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*

Alzheimer's disease (AD) poses one of the most urgent medical challenges in the 21st century as it affects millions of people. Unfortunately, the etiopathogenesis of AD is not yet fully understood and the current pharmacotherapy options are somewhat limited. Here, we report a novel inhibitor, Compound 44, for targeting cholinesterases, amyloid-β (Aβ) aggregation, and glycogen synthase kinase 3β (GSK-3β) simultaneously with the aim of achieving symptomatic relief and disease modification in AD therapy. We found that Compound 44 had good inhibitory effects on all intended targets with IC₅₀s of submicromolar or better, significant neuroprotective effects in cell models, and beneficial improvement of cognitive deficits in the triple transgenic AD (3 × Tg AD) mouse model. Moreover, we showed that Compound 44 acts as an autophagy regulator by inducing nuclear translocation of transcription factor EB through GSK-3β inhibition, enhancing the biogenesis of lysosomes and elevating autophagic flux, thus ameliorating the amyloid burden and tauopathy, as well as mitigating the disease phenotype. Our results suggest that triple-target inhibition via Compound 44 could be a promising strategy that may lead to the development of effective therapeutic approaches for AD.
Animals ; Alzheimer Disease/genetics* ; Mice, Transgenic ; Glycogen Synthase Kinase 3 beta/metabolism* ; Disease Models, Animal ; Mice ; Amyloid beta-Peptides/metabolism* ; Cholinesterase Inhibitors/therapeutic use* ; Humans ; Autophagy/drug effects* ; Cognitive Dysfunction/pathology* ; Neuroprotective Agents/pharmacology*

OBJECTIVE: This study investigated the effects of bis (2-butoxyethyl) phthalate (BBOP) on the onset of male puberty by affecting Leydig cell development in rats. METHODS: Thirty 35-day-old male Sprague-Dawley rats were randomly allocated to five groups mg/kg bw per day that were gavaged for 21 days with BBOP at 0, 10, 100, 250, or 500 mg/kg bw per day. The hormone profiles; Leydig cell morphological metrics; mRNA and protein levels; oxidative stress; and AKT, mTOR, ERK1/2, and GSK3β pathways were assessed. RESULTS: BBOP at 250 and/or 500 mg/kg bw per day decreased serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels mg/kg bw per day (P < 0.05). BBOP at 500 mg/kg bw per day decreased Leydig cell number mg/kg bw per day and downregulated Cyp11a1, Insl3, Hsd11b1, and Dhh in the testes, and Lhb and Fshb mRNAs in the pituitary gland (P < 0.05). The malondialdehyde content in the testis significantly increased, while Sod1 and Sod2 mRNAs were markedly down-regulated, by BBOP treatment at 250-500 mg/kg bw per day (P < 0.05). Furthermore, BBOP at 500 mg/kg bw per day decreased AKT1/AKT2, mTOR, and ERK1/2 phosphorylation, and GSK3β and SIRT1 levels mg/kg bw per day (P < 0.05). Finally, BBOP at 100 or 500 μmol/L induced ROS and apoptosis in Leydig cells after 24 h of treatment in vitro (P < 0.05). CONCLUSION: BBOP delays puberty onset by increasing oxidative stress and apoptosis in Leydig cells in rats. UNLABELLED The graphical abstract is available on the website www.besjournal.com.
Rats ; Male ; Animals ; Leydig Cells/metabolism* ; Testosterone ; Glycogen Synthase Kinase 3 beta/pharmacology* ; Rats, Sprague-Dawley ; Sexual Maturation ; Testis ; Oxidative Stress ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis

OBJECTIVE: To explore the specific pharmacological molecular mechanisms of Kai Xin San (KXS) on treating Alzheimer's disease (AD) based on network pharmacology and experimental validation. METHODS: The chemical compounds of KXS and their corresponding targets were screened using the Encyclopedia of Traditional Chinese Medicine (ETCM) database. AD-related target proteins were obtained from MalaCards database and DisGeNET databases. Key compounds and targets were identified from the compound-target-disease network and protein-protein interaction (PPI) network analysis. Functional enrichment analysis predicted the potential key signaling pathways involved in the treatment of AD with KXS. The binding affinities between key ingredients and targets were further verified using molecular docking. Finally, the predicted key signaling pathway was validated experimentally. Positioning navigation and space search experiments were conducted to evaluate the cognitive improvement effect of KXS on AD rats. Western blot was used to further examine and investigate the expression of the key target proteins related to the predicted pathway. RESULTS: In total, 38 active compounds and 469 corresponding targets of KXS were screened, and 264 target proteins associated with AD were identified. The compound-target-disease and PPI networks identified key active ingredients and protein targets. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested a potential effect of KXS in the treatment of AD via the amyloid beta (A β)-glycogen synthase kinase-3 beta (GSK3 β)-Tau pathway. Molecular docking revealed a high binding affinity between the key ingredients and targets. In vivo, KXS treatment significantly improved cognitive deficits in AD rats induced by Aβ_1-42, decreased the levels of Aβ, p-GSK3β, p-Tau and cyclin-dependent kinase 5, and increased the expressions of protein phosphatase 1 alpha (PP1A) and PP2A (P<0.05 or P<0.01). CONCLUSION KXS exerted neuroprotective effects by regulating the Aβ -GSK3β-Tau signaling pathway, which provides novel insights into the therapeutic mechanism of KXS and a feasible pharmacological strategy for the treatment of AD.
Rats ; Animals ; Alzheimer Disease/drug therapy* ; Amyloid beta-Peptides ; Glycogen Synthase Kinase 3 beta ; Network Pharmacology ; Molecular Docking Simulation ; Glycogen Synthase Kinase 3/therapeutic use* ; Drugs, Chinese Herbal/therapeutic use*

OBJECTIVE: To investigate the role of hippocampal neurodevelopment in the antidepressant effect of baicalin. METHODS: Forty male Institute of Cancer Research mice were divided into control, corticosterone (CORT, 40 mg/kg), CORT+baicalin-L (25 mg/kg), CORT+baicalin-H (50 mg/kg), and CORT+fluoxetine (10 mg/kg) groups according to a random number table. An animal model of depression was established by chronic CORT exposure. Behavioral tests were used to assess the reliability of depression model and the antidepressant effect of baicalin. In addition, Nissl staining and immunofluorescence were used to evaluate the effect of baicalin on hippocampal neurodevelopment in mice. The protein and mRNA expression levels of neurodevelopment-related factors were detected by Western blot analysis and real-time polymerase chain reaction, respectively. RESULTS: Baicalin significantly ameliorated the depressive-like behavior of mice resulting from CORT exposure and promoted the development of dentate gyrus in hippocampus, thereby reversing the depressive-like pathological changes in hippocampal neurons caused by CORT neurotoxicity. Moreover, baicalin significantly decreased the protein and mRNA expression levels of glycogen synthase kinase 3β (GSK3β), and upregulated the expression levels of cell cycle protein D1, p-mammalian target of rapamycin (mTOR), doublecortin, and brain-derived neurotrophic factor (all P<0.01). There were no significant differences between baicalin and fluoxetine groups (P>0.05). CONCLUSION Baicalin can promote the development of hippocampal neurons via mTOR/GSK3β signaling pathway, thus protect mice against CORT-induced neurotoxicity and play an antidepressant role.
Male ; Animals ; Mice ; Corticosterone ; Fluoxetine/metabolism* ; Depression/chemically induced* ; Glycogen Synthase Kinase 3 beta/metabolism* ; Reproducibility of Results ; Antidepressive Agents/pharmacology* ; Hippocampus ; TOR Serine-Threonine Kinases/metabolism* ; RNA, Messenger/genetics* ; Behavior, Animal ; Disease Models, Animal ; Mammals/metabolism*

OBJECTIVE: To explore the proliferation inhibitory effect of quinones from Blaps rynchopetera defense secretion on colorectal tumor cell lines. METHODS: Human colorectal cancer cell HT-29, human colorectal adenocarcinoma cell Caco-2 and normal human colon epithelial cell CCD841 were chosen for the evaluation of inhibitory activity of the main quinones of B. rynchopetera defense secretion, including methyl p-benzoquinone (MBQ), ethyl p-benzoquinone (EBQ), and methyl hydroquinone (MHQ), through methyl thiazolyl tetrazolium assay. The tumor-related factors, cell cycles, related gene expressions and protein levels were detected by enzyme-linked immunosorbent assy, flow cytometry, RT-polymerase chain reaction and Western blot, respectively. RESULTS: MBQ, EBQ, and MHQ could significantly inhibit the proliferation of Caco-2, with half maximal inhibitory concentration (IC₅₀) values of 7.04 ± 0.88, 10.92 ± 0.32, 9.35 ± 0.83, HT-29, with IC₅₀ values of 14.90 ± 2.71, 20.50 ± 6.37, 13.90 ± 1.30, and CCD841, with IC₅₀ values of 11.40 ± 0.68, 7.02 ± 0.44 and 7.83 ± 0.05 µg/mL, respectively. Tested quinones can reduce the expression of tumor-related factors tumor necrosis factor α, interleukin (IL)-10, and IL-6 in HT-29 cells, selectively promote apoptosis, and regulate the cell cycle which can reduce the proportion of cells in the G₁ phase and increase the proportion of the S phase. Meanwhile, tested quinones could up-regulate mRNA and protein expression of GSK-3β and APC, while down-regulate that of β-catenin, Frizzled1, c-Myc, and CyclinD1 in the Wnt/β-catenin pathway of HT-29 cells. CONCLUSION Quinones from B. rynchopetera defense secretion could inhibit the proliferation of colorectal tumor cells and reduce the expression of related factors, which would be functioned by regulating cell cycle, selectively promoting apoptosis, and affecting Wnt/β-catenin pathway-related mRNA and protein expressions.
Humans ; beta Catenin/metabolism* ; Caco-2 Cells ; Quinones/pharmacology* ; Glycogen Synthase Kinase 3 beta/metabolism* ; Cell Proliferation ; Colorectal Neoplasms/metabolism* ; Cell Line, Tumor ; Apoptosis ; Benzoquinones/pharmacology* ; RNA, Messenger ; Wnt Signaling Pathway

Oxidative stress plays a crucial role in cadmium (Cd)-induced myocardial injury. Mitsugumin 53 (MG53) and its mediated reperfusion injury salvage kinase (RISK) pathway have been demonstrated to be closely related to myocardial oxidative damage. Potentilla anserina L. polysaccharide (PAP) is a polysaccharide with antioxidant capacity, which exerts protective effect on Cd-induced damage. However, it remains unknown whether PAP can prevent and treat Cd-induced cardiomyocyte damages. The present study was desgined to explore the effect of PAP on Cd-induced damage in H9c2 cells based on MG53 and the mediated RISK pathway. For in vitro evaluation, cell viability and apoptosis rate were analyzed by CCK-8 assay and flow cytometry, respectively. Furthermore, oxidative stress was assessed by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining and using superoxide dismutase (SOD), catalase (CAT), and glutathione/oxidized glutathione (GSH/GSSG) kits. The mitochondrial function was measured by JC-10 staining and ATP detection assay. Western blot was performed to detect the expression of proteins related to MG53, the RISK pathway, and apoptosis. The results indicated that Cd increased the levels of reactive oxygen species (ROS) in H9c2 cells. Cd decreased the activities of SOD and CAT and the ratio of GSH/GSSG, resulting in decreases in cell viability and increases in apoptosis. Interestingly, PAP reversed Cd-induced oxidative stress and cell apoptosis. Meanwhile, Cd reduced the expression of MG53 in H9c2 cells and inhibited the RISK pathway, which was mediated by decreasing the ratio of p-Akt^Ser473/Akt, p-GSK3β^Ser9/GSK3β and p-ERK1/2/ERK1/2. In addition, Cd impaired mitochondrial function, which involved a reduction in ATP content and mitochondrial membrane potential (MMP), and an increase in the ratio of Bax/Bcl-2, cytoplasmic cytochrome c/mitochondrial cytochrome c, and Cleaved-Caspase 3/Pro-Caspase 3. Importantly, PAP alleviated Cd-induced MG53 reduction, activated the RISK pathway, and reduced mitochondrial damage. Interestingly, knockdown of MG53 or inhibition of the RISK pathway attenuated the protective effect of PAP in Cd-induced H9c2 cells. In sum, PAP reduces Cd-induced damage in H9c2 cells, which is mediated by increasing MG53 expression and activating the RISK pathway.
Cadmium/metabolism* ; Caspase 3/metabolism* ; Potentilla/metabolism* ; Glycogen Synthase Kinase 3 beta/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Cytochromes c/metabolism* ; Glutathione Disulfide/pharmacology* ; Oxidative Stress ; Myocytes, Cardiac ; Reactive Oxygen Species/metabolism* ; Reperfusion Injury/metabolism* ; Apoptosis ; Polysaccharides/pharmacology* ; Adenosine Triphosphate/metabolism*

Objective: To investigate the effects and mechanism of hydrogen peroxide (HP) pretreatment with low molarity on oxidative stress induced apoptosis of mouse bone marrow mesenchymal stem cells (BMSCs). Methods: The experimental research methods were used. BMSCs were isolated and cultured from two 2-week-old male BALB/c mice by the whole bone marrow culture method. The 3^rd-7^th passages of cells in logarithmic growth phase were used for the experiments after identification. According to the random number table (the same grouping method below), the cells were divided into 0 μmol/L HP group (without HP, the same below), 25 μmol/L HP group, 50 μmol/L HP group, 100 μmol/L HP group, 150 μmol/L HP group, 200 μmol/L HP group, 250 μmol/L HP group, and 300 μmol/L HP group in which cells were treated by the corresponding final molarity of HP, respectively. The apoptosis rate was detected by flow cytometry (n=4) after 24 hours of culture. The cells were divided into 0 μmol/L HP group, 25 μmol/L HP group, 50 μmol/L HP group, and 100 μmol/L HP group in which cells were treated by the corresponding final molarity of HP, respeclively. After 24 hours of culture, the protein expressions of B-lymphoma-2 (Bcl-2) and Bcl-2-related X protein (Bax) were detected by Western blotting, and the Bcl-2/Bax ratio was calculated (n=3). The cells were divided into 0 μmol/L HP group, 25 μmol/L HP group, 50 μmol/L HP group, 100 μmol/L HP group, 200 μmol/L HP group, and 300 μmol/L HP group in which cells were treated by the corresponding final molarity of HP, respectively. After 24 hours of culture, the protein expressions of glycogen synthase kinase-3β (GSK-3β) and phosphorylated GSK-3β (p-GSK-3β) were detected by Western blotting (n=3). The cells were divided into 0 μmol/L HP group, 50 μmol/L HP group, and 300 μmol/L HP group in which cells were treated by the corresponding final molarity of HP, respeclively, and HP pretreatment group with 50 μmol/L HP being added in advance for 12 h and then 300 μmol/L HP being added. After 24 hours of culture, the morphology and growth of cells were observed by inverted fluorescence microscopy (non-fluorescent condition) and immunofluorescence method, the apoptosis rate was detected by flow cytometry, the protein expressions of Bcl-2, Bax, cysteine aspartic acid specific protease-3 (caspase-3), caspase-9, cleavage caspase-3, cleavage caspase-9, GSK-3β, and p-GSK-3β were detected by Western blotting, and the Bcl-2/Bax ratio was calculated, with all the number of samples being 3. Data were statistically analyzed with one-way analysis of variance and Bonferroni test. Results: After 24 hours of culture, compared with that in 0 μmol/L HP group, the apoptosis rate of cells did not change significantly in 25 μmol/L HP group, 50 μmol/L HP group, or 100 μmol/L HP group (P>0.05) but increased significantly in 150 μmol/L HP group, 200 μmol/L HP group, 250 μmol/L HP group, and 300 μmol/L HP group (P<0.01). After 24 hours of culture, compared with that in 0 μmol/L HP group, the Bcl-2/Bax ratio of cells increased significantly in 25 μmol/L HP group and 50 μmol/L HP group (P<0.05 or P<0.01) but decreased significantly in 100 µmol/L HP group (P<0.05). After 24 hours of culture, compared with those in 0 μmol/L HP group, the protein expression of GSK-3β in cells showed no significant change in 25 μmol/L HP group and 50 μmol/L HP group (P>0.05), the protein expressions of p-GSK-3β in cells significantly increased in 25 μmol/L HP group and 50 μmol/L HP group (P<0.01), the protein expressions of GSK-3β and p-GSK-3β in cells in 100 μmol/L HP group showed no significant change (P>0.05), the protein expressions of GSK-3β in cells in 200 μmol/L HP group and 300 μmol/L HP group were significantly increased (P<0.05). but the protein expression of p-GSK-3β in cells in 200 μmol/L HP group and 300 μmol/L HP group was significantly decreased (P<0.05). After 24 hours of culture, the morphology and growth of cells in 0 μmol/L HP group and 50 μmol/L HP group were similar and normal; in contrast, the cells in 300 µmol/L HP group became smaller and round, with the cell protrusions being shorter or disappeared, the nucleus being cavitated, and the cell abscission being increased significantly; the morphology of most cells in HP pretreatment group was normal, with the shedding of cells being less than that in 300 µmol/L HP group, and the morphology of nucleus being normal. After 24 hours of culture, the protein expression of caspase-9 was similar among the four groups (P>0.05). Compared with that in 0 μmol/L HP group, the apoptosis rate and the protein expressions of cleavage caspase-9, caspase-3, and cleavage caspase-3 of cells in 50 μmol/L HP group showed no significant changes (P>0.05), the Bcl-2/Bax ratio of cells in 50 μmol/L HP group increased significantly (P<0.05), the apoptosis rate and the protein expressions of cleavage caspase-9, caspase-3, and cleavage caspase-3 of cells in 300 μmol/L HP group were significantly increased (P<0.01), while the Bcl-2/Bax ratio of cells in 300 μmol/L HP group was significantly decreased (P<0.05). Compared with those in 300 μmol/L HP group, the apoptosis rate and the protein expressions of cleavage caspase-9, caspase-3, and cleavage caspase-3 of cells were significantly decreased in HP pretreatment group (P<0.05 or P<0.01), while the Bcl-2/Bax ratio of cells was significantly increased in HP pretreatment group (P<0.01). After 24 hours of culture, the protein expressions of GSK-3β and p-GSK-3β of cells in 0 μmol/L HP group, 50 μmol/L HP group, 300 μmol/L HP group, and HP pretreatment group were 1.09±0.14, 0.62±0.17, 1.35±0.21, 0.74±0.34, 0.68±0.03, 0.85±0.08, 0.38±0.10, and 0.54±0.09, respectively. Compared with those in 0 μmol/L HP group, the protein expression of p-GSK-3β of cells was significantly increased in 50 μmol/L HP group (P<0.05) but significantly decreased in 300 μmol/L HP group (P<0.01), while the protein expression of GSK-3β of cells was significantly increased in 300 μmol/L HP group (P<0.05). Compared with those in 300 μmol/L HP group, the protein expression of GSK-3β of cells was significantly decreased in HP pretreatment group (P<0.01), while the protein expression of p-GSK-3β of cells was significantly increased in HP pretreatment group (P<0.01). Conclusions: The molarity of 50 μmol/L may be the optimal molarity of HP to pretreat mouse BMSCs, and 50 μmol/L HP pretreatment can antagonize mitochondrial pathway of oxidative stress induced apoptosis by inhibiting the activity of GSK-3β.
Animals ; Apoptosis ; Glycogen Synthase Kinase 3 beta/pharmacology* ; Hydrogen Peroxide/pharmacology* ; Male ; Mesenchymal Stem Cells ; Mice ; Oxidative Stress

Carthamus tinctorius is proved potent in treating ischemic stroke. Flavonoids, such as safflower yellow, hydroxysafflor yellow A(HSYA), nicotiflorin, safflower yellow B, and kaempferol-3-O-rutinoside, are the main substance basis of C. tinctorius in the treatment of ischemic stroke, and HSYA is the research hotspot. Current studies have shown that C. tinctorius can prevent and treat ischemic stroke by reducing inflammation, oxidative stress, and endoplasmic reticulum stress, inhibiting neuronal apoptosis and platelet aggregation, as well as increasing blood flow. C. tinctorius can regulate the pathways including nuclear factor(NF)-κB, mitogen-activated protein kinase(MAPK), signal transducer and activator of transcription protein 3(STAT3), and NF-κB/NLR family pyrin domain containing 3(NLRP3), and inhibit the activation of cyclooxygenase-2(COX-2)/prostaglandin D2/D prostanoid receptor pathway to alleviate the inflammatory development during ischemic stroke. Additionally, C. tinctorius can relieve oxidative stress injury by inhibiting oxidation and nitrification, regulating free radicals, and mediating nitric oxide(NO)/inducible nitric oxide synthase(iNOS) signals. Furthermore, mediating the activation of Janus kinase 2(JAK2)/STAT3/suppressor of cytokine signaling 3(SOCS3) signaling pathway and phosphoinositide 3-kinase(PI3 K)/protein kinase B(Akt)/glycogen synthase kinase-3β(GSK3β) signaling pathway and regulating the release of matrix metalloproteinase(MMP) inhibitor/MMP are main ways that C. tinctorius inhibits neuronal apoptosis. In addition, C. tinctorius exerts the therapeutic effect on ischemic stroke by regulating autophagy and endoplasmic reticulum stress. The present study reviewed the molecular mechanisms of C. tinctorius in the treatment of ischemic stroke to provide references for the clinical application of C. tinctorius.
Carthamus tinctorius/chemistry* ; Chalcone/therapeutic use* ; Cyclooxygenase 2/metabolism* ; Cytokines/metabolism* ; Flavonoids/therapeutic use* ; Glycogen Synthase Kinase 3 beta/metabolism* ; Humans ; Ischemic Stroke/drug therapy* ; Janus Kinase 2/metabolism* ; Mitogen-Activated Protein Kinases/metabolism* ; NF-kappa B/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Nitric Oxide/metabolism* ; Nitric Oxide Synthase Type II/metabolism* ; Phosphatidylinositol 3-Kinase/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Prostaglandin D2 ; Proto-Oncogene Proteins c-akt/metabolism* ; Quinones/pharmacology*

The present study explored the main active ingredients and the underlying mechanism of Spatholobi Caulisin the treatment of ovarian cancer(OC) by network pharmacology, molecular docking, and in vitro cell experiments. The active ingredients and their predicted targets(AITs) were first acquired online with the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). Theoretical disease targets(DTs) were obtained through professional databases including GeneCards, OMIM, PharmGkb, TTD, and DrugBank. The common targets in the intersection of AITs and DTs were used for the construction of a "drug-ingredient-disease-target" network by Cytoscape 3.7.1. STRING database was used to construct a protein-protein interaction(PPI) network. R 4.0.5 was used for GO and KEGG functional enrichment analyses. Schr9 dinger Maestro was used to perform and optimize the molecular docking and virtual screening.Twenty-three active ingredients of Spatholobi Caulis were screened out, involving 75 OC targets and 178 signaling pathways.Network analysis revealed that Spatholobi Caulis presumedly exerted an anti-OC effect by acting on key protein targets such as GSK-3β, Bcl-2, and Bax. Molecular docking showed that GSK-3β possessed goodbinding activity to prunetin. In vitro cell experiments preliminarily verified the core targets and pathways of prunetin, the active ingredient of Spatholobi Caulis against human OC SKOV3 cells.CCK-8 assay was used to detect the cell proliferation, and flow cytometry was used to detect the effect of prunetin on apoptosis of human OC SKOV3 cells.The expression of prunetin targets and related regulatory proteins was detected by Western blot.In vitro cell experiments demonstrated that prunetindisplayed significant inhibitory effects on the proliferation of OC cells and could induce apoptosis of SKOV3 cells. Western blot showed that prunetin could induce SKOV3 cell apoptosis by inhibiting GSK-3β phosphorylation and regulating the expression of downstream Bcl-2 and Bax proteins. This study reveals the scientific nature of network pharmacology in the prediction and guidance of experimental design, confirming that prunetin can treat OC by blocking the GSK-3β/Bcl-2/Bax cell signal transduction pathway. The findings are expected to provide a basis for the investigation of the mechanism of Spatholobi Caulis in the treatment of OC.
Drugs, Chinese Herbal/pharmacology* ; Glycogen Synthase Kinase 3 beta/genetics* ; Humans ; Medicine, Chinese Traditional ; Molecular Docking Simulation ; Network Pharmacology ; Ovarian Neoplasms/genetics*