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The KunMingShanHaiTang Formula (KMSHTF), adjusted by Professor Zhong Chuanhua for the treatment of ulcerative colitis (UC), is the work of a renowned veteran practitioner of Chinese medicine. However, its specific mechanism remains unknown. Consequently, it is intriguing to investigate the molecular mechanism by which KMSHTF treats UC. To elucidate the mechanism of KMSHTF in the treatment of UC in rats. Initially, the active ingredients and key target genes of KMSHTF in treating UC were analyzed using network pharmacology. Protein-Protein interaction and gene enrichment analyses were performed to predict key targets and pathways. Subsequently, UC rats were treated with KMSHTF, and the expression proteins in intestinal tissue were detected. Finally, the active compounds of KMSHTF intreating ulcerative colitis were further screened using Molecular Docking, and their pharmacological effects were validated through cell experiments. A total of 47 active compounds and 365 key target genes of KMSHTF for UC treatment were identified through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,along with the GeneCards database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Enrichment Analysis revealed that KMSHTF exerted its therapeutic effects on UC through regulating multiple pathways. In this study, the HIF-1α pathway was selected as the main molecular pathway of KMSHTF treating UC, and further validation was conducted through in vivo and in vitro experiments.Animal studies revealed that KMSHTF significantly ameliorated UC symptoms in rats, including diarrhea,rectal bleeding and specific pathological alterations in the intestinal wall. Furthermore, KMSHTF reduced pro-inflammatory cytokines IL-6 and TNF-α, up-regulated IL-4 of M2 macrophages and down-regulated iNOS and IL-1β of M1 macrophages. Additionally, it decreased the expression levels of HKII and GLUT1 related HIF-1α pathway. The three active compounds of KMSHTF, Baicalein, Palmatine and Triptonide-were selected based on their strong binding affinity with HIF-1α and HKII through computational molecular docking. Cellular experiments demonstrated that each of these compounds downregulated the protein expression levels of HIF-1α, HKII, GLUT1 and IL-6 in an intestinal wall cell model. Of Note, Baicalein exhibited the most pronounced effect. However, the overexpression of HIF-1α reversed the Baicalein-induced downregulation of HKII, GLUT1 and IL-6 at the protein level in vitro. KMSHTF may modulate macrophage metabolism to promote M2 polarization through the HIF-1α pathway, thereby contributing to its therapeutic efficacy in ulcerative colitis (UC). Baicalein, Palmatine, and Triptonide are the three core active compounds of KMSHTF that primarily contribute to this hypothesis.
To investigate the role and molecular mechanisms of the RNA-binding protein MATR3 in myocardial fibrosis of atrial fibrillation (AF). Expression of MATR3 and MSI2 in AF patients was analyzed using GEO datasets (GSE79768, GSE14975, GSE31821). Human atrial fibroblasts (HAFs) induced by Angiotensin II (Ang-II) were used as an in vitro cellular model of myocardial fibrosis. Expression and interactions of MATR3, METTL3, and MSI2 were validated by qRT-PCR and Western blot. The binding between MATR3 and METTL3 was confirmed by co-immunoprecipitation (Co-IP). The m6A modification level of MSI2 mRNA was detected by methylated RNA immunoprecipitation (MeRIP-qPCR). Cell proliferation, migration, and fibrotic phenotypes were evaluated by CCK-8, EdU, scratch, and Transwell assays, as well as detection of fibrosis markers. An Ang-II-induced mouse model of atrial fibrosis was constructed, and the in vivo effects of MATR3 were verified by HE staining, Masson's trichrome staining, and molecular detection. Analysis of GEO datasets showed that both MATR3 and MSI2 were highly expressed in AF patients. Ang-II treatment significantly upregulated the expression of MATR3 in HAFs, while knockdown of MATR3 inhibited Ang-II-induced proliferation, migration, and pro-fibrotic phenotypic changes in HAFs (reducing the expression of α-SMA, collagen I/III). Mechanistically, MATR3 interacted endogenously with METTL3 and stabilized the METTL3 protein by inhibiting proteasomal degradation. METTL3 mediated the m6A modification of MSI2 mRNA, enhancing its stability and promoting its expression. MSI2 exerted a pro-fibrotic effect by activating the Wnt/β-Catenin pathway. In vivo experiments confirmed that silencing MATR3 downregulated the expression of METTL3 and MSI2, inhibited the activation of the Wnt pathway, and alleviated Ang-II-induced atrial fibrosis in mice. MATR3 promotes myocardial fibrosis and exacerbates AF by regulating METTL3-mediated m6A modification of MSI2 mRNA to activate the Wnt/β-Catenin pathway. Targeting MATR3 may represent a potential therapeutic strategy for AF.
Cerebral infarction is a leading cause of severe long-term disability and functional and cognitive impairment. With the advancement of acute stroke treatment, more patients are now experiencing stroke with varying degrees of impairment. The present study was conducted to determine the effects of 1-week 3,4-dihydroxyflavonol (DiOHF) administration during transient experimental cerebral ischemia-reperfusion (I/R) in rats on NeuN, Tuba1a, Tubb3, and calbindin were evaluated as markers of neuronal phenotype and cytoskeletal organization, while ICAM and BDNF were assessed in relation to inflammatory and neurotrophic processes. Changes in these markers indicate alterations in neuronal marker expression, cytoskeletal integrity, and neurotrophic and inflammatory status. In this study, a total of 28 male Wistar albino rats, aged 10-12 weeks and weighing 300-400 g, were used. 1-Control Group (n = 6): The animals in this group received no anaesthesia or surgical procedures. 2-Sham Group (n = 6): After general anaesthesia was induced in the animals in this group, the carotid artery regions were opened and closed. 3- Ischemia-Reperfusion Group (n = 8): Under general anaesthesia, the carotid arteries of the rats were isolated and ligated for 30 min, followed by ischemia. 4- Ischemia-Reperfusion + DiOHF Group (n = 8): Under general anaesthesia, the carotid arteries of the rats were ligated for 30 min, followed by ischemia. Reperfusion was then allowed. Tuba1A, Tubb3, ICAM and Calbindin were analyzed by real-time PCR, BDNF by a commercial ELISA kit, and NeuN by immunohistochemistry. I/R decreased the levels of Neu N, Tuba1a, Tubb3, calbindin and BDNF in the striatum tissues and increased ICAM levels. DiOHF supplementation halted the decrease in expression level in Tuba1a, Tubb3, Calbindin, BDNF; the Increase in Icam level.Also DiOHF supplementation prevented the decrease in the level of anti-NeuN antibody and led to an increase. The study results revealed that one week of transient I/R in rats suppressed NeuN, TUBA1A, TUBB3, calbindin and BDNF levels, which are important in neuronal phenotype and cytoskeletal organization inflammatory and neurotrophic processes. However, a week DiOHF treatment significantly corrected the distortions caused by I/R.
This study aimed to investigate the therapeutic effects of Sini Decoction on a murine model of peripheral arterial disease (PAD) and to explore its potential mechanisms of action related to mitochondrial autophagy and M1 macrophage polarization. A total of 36 specific-pathogen-free Kunming mice were used to establish a PAD model and were randomly assigned into four groups: the experimental group (EG, administered Sini Decoction via gavage), the control group (CG, administered rapamycin via gavage), the model group (MG, administered 0.9% sodium chloride solution via gavage), and the normal group (NG, administered 0.9% sodium chloride solution via gavage). Serum inflammatory cytokines, mitochondrial autophagy-related proteins (LC3bII and p62), M1 macrophage markers (iNOS and COX2), key proteins in the mitochondrial autophagy pathway (PINK1 and Parkin), relative mitochondrial DNA (mtDNA) content, and mitochondrial function indicators [oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)] were measured and analyzed. The serum levels of IL-6, IL-1β, TNF-α, IL-10, and MCP-1 were significantly decreased in both the EG and CG compared to the MG (P < 0.05), with the EG showing considerably greater reductions than the CG (P < 0.05). Compared with the CG, the EG exhibited significantly increased protein and mRNA expression levels of LC3bII, p62, iNOS, and COX2 (P < 0.05), considerably elevated mitochondrial OCR, and considerably reduced ECAR (P < 0.05). Additionally, the relative mtDNA content and the percentage of atherosclerotic lesion area were markedly lower in the EG than in the CG (P < 0.05). Moreover, the expression level of PINK1 and Parkin proteins were significantly increased in both the EG and CG compared to the MG (P < 0.05). Sini Decoction demonstrated superior efficacy in ameliorating PAD compared to the autophagy inducer rapamycin. Its therapeutic effects may be associated with the promotion of mitochondrial autophagy and the induction of M1 macrophage polarization.
Cataracts are a significant cause of vision loss, adversely affecting the quality of human life. Numerous studies have reported that lens epithelial cells (LECs) play a crucial role in age-related cataract (ARC). However, the roles of carboxypeptidase B 1 (CPB1) and transcription factor BTB and CNC homologue 2 (BACH2) in the pathogenesis of ARC remain unclear. In this study, we aim to explore the contributions of CPB1 and BACH2 to the development of ARC. The Gene Expression Omnibus (GEO) was utilized to screen for differentially expressed genes. mRNA and protein levels were assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analysis. Flow cytometry was conducted to analyze apoptosis. The levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and malondialdehyde (MDA) were measured using a commercial kit. Dual-luciferase reporter assays and chromatin immunoprecipitation (CHIP) were performed to investigate the interaction between CPB1 and BACH2. The methylation site of BACH2 was analyzed using the RNA-protein binding sites prediction suite and the sequence-based RNA adenosine methylation site predictor suite. Methylated RNA immunoprecipitation (Me-RIP) was employed to detect m6A modification level of BACH2. In ARC and H2O2-induced human lens epithelial cells (HLECs), CPB1, BACH2, and METTL3 were found to be up-regulated. Silencing CPB1 reduced apoptosis and MDA levels while enhancing the activities of SOD and GSH-PX in H2O2-induced HLECs. Additionally, CPB1 was shown to bind to BACH2, and knockdown of BACH2 attenuated apoptosis and oxidative stress in H2O2-induced HLECs by targeting CPB1. Notably, METTL3 promoted the BACH2 expression by enhancing CPB1 expression in H2O2-induced HLECs. Finally, silencing METTL3 inhibited apoptosis and oxidative stress in H2O2-induced HLECs by hampering BACH2 expression. METTL3 facilitates apoptosis and oxidative stress in H2O2-induced HLECs by promoting the modification of BACH2 and CPB1 expression.
Renal ischemia/reperfusion injury (RIRI), a common complication of renal transplantation, partial nephrectomy, and transient hypoperfusion, is a major etiological factor of acute kidney injury (AKI) with limited treatment options. Total flavonoids from Desmodium styracifolium (TFDS), a traditional Chinese medicinal herb used in urinary disorders, have shown promising renoprotective properties. This study aimed to investigate the efficacy of TFDS against RIRI and elucidate its underlying mechanisms, with a particular focus on oxidative stress and ferroptosis. A RIRI model was established in C57BL/6J mice, and the effects of TFDS were evaluated in both in vivo and in vitro hypoxia/reoxygenation (H/R) models. Evaluation of renal function was performed by measuring serum blood urea nitrogen (BUN) and creatinine levels. Histopathological and ultrastructural alterations were examined using hematoxylin-eosin (H&E) staining and transmission electron microscopy (TEM). Oxidative stress and ferroptosis were evaluated by determining glutathione (GSH) levels, malondialdehyde (MDA) content, reactive oxygen species (ROS) levels, and iron accumulation. Potential therapeutic targets and pathways were predicted by network pharmacology and further validated through Western blot (WB) and immunofluorescence analyses. In vivo, TFDS administration markedly improved renal function in RIRI mice, as evidenced by significant reductions in serum BUN and creatinine levels, and attenuated histopathological damage, including tubular epithelial cell loss and mitochondrial structural disruption. TFDS also decreased tissue iron and malondialdehyde (MDA) levels while restoring GSH content, thereby alleviating oxidative stress and ferroptosis. In vitro, TFDS enhanced HK-2 cell viability after hypoxia/reoxygenation injury, reduced intracellular ROS, iron, and MDA accumulation, and preserved mitochondrial morphology. Network pharmacology and molecular docking identified TP53 as a central target, with vicenin-2, schaftoside, and isovitexin exhibiting strong binding affinity to P53. Mechanistically, TFDS downregulated P53 expression and upregulated SLC7A11 and GPX4 both in vivo and in vitro, effects that were abolished by the P53 agonist Kevetrin, confirming the involvement of the P53/SLC7A11/GPX4 axis in TFDS-mediated ferroptosis suppression. TFDS alleviates kidney injury following RIRI by attenuating oxidative stress and suppressing ferroptosis, effects mediated at least in part through modulation of the P53/SLC7A11/GPX4 signaling axis. These findings identify TFDS as a promising therapeutic candidate for ischemic kidney injury and provide mechanistic insight supporting its potential clinical application.
Metabolic associated fatty liver disease (MAFLD) is a highly prevalent global chronic liver disease. While abnormal expression of RNA-binding proteins (RBPs) has been implicated in MAFLD, their functional roles-particularly in regulating alternative splicing (AS)-remain poorly characterized. This study aimed to investigate the abnormal expression and regulatory mechanism of RBPs in MAFLD. The source data were obtained from the GSE130970 dataset of the Gene Expression Omnibus (GEO) database. Then, we utilized differential expression analysis to acquire the differentially expressed genes (DEGs) between different stages of MAFLD patients and normal patients. Alternative splicing analysis was performed via the ABLas pipeline to explore the alternative splicing events that may enhance and regulate the development of MAFLD. The Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were utilized via KOBAS2.0. Finally, quantitative real-time Polymerase Chain Reaction (RT-qPCR) and Western blotting analysis were performed to confirm the expression of significant RBPs. We observed a significant increase in the number of DEGs as the stages of MAFLD progressed. Furthermore, co-expression analysis suggested that abnormally expressed RBPs such as S100A4, CYCS, and JUN might participate in MAFLD development by potentially influencing the AS of downstream metabolism-related genes such as FAT1, SLCO2B1, and C4BPB. Moreover, we confirmed that the expression of the three RBPs (S100A4, CYCS and JUN) is significantly up-regulated in the liver through validation experiments. The abnormal up-regulated expression of RBPs (S100A4, CYCS and JUN) might contribute to the progression of MAFLD and hence they can be further regarded as potential therapeutic targets for MAFLD.
Prolonged glucocorticoid exposure leads to oxidative stress, mitochondrial damage and impaired myogenesis reducing the overall health of the skeletal muscles. Dexamethasone (dex), a synthetic glucocorticoid, induces proteolysis and inflammation by disrupting cellular energetics and mitochondrial function. Vitamin B3 (vit B3), an NAD+ precursor, is known to be a natural antioxidant and anti-inflammatory compound. This study investigates the protective role of vit B3 against dex-induced skeletal muscle damage, focusing on mitochondrial homeostasis and the IKK/FoxO3a signalling axis. C2C12 myoblasts were treated with dex (200 µM) and/or vit B3 (1 mM). Oxidative stress, mitochondrial potential and DNA damage was evaluated using DCFDA, JC1, and γH2AX immunostaining, respectively. Gene expression analysis was performed to assess the mitochondrial fission/fusion and the extent of electron transport chain (ETC) gene expression. Protein expression of inflammatory (IKKα/β, NFκB) and atrophy markers were analysed using immunoblotting and flow cytometry. The extent of myogenic differentiation was evaluated using MyoD and MyHC1 immunostaining along with measurement of the morphometric parameters. Vit B3 treatment significantly enhanced C2C12 viability and reduced dex-induced ROS production while restoring Nrf2 expression. It prevented DNA damage and preserved mitochondrial membrane potential. The results also implicated increased mitochondrial fusion upon vit B3 treatment as seen by the elevated gene expression of Mfn1, Mfn2 and Opa1 and decreased fission as observed by the reduced expression of Fis1 and Drp1. The NADH levels were also seen to be rescued by vit B3 supplementation which translates to better energy production by the electron transport system. Additionally, vit B3 was observed to suppress inflammation and prevent muscle proteolysis by modulating an IKK/FoxO3a axis. Finally, vit B3 was able to improve differentiation as seen by the levels of MyoD and MyHC1 expression in the cells. Vit B3 acts in a multifaceted manner and reduces dex-induced skeletal muscle atrophy which is primarily a result of reduced oxidative stress and restored mitochondrial homeostasis. These findings highlight vit B3 as a potential therapeutic and nutritional supplement for maintaining the skeletal muscle health under myopathic conditions.
Myocardial fibrosis (MF) is a key pathological process driving heart failure, characterized by excessive extracellular matrix (ECM) deposition and impaired cardiac function. Although myocyte-specific enhancer factor 2 A (MEF2A) is implicated in cardiac fibroblast activation, its role in MF remains unclear. We manipulated MEF2A expression in cardiac fibroblasts (CFs) through knockdown and overexpression, and assessed fibrosis markers, migration, and RhoA signaling. Binding of MEF2A to the Snail1 promoter was predicted using JASPAR and validated by chromatin immunoprecipitation (ChIP) and luciferase reporter assays. Rescue experiments with Snail1 overexpression and RhoA inhibition were performed. An angiotensin II (Ang II)-induced MF mouse model was used to evaluate cardiac function by echocardiography and to assess collagen deposition through picrosirius red (PSR) staining. MEF2A was significantly upregulated in Ang II-induced fibrotic hearts and CFs. MEF2A knockdown reduced α-SMA and Col1a1 expression, inhibited CF migration, and suppressed activation of the Snail1/RhoA/α-SMA pathway. ChIP and luciferase assays confirmed the direct binding of MEF2A to the Snail1 promoter. Inhibition of RhoA signaling reversed MEF2A-induced myofibroblast activation and migration. Rescue experiments showed that Snail1 overexpression restored the fibrotic phenotype suppressed by MEF2A knockdown. In vivo, MEF2A knockdown improved left ventricular function, reduced collagen deposition (PSR staining), and lowered heart weight/tibia length ratios. MEF2A promotes myocardial fibrosis by directly activating Snail1 and engages the RhoA/α-SMA pathway. Targeting MEF2A offers a promising therapeutic strategy to attenuate MF and improve heart function.
Ischemia/reperfusion-induced myocardial dysfunction remains a clinical problem and is associated to poor outcomes in patients with cardiovascular disorders, such as myocardial infarction. Shionone is a triterpenoid extracted from the herbal medicine Radix Asteris which has health benefits. This study aimed to explore the roles and functional mechanism of Shionone in regulating myocardial ischemia/reperfusion injury. The network pharmacology was performed to analyze the potential pathway interacted with Shionone in myocardial ischemia/reperfusion injury. The oxygen-glucose deprivation and reperfusion (OGD/R)-treated H9c2 cardiomyocytes and ischemia/reperfusion-induced murine models were regarded as in vitro and in vivo models. Lactate dehydrogenase (LDH), reactive oxygen species (ROS), glutathione (GSH) and iron levels were analyzed using specific kits. Related protein levels were detected by immunofluorescence staining and western blotting assays. Heart infarct in mice was investigated via TTC staining. Network pharmacology predicted LCN2 and PI3K/Akt signaling might be required by Shionone to involve in myocardial ischemia/reperfusion injury. Shionone mitigated OGD/R-induced ferroptosis through decreasing LDH release, ROS generation, iron and ACSL4 levels, and enhancing GSH, SLC7A11 an GPX4 levels in cardiomyocytes. Shionone attenuated OGD/R-induced endoplasmic reticulum stress through reducing CHOP, GRP-78, and phosphorylation levels of PERK and eIF2α. Endoplasmic reticulum stress inducer reversed the effects of Shionone on cardiomyocyte ferroptosis. Shionone decreased LCN2 expression and enhanced activation of PI3K/Akt signaling. Overexpressed LCN2 reversed the effects of Shionone on cardiomyocyte ferroptosis and endoplasmic reticulum stress in OGD/R-treated cardiomyocytes, and this function was mitigated via PI3K/Akt signaling activation. Shionone mitigated ischemia/reperfusion damage in murine heart by reducing heart infarct. Shionone attenuated endoplasmic reticulum stress-associated ferroptosis in cardiomyocytes through decreasing LCN2 and activating PI3K/Akt signaling, offering a basis for understanding the potentially cardioprotective potential of Shionone post ischemia/reperfusion injury.
Colorectal adenocarcinoma (COAD) poses a serious threat to the life of the patient. Notably, Uroplakin 1 A (UPK1A) is a prognostic biomarker for a variety of tumors. However, the role played by UPK1A in the occurrence and development of COAD and its associated molecular mechanisms still lacks a clear and in-depth understanding. The relationship between UPK1A expression and clinicopathological features, as well as patient prognosis, was examined through the use of online databases. Differences in UPK1A expression in COAD tissues and adjacent normal tissues were assessed in clinical samples. The effects of knocking down UPK1A under Escherichia coli (E. coli) co-culture/non-co-culture conditions on COAD cell proliferation, cell invasion, and apoptosis were investigated. In vivo subcutaneous tumor xenograft model, we knocked down the UPK1A gene in a tumor mouse model and assessed tumor growth. The effects of UPK1A and E. coli on glycolysis were investigated by detecting mRNA expression of glucose consumption, lactate production, HIF-1α, and glycolytic enzymes (GLUT1, LDHA, and PDK1). UPK1A was highly expressed in COAD tissues and showed a positive association with unfavorable outcomes in colorectal cancer patients. By knocking down UPK1A, co-culture conditions with E. coli inhibited COAD cell proliferation and invasion, promoted apoptosis, and reduced tumor growth. Knockdown of UPK1A inhibited COAD cell glycolysis by modulating HIF-1α signaling under E. coli co-culture conditions. It is suggested that UPK1A and E. coli synergistically promoted COAD cell proliferation, invasion, and tumor growth and inhibited apoptosis. By regulating HIF-1α signaling, UPK1A and E. coli were able to promote glycolysis in COAD cells. UPK1A and E. coli synergistically interfered with junctional COAD processes.
Impaired osteogenic differentiation in bone marrow-derived mesenchymal stem cells (BMSCs) is involved in the pathogenesis of osteoporosis. ZC3H13, a crucial m6A writer, promotes BMSC osteogenic differentiation by inhibiting ferroptosis. However, its molecular underpinnings remain largely unexplored. Expression analysis was performed by quantitative PCR and immunoblotting. Cell proliferation was assessed by EdU assay. Osteogenic differentiation was assessed using alkaline phosphatase (ALP) expression/activity assays and Alizarin Red staining. Ferroptosis alteration was evaluated by detecting ROS, Fe2+, and MDA levels. RNA immunoprecipitation (RIP), methylated RIP (MeRIP), luciferase, and mRNA stability assays were used to validate the regulation of ZC3H13 in SLC3A2. The relationship between FOXO3 and ZC3H13 was confirmed by chromatin immunoprecipitation (ChIP) and luciferase experiments. ZC3H13 was downregulated in the bone marrow of osteoporosis patients. ZC3H13 deficiency inhibited BMSC proliferation and osteogenic differentiation while enhancing ferroptosis. Conversely, ZC3H13 overexpression promoted proliferation, differentiation and suppressed ferroptosis. Mechanistically, ZC3H13 mediated the m6A modification and stability of SLC3A2 in an IGF2BP2-dependent manner. ZC3H13 silencing suppressed BMSC proliferation and osteogenic differentiation while enhancing ferroptosis through SLC3A2 reduction. Furthermore, FOXO3 acted as a transcriptional activator of ZC3H13. Depletion of FOXO3 suppressed BMSC proliferation and osteogenic differentiation while enhancing ferroptosis through downregulation of ZC3H13. Additionally, FOXO3 regulated SLC3A2 expression via ZC3H13. This study delineates a novel FOXO3/ZC3H13/SLC3A2 regulatory cascade that safeguards BMSCs from ferroptosis and is compromised in osteoporosis, providing a new conceptual framework for therapeutic development.
Metabolic reprogramming characterized by aerobic glycolysis is observed in various cancers, including breast cancer (BC), exerting essential influence on maintaining cancer stemness. The abnormal expression of SPC24 is linked to the occurrence and development of various cancers, but its role in BC remains unelucidated. Bioinformatics analysis was undertaken to determine the levels of SPC24 and E2F7 in BC, and the enriched signaling pathways of SPC24 with differential expression, which were validated through cell experiments. The transcriptional regulatory relationship between E2F7 and SPC24 was also assessed through bioinformatics analysis, with validation completed by dual luciferase assay and chromatin immunoprecipitation (ChIP). To evaluate BC stemness, we employed the western blot (WB) to detect the levels of CD44, CD133, Oct-4, and ALDH1A1, and conducted the cell sphere formation. Flow cytometry was used to detect the proportion of stem cells. To assess the level of glycolysis in BC cells, we detected the expression of key proteins LDHA, HK2, and GLUT1 through WB, and measured the extracellular acidification rate and oxygen consumption rate with kits. Cell experiments combining bioinformatics analysis demonstrated that both E2F7 and SPC24 were greatly upregulated in BC, with SPC24 primarily enriched in the glycolysis metabolic pathway. Further experiments manifested that SPC24 reinforced cell stemness through aerobic glycolysis reprogramming, and SPC24 was modulated by transcription factor E2F7. E2F7 transcriptionally activates the upregulation of SPC24 in BC, which boosts stemness through aerobic glycolysis reprogramming.
Temporal lobe epilepsy (TLE) is one of the most common types of epilepsy, with frequent seizures often leading to cognitive, emotional, and psychiatric issues. A prominent pathological change associated with TLE is hippocampal sclerosis (HS), characterized by neuronal loss, gliosis, and increased neuron fibre density. However, the pathogenesis of Temporal lobe epilepsy with hippocampal sclerosis (TLE-HS) remains unclear. This study aimed to investigate the abnormal expression and regulatory mechanism of hub genes in TLE-HS. The source data were obtained from the epilepsy dataset (GSE256068) of the Gene Expression Omnibus GEO database. Then, differential expression gene (DEG) analysis and weighted gene coexpression network analysis (WGCNA) were employed to screen for module-related DEGs in TLE-HS, followed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Subsequently, these intersected targets were subjected to cross-validation using three machine learning algorithms- LASSO regression, SVM-RFE, and RF, ultimately identifying three hub genes. Finally, CIBERSORT and ssGSEA algorithms were used to analyze the infiltration status of different immune cell populations in TLE-HS patients, followed by assessing the association between hub genes and immune cell populations. The expression of hub genes was determined using RT-qPCR and western blot. Functional experiments were performed using CCK-8, flow cytometry, and special kits. Results indicated that three hub genes, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 4-like 2 (NDUFA4L2), Protein-tyrosine Phosphatase 4A3 (PTP4A3), and Zinc-alpha-2-glycoprotein (AZGP1), were identified in TLE-HS, which are associated with the infiltration of specific immune cells. Besides, NDUFA4L2 expression was reduced in kainic acid (KA)-induced HT22 cells compared to the other two hub genes. Thus, NDUFA4L2 was selected for this research. Moreover, NDUFA4L2 overexpression alleviated KA‑induced HT22 cell neurotoxicity, apoptosis, oxidative stress, and mitochondrial dysfunction. In conclusion, NDUFA4L2 upregulation could alleviate KA-induced neurotoxicity oxidative stress, which provided a theoretical foundation and a potential therapeutic target for epilepsy.
Cold atmospheric plasma (CAP) recently it has been introduced as an innovative therapeutic approach for cancer cell treatment. However the cancer treatment faces questions about the selective anti-cancer capacity of CAP, the distinct molecular responses between cancer and normal cells. In present work 3T3 fibroblast and MCF-7 breast cancer epithelial cells were subjected to treatment of CAP with atmospheric discharge with runaway electrons. We have shown that a decrease in the 3T3 and MCF-7 cell viability under the influence of CAP. In addition, there was an increase in lactate dehydrogenase activity and an increase in the amount of NAD(P)H. An increase in the duration and dose of cold plasma exposure to living systems leaded to a change in the metabolic activity of cells. It was noted that after exposure to the culture of normal and cancer cells, there variability in biochemical and metabolic effects (lactate and growth of free form NAD(P)H), which was primarily accompanied shift in the equilibrium between oxidative phosphorylation and glycolysis. Therefore, cold plasma, at the same dose of radiation, has a stimulating effect on 3T3 cells and an apoptotic effect on MCF-7 cells, leading to a reduction in their metabolic activity. This results in a shift in the metabolic balance towards glycolysis for both 3T3 and MCF-7 cell cultures.
Clinical evidence points to the Traditional Chinese Medicine Fuzheng Huayu recipe (FZHYR) as an anti-fibrosis drug. Our previous studies have shown that FZHYR regulates macrophage polarization and the expression of NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 2 (NDUFA2) to inhibit pulmonary fibrosis. This study aims to explore the mechanism of FZHYR regulates macrophage polarization and NDUFA2 expression in the treatment of pulmonary fibrosis. NR8383 alveolar macrophages polarizing to M1 or M2 polarization by stimulation with LPS/IFN-γ or IL-14/IL-13 and received FZHYR treatment. Macrophage polarization was verified by detecting the levels of transmembrane protein that specific expression using flow cytometry and levels of inflammatory factors. Oxidative phosphorylation change was reflected by mitochondrial ROS and oxygen consumption rate. The effect of FZHYR on m6A of Ndufa2 mRNA and the involvement of m6A modification enzymes (METTL3 and IGF2BP1) was investigated. FZHYR promoted macrophage M1 polarization and inhibited macrophage M2 polarization. FZHYR inhibited oxidative phosphorylation and NDUFA2 expression in M2 macrophages. Ndufa2 silencing inhibited macrophage M2 polarization and oxidative phosphorylation. M2 macrophage polarization and oxidative phosphorylation induced by Ndufa2 overexpression were reversed by FZHYR. Mechanistically, METTL3 induced Ndufa2 m6A methylation in an IGF2BP1-dependent manner in FZHYR-treated M2 macrophage. Moreover, the inhibition of METTL3 suppressed macrophage M2 polarization and oxidative phosphorylation. FZHYR inhibits M2 macrophage polarization through the inhibition of METTL3-mediated m6A modification and downregulation of NDUFA2 and oxidative phosphorylation.
As an important transcription factor, activin receptor-like kinase 5 (ALK5) plays a crucial role in the development of various diseases. However, there have been no reports on whether ALK5 is involved in the pathogenesis of asthma, and further exploration is needed. An in vitro asthma model was constructed using house dust mite (HDM). Quantitative real-time polymerase chain reaction and western blot were used to detect the expression of ALK5. Enzyme-linked immunosorbent assays were used to measure the levels of inflammatory factors. Oxidative stress-related factors and glycolysis-related indicators were analysed using commercial kits. Co-immunoprecipitation was used to assess the binding activity of ALK5 and Krüppel-like factor 4 (KLF4). Finaly, an HDM-induced asthmatic mouse model was established. H&E and PAS staining were used to evaluate the pathological status of mouse lungs, immunohistochemistry (IHC) was used to detect the expression of ALK5 and KLF4 in mouse lung tissue, and Masson staining was used to detect collagen deposition. The results showed that ALK5 was up-regulated in HDM-induced BEAS-2B cells. Silencing ALK5 suppressed inflammation and apoptosis in BEAS-2B cells. Furthermore, knockdown of ALK5 inhibited oxidative stress and promoted glycolysis in BEAS-2B cells. ALK5 specifically bound to KLF4 and promoted its protein degradation. Mechanistically, KLF4-mediated glycolysis was involved in the regulation of ALK5 in BEAS-2B cells. In vivo, ALK5 knockdown attenuated airway inflammation, reduced inflammatory cell infiltration, decreased collagen deposition, and improved lung histopathological damage. In conclusion, inhibiting ALK5 suppresses inflammation, apoptosis, and oxidative stress by regulating KLF4-mediated glycolysis in BEAS-2B cells, thus inhibiting the further development of asthma. Therefore, ALK5 may be a potential target for the clinical treatment of asthma.
Thyroid eye disease (TED) is the most common extra-thyroidal complication of Graves' disease, but its molecular pathogenesis is not fully understood. This study explored the mechanism associated with methyltransferase like 3 (METTL3), NOP2/Sun RNA methyltransferase 4 (NSUN4), and solute carrier family 2 member 3 (SLC2A3) in an in vitro TED model. Human normal orbital fibroblasts were stimulated with platelet-derived growth factor BB (PDGF-BB) to establish in vitro TED model. Differentially expressed genes were screened using online databases. Western blotting was performed for protein detection, and qPCR was used for mRNA quantification. CCK-8 assay and EdU assay were conducted to examine metabolic viability and proliferation, respectively. Hyaluronan (HA) production and glycolysis metabolism were assessed via commercial kits. Methylated RNA immunoprecipitation (MeRIP), RIP, dual-luciferase reporter assay, and RNA-protein pull-down assay were utilized for interaction analysis. Bioinformatics screening obtained 1139 differential genes and 45 glycolytic genes, yielding 2 overlapping genes that were considered as glycolysis-related genes in TED. SLC2A3 was highly expressed in TED and PDGF-BB-stimulated orbital fibroblasts, and silencing SLC2A3 suppressed PDGF-BB-induced cell proliferation, HA production and glycolysis. NSUN4 mediated the m5C methylation modification to increase SLC2A3 expression in an YBX1-dependent manner. NSUN4 inhibition restrained PDGF-BB-induced effects via downregulating SLC2A3. METTL3 mediated m6A methylation modification to reduce NSUN4 expression, with YTHDF2 as a reader protein. The influences of PDGF-BB on human orbital fibroblasts were significantly alleviated by METTL3 overexpression to down-regulate NSUN4. NSUN4 downregulation caused by METTL3-induced m6A modification of NSUN4 mRNA regulated m5C modification of SLC2A3 mRNA, thereby ameliorating PDGF-BB-induced proliferation, HA generation and glycolysis of human orbital fibroblasts in TED. The evidence uncovered the specific molecular mechanism underlying TED pathogenesis.
Acute lung injury characterized by overactive pulmonary inflammation is a common and serious complication of sepsis. Esomeprazole (ESO), a potent proton pump inhibitor (PPI), has been demonstrated as a promising anti-inflammatory agent in treating sepsis at high concentrations, the efficacy of which in sepsis-induced lung injury has not been explored. This research aimed to investigate the role of ESO in septic lung injury and the potential mechanism. The mice were pretreated by ESO prior to the construction of cecal ligation and puncture (CLP) sepsis model. MH-S lung macrophages were exposed to lipopolysaccharide (LPS) to induce inflammatory injury. The severity of lung damage was detected by H&E staining, measurement of lactic dehydrogenase (LDH) and lung wet/dry weight (W/D) ratio. The levels of inflammatory cytokines were detected by ELISA and Western blotting. The number of inflammatory cells was counted. Macrophage distribution was measured by immunohistochemical staining of macrophage markers. Western blotting also determined the expression of endoplasmic reticulum stress (ERS) and NLR family pyrin domain containing 3 (NLRP3) inflammasome-related proteins. CCK-8 method was used to detect cell viability. ESO concentration-dependently mitigated the pathological damage of lung tissues, reduced LDH activity, lung W/D ratio, decreased inflammatory cell counts and F4/80 expression in the lung tissues of sepsis mice. Besides, ESO suppressed inflammatory response, NLRP3 inflammasome activation and inactivated activating transcription factor 6 (ATF6)-CCAAT-enhancer-binding protein homologous protein (CHOP)-mediated ERS signaling both in vitro and in vivo. ATF6 overexpression partially reversed the impacts of ESO on NLRP3 inflammasome and the levels of inflammatory cytokines in LPS-induced MH-S cells. Anyway, ESO may inhibit ATF6/CHOP pathway to protect against inflammation in septic lung injury.