In Vitro Cellular & Developmental Biology. Animal最新文献

To develop a biological pacemaker by differentiating rabbit BMSCs into vascular endothelial cells (VECs) and pacemaker-like cells, and constructing a 3D vascularized sinoatrial node (SAN) in vitro. BMSCs were isolated via whole bone marrow adherence. Sinoatrial node cells were purified using differential adhesion with 5-BrdU treatment, and their lysate was prepared by freeze-thaw cycling. BMSCs were induced into VECs (CD31+/CD34+) using endothelial medium and into pacemaker-like cells (HCN2+/cTnT+) using SAN lysate. These cells were co-cultured on Matrigel at 1:1, 1:2, and 2:1 ratios to form 3D vascularized constructs. Cell distribution was analyzed via frozen sectioning and H&E staining. BMSCs successfully differentiated into VECs and pacemaker-like cells, confirmed by marker expression. The 1:1 co-culture ratio optimally promoted uniform cell distribution, network formation, and angiogenesis in the Matrigel scaffold. This study demonstrates that BMSCs can be differentiated into functional pacemaker-like cells and VECs, enabling the in vitro construction of a vascularized tissue-engineered SAN-a promising step toward biological pacemaker development.

This study aimed to explore the function of miR-495-3p in cerebral ischemia-reperfusion injury (CI/RI) and reveal its potential molecular mechanism. In vivo and in vitro models of CI/RI were established by MACO/R and OGD/R, respectively. Neural function scores, HE staining, and TUNEL staining assessed the degree of brain tissue injury in mice. LDH assay, MTT assay, and flow cytometry evaluated neuronal toxicity, viability, and apoptosis rate. ELISA and Western blot evaluated inflammatory factors and the NF-κB pathway. Dual-luciferase reporting assay and RIP explored the targeting relationship between miR-495-3p and CCL2. miR-495-3p was abnormally low in MACO/R mouse brain tissue and OGD/R-damaged neurons, while CCL2 was highly expressed. miR-495-3p overexpression improved neuronal apoptosis and inflammation in the brain tissue of MACO/R mice. Consistent results were also obtained in in vitro experiments. Enhancing CCL2 or knocking down miR-495-3p aggravated OGD/R-induced neuronal damage. The deleterious effects of miR-495-3p knockdown were prevented by the knockdown of CCL2. miR-495-3p targeted CCL2. miR-495-3p improves CI/R-mediated neuronal apoptosis and inflammation through targeted regulation of CCL2 expression. These results provide data support for CI/RI-targeting drugs and the understanding of disease mechanisms.

Acute liver failure (ALF), a life-threatening condition marked by rapid hepatocyte death and systemic inflammation, poses significant clinical challenges due to its high mortality. The crosstalk between necrotic hepatocytes and infiltrating immune cells is hypothesized to drive disease progression. To investigate this interplay, we developed a sequential "two-hit" murine model using concanavalin A (Con A) challenges and compared its pathophysiological outcomes with the conventional single-dose "one-hit" approach. The results demonstrated that the "two-hit" model induced more severe hepatic coagulation dysfunction, extensive hepatocellular necrosis, destruction of liver lobular architecture, and inflammatory responses. Furthermore, serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) were markedly elevated in the "two-hit" group. Inflammatory cytokines including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were also significantly elevated. Moreover, substantial recruitment of macrophages was observed in the "two-hit" model, indicating that these cells are key determinants in the interaction with dying hepatocytes for the progression of ALF. Complementary ex vivo experiments revealed that Raw264.7 cells subjected to a "two-hit" stimulation with Con A and hepatocyte debris produced a robust inflammatory response through the classical NF-κB signaling pathway.

Intervertebral disc degeneration (IVDD), a natural aging process, leads to intervertebral disc pain, with lumbar resection providing pain relief but potential postoperative complications affecting outcomes. The chitosan-collagen-salvianolic (CCS) membrane has shown promise in reducing scar formation after lumbar resection, though the mechanisms remain unclear. A lumbar laminectomy model was used, with materials applied to the surgical site. Tissue samples were collected at 4 and 8 wk post-surgery for histological evaluation to assess pathological changes and apoptosis. Western blot analysis examined protein expression in the Wnt/β-catenin signaling pathway in scar tissue. Rabbit vascular adventitia fibroblast (VAF) cells were isolated, and a Wnt3a overexpression vector was created. Various composite membrane materials and pathway inhibitors were tested, with assessments of cell invasion, apoptosis, viability, cytokine levels, and protein expression. Results showed that lumbar laminectomy activated the Wnt/β-catenin pathway. The CCS membrane facilitated healing by inhibiting this pathway. It enhanced VAF cell activity, reduced apoptosis, and suppressed Wnt3a pathway activity. Additionally, it protected cells from Wnt3a overexpression-induced damage while maintaining normal function. In conclusion, the CCS composite membrane promoted bone tissue repair after lumbar laminectomy by inhibiting Wnt/β-catenin pathway activation and preserving extracellular matrix fibroblast function.

Cellular metabolic reprogramming is intimately linked to various physiological and pathological processes. For instance, calcium (Ca²⁺)-mediated signaling pathways are essential for maintaining the homeostasis of critical cellular organelles. Stromal interaction molecule 1 (STIM1)-mediated store-operated calcium entry (SOCE) is a primary pathway for Ca²⁺ influx in non-excitable cells. This study aims to elucidate the role of STIM1 in podocyte injury. An STIM1 eukaryotic overexpression plasmid (p-STIM1) and small interfering RNA (si-STIM1) were constructed and separately transfected into mouse podocytes (MPC5). Flow cytometry was used to assess apoptotic rates, Fluo-3/AM calcium imaging to measure intracellular Ca²⁺ levels, and Western blotting to analyze the expression of endoplasmic reticulum stress (ERS)-related proteins. Additionally, mitochondrial morphology, membrane potential (MMP), reactive oxygen species (ROS) levels, and mitochondrial DNA (mtDNA) copy numbers were evaluated. Compared to STIM1 deficiency, STIM1 overexpression led to a marked increase in the apoptotic rate of Adriamycin-induced injured podocytes in vitro. This was associated with a significant rise in intracellular Ca²⁺ concentration and upregulation of ERS-related proteins, including GRP78, GRP94, and CHOP. Mitochondria displayed pronounced swelling and vacuole-like changes, a notable reduction in MMP, elevated ROS levels, and a decrease in mtDNA copies. STIM1 exacerbates podocyte injury by promoting intracellular Ca²⁺ influx, intensifying ERS, and inducing significant morphological and functional mitochondrial alterations. These findings suggest that targeting STIM1-mediated pathways could be a potential therapeutic strategy for podocyte-related kidney diseases.

Stress is a key trigger of gastric dysmotility, partly via mitochondrial dysfunction and disordered gut-brain hormonal signaling. Xinwei Tang (XWT) is a multi-herb formula used empirically for upper gastrointestinal symptoms, but its mechanisms remain unclear. This study aimed to determine whether XWT alleviates water-immersion restraint stress (WIRS)-induced gastric dysmotility and to delineate underlying mitochondrial and metabolic pathways using integrated in vivo, in vitro and multi-omics approaches. Male rats underwent 7-d WIRS and received vehicle, domperidone (3 mg/kg) or XWT (3, 6, 12 g/kg). Gastric emptying, serum motilin/gastrin, oxidative stress indices and PINK1/Parkin-LC3/p62 proteins were assessed, and H₂O₂-injured GES-1 cells were treated with XWT-medicated serum. Gastric antra from MOD and XWT-H rats were analyzed by RNA-seq and DIA proteomics (n = 3/group). WIRS reduced gastric emptying by roughly half and lowered motilin/gastrin, increased ROS/MDA and disrupted PINK1/Parkin-LC3/p62 profiles; XWT dose-dependently reversed these changes, with XWT-H approximating domperidone. Omics revealed XWT-associated downregulation of inflammatory/protease and acute-phase genes/proteins and enrichment of oxidative phosphorylation, tricarboxylic-acid cycle and other metabolic pathways, without global activation of canonical autophagy/mitophagy gene sets. These preclinical data indicate that XWT ameliorates stress-induced gastric dysmotility via mitochondria- and metabolism-centred protection with selective tuning of mitophagy-related proteins.

Lipocalin-2 (LCN-2) is an adipocytokine secreted mainly by adipose tissue. Increasing evidences suggest that LCN-2 is an inflammatory factor associated with insulin resistance, obesity, and its complications. The precise mechanism of the development of obesity-related disorders induced by LCN-2 is not very clear. This study evaluated the expression of LCN-2 in pig tissues and its molecular mechanism of regulating preadipocyte differentiation in porcine intramuscular preadipocytes. LCN-2 expression in tissues of Tongcheng pigs, intramuscular adipose tissue of Tongcheng pigs (fat type) and Landrace pigs (lean type) in embryonic stage and growth stage, and adipocyte differentiation-induced porcine intramuscular preadipocytes was detected using reverse transcription polymerase chain reaction (qRT-PCR). After LCN-2 treatment, cell viability was measured by the methyl thiazolyl tetrazolium (MTT) method, mRNA expression of CCAAT/enhancer binding protein-ɑ (C/EBPɑ), adipocyte determination and differentiation factor-1 (ADD1), fatty acid desaturase (FAD), fatty acid synthase (FAS), and glucose transporter (GLUT) 1,4 was determined by qRT-PCR, protein expression of LCN-2 and peroxisome proliferator-activated receptor-γ (PPARγ) was analyzed by Western blot. Results of qRT-PCR indicated that LCN-2 showed significantly higher expression in high-intramuscular fat (IMF) pigs compared to low-IMF pigs (P < 0.05). LCN-2 expression in porcine intramuscular preadipocytes was significantly upregulated after adipocytic differentiation induction (P < 0.05). Silencing of LCN-2 with LCN-2 siRNA (siLCN-2) inhibited cell viability, lipid droplets, protein expression of PPARγ, and mRNA expression of C/EBPa, ADD1, FAD, FAS, and GLUT 1,4. siLCN-2 treated cells also showed a lower content of triglyceride and release of glucose. Moreover, LCN-2-induced downregulation of cell viability, adipocytic differentiation, adipogenesis, and glycometabolism of porcine intramuscular preadipocytes was partially blocked by the PPARγ inhibitor (GW9662). It is indicated that LCN-2 silencing suppresses cell viability, adipocytic differentiation and adipogenesis, glycometabolism, and fat deposition of porcine intramuscular preadipocytes through suppression of the PPARγ signaling pathway.

The induction of osteogenic differentiation in preadipocytes may serve as a potential therapeutic approach for treating osteoporosis and osteoporotic fractures. All-trans retinoic acid (ATRA) promotes the bone morphogenetic protein 9 (BMP9)-induced osteogenic differentiation of preadipocytes. The present study further investigated whether vascular endothelial growth factor A (VEGFA) may play a role in this process and the effect of ATRA and BMP9 on osteoporotic fracture healing in rats. The results indicated that ATRA and BMP9 synergistically upregulated VEGFA expression in preadipocytes. Furthermore, knockdown of VEGFA expression abolished the stimulatory effect of ATRA on the BMP9-induced early and late osteogenic differentiation of preadipocytes in vitro, as evidenced by a decrease in alkaline phosphatase (ALP) activity, osteopontin and osteocalcin expression as well as mineralization. The in vivo cell implantation assay showed that ATRA failed to augment BMP9-induced ectopic bone formation in the absence of VEGFA. Subsequently, an osteoporotic femoral fracture rat model was established and micro-CT scans, alongside quantitative analysis, revealed that ATRA effectively promoted BMP9-stimulated callus formation during osteoporotic fracture healing. Moreover, ATRA and BMP9 acted together to significantly elevate VEGFA expression in bone calluses. Mechanistically, ATRA and BMP9 synergistically stimulated the osteogenic transcription factor, runt-related transcription factor 2 (Runx2). Transcriptomic and bioinformatic analyses further revealed that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway may be crucial for mediating the synergistic effects of ATRA and BMP9, as validated by detecting the phosphorylation of PI3K and Akt. However, the increases in Runx2 expression and Akt phosphorylation induced by the combination of ATRA and BMP9 were inhibited by VEGFA silencing. ATRA also failed to increase the BMP9-induced ALP activity in preadipocytes treated with an Akt inhibitor. These findings suggest that VEGFA may influence the potentiation effect of ATRA on the BMP9-mediated osteogenesis of preadipocytes and in osteoporotic fracture healing through Runx2 and the PI3K/Akt signaling pathway.

The modified Taohong Siwu decoction (MTHSWD), a traditional prescription, demonstrates notable efficacy in treating cardiovascular diseases. However, its potential mechanisms in addressing atherosclerosis (AS) require further exploration. Investigating the underlying mechanisms of MTHSWD in treating AS is imperative. AS mouse models received MTHSWD treatment, with vascular pathology and fibrosis assessed through histopathological staining. Network pharmacology identified bioactive components and potential targets, followed by molecular docking verification of key compound-target interactions. Biochemical validation included Western blot analysis. MTHSWD significantly ameliorated vascular structural abnormalities and fibrosis in AS models. Network pharmacology analysis revealed 133 active components targeting 295 proteins, intersecting with 3309 AS-related genes to form 191 overlapping targets. Twenty-two components (e.g., quercetin) demonstrated multi-target activity (≥ 15 targets). PPI network topology identified 32 hub targets including AKT1, MAPK1/8/14. Functional analysis showed that apoptosis, MAPK signaling, and lipid metabolism pathways were significantly enriched. The pathway-target network highlighted AKT1 and MAPK family members as central regulators. Molecular docking confirmed strong interactions (binding energy < -9.0 kcal/mol) between AKT1-stigmasterol/anhydroicaritin, MAPK1-emodin, and MAPK8/14-β-carotene. Experimental validation demonstrated MTHSWD's regulation of AKT1, MAPK1/14, cleaved-caspase-3, and Bcl-2 expression, enhancing endothelial viability while suppressing apoptosis. This integrated approach reveals that MTHSWD exerts anti-AS effects through multi-target modulation of PI3K/AKT and MAPK signaling pathways, promoting endothelial survival and attenuating apoptotic processes, ultimately alleviating AS-related vascular pathology.

The development of a reliable ex vivo intestinal model is essential for studying gut physiology and host-microbe interactions under controlled conditions. This study evaluated the temporal dynamics of viability, morphology, and apoptotic regulation in chicken ileal explants cultured ex vivo. Ileal tissues from 21-d-old broiler chickens were incubated for 0, 4, and 8 h and analyzed using metabolic assays (MTS, CCK-8), histological staining (hematoxylin and eosin (H&E), Periodic Acid-Schiff (PAS)), and quantitative PCR for apoptosis-related genes (BCL2L11, cytochrome c, caspase-3, caspase-8, and caspase-9). After 4 h, explants retained structural integrity with well-developed villi and active mucus secretion. Metabolic assays confirmed preserved cell viability, while transcriptional analysis revealed moderate upregulation of cytochrome c and downregulation of BCL2L11, suggesting transient adaptation to culture stress. After 8 h, villi appeared shortened with epithelial exfoliation and reduced PAS reactivity, indicating progressive tissue deterioration. Correspondingly, BCL2L11 and cytochrome c were significantly upregulated together with caspase-8, reflecting activation of intrinsic and extrinsic apoptotic pathways. Metabolic activity declined markedly, confirming reduced tissue vitality. These findings demonstrate that chicken ileal explants with a diameter of 4 mm remain viable and functionally stable up to approximately 4 h of ex vivo culture, after which apoptotic processes intensify, compromising tissue integrity. The results define the practical viability window for short-term intestinal explant studies and provide methodological guidance for future investigations on host-pathogen or probiotic interactions in poultry gut models.