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 To investigate the role and possible mechanism of glycogen synthase kinase-3 beta (GSK-3β)/cAMP response element binding protein (CREB) signaling pathway in regulating macrophage pyroptosis in the pathogenesis and development of diabetic foot ulcer (DFU). Methods Thirty rats were randomly divided into control group, DFU group and GSK-3β inhibited group, with 10 rats in each group. Fasting blood glucose (FBG) was detected by dynamic blood glucose detector. The wound healing of each group was observed and recorded. The histopathologic changes of the wound were detected by HE staining. The level of wound fibrosis was detected by Masson staining. The protein levels of GSK-3β, CREB, gasdermin E (GSDME) and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) in wound tissue were detected by Western blotting. The co-expression of F4/80, GSDME and NLRP3 in wound tissue was detected by immunofluorescence staining. The serum levels of IL-1β and IL-18 were detected by ELISA. Results Compared with the control group, FBG in DFU group was increased. Compared with DFU group, FBG in GSK-3β inhibition group was decreased. The wound healing rate of rats in the inhibited GSK-3β group was higher than that in the DFU group from day 3 to day 14, and the difference was significant on day 14. Therefore, samples from day 14 were used in the follow-up experiment. Compared with the control group, the wound tissue of rats in DFU group was significantly damaged with collagen deposition defect, and the expressions of GSK-3β, CREB and apoptosis-related proteins GSDME and NLRP3 were increased, and the co-expressions of F4/80 and GSDME, F4/80 and NLRP3 were increased. Serum levels of IL-1β and IL-18 were increased. Compared with DFU group, most of the wound tissues of rats in GSK-3β group were healed. Collagen deposition at the fracture was increased. The expressions of GSK-3β, CREB and GSDME, NLRP3 were decreased. The expression levels of F4/80 and GSDME were reduced, along with a decrease in the co-expression of F4/80 and NLRP3. Additionally, there was a reduction in serum concentrations of IL-1β and IL-18. Conclusion GSK-3β/CREB signaling pathway and macrophage pyroptosis are significantly up-regulated in DFU rats. Inhibition of this pathway can promote DFU healing and down-regulate macrophage pyroptosis level.
Animals ; Pyroptosis ; Diabetic Foot/metabolism* ; Glycogen Synthase Kinase 3 beta/metabolism* ; Signal Transduction ; Male ; Rats ; Cyclic AMP Response Element-Binding Protein/metabolism* ; Macrophages/metabolism* ; Rats, Sprague-Dawley ; Wound Healing ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Interleukin-1beta/metabolism*

OBJECTIVE: Physical exercise, a common non-drug intervention, is an important strategy in cancer treatment, including hepatocellular carcinoma (HCC). However, the mechanism remains largely unknown. Due to the importance of hypoxia and cancer stemness in the development of HCC, the present study investigated whether the anti-HCC effect of physical exercise is related to its suppression on hypoxia and cancer stemness. METHODS: A physical exercise intervention of swimming (30 min/d, 5 d/week, for 4 weeks) was administered to BALB/c nude mice bearing subcutaneous human HCC tumor. The anti-HCC effect of swimming was assessed in vivo by tumor weight monitoring, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) detection of proliferating cell nuclear antigen (PCNA) and Ki67. The expression of stemness transcription factors, including Nanog homeobox (NANOG), octamer-binding transcription factor 4 (OCT-4), v-Myc avian myelocytomatosis viral oncogene homolog (C-MYC) and hypoxia-inducible factor-1α (HIF-1α), was detected using real-time reverse transcription polymerase chain reaction. A hypoxia probe was used to explore the intratumoral hypoxia status. Western blot was used to detect the expression of HIF-1α and proteins related to protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling pathway. The IHC analysis of platelet endothelial cell adhesion molecule-1 (CD31), and the immunofluorescence co-location of CD31 and desmin were used to analyze tumor blood perfusion. SMMC-7721 cells were treated with nude mice serum. The inhibition effect on cancer stemness in vitro was detected using suspension sphere experiments and the expression of stemness transcription factors. The hypoxia status was inferred by measuring the protein and mRNA levels of HIF-1α. Further, the expression of proteins related to Akt/GSK-3β/β-catenin signaling pathway was detected. RESULTS: Swimming significantly reduced the body weight and tumor weight in nude mice bearing HCC tumor. HE staining and IHC results showed a lower necrotic area ratio as well as fewer PCNA or Ki67 positive cells in mice receiving the swimming intervention. Swimming potently alleviated the intratumoral hypoxia, attenuated the cancer stemness, and inhibited the Akt/GSK-3β/β-catenin signaling pathway. Additionally, the desmin⁺/CD31⁺ ratio, rather than the number of CD31⁺ vessels, was significantly increased in swimming-treated mice. In vitro experiments showed that treating cells with the serum from the swimming intervention mice significantly reduced the formation of SMMC-7721 cell suspension sphere, as well as the mRNA expression level of stemness transcription factors. Consistent with the in vivo results, HIF-1α and Akt/GSK-3β/β-catenin signaling pathway were also inhibited in cells treated with serum from swimming group. CONCLUSION Swimming alleviated hypoxia and attenuated cancer stemness in HCC, through suppression of the Akt/GSK-3β/β-catenin signaling pathway. The alleviation of intratumoral hypoxia was related to the increase in blood perfusion in the tumor. Please cite this article as: Xiao CL, Zhong ZP, Lü C, Guo BJ, Chen JJ, Zhao T, Yin ZF, Li B. Physical exercise suppresses hepatocellular carcinoma progression by alleviating hypoxia and attenuating cancer stemness through the Akt/GSK-3β/β-catenin pathway. J Integr Med. 2023; 21(2): 184-193.
Humans ; Animals ; Mice ; Carcinoma, Hepatocellular/drug therapy* ; Proto-Oncogene Proteins c-akt/metabolism* ; Proliferating Cell Nuclear Antigen/therapeutic use* ; Mice, Nude ; Glycogen Synthase Kinase 3 beta/genetics* ; beta Catenin/therapeutic use* ; Liver Neoplasms/drug therapy* ; Desmin/therapeutic use* ; Ki-67 Antigen ; Cell Line, Tumor ; Hypoxia ; RNA, Messenger/therapeutic use* ; Cell Proliferation

Marsdenia tenacissima injection, a standard Marsdenia tenacissima extract (MTE), has been approved as an adjuvant therapeutic agent for various cancers. Our previous study showed that MTE inhibited the proliferation and metastasis of prostate cancer (PCa) cells. However, the underlying mechanisms and active ingredients of MTE against PCa were not completely understood. This study revealed that MTE induced significant decreases in cell viability and clonal growth in PCa cells. In addition, MTE induced the apoptosis of DU145 cells by reducing the mitochondrial membrane potential and increasing the expression of Cleaved Caspase 3/7, Cyt c, and Bax. In vivo, DU145 xenografted NOD-SCID mice treated with MTE showed significantly decreased tumor size. TUNEL staining and Western blot confirmed the pro-apoptotic effects of MTE. Network pharmacology analysis collected 196 ingredients of MTE linked to 655 potential targets, and 709 PCa-associated targets were retrieved, from which 149 overlapped targets were screened out. Pathway enrichment analysis showed that the HIF-1, PI3K-AKT, and ErbB signaling pathways were closely related to tumor apoptosis. Western blot results confirmed that MTE increased the expression of p-AKT^Ser473 and p-GSK3β^Ser9, and decreased the expression of p-STAT3^Tyr705in vitro and in vivo. A total of 13 compounds in MTE were identified by HPLC-CAD-QTOF-MS/MS and UPLC-QTOF-MS/MS. Molecular docking analysis indicated that six compounds may interact with AKT, GSK3β, and STAT3. In conclusion, MTE induces the endogenous mitochondrial apoptosis of PCa by regulating the AKT/GSK3β/STAT3 signaling axis, resulting in inhibition of PCa growth in vitro and in vivo.
Mice ; Animals ; Male ; Humans ; Mice, Inbred NOD ; Mice, SCID ; Marsdenia ; Proto-Oncogene Proteins c-akt ; Glycogen Synthase Kinase 3 beta ; Molecular Docking Simulation ; Phosphatidylinositol 3-Kinases ; Tandem Mass Spectrometry ; Prostatic Neoplasms ; Apoptosis ; STAT3 Transcription Factor

Objective: To investigate the effect and mechanism of miR-96-5p on apoptosis of PC12 cells induced by maltol aluminum. Methods: In January 2021, PC12 cells at logarithmic growth phase were divided into blank control group and low, medium and high dose group. Cells in each group were treated with 0, 100, 200 and 400 μmol/L maltol aluminum for 24 hours respectively. Cells were collected and cell apoptosis rates were detected by flow cytometry, miR-96-5p and insulin receptor substrate 1 (IRS1) mRNA expressions were detected by qRT-PCR, and the protein expression levels of cysteine protease 3 (Caspase3) 、activated cysteine protease 3 (Cleaved-caspase3) 、IRS1、phosphorylated protein kinase B (p-AKT) and phosphorylated glucose synthesis kinase 3β (p-GSK3β) were detected by western blotting. The target binding relationship between miR-96-5p and IRS1 was detected by double luciferase reporter gene experiment. The miR-96-5p inhibitor cells and negative control cells were constructed after transfecting PC12 cells with miR-96-5p inhibitor for 24 hours. The cells were divided into blank control group, negative control group, aluminum exposure group, aluminum exposure+negative control group, aluminum exposure+miR-96-5p inhibition group, and miR-96-5p inhibition group. After transfecting PC12 cells with miR-96-5p inhibition and IRS1 siRNA for 24 h, the cells were divided into aluminum exposure+miR-96-5p inhibition+negative control group and aluminum exposure+miR-96-5p inhibition+IRS1 inhibition group. The control group was cultured in complete culture medium, and cells in the aluminum exposure group were treated with 200 μmol/L maltol aluminum for 24 hours. Cells in each group were collected and the apoptosis rate, miR-96-5p and IRS1 mRNA expression levels, as well as protein expression levels of Caspase3, Cleaved-caspase3, IRS1, p-AKT, and p-GSK3β were measured. Results: After 24 hours of exposure, compared with blank control group and low-dose group, the apoptosis rates, relative expressions of Caspase3 and Cleaved-caspase3 proteins, and relative expressions of miR-96-5p in the medium and high-dose groups of PC12 cells were significantly increased, while the relative expression levels of IRS1 mRNA, IRS1, p-AKT and p-GSK3β proteins were significantly decreased (P<0.05). Targetscan prediction and double luciferase report experiment both proved that IRS1 was a direct target gene of miR-96-5p. In the transfection experiment, compared with the aluminum exposure group, the apoptosis rate, the relative expressions of Caspase3 and Cleaved-caspase3 proteins, the relative expression of miR-96-5p in the aluminum exposure+miR-96-5p inhibition group were significantly decreased, while the relative expression levels of IRS1 mRNA and IRS1, p-AKT and p-GSK3β proteins were significantly increased (P<0.05). In the IRS1 low expression experiment, compared with the aluminum exposure+miR-96-5p inhibition+negative control group, the apoptosis rate, the relative expressions of Caspase3 and Cleaved-caspase3 proteins in the aluminum exposure+miR-96-5p inhibition+IRS1 inhibition group were significantly increased, while the relative expression levels of IRS1 mRNA and IRS1, p-AKT and p-GSK3β proteins were significantly decreased (P<0.05) . Conclusion: The increased expression of miR-96-5p and the targeted inhibition of IRS1 may be one of the mechanisms of apoptosis of PC12 cells induced by maltol aluminum exposure.
Animals ; Rats ; Aluminum/toxicity* ; Apoptosis ; Cell Proliferation ; Glycogen Synthase Kinase 3 beta/metabolism* ; Insulin Receptor Substrate Proteins/metabolism* ; MicroRNAs/metabolism* ; PC12 Cells ; Proto-Oncogene Proteins c-akt/metabolism* ; RNA, Messenger

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 observe the effects of Yizhi Tiaoshen (benefiting mental health and regulating the spirit) acupuncture on learning and memory function, and the expression of phosphorylated tubulin-associated unit (tau) protein in the hippocampus of Alzheimer's disease (AD) model rats, and explore the effect mechanism of this therapy on AD. METHODS: A blank group and a sham-operation group were randomly selected from 60 male SD rats, 10 rats in each one. AD models were established in the rest 40 rats by the intraperitoneal injection of D-galactose and okadaic acid in the CA1 region of the bilateral hippocampus. Thirty successfully-replicated model rats were randomly divided into a model group, a western medication group and an acupuncture group, 10 rats in each one. In the acupuncture group, acupuncture was applied to "Baihui" (GV 20), "Sishencong" (EX-HN 1), "Neiguan" (PC 6), "Shenmen" (HT 7), "Xuanzhong" (GB 39) and "Sanyinjiao" (SP 6); and the needles were retained for 10 min. Acupuncture was given once daily. One course of treatment was composed of 6 days, with the interval of 1 day; the completion of treatment included 4 courses. In the western medication group, donepezil hydrochloride solution (0.45 mg/kg) was administrated intragastrically, once daily; it took 7 days to accomplish one course of treatment and a completion of intervention was composed of 4 courses. Morris water maze (MWM) and novel object recognition test (NORT) were used to assess the learning and memory function of the rats. Using HE staining and Nissl staining, the morphological structure of the hippocampus was observed. With Western blot adopted, the protein expression of the tau, phosphorylated tau protein at Ser198 (p-tau Ser198), protein phosphatase 2A (PP2A) and glycogen synthase kinase-3β (GSK-3β) in the hippocampus was detected. RESULTS: There were no statistical differences in all of the indexes between the sham-operation group and the blank group. Compared with the sham-operation group, in the model group, the MWM escape latency was prolonged (P<0.05), the crossing frequency and the quadrant stay time in original platform were shortened (P<0.05), and the NORT discrimination index (DI) was reduced (P<0.05); the hippocampal cell numbers were declined and the cells arranged irregularly, the hippocampal neuronal structure was abnormal and the numbers of Nissl bodies decreased; the protein expression of p-tau Ser198 and GSK-3βwas increased (P<0.05) and that of PP2A decreased (P<0.05). When compared with the model group, in the western medication group and the acupuncture group, the MWM escape latency was shortened (P<0.05), the crossing frequency and the quadrant stay time in original platform were increased (P<0.05), and DI got higher (P<0.05); the hippocampal cell numbers were elevated and the cells arranged regularly, the damage of hippocampal neuronal structure was attenuated and the numbers of Nissl bodies were increased; the protein expression of p-tau Ser198 and GSK-3β was reduced (P<0.05) and that of PP2A was increased (P<0.05). There were no statistically significant differences in the above indexes between the acupuncture group and the western medication group (P>0.05). CONCLUSION Acupuncture therapy of "benefiting mental health and regulating the spirit" could improve the learning and memory function and alleviate neuronal injure of AD model rats. The effect mechanism of this therapy may be related to the down-regulation of GSK-3β and the up-regulation of PP2A in the hippocampus, and then to inducing the inhibition of tau protein phosphorylation.
Male ; Animals ; Rats ; Rats, Sprague-Dawley ; Glycogen Synthase Kinase 3 beta ; Tubulin ; Alzheimer Disease/therapy* ; tau Proteins/genetics* ; Acupuncture Therapy ; Hippocampus

OBJECTIVE: Programmed cell death 6 (PDCD6), a Ca ²⁺-binding protein, has been reported to be aberrantly expressed in all kinds of tumors. The aim of this study was to explore the role and mechanism of PDCD6 in hepatocellular carcinomas (HCCs). METHODS: The expression levels of PDCD6 in liver cancer patients and HCC cell lines were analyzed using bioinformatics and Western blotting. Cell viability and metastasis were determined by methylthiazol tetrazolium (MTT) and transwell assays, respectively. And Western blotting was used to test related biomarkers and molecular pathway factors in HCC cell lines. LY294002, a PI3K inhibitor inhibiting AKT, was used to suppress the AKT/GSK3β/β-catenin pathway to help evaluate the role of this pathway in the HCC carcinogenesis associated with PDCD6. RESULTS: The analysis of The Cancer Genome Atlas Database suggested that high PDCD6 expression levels were relevant to liver cancer progression. This was consistent with our finding of higher levels of PDCD6 expression in HCC cell lines than in normal hepatocyte cell lines. The results of MTT, transwell migration, and Western blotting assays revealed that overexpression of PDCD6 positively regulated HCC cell proliferation, migration, and invasion. Conversely, the upregulation of PDCD6 expression in the presence of an AKT inhibitor inhibited HCC cell proliferation, migration, and invasion. In addition, PDCD6 promoted HCC cell migration and invasion by epithelial-mesenchymal transition. The mechanistic investigation proved that PDCD6 acted as a tumor promoter in HCC through the AKT/GSK3β/β-catenin pathway, increasing the expression of transcription factors and cellular proliferation and metastasis. CONCLUSION PDCD6 has a tumor stimulative role in HCC mediated by AKT/GSK3β/β-catenin signaling and might be a potential target for HCC progression.
Humans ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; Proto-Oncogene Proteins c-akt/metabolism* ; beta Catenin/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Glycogen Synthase Kinase 3 beta/metabolism* ; Cell Line ; Cell Proliferation ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Calcium-Binding Proteins/metabolism* ; Apoptosis Regulatory Proteins/genetics*

Proto-Oncogene Proteins c-akt/metabolism* ; Butyrates ; Ligands ; Glucose ; Receptors, G-Protein-Coupled/metabolism* ; Glycogen Synthase Kinases/metabolism* ; Glycogen Synthase Kinase 3 beta ; Phosphorylation