ObjectiveSpinal cord injury (SCI) directly impairs the regulatory function of the autonomic nervous system, induces intestinal dysfunction, and significantly reduces patients’ quality of life. Preclinical studies have shown that electroacupuncture (EA) therapy can regulate the brain-gut axis and is used to treat central nervous system diseases such as major depressive disorder, Alzheimer’s disease and Parkinson’s disease. Recent research has established that fecal microbiota transplantation (FMT) from EA-treated SCI rats restored intestinal motility and colonic morphology. However, it remains unclear whether the regulation of gut microbiota by EA therapy directly contributes to neural repair after SCI. This study aims to explore whether gut microbiota mediates the neuroprotective effect of EA in the treatment of SCI and its possible mechanism. MethodsThe study employed RNA transcriptome analysis of spinal cord tissue to characterize gene expression profiles and to identify key signaling pathways following EA treatment for SCI. Hematoxylin-Eosin (HE) staining and Nissl staining were used to observe the morphological changes in spinal cord tissue. Western blot (WB) and enzyme-linked immunosorbent assay (ELISA) were applied to detect the effects of EA on the expression of proteins related to nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3) -dependent pyroptosis. Using 16S rDNA sequencing, the study observed alterations in gut microbiota diversity and community composition in SCI rats. Prior to establishing SCI models, rats were pretreated with an antibiotic cocktail to induce gut dysbiosis, and the effects on intestinal function and spinal cord neural repair were evaluated. FMT was performed to investigate the regulatory effects of post-EA FMT on motor function, general status, liver and spleen indices, and NLRP3-mediated pyroptosis in SCI rats. ResultsEA improved motor function and reduced regulated neuronal cell death in SCI rats. Transcriptomic analysis demonstrated the activation of immune- and inflammation-related pathways post-SCI, including NOD-like receptors, nuclear factor-kappa B(NF-κB), and Toll-like receptor (TLR) pathways. EA primarily influenced intestinal inflammation and autoimmune functions. 16S rDNA sequencing illustrated that EA did not alter the diversity of gut microbiota. However, EA altered the gut microbiota composition in SCI rats, increasing Lactobacillus and Akkermansia genera while rebalancing the Firmicutes/Bacteroidetes ratio. Furthermore, depletion of gut microbiota by antibiotics disrupted the intestinal barrier, reduced the expression of intestinal barrier proteins Zonula Occludens-1 (ZO-1) and Occludin, elevated serum lipopolysaccharide-binding protein (LBP) levels, exacerbated spinal cord tissue damage, and hindered motor function recovery in SCI rats. FMT from donors treated with EA reduced LBP levels in the intestine, blood, and spinal cord of rats, inhibited the TLR4 myeloid differentiation primary response protein 88 (MyD88)-NF‑κB pathway and NLRP3-dependent pyroptosis, and improved motor function. On the other hand, FMT treatment resulted in decreased body weight and food intake, whereas FMT using EA-treated donors effectively alleviated these alterations. ConclusionEA effectively alleviated neuroinflammatory responses in rats with SCI, primarily through regulating the gut microbiota and suppressing the NLRP3-dependent pyroptosis signaling pathway.

ObjectiveSpinal cord injury (SCI) directly impairs the regulatory function of the autonomic nervous system, induces intestinal dysfunction, and significantly reduces patients’ quality of life. Preclinical studies have shown that electroacupuncture (EA) therapy can regulate the brain-gut axis and is used to treat central nervous system diseases such as major depressive disorder, Alzheimer’s disease and Parkinson’s disease. Recent research has established that fecal microbiota transplantation (FMT) from EA-treated SCI rats restored intestinal motility and colonic morphology. However, it remains unclear whether the regulation of gut microbiota by EA therapy directly contributes to neural repair after SCI. This study aims to explore whether gut microbiota mediates the neuroprotective effect of EA in the treatment of SCI and its possible mechanism. MethodsThe study employed RNA transcriptome analysis of spinal cord tissue to characterize gene expression profiles and to identify key signaling pathways following EA treatment for SCI. Hematoxylin-Eosin (HE) staining and Nissl staining were used to observe the morphological changes in spinal cord tissue. Western blot (WB) and enzyme-linked immunosorbent assay (ELISA) were applied to detect the effects of EA on the expression of proteins related to nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3) -dependent pyroptosis. Using 16S rDNA sequencing, the study observed alterations in gut microbiota diversity and community composition in SCI rats. Prior to establishing SCI models, rats were pretreated with an antibiotic cocktail to induce gut dysbiosis, and the effects on intestinal function and spinal cord neural repair were evaluated. FMT was performed to investigate the regulatory effects of post-EA FMT on motor function, general status, liver and spleen indices, and NLRP3-mediated pyroptosis in SCI rats. ResultsEA improved motor function and reduced regulated neuronal cell death in SCI rats. Transcriptomic analysis demonstrated the activation of immune- and inflammation-related pathways post-SCI, including NOD-like receptors, nuclear factor-kappa B(NF-κB), and Toll-like receptor (TLR) pathways. EA primarily influenced intestinal inflammation and autoimmune functions. 16S rDNA sequencing illustrated that EA did not alter the diversity of gut microbiota. However, EA altered the gut microbiota composition in SCI rats, increasing Lactobacillus and Akkermansia genera while rebalancing the Firmicutes/Bacteroidetes ratio. Furthermore, depletion of gut microbiota by antibiotics disrupted the intestinal barrier, reduced the expression of intestinal barrier proteins Zonula Occludens-1 (ZO-1) and Occludin, elevated serum lipopolysaccharide-binding protein (LBP) levels, exacerbated spinal cord tissue damage, and hindered motor function recovery in SCI rats. FMT from donors treated with EA reduced LBP levels in the intestine, blood, and spinal cord of rats, inhibited the TLR4 myeloid differentiation primary response protein 88 (MyD88)-NF‑κB pathway and NLRP3-dependent pyroptosis, and improved motor function. On the other hand, FMT treatment resulted in decreased body weight and food intake, whereas FMT using EA-treated donors effectively alleviated these alterations. ConclusionEA effectively alleviated neuroinflammatory responses in rats with SCI, primarily through regulating the gut microbiota and suppressing the NLRP3-dependent pyroptosis signaling pathway.

Background and aims The mechanism of cholestatic liver injury (CLI) is unclear, and effective therapies are lacking. While peroxisome proliferator-activated receptor alpha (PPARα) agonists show potential hepatoprotective effect and pyroptosis is implicated in hepatocellular damage, how PPARα activation mitigates lithocholic acid (LCA)-induced pyroptosis remains unknown. Methods The hepatoprotective effect of PPARα agonists was evaluated in a mouse model of intrahepatic cholestasis induced by LCA. Liver injury was assessed via serum biochemistry, hematoxylin and eosin and TUNEL staining, and electron microscopy. Pyroptosis pathways were analyzed using real-time quantitative polymerase chain reaction, Western blot, and co-immunoprecipitation. Results Combined morphological, histopathological, and biochemical analyses confirmed that PPARα activation protects against CLI. Compared with LCA treatment alone, PPARα activation significantly attenuated the elevation of serum lactate dehydrogenase (LDH), the increased TUNEL-positive cells, and the formation of hepatocyte membrane pores. Mechanistically, PPARα activation suppressed both NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis and apoptosis protease-activating factor-1 (APAF-1)/CASPASE-3/GSDME-mediated pyroptosis. Furthermore, PPARα agonist pretreatment inhibited activation of the nuclear factor-kappa B (NF-κB) and forkhead box O1 (FOXO1) signaling pathways. Conclusions PPARα protects against LCA-induced CLI by inhibiting both NLRP3 inflammasome-mediated pyroptosis associated with NF-κB and APAF-1/CASPASE-3/GSDME-mediated pyroptosis associated with the FOXO1 signaling pathway.

RNA modifications constitute a crucial class of post-transcriptional chemical alterations that profoundly influence RNA stability and translational efficiency, thereby shaping cellular protein expression profiles. These diverse chemical marks are ubiquitously involved in key biological processes, including cell proliferation, differentiation, apoptosis, and metastatic potential, and they exert precise regulatory control over these functions. A major advance in the field is the recognition that RNA modifications do not act in isolation. Instead, they participate in complex, dynamic interactions—through synergistic enhancement, antagonism, competitive binding, and functional crosstalk—forming what is now termed the “RNA modification interactome” or “RNA modification interaction network.” The formation and functional operation of this interactome rely on a multilayered regulatory framework orchestrated by RNA-modifying enzymes—commonly referred to as “writers,” “erasers,” and “readers.” These enzymes exhibit hierarchical organization within signaling cascades, often functioning in upstream-downstream sequences and converging at critical regulatory nodes. Their integration is further mediated through shared regulatory elements or the assembly into multi-enzyme complexes. This intricate enzymatic network directly governs and shapes the interdependent relationships among various RNA modifications. This review systematically elucidates the molecular mechanisms underlying both direct and indirect interactions between RNA modifications. Building upon this foundation, we introduce novel quantitative assessment frameworks and predictive disease models designed to leverage these interaction patterns. Importantly, studies across multiple disease contexts have identified core downstream signaling axes driven by specific constellations of interacting RNA modifications. These findings not only deepen our understanding of how RNA modification crosstalk contributes to disease initiation and progression, but also highlight its translational potential. This potential is exemplified by the discovery of diagnostic biomarkers based on interaction signatures and the development of therapeutic strategies targeting pathogenic modification networks. Together, these insights provide a conceptual framework for understanding the dynamic and multidimensional regulatory roles of RNA modifications in cellular systems. In conclusion, the emerging concept of RNA modification crosstalk reveals the extraordinary complexity of post-transcriptional regulation and opens new research avenues. It offers critical insights into the central question of how RNA-modifying enzymes achieve substrate specificity—determining which nucleotides within specific RNA transcripts are selectively modified during defined developmental or pathological stages. Decoding these specificity determinants, shaped in large part by the modification interactome, is essential for fully understanding the biological and pathological significance of the epitranscriptome.

The current study aimed to clarify the roles of apolipoprotein A5 (ApoA5) and milk fat globule-epidermal growth factor 8 (Mfge8) in regulating myocardial lipid deposition and the regulatory relationship between them. The serum levels of ApoA5 and Mfge8 in obese and healthy people were compared, and the obesity mouse model induced by the high-fat diet (HFD) was established. In addition, primary cardiomyocytes were purified and identified from the hearts of suckling mice. The 0.8 mmol/L sodium palmitate treatment was used to establish the lipid deposition cardiomyocyte model in vitro. ApoA5-overexpressing adenovirus was used to observe its effects on cardiac function and lipids. The expressions of the fatty acid uptake-related molecules and Mfge8 on transcription or translation levels were detected. Co-immunoprecipitation was used to verify the interaction between ApoA5 and Mfge8 proteins. Immunofluorescence was used to observe the co-localization of Mfge8 protein with ApoA5 or lysosome-associated membrane protein 2 (LAMP2). Recombinant rMfge8 was added to cardiomyocytes to investigate the regulatory mechanism of ApoA5 on Mfge8. The results showed that participants in the simple obesity group had a significant decrease in serum ApoA5 levels (P < 0.05) and a significant increase in Mfge8 levels (P < 0.05) in comparison with the healthy control group. The adenovirus treatment successfully overexpressed ApoA5 in HFD-fed obese mice and palmitic acid-induced lipid deposition cardiomyocytes, respectively. ApoA5 reduced the weight of HFD-fed obese mice (P < 0.05), shortened left ventricular isovolumic relaxation time (IVRT), increased left ventricular ejection fraction (LVEF), and significantly reduced plasma levels of triglycerides (TG) and cholesterol (CHOL) (P < 0.05). In myocardial tissue and cardiomyocytes, the overexpression of ApoA5 significantly reduced the deposition of TG (P < 0.05), transcription of fatty acid translocase (FAT/CD36) (P < 0.05), fatty acid-binding protein (FABP) (P < 0.05), and fatty acid transport protein (FATP) (P < 0.05), and protein expression of Mfge8 (P < 0.05), while the transcription levels of Mfge8 were not significantly altered (P > 0.05). In vitro, the Mfge8 protein was captured using ApoA5 as bait protein, indicating a direct interaction between them. Overexpression of ApoA5 led to an increase in co-localization of Mfge8 with ApoA5 or LAMP2 in cardiomyocytes under lipid deposition status. On this basis, exogenous added recombinant rMfge8 counteracted the improvement of lipid deposition in cardiomyocytes by ApoA5. The above results indicate that the overexpression of ApoA5 can reduce fatty acid uptake in myocardial cells under lipid deposition status by regulating the content and cellular localization of Mfge8 protein, thereby significantly reducing myocardial lipid deposition and improving cardiac diastolic and systolic function.
Animals ; Humans ; Mice ; Myocytes, Cardiac/metabolism* ; Obesity/physiopathology* ; Male ; Apolipoprotein A-V/blood* ; Lipid Metabolism/physiology* ; Milk Proteins/blood* ; Myocardium/metabolism* ; Diet, High-Fat ; Antigens, Surface/physiology* ; Mice, Inbred C57BL ; Cells, Cultured ; Female

Intelligence encompasses various abilities, including logical reasoning, comprehension, self-awareness, learning, planning, creativity, and problem-solving. Extensive research and practical experience suggest that there are sex differences in intellectual development, with females typically maturing earlier than males. However, the key genes and molecular network mechanisms underlying these sex differences in intellectual development remain unclear. To date, Genome-Wide Association Studies (GWAS) have identified 507 genes that are significantly associated with intelligence. This study first analyzed RNA sequencing data from different stages of brain development (from BrainSpan), revealing that during the late embryonic stage, the average expression levels of intelligence-related genes are higher in males than in females, while the opposite is observed during puberty. This study further constructed interaction networks of intelligence-related genes with sex-differential expression in the brain, including the prenatal male network (HELP-M: intelligence genes with higher expression levels in prenatal males) and the pubertal female network (HELP-F: intelligence genes with higher expression levels in pubertal females). The findings indicate that the key genes in both networks are Ep300 and Ctnnb1. Specifically, Ep300 regulates the transcription of 53 genes in both HELP-M and HELP-F, while Ctnnb1 regulates the transcription of 45 genes. Ctnnb1 plays a more prominent role in HELP-M, while Ep300 is more crucial in HELP-F. Finally, this study conducted sequencing validation on rats at different developmental stages, and the results indicated that in the prefrontal cortex of female rats during adolescence, the expression levels of the intelligence genes in HELP-F, as well as key genes Ep300 and Ctnnb1, were higher than those in male rats. These genes were also involved in neurodevelopment-related biological processes. The findings reveal a sex-differentiated intelligence gene network and its key genes, which exhibit varying expression levels during the neurodevelopmental process.
Female ; Intelligence/physiology* ; Male ; Sex Characteristics ; Animals ; Brain/growth & development* ; E1A-Associated p300 Protein/physiology* ; beta Catenin/physiology* ; Transcriptome ; Rats ; Gene Expression Profiling ; Genome-Wide Association Study

The development of bone in human body and the maintenance of bone mass in adulthood are regulated by a variety of biological factors. Myocyte enhancer factor 2C (MEF2C), as one of the many factors regulating bone tissue development and balance, has been shown to play a key role in bone development and metabolism. However, there is limited systematic analysis on the effects of MEF2C on bone tissue. This article reviews the role of MEF2C in bone development and metabolism. During bone development, MEF2C promotes the development of neural crest cells (NC) into craniofacial cartilage and directly promotes cartilage hypertrophy. In terms of bone metabolism, MEF2C exhibits a differentiated regulatory model across different types of osteocytes, demonstrating both promoting and other potential regulatory effects on bone formation, with its stimulating effect on osteoclasts being determined. In view of the complex roles of MEF2C in bone tissue, this paper also discusses its effects on some bone diseases, providing valuable insights for the physiological study of bone tissue and strategies for the prevention of bone diseases.
Humans ; MEF2 Transcription Factors/physiology* ; Bone and Bones/metabolism* ; Animals ; Bone Development/physiology* ; Osteogenesis/physiology* ; Myogenic Regulatory Factors/physiology*

Guided by the theory of "the kidney storing essence, governing the bones, and producing marrow", this study explored the mechanism of icariin(ICA) in regulating the osteogenic differentiation of rat bone mesenchymal stem cells(BMSCs) through caveolin-1(Cav1) via in vitro and in vivo experiments, aiming to provide a theoretical basis for the prevention and treatment of postmenopausal osteoporosis with traditional Chinese medicine(TCM). Primary cells were obtained from 4-week-old female SD rats using the whole bone marrow adherent method. Flow cytometry was used to detect the expression of surface markers CD29, CD90, CD11b, and CD45. The potential for osteogenic and adipogenic differentiation was assessed. The effect of ICA on cell viability was determined using the CCK-8 assay, and the impact of ICA on the formation of mineralized nodules was verified by alizarin red staining. A stable Cav1-silenced cell line was constructed using lentivirus. The effect of Cav1 silencing on osteogenic differentiation was observed via alizarin red staining. Western blot analysis was conducted to detect the expression of Cav1, Hippo/TAZ, and osteogenic markers such as Runt-related transcription factor 2(RUNX2) and alkaline phosphatase(ALP). The results showed that primary cells were successfully obtained using the whole bone marrow adherent method, positively expressing surface markers of rat BMSCs and possessing the potential for both osteogenic and adipogenic differentiation. The CCK-8 assay and alizarin red staining results indicated that 1×10~(-7) mol·L~(-1) was the optimal concentration of ICA for intervention in this experiment(P<0.05). During osteogenic induction, ICA inhibited Cav1 expression(P<0.05) while promoting TAZ expression(P<0.05). Alizarin red staining demonstrated that Cav1 silencing significantly promoted the osteogenic differentiation of BMSCs. After ICA intervention, TAZ expression was activated, and the expression of osteogenic markers ALP and RUNX2 was increased. In conclusion, Cav1 silencing significantly promotes the osteogenic differentiation of BMSCs, and ICA promotes this differentiation by inhibiting Cav1 and regulating the Hippo/TAZ signaling pathway.
Animals ; Mesenchymal Stem Cells/metabolism* ; Caveolin 1/genetics* ; Osteogenesis/drug effects* ; Rats, Sprague-Dawley ; Rats ; Cell Differentiation/drug effects* ; Female ; Signal Transduction/drug effects* ; Flavonoids/administration & dosage* ; Protein Serine-Threonine Kinases/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Cells, Cultured ; Humans

Metal ion-promoted chemodynamic therapy(CDT) combined with photodynamic therapy(PDT) offers broad application prospects for enhancing anti-tumor effects. In this study, glycyrrhizic acid(GA), copper ions(Cu~(2+)), and norcantharidin(NCTD) were co-assembled to successfully prepare a natural small-molecule, carrier-free hydrogel(NCTD Gel) with excellent material properties. Under 808 nm laser irradiation, NCTD Gel responded to the tumor microenvironment(TME) and acted as an efficient Fenton reagent and photosensitizer, catalyzing the conversion of endogenous hydrogen peroxide(H_2O_2) within the tumor into oxygen(O_2), and hydroxyl radicals(·OH, type Ⅰ reactive oxygen species) and singlet oxygen(~1O_2, type Ⅱ reactive oxygen species), while depleting glutathione(GSH) to stabilize reactive oxygen species and alleviate tumor hypoxia. In vitro and in vivo experiments demonstrated that NCTD Gel exhibited significant CDT/PDT synergistic therapeutic effects. Further safety evaluation and metabolic testing confirmed its good biocompatibility and safety. This novel hydrogel is not only simple to prepare, safe, and cost-effective but also holds great potential for clinical transformation, providing insights and references for the research and development of metal ion-mediated hydrogel-based anti-tumor therapies.
Hydrogels/chemistry* ; Animals ; Photochemotherapy ; Humans ; Mice ; Antineoplastic Agents/administration & dosage* ; Photosensitizing Agents/chemistry* ; Neoplasms/metabolism* ; Female ; Copper/chemistry* ; Reactive Oxygen Species/metabolism* ; Tumor Microenvironment/drug effects* ; Cell Line, Tumor ; Male

This study investigated the effect of Wuling San on transforming growth factor-β1(TGF-β1)-induced fibrosis, inflammation, and oxidative stress in human renal tubular epithelial cells(HK-2) and its mechanism of antioxidant stress injury. HK-2 cells were cultured in vitro and divided into a control group, a TGF-β1 model group, and three treatment groups receiving Wuling San-containing serum at low(2.5%), medium(5.0%), and high(10.0%) doses. TGF-β1 was used to establish the model in all groups except the control group. CCK-8 was used to analyze the effect of different concentrations of Wuling San on the activity of HK-2 cells with or without TGF-β1 stimulation. The expression of key fibrosis molecules, including actin alpha 2(Acta2), collagen type Ⅰ alpha 1 chain(Col1α1), collagen type Ⅲ alpha 1 chain(Col3α1), TIMP metallopeptidase inhibitor 1(Timp1), and fibronectin 1(Fn1), was detected using qPCR. The expression levels of inflammatory cytokines, including tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-8(IL-8), and interleukin-4(IL-4), were measured using ELISA kits. Glutathione peroxidase(GSH-Px), malondialdehyde(MDA), catalase(CAT), and superoxide dismutase(SOD) biochemical kits were used to analyze the effect of Wuling San on TGF-β1-induced oxidative stress injury in HK-2 cells, and the expression of nuclear factor E2-related factor 2(Nrf2), heme oxygenase 1(HO-1), and NAD(P)H quinone oxidoreductase 1(NQO1) was analyzed by qPCR and immunofluorescence. The CCK-8 results indicated that the optimal administration concentrations of Wuling San were 2.5%, 5.0%, and 10.0%. Compared with the control group, the TGF-β1 model group showed significantly increased levels of key fibrosis molecules(Acta2, Col1α1, Col3α1, Timp1, and Fn1) and inflammatory cytokines(TNF-α, IL-1β, IL-6, IL-8, and IL-4). In contrast, the Wuling San administration groups were able to dose-dependently inhibit the expression levels of key fibrosis molecules and inflammatory cytokines compared with the TGF-β1 model group. Wuling San significantly increased the activities of GSH-Px, CAT, and SOD enzymes in TGF-β1-stimulated HK-2 cells and significantly inhibited the level of MDA. Furthermore, compared with the control group, the TGF-β1 model group exhibited a significant reduction in the expression of Nrf2, HO-1, and NQO1 genes and proteins. After Wuling San intervention, the expression of Nrf2, HO-1, and NQO1 genes and proteins was significantly increased. Correlation analysis showed that antioxidant stress enzymes(GSH-Px, CAT, and SOD) and Nrf2 signaling were significantly negatively correlated with key fibrosis molecules and inflammatory cytokines in the TGF-β1-stimulated HK-2 cell model. In conclusion, Wuling San can inhibit TGF-β1-induced fibrosis in HK-2 cells by activating the Nrf2 signaling pathway, improving oxidative stress injury, and reducing inflammation.
Humans ; Oxidative Stress/drug effects* ; Transforming Growth Factor beta1/metabolism* ; Fibrosis/genetics* ; Cell Line ; Drugs, Chinese Herbal/pharmacology* ; Epithelial Cells/immunology* ; Inflammation/metabolism*