OBJECTIVE: To investigate the anti-tumor effects of cinobufacini (CINO) on hepatocellular carcinoma (HCC) induced by des-gamma-carboxy-prothrombin (DCP) and to uncover the underlying mechanisms. METHODS: The inhibitory effect of CINO on HCC cell proliferation was evaluated using the cell counting kit-8 method, and the apoptosis rate was quantified using flow cytometry. Immunofluorescence and Western blot analyses were used to investigate the differential expression of proteins associated with cell growth, apoptosis, migration, and invasion pathways after CINO treatment. The therapeutic potential of CINO for HCC was confirmed, and the possibility of combining cinobufacini with c-Met inhibitor for the treatment of primary HCC was further validated by in vivo experiments. RESULTS: Under the induction of DCP, CINO inhibited the activity of HCC cells, induced apoptosis, and inhibited migration and invasion. Upon the induction of DCP, CINO regulated c-Met activation and the activation of the phosphatidylinositol-3 kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathways. In a mouse model of HCC, CINO exhibited significant antitumor effects by inhibiting the phosphorylation of c-Met and the downstream PI3K/AKT and MEK/ERK pathways in tumor tissues. CONCLUSIONS CINO inhibited HCC cell growth, promoted apoptosis, and suppressed HCC cell invasion and migration by targeting c-Met and PI3K/AKT and MEK/ERK signaling pathways under DCP induction.
Carcinoma, Hepatocellular/drug therapy* ; Proto-Oncogene Proteins c-met/metabolism* ; Liver Neoplasms/drug therapy* ; Signal Transduction/drug effects* ; Animals ; Humans ; Cell Movement/drug effects* ; Apoptosis/drug effects* ; Cell Proliferation/drug effects* ; Amphibian Venoms/therapeutic use* ; Cell Line, Tumor ; Neoplasm Metastasis ; Cell Survival/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Neoplasm Invasiveness ; Mice, Inbred BALB C ; Mice, Nude ; Mice ; Male ; Bufanolides/therapeutic use* ; Protein Precursors ; Prothrombin ; Biomarkers

OBJECTIVE: To explore the mechanism of action of asperosaponin VI (AVI) in the treatment of rheumatoid arthritis (RA) and validate it in ex vivo experiments using network pharmacology and molecular docking methods. METHODS: The predicted targets of AVI were obtained from PharmMaper, UniProt and SwissTarget Prediction platforms, the disease targets were collected from Online Mendelian Inheritance in Man, Therapeutic Target Database and GeneCards databases, the intersection targets of AVI and RA were obtained from Venny 2.1.0, and the protein-protein interaction (PPI) network was obtained from STRING database, which was analyzed by Cytoscape software and screened to obtain the core targets. Cytoscape software was used to analyze PPI network and screen the core targets. Based on the Database for Annotation, Visualization and Integrated Discovery database, Gene Ontology functional and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed, and Cytoscape software was used to construct the "Disease-Pathway-Target-Drug" network, which was finally verified by molecular docking and animal experiments. RESULTS: Network pharmacological studies showed that AVI was able to modulate 289 targets, with 102 targets for the potential treatment of RA, with the core pathway being the AKT/PI3K signaling pathway, and the core targets being the epidermal growth factor receptor (EGFR) and matrix metalloproteinase 9 (MMP9). Molecular docking results showed that AVI could produce strong binding with both of the 2 core targets. In vitro cellular experiments showed that AVI reduced nitric oxide, prostaglandin E₂, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-1 β levels (P<0.05) and inhibited cyclooxygenase-2, nitric oxide synthase, EGFR, MMP9, phosphorylated phosphoinositide 3-kinase (p-PI3K), and phosphorylated serine-threonine kinase (p-AKT) proteins (P<0.05). The results of in vivo studies showed that AVI improved RA score and foot swelling thickness and decreased TNF-α, IL-6, p-PI3K and p-AKT levels in RA rats (P<0.05). CONCLUSION AVI exerts anti-inflammatory and anti-RA effects which might be related to the EGFR/MMP9/AKT/PI3K pathway.
Saponins/chemistry* ; ErbB Receptors/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Signal Transduction/drug effects* ; Molecular Docking Simulation ; Animals ; Arthritis, Rheumatoid/drug therapy* ; Proto-Oncogene Proteins c-akt/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; Protein Interaction Maps/drug effects* ; Humans ; Network Pharmacology ; Male ; Rats

OBJECTIVE: To explore the molecular mechanism of Shenmai Injection (SMI) against doxorubicin (DOX) induced cardiomyocyte apoptosis. METHODS: A total of 40 specific pathogen-free (SPF) male Sprague Dawley (SD) male rats were divided into 5 groups based on the random number table, including the control group, the model group, miR-30a agomir group, SMI low-dose (SMI-L) group, and SMI high-dose (SMI-H) group, with 8 rats in each group. Except for the control group, the rats were injected weekly with DOX (2 mg/kg) in the tail vein for 4 weeks to induce myocardial injury, and were given different regimens of continuous intervention for 2 weeks. Cardiac function was detected by echocardiography and myocardial pathological changes were observed by Van Gieson (VG) staining. Myocardial injury serum markers, including creatine kinase (CK), lactate dehydrogenase (LDH), troponin T (cTnT), N-terminal pro-brain natriuretic peptide (NT-proBNP), soluble ST2 (sST2), and growth differentiation factor-15 (GDF-15) were detected by enzyme linked immunosorbent assay (ELISA). Cardiomyocyte apoptosis was observed by terminal deoxynucleotidyl transferase-mediated biotinylated dUTP triphosphate nick end labeling (TUNEL) and transmission electron microscopy, and the expressions of target proteins and mRNA were detected by Western blot and quantitative real time polymerase chain reaction (qRT-RCR), respectively. RESULTS: The treatment with different doses of SMI reduced rat heart mass index and left ventricular mass index (P<0.05), significantly improved the left ventricular ejection fraction (P<0.05), decreased the levels of serum CK, LDH, cTnT, and NT-proBNP (P<0.05 or P<0.01), reduced the levels of serum sST2 and GDF-15 (P<0.05 or P<0.01), decreased the collagen volume fraction, reduced the expressions of rat myocardial type I and type III collagen (P<0.05 or P<0.01), and effectively alleviated myocardial fibrosis. And the study found that SMI promoted the expression levels of miR-30a and Bcl-2 in myocardium, and down-regulated the expression of Bax, which inhibited the activation of Caspase-3 and Caspase-9 (P<0.05 or P<0.01), and improved myocardial cell apoptosis. CONCLUSIONS SMI can alleviate myocardial injury and apoptosis caused by DOX, and its mechanism possibly by promoting the targeted expression of myocardial Bcl-2 protein through miR-30a.
Animals ; Myocytes, Cardiac/metabolism* ; Apoptosis/drug effects* ; MicroRNAs/genetics* ; Rats, Sprague-Dawley ; Male ; Drugs, Chinese Herbal/administration & dosage* ; Doxorubicin/pharmacology* ; Proto-Oncogene Proteins c-bcl-2/genetics* ; Drug Combinations ; Injections ; Rats

OBJECTIVE: To elucidate the modulation mechanism of Suanzaoren Decoction (SZRD) on basolateral amygdala (BLA) neuronal activity to alleviate chronic restraint stress (CRS)-related behavioral deficits. METHODS: The male C57BL/6J mice were assigned to 4 groups using the complete randomization method, including control (CON, n=19), CRS (n=19), SZRD (n=21), and fluoxetine (Flu, n=22) groups. Mice were restrained for 6 h per day, over a 21-d period to establish CRS models. The CON group remained in their cages without food or water during the 6-h matching period. SZRD and Flu groups received intragastric administration of SZRD (4.68 g/kg) and Flu (20 mg/kg) daily, respectively, 30 min before restraint for 21 consecutive days. The therapeutic effects of SZRD were evaluated using behavioral tests including the tail suspension test, elevated plus maze test, and forced swimming test. The cellular Fletcher B. Judson murine osteosarcoma proto-oncogene (c-Fos) expression in the BLA was measured using immunofluorescence, while action potential (AP) firing and synaptic transmission in BLA pyramidal neurons were evaluated using whole-cell patch-clamp recordings. RESULTS: SZRD administration significantly increased time spent in the open arms and open-arm entries while reducing immobility time (P<0.05 or P<0.01). It downregulated CRS-induced c-Fos expression and AP firing of pyramidal neurons in the BLA (P<0.01). Additionally, SZRD selectively attenuated excitatory (P<0.01), but not inhibitory, synaptic transmission onto BLA pyramidal neurons. CONCLUSION SZRD alleviated CRS-induced anxiety- and depression-like behaviors in mice by modulating the excitability and synaptic transmission of BLA pyramidal neurons.
Animals ; Drugs, Chinese Herbal/therapeutic use* ; Depression/complications* ; Pyramidal Cells/pathology* ; Male ; Mice, Inbred C57BL ; Basolateral Nuclear Complex/pathology* ; Restraint, Physical ; Anxiety/complications* ; Behavior, Animal/drug effects* ; Stress, Psychological/physiopathology* ; Mice ; Proto-Oncogene Proteins c-fos/metabolism* ; Action Potentials/drug effects* ; Synaptic Transmission/drug effects*

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