Bergapten (BG), a furanocoumarin compound, has demonstrated diverse pharmacological properties, including the reduction of oxidative stress and inflammation in osteoarthritic chondrocytes. However, the mechanisms underlying BG's effects on chondrocyte differentiation and proliferation remain incompletely understood. This study aimed to elucidate BG's role in chondrocyte differentiation, proliferation, and inflammation prevention using in vitro and in vivo osteoarthritis (OA) models. An OA model was established by treating rabbit primary chondrocytes with sodium nitroprusside (SNP) to induce apoptosis and inflammation. BG treatment significantly upregulated chondrogenic differentiation markers, including type II collagen, SOX9, and aggrecan, and histological staining revealed increased proteoglycan accumulation in OA-induced cells treated with BG. Importantly, BG exhibited pronounced anti-inflammatory effects by modulating the NF-κB pathway: it increased IκBα expression and reduced cyclooxygenase-2 (COX-2) and p50 expression, thereby preventing inflammation in chondrocytes. BG also activated p38 kinase signaling, suggesting its contribution to Chondroprotective effects. Flow cytometry demonstrated that BG reduced SNP-induced apoptosis from 22 to 4%. Western blot analysis confirmed that BG downregulated apoptotic proteins p53 and BAX while upregulating the anti-apoptotic protein Bcl-2. In vivo validation using a zebrafish model showed that BG rescued SNP-induced craniofacial cartilage structural defects. These findings provide novel insights into BG's pharmacological role in promoting chondrocyte differentiation and survival, as well as preventing inflammation, by modulating key inflammatory pathways, apoptotic proteins, and chondrogenic markers. Given its clinical potential, BG may hold promise as a therapeutic agent for OA treatment.

Skeletal muscle tissue consists of not only myofibers, i.e., muscle cells, but also intramuscular adipocytes. Our previous study demonstrated that adipocytes produce secretory factors during differentiation, leading us to hypothesize that soluble factors derived from adipocytes regulate gene expression and cellular function in muscle cells. Yet the mechanism by which coexisting adipocytes influence muscle cells remains unclear. Here, microarray analysis was used to examine transcriptional changes in muscle cells under two co-culture conditions: myoblasts co-cultured with differentiated adipocytes and myotubes co-cultured with preadipocytes. Gene Ontology terms related to cell adhesion, extracellular matrix (ECM) organization, and metabolic processes were significantly enriched in both conditions. We also assessed the influence of adipocyte co-culture on myogenic differentiation and fiber type-specific gene expression. In myoblasts, co-culture with differentiated adipocytes had no significant effect on the expression of myogenic regulatory factors, whereas Myh2 and Myh4 expression was markedly increased in myotubes co-cultured with preadipocytes. These results indicate that adipocyte-derived soluble factors alter the transcriptional landscape of muscle cells, especially genes involved in ECM remodeling and metabolic regulation. This intercellular communication likely contributes to structural and metabolic adaptations in skeletal muscle tissue in vivo.

This study aimed to evaluate the effects of conditioned medium derived from murine skeletal muscle (SMCM) on oxidative stress and testicular morphology in vitro. Initially, Wistar rats underwent treadmill familiarization and a maximal incremental test (MIT). Animals were then submitted to a single exercise session at 60% of the maximum speed established by MIT. In Experiment 1, femoral muscles from trained animals were cultured in αMEM supplemented with 1.25 mg/mL BSA to produce SMCM. In Experiment 2, testes from sedentary rats were fragmented and cultured for 24 h in αMEM alone or αMEM added to irisin at 100 ng/mL or αMEM added to SMCM at 25, 50, 75, or 100%. HPLC confirmed the presence of irisin in SMCM. Oxidative stress analyses demonstrated catalase activity was higher in irisin and 75% of SMCM treatments, while glutathione peroxidase (GPX) activity was significantly higher in the irisin when compared to fresh control. It is important to highlight that 25% of SMCM was similar to fresh control in GPX activity and thiol content. Histological assessment revealed structural alterations in cultured testes, although overall tubular organization was preserved. These findings suggest that skeletal muscle SMCM modulates oxidative balance and testicular structure, with the 25% concentration yielding the most favorable antioxidant profile.

Culturing stem cells in species-specific serum ensures physiological relevance, reduces variability, and supports safer clinical use, highlighting the urgent need to develop reliable, species-matched systems for research and therapy. To address the concept of species specificity in cell culture, we investigated the effects of goat serum (GS) on the derivation of goat muscle stem cells (MuSCs). First, MuSCs were isolated from goat muscle tissue and cultured in media supplemented with either 10% goat GS or fetal bovine serum (FBS). Next, the isolated cells underwent characterization and differentiation. Finally, the effects of varying concentrations of GS and FBS on cell proliferation were evaluated. The results demonstrated that goat MuSCs grew in a GS-containing medium and were positively immunostained for CD29 and Pax7. Gene expression analysis revealed no significant differences in the expression of Pax7, MyoD, and MyoG genes between goat MuSCs grown in GS- or FBS-containing medium. Cells grown in GS-containing medium showed more efficient differentiation toward myogenic and adipogenic lineages than those grown in FBS. Supplementing the culture medium with 10% GS resulted in the greatest enhancement of goat MuSC proliferation, as evidenced by the MTT assay, increased Ki67 expression, and a higher number of colony-forming units. This study demonstrated that GS supplementation is notably beneficial for the proliferation of goat MuSCs.

ARP2/3 is a seven-subunit protein complex involved in the formation of actin filament branching, which is essential for the formation of membrane protrusions, cell migration, and establishment of cell polarity. Among these functions, ARP2/3 has been implicated in myoblast fusion. Since myogenesis is a complex multistep process, here we decided to explore deeper the distribution patterns and functions of ARP2/3 during the initial steps of embryonic chicken skeletal myogenesis. The chosen biological experimental model was the cell culture of pectoralis muscle obtained from 11-d-old chick embryos, which is composed of myoblasts, multinucleated myotubes, and muscle fibroblasts. Our results show that ARP2/3 was found in myoblasts, myotubes, and muscle fibroblasts in four main distributions: the perinuclear region, in small puncta in the cytoplasm, along F-actin structures in the cytoplasm, and in circular structures in myotubes. Inhibition of ARP2/3 function by CK-666 led to a significant reduction in several parameters of skeletal myogenesis, including the area of muscle cells (desmin-positive cells), myotube thickness, the number of myoblasts, the number of nuclei within myotubes, the number of fibroblasts, the total number of nuclei (including nuclei in myoblasts, myotubes, and fibroblasts), and the myoblast fusion index. Interestingly, CK-666 reduced myotube formation and induced the formation of spindle-shaped myoblasts. Live cell video microscopy showed that inhibition of ARP2/3 induced a decrease in myoblast cell migration and the formation of blebs in the membranes of cells. The collection of our results shows that ARP2/3 is essential for the initial steps of embryonic chick skeletal myogenesis, and its inhibition leads to a major reduction in myoblast proliferation, migration, fusion, and muscle fiber formation.

Immunoglobulin A (IgA) nephropathy (IgAN) is characterized by the deposition of IgA1 in the glomerular mesangium, which induces secondary glomerular and tubulointerstitial inflammation and subsequently leads to podocyte apoptosis and fibrosis. This condition often progresses to end-stage renal disease and lacks effective targeted treatment. Our study aimed to explore the role of M2 macrophage-mediated Ubiquitin C-terminal hydrolase L1 (UCHL1) expression in podocytes and its potential impact on the progression of IgAN. This study established an IgAN cellular model by exposing podocytes to aggregated IgA1 (aIgA1)-treated glomerular mesangial cells supernatants and assessed the impact of M2 macrophage polarization on UCHL1 expression and podocyte apoptosis. Additionally, we utilized siRNA technology and overexpression constructs to investigate the direct effects of UCHL1 modulation on podocyte apoptosis. The supernatant from aIgA1-treated glomerular mesangial cells significantly induced apoptosis in podocytes. Based on this, M2 macrophage polarization was induced using interleukin (IL)-4. The results showed that M2 macrophages (CD163) effectively alleviated podocyte apoptosis by reducing the secretion of inflammatory cytokines IL-6, tumor necrosis factor (TNF)-α, and IL-1β, as well as downregulating the expression of apoptosis-related proteins. Notably, M2 macrophages (CD163) inhibited the expression of UCHL1 in podocytes. Blockade of UCHL1 promoted podocyte proliferation, reduced apoptosis, and downregulated the protein expression of the fibrotic markers vascular endothelial growth factor and collagen type IV. Overexpression of UCHL1 reversed the protective effects of M2 macrophages on podocyte apoptosis. M2 macrophage (CD163)-mediated UCHL1 downregulation in podocytes presents a potential therapeutic approach for IgAN by alleviating apoptosis.

Polycystic ovary syndrome (PCOS), a common endocrine disorder affecting over 10% of women, is characterized by hyperandrogenism and ovarian dysfunction. While linked to chronic inflammation and granulosa cell apoptosis, its molecular mechanisms remain unclear. In this study, we elucidated the novel role of Krüppel-like factor 5 (KLF5) in the pathogenesis of PCOS and its regulatory role with thioredoxin interacting protein (TXNIP). In a mouse model of PCOS induced by dehydroepiandrosterone (DHEA), KLF5 expression was significantly elevated in ovarian tissues (up-regulated 2.62-fold, P < 0.001), correlating with hyperandrogenism (testosterone: up-regulated 2.83-fold, P < 0.001) and cystic follicle formation. The proliferative capacity of testosterone-treated KGN cells was reduced to 59% after KLF5 was knocked down (P < 0.01), attenuated apoptosis by inhibiting the increase of Bax and Cleaved-caspase 3 proteins and thus attenuated inflammation by down-regulating NLRP3 and Interleukin- (IL-) 1β. Most importantly, dual luciferase assay showed that KLF5 transcriptionally activated TXNIP, resulting in a 3.04-fold enhancement of its promoter activity (P < 0.001). Meanwhile, overexpression of TXNIP reversed the silencing effect of KLF5, resulting in a significant increase in apoptosis and secretion of inflammatory factors. These results reveal a previously unrecognized KLF5/TXNIP axis driving granulosa cell (GC) dysfunction in polycystic ovary syndrome, in which KLF5 transcriptionally upregulates TXNIP to promote apoptosis and NLRP3 inflammasome activation. Our findings provide the first evidence linking KLF5 to the pathogenesis of PCOS and establish this pathway as a potential therapeutic target, bridging a significant gap in understanding the molecular basis of the disease and providing compelling evidence for clinical drug development.

Obesity resulting from chronic overnutrition and physical inactivity promotes the development of metabolic disorders by disrupting physiological processes in metabolically active organs, including skeletal muscles. To investigate whether skeletal muscle stem cells (satellite cells, SCs) are affected by systemic metabolic stress, we established primary SC cultures from male mice fed a high-fat diet (HFD) for 8 wk, and from control mice fed a standard chow (CTL). This model allowed us to assess diet-induced obesity (DIO)-related changes in SC-specific molecular and cellular signatures. Although body weight, body fat composition, and adipose tissue-associated macrophages differed significantly between DIO and CTL ex vivo, we observed no differences in the in vitro behaviour of primary SC-derived myoblasts from either group. Parameters such as proliferation and differentiation following serum deprivation were comparable. Expression levels and distribution patterns of myogenic regulatory factors (MRF), SC-specific markers (Pax7, CD56, Itga7), and hallmarks for senescence (GLB1), autophagy (p62, LC3B), and oxidative stress (ALDH1A1, ALDH1A3) remained unchanged. Thus, potential differences in the signatures of SC-derived myoblasts after 8 wk of a high-fat diet cannot be depicted in vitro. However, future experiments should address whether prolonged and metabolically more susceptible diets will exert long-term effects on myogenesis in vitro or not. Overall, we propose that primary SC cultures are better suited for acute in vitro testing regarding the molecular and cellular plasticity in metabolic shifts as induced by pharmacological treatments or genetical modifications, rather than for modeling long-term dietary effects.

The skin is the largest organ of the human body, capable of protecting it from external harm. However, due to trauma, paralysis, and other external factors, skin damage can occur, and scars may form. Exosomes have regenerative functions and, as a cell-free therapy, show great potential for wound healing. In this study, we aimed to investigate whether thrombin-preconditioned umbilical cord mesenchymal stem cells (T-pre-UCMSCs) increase the production of exosomes. Different umbilical cord mesenchymal stem cell exosomes can accelerate the healing of skin. In our study, umbilical cord mesenchymal stem cells (UCMCs) were cultured in DMEM/F12 medium without fetal bovine serum (FBS) for 72 h with 200U/ml thrombin. Exosomes were isolated from the supernatant by ultracentrifugation. There are two kinds of exosomes: UCMCs culture supernatant-derived exosomes (UCMSCs-Exos) and T-pre-UCMSCs culture supernatant-derived exosomes (T-UCMSCs-Exos). The skin injury cell model was constructed by treating HaCats with a tip. Additionally, the wound healing capacity of exosomes was evaluated in vivo using a mouse skin injury model. Compared to UCMSCs-Exos, T-UCMSCs-Exos significantly promoted cell proliferation and migration of cells. In vivo experiments demonstrated that T-UCMSCs-Exos can accelerate wound closure and enhance collagen maturation, promoting angiogenesis in the vascularized wound area. These results indicate that T-UCMSCs-Exos have good potential for accelerating wound healing and minimizing scar formation. Our research indicates that thrombin pre-UCMSCs significantly increased the production of exosomes. These findings demonstrate that T-UCMSCs-Exos for skin wounds are a promising cell-free therapy that can be applied in the treatment of skin injuries.

Research shows that transplanted bone marrow mesenchymal stem cells (BMSCs) have been shown to improve functional outcomes in mice with spinal cord injury (SCI). Many experimental centers have demonstrated that systemic delivery of MSCs in mice can treat neurological diseases, but whether or how it works in acute spinal cord injury is not understood. Various methods such as Basso, Beattie, and Bresnahan (BBB) locomotor rating scale score and biological detection of inflammatory factors were used to test the changes in inflammatory factors of spinal cord injury at different time points in 24 h, 7 d, and 14 d. Twenty-four hours after injury, the functional measurement results of the injured group were significantly weakened compared with the control rats. The functional results of the BMSCs injection injured group were also significantly weakened compared with the control rats. There was no statistical difference between the injured group and the BMSCs injection group. However, the injury group had the highest mortality rate (p < 0.05). Biochemical results showed that compared with the control group, the expression of high-mobility group box 1 (HMGB1) and receptor for advanced glycation end-products (RAGE) and related inflammatory factors in the injury group and BMSCs injection group increased significantly at 7 d after the experiment in Western blot. Similarly, the expression of HMGB1 and RAGE in the injury group was also greater than that in the injection group, and there was a statistical difference in immunohistochemical assessment. Injecting BMSCs into rats with acute spinal cord injury could reduce rat mortality and improve prognostic functional measurements after SCI. BMSCs may promote spinal cord re-repair by inhibiting the HMGB1/RAGE signaling pathway after acute contusive spinal cord injury.

This study aimed to extract and isolate endometrial stromal cells from Arabian mares and investigate their growth and differentiation potential. Endometrial biopsies were obtained from three healthy 6-year-old Arabian mares using a standardized, minimally invasive protocol. The isolated cells were characterized using flow cytometry and differentiation analysis. Flow cytometry revealed mesenchymal markers CD90 (95.2%) and CD105 (97.4%) and hematopoietic markers CD34 (1.17%) and CD45 (0.339%). The cells exhibited differentiation potential into adipocytes, osteoblasts, and chondrocytes. The findings suggest that endometrial cells from Arabian mares represent a promising autologous source of MSCs, particularly suited for regenerative applications in musculoskeletal disorders.

The molecular mechanisms underlying growth hormone (GH) therapy in children with idiopathic short stature (ISS) remain incompletely understood. This study investigated how GH promotes bone growth in children with ISS, focusing on insulin-like growth factor-binding protein 2 (IGFBP2) and thrombospondin-1 (THBS1). Analysis of ISS patient plasma showed downregulated IGFBP2, predicted to interact strongly with THBS1. Experiments using human chondrocytes revealed that GH treatment stimulated cell proliferation, accelerated the cell cycle, and induced hypertrophic differentiation, marked by increased expression of proteins like COL10A1, RUNX2, OCN, OPN, and alkaline phosphatase activity. GH also elevated IGFBP2 and insulin-like growth factor-1 (IGF-1) while suppressing THBS1. Crucially, knocking down IGFBP2 blocked these GH effects, reducing proliferation, halting cell cycle progression, decreasing differentiation markers and IGF-1, while increasing THBS1. Conversely, overexpressing IGFBP2 mimicked GH's effects. Importantly, silencing IGFBP2 partially prevented GH-induced proliferation, differentiation, and IGF-1 secretion. This demonstrates that IGFBP2 acts as a key mediator of GH's action by inhibiting THBS1, which subsequently activates the IGF-1 pathway to drive chondrocyte proliferation and hypertrophic differentiation. The IGFBP2-THBS1 axis is thus a core mechanism for GH therapy in ISS, offering a novel therapeutic target for improving treatment.

Cisplatin-based chemotherapy is the first-line treatment for lung cancer. However, cisplatin resistance (CR) remains a major challenge, leading to treatment failure. A key driver of CR is enhanced DNA damage repair. Although males absent on the first (MOF) participate in DNA repair, their specific role in mediating CR remains unclear. In this study, CR models were established in PC9 and A549 lung cancer cell lines. Our results showed that high expression of Williams syndrome transcription factor (WSTF) in lung cancer cells was associated with CR. WSTF knockdown inhibited proliferation and promoted apoptosis, DNA damage, and γ-H2AX levels in CR cells. Moreover, MOF was highly expressed in lung cancer cells and regulated by WSTF acetylation. Furthermore, MOF knockdown downregulated H4K16ac levels in CR cells. MOF overexpression significantly upregulated H4K16ac levels, enhanced proliferation, and suppressed apoptosis in CS cells, concomitant with DNA damage repair and reduced γ-H2AX expression. Notably, transfection with the K46R attenuated these MOF-mediated effects in CS cells. Collectively, our study demonstrates that MOF promotes DNA damage repair and enhances CR in lung cancer cells via H4K16ac-mediated WSTF acetylation. These findings provide valuable insights for overcoming chemoresistance and improving patient outcomes.

Tissue densification, as a hallmark of development, injury, and fibrosis, alters the mechanical and structural properties of the extracellular matrix (ECM). However, its specific effects on neural cell behavior remain poorly understood. To address this, we developed a 3D in vitro microtissue model composed of collagen and Matrigel, incorporating co-cultures of N2A neuroblastoma and 3T3 fibroblast cells. By modulating fibroblast-driven contraction through scaffold-guided constraint, we generated microtissues with distinct levels of densification with Young's moduli ranging from approximately 0.5 to 1 kPa. Our results demonstrated that increased microtissue densification significantly enhanced N2A migration and aggregate formation, indicating that mechanical compaction facilitates neuronal clustering. Furthermore, higher densification promoted N2A cell proliferation, while apoptosis remained at relatively low baseline levels, suggesting that dense environments support cell expansion without affecting overall viability. Additionally, higher densification suppressed the proportion of neurite-bearing cells without affecting neurite length, implying impaired initiation of neuronal differentiation but not morphological maturation. Together, these findings reveal that ECM densification serves as a critical mechanical cue shaping multiple aspects of neural cell behavior. Compared to conventional hydrogel systems, our collagen-based microtissue model provides a more physiologically relevant in vitro platform for studying neurodevelopmental mechanobiology and guiding biomaterial design for neural tissue engineering.

Coho salmon (Oncorhynchus kisutch) is an important salmonid species differing from other salmonids in its tolerance and response to pathogens endemic to the aquaculture industry, such as infectious pancreatic necrosis virus (IPNV) and infectious salmon anaemia virus (ISAV). Consequently, coho salmon has become a subject of increased scientific interest to investigate the underlying genetic mechanisms behind these and other host-pathogen interactions. Currently, most research studying coho salmon has been conducted using live animal models as there have been few in vitro tools readily available. Here, we present the first cell line from an adult coho salmon, Coho Salmon Fibroblast-Like 1 Norway-Canada (CSFL-1NC) and its preliminary characterisation. CSFL-1NC is a homogenous, spontaneously immortalised cell line from the pectoral fin of a wild adult coho salmon, with a consistent and stable fibroblastic morphology. The cell line has a relatively stable transcriptome across several passages, with high expression of key fibroblastic marker genes, displays rapid migration, and can be genetically manipulated both by transfection and transduction with varying efficiency using plasmids, lentivirus, and/or CRISPR methodology. Virus challenges show clear susceptibility to IPNV as evidenced by cytopathic effects and efficient viral replication, yet it shows little to no response when exposed to ISAV (HPRD).

The current study aimed to explore the effect of Cadmium (Cd) on nucleus pulposus derived mesenchymal stem cells (NPMSCs) and the possible mechanism of IVDD caused by Cd. In this study, cell viability assay, EdU assay, TUNEL staining, flow cytometry assay, mRNA transcriptome sequencing, quantitative real-time polymerase chain reaction (PCR) assay, immunofluorescence assay and western blot assay were used to prove that Cadmium induces apoptosis of NPMSCs. Cd impaired the proliferation of NPMSCs and promoted cell apoptosis, and this effect was time and concentration dependent. Further study also found that the expression levels of senescence-related molecules (P16, P21 and P53) in the Cd group were up-regulated and the expression levels of pro-apoptotic molecules Bax and Caspase-3 in the Cd group were significantly up-regulated, while the expression level of anti-apoptotic molecule Bcl-2 was significantly down-regulated compared with those of the Control group. The MAPK signaling pathway-related proteins were detected, and the results found that the ratios of p-P38/P38 and p-JNK/JNK in the Cd group were significantly increased, while the ratios of p-ERK/ERK was significantly less compared with the control group, and it was in a concentration-dependent relationship. Cd can inhibit the activity and proliferation of NPMSCs in a dose and time-dependent manner, and promote cell aging and apoptosis. Cd may promote the apoptosis of NPMSCs by activating MAPK signaling pathway.

Endothelial cell damage often results in apoptosis and is thus a crucial factor for the development of coronary artery disease (CAD). However, the mechanisms underlying endothelial cell apoptosis remain unclear. Although circular (circ) RNAs have been implicated in apoptosis, the involvement of hsa_circ_0124644 is uncertain. Therefore, the aim of this study was to investigate the effect of hsa_circ_0124644 on endothelial cell apoptosis and to elucidate the underlying molecular processes. We treated endothelial cells with tumor necrosis factor alpha (TNF-α) to simulate the microenvironment of CAD and generate an apoptosis model. We measured cell apoptosis in conjunction with hsa_circ_0124644 expression. After hsa_circ_0124644 overexpression or inhibition, we assessed apoptosis levels using flow cytometry, RT-qPCR, and western blotting. We found that hsa_circ_0124644 overexpression lowered the apoptosis rate and increased cell viability. Similarly, overexpression also upregulated the expression of an anti-apoptotic protein (Bcl-2) and downregulated that of a pro-apoptotic protein (Bax). In conclusion, our findings suggested that hsa_circ_0124644 mediates endothelial cell apoptosis in CAD. These findings have important implications for developing effective treatments of cardiovascular conditions with excessive apoptosis.

Resistance to radiation therapy (RT) poses a significant challenge in managing non-small cell lung cancer (NSCLC). Despite research into how tumor-sourced exosome (Exo) miRNAs influence tumor RT resistance and macrophage M2 polarization, the process through which Exos with miR-616-3p modulate macrophage polarization to impact NSCLC RT resistance is still not well understood. The objective of this research was to investigate the molecular processes by which RT regulates M2 polarization of macrophages via the Exos miR-616-3p derived from NSCLC cells. Identification of Exos was conducted using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Flow cytometry, immunofluorescence, and ELISA were employed to verify the macrophage phenotype. The expression of miR-616-3p was identified using RT-qPCR, and the targeting relationship between miR-616-3p and PTEN was confirmed through dual-luciferase reporter gene tests and RIP identification. In NSCLC, miR-616-3p showed high expression levels and was linked to RT and M2 polarization in macrophages. Subsequent research indicated that RT prompted the influx of Exos-miR-616-3p from NSCLC cells into macrophages. Both the H1299 lung cancer cell line and the M0 macrophages underwent co-culture. Findings indicated that NSCLC cells induced by RT and Exos elevated the proportion of CD163 + CD206 + positive cells in macrophages via miR-616-3p and augmented Arg1, IL-10, TGF-β1, and VEGF levels, and enhanced M2 polarization in macrophages. Regarding the molecular process, miR-616-3p suppressed PTEN protein expression while concurrently boosting the levels of p-PI3K/PI3K and p-AKT/AKT; either amplifying PTEN or suppressing PI3K could markedly weaken the impact of ionizing radiation (IR), inhibiting the impact of NSCLC cell Exos on macrophages' M2 polarization. This research reveals that in NSCLC cells induced by IR, Exos with miR-616-3p expression reduce PTEN levels and enhance the PI3K/AKT signaling pathway, leading to increased M2 polarization in macrophages and worsening NSCLC progression.

Rapid growth of the aquaculture industry is hampered by infectious diseases in marine invertebrates, causing economic losses. Marine invertebrate cell cultures offer tools to evaluate biological properties and cellular responses in different conditions. Long-term culture aims to isolate tissue-specific cells and identify bioactive compounds from stem cells. Echinometra mathaei, known as Persian Gulf sea urchin, has lots of benefits in various fields including aquaculture, embryology, and evolutionary biology. However, its cell culture faces challenges due to poorly characterized microenvironmental and specific cultivation requirements. This study aims to establish and optimize a long-term cell culture for coelomocyte derived from E. mathaei, focusing on the characterization of microenvironment conditions to overcome the limitations of current marine invertebrate cell culture. After the collection of E. mathaei from Lark Island, Persian Gulf, Iran, and their acclimatization in artificial seawater, coelomocytes were isolated from different sources including the coelomic fluid, the coelomic epithelium, and the axial organ. Various cell dissociation methods, culture media, growth supplements, culture dishes, and physical conditions were tested to determine optimal conditions for coelomocyte in vitro culture. Moreover, coelomocytes were differentiated to pigment-producing cells, and naphthoquinone pigments were extracted and identified using spectrophotometry. Light microscopy identified several coelomocyte types, including petaloid, filopodial, vibratile cells, and spherulocytes. The HCCM medium supplemented with coelomic fluid proved most effective for cell growth and viability. Moreover, coelomic fluid is the best culture media for differentiation of coelomocyte into the cell producing naphthoquinone pigments. These findings contribute to developing in vitro cell culture methods for sea urchin, providing a foundation for further research on sea urchin immunology, cell biology, and cellular responses to pathogens and other biological stress.