Although traumatic brain injury (TBI), particularly recurrent concussions during adolescence, is strongly associated with post-traumatic hyperactivity, the role of mitochondrial dysfunction in driving this behavioral alteration remains poorly understood and insufficiently characterized. In this context, we investigated the effects of varying frequencies of recurrent concussions on exploratory behavior, hippocampal mitochondrial bioenergetics, and nitrosative stress in adolescent male Swiss mice (32 days old) subjected to a weight-drop model over 10 days and allocated to Control, 4, 7, and 10-concussion groups. Exploratory behavior was assessed using the open field test 24 h post-injury, while hippocampal mitochondrial respiration (high-resolution respirometry) and nitrosative stress markers (3-nitrotyrosine and iNOS) were evaluated 6 days after the final injury. Twenty-four hours after the final injury, behavioral hyperactivity peaked following 7 concussions. While this phenotype was attenuated after 10 recurrent concussions, exploratory alterations (increased proportion of mobile time, greater time spent in the center, and fewer immobile episodes) persisted relative to controls. Respirometry at 6 days post-injury indicated a threshold effect for mitochondrial dysfunction: although 7 concussions compromised maximal electron transfer capacity, basal and phosphorylation states were impaired only after 10 concussions, resulting in a reduction in ATP synthase activity. The severe bioenergetic failure coincided with increased hippocampal protein nitrosylation (3-NT), indicating a progressive metabolic deterioration subsequent to the early behavioral hyperactivity. Our findings suggest that early hyperactivity precedes bioenergetic failure, reflecting the sequential progression of the injury. Thus, this transient behavioral phenotype may serve as a predictive marker for subsequent mitochondrial collapse. Dose-dependent neurometabolic and behavioral progression following recurrent concussions in the hippocampus of young mice. The diagram illustrates the sequential relationship between the acute behavioral phenotype observed 24 h post-injury and the subacute mitochondrial dysfunction evaluated 6 days post-injury. Initially, exposure to 4 concussions induces early exploratory alterations, characterized by an increased proportion of mobility time in the open field, but without significant impairments in the respiratory chain complexes, indicating an early mitigation of metabolic stress. Subsequently, 7 concussions trigger a peak in motor hyperactivity, evidenced by a significant increase in line crossings. This behavioral peak precedes the onset of mitochondrial stress, which is marked by a compromised maximal electron transfer capacity (ETS) and early Complex I inhibition. Finally, the cumulative damage of 10 concussions leads to an attenuation of the hyperactive locomotor phenotype, suggesting a threshold effect. Concurrently, this stage is characterized by a severe bioenergetic collapse featuring the sustained inhibition of NADH dehydrogenase (Complex I) and a drastic reduction in ATP synthase activity. This severe metabolic failure directly coincides with elevated hippocampal nitrosative stress, demonstrated by the significant accumulation of 3-nitrotyrosine (3-NT). Collectively, these findings raise the possibility that acute hyperactivity could serve as an early behavioral correlate to the later development of mitochondrial exhaustion and nitrosative damage.
Breast cancer metastasis is driven by metabolic changes and cellular plasticity, particularly epithelial-mesenchymal transition (EMT). Increasing evidence suggests that increased plasticity due to EMT can be used to reprogram malignant cells toward terminal differentiation. This study investigates the therapeutic potential of Ppar-γ agonists in regulating lipid metabolism and transforming breast cancer cells into post-mitotic, adipocyte-like phenotypes. Using the Py2T mouse breast cancer cell model, EMT was induced with TGF-β, followed by treatment of mesenchymal-like cells with a combination of rosiglitazone (Ppar-γ agonist), trametinib (MEK inhibitor), and paclitaxel (conventional chemotherapeutic agent). Adipogenic phenotypic conversion of the cells was assessed by histological staining (Oil Red O), quantitative PCR, and RNA sequencing targeting 180 genes associated with lipid metabolism and signaling. Our findings demonstrate that mesenchymal breast cancer cells retain significant metabolic plasticity and can be successfully directed towards an adipocyte-like lineage. The combination of rosiglitazone and trametinib significantly increased intracellular lipid accumulation and notably elevated adipogenic markers, including Fabp4 and Ppar-γ. Transcriptomic analysis revealed a coordinated metabolic reprogramming process integrating lipid biosynthesis with cellular motility programs (Itgb3-Itgav) and centered around the Fasn-Srebp1 axis. Furthermore, the addition of paclitaxel consistent with its known antiproliferative pharmacological action, did not further enhance lipid accumulation beyond that observed with rosiglitazone and trametinib alone, while the adipocyte-like phenotype was preserved. In conclusion, this study identifies coordinated changes in lipid metabolism-associated genes during EMT and adipogenic reprogramming of Py2T breast cancer cells. These findings are consistent with a dynamic metabolic regulatory network centered on lipid and sterol metabolism and that warrant further mechanistic validation. Furthermore, they suggest that differentiation-based approaches to target metabolic vulnerabilities and suppress cellular plasticity in aggressive breast cancer subtypes may represent a promising therapeutic strategy that merits further investigation.
Oral submucous fibrosis (OSF) is an oral potentially malignant disorder strongly associated with the areca nut alkaloids. Although lysyl oxidase-like 2 (LOXL2) is implicated in pathological collagen accumulation and fibrosis, its regulatory role in OSF progression remains unclear. Hematoxylin-eosin, Masson and immunofluorescent staining were performed on rat oral mucosal tissues to assess fibrotic and senescent phenotypes. Senescence of human oral keratinocytes (HOKs) was assessed by senescence-associated β-galactosidase staining, flow cytometry and western blotting. LOXL2 N6-methyladenosine (m6A) methylation levels in tissues and cells were then detected, and methyltransferase-like 3 (METTL3)-LOXL2 interactions were validated using RNA immunoprecipitation and photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation assays. LOXL2 mRNA stability was further assessed after actinomycin D treatment. In addition, lactate levels were measured to explore epithelial-fibroblast crosstalk between HOKs and human oral mucosal fibroblasts. Arecoline-treated rats exhibited progressive epithelial thinning and collagen deposition in oral mucosal tissues, accompanied by upregulated expression of fibronectin, collagen type I, α-smooth muscle actin, p21, and p16. Arecoline exposure elevated reactive oxygen species levels and promoted co-localization of the senescence markers p16/p21 with the epithelial marker cytokeratin. LOXL2 was upregulated in arecoline-induced senescence models, and LOXL2 silencing suppressed fibroblast activation by inhibiting HOKs senescence. Mechanistically, METTL3 stabilized LOXL2 mRNA through m6A-dependent interactions. Downregulation of the METTL3/LOXL2 axis reduced lactate secretion from senescent HOKs, thereby attenuating fibroblast activation. Similar findings were observed in OSF rats. The METTL3/LOXL2 axis may mediate lactate secretion from senescent epithelial cells and regulate epithelial-fibroblast crosstalk during OSF pathogenesis. This study reveals a potential mechanism and candidate targets for further evaluation in OSF.
Renal cell carcinoma (RCC) exhibits marked intratumoral heterogeneity that contributes to disease progression and therapeutic resistance. Although ROCK2 has been implicated in RCC progression, its role in regulating tumor cell heterogeneity and cell-state plasticity remains poorly understood. Using single-cell RNA sequencing, we identified a ROCK2-associated stress-responsive transcriptional state characterized by inflammatory signaling and enhanced calcium pathway activity. ROCK2 knockdown was associated with a lower representation of this transcriptional state together with proliferative tumor cells, and integration with the TCGA-KIRC cohort supported the presence of this stress-associated transcriptional program in bulk RCC tumors. Mechanistically, ROCK2 depletion reduced intracellular calcium levels, suppressed CALM1 and CREB1 expression, promoted apoptosis, and impaired cell-cycle progression and proliferation. Pharmacological restoration of calcium signaling with Bay K8644, together with CALM1 or CREB1 overexpression, partially rescued these defects, supporting functional interactions among ROCK2, calcium signaling, CALM1, and CREB1. Collectively, our findings identify a ROCK2-associated stress-responsive transcriptional state linked to altered calcium signaling and provide new mechanistic insight into RCC cellular heterogeneity, highlighting the functional relationship between ROCK2 and calcium signaling as a potential therapeutic vulnerability in RCC.
Chronic pancreatitis is a progressive fibroinflammatory disease in which activated pancreatic stellate cells and extracellular matrix deposition drive irreversible tissue remodeling. Octreotide has anti-secretory and anti-inflammatory properties, but its anti-fibrotic mechanism remains unclear. To determine whether octreotide attenuates DBTC-induced pancreatic fibrosis through suppression of high-mobility group box 1 (HMGB1) and downstream Wnt/β-catenin signaling. Male Sprague–Dawley rats received dibutyltin dichloride (DBTC, 7 mg/kg) to induce chronic pancreatitis and were assigned to seven groups (n = 10/group) comprising untreated controls, DBTC model rats, and DBTC rats treated with octreotide alone or in combination with HMGB1 overexpression, HMGB1 knockdown, a vector control, or pharmacological Wnt/β-catenin activation. Octreotide was administered at 10 µg/kg/day for 21 days. Pancreatic injury, collagen deposition, stellate cell activation, HMGB1 inflammatory signaling, and Wnt/β-catenin activity were assessed by biochemical assays, histology, hydroxyproline measurement, immunofluorescence, Western blotting, qPCR, and ELISA. DBTC caused weight loss, pancreatic atrophy, elevated amylase/lipase, severe collagen accumulation, and marked increases in collagen I, α-smooth muscle actin (α-SMA), HMGB1, TLR4/RAGE, phospho-NF-κB p65, inflammatory cytokines, active/nuclear β-catenin, Wnt1, phospho-GSK3β, Axin2, c-Myc, and Cyclin D1 (all p < 0.0001 vs. Control). Octreotide substantially reversed these changes, halving fibrosis and hydroxyproline content and suppressing HMGB1 translocation/release (all p < 0.0001 vs. Model). HMGB1 overexpression weakened, whereas HMGB1 knockdown mimicked, octreotide-mediated protection. Wnt activation restored β-catenin activity and fibrosis without significantly changing HMGB1 or TLR4/RAGE/NF-κB signaling (all p > 0.15), a pattern consistent with HMGB1 acting upstream of Wnt/β-catenin. Octreotide limits experimental pancreatic fibrosis by suppressing HMGB1-driven inflammation and downstream Wnt/β-catenin activation, identifying the HMGB1–Wnt/β-catenin axis as a promising anti-fibrotic target.
