Search Results (533)

The mechanisms underlying wound healing mediated by cannabinoid receptor 1 (CB1)—known for its neuromodulatory functions—remain incompletely understood. Therefore, we investigated the impact of activating CB1 using specific agonists, both in vitro and in vivo, with a focus on wound healing. In the in vitro study, fibroblasts were isolated and cultured from the dermis of human skin and treated with a CB1 agonist, 2-arachidonyl glyceryl ether (2-AGE). In the in vivo study, a mouse acute wound model was created using a skin biopsy punch and treated with the CB1 agonist arachidonoyl 2′-chloroethylamide (ACEA). The in vitro study revealed that 2-AGE increased cell proliferation and differentiation, upregulated the expression of alpha-smooth muscle actin (α-SMA), N-cadherin, and vimentin, and enhanced cell migration as well as the synthesis of type I and III collagen and fibronectin in normal human dermal fibroblasts. The CB1 antagonist AM251 abolished 2-AGE-induced expression of α-SMA, type I collagen, and fibronectin. In vivo, ACEA treatment accelerated wound closure, increased expression of α-SMA, type I collagen, and fibronectin, and ultimately increased epidermal and dermal thickness. Overall, these findings suggest that the activation of CB1 promotes wound healing and provides evidence for the therapeutic potential of CB1 agonists in wound treatment. Full article

Activation of hepatic stellate cells (HSCs) featuring upregulated expression of α-smooth muscle actin (α-SMA) is recognized as a key driver for hepatic fibrosis, which provides a promising strategy for seeking anti-liver fibrogenic agents via suppressing the activation event. In this study, we designed and synthesized twenty-eight cordycepin derivatives through structural modifications at the C2 position and the C6-NH₂ group of the purine moiety. These compounds were screened for their inhibitory effects on HSC activation by detecting the mRNA expression of α-SMA using quantitative real-time polymerase chain reaction (qPCR) in the LX-2 cell model. Most compounds displayed inhibitory activity comparable to cordycepin, with compound 3a bearing a C2-chloro and a N6-methyl-N6-(2-chlorobenzyl) substituent, demonstrating enhanced in vitro anti-fibrotic effect. This compound was able to dose-dependently downregulate α-SMA and collagen-I at both mRNA and protein levels, inhibited LX-2 cell migration, and exhibited improved metabolic stability in liver microsomes. The Western blotting result also indicated that 3a could activate the AMPK signaling pathway. Overall, these results suggest 3a may serve as a lead compound for further investigation. Full article

Background: The anterior cruciate ligament (ACL) and medial collateral ligament (MCL) display strikingly different healing behaviors, despite their similar structural roles within the knee. The epiligament (EL)—a vascular and cellular envelope surrounding each ligament—has emerged as a critical determinant of repair capacity. The aim of this study was to perform a region-specific, comparative analysis of EL molecular profiles in the ACL and MCL to elucidate the mechanisms underlying their contrasting reparative outcomes. Methods: Human ACL and MCL specimens were obtained from 12 fresh knee joints. Immunohistochemical labeling for CD34, α-smooth muscle actin (α-SMA), and vascular endothelial growth factor (VEGF) was performed across proximal, mid-substance, and distal EL regions. Quantitative image analysis using IHC Profiler for ImageJ generated semiquantitative (negative, low-positive, positive) distributions, and inter-ligament comparisons were quantified using t-tests (p  <  0.05). Results: Distinct, region-specific EL signatures were identified. The ACL EL exhibited strong proximal α-SMA expression (0% neg/66.8% low+/33.2%+) and notable distal CD34 positivity (0% neg/83.3% low+/16.7%+), while VEGF expression was confined to the mid-substance (≈55% low+/26%+). In contrast, the MCL EL was largely negative for CD34 and VEGF across all regions, showing a homogeneous but functionally oriented α-SMA profile: proximally negative, sparse mid positivity, and high distal low-positive staining (93.4% low+). Differences in proximal and distal CD34 and α-SMA expression between the ACL and MCL were highly significant (p  <  0.0001–0.001), confirming a mechanistic divergence in EL organization. Conclusions: The ACL EL is regionally heterogeneous, vascularly biased, and enriched in contractile α-SMA+ cells, suggesting localized but poorly coordinated reparative potential. In contrast, the MCL EL is structurally uniform, with distributed α-SMA activity supporting stable wound contraction and tissue continuity, despite limited angiogenic signaling. These findings indicate that the ACL’s failure to heal is not attributable to the absence of progenitor or angiogenic factors, but rather to its fragmented spatial organization and dominant contractile phenotype. Therapeutically, preserving and modulating the EL, particularly its CD34+ and α-SMA+ compartments, could be key to enhancing intrinsic ACL repair and improving outcomes in ligament reconstruction and regeneration. Full article

Dietary pattern characterized by low intake of vegetables and fruits and high consumption of fat, soft drink and desserts are associated with an increased risk of chronic diseases. To investigate the effects of folate status and fructose intake on adenine-induced chronic kidney disease (CKD), seven-week-old C57BL/6 mice were divided into six groups and fed either a control diet (Ctrl), a 26% (w/w) high-fructose diet (Hfru), Ctrl plus 0.15% adenine (Ctrl+ade), Hfru+ade, Hfru with folate deficiency plus adenine (Hfru−f+ade), or Hfru with tenfold folate supplementation plus adenine (Hfru+f10+ade). After 10 weeks on the assigned diets, adenine was administrated to the +ade groups for 7 weeks. The results showed that all adenine-treated mice exhibited increased fasting blood glucose, urinary glucose, and elevated renal expression of collagen 1a1 (Col1a1), fibronectin (Fn1), and smooth muscle α-actin (Acta2). Compared with Ctrl mice, Hfru-fed mice showed significantly higher serum creatinine, increased urinary protein, and reduced creatinine clearance. Adenine induced kidney injury in all +ade groups, with the most severe damage observed in Hfru−f+ade mice, as indicated by elevated blood urine nitrogen (BUN), urinary protein, neutrophil gelatinase-associated lipocalin (NGAL), and renal fibrosis. In contrast, Hfru+f10+ade mice showed the lowest levels of these renal injury markers. The Hfru+ade diets increased renal Hif1α and iNos gene expression, which was further exacerbated by folate deficiency. Secretion of the anti-inflammatory cytokine interleukin (IL-10) by splenocytes was significantly reduced under folate-deficient conditions. Renal IL-10 levels were suppressed in all +ade groups but were significantly increased by folate supplementation. Renal IL-10 levels were negatively correlated with the inflammatory chemokine monocyte chemoattractant protein (MCP-1) and transforming growth factor (TGF)-β, whereas renal MCP-1 levels showed positive correlations with TGF-β and IL-6. Overall, these findings suggest that high fructose consumption in the absence of adequate folate intake may be of concern for CKD progression. Full article

Idiopathic pulmonary fibrosis (IPF) is a chronic and irreversible interstitial lung disease characterized by progressive scarring of the lungs. The available therapeutic strategies are limited and primarily focus on slowing disease progression rather than achieving fibrosis reversal. Cardamonin (CDN), a food-derived natural chalcone, has exhibited anti-fibrotic activity in liver and kidney fibrosis models; however, its role and underlying mechanism in IPF remain unelucidated. Herein, we integrated network pharmacology, machine learning, molecular simulations, and in vitro experiments. Network pharmacology identified 135 overlapping targets between CDN and IPF, which demonstrated a significant enrichment in the Phosphatidylinositol 3-Kinase/Protein Kinase B signaling pathway (PI3K/AKT). Machine learning further prioritized 6 core targets, with IGF1 emerging as a key candidate. Molecular docking revealed a favorable binding energy of −7.9 kcal/mol for the CDN-IGF1 complex. Subsequent 100 ns molecular dynamics simulations further confirmed its robust binding stability, yielding a mean binding free energy of −150.978 kcal/mol. In vitro, CDN significantly mitigated fibrosis in bleomycin (BLM)-challenged A549 cells, downregulating the expression of α-smooth muscle actin (α-SMA) and fibronectin. This effect was accompanied by a beneficial reversal of epithelial–mesenchymal transition (EMT), as indicated by increased E-cadherin levels and decreased vimentin expression. Mechanistically, CDN significantly suppressed the IGF1/PI3K/AKT axis; this inhibitory effect was partially reversed by exogenous IGF1 supplementation and further enhanced by the PI3K-specific inhibitor LY294002. This work provides the evidence that CDN alleviates BLM-induced pulmonary fibrosis by targeting the IGF1/PI3K/AKT-EMT axis. These findings lend support to a robust mechanistic basis for developing CDN as a potential therapeutic candidate for IPF. It should be noted that these conclusions are drawn from in vitro experiments using A549 cells, and further validation in primary alveolar epithelial cells and animal models is warranted to confirm their physiological relevance. Full article

Background/Objectives: Amlodipine, a widely prescribed calcium channel blocker, has been associated with gingival enlargement, yet the mechanisms underlying this adverse effect remain unclear. The present study aimed to explore molecular and histopathological factors potentially contributing to gingival changes in patients receiving amlodipine therapy, with a particular focus on molecules implicated in extracellular matrix turnover and tissue remodeling. Methods: The study included three groups of participants: patients with amlodipine-induced gingival enlargement, patients with gingival enlargement of inflammatory origin, and amlodipine-treated patients without gingival overgrowth. Gingival tissue samples were analyzed using hematoxylin-eosin staining to assess inflammatory changes and general tissue architecture, and Picrosirius Red staining to visualize collagen fibers. Relative gene expression of alpha-smooth muscle actin (α-SMA), IL-13, MMP-1, and procollagen was determined by real-time PCR, while collagen content was quantified using ImageJ software. Results: Histopathological evaluation revealed a less pronounced inflammatory response in amlodipine-related gingival enlargement compared to those who did not use amlodipine. The highest expression of α-SMA was detected in patients who did not receive amlodipine, whereas IL-13 and procollagen expression were markedly elevated in the amlodipine-induced group compared to others. MMP-1 expression was significantly lower in amlodipine-treated patients relative to those who did not use amlodipine, suggesting impaired collagen degradation. These findings, together with our previous results indicating enhanced expression of profibrotic mediators, suggest that altered extracellular matrix metabolism is potentially dominant in this condition. Conclusions: Amlodipine-induced gingival enlargement appears to involve a multifactorial process characterized by a prominent fibrotic component, reduced matrix degradation, and secondary inflammation. Full article

Background: Radiation-induced pulmonary fibrosis (RPF) remains a major burden of successful lung cancer radiotherapy. Clinically validated drugs targeting RPF remains scarce. Methods: We employed a transcriptome-based drug repurposing approach using REMEDY, a computational platform built on the Library of Integrated Network-Based Cellular Signatures (LINCS). Differentially expressed genes (DEGs) derived from radiation-induced lung injury (RILI) models were used as a query to identify compounds capable of reversing pro-fibrotic expression profile. Among top-ranked candidates, homoharringtonine (HHT), an FDA-approved protein synthesis inhibitor, was selected for experimental validation. Anti-fibrotic effects of HHT were assessed using an optimized in vitro fibrotic model based on activation of MRC-5 human lung fibroblasts. Complementary in silico molecular docking analyses were also conducted to explore the mechanistic basis of HHT’s actions. This represents the first transcriptome-guided, LINCS-based drug repurposing study applied specifically to radiation-induced pulmonary fibrosis, utilizing RPF-derived molecular signatures rather than general fibrosis-related datasets. Results: HHT significantly attenuated key fibrotic phenotypes, including fibroblast proliferation, myofibroblast differentiation, and extracellular matrix (ECM) production. Notably, HHT suppressed expression of cyclin D1 and α-smooth muscle actin (α-SMA), and reduced collagen deposition. Mechanistic investigations revealed that HHT modulates two pro-fibrotic pathways: RhoA/ROCK and Wnt/β-catenin signaling. Molecular docking further suggested that HHT may directly interact with fibrosis-related receptors such as integrins and Frizzled, providing structural insight into its anti-fibrotic potential. These findings underscore the novelty of reassigning HHT to a radiation-specific fibrotic context using a signature-reversal strategy uniquely tailored to RPF biology. Conclusions: Our findings identify HHT as a promising treatment of RPF, offering a dual mechanism of action—interruption of protein synthesis and targeted inhibition of fibrotic signaling pathways. This study highlights the value of computational drug repurposing platforms for accelerating therapeutic discovery. Further preclinical investigations are warranted to evaluate HHT’s in vivo efficacy and clinical applicability in RPF. Full article

Aging is a critical factor influencing susceptibility to hepatic injury. In this study, the spontaneous development of liver injury with advancing age and potential sex-related differences in these processes are examined. This study focuses on key mechanisms such as fatty acid metabolism, immune response, and cellular stress in male and female C57BL/6 mice. Aged male and female mice (20 to 22 months old) exhibited higher body weight and an altered metabolic profile and fatty acid metabolism compared to their younger counterparts (8 to 10 weeks old). In addition, increased oxidative stress, cellular senescence, expression of inflammatory markers, and cytokines/chemokines levels were also observed in aged male and female mice compared to younger mice. Furthermore, the aged mice exhibited increased indices of hepatic fibrosis, evident from the upregulation of smooth muscle actin-α, collagen, and transforming growth factor-β. In conclusion, aging promotes spontaneous liver injury by increasing indices of oxidative stress, steatosis, inflammation, and fibrosis. These results highlight the impact of chronological age on the liver that can increase its susceptibility to secondary hepatic stressors such as alcohol, high-calorie diet, or hepatotropic infections. Understanding how metabolic and inflammatory pathways change with aging in males and females is essential for elucidating the mechanisms that drive chronic liver disease progression. These insights are particularly important for developing targeted, sex-specific prevention and therapeutic strategies for the aging population. Full article

This study investigated the region-specific roles of transforming growth factor-β2 (TGF-β2) and angiotensin II (AngII) in extracellular matrix (ECM) remodeling and inflammatory responses within scleral tissues surrounding the optic nerve head (ONH), using primary human fibroblasts from posterior sclera, peripapillary sclera (ppScl), and fibroblast-like cells from lamina cribrosa (LC). In vivo validation was performed in a chronic ocular hypertension rat model. Fibrotic and inflammatory markers were analyzed by Western blotting, quantitative PCR, and immunocytochemistry following TGF-β2 or AngII stimulation, and in vivo effects were assessed after subtenon injection of pathway-specific inhibitors. TGF-β2 induced robust upregulation of α-smooth muscle actin, collagen type I, and fibronectin across all scleral regions, whereas AngII elicited regionally confined pro-inflammatory responses, particularly in the LC and ppScl, characterized by increased cyclooxygenase-2 expression. Inhibition of either pathway reduced ECM deposition in vivo, but only AngII blockade significantly attenuated glial activation and preserved retinal ganglion cells. These findings demonstrate that TGF-β2 predominantly drives fibrosis, while AngII promotes region-specific neuroinflammation, and that inflammation, rather than fibrosis alone, plays a critical role in glaucomatous neurodegeneration. Targeting both fibrotic and inflammatory mechanisms in a region-specific manner may offer improved neuroprotection in glaucoma. Full article

The myometrium is the smooth muscle layer of the uterus, whose dysfunctions are involved in various pathologies leading to infertility, such as adenomyosis and uterine fibroids. Developing relevant in vitro models of the myometrium is crucial for investigating the pathogenesis of these diseases. In this study, we propose a novel approach for cultivating mouse myometrial smooth muscle cells (SMCs) using plant-derived cellulose scaffolds. The scaffolds were obtained through the decellularization of green onion leaf, celery stalk, or bluegrass leaf, subsequently coated with collagen type I. We found that the structure of the green onion leaf scaffold provides unidirectional orientation of cultured cells, mimicking the tissue-specific organization of mouse myometrial SMCs in vivo. The mouse myometrial SMCs, cultured on this scaffold, proliferated, maintained viability up to 2.5 months, and retained the expression of the main markers of smooth muscle contractility (α-smooth muscle actin, transgelin, calponin, smooth muscle myosin heavy chains, connexin-43). To reproduce the native myometrium structure, a multilayered cultivation system was created. In a system of two overlaying scaffolds, cells also retained the viability and expression of smooth muscle contractility markers. The developed approach can be used for three-dimensional myometrium modeling to study the pathogenesis of myometrium-associated diseases. Full article

Ovarian hormones are essential regulators of vascular homeostasis, yet their deficiency’s effects on the meningeal microvasculature remain incompletely understood. We used high-resolution imaging to assess the cranial dura mater (CDM) blood and lymphatic microvasculature in ovariectomized (OVX) and control (intact or sham-operated) mice, followed by morphometric analysis of microvessel architecture. Immunofluorescent staining and Western blotting were employed to evaluate markers of vascular remodeling and profibrotic signaling. Blood microvascular quantification revealed a significant reduction in total microvessel length two weeks post-OVX, primarily due to arteriolar, but not venular, shortening. At the same time, the lengths of individual segments of both arterioles and venules were also significantly decreased, indicating microvascular fragmentation. Despite these changes, total vessel surface area remained preserved, suggesting compensatory dilation, particularly in arterioles. OVX also increased overall vessel tortuosity, again selectively affecting arterioles. Region-specific analysis of lymphatic networks associated with the coronal suture (CS) showed significantly increased surface area of podoplanin-positive lymphatic vessels. Elevated α-smooth muscle actin (α-SMA) expression in vascular and stromal compartments in OVX animals, along with increased transforming growth factor beta (TGF-β) levels, indicated early profibrotic changes. These findings highlight the selective vulnerability of arterial and lymphatic microvascular structures to hormonal deficiency post-OVX and suggest an association between hormonal status, microvascular remodeling, and profibrotic alterations in the CDM. Full article

Corneal fibrosis, a significant source of visual impairment, can result from keratocyte-to-myofibroblast transdifferentiation during wound healing. This study investigated the antifibrotic role of 1,25-dihydroxyvitamin D₃ (1,25 Vit D) and the lesser-known vitamin D, 24,25-dihydroxyvitamin D₃ (24,25 Vit D), in human and mouse corneal stromal cells (HSCs and MSCs) and in a Vit D receptor knockout (VDR KO) mouse model. Cells were treated with TGF-β1 ± Vit D metabolites and the expression of fibrotic and antifibrotic genes and proteins was evaluated. Both metabolites significantly reduced α-smooth muscle actin levels in HSCs, MSCs and organ-cultured mouse corneas (p < 0.05). They also upregulated the mRNA expression of BMP2, BMP6, BMPR2, and TGF-β3, as well as the protein expression of BMP6 and TGF-β3. VDR KO corneas subjected to alkali injury exhibited increased fibrotic responses and reduced CD45+ immune cell infiltration compared to wild-type controls. Notably, 24,25 Vit D exerted antifibrotic effects even in VDR KO cells, and the alternative 24,25 Vit D receptor FAM57B was expressed in all corneal cell layers. These results reveal consistent antifibrotic effects of both 1,25 and 24,25 Vit D across species, support the existence of VDR-independent mechanisms in the cornea, and offer new insights into potential therapeutic strategies for preventing corneal fibrosis. Full article

Age-related macular degeneration (AMD) progresses to vision-threatening dry and wet forms, with no effective dry AMD treatments available. The sulfated polysaccharide (GNP, 25.8 kDa) derived from Gelidium crinale exhibits diverse biological activities and represents a potential source of novel therapeutic agents. This study employed a hydrogen peroxide (H₂O₂)-induced oxidative stress and epithelial–mesenchymal transition (EMT) model in retinal pigment epithelial (RPE) cells to investigate GNP’s protective mechanisms against both oxidative damage and EMT. The results demonstrated that GNP effectively suppressed oxidative stress, with the 600 μg/mL dose significantly inhibiting excessive reactive oxygen species (ROS) generation to levels comparable to untreated controls. Concurrently, at concentrations of 200–600 μg/mL, GNP inhibited NF-κB signaling and increased the Bax/Bcl-2 ratio, effectively counteracting H₂O₂-induced oxidative damage and cell apoptosis. Furthermore, in H₂O₂-treated ARPE-19 cells, 600 μg/mL GNP significantly reduced the secretion of N-cadherin (N-cad), Vimentin (Vim), and α-smooth muscle actin (α-SMA), while increasing E-cadherin (E-cad) expression, consequently inhibiting cell migration. Mechanistically, GNP activated the Nrf2/HO-1 pathway, thereby mitigating oxidative stress. These findings suggest that GNP may serve as a potential therapeutic agent for dry AMD. Full article

A subset of chondrocytes in various human cartilage tissues, including neoplastic, regenerative, and normal cartilage, expresses α-smooth muscle actin (α-SMA), a protein typically found in smooth muscle cells. These α-SMA-containing chondrocytes, termed myochondrocytes and myochondroblasts, may play important roles in cartilage physiology, regeneration, and structural integrity, particularly in auricular and articular cartilage. This review synthesizes current knowledge regarding the terminology, distribution, and biological significance of these cells across normal, osteoarthritic, transplanted, and neoplastic cartilage. We summarize key findings from immunohistochemical studies using markers such as S-100, α-SMA, and SOX9, along with ultrastructural confirmation of myofilament bundles via electron microscopy. Current evidence suggests that myochondrocytes exhibit enhanced regenerative potential and contribute to matrix remodeling. Furthermore, their presence reflects the inherent cellular heterogeneity of cartilage, potentially arising from transdifferentiation processes involving fibroblasts, mesenchymal stem cells, or chondroblasts. Finally, TGF-β1 and PDGF-BB are identified as a critical modulator of α-SMA expression and chondrocyte phenotype. A deeper understanding of nature and function of myochondrocytes and myochondroblasts may improve interpretations of cartilage pathology and inform strategies for tissue engineering and cartilage repair. This review highlights the need for further investigation into the molecular regulation and functional roles of these cells in both physiological and pathological contexts. Full article

Background/Objectives: Abnormal differentiation of human skin fibroblasts into myofibroblasts contributes to fibrotic skin disorders such as hypertrophic scars, keloids, and Dupuytren’s disease. This process is characterized by increased fibroblast proliferation, enhanced differentiation into myofibroblasts, and reduced programmed cell death (apoptosis). We previously demonstrated that blue light irradiation (λ = 453 nm) significantly and dose-dependently inhibits both spontaneous and TGF-β-induced fibroblast differentiation. Methods: Because fibroblast differentiation depends on cellular energy metabolism, we investigated whether the inhibitory effect of blue light is linked to changes in the cells’ energy balance. Results: We found that blue light reduced TGF-β-induced differentiation, as shown by decreased levels of α-SMA and EDA-fibronectin, key markers of myofibroblast formation. This effect was strongly associated with almost complete inhibition of mitochondrial respiration, reduced glycolysis, a lower NAD⁺/NADH ratio, and decreased ATP production. ATP-dependent processes, including endocytosis and lysosomal activity, both essential parameters of fibroblast differentiation, were also strongly suppressed. Importantly, all these changes were fully reversible within 24 h after the last irradiation. Conclusions: Mechanistically, we propose that blue light triggers photochemical reduction in flavins in proteins of the respiratory chain and possibly the Krebs cycle, which temporarily alters cellular energy metabolism. These findings suggest that non-toxic blue light therapy (80 J/cm²) can effectively prevent factor-induced fibroblast differentiation and may serve as a standalone or supportive treatment to reduce fibrotic events such as scarring and keloid formation. Furthermore, our results indicate that targeting cellular energy metabolism, whether physically or pharmacologically, could be a promising strategy to prevent sclerotic skin disorders. Full article