OBJECTIVES: Oxaliplatin (OXA) and 5-fluorouracil (5-FU) are 2 commonly used chemotherapeutic agents for colorectal cancer (CRC). MicroRNAs (miRNAs, miRs) play crucial roles in the development of chemoresistance in various cancers. However, the role and mechanism of miR-224-5p in regulating CRC chemoresistance remain unclear. This study aims to investigate the function of miR-224-5p in chemoresistant CRC cells and the underlying mechanisms. METHODS: CRC datasets GSE28702 and GSE69657 were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed miRNAs between drug-sensitive and resistant groups (OXA or 5-FU) were analyzed, and miR-224-5p was identified as the target miRNA. Chemoresistant cell lines HCT15-OXR, HCT15-5-FU, SW480-OXR, and SW480-5-FU were established. Transient transfections were performed using miR-224-5p mimics, inhibitors, and their respective negative controls (control mimic, control inhibitor) in these cell lines. Cells were treated with different concentrations of OXA or 5-FU post-transfection, and the half-maximal inhibitory concentration (IC₅₀) was determined using the cell counting kit-8 (CCK-8) assay. Cell proliferation was assessed by CCK-8 and colony formation assays. The expression levels of miR-224-5p, LC3, and P62 were measured by real-time polymerase chain reaction (real-time PCR) and/or Western blotting. Autophagic flux was assessed using a tandem fluorescent-tagged LC3 reporter assay. TargetScan 8.0, miRTarBase, miRPathDB, and HADb were used to predict B-cell lymphoma-2 (Bcl-2) as a potential miR-244-5p target, which was further validated by dual-luciferase reporter assays. RESULTS: Chemoresistant CRC cells exhibited down-regulated miR-224-5p expression, whereas up-regulation of miR-224-5p enhanced chemotherapy sensitivity. Exposure to OXA or 5-FU significantly increased autophagic activity in chemoresistant CRC cells, which was reversed by miR-224-5p overexpression. Dual-luciferase assays verified Bcl-2 as a direct target of miR-224-5p. CONCLUSIONS MiR-224-5p regulates chemoresistance in CRC by modulating autophagy through direct targeting of Bcl-2.
Humans ; MicroRNAs/physiology* ; Colorectal Neoplasms/drug therapy* ; Drug Resistance, Neoplasm/genetics* ; Autophagy/drug effects* ; Fluorouracil/pharmacology* ; Oxaliplatin ; Cell Line, Tumor ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Gene Expression Regulation, Neoplastic

OBJECTIVES: There is currently no consensus on whether extra-gastrointestinal stromal tumors (EGISTs) and gastrointestinal stromal tumors (GISTs) are the same type of tumor, and whether the diagnosis and treatment of EGISTs can directly replicate the current diagnostic and treatment standards for GISTs. This study aims to further elucidate the clinical and pathological characteristics, diagnosis, treatment, and prognosis of EGISTs by analyzing the research results of domestic scholars in the field of EGISTs in the past decade. METHODS: A review was conducted on original Chinese and English research articles published from 2013 to 2022 focusing on EGISTs. A descriptive approach was used to extract key information from the literature, including patient demographics, tumor location, tumor diameter, mitotic figures, risk stratification, immunohistochemical markers, cell type, and prognostic factors. The data were subjected to statistical analysis. RESULTS: A total of 12 articles containing 780 EGIST patients were included. The male-to-female incidence of EGISTs was 0.92꞉1. The most common sites of EGISTs were mesentery (30.96%), peritoneum or retroperitoneum (28.53%), omentum (20.32%), and pelvic cavity (12.52%). 52.77% of EGISTs had tumor diameters greater than 10 cm, and the proportions of EGISTs with nuclear fission patterns greater than 5/50 high power field (HPF) and greater than 10/50 HPF were 51.24% and 26.11%, respectively. The proportion of high-risk EGISTs was 79.05%. The positive rates of immune markers CD117, CD34, and DOG-1 in EGISTs were 82.3%, 69.0%, and 79.5%, respectively. The proportion of Ki-67 >5% was 49.2%, and the proportion of Ki-67 >10% was 24.8%. The proportions of EGISTs in spindle cells, epithelial cells, and mixed cells were 74.4%, 14.8%, and 13.1%, respectively. The diameter of the tumor, resection method, risk level, Ki-67 index, mitotic counts, presence of rupture/bleeding/necrosis/peripheral tissue invasion/recurrence and metastasis, as well as the use of imatinib treatment after surgery were important factors affecting the prognosis of EGISTs. CONCLUSIONS Current medical research is relatively well cognizant of GISTs with primary sites in the gastrointestinal tract. Compared with GISTs, EGISTs have large tumor diameters, high mitotic counts, a high percentage of high-risk grades, relatively unique molecular expression, and high aggressiveness. EGISTs differ from GISTs in clinicopathological characteristics. Whether EGISTs and GISTs share a common origin remains unclear. If they are distinct tumor entities, separate diagnostic and treatment guidelines for EGISTs should be established. If EGISTs are ultimately confirmed to be a special subtype of GISTs, then directly applying existing GIST-based standards to EGISTs may be inappropriate. A more scientific approach would involve subclassifying EGISTs based on anatomical location and then tailoring treatment strategies accordingly with reference to GIST guidelines.
Humans ; Gastrointestinal Stromal Tumors/epidemiology* ; Male ; Female ; Prognosis ; Proto-Oncogene Proteins c-kit/metabolism*

OBJECTIVES: To investigate the role of the BNIP3-PI3K/Akt signaling pathway in mediating the inhibitory effect of Buyang Huanwu Decoction (BYHWT) on mitochondrial autophagy in human synovial fibroblasts from rheumatoid arthritis patients (FLS-RA) cultured under a hypoxic condition. METHODS: Forty normal Wistar rats were randomized into two groups (n=20) for daily gavage of BYHWT or distilled water for 7 days to prepare BYHWT-medicated or control sera. FLS-RA were cultured in routine condition or exposed to hypoxia (10% O₂) for 24 h wigh subsequent treatment with IL-1β, followed by treatment with diluted BYHWT-medicated serum (5%, 10% and 20%) or control serum. AnnexinV-APC/7-AAD double staining and T-AOC kit were used for detecting apoptosis and total antioxidant capacity of the cells, and the changes in ROS, ATP level, mitochondrial membrane potential and Ca²⁺ homeostasis were analyzed. The changes in mRNA and protein expressions of BNIP3, PI3K and AKT and mRNA expressions of LC3, Beclin-1 and P62 were detected using RT-qPCR and Western blotting. RESULTS: Treatment with BYHWT-medicated serum dose-dependently lowered apoptosis rate of IL-1β-induced FLS-RA with hypoxic exposure. The treatment significantly decreased T-AOC concentration, increased ROS production, autophagosome formation and ATPase levels, and lowered mitochondrial membrane potential and Ca²⁺ level in the cells. In IL-1β-induced FLS-RA with hypoxic exposure, treatment with BYHWT-medicated serum significantly increased BNIP3 protein expression, decreased the protein expressions of PI3K and AKT, increased the mRNA expressions of BNIP3 and P62, and lowered the mRNA expressions of PI3K, AKT, LC3 and Beclin-1 without significantly affecting Beclin-1 protein expression. The cells treated with 5% and 10% BYHWT-medicated serum showed no significant changes in LC3 expression. CONCLUSIONS BYHWT inhibits mitochondrial autophagy in IL-1β-induced FLS-RA with hypoxic exposure possibly by inhibiting BNIP3-mediated PI3K/AKT signaling pathway.
Drugs, Chinese Herbal/pharmacology* ; Arthritis, Rheumatoid/pathology* ; Animals ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Autophagy/drug effects* ; Humans ; Fibroblasts/cytology* ; Rats, Wistar ; Membrane Proteins/metabolism* ; Rats ; Phosphatidylinositol 3-Kinases/metabolism* ; Mitochondria/metabolism* ; Cells, Cultured ; Proto-Oncogene Proteins/metabolism* ; Apoptosis/drug effects* ; Cell Hypoxia ; Synovial Membrane/cytology* ; Male ; Mitochondrial Proteins

OBJECTIVES: To investigate the mechanism mediating the regulatory effect of miR-155-5p on proliferation of human submandibular gland epithelial cells (HSGECs) in primary Sjogren's syndrome (pSS). METHODS: Dual luciferase reporter assay was used to verify the targeting relationship between miR-155-5p and the PI3K/AKT pathway. In a HSGEC model of pSS induced by simulation with TRAIL and INF-γ, the effects of miR-155-inhibitor-NC or miR-155 inhibitor on cell viability, cell cycle, apoptosis and proliferation were evaluated using CKK8 assay, flow cytometry and colony formation assay. ELISA and RT-PCR were used to detect the expressions of inflammatory cytokines and miR-155-5p mRNA in the cells; Western blotting was performed to detect the expressions of proteins in the PI3K/AKT signaling pathway. RESULTS: Dual luciferase assay showed that miR-155-5p targets the PI3K/AKT pathway via PIK3R1 mRNA. The HSGEC model of pSS showed significantly decreased cell viability, cell clone formation ability and expressions IL-10 and IL-4 and increased cell apoptosis, cell percentage in G2 phase, expressions of TNF‑α, IL-6, miR-155-5p and PIK3R1 mRNA, p-PI3K/PI3K ratio, p-Akt/AKT ratio, and PIK3R1 protein expression. Treatment of the cell models with miR-155 inhibitor significantly increased the cell viability, G1 phase cell percentage, colony formation ability, and expressions of IL-10 and IL-4 levels, and obviously reduced cell apoptosis rate, G2 phase cell percentage, expressions of TNF-α, IL-6, miR-155-5p and PIK3R1 mRNA, p-PI3K/PI3K ratio, p-AKT/AKT ratio, and PIK3R1 protein expression. CONCLUSIONS In HSGEC model of pSS, inhibition of miR-155-5p can promote cell proliferation and reduced cell apoptosis by targeting PI3K1 mRNA to negatively regulate the overexpression of PI3K/AKT signaling pathway.
Humans ; MicroRNAs/genetics* ; Cell Proliferation ; Signal Transduction ; Proto-Oncogene Proteins c-akt/metabolism* ; Sjogren's Syndrome/pathology* ; Epithelial Cells/cytology* ; Submandibular Gland/cytology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Apoptosis ; Class Ia Phosphatidylinositol 3-Kinase ; Cells, Cultured

OBJECTIVES: To investigate the therapeutic mechanism of Thesium chinense Turcz. (TCT) for antibiotic-associated diarrhea (AAD). METHODS: Network pharmacology, KEGG pathway enrichment analysis and molecular docking were used to identify the shared targets and genes of TCT and AAD, the key signaling pathways and the binding between the active components in TCT and the core protein targets. In a Kunming mouse model of AAD established by intragastric administration of lincomycin hydrochloride, the effects of daily gavage of 1% carboxymethyl cellulose sodium or TCT gel solutions at 1.5 g/kg and 3 g/kg (n=10) on body weight and diarrhea were observed. HE staining, ELISA, 16S rRNA sequencing, and Western blotting were used to examine pathologies, expression levels of IL-6 and TNF-α, changes in gut microbiota, and protein expressions of EGFR, p-EGFR, PI3K, p-PI3K, Akt, and p-Akt in the colon tissues of the mice. RESULTS: We identified a total of 66 active components of TCT and 68 core targets including EGFR, STAT3 and PIK3CA. KEGG pathway enrichment analysis suggested that the therapeutic effects of TCT was mediated primarily through the PI3K/Akt signaling pathway. Molecular docking showed that EGFR had the highest binding affinity with coniferin, and the EGFR-coniferin complex maintained a stable conformation at 10 ns, whose stability was also confirmed by Gibbs free energy analysis. In the mouse models of AAD, treatment with TCT significantly improved colonic tissue morphology, decreased colonic levels of TNF-α and IL-6, increased gut microbiota diversity, and modulated the relative abundances of the key genera including Lactobacillus and Bacteroides. TCT treatment also markedly reduced protein expressions of p-EGFR, p-PI3K and p-Akt in the colon tissues of the mice. CONCLUSIONS TCT can alleviate AAD in mice by modulating gut microbiota composition, regulating the EGFR/PI3K/Akt signaling pathway, and reducing TNF‑α and IL-6 expressions.
Animals ; Gastrointestinal Microbiome/drug effects* ; Signal Transduction/drug effects* ; Mice ; ErbB Receptors/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Diarrhea/drug therapy* ; Phosphatidylinositol 3-Kinases/metabolism* ; Anti-Bacterial Agents/adverse effects* ; Drugs, Chinese Herbal/therapeutic use* ; Molecular Docking Simulation

OBJECTIVES: To explore the molecular mechanism by which miR-124 affects cognitive function of sleep-deprived rats. METHODS: Fifty-four adult male SD rats were randomized into 6 groups (n=9), including a normal control group, a sleep deprivation (SD) model group, and 4 intracerebral microinjection groups in which the rats were subjected to stereotactic injection of miR-124 agomir, miR-124 agomir NC, miR-124 antagomir, or miR-124 antagomir into the lateral ventricle 7 days before SD modeling. The cognitive functions of the rats were evaluated with Morris water maze test, and pathological changes in the hippocampus were observed using HE staining. The expression level of miR-124 in hippocampal tissues of the rats was detected with qRT-PCR, and the expression level of apoptosis-related proteins and signaling pathway proteins were determined using Western blotting. RESULTS: In Morris water maze test, the SD rat models treated with miR-124 agomir showed a significantly shorter escape latency and fewer platform crossings with increased percentage of time and swimming distance in the fourth quadrant as compared with those in SD model group, while the rats treated with miR-124 antagomir exhibited worsened performance in the test. In the SD rat models, treatment with miR-124 agomir obviously lessened pathological changes in the hippocampus, while treatment with miR-124 antagomir significantly worsened the pathological changes. Compared with those in SD model group, the miR-124 agomir-treated rats showed an increased hippocampal expression of miR-124 with upregulated protein expressions of PI3K, p-AKT/AKT, and Bcl-2 and downregulated expressions of Bax and caspase-3 proteins, while rats treated with miR-124 antagomir showed significantly decreased hippocampal expression of miR-124 with lowered expressions of PI3K, p-AKT/AKT and Bcl-2 proteins and increased Bax and caspase-3 protein expressions. CONCLUSIONS High expression of miR-124 alleviates SD-induced cognitive decline and neuronal apoptosis in rats by activating the PI3K/AKT signaling pathway.
Animals ; MicroRNAs/metabolism* ; Signal Transduction ; Proto-Oncogene Proteins c-akt/metabolism* ; Male ; Rats, Sprague-Dawley ; Hippocampus/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Cognition ; Rats ; Sleep Deprivation/metabolism* ; Apoptosis ; Maze Learning

OBJECTIVES: To investigate the effect of Didang Decoction-medicated serum on autophagy in high glucose (HG)-induced rat glomerular endothelial cells (RGECs) and explore the pathway that mediates its effect. METHODS: Primary RGECs were isolated and cultured using sequential sieving combined with collagenase digestion, followed by identification using immunofluorescence assay for factor VIII. High glucose medium was used to induce RGECs to simulate a diabetic environment, and the effects of Didang Decoction-medicated serum and 3-MA (an autophagy inhibitor), either alone or in combination, on autophagy of HG-exposed cells were evaluated by observing autophagic vacuoles using monodansylcadaverine (MDC) staining. RT-qPCR and Western blotting were employed to measure mRNA and protein expression levels of Beclin-1, p62, LC3B, p-PI3K, p-Akt, and p-mTOR. RESULTS: Compared with the control cells, the HG-exposed RGECs showed significantly reduced autophagic fluorescence intensity, decreased Beclin-1 mRNA expression, increased p62 mRNA expression, downregulated Beclin-1 protein and LC3-II/I ratio, and upregulated p62, p-PI3K, p-Akt, and p-mTOR protein levels. Didang Decoction-medicated serum significantly enhanced autophagic fluorescence intensity in HG-exposed cells, increased Beclin-1 mRNA expression, decreased p62 mRNA expression, upregulated Beclin-1 protein, and downregulated p62, p-PI3K, p-Akt, and p-mTOR protein levels. CONCLUSIONS Didang Decoction-medicated serum enhances autophagy in HG-exposed RGECs by regulating the PI3K/Akt/mTOR signaling pathway, which sheds light on a new therapeutic strategy for diabetic nephropathy.
Animals ; Autophagy/drug effects* ; Signal Transduction/drug effects* ; Rats ; TOR Serine-Threonine Kinases/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Endothelial Cells/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Glucose ; Cells, Cultured ; Kidney Glomerulus/cytology* ; Rats, Sprague-Dawley

OBJECTIVES: To investigate the antioxidative mechanism of snake venom-derived protein C activator (PCA) in mitigating vascular endothelial cell injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in DMEM containing 1.0 g/L D-glucose and exposed to hypoxia (1% O₂) for 6 h followed by reoxygenation for 2 h to establish a cell model of oxygen-glucose deprivation/reoxygenation (OGD/R). The cell model was treated with 2 μg/mL PCA alone or in combination with 2-ME2 (a HIF-1α inhibitor) or DMOG (a HIF-1α stabilizer), and intracellular production of reactive oxygen species (ROS) and protein expression levels of HIF-1α, BNIP3, and Beclin-1 were detected using DCFH-DA fluorescence probe, flow cytometry, and Western blotting. The OGD/R cell model was transfected with a BNIP3-specific siRNA or a scrambled control sequence prior to PCA treatment, and the changes in protein expressions of HIF-1α, BNIP3 and Beclin-1 and intracellular ROS production were examined. RESULTS: In the OGD/R cell model, PCA treatment significantly upregulated HIF-1α, BNIP3 and Beclin-1 expressions and reduced ROS production. The effects of PCA were obviously attenuated by co-treatment with 2-ME2 but augmented by treatment with DMOG (a HIF-1α stabilizer). In the cell model with BNIP3 knockdown, PCA treatment increased BNIP3 expression and decreased ROS production without causing significant changes in HIF-1α expression. Compared with HUVECs with PCA treatment only, the cells with BNIP3 knockdown prior to PCA treatment showed significantly lower Beclin-1 expression and higher ROS levels. CONCLUSIONS Snake venom PCA alleviates OGD/R-induced endothelial cell injury by upregulating HIF-1α/BNIP3 signaling to suppress ROS generation, suggesting its potential as a therapeutic agent against oxidative stress in vascular pathologies.
Humans ; Reactive Oxygen Species/metabolism* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Human Umbilical Vein Endothelial Cells/drug effects* ; Membrane Proteins/metabolism* ; Proto-Oncogene Proteins/metabolism* ; Up-Regulation ; Cell Hypoxia ; Cells, Cultured ; Snake Venoms/chemistry* ; Beclin-1

OBJECTIVES: To investigate the effect of monotropein on motor function recovery of mice with spinal cord injury (SCI) and explore the underlying mechanism. METHODS: Forty-five adult female C57BL/6 mice were randomized equally into sham operation group, SCI group, and SCI group with daily intraperitoneal monotropein injection. The mice in the former two groups received daily saline injections. Motor function of the mice was evaluated using BMS scores, slant plate test, and footprint analyses. Pathological changes and neuronal counts in the spinal cord were observed using HE, LFB, and Nissl staining. The biological functions of monotropein were explored using GO and KEGG enrichment analyses. NeuN/cleaved caspase-3 immunofluorescence assay and Western blotting were used to detect neuronal apoptosis in the spinal cord of the mice. In cultured HT22 cells, the effect of monotropein on TNF-α-induced cell apoptosis was evaluated using TUNEL staining and Western blotting. In monotropein-treated HT22 cells and SCI mice, the changes in the PI3K/AKT pathway were examined, and the effect of a PI3K/AKT pathway activator (IGF-1) on HT22 cell apoptosis and motor function recovery of SCI mice were observed. RESULTS: SCI mice with monotropein treatment showed significantly improved motor functions with reduced SCI areas and increased myelin retention and neuron counts in the spinal cord. Bioinformatics analysis suggested a role of PI3K/AKT signaling pathway in mediating the anti-apoptotic effects of monotropein. In SCI mice, monotropein obviously reduced apoptotic neurons, decreased expressions of cleaved caspase-3 and Bax and increased Bcl-2 expression in the spinal cord. In HT22 cells, monotropein significantly inhibited TNF-α-induced apoptosis and PI3K/AKT pathway activation. Treatment with IGF-1 obviously increased apoptosis of HT22 cells and exacerbated locomotor dysfunction in SCI mice. CONCLUSIONS Monotropein promotes motor function recovery in SCI mice by reducing neuronal apoptosis possibly by inhibiting the PI3K/AKT signaling pathway.
Animals ; Spinal Cord Injuries/metabolism* ; Apoptosis/drug effects* ; Signal Transduction/drug effects* ; Mice, Inbred C57BL ; Mice ; Proto-Oncogene Proteins c-akt/metabolism* ; Female ; Phosphatidylinositol 3-Kinases/metabolism* ; Neurons/pathology* ; Recovery of Function

OBJECTIVES: To investigate the effect of moslosooflavone (MOS) for ameliorating dextran sulfate sodium (DSS)-induced colitis in mice and the underlying molecular mechanism. METHODS: C57BL/6J mice with or without DSS exposure in the drinking water were both randomized into two groups for treatment with intraperitoneal injections with MOS (200 mg/kg) or normal saline for 7 days (n=6). Disease severity of the mice was assessed by observing changes in body weight, colon length, histopathology (HE staining), intestinal barrier function, and TUNEL staining. In the in vitro studies, lipopolysaccharide (LPS)-stimulated mouse colon organoids were treated with MOS (120 μmol/L) for 24 h, and the changes in barrier dysfunction and inflammation were analyzed. Network pharmacology and Western blotting were employed to identify functional pathways and apoptotic protein regulation associated with the therapeutic effect of MOS on colitis. RESULTS: In the mouse models of DSS-indcued colitis, MOS treatment significantly reduced body weight loss, disease activity index (DAI) scores and colon shortening, ameliorated colonic histopathological changes and inflammation, and lowered pro-inflammatory cytokine levels (TNF-α, IL-1β, IL-6, and IFN-γ). MOS effectively restored intestinal barrier integrity in the mice by reducing serum FITC-dextran and I-FABP concentrations while enhancing the tight junction proteins (ZO-1 and claudin-1). In the colon organoids, MOS significantly suppressed LPS-induced inflammatory responses and epithelial barrier disruption. Western blotting revealed that MOS downregulated C-caspase-3 and BAX and upregulated Bcl-2 expressions in both models. Mechanistically, MOS suppressed PI3K and AKT phosphorylation in both DSS-treated mouse colonic tissues and LPS-stimulated organoids. CONCLUSIONS MOS alleviates experimental colitis in mice by inhibiting intestinal epithelial apoptosis via inhibiting the PI3K/AKT pathway, thereby restoring intestinal barrier integrity and reducing inflammation.
Animals ; Dextran Sulfate ; Mice, Inbred C57BL ; Colitis/metabolism* ; Mice ; Signal Transduction/drug effects* ; Intestinal Mucosa/metabolism* ; Apoptosis/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Flavones/pharmacology* ; Male