of Lachnospiraceae (P<0.05), Rikenellaceae (P<0.01), Ruminococcaceae (P<0.01), Lachnoclostridium (P<0.05), and unclassified_f__Ruminococcaceae (P<0.05). Kyoto Encyclopedia of Genes and Genomes analysis revealed enrichment in carbohydrate, amino acid, and lipid metabolism pathways. Masson’s trichrome staining revealed that compared to the untreated group, the Shaofu Zhuyu decoction group exhibited significantly reduced collagen deposition areas (P<0.001), lower TGF-β1 (P<0.001), COL1A1 (P<0.05), and α-SMA (P<0.01) expression in ectopic lesions, along with increased serum adiponectin (P<0.05), decreased serum leptin (P<0.05), TGF-β1 (P<0.001), and CTGF (P<0.05). Shaofu Zhuyu decoction regulates the intestinal flora of mice with endometriosis while also reducing fibrosis at the lesion site. These findings highlight novel mechanisms for its efficacy in alleviating endometriosis.

Key Genes Involved in the Beneficial Mechanism of Hyperbaric Oxygen for Glioblastoma and Predictive Indicators of Hyperbaric Oxygen Prolonging Survival in Glioblastoma Patients.

The prognosis of glioblastoma is poor, and therapy-resistance is largely attributed to intratumor hypoxia. Hyperbaric oxygen (HBO) effectively alleviates hypoxia. However, the sole role of HBO in glioblastoma remains controversial. We previously reported that HBO can promote apoptosis, shorten protrusions, and delay growth of glioblastoma, but the molecular mechanism is unclear. We aimed to test candidate genes in HBO-exposed glioblastoma cells and to analyze their correlation with the survival of glioblastoma patients. Glioblastoma cell lines exposed to repetitive HBO or normobaric air (NBA) were collected for RNA isolation and microarray data analysis. GO analysis, KEGG pathway analysis and survival analysis of the differentially expressed genes (DEGs) were performed. HBO not only inhibited hypoxia-inducing genes including CA9, FGF11, PPFIA4, TCAF2 and SLC2A12, but also regulated vascularization by downregulating the expression of COL1A1, COL8A1, COL12A1, RHOJ and FILIP1L, ultimately attenuated hypoxic microenvironment of glioblastoma. HBO attenuated inflammatory microenvironment by reducing the expression of NLRP2, CARD8, MYD88 and CD180. HBO prevented metastasis by downregulating the expression of NTM, CXCL12, CXCL13, CXCR4, CXCR5, CDC42, IGFBP3, IGFBP5, GPC6, MMP19, ADAMTS1, EFEMP1, PTBP3, NF1 and PDCD1. HBO upregulated the expression of BAK1, PPIF, DDIT3, TP53I11 and FAS, whereas downregulated the expression of MDM4 and SIVA1, thus promoting apoptosis. HBO upregulated the expression of CDC25A, MCM2, PCNA, RFC33, DSCC1 and CDC14A, whereas downregulated the expression of ASNS, CDK6, CDKN1B, PTBP3 and MAD2L1, thus inhibiting cell cycle progression. Among these DEGs, 17 indicator-genes of HBO prolonging survival were detected. HBO is beneficial for glioblastoma. Glioblastoma patients with these predictive indicators may prolong survival with HBO therapy. These potential therapeutic targets especially COL1A1, ADAMTS1 and PTBP3 deserve further validation.

Spatial transcriptomic analysis deciphers adipocyte-to-fibroblast transformation in bleomycin-induced murine skin fibrosis.

Scleroderma is characterized by inflammation and fibrosis, predominantly occurring in the skin and extending to various parts of the body. The pathophysiology of scleroderma is multifaceted, with the current understanding including endothelial damage, inflammatory cell infiltration, and fibroblast activation in its progression. Nonetheless, the mechanism of cellular interactions and the precise spatial distribution of these cellular events within the fibrotic tissues remain elusive, highlighting a critical gap in our comprehensive understanding of scleroderma's pathogenesis. In this study, we administered bleomycin intradermally to the dorsal skin of four individual murine models. Subsequently, skin tissues were harvested at predetermined intervals for comprehensive spatial transcriptomic analysis to determine the spatial dynamics influencing scleroderma pathogenesis. To validate the possible results from bioinformatic analysis, further in vitro and in vivo experiments were conducted. Analysis of the spatial transcriptome revealed significant alterations in cell clusters during the progression of scleroderma. Gene Ontology analysis identified disruptions in lipid metabolism as the disease advanced. Pseudotime analysis provided evidence for a phenotypic transition from adipocytes to fibroblasts. In vitro studies demonstrated increased expression of Col1a1 and α-SMA as the disease progressed. These fibroblasts have been identified as key contributors to the increasing inflammation. Co-culturing TGF-β induced adipocytes with RAW264.7 cells resulted in overexpression of pro-inflammatory cytokines in the RAW264.7 cells. Both in vitro and in vivo experiments confirmed adipocyte loss and fibroblast formation, with transformed fibroblasts showing pronounced pro-inflammatory characteristics, highlighting their crucial role in the disease mechanism. Our study showed the spatial distribution and dynamic alterations of various cell types during scleroderma progression. Crucially, we identified the transformation of adipocytes into fibroblasts as a key factor promoting disease advancement. These emergent fibroblasts intensify inflammation, indicating that research on these cell clusters could reveal key scleroderma mechanisms and guide future therapies.

Small extracellular vesicles derived from synovial fibroblasts contain distinct miRNA profiles and contribute to chondrocyte damage in osteoarthritis

BackgroundSmall extracellular vesicles (sEV) derived from synovial fibroblasts (SF) represent a novel molecular mechanism regulating cartilage erosion in osteoarthritis (OA). However, a comprehensive evaluation using disease relevant cells has not been undertaken. The aim of this study was to isolate and characterise sEV from OA SF and to look at their ability to regulate OA chondrocyte effector responses relevant to disease. Profiling of micro (mi) RNA signatures in sEV and parental OA SF cells was performed.MethodsSF and chondrocytes were isolated from OA synovial membrane and cartilage respectively (n = 9). sEV were isolated from OA SF (± IL-1β) conditioned media by ultracentrifugation and characterised using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Particle size was confirmed by nanoparticle tracking analysis (NTA). sEV regulation of OA chondrocyte and cartilage effector response was evaluated using qPCR, ELISA and sulphated glycosaminoglycan assay (sGAG). RNA-sequencing was used to establish miRNA signatures in isolated sEV from OA SF.ResultsOA SF derived sEV were readily taken up by OA chondrocytes, with increased expression of the catabolic gene MMP 13 (p < 0.01) and decreased expression of the anabolic genes aggrecan and COL2A1 (p < 0.01) observed. Treatment with sEV derived from IL-1β stimulated OA SF significantly decreased expression of aggrecan and COL2A1 (p < 0.001) and increased SOX 9 gene expression (p < 0.05). OA chondrocytes cultured with sEV from either non-stimulated or IL-1β treated OA SF, resulted in a significant increase in the secretion of IL-6, IL-8 and MMP-3 (p < 0.01). Cartilage explants cultured with sEV from SF (± IL-1β) had a significant increase in the release of sGAG (p < 0.01). miRNA signatures differed between parental SF cells and isolated sEV. The recently identified osteoclastogenic regulator miR182, along with miR4472-2, miR1302-3, miR6720, miR6087 and miR4532 were enriched in sEV compared to parental cells, p < 0.01. Signatures were similar in sEVs derived from non-stimulated or IL-1β stimulated SF.ConclusionsOA SF sEV regulate chondrocyte inflammatory and remodelling responses. OA SF sEV have unique signatures compared to parental cells which do not alter with IL-1β stimulation. This study provides insight into a novel regulatory mechanism within the OA joint which could inform future targeted therapy.

Oenothera biennis improves pregnancy outcomes by suppressing inflammation and fibrosis in an intra-uterine adhesion rat model

Intrauterine adhesion (IUA), also referred to as Asherman’s syndrome, is characterized by fibrosis, inflammation, and can cause amenorrhea and infertility due to abnormal endometrial healing. Histological and Molecular methods were used to evaluate the efficacy of EPO, which is traditionally known for its anti-inflammatory and fibrinolytic properties, in preventing the formation of IUA. Oral administration of EPO reduced the formation of adhesion bands and promoted endometrial regeneration. EPO administration decreased extracellular matrix accumulation, evidenced by the down-regulation of tissue COL1A1 and COL3A1 expression. The anti-inflammatory effect of EPO was confirmed by a reduction in oxidants and down-regulation of pro-inflammatory cytokines including TNF-α, IL-6, IFN-γ, and IL-1β. Furthermore, EPO improved embryonic development parameters, including size and weight of embryo, as well as increased embryo count and live embryo percentage in the rat IUA model. EPO also positively enhanced implantation markers, particularly enlargement and mass gain in the placenta of the treated group, consequently improving pregnancy outcomes such as the number of babies, percent of live babies, baby weight and gestation time. Histopathological investigation provides evidence that oral administration of EPO showed no toxicity on the main three organs including liver, kidney and heart. These results showed that EPO can be considered as a safe and natural product with potent anti-inflammatory and fibrinolytic properties without any observed side effects for the treatment of IUA.

Whole-transcriptome analyses of ovine lung microvascular endothelial cells infected with bluetongue virus

Bluetongue virus (BTV) infection induces profound and intricate changes in the transcriptional profile of the host to facilitate its survival and replication. However, there have been no whole-transcriptome studies on ovine lung microvascular endothelial cells (OLMECs) infected with BTV. In this study, we comprehensively analysed the whole-transcriptome sequences of BTV-1 serotype-infected and mock-infected OLMECs and subsequently performed bioinformatics differential analysis. Our analysis revealed 1215 differentially expressed mRNA transcripts, 82 differentially expressed long noncoding RNAs (lncRNAs) transcripts, 63 differentially expressed microRNAs (miRNAs) transcripts, and 42 differentially expressed circular RNAs (circRNAs) transcripts. Annotation from Gene Ontology, enrichment from the Kyoto Encyclopedia of Genes and Genomes, and construction of endogenous competing RNA network analysis revealed that the differentially expressed RNAs primarily participated in viral sensing and signal transduction pathways, antiviral and immune responses, inflammation, and extracellular matrix (ECM)-related pathways. Furthermore, protein‒protein interaction network analysis revealed that BTV may regulate the conformation of ECM receptor proteins and change their biological activity through a series of complex mechanisms. Finally, on the basis of real-time fluorescence quantitative polymerase chain reaction results, the expression trends of the differentially expressed RNA were consistent with the whole-transcriptome sequencing data, such as downregulation of the expression of COL4A1, ITGA8, ITGB5, and TNC and upregulation of the expression of CXCL10, RNASEL, IRF3, IRF7, and IFIHI. This study provides a novel perspective for further investigations of the mechanism of the ECM in the BTV-host interactome and the pathogenesis of lung microvascular endothelial cells.

Photobiomodulation associated with alginate-based engineered tissue on promoting chondrocytes-derived biological responses for cartilage regeneration

Articular cartilage is a connective tissue with limited self-healing potential, frequently affected by trauma and degenerative changes, leading to osteoarthritis. Photobiomodulation paired with engineered tissue can improve cartilage's poor intrinsic healing and overcome its restricted self-regeneration. In this study, alginate-based scaffolds were fabricated with varying concentrations of CaCl₂ to achieve optimal mechanical, biocompatible, and biodegradable properties. The fluence-dependence of near-infrared (NIR) laser irradiation (830 nm) on chondrocyte viability and proliferation was investigated in a 2D environment across fluences (2.5–10 J/cm2). Optimal conditions of 3 % w/v CaCl₂ and 5 J/cm2 were identified to construct alginate scaffolds and promote chondrocyte growth in 2D and 3D cultures. Single PBM (830 nm, 5 J/cm2) further exhibited a significant relative intensity of collagen type II immunostaining and stimulation of Col2a1 expression in 2D culture. Multiple PBM sessions (830 nm, 5 J/cm2) significantly enhanced chondrocyte proliferation and glycosaminoglycan production in alginate scaffolds, with a protocol of one session every four days being the most effective. Scanning electron microscopy revealed PBM-induced secretory granule formation, corresponding to a significant increase in extracellular vesicle release. Consequently, integrating PBM and alginate-based scaffolds is a promising technique for accelerating and optimizing cartilage regeneration, with potential application in tissue engineering.

Modulation of regenerative responses by retinoic and ascorbic acids in human apical papilla cells

ObjectiveThis study investigated the bioactive effects of retinoic acid and ascorbic acid on hSCAPs in vitro. DesignCells were obtained from human third molars (n=4) and characterized for mesenchymal stem cell markers by flow cytometry. The experimental groups: control (α-MEM); vehicle control group (α-MEM + 0.17 % DMSO); retinoic acid 0.1, 1, and 10 µM; and ascorbic acid 3, 30, and 300 µM (n=8) were tested for cell viability (alamarBlue; 1, 3, and 7 days), total collagen synthesis (Sirius Red; 1 and 7 days), mineralized matrix formation (Alizarin red; 14 days), and the regulation of gene expression related to mineralization (ALPL and DSPP), cell migration (ITGAV and CXCL12) angiogenesis (VEGFA) and collagen synthesis (COL1A1 and COL3A1; RT-qPCR) on 1 and 7 days. ACTB and GAPDH were used as reference genes. Data were analyzed by ANOVA and complementary tests at a 5 % significance level. ResultsAscorbic acid 300 µM increased viability, and retinoic acid reduced it dose-dependently. Retinoic acid 0.1 µM and ascorbic acid 30 and 300 µM increased mineralized matrix formation and total collagen synthesis, and retinoic acid 10 µM decreased. On day 1, 0.1 µM retinoic acid upregulated the gene expression of COL1A1, COL3A1, VEGFA, CXCL12, ALPL, DSPP e ITGAV, and 300 µM ascorbic acid upregulated COL1A1, COL3A1 and DSPP. However, on day 7, retinoic acid downregulated ALPL, COL3A1, CXCL12, and VEGFA and downregulated ITGAV and VEGFA. ConclusionRetinoic acid 0.1 µM and ascorbic acid 300 µM biostimulated hSCAPs to differentiate into pro-regenerative phenotypes with potential application for REPs.

Effect of Ritanserin and Duloxetine on the Gene Expression of Primary Aniridia and Healthy Human Limbal Stromal Cells, In Vitro.

In congenital aniridia caused by mutations in paired box 6 (PAX6), PAX6 influences the migration and differentiation of limbal epithelial cells (LECs), thereby playing a pivotal role in aniridia-associated keratopathy. The antidepressants ritanserin and duloxetine affect PAX6 expression in LECs. Limbal stromal cells, which support limbal epithelial stem cells, are crucial in the limbal stem cell niche. This study explores how ritanserin and duloxetine influence gene expression in primary human limbal stromal cells from subjects with congenital aniridia and from healthy subjects, in vitro. Primary human limbal stromal cells from corneas affected by aniridia (AN-LSCs) (n = 8) and from healthy corneas (LSCs) (n = 8) were isolated and cultured in either low-glucose serum-free (LGSF) or normal-glucose serum-containing (NGSC) media. Cells were treated with 4µM ritanserin or duloxetine for 24h. Quantitative PCR (qPCR) and western blot were used to assess the expression of PAX6, FOSL2, TGF-β1, ACTA2A1, LUM, COL1A1, COL5A1, DSG1, FABP5 and ADH7. In AN-LSCs with LGSF-medium, ritanserin increased PAX6 messenger RNA (mRNA) (p = 0.007) and decreased TGF-β1 and FOSL2 mRNA levels (P = 0.005, P = 0.038). In addition, TGF-β1 protein levels decreased with both treatments (P = 0.02, P = 0.007), and FABP5 protein level increased, using ritanserin (P = 0.019). In LSCs with LGSF-medium, ACTA2A1 mRNA levels decreased using ritanserin and duloxetine (P = 0.028; P = 0.031), while FABP5 mRNA levels increased with ritanserin treatment (P = 0.003). Also, duloxetine use reduced α-SMA protein (P = 0.013) and increased FABP5 protein levels (P = 0.029). In LSCs with NGSC-medium, ritanserin elevated LUM, FABP5 and ADH7 mRNA and protein levels (P = 0.025, P = 0.003, P = 0.047, P = 0.024, P = 0.013, P = 0.039). The results of our study confirmed that the antipsychotropic drugs ritanserin and duloxetine alter PAX6 and TGF-β1 gene expression in AN-LSCs cultured in LGSF-medium. These drugs were found to have an impact on retinoic acid signaling pathways and keratocyte characteristic markers both in LSCs and AN-LSCs, using different culture media.

Regulation of Skeletal Development and Maintenance by Runx2 and Sp7.

Runx2 (runt related transcription factor 2) and Sp7 (Sp7 transcription factor 7) are crucial transcription factors for bone development. The cotranscription factor Cbfb (core binding factor beta), which enhances the DNA-binding capacity of Runx2 and stabilizes the Runx2 protein, is necessary for bone development. Runx2 is essential for chondrocyte maturation, and Sp7 is partly involved. Runx2 induces the commitment of multipotent mesenchymal cells to osteoblast lineage cells and enhances the proliferation of osteoprogenitors. Reciprocal regulation between Runx2 and the Hedgehog, fibroblast growth factor (Fgf), Wnt, and parathyroid hormone-like hormone (Pthlh) signaling pathways and Dlx5 (distal-less homeobox 5) plays an important role in these processes. The induction of Fgfr2 (Fgf receptor 2) and Fgfr3 expression by Runx2 is important for the proliferation of osteoblast lineage cells. Runx2 induces Sp7 expression, and Runx2+ osteoprogenitors become Runx2+Sp7+ preosteoblasts. Sp7 induces the differentiation of preosteoblasts into osteoblasts without enhancing their proliferation. In osteoblasts, Runx2 is required for bone formation by inducing the expression of major bone matrix protein genes, including Col1a1 (collagen type I alpha 1), Col1a2, Spp1 (secreted phosphoprotein 1), Ibsp (integrin binding sialoprotein), and Bglap (bone gamma carboxyglutamate protein)/Bglap2. Bglap/Bglap2 (osteocalcin) regulates the alignment of apatite crystals parallel to collagen fibrils but does not function as a hormone that regulates glucose metabolism, testosterone synthesis, and muscle mass. Sp7 is also involved in Co1a1 expression and regulates osteoblast/osteocyte process formation, which is necessary for the survival of osteocytes and the prevention of cortical porosity. SP7 mutations cause osteogenesis imperfecta in rare cases. Runx2 is an important pathogenic factor, while Runx1, Runx3, and Cbfb are protective factors in osteoarthritis development.

Quantitative tissue analysis reveals AK2, COL1A1, & PLG protein signatures: Targeted therapeutics for meningioma.

Meningioma is the most prevalent primary intracranial brain tumor and accounts for one-third of all CNS tumors. Meningioma is known to be the most common yet life-threatening brain tumor with a higher recurrence rate. Globally, there is an increase in the healthcare burden due to meningioma and hence in its research. The present clinical approach includes surgical resection, chemotherapy, and radiation therapies to which the malignancy does not seem to respond efficiently. Targeted therapies and molecular markers provide elite patient treatment and care for individuals suffering from meningiomas as compared to conventional measures. Although there is proteomic data on meningioma the knowledge of potential biomarkers differentiating the grades is scarce. To identify the best set of biomarkers, validation of reported markers in large and independent sample cohorts in the future is necessary. A total of 12 samples, 3 each of control (which made pool 1) Meningioma grade I (which made 2 sets: pool 2 and pool 3), and Meningioma grade II (which made pool 4) were taken for LC-MS/MS. After this, the expression of three proteins was checked by immunocytochemistry, flow cytometry, and western blotting. Protein expression was analyzed using various techniques like mass spectrometry, immunocytochemistry, flow cytometry, and western blotting. Mass spectrometry is the most commonly used standard and reliable technique for identifying and quantifying protein expression. We got three highly upregulated proteins namely AK2, COL1A1, and PLG using this technique. The biomarker potential of these proteins was further checked by ICC, western blotting, and flow cytometry. Three important proteins were found to be upregulated namely AK2 (Adenylate Kinase 2), COL1A1 (Collagen 1A1), and PLG (Plasminogen). The order of increased protein expression was control < MG grade I < MG grade II according to mass spectrometry and western blotting. In immunocytochemistry, we found that COL1A1 expression increases significantly with grades in comparison to control. Similarly, AK2 and PLG also showed little increase but not as much as COL1A1. In flow cytometry, PLG showed higher upregulation in grades than control. While AK2 and COL1A1 showed little increase in expression in grades than control. All techniques, especially mass spectrometry and western blotting presented higher expression of these proteins in grades as compared to control. In the quest to find a suitable therapeutic marker, this study incorporates quantitative labeling and detection followed by flow cytometry, Immunocytochemistry, and western blotting for early diagnosis and treatment of meningioma. The article further explores the efficacy of some proteins namely AK2, COL1A1 & PLG to be the targeted molecules.

Systemic low-dose anti-fibrotic treatment attenuates ovarian aging in the mouse.

The female reproductive system is one of the first to age in humans, resulting in infertility and endocrine disruptions. The aging ovary assumes a fibro-inflammatory milieu which negatively impacts gamete quantity and quality as well as ovulation. Here, we tested whether the systemic delivery of anti-inflammatory (Etanercept) or anti-fibrotic (Pirfenidone) drugs attenuates ovarian aging in mice. We first evaluated the ability of these drugs to decrease the expression of fibro-inflammatory genes in primary ovarian stromal cellstreated with a pro-fibrotic or a pro-inflammatory stimulus. Whereas Etanercept did not block Tnf expression in ovarian stromal cells, Pirfenidone significantly reduced Col1a1 expression. We then tested Pirfenidone in vivo where the drug was delivered systemically via mini-osmotic pumps for 6weeks. Pirfenidone mitigated the age-dependent increase in ovarian fibrosis without impacting overall health parameters. Ovarian function was improved in Pirfenidone-treated mice as evidenced by increased follicle and corpora lutea number, AMH levels, and improved estrous cyclicity. Transcriptomic analysis revealed that Pirfenidone treatment resulted in an upregulation of reproductive function-related genes at 8.5months and a downregulation of inflammatory genes at 12months of age. These findings demonstrate that reducing the fibroinflammatory ovarian microenvironment improves ovarian function, thereby supporting modulating the ovarian environment as a therapeutic avenue to extend reproductive longevity.

Properties of Skin Collagen from Southern Catfish (Silurus meridionalis) Fed with Raw and Cooked Food.

The southern catfish (Silurus meridionalis) is an economically important carnivorous freshwater fish in China. In this study, we compared the properties of skin collagen from southern catfish fed with raw food (RF) and cooked food (CF). The skin collagen yield in the RF group (8.66 ± 0.11%) was significantly higher than that of the CF group (8.00 ± 0.27%). SDS-PAGE, circular dichroism spectroscopy, and FTIR analyses revealed that the collagen extracted from southern catfish skin in both groups was type I collagen, with a unique triple helix structure and high purity. The thermal denaturation temperature of collagen in the RF group (35.20 ± 0.11 °C) was significantly higher than that of the CF group (34.51 ± 0.25 °C). The DPPH free radical scavenging rates were 68.30 ± 2.41% in the RF collagen and 61.78 ± 3.91% in the CF collagen, which was higher than that found in most fish collagen. Both the RF and CF groups had high ability to form fibrils in vitro. Under the same conditions, the CF group exhibited faster fibril formation and a thicker fibril diameter (p < 0.05). In addition, the RF group exhibited significantly higher expression of col1a1 compared to the CF group. These results indicated that feeding southern catfish raw food contributed to collagen production, and the collagen from these fish may have potential in biomaterial applications.