This study explores the therapeutic differences and mechanisms of salt-processed Phellodendri Chinensis Cortex in improving insulin resistance(IR) based on network pharmacology, molecular docking, and cellular experiments. The components and intersection targets of Phellodendri Chinensis Cortex in improving IR were collected from databases, and a "drug-component-target-disease" network and protein-protein interaction(PPI) network were constructed to screen core components and targets. A total of 29 active components and 240 intersection targets were identified, of which 13 were core targets. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were used to identify key signaling pathways, and molecular docking was performed to validate the binding activity between core components and targets. An IR model in HepG2 cells was induced using insulin combined with high glucose, and the effects of Phellodendri Chinensis Cortex before and after salt-processing on cell glucose consumption were evaluated. The expression of proteins related to the mitogen-activated protein kinase(MAPK) and phosphatidylinositol 3-kinase(PI3K)-protein kinase B(AKT) signaling pathways was detected by Western blot. The cellular experimental results showed that, compared with the model group, glucose consumption in the drug-treated groups was significantly increased(P<0.01), the phosphorylation level of extracellular regulated protein kinase(ERK) was decreased(P<0.05), the phosphorylation levels of PI3K and AKT were increased, and the expression of glucose transporter 4(GLUT4) was also upregulated(P<0.05). Furthermore, the effect of salt-processed Phellodendri Chinensis Cortex was better than that of raw Phellodendri Chinensis Cortex. The study demonstrates that Phellodendri Chinensis Cortex, both before and after salt-processing, improves IR by regulating the expression of related proteins in the MAPK and PI3K-AKT signaling pathways, with enhanced effects after salt-processing.
Humans ; Network Pharmacology ; Phellodendron/chemistry* ; Insulin Resistance ; Drugs, Chinese Herbal/chemistry* ; Hep G2 Cells ; Signal Transduction/drug effects* ; Molecular Docking Simulation ; Protein Interaction Maps/drug effects* ; Proto-Oncogene Proteins c-akt/genetics* ; Phosphatidylinositol 3-Kinases/genetics* ; Glucose/metabolism*

To investigate the mechanism of action of Syngnathus extract in treating knee osteoarthritis of rats, forty-eight male SD rats were randomly divided into the blank group, model group, positive drug group, as well as low-dose, medium-dose, and high-dose groups of Syngnathus extract. The rat model of knee osteoarthritis was constructed by intra-articular injection of sodium iodoacetate. After successful modeling, celecoxib(18 mg·kg~(-1)·d~(-1)) and Syngnathus extract(0.4, 0.8, and 1.6 g·kg~(-1)·d~(-1)) were given in different groups by gavage intervention for two weeks. Hematoxylin-eosin(HE) staining was used to observe the histopathological changes of cartilage in knee joints, and enzyme-linked immunosorbent assay(ELISA) was used to detect the expression level of inflammatory factors in serum. Real-time fluorescence quantitative PCR, Western blot, and immunohistochemistry were used to detect the levels of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target protein of rapamycin(mTOR) pathway-related mRNA and protein expression. The results showed that, comparied with the blank group, the cartilage surface of the knee joints of rats in the model group was uneven, with disorganized levels and defective cartilage tissue. The serum levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) and the mRNA levels of PI3K, Akt, and mTOR in cartilage tissue, as well as the protein expression levels of phosphorylated PI3K(p-PI3K)/PI3K, phosphorylated Akt(p-Akt)/Akt, phosphorylated mTOR(p-mTOR)/mTOR, and P62 were significantly increased. Beclin1 protein expression was decreased. Comparied with the model group, the number of chondrocytes in the knee joint of rats in each group of Syngnathus extract increased, and the arrangement of chondrocytes was relatively neat. The cartilage layer was restored, and the serum levels of IL-1β, IL-6, and TNF-α, as well as the mRNA expression levels of PI3K, Akt, and mTOR in cartilage tissue were significantly reduced. The protein expression levels of p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR, and P62 were significantly reduced in the rats in the middle-dose and high-dose groups of Syngnathus extract, and the Beclin1 protein expression was significantly increased. The protein expression levels of p-PI3K/PI3K, p-Akt/Akt, and P62 in rats in the low-dose group of Syngnathus extract were significantly reduced. In summary, Syngnathus extract may be used to treat knee osteoarthritis by inhibiting the expression of PI3K/Akt/mTOR signaling pathway, so as to alleviate the inflammatory response in the organism, enhance the autophagy activity of chondrocytes, and reduce the apoptosis of chondrocytes.
Animals ; TOR Serine-Threonine Kinases/genetics* ; Male ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/genetics* ; Rats ; Osteoarthritis, Knee/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Phosphatidylinositol 3-Kinases/genetics* ; Humans

This study aims to investigate the optimal ratio of Astragali Radix-Curcumae Rhizoma(AC) for inhibiting the proliferation of 143B osteosarcoma cells, and to investigate the mechanism by which AC inhibits osteosarcoma growth and metastasis through angiogenesis and cell adhesion mediated by the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/hypoxia inducible factor-1α(HIF-1α) pathway. A subcutaneous 143B tumor-bearing nude mouse model was successfully established and randomly divided into the model group, and the AC 1∶1, 2∶1, and 4∶1 groups. Body weight, tumor volume, and tumor weight were recorded. Real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot were used to detect the mRNA and protein expression levels of PI3K, Akt, phosphorylated Akt(p-Akt), HIF-1α, vascular endothelial growth factor A(VEGFA), transforming growth factor-β1(TGF-β1), epithelial cadherin(E-cadherin), neural cadherin(N-cadherin), vimentin, matrix metalloproteinase 2(MMP2), matrix metalloproteinase 9(MMP9), B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), and caspase-3 in the hypoxic core region of the tumor tissue. A cell hypoxia model was established, and the effects of AC-medicated serum(model group, AC 1∶1, 2∶1, and 4∶1 groups) on angiogenesis, proliferation, adhesion, invasion, and migration of 143B osteosarcoma cells were examined through CCK-8, flow cytometry, Transwell assay, cell adhesion assay, and HUVEC tube formation assay. The results showed that compared with the model group, the tumor weight and volume were smallest in the 2∶1 group. The expression levels of PI3K, Akt, p-Akt, HIF-1α, VEGFA, and TGF-β1 were significantly decreased, and the protein expression of E-cadherin was significantly increased, while the protein expression of N-cadherin, vimentin, MMP2, and MMP9 was significantly decreased. Additionally, the protein expression of Bax and caspase-3 was significantly increased, and Bcl-2 protein expression was significantly decreased. In vitro experiments showed that after intervention with AC-medicated serum at a 2∶1 ratio, the cell activity, adhesion, invasion, and migration of 143B cells were significantly reduced, apoptosis was significantly increased, and HUVEC tube formation was significantly decreased. In conclusion, the 2∶1 ratio of AC showed the most effective inhibition of 143B cell growth. AC can inhibit the growth and metastasis of osteosarcoma 143B cells by regulating the PI3K/Akt/HIF-1α signaling pathway, inhibiting angiogenesis and reducing cell adhesion, invasion, and migration.
Osteosarcoma/pathology* ; Animals ; Proto-Oncogene Proteins c-akt/genetics* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Humans ; Mice ; Cell Adhesion/drug effects* ; Cell Proliferation/drug effects* ; Neovascularization, Pathologic/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Phosphatidylinositol 3-Kinases/genetics* ; Cell Line, Tumor ; Mice, Nude ; Signal Transduction/drug effects* ; Astragalus Plant/chemistry* ; Bone Neoplasms/physiopathology* ; Male ; Rhizome/chemistry* ; Mice, Inbred BALB C ; Angiogenesis

This study elucidates the mechanism of Rhodiolae Crenulatae Radix et Rhizoma(RCRR) in protecting brain microvascular endothelial cells from oxygen-glucose deprivation(OGD) injury and reveals the modern pharmacological mechanism of RCRR's traditional use in nourishing Qi and promoting blood circulation to protect endothelial cells. The scratch assay was employed to assess the migratory capacity of endothelial cells. Immunofluorescence and Western blot techniques were employed to assess the protein expression of tight junction proteins zonula occludens-1(ZO-1), occludin, claudin-5, and proteins of the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)/glycogen synthase kinase-3beta(GSK3β) pathway. The results demonstrated that 63 bioactive components and 125 potential core targets of RCRR were identified from the ETCM, TCMBank, and SwissTargetPrediction databases, as well as from the literature. A total of 1 708 brain microvascular endothelial cell-related targets were identified from the GeneCards and OMIM databases, and 52 targets were obtained by intersecting drug components with cell targets. The protein-protein interaction(PPI) network analysis revealed that AKT1, epidermal growth factor receptor(EGFR), matrix metalloproteinase 9(MMP9), estrogen receptor 1(ESR1), proto-oncogene tyrosine-protein kinase(SRC), peroxisome proliferator-activated receptor gamma(PPARG), GSK3β, and matrix metalloproteinase 2(MMP2) were considered hub genes. The KEGG enrichment analysis identified the PI3K/AKT pathway as the primary signaling pathway. Cell experiments demonstrated that RCRR-containing serum could enhance the migratory capacity of brain microvascular endothelial cells and the expression of tight junction proteins following OGD injury, which may be associated with the downregulation of the PI3K/AKT/GSK3β pathway. This study elucidates the pharmacological mechanism of RCRR in protecting brain microvascular endothelial cells through network pharmacology, characterized by multiple components and targets. These findings were validated through in vitro experiments and provide important ideas and references for further research into the molecular mechanisms of RCRR in protecting brain microvascular endothelial cells.
Endothelial Cells/cytology* ; Glycogen Synthase Kinase 3 beta/genetics* ; Proto-Oncogene Proteins c-akt/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Phosphatidylinositol 3-Kinases/genetics* ; Signal Transduction/drug effects* ; Brain/metabolism* ; Humans ; Animals ; Rhizome/chemistry* ; Microvessels/metabolism* ; Brain Ischemia/drug therapy*

Objective To investigate the effects and molecular mechanism of Homer protein homolog 1a (Homer 1a) overexpression on nerve injury in mice with traumatic brain injury (TBI). Methods Sixty male C57BL/6 mice were randomly divided into five groups: sham group, TBI group, empty lentivirus (Lv-NC) group, Homer 1a overexpression lentivirus (Lv-Homer 1a) group and Lv-Homer 1a + 740 Y-P group, with 12 mice in each group. The lentivirus was orthotopic injected into the cerebral cortex of mice 5 d before modeling, while 740 Y-P was injected intraperitoneally 1 d before modeling. The TBI model was established using the free-fall impact method, and the modified neurological severity scores (mNSS) of the mice was assessed 72 h post-surgery. The water content of brain tissue was quantified, and the histopathological damage and neuronal loss in brain tissue were assessed using HE staining and Nissl staining respectively. The formation of autophagosomes in brain tissue was observed by transmission electron microscopy. The protein expression levels of Homer 1a, microtubule-associated protein 1 light chain 3B (LC3B), Beclin 1, phosphatidylinositol 3-kinase (PI3K), phosphorylation PI3K(p-PI3K), protein kinase B (AKT), p-AKT, mammalian target of rapamycin (mTOR), and p-mTOR in brain tissue were detected by Western blot analysis. Results Compared to the sham group, the mice in the TBI group exhibited a significant increase in mNSS and cerebral water content. Moreover, severe brain tissue pathological damage was observed, accompanied by a substantial loss of neurons and an increase in autophagosome formation. The protein expressions of Homer 1a and Beclin 1, as well as the protein ratio of LC3B-II/LC3B-I, in brain tissues were significantly elevated, while the protein ratios of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR were significantly reduced. Compared to the TBI group, the Lv-Homer 1a group exhibited reduced mNSS and brain water content. Additionally, there was an improvement in pathological brain tissue damage and neuron loss. Furthermore, there was an increase in autophagosome formation and expression of autophagy-related proteins, while the protein ratios of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR were decreased. Compared to the Lv-Homer 1a group, the nerve injury in the Lv-Homer 1a+740 Y-P group was exacerbated, accompanied by a reduction in autophagosome formation and expression of autophagy-related proteins, while the PI3K/AKT/mTOR signaling pathway was activated. Conclusion Overexpression of Homer 1a effectively mitigates neurological damage in TBI mice, potentially through modulation of autophagy mediated by the PI3K/AKT/mTOR signaling pathway.
Animals ; TOR Serine-Threonine Kinases/genetics* ; Autophagy ; Brain Injuries, Traumatic/pathology* ; Male ; Proto-Oncogene Proteins c-akt/genetics* ; Phosphatidylinositol 3-Kinases/genetics* ; Homer Scaffolding Proteins/metabolism* ; Mice, Inbred C57BL ; Signal Transduction ; Mice

Objective To investigate the effect of basic helix-loop-helix family member E40 (BHLHE40) on the invasion and migration of osteosarcoma (OS) cells, and to explore the role of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway in the biological behavior of OS mediated by BHLHE40, providing a scientific basis for targeted therapy of OS. Methods On the basis of clinical OS samples and OS cell lines, the expression differences of BHLHE40 between OS and adjacent tissues, as well as those between OS cells and normal osteoblast cell lines, were analyzed. BHLHE40 knockdown OS cells were obtained through shRNA transfection. The effects of BHLHE40 on OS cell proliferation, migration, and invasion were examined using CCK-8, EdU staining, wound healing, and Transwell assays. The involvement of the PI3K/AKT signaling pathway was assessed by Western blotting. Further validation was conducted in vivo experiments. Results The expression of BHLHE40 was significantly higher in OS tissues compared to adjacent tissues. In OS cell lines, BHLHE40 protein expression levels were increased compared to normal osteoblasts, and the cell line with the highest BHLHE40 expression was selected for subsequent knockdown experiments. Compared with the knockdown control group, the BHLHE40 knockdown group exhibited reduced cell viability, EdU-positive cell count, colony number, cell migration, and invasion abilities, along with downregulation of phosphorylated PI3K(p-PI3K)/PI3K and p-AKT/AKT protein expression. The aforementioned functions of BHLHE40 were also reproduced in in vivo experiments. Conclusion BHLHE40 is highly expressed in OS tissues, and its knockdown can significantly inhibit OS cell proliferation, migration, and invasion, while reducing PI3K/AKT signaling pathway activity. This suggests that BHLHE40 could serve as a novel therapeutic target for OS.
Osteosarcoma/metabolism* ; Humans ; Proto-Oncogene Proteins c-akt/genetics* ; Cell Proliferation/genetics* ; Cell Movement/genetics* ; Signal Transduction/genetics* ; Phosphatidylinositol 3-Kinases/genetics* ; Cell Line, Tumor ; Animals ; Neoplasm Invasiveness ; Basic Helix-Loop-Helix Transcription Factors/metabolism* ; Bone Neoplasms/metabolism* ; Mice ; Gene Knockdown Techniques ; Male ; Female ; Mice, Nude

Objective To investigate whether miR-15b-5p can alleviate airway inflammation in asthma by negatively regulating ubiquitin specific peptidase 9X (USP9X) to down-regulate the expression of phosphatidylinositol 4, 5-diphosphate 3-kinase catalytic subunit α/AKT serine/threonine kinase 1 (PIK3CA/AKT1) pathway. Methods USP9X was predicted to be a direct target of miR-15b-5p by using an online database (miRWalk), and the luciferase reporter gene assay was performed to verify it. Co-immunoprecipitation (CO-IP) was used to verify the direct binding between USP9X and PIK3CA and the role of USP9X and its small molecule inhibitor WP1130 in the deubiquitination of PIK3CA. C57 mice were randomly divided into Control group, OVA group, OVA combined with NC group and miR-15b-5p agomir group, with 10 mice in each group. BEAS-2B cells were induced with interleukin 13 (IL-13) and treated with miR-15b-5p mimic. HE, Masson, PAS, immunohistochemistry, immunofluorescence staining, flow cytometry, Western blot and quantitative real-time PCR(qRT-PCR) were performed. Results It was found that the administration of miR-15b-5p agomir and mimic could reduce peribronchial inflammatory cells and improve airway inflammation, and miR-15b-5p could target negative regulation of USP9X. USP9X could directly bind to PIK3CA and regulate PIK3CA level in a proteasome-dependent manner, and USP9X could deubiquitinate K29-linked PIK3CA protein. Down-regulation of USP9X could increase PIK3CA ubiquitination level. WP1130, a small molecule inhibitor of USP9X, has the same effect as knockdown of USP9X, both of which could increase the ubiquitination level of PIK3CA and reduce the protein level of PIK3CA. Conclusion The miR-15b-5p/USP9X/PIK3CA/AKT1 signaling pathway may provide potential therapeutic targets for asthma.
Animals ; MicroRNAs/metabolism* ; Asthma/pathology* ; Class I Phosphatidylinositol 3-Kinases/genetics* ; Ubiquitin Thiolesterase/metabolism* ; Proto-Oncogene Proteins c-akt/genetics* ; Mice ; Signal Transduction ; Mice, Inbred C57BL ; Humans ; Inflammation/genetics* ; Cell Line ; Female ; Male

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 evaluate the protective effect of Lycium barbarum polysaccharides (LBP) against cisplatin-induced ovarian granulosa cell injury and investigate its possible mechanisms. METHODS: Human granulosa-like tumor cell line (KGN) were treated with 2.5 µg/mL cisplatin for 24 h, followed by treatment with 100, 500, and 1000 mg/L LBP, and the changes in cell viability, apoptosis, level of anti-Müllerian hormone (AMH), and cell ultrastructure were detected with CCK-8 assay, flow cytometry, ELISA and transmission electron microscopy. The cellular expressions of Bax, caspase-3, Bcl-2, and the PI3K/AKT pathway proteins were analyzed using Western blotting, and the expression of miR-23a was detected with RT-qPCR. KGN cell models with lentivirus-mediated miR-23a overexpression or knockdown were used to verify the therapeutic mechanism of LBP. RESULTS: Cisplatin treatment significantly inhibited cell viability, induced apoptosis, decreased AMH level, caused ultrastructural abnormalities, increased Bax and caspase-3 expression, and lowered Bcl-2 expression in KGN cells. Cisplatin also suppressed the activation of the PI3K/AKT signaling pathway and upregulated miR-23a expression in the cells. LBP intervention obviously alleviated cisplatin-induced injuries in KGN cells, and in particular, LBP treatment at the medium dose for 24 h significantly improved KGN cell viability, reduced apoptosis, enhanced their endocrine function, and ameliorated ultrastructural abnormalities. Mechanistically, medium-dose LBP obviously activated the PI3K/AKT pathway by downregulating miR-23a in cisplatin-treated cells, subsequently inhibiting Bax and caspase-3 while upregulating Bcl-2. Overexpression of miR-23a weakened while knockdown of miR-23a significantly enhanced the protective effects of LBP. CONCLUSIONS LBP alleviates cisplatin-induced apoptosis in KGN cells by inhibiting miR-23a expression and activating the PI3K/AKT pathway, suggesting a potential therapeutic strategy for ovarian function preservation.
Humans ; Cisplatin/adverse effects* ; MicroRNAs/genetics* ; Female ; Granulosa Cells/cytology* ; Apoptosis/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Down-Regulation ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Cell Line, Tumor ; Cell Survival/drug effects*

The angiogenic response is essential for the repair of ischemic brain tissue. Integrin α6 (Itga6) expression has been shown to increase under hypoxic conditions and is expressed exclusively in vascular structures; however, its role in post-ischemic angiogenesis remains poorly understood. In this study, we demonstrate that mice with endothelial cell-specific knockout of Itga6 exhibit reduced neovascularization, reduced pericyte coverage on microvessels, and accelerated breakdown of microvascular integrity in the peri-infarct area. In vitro, endothelial cells with ITGA6 knockdown display reduced proliferation, migration, and tube-formation. Mechanistically, we demonstrated that ITGA6 regulates post-stroke angiogenesis through the PI3K/Akt-eNOS-VEGFA axis. Importantly, the specific overexpression of Itga6 in endothelial cells significantly enhanced neovascularization and enhanced the integrity of microvessels, leading to improved functional recovery. Our results suggest that endothelial cell Itga6 plays a crucial role in key steps of post-stroke angiogenesis, and may represent a promising therapeutic target for promoting recovery after stroke.
Animals ; Nitric Oxide Synthase Type III/metabolism* ; Mice ; Proto-Oncogene Proteins c-akt/metabolism* ; Integrin alpha6/genetics* ; Endothelial Cells/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Stroke/pathology* ; Vascular Remodeling/physiology* ; Vascular Endothelial Growth Factor A/metabolism* ; Mice, Knockout ; Signal Transduction/physiology* ; Mice, Inbred C57BL ; Male ; Neovascularization, Physiologic/physiology*