Capsaicin combined with stem cells improved mitochondrial dysfunction in PIG3V cells, an immortalized human vitiligo melanocyte cell line, by inhibiting the HSP70/TLR4/mTOR/FAK signaling axis.

BACKGROUND: Vitiligo is a common autoimmune skin disease. Capsaicin has been found to exert a positive effect on vitiligo treatment, and mesenchymal stem cells (MSCs) are also confirmed to be an ideal cell type. This study aimed to explore the influence of capsaicin combined with stem cells on the treatment of vitiligo and to confirm the molecular mechanism of capsaicin combined with stem cells in treating vitiligo. METHODS AND RESULTS: PIG3V cell proliferation and apoptosis were detected using CCK-8 and TUNEL assays, MitoSOX Red fluorescence staining was used to measure the mitochondrial ROS level, and JC-1 staining was used to detect the mitochondrial membrane potential. The expression of related genes and proteins was detected using RT‒qPCR and Western blotting. Coimmunoprecipitation was used to analyze the protein interactions between HSP70 and TLR4 or between TLR4 and mTOR. The results showed higher expression of HSP70 in PIG3V cells than in PIG1 cells. The overexpression of HSP70 reduced the proliferation of PIG3V cells, promoted apoptosis, and aggravated mitochondrial dysfunction and autophagy abnormalities. The expression of HSP70 could be inhibited by capsaicin combined with MSCs, which increased the levels of Tyr, Tyrp1 and DCT, promoted the proliferation of PIG3V cells, inhibited apoptosis, activated autophagy, and improved mitochondrial dysfunction. In addition, capsaicin combined with MSCs regulated the expression of TLR4 through HSP70 and subsequently affected the mTOR/FAK signaling pathway CONCLUSIONS: Capsaicin combined with MSCs inhibits TLR4 through HSP70, and the mTOR/FAK signaling pathway is inhibited to alleviate mitochondrial dysfunction and autophagy abnormalities in PIG3V cells.

Astragaloside-IV promotes autophagy via the Akt/mTOR pathway to improve cellular lipid deposition.

The current study aimed to investigate the potential role of astragaloside IV (AS-IV) in improving cellular lipid deposition and its underlying mechanism. A fatty liver cell model was established by treating hepatoma cells with palmitic acid. AS-IV and SC79 were used for treatment. Oil Red O staining was applied to detect intracellular lipid deposition, and transmission electron microscopy was utilized to assess autophagosome formation. Immunofluorescence double staining was applied to determine microtubule-associated proteins 1A/1B light chain 3 (LC3) expression. Western blot analysis was performed to detect the expression of LC3, prostacyclin, Beclin-1, V-akt murine thymoma viral oncogene homolog (Akt), phosphorylated Akt, mTOR, and phosphorylated mTOR. Oil Red O staining revealed that AS-IV reduced intracellular lipid accumulation. Further, it increased autophagosome synthesis and the expression of autophagy proteins LC3 and Beclin-1 in the cells. It also reduced the phosphorylation levels of Akt and mTOR and the levels of prostacyclin. However, the effects of AS-IV decreased with SC79 treatment. In addition, LC3B + BODIPY493/503 fluorescence double staining showed that AS-IV reduced intracellular lipid deposition levels by enhancing autophagy. AS-IV can reduce lipid aggregation in fatty liver cells, which can be related to enhanced hepatocyte autophagy by inhibiting the Akt/mTOR signaling pathway.

The polysaccharides from Balanophora polyandra enhanced neuronal autophagy to ameliorate brain function decline in natural aging mice through the PI3K/AKT/mTOR signaling pathway.

The decline of aging brain neurons is the main cause of various neurodegenerative disease. This study aimed to examine the impact of Balanophora polyandra polysaccharides (BPP) against aging related neuronal deterioration. C57BL/6 mice were fed with regular feed for 27 months to establish a natural aging mouse model. From 3 months of age, mice in the drug-treated group were respectively fed with feed containing 0.05 or 0.18% BPP until 27 months of age. The effects of BPP treatment on the pathological changes of neurons in mice brain were evaluated, as well as autophagy-related and signaling pathway proteins. BPP treatment had a notable positive impact on the pathological injury of cortical and hippocampal neurons, alleviated neuronal degeneration, and enhanced the staining of Nissl bodies in natural aging mice. Furthermore, BPP upregulated autophagy-related proteins LC3 II/I, Parkin, and PINK1 in the cortex and hippocampus of aging mice, and significantly decreased the expression of p62, PI3K, p-protein Kinase B (AKT), and p-mTOR. Immunofluorescence results showed a reduction in the brightness of LC3, which mainly coexpressed with NeuN in natural aging mice brain, and increased LC3-positive neurons were observed after BPP treatment. Collectively, BPP treatment enhanced neuronal autophagy to improve brain functional degradation through the PI3K/AKT/mTOR signaling in natural aging mice. These finding suggested that BPP has potential to mitigate or delay the neurodegeneration associated with aging and further investigation was needed to validate its efficacy in elderly populations.

Total flavonoids of Broussonetia papyrifera alleviate non-alcohol fatty liver disease via regulating Nrf2/AMPK/mTOR signaling pathways.

BACKGROUNDS: Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic liver diseases. The leaves of Broussonetia papyrifera contain a large number of flavonoids, which have a variety of biological functions. METHODS: In vitro experiments, free fatty acids were used to stimulate HepG2 cells. NAFLD model was established in vivo in mice fed with high fat diet (HFD) or intraperitoneally injected with Tyloxapol (Ty). At the same time, Total flavonoids of Broussonetia papyrifera (TFBP) was used to interfere with HepG2 cells or mice. RESULTS: The results showed that TFBP significantly decreased the lipid accumulation induced by oil acid (OA) with palmitic acid (PA) in HepG2 cells. TFBP decreased the total cholesterol (TC), the triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and increased high-density lipoprotein cholesterol (HDLC) in serum. TFBP could also effectively inhibit the generation of reactive oxygen species (ROS) and restrained the level of myeloperoxidase (MPO), and enhance the activity of superoxide dismutase (SOD) to alleviate the injury from oxidative stress in the liver. Additionally, TFBP activated nuclear factor erythroid-2-related factor 2 (Nrf2) pathway to increasing the phosphorylation of AMP-activated protein kinase (AMPK). Meanwhile, protein levels of mTORC signaling pathway were evidently restrained with the treatment of TFBP. CONCLUSION: Our experiments proved that TFBP has the therapeutic effect in NAFLD, and the activation of Nrf2 and AMPK signaling pathways should make sense.

High-dose Vitamin C injection ameliorates against sepsis-induced myocardial injury by anti-apoptosis, anti-inflammatory and pro-autophagy through regulating MAPK, NF-κB and PI3K/AKT/mTOR signaling pathways in rats.

AIMS: This study aimed to evaluate the effects of VC on SIMI in rats. METHODS: In this study, the survival rate of high dose VC for SIMI was evaluated within 7 days. Rats were randomly assigned to three groups: Sham group, CLP group, and high dose VC (500 mg/kg i.v.) group. The animals in each group were treated with drugs for 1 day, 3 days or 5 days, respectively. Echocardiography, myocardial enzymes and HE were used to detect cardiac function. IL-1ß, IL-6, IL-10 and TNF-α) in serum were measured using ELISA kits. Western blot was used to detect proteins related to apoptosis, inflammation, autophagy, MAPK, NF-κB and PI3K/Akt/mTOR signaling pathways. RESULTS: High dose VC improved the survival rate of SIMI within 7 days. Echocardiography, HE staining and myocardial enzymes showed that high-dose VC relieved SIMI in rats in a time-dependent manner. And compared with CLP group, high-dose VC decreased the expressions of pro-apoptotic proteins, while increased the expression of anti-apoptotic protein. And compared with CLP group, high dose VC decreased phosphorylation levels of Erk1/2, P38, JNK, NF-κB and IKK α/ß in SIMI rats. High dose VC increased the expression of the protein Beclin-1 and LC3-II/LC3-I ratio, whereas decreased the expression of P62 in SIMI rats. Finally, high dose VC attenuated phosphorylation of PI3K, AKT and mTOR compared with the CLP group. SIGNIFICANCE: Our results showed that high dose VC has a good protective effect on SIMI after continuous treatment, which may be mediated by inhibiting apoptosis and inflammatory, and promoting autophagy through regulating MAPK, NF-κB and PI3K/AKT/mTOR pathway.

Integrating network pharmacology and experimental validation reveals therapeutic effects of D-mannose on NAFLD through mTOR suppression.

Non-alcoholic fatty liver disease (NAFLD), a chronic liver condition and metabolic disorder, has emerged as a significant health issue worldwide. D-mannose, a natural monosaccharide widely existing in plants and animals, has demonstrated metabolic regulatory properties. However, the effect and mechanism by which D-mannose may counteract NAFLD have not been studied. In this study, network pharmacology followed by molecular docking analysis was utilized to identify potential targets of mannose against NAFLD, and the leptin receptor-deficient, genetically obese db/db mice was employed as an animal model of NAFLD to validate the regulation of D-mannose on core targets. As a result, 67 targets of mannose are predicted associated with NAFLD, which are surprisingly centered on the mechanistic target of rapamycin (mTOR). Further analyses suggest that mTOR signaling is functionally enriched in potential targets of mannose treating NAFLD, and that mannose putatively binds to mTOR as a core mechanism. Expectedly, repeated oral gavage of supraphysiological D-mannose ameliorates liver steatosis of db/db mice, which is based on suppression of hepatic mTOR signaling. Moreover, daily D-mannose administration reduced hepatic expression of lipogenic regulatory genes in counteracting NAFLD. Together, these findings reveal D-mannose as an effective and potential NAFLD therapeutic through mTOR suppression, which holds translational promise.

The mechanism of curcumin to protect mouse ovaries from oxidative damage by regulating AMPK/mTOR mediated autophagy.

BACKGROUND: Oxidative stress is considered the main cause of granulosa cell apoptosis in ovarian disease. Curcumin has various biological roles, but its potential role in protecting granulosa cells from oxidative damage remains unidentified. PURPOSE: The study revealed the protective effect of curcumin on granulosa cell survival under oxidative stress, and explored its mode of action. STUDY DESIGN: The protective effect of curcumin on oxidative stress-induced ovarian cell apoptosis was evaluated in vivo and in vitro, and the role of autophagy and AMPK/mTOR signaling pathway in this process was also demonstrated. METHODS: First, mice were injected to 3-nitropropionic acid (3-NPA, 20 mg/kg/day) for 14 consecutive days to establish the ovarian oxidative stress model, at same time, curcumin (50, 100, 200 mg/kg/day) was given orally. Thereafter, functional changes, cell apoptosis, and autophagy in ovarian tissue were evaluated by hematoxylin-eosin staining, enzyme-linked immunosorbent assay, western blotting, TUNEL assays, and transmission electron microscopy. Finally, oxidative stress model of granulosa cells was established with H2O2in vitro and treated with curcumin. The underlying mechanisms of curcumin to protect the apoptosis under oxidative stress in vitro were determined using western blotting and TUNEL assays. RESULTS: In our study, after curcumin treatment, the mouse ovarian function disorder under 3-nitropropionic acid-induced oxidative stress recovered significantly, and ovarian cell apoptosis decreased. H2O2 induced granulosa cell apoptosis in vitro, and curcumin antagonized this process. Autophagy contributes to tissue and cell survival under stress. We therefore examined the role of autophagy in this process. According to the in vivo and in vitro results, curcumin restored autophagy under oxidative stress. The autophagy inhibitor (chloroquine) exhibited the same effect as curcumin, whereas the autophagy activator (rapamycin) antagonized the effect of curcumin. In addition, the study found that the AMPK/mTOR pathway plays a crucial role in curcumin- mediated autophagy to protect against oxidative stress-induced apoptosis. CONCLUSION: Our findings for the first time systematically revealed a new mechanism through which curcumin protects ovarian granulosa cells from oxidative stress-induced damage through AMPK/mTOR-mediated autophagy and suggested that it can be a new therapeutic direction for female ovarian diseases.

Investigation of prunetrin induced G2/M cell cycle arrest and apoptosis via Akt/mTOR/MAPK pathways in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) stands as a leading cause of mortality, and despite recent advancements in the overall survival rates, the prognosis remains dismal. Prunetin 4-O-glucoside (Prunetrin or PUR), an active compound derived from Prunus sp., was explored for its impact on HepG2 and Huh7 cells. The cytotoxicity assessment revealed a notable reduction in cell viability in both cell lines, while exhibiting non-toxicity towards HaCaT cells. Colony formation studies underscored PUR's inhibitory effect on cell proliferation, dose-dependently. Mechanistically, PUR downregulated cell cycle proteins (CDC25c, Cdk1/CDC2, and Cyclin B1), inducing G2/M phase arrest, corroborated by flow cytometry. Western blot analyses exhibited dose-dependent cleavages of PARP and caspase 3, indicative of apoptosis. Treatment with the apoptotic inhibitor z-vmd-fmk provided evidence of PUR-induced apoptosis. Annexin V and PI flow cytometry further affirmed apoptotic induction. Enhanced expression of cleaved-caspase 9 and the pro-apoptotic protein Bak, coupled with reduced anti-apoptotic Bcl-xL, and affirmed PUR's induction of intrinsic apoptosis. Additionally, PUR activated the MAPK pathway, evidenced by elevated phospho p38 and phospho ERK expressions in both cell lines. Notably, a concentration-dependent decrease in mTOR and Akt expressions indicated PUR's inhibition of the Akt/mTOR pathway in HepG2 and Huh7 cells. These findings illuminate PUR's multifaceted impact, revealing its potential as a promising therapeutic agent against HepG2 and Huh7 cells through modulation of cell cycle, apoptosis, and key signaling pathways.

Interleukin-22 Alleviates Caerulein-Induced Acute Pancreatitis by Activating AKT/mTOR Pathway.

BACKGROUND: Acute pancreatitis (AP) is one of the most common acute abdominal disorders; due to the lack of specific treatment, the treatment of acute pancreatitis, especially serious acute pancreatitis (SAP), is difficult and challenging. We will observe the changes of Interleukin -22 levels in acute pancreatitis animal models, and explore the mechanism of Interleukin -22 in acute pancreatitis. OBJECTIVE: This study aims to assess the potential protective effect of Interleukin -22 on caerulein-induced acute pancreatitis and to explore its mechanism. METHODS: Blood levels of amylase and lipase and Interleukin -22 were assessed in mice with acute pancreatitis. In animal model and cell model of caerulein-induced acute pancreatitis, the mRNA levels of P62 and Beclin-1 were determined using PCR, and the protein expression of P62, LC3-II, mTOR, AKT, p-mTOR, and p-AKT were evaluated through Western blot analysis. RESULTS: Interleukin -22 administration reduced blood amylase and lipase levels and mitigated tissue damage in acute pancreatitis mice model. Interleukin -22 inhibited the relative mRNA levels of P62 and Beclin-1, and the Interleukin -22 group showed a decreased protein expression of LC3-II and P62 and the phosphorylation of the AKT/mTOR pathway. Furthermore, we obtained similar results in the cell model of acute pancreatitis. CONCLUSION: This study suggests that Interleukin -22 administration could alleviate pancreatic damage in caerulein-induced acute pancreatitis. This effect may result from the activation of the AKT/mTOR pathway, leading to the inhibition of autophagy. Consequently, Interleukin -22 shows potential as a treatment.

CoCl2-Induced hypoxia promotes hPDLSCs osteogenic differentiation through AKT/mTOR/4EBP-1/HIF-1α signaling and facilitates the repair of alveolar bone defects.

Bone defects are characterized by a hypoxic environment, which affects bone tissue repair. However, the role of hypoxia in the repair of alveolar bone defects remains unclear. Human periodontal ligament stem cells (hPDLSCs) are high-quality seed cells for repairing alveolar bone defects, whose behavior changes under hypoxia. However, their mechanism of action is not known and needs to be elucidated. We hypothesized that hypoxia might be beneficial to alveolar bone defect repair and the osteogenic differentiation of hPDLSCs. To test this hypothesis, cobalt chloride (CoCl2) was used to create a hypoxic environment, both in vitro and in vivo. In vitro study, the best osteogenic effect was observed after 48 h of hypoxia in hPDLSCs, and the AKT/mammalian target of rapamycin/eukaryotic translation initiation factor 4e-binding protein 1 (AKT/mTOR/4EBP-1) signaling pathway was significantly upregulated. Inhibition of the AKT/mTOR/4EBP-1 signaling pathway decreased the osteogenic ability of hPDLSCs under hypoxia and hypoxia-inducible factor 1 alpha (HIF-1α) expression. The inhibition of HIF-1α also decreased the osteogenic capacity of hPDLSCs under hypoxia without significantly affecting the level of phosphorylation of AKT/mTOR/4EBP-1. In vitro study, Micro-CT and tissue staining results show better bone regeneration in hypoxic group than control group. These results suggested that hypoxia promoted alveolar bone defect repair and osteogenic differentiation of hPDLSCs, probably through AKT/mTOR/4EBP-1/HIF-1α signaling. These findings provided important insights into the regulatory mechanism of hypoxia in hPDLSCs and elucidated the effect of hypoxia on the healing of alveolar bone defects. This study highlighted the importance of physiological oxygen conditions for tissue engineering.

Shenmai Injection Alleviates Myocardial Ferroptosis via Activating the AKT1/mTOR Pathway in Rats with Acute Myocardial Infarction.

OBJECTIVE: Acute myocardial infarction (AMI) poses a serious burden on public health. Shenmai Injection (SMI) has been reported to have a cardioprotective effect and is used clinically attributed to its targeting of ferroptosis. This study aims to explore the underlying mechanisms of SMI in treating AMI through the application of network pharmacology analysis. METHODS: This study utilized network pharmacology to identify the bioactive ingredients and potential targets of SMI in treating AMI. A rat model of AMI was created by ligating the coronary arteries of rats, and a cell model was established by subjecting H9c2 cells to oxygen-glucose deprivation (OGD) to reveal the cardioprotective effects of SMI. Western blotting was employed to measure protein expressions, while hematoxylin-eosin staining was used to observe relevant pathological changes. Enzyme linked immunosorbent assay was conducted to measure the levels of biomarkers associated with cardiac injury and oxidative stress. RESULTS: A comprehensive analysis revealed a total of 225 putative targets of SMI in the context of AMI which exerted regulatory effects on numerous pathways and targeted multiple biological processes. AKT1 was identified as a core target mediating the effects of SMI on AMI by topological analysis. In vivo experiments revealed that SMI attenuated myocardial injury, oxidative stress, and ferroptosis in rats with AMI. Furthermore, SMI was found to enhance the expression levels of p-AKT1 and p-mTOR proteins in the myocardial tissues of rats afflicted with AMI. Similar findings were also observed in H9c2 cells subjected to OGD. Of particular interest, the suppression of OGD-induced iron accumulation, oxidative stress, and ferroptosis-associated proteins by SMI in H9c2 cells was reversed upon inhibition of the AKT1/mTOR pathway via MK2206. CONCLUSION: This study revealed that SMI exerts a protective effect against myocardial injury and ferroptosis caused by AMI via the activation of the AKT1/mTOR pathway.

Emodin activates autophagy to suppress oxidative stress and pyroptosis via mTOR-ULK1 signaling pathway and promotes multi-territory perforator flap survival.

BACKGROUND: Multi-territory perforator flap reconstruction has been proven effective in treating large skin and soft tissue defects in clinical settings. However, in view of that the multi-territory perforator flap is prone to partial postoperative necrosis, increasing its survival is the key to the success of reconstruction. In this study, we aimed to clarify the effect of emodin on multi-territory perforator flap survival. METHODS: Flap survival was assessed by viability area analysis, infrared laser imaging detector, HE staining, immunohistochemistry, and angiography. Western blotting, immunofluorescence assays, and real-time fluorescent quantitative PCR were performed to detect the indicators of oxidative stress, pyroptosis and autophagy. RESULTS: After emodin treatment, the multi-territory perforator flap showed a significantly increased survival rate, which was shown to be closely related to the inhibition of oxidative stress and pyroptosis and enhanced autophagy. Meanwhile, the use of autophagy inhibitor 3 MA was found to reverse the inhibitory effects of emodin on oxidative stress and pyroptosis and weaken the improving effect of emodin on flap survival, suggesting that autophagy plays a critical role in emodin-treated flaps. Interestingly, our mechanistic investigations revealed that the positive effect of emodin on multi-territory perforator flap was attributed to the mTOR-ULK1 signaling pathway activation. CONCLUSIONS: Emodin can inhibit oxidative stress and pyroptosis by activating autophagy via the mTOR-ULK1 pathway, thereby improving the multi-territory perforator flap survival.

DT-13 inhibits the proliferation of pancreatic cancer by inducing apoptosis via AMPK-mTOR signaling.

BACKGROUND/OBJECTIVE: DT-13, the principal active component of Mysidium shortscapes from the Liliaceae family, has garnered substantial interest in cancer therapy owing to its potential anticancer properties. This study investigated the effects of DT-13 on the proliferation and apoptosis of human pancreatic cancer cell lines and aimed to elucidate the underlying mechanisms. METHODS: PANC1 and CFPAC1 cells were exposed to DT-13 and their proliferation was assessed using RTCA and clone formation assays. Apoptotic protein expression was analyzed by western blotting, and apoptotic cells were identified by flow cytometry. RNA was extracted from DT-13 treated and untreated PANC1 cells for RNA sequencing. Differentially expressed genes were identified and subjected to GO bioprocess, KEGG pathway analysis, and western blotting. Finally, to evaluate tumor growth, CFPAC1 cells were subcutaneously injected into BALB/c nude mice. RESULTS: DT-13 inhibited proliferation and induced apoptosis of PANC1 and CFPAC1 cells by activating the AMPK/mTOR pathway and suppressing p70 S6K. Moreover, DT-13 hindered the growth of CFPAC1 xenograft tumors in nude mice. CONCLUSIONS: DT-13 effectively inhibited the growth of human pancreatic cancer cells.

Rapamycin promotes hematoma resorption and enhances endothelial cell function by suppressing the mTOR/STAT3 signaling in chronic subdural hematoma.

Chronic subdural hematoma (CSDH) remains a neurosurgical condition and a healthy burden especially in elderly patients. This study focuses on the functions of rapamycin and its related molecular mechanisms in CSDH management. A rat model of CSDH was induced, which developed significant hematoma on day 5 after operation. The rats were treated with rapamycin or atorvastatin, a drug with known effect on hematoma alleviation, or treated with rapamycin and atorvastatin in combination. The atorvastatin or rapamycin treatment reduced the hematoma development, blood-brain barrier permeability, neurological dysfunction in CSDH rats, and the combination treatment showed more pronounced effects. Human brain microvascular endothelial cells hCMEC/D3 were stimulated by hematoma samples to mimic a CSDH condition in vitro. The drug treatments elevated the cell junction-related factors and reduced the pro-inflammatory cytokines both in rat hematoma tissues and in hCMEC/D3 cells. Rapamycin suppressed the mTOR and STAT3 signaling pathways. Overexpression of mTOR or the STAT3 agonist suppressed the alleviating effects of rapamycin on CSDH. In summary, this study demonstrates that rapamycin promotes hematoma resorption and enhances endothelial cell function by suppressing the mTOR/STAT3 signaling.

Metformin and thymoquinone co-treatment enhance 5-fluorouracil cytotoxicity by suppressing the PI3K/mTOR/HIF1α pathway and increasing oxidative stress in colon cancer cells.

This study investigated the chemotherapeutic effects of 5-fluorouracil (5-FU), metformin (Met), and/or thymoquinone (TQ) single/dual/triple therapies in the HT29, SW480 and SW620 colon cancer (CRC) cell lines. Cell cycle/apoptosis were measured by flow cytometry. The gene and protein expression of apoptosis (PCNA/survivin/BAX/Cytochrome-C/Caspase-3) and cell cycle (CCND1/CCND3/p21/p27) molecules, the PI3K/mTOR/HIF1α oncogenic pathway, and glycolysis regulatory enzymes were measured by quantitative-PCR and Western blot. Markers of oxidative stress were also measured by colorimetric assays. Although all treatments induced anti-cancer effects related to cell cycle arrest and apoptosis, the triple therapy showed the highest pro-apoptotic actions that coincided with the lowest expression of CCND1/CCND3/PCNA/survivin and the maximal increases in p21/p27/BAX/Cytochrome-C/Caspase-3 in all cell lines. The triple therapy also revealed the best suppression of the PI3K/mTOR/HIF1α pathway by increasing its endogenous inhibitors (PTEN/AMPKα) in all cell lines. Moreover, the lowest expression of lactate dehydrogenase and pyruvate dehydrogenase kinase-1 with the highest expression of pyruvate dehydrogenase were seen with the triple therapy, which also showed the highest increases in oxidative stress markers (ROS/RNS/MDA/protein carbonyl groups) alongside the lowest antioxidant levels (GSH/CAT) in all cell lines. In conclusion, this is the first study to reveal enhanced anti-cancer effects for metformin/thymoquinone in CRC that were superior to all monotherapies and the other dual therapies. However, the triple therapy approach showed the best tumoricidal actions related to cell cycle arrest and apoptosis in all cell lines, possibly by enhancing oxidative glycolysis and augmenting oxidative stress through stronger modulation of the PI3K/mTOR/HIF1α oncogenic network.

Schaftoside improves cerebral ischemia-reperfusion injury by enhancing autophagy and reducing apoptosis and inflammation through the AMPK/mTOR pathway.

BACKGROUND: As a flavonoid compound, schaftoside (SS) possesses a wide range of pharmaceutical activities. Nonetheless, it is unclear whether SS has a neuroprotective effect in cerebral ischemia-reperfusion injury (CI/RI). OBJECTIVES: To examine the neuroprotective effect of SS in CI/RI and explore the underlying mechanism. MATERIAL AND METHODS: An in vivo middle cerebral artery occlusion (MCAO) was used to simulate CI/RI in rats. Oxygen glucose deprivation/reperfusion (OGD/R) of HT22 cells was used to establish a cellular model of CI/RI in vitro. Pathological changes were evaluated with hematoxylin and eosin (H&E) staining, apoptosis was measured using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and flow cytometry, and inflammatory factors were assessed using enzyme-linked immunosorbent assay (ELISA). Protein expression was detected using western blot or immunofluorescence. RESULTS: Our results indicated that SS attenuated CI/RI by improving neurologic deficits and reducing brain edema. Moreover, SS treatment blocked apoptosis and inflammation and enhanced autophagy in MCAO rats. Schaftoside was found to amplify the activation of adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway induced by MCAO. Similarly, SS pretreatment increased cell viability and autophagy, and reduced apoptosis and inflammation in OGD/R-induced HT22 cells. The OGD/R enlarges the p-AMPK/AMPK ratio while restricting the p-mTOR/mTOR ratio, and it was found that SS further enhanced the effect of OGD/R on the AMPK/mTOR pathway. Rapamycin promoted the effect of SS on OGD/R-induced HT22 cells, while compound C produced the opposite results. Mechanistically, SS promoted autophagy and reduced apoptosis and inflammation through the regulation of the AMPK/mTOR signaling pathway. CONCLUSIONS: The obtained results showed that SS protected against CI/RI through an autophagy-mediated AMPK/mTOR pathway when accessed in vitro and in vivo.

Metformin inhibits the development and metastasis of colorectal cancer.

Metformin is a commonly used drug for the treatment of diabetes. Accumulating evidence suggests that it exerts anti-cancer effects in many cancers, including colorectal cancer. However, the underlying molecular mechanisms of colorectal cancer metastasis remain unclear. Colorectal cancer cell lines were treated with metformin, and cell proliferation, invasion, and migration were analyzed in vitro. The relationship between metformin and the AMPK-mTOR axis was assessed by Western blot analysis and transfection with small interfering RNA. A colorectal cancer xenograft mouse model was used to observe the effects of metformin on liver metastasis. Immunohistochemical analysis was performed on liver metastatic tumors. In in vitro experiments, metformin significantly inhibited the proliferation, migration, and invasion only in HCT116 and SW837 cells, but not in HCT8 and Lovo cells. Only in HCT116 and SW837, a change in AMPK-mTOR expression was observed in a dose-dependent manner. In colorectal cancer xenograft mice, the liver metastatic rate (10% vs. 50%, p = 0.05) and the number of liver metastatic nodules (0.1/body vs. 1.2/body, p = 0.04) were significantly lower in the metformin group. Tumor proliferation and EMT were decreased and apoptosis was promoted only in metastatic liver tumors of mice treated with metformin. The molecular mechanism of the anti-cancer effects of metformin involves repression of mTOR pathways via AMPK activation. Moreover, the differences in metformin sensitivity depend on the response of the AMPK-mTOR pathway to metformin. Our study provides a theoretical basis for the anti-metastatic treatment of colorectal cancer using metformin.

Zexie Tang targeting FKBP38/mTOR/SREBPs pathway improves hyperlipidemia.

ETHNOPHARMACOLOGICAL RELEVANCE: Zexie Tang (ZXT), only two consists with Alismatis Rhizoma (AR) and Atractylodes macrocephala Rhizoma (AM), a classical Chinese medicine formula from Synopsis of the Golden Chamber with a history of 2000 years. Clinical observation in recent years has found that ZXT has excellent lipid-lowering effect. AIM OF THE STUDY: To explore the potential mechanism of ZXT ameliorates hyperlipidemia based on FKBP38/mTOR/SREBPs pathway. MATERIALS AND METHODS: WD-induced hyperlipidemia mice and oleic acid induced cell lipid accumulation model were used to investigate pharmacodynamic. The effect of ZXT on the transcriptional activity of SREBPs was detected by reporter gene assay. Proteins and downstream genes of mTOR/SREBPs pathway were detected in vivo and in vitro. Combined with network pharmacology and HPLC-Q-TOF/MS, the active ingredients were screened and identified. The interaction between active compounds of ZXT and FKBP38 protein were analyzed by docking analysis. RESULTS: ZXT decreased TC, TG and LDL-c levels in blood of WD-induced hyperlipidemia mouse model, and improved insulin resistance in vivo. ZXT also reduced TC, TG and lipid accumulation in cells line, and inhibited SREBPs luciferase activity, protein and its target genes expression such as FASN, HMGCR, etc. Meanwhile, ZXT inhibited protein expression levels of p-mTOR, p-S6K, etc in vitro and in vivo. Combined with network pharmacology and HPLC-Q-TOF/MS, 16 active ingredients were screened and identified. Docking results showed that active compounds of ZXT binding to FKBP38 and formed hydrogen bond. CONCLUSION: Our findings highlighted that ZXT ameliorates hyperlipidemia, in which FKBP/mTOR/SREBPs pathway might be the potential regulatory mechanism.

The novel role of circular RNA ST3GAL6 on blocking gastric cancer malignant behaviours through autophagy regulated by the FOXP2/MET/mTOR axis.

Gastric cancer (GC) ranks third in mortality among all cancers worldwide. Circular RNAs (circRNAs) play an important role in the occurrence and development of gastric cancer. Forkhead box P2 (FOXP2), as a transcription factor, is closely associated with the development of many types of tumours. However, the regulatory network between FOXP2 and circRNAs remains to be explored. In our study, circST3GAL6 was significantly downregulated in GC and was associated with poor prognosis in GC patients. Overexpression of circST3GAL6 inhibited the malignant behaviours of GC cells, which was mediated by inducing apoptosis and autophagy. In addition, we demonstrated that circST3GAL6 regulated FOXP2 through the mir-300 sponge. We further found that FOXP2 inhibited MET Proto-Oncogene (MET), which was the initiating factor that regulated the classic AKT/mTOR pathway of autophagy. In conclusion, our results suggested that circST3GAL6 played a tumour suppressive role in gastric cancer through miR-300/FOXP2 axis and regulated apoptosis and autophagy through FOXP2-mediated transcriptional inhibition of the MET axis, which may become a potential target for GC therapy.

Modulation of mTOR signaling by radiation and rapamycin treatment in canine mast cell cancer cells.

Rapamycin has been reported to reduce cancer cell survival in certain tumors following radiation therapy, but the mechanisms driving this phenomenon are unclear. Rapamycin inhibits mTOR signaling, a pathway responsible for several essential cell functions. The objective of this study was to investigate the effects of rapamycin and radiation on the activation and inhibition of mTOR signaling and the relationship between mTOR signaling and DNA damage response in vitro using canine mast cell tumor (MCT) cancer cell lines. Rapamycin rapidly inhibited S6K phosphorylation in a dose-dependent manner. Ionizing radiation (3, 6, or 10 Gy) was able to activate mTOR signalling, but the combination of radiation and rapamycin maintained mTOR inhibition. The comet assay revealed that co-treatment with rapamycin induced modest increases in the severity of DNA damage to MCT cells, but that these differences were not statistically significant. Although the relationship between mTOR and DNA damage response in MCT cancer cell lines remains unclear, our findings suggest the possibility of interaction, leading to enhancement of radiation response.

Il a été rapporté que la rapamycine réduisait la survie des cellules cancéreuses dans certaines tumeurs après une radiothérapie, mais les mécanismes à l'origine de ce phénomène ne sont pas clairs. La rapamycine inhibe la signalisation mTOR, une voie responsable de plusieurs fonctions cellulaires essentielles. L'objectif de cette étude était d'étudier les effets de la rapamycine et des radiations sur l'activation et l'inhibition de la signalisation mTOR et la relation entre la signalisation mTOR et la réponse aux dommages à l'ADN in vitro à l'aide de lignées cellulaires cancéreuses de tumeurs mastocytaires canines (MCT). La rapamycine a rapidement inhibé la phosphorylation de S6K de manière dose-dépendante. Le rayonnement ionisant (3, 6 ou 10 Gy) a pu activer la signalisation mTOR, mais la combinaison de rayonnement et de rapamycine a maintenu l'inhibition de mTOR. Le test des comètes a révélé que le co-traitement avec la rapamycine induisait des augmentations modestes de la gravité des dommages à l'ADN des cellules MCT, mais que ces différences n'étaient pas statistiquement significatives. Bien que la relation entre mTOR et la réponse aux dommages à l'ADN dans les lignées cellulaires cancéreuses MCT reste incertaine, nos résultats suggèrent la possibilité d'une interaction, conduisant à une amélioration de la réponse aux radiations.(Traduit par Docteur Serge Messier).