Qili Qiangxin, a compound herbal medicine formula, alleviates hypoxia-reoxygenation-induced apoptotic and autophagic cell death via suppression of ROS/AMPK/mTOR pathway in vitro / 中西医结合学报

OBJECTIVE@#Qili Qiangxin (QLQX), a compound herbal medicine formula, is used effectively to treat congestive heart failure in China. However, the molecular mechanisms of the cardioprotective effect are still unclear. This study explores the cardioprotective effect and mechanism of QLQX using the hypoxia-reoxygenation (H/R)-induced myocardial injury model.@*METHODS@#The main chemical constituents of QLQX were analyzed using high-performance liquid chromatography-evaporative light-scattering detection. The model of H/R-induced myocardial injury in H9c2 cells was developed to simulate myocardial ischemia-reperfusion injury. Apoptosis, autophagy, and generation of reactive oxygen species (ROS) were measured to assess the protective effect of QLQX. Proteins related to autophagy, apoptosis and signalling pathways were detected using Western blotting.@*RESULTS@#Apoptosis, autophagy and the excessive production of ROS induced by H/R were significantly reduced after treating the H9c2 cells with QLQX. QLQX treatment at concentrations of 50 and 250 μg/mL caused significant reduction in the levels of LC3II and p62 degradation (P < 0.05), and also suppressed the AMPK/mTOR signalling pathway. Furthermore, the AMPK inhibitor Compound C (at 0.5 μmol/L), and QLQX (250 μg/mL) significantly inhibited H/R-induced autophagy and apoptosis (P < 0.01), while AICAR (an AMPK activator, at 0.5 mmol/L) increased cardiomyocyte apoptosis and autophagy and abolished the anti-apoptotic effect of QLQX. Similar phenomena were also observed on the expressions of apoptotic and autophagic proteins, demonstrating that QLQX reduced the apoptosis and autophagy in the H/R-induced injury model via inhibiting the AMPK/mTOR pathway. Moreover, ROS scavenger, N-Acetyl-L-cysteine (NAC, at 2.5 mmol/L), significantly reduced H/R-triggered cell apoptosis and autophagy (P < 0.01). Meanwhile, NAC treatment down-regulated the ratio of phosphorylation of AMPK/AMPK (P < 0.01), which showed a similar effect to QLQX.@*CONCLUSION@#QLQX plays a cardioprotective role by alleviating apoptotic and autophagic cell death through inhibition of the ROS/AMPK/mTOR signalling pathway.

Ershiwuwei Shanhu Pills regulate Akt/mTOR/GSK-3β signaling pathway to alleviate Alzheimer's disease mice / 中国中药杂志

The present study investigated the mechanism of the Tibetan patent medicine Ershiwuwei Shanhu Pills(ESP) in alleviating Alzheimer's disease in mice via Akt/mTOR/GSK-3β signaling pathway. BALB/c mice were randomly assigned into a blank control group, a model group, low(200 mg·kg~(-1)), medium(400 mg·kg~(-1)) and high(800 mg·kg~(-1)) dose groups of ESP, and donepezil hydrochloride group. Except the blank control group, the other groups were given 20 mg·kg~(-1) aluminum chloride by gavage and 120 mg·kg~(-1) D-galactose by intraperitoneal injection for 56 days to establish Alzheimer's disease model. Morris water maze was used to detect the learning and memory ability of mice. The level of p-tau protein in mouse hippocampus and the levels of superoxide dismutase(SOD), malondialdehyde(MDA), catalase(CAT), and total antioxidant capacity(T-AOC) in hippocampus and serum were detected. Hematoxylin-eosin staining and Nissl staining were performed for the pathological observation of whole brain in mice. TdT-mediated dUTP nick-end labeling(TUNEL) staining was employed for the observation of apoptosis in mouse cortex. Western blot was adopted to detect the protein levels of p-mTOR, p-Akt, and GSK-3β in the hippocampus. Compared with the model group, the ESP groups showcased alleviated pathological damage of the whole brain, decreased TUNEL positive cells, reduced level of p-tau protein in hippocampus, and risen SOD, CAT, and T-AOC levels and declined MDA level in hippocampus and serum. Furthermore, the ESP groups had up-regulated protein levels of p-mTOR and p-Akt while down-regulated protein level of GSK-3β in hippocampus. Therefore, ESP can alleviate the learning and memory decline and oxidative damage in mice with Alzheimer's disease induced by D-galactose combined with aluminum chloride, which may be related to Akt/mTOR/GSK-3β signaling pathway.

Influence of 6-shogaol potentiated on 5-fluorouracil treatment of liver cancer by promoting apoptosis and cell cycle arrest by regulating AKT/mTOR/MRP1 signalling / 中国天然药物

Currently, chemoresistance seriously attenuates the curative outcome of liver cancer. The purpose of our work was to investigate the influence of 6-shogaol on the inhibition of 5-fluorouracil (5-FU) in liver cancer. The cell viability of cancer cells was determined by MTT assay. Liver cancer cell apoptosis and the cell cycle were examined utilizing flow cytometry. Moreover, qRT-PCR and western blotting was used to analyse the mRNA and protein expression levels, respectively. Immunohistochemistry assays were used to examine multidrug resistance protein 1 (MRP1) expression in tumour tissues. In liver cancer cells, we found that 6-shogaol-5-FU combination treatment inhibited cell viability, facilitated G0/G1 cell cycle arrest, and accelerated apoptosis compared with 6-shogaol or 5-FU treatment alone. In cancer cells cotreated with 6-shogaol and 5-FU, AKT/mTOR pathway- and cell cycle-related protein expression levels were inhibited, and MRP1 expression was downregulated. AKT activation or MRP1 increase reversed the influence of combination treatment on liver cancer cell viability, apoptosis and cell cycle arrest. The inhibition of AKT activation to the anticancer effect of 6-shogaol-5-FU could be reversed by MRP1 silencing. Moreover, our results showed that 6-shogaol-5-FU combination treatment notably inhibited tumour growth in vivo. In summary, our data demonstrated that 6-shogaol contributed to the curative outcome of 5-FU in liver cancer by inhibiting the AKT/mTOR/MRP1 signalling pathway.

EGCG and ECG induce apoptosis and decrease autophagy via the AMPK/mTOR and PI3K/AKT/mTOR pathway in human melanoma cells / 中国天然药物

Catechins have been proven to exert antitumor effects in different kinds of cancers. However, the underlying mechanisms have not been completely clarified yet. This study aimed to assess the effects and mechanisms of (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG) on human melanoma skin A375 cells. Results showed that EGCG and ECG inhibited the proliferation of A375 cells and ECG showed better inhibitory effect. Flow cytometry analysis had shown that EGCG and ECG induced apoptosis and led to cell cycle arrest. EGCG and ECG decreased Bcl-2 expression and upregulated Caspase-3 protein level, indicating the development of apoptosis. Furthermore, EGCG and ECG could decreased mitochondrial membrane potential of A375 cells. In addition, the expression of Beclin-1, LC3 and Sirt3 were downregulated at protein levels, which known to be associated with autophagy. After autophagy was increased by rapamycin, the apoptotic trend was not change, indicating that apoptosis and autophagy are independent. Mechanistically, EGCG and ECG treatments decreased phosphorylated-AMPK (p-AMPK) and increased the ratios of p-PI3K, p-AKT and p-mTOR in melanoma cells. Conclusively, EGCG and ECG induced apoptosis via mitochondrial signaling pathway, downregulated autophagy through modulating the AMPK/mTOR and PI3K/AKT/mTOR signaling pathway. It indicated that EGCG and ECG may be utilized in human melanoma treatment.

Relationship of PI3K-Akt/mTOR/AMPK signaling pathway genetic mutation with efficacy and prognosis in nasopharyngeal carcinoma / 中南大学学报(医学版)

OBJECTIVES@#Genetic mutation is one of the important causes for tumor genesis and development, but genetic mutation in nasopharyngeal carcinoma (NPC) has rarely been reported. This study explored the role of phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt), mammalian target of rapamycin (mTOR), and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway in the efficacy and prognosis in patients with NPC.@*METHODS@#A total of 31 patients with advanced NPC, who came from the Affiliated Cancer Hospital of Xiangya School of Medicine of Central South University/Hunan Provincial Cancer Hospital, were enrolled. All of the exons of 288 genes, introns of 38 genes and promoters or fusion breakpoint regions from the nasopharyngeal biopsy tissues before treatment were detected by the gene sequencing platform Illumina NextSeq CN500. The coding regions of 728 genes were carried out a high-depth sequencing of target region capture, and the 4 variant types of tumor genes (including point mutations, insertion deletions of small fragments, copy number variations, and currently known fusion genes) were detected. All of 31 patients received platinum-based induction chemotherapy combined with concurrent chemoradiotherapy and were followed up for a long time.@*RESULTS@#The 3-year regional failure-free survival (RFFS) and disease-free survival (DFS) in patients with PI3K-Akt pathway mutation were significantly lower than those in unmutated patients (χ2=6.647, P<0.05). The 3-year RFFS and DFS in patients with mTOR pathway mutations were significantly lower than those in unmutated patients, and there was significant difference (χ2=5.570, P<0.05). The rate of complete response (CR) in patients with unmutated AMPK pathway was significantly higher than that in patients with mutation at 3 months after treatment (P<0.05), and the 3-year RFFS and DFS in patients with AMPK pathway mutation were significantly lower than those in unmutated patients (χ2=4.553, P<0.05). PI3K-Akt/mTOR/AMPK signaling pathway mutations and pre-treatment EB virus DNA copy numbers were independent prognostic factors for 3-year RFFS and DFS in patients with NPC (both P<0.05).@*CONCLUSIONS@#The NPC patients with PI3K-Akt/mTOR/AMPK signaling pathway mutation have poor prognosis, and the detection of PI3K-Akt, mTOR, AMPK driver genes and signaling pathways by next-generation sequencing is expected to provide new idea for basic research and targeted therapy of NPC.

Leptin relieves ischemia/reperfusion induced acute kidney injury through inhibiting apoptosis and autophagy / 中南大学学报(医学版)

OBJECTIVES@#Acute kidney injury (AKI) can be caused by ischemia/reperfusion (I/R), nephrotoxin, and sepsis, with poor prognosis and high mortality. Leptin is a protein molecule that regulates the body's energy metabolism and reproductive activities via binding to its specific receptor. Leptin can inhibit cardiomyocyte apoptosis caused by I/R, but its effect on I/R kidney injury and the underlying mechanisms are still unclear. This study aims to investigate the effect and mechanisms of leptin on renal function, renal histopathology, apoptosis, and autophagy during acute I/R kidney injury.@*METHODS@#Healthy adult male mice were randomly divided into 4 groups: a sham+wild-type mice (ob/+) group, a sham+leptin gene-deficient mice (ob/ob) group, an I/R+ob/+ group, and an I/R+ob/ob group (n=8 per group). For sham operation, a longitudinal incision was made on the back of the mice to expose and separate the bilateral kidneys and renal arteries, and no subsequent treatment was performed. I/R treatment was ischemia for 30 min and reperfusion for 48 h. The levels of BUN and SCr were detected to evaluate renal function; HE staining was used to observe the pathological changes of renal tissue; TUNEL staining was used to observe cell apoptosis, and apoptosis-positive cells were counted; Western blotting was used to detect levels of apoptosis-related proteins (caspase 3, caspase 9), autophagy-related proteins [mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), LC3 I, LC3 II], mTOR-dependent signaling pathway proteins [phosphate and tension homology (PTEN), adenosine monophosphate-activated protein kinase (AMPK), protein kinase B (AKT), extracellular regulated protein kinase (ERK), phosphorylated PTEN (p-PTEN), phosphorylated AMPK (p-AMPK), phosphorylated AKT (p-AKT), phosphorylated ERK (p-ERK)].@*RESULTS@#There was no significant difference in the levels of BUN and SCr between the sham+ob/+ group and the sham+ob/ob group (both P>0.05). The levels of BUN and SCr in the I/R+ob/+ group were significantly higher than those in the sham+ob/+ group (both P<0.05). Compared with the mice in the sham+ob/ob group or the I/R+ob/+ group, the levels of BUN and SCr in the I/R+ob/ob group were significantly increased (all P<0.05). There was no obvious damage to the renal tubules in the sham+ob/+ group and the sham+ob/ob group. Compared with sham+ob/+ group and sham+ob/ob group, both the I/R+ob/+ group and the I/R+ob/ob group had cell damage such as brush border shedding, vacuolar degeneration, and cast formation. Compared with the I/R+ob/+ group, the renal tubules of the mice in the I/R+ob/ob group were more severely damaged. The pathological score of renal tubular injury showed that the renal tubular injury was the most serious in the I/R+ob/ob group (P<0.05). Compared with the sham+ob/+ group, the protein levels of caspase 3, caspase 9, PTEN, and LC3 II were significantly up-regulated, the ratio of LC3 II to LC3 I was significantly increased, and the protein levels of p-mTOR, p-PTEN, p-AMPK, p-AKT, and p-ERK were significantly down-regulated in the I/R+ob/+ group (all P<0.05). Compared with the sham+ob/ob group, the protein levels of caspase 3, caspase 9, PTEN, and LC3 II were significantly up-regulated, and the ratio of LC3 II to LC3 I was significantly increased, while the protein levels of p-mTOR, p-PTEN, p-AMPK, p-AKT, and p-ERK were significantly down-regulated in the I/R+ob/ob group (all P<0.05). Compared with the I/R+ob/+ group, the levels of p-mTOR, p-PTEN, p-AMPK, p-AKT were more significantly down-regulated, while the levels of caspase 3, caspase 9, PTEN, and LC3 II were more significantly up-regulated, and the ratio of LC3 II to LC3 I was more significantly increase in the I/R+ob/ob group (all P<0.05).@*CONCLUSIONS@#Renal function and tubular damage, and elevated levels of apoptosis and autophagy are observed in mice kidneys after acute I/R. Leptin might relieve I/R induced AKI by inhibiting apoptosis and autophagy that through a complex network of interactions between mTOR-dependent signaling pathways.

Total Saponins of Panax notoginseng Activate Akt/mTOR Pathway and Exhibit Neuroprotection in vitro and in vivo against Ischemic Damage / 中国结合医学杂志

OBJECTIVE@#To reveal the neuroprotective effect and the underlying mechanisms of a mixture of the main components of Panax notoginseng saponins (TSPN) on cerebral ischemia-reperfusion injury and oxygen-glucose deprivation/reoxygenation (OGD/R) of cultured cortical neurons.@*METHODS@#The neuroprotective effect of TSPN was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry and live/dead cell assays. The morphology of dendrites was detected by immunofluorescence. Middle cerebral artery occlusion (MCAO) was developed in rats as a model of cerebral ischemia-reperfusion. The neuroprotective effect of TSPN was evaluated by neurological scoring, tail suspension test, 2,3,5-triphenyltetrazolium chloride (TTC) and Nissl stainings. Western blot analysis, immunohistochemistry and immunofluorescence were used to measure the changes in the Akt/mammalian target of rapamycin (mTOR) signaling pathway.@*RESULTS@#MTT showed that TSPN (50, 25 and 12.5 µ g/mL) protected cortical neurons after OGD/R treatment (P<0.01 or P<0.05). Flow cytometry and live/dead cell assays indicated that 25 µ g/mL TSPN decreased neuronal apoptosis (P<0.05), and immunofluorescence showed that 25 µ g/mL TSPN restored the dendritic morphology of damaged neurons (P<0.05). Moreover, 12.5 µ g/mL TSPN downregulated the expression of Beclin-1, Cleaved-caspase 3 and LC3B-II/LC3B-I, and upregulated the levels of phosphorylated (p)-Akt and p-mTOR (P<0.01 or P<0.05). In the MCAO model, 50 µ g/mL TSPN improved defective neurological behavior and reduced infarct volume (P<0.05). Moreover, the expression of Beclin-1 and LC3B in cerebral ischemic penumbra was downregulated after 50 µ g/mL TSPN treatment, whereas the p-mTOR level was upregulated (P<0.05 or P<0.01).@*CONCLUSION@#TSPN promoted neuronal survival and protected dendrite integrity after OGD/R and had a potential therapeutic effect by alleviating neurological deficits and reversing neuronal loss. TSPN promoted p-mTOR and inhibited Beclin-1 to alleviate ischemic damage, which may be the mechanism that underlies the neuroprotective activity of TSPN.

Ethyl Acetate Fraction of Dicliptera chinensis (L.) Juss. Ameliorates Liver Fibrosis by Inducing Autophagy via PI3K/AKT/mTOR/p70S6K Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate the molecular mechanism underlying the anti-hepatic fibrosis activity of ethyl acetate fraction Dicliptera chinensis (L.) Juss. (EDC) in human hepatic stellate cells (HSCs) in vitro and in a carbon tetrachloride (CCl4)-induced hepatic fibrosis mouse model in vivo.@*METHODS@#For in vitro study, HSCs were pre-treated with platelet-derived growth factor (10 ng/mL) for 2 h to ensure activation and treated with EDC for 24 h and 48 h, respectively. The effect of EDC on HSCs was assessed using cell counting kit-8 assay, EdU staining, transmission electron microscopy, immunofluorescence staining, and Western blot, respectively. For in vivo experiments, mice were intraperitoneally injected with CCl4 (2 ° L/g, adjusted to a 25% concentration in olive oil), 3 times per week for 6 weeks, to develop a hepatic fibrosis model. Forty 8-week-old male C57BL/6 mice were divided into 4 groups using a random number table (n=10), including control, model, positive control and EDC treatment groups. Mice in the EDC and colchicine groups were intragastrically administered EDC (0.5 g/kg) or colchicine (0.2 mg/kg) once per day for 6 weeks. Mice in the control and model groups received an equal volume of saline. Biochemical assays and histological examinations were used to assess liver damage. Protein expression levels of α -smooth muscle actin (α -SMA) and microtubule-associated protein light chain 3B (LC3B) were measured by Western blot.@*RESULTS@#EDC reduced pathological damage associated with liver fibrosis, downregulated the expression of α -SMA and upregulated the expression of LC3B (P<0.05), both in HSCs and the CCl4-induced liver fibrosis mouse model. The intervention of bafilomycin A1 and rapamycin in HSCs strongly supported the notion that inhibition of autophagy enhanced α -SMA protein expression levels (P<0.01). The results also found that the levels of phosphoinositide (PI3K), p-PI3K, AKT, p-AKT, mammalian target of rapamycin (mTOR), p-mTOR, and p-p70S6K all decreased after EDC treatment (P<0.05).@*CONCLUSIONS@#EDC has anti-hepatic fibrosis activity by inducing autophagy and might be a potential drug to be further developed for human liver fibrosis therapy.

Modified Linggui Zhugan Decoction () Ameliorates Glycolipid Metabolism and Inflammation via PI3K-Akt/mTOR-S6K1/AMPK-PGC-1 α Signaling Pathways in Obese Type 2 Diabetic Rats / 中国结合医学杂志

OBJECTIVE@#To investigate the protective effects of modified Linggui Zhugan Decoction (, MLZD), a traditional Chinese medicine formula, on obese type 2 diabetes mellitus (T2DM) rats.@*METHODS@#Fifty Sprague-Dawley rats were randomly divided into 5 groups by a random number table, including normal, obese T2DM (ob-T2DM), MLZD low-dose [MLDZ-L, 4.625 g/(kg·d)], MLZD middle-dose [MLD-M, 9.25 g/(kg·d) ] and MLZD high-dose [MLD-H, 18.5 g/(kg·d)] groups, 10 rats in each group. After 4-week intervention, blood samples and liver, pancreas, muscle tissues were collected to assess the insulin resistance (IR), blood lipid, adipokines and inflammation cytokines. The alteration of phosphatidylinositol 3 kinase (PI3K)-protein kinase B (PKB or Akt)/the mammalian target of rapamycin (mTOR)-ribosome protein subunit 6 kinase 1 (S6K1 )/AMP-activated protein kinase (AMPK)-peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 α) pathways were also studied.@*RESULTS@#MLZD dose-dependently reduced fasting blood glucose, fasting insulin, homeostasis model of assessment for IR index and increased insulin sensitive index compared with ob-T2DM rats (P<0.05). Similarly, total cholesterol, triglyceride, low-density lipoprotein cholesterol and free fatty acids were also decreased compared with ob-T2DM rats after 4-week treatment (P<0.05 or P<0.01). Improvements in adipokines and inflammatory cytokines were observed with a raised level of adiponectin and a reduced level of leptin, resistin, tumor necrosis factor-α and interleukin-6 (P<0.05 or P<0.01). MLZD regulated the PI3K-Akt/mTOR-S6K1/AMPK-PGC-1 α pathways and restored the tissue structure of liver and pancreas (P<0.05 or P<0.01).@*CONCLUSIONS@#MLZD ameliorated glycolipid metabolism and inflammation, which may be attributed to the regulation of PI3K-Akt/mTOR-S6K1/AMPK-PGC-1 α pathways.

Effect of γδ T cells on the Proliferation, Apoptosis and Autophagy of Multiple Myeloma Cells / 中国实验血液学杂志

AbstractObjective: To investigate the effect of γδ T cells on the proliferation, apoptosis and autophagy of multiple myeloma cells.@*METHODS@#Peripheral blood mononuclear cells (PBMNC) were isolated from healthy volunteers, and stimulated with zoledronic acid (Zol) in combination with rhIL-2. Flow cytometry analysis was used to detected the purity of γδ T cells. γδ T cells were collected and co-cultured with RPMI-8226 or U-266 cells at different effector target ratios. The proliferation of RPMI-8226 or U-266 cell lines were detected by CCK-8. Cell cycle and cell apoptosis were detected by flow cytometry and Western blot．The expressions of autophagy-related proteins were detected by Western blot.@*RESULTS@#γδ T cells can be expanded in vitro. γδ T cells could inhibit the proliferation of RPMI-8226 or U-266 cells, induced cell cycle arrest and promoted apoptosis in an effector target-dependent manner. In addition, γδ T cells could induce autophagy of myeloma cells, inhibited the expression of autophagy-related PI3K, P-AKT and P-mTOR, while increased the expression of AMPK and Beclin-1.@*CONCLUSION@#γδ T cells can inhibit the proliferation of RPMI-8226 and U-266 myeloma cells, induce cell cycle arrest, promote apoptosis, and enhance autophagy in vitro. The mechanism may be related to inhibition of PI3K/AKT/mTOR signaling pathway and/or activation of AMPK/Beclin-1 signaling pathway.

Expert consensus on the clinical application of PI3K/AKT/mTOR inhibitors in the treatment of advanced breast cancer / 中华肿瘤杂志

Phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway (PAM pathway) plays an important role in the development of breast cancer and are closely associated with the resistance to endocrine therapy in advanced breast cancer. Therefore, anti-cancer treatment targeting key molecules in this signaling pathway has become research hot-spot in recent years. Randomized clinical trials have demonstrated that PI3K/AKT/mTOR inhibitors bring significant clinical benefit to patients with advanced breast cancer, especially to those with hormone receptor (HR)-positive, human epidermal growth factor receptor (HER) 2-negative advanced breast cancer. Alpelisib, a PI3K inhibitor, and everolimus, an mTOR inhibitor, have been approved by Food and Drug Administration. Based on their high efficacy and relatively good safety profile, expanded indication of everolimus in breast cancer have been approved by National Medical Products Administration. Alpelisib is expected to be approved in China in the near future. The members of the consensus expert panel reached this consensus to comprehensively define the role of PI3K/AKT/mTOR signaling pathway in breast cancer, efficacy and clinical applications of PI3K/AKT/mTOR inhibitors, management of adverse reactions, and PIK3CA mutation detection, in order to promote the understanding of PI3K/AKT/mTOR inhibitors for Chinese oncologists, improve clinical decision-making, and prolong the survival of target patient population.

P4HA2 promotes occurrence and progression of liver cancer by regulating the PI3K/Akt/mTOR signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the role of proline 4-hydroxylase Ⅱ (P4HA2) in the occurrence and progression of liver cancer.@*METHODS@#GEPIA and Human Protein Atlas database were used to predict the expression of P4HA2 in hepatocellular carcinoma (HCC), and K-M plotter online database was used to analyze the relationship between P4HA2 expression and the prognosis of HCC. We also examined the expressions of P4HA2 in HCC cells and normal hepatocytes using qRT-PCR and Western blotting. With lentivirus-mediated RNA interference, P4HA2 expression was knocked down in hepatoma SNU-449 and Hep-3B cells, and the changes in cell proliferation, migration and invasion were assessed using cell counting kit-8 (CCK-8) assay, colony formation test, scratch test and Transwell assay. The changes in the expressions of epithelial-mesenchymal transition (EMT) and PI3K/Akt/mTOR signal pathway-related proteins were detected using Western blotting.@*RESULTS@#Online database analysis showed that the expression of P4HA2 was significantly higher in HCC tissues than in normal liver tissues (P < 0.05). The expression levels of P4HA2 mRNA and protein were also significantly higher in HCC cell lines than in normal hepatocytes (P < 0.01). Lentivirus-mediated RNA interference of P4HA2 significantly lowered the expression levels of P4HA2 mRNA and protein in the hepatoma cells (P < 0.05) and caused obvious inhibition of cell proliferation, migration and invasion. P4HA2 knockdown significantly increased the expression of E-cadherin protein, lowered the expressions of N-cadherin and Snail, and obviously decreased the expressions of phosphorylated PI3K, AKT and mTOR (P < 0.05).@*CONCLUSION@#P4HA2 enhances the proliferation, migration, invasion, and EMT of hepatoma cells by activating the PI3K/Akt/mTOR signaling pathway to promote the occurrence and progression of liver cancer.

Dihydromyricetin reduces lipid accumulation in LO2 cells via AMPK/mTOR-mediated lipophagy pathway and inhibits HepG2 cell proliferation in vitro / 南方医科大学学报

OBJECTIVE@#To explore the mechanism underlying the hepatoprotective effect of dihydromyricetin (DMY) against lipid accumulation in light of the lipophagy pathway and the inhibitory effect of DMY on HepG2 cell proliferation.@*METHODS@#LO2 cells were cultured in the presence of 10% FBS for 24 h and treated with 100 μg/mL DMY, or exposed to 50% FBS for 24 h followed by treatment with 50, 100, or 200 μg/mL DMY; the cells in recovery group were cultured in 50% FBS for 24 h and then in 10% FBS for another 24 h. Oil red O staining was used to observe the accumulation of lipid droplets in the cells, and the levels of TC, TG, and LDL and activities of AST, ALT and LDH were measured. The expression of LC3 protein was detected using Western blotting. AO staining and transmission electron microscopy were used to determine the numbers of autophagolysosomes and autophagosomes, respectively. The formation of autophagosomes was observed with MDC staining, and the mRNA expression levels of LC3, ATG7, AMPK, mTOR, p62 and Beclin1 were determined with q-PCR. Flow cytometry was performed to analyze the effect of 50, 100, and 200 μg/mL DMY on cell cycle and apoptosis of HepG2 cells; DNA integrity in the treated cells was examined with cell DNA fragmentation test.@*RESULTS@#DMY treatment and pretreatment obviously inhibited lipid accumulation and reduced the levels of TC, TG, LDL and enzyme activities of AST, ALT and LDH in LO2 cells (P < 0.05). In routinely cultured LO2 cells, DMY significantly promoted the formation of autophagosomes and autophagolysosomes and upregulated the expression of LC3 protein. DMY obviously attenuated high FBS-induced inhibition of autophagosome formation in LO2 cells, up- regulated the mRNA levels of LC3, ATG7, Beclin1 and AMPK, and downregulated p62 and mTOR mRNA levels (P < 0.05 or 0.01). In HepG2 cells, DMY caused obvious cell cycle arrest, inhibited cell proliferation, and induced late apoptosis and DNA fragmentation.@*CONCLUSION@#DMY reduces lipid accumulation in LO2 cells by regulating the AMPK/ mTOR-mediated lipophagy pathway and inhibits the proliferation of HepG2 by causing cell cycle arrest and promoting apoptosis.

Hyperactivation of PI3K/AKT/mTOR signal pathway impairs TNF-α-induced autophagy in mesenchymal stem cells from patients with ankylosing spondylitis / 南方医科大学学报

OBJECTIVE@#To investigate the changes in autophagy of mesenchymal stem cells (MSCs) from patients with ankylosing spondylitis and explore the mechanism for decreased autophagy in ASMSCs.@*METHODS@#MSCs collected from 14 patients with AS (ASMSCs) and from 15 healthy donors (HDMSCs) were cultured in the absence or presence of 25 ng/mL TNF-α for 6 h. Autophagy of the cells was determined by immunofluorescence staining of GFP-LC3B, and the results were confirmed by detecting the protein expressions of autophagy markers LC3 II/LC3 I and P62. The mRNA expressions of the related genes were detected using qRT-PCR, and the protein expressions of the autophagy markers and signaling pathway-related molecules were determined with Western blotting. TG100713 was used to block the PI3K/AKT/mTOR signal pathway, and its effect on autophagy of ASMSCs was evaluated.@*RESULTS@#ASMSCs showed significantly weaker GFP-LC3B puncta staining and lower protein expression levels of LC3 II/LC3 I but higher levels of P62 protein (P < 0.05), indicating a decreased autophagy capacity as compared with HDMSCs. TNF-α-induced ASMSCs showed significantly higher protein expressions of p-PI3K/ PI3K, p-AKT/AKT and p-mTOR/mTOR than HDMSCs (P < 0.05), suggesting hyperactivation of the PI3K/AKT/mTOR signaling pathway in ASMSCs. Blocking PI3K/AKT/mTOR signaling with TG100713 eliminated the difference in TNF-α-induced autophagy between HDMSCs and ASMSCs.@*CONCLUSION@#In patients with AS, hyperactivation of the PI3K/AKT/mTOR signaling pathway results in decreased autophagy of the MSCs and potentially contributes to chronic inflammation.

Hepatocyte growth factor protects pulmonary endothelial barrier against oxidative stress and mitochondria-dependent apoptosis / 中华医学杂志(英文版)

BACKGROUND@#Pulmonary microvascular endothelial cells (PMVECs) were not complex, and the endothelial barrier was destroyed in the pathogenesis progress of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Previous studies have demonstrated that hepatocyte growth factor (HGF), which was secreted by bone marrow mesenchymal stem cells, could decrease endothelial apoptosis. We investigated whether mTOR/STAT3 signaling acted in HGF protective effects against oxidative stress and mitochondria-dependent apoptosis in lipopolysaccharide (LPS)-induced endothelial barrier dysfunction and ALI mice.@*METHODS@#In our current study, we introduced LPS-induced PMEVCs with HGF treatment. To investigate the effects of mammalian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) pathway in endothelial oxidative stress and mitochondria-dependent apoptosis, mTOR inhibitor rapamycin and STAT3 inhibitor S3I-201 were, respectively, used to inhibit mTOR/STAT3 signaling. Moreover, lentivirus vector-mediated mTORC1 (Raptor) and mTORC2 (Rictor) gene knockdown modifications were introduced to evaluate mTORC1 and mTORC1 pathways. Calcium measurement, reactive oxygen species (ROS) production, mitochondrial membrane potential and protein, cell proliferation, apoptosis, and endothelial junction protein were detected to evaluate HGF effects. Moreover, we used the ALI mouse model to observe the mitochondria pathological changes with an electron microscope in vivo.@*RESULTS@#Our study demonstrated that HGF protected the endothelium via the suppression of ROS production and intracellular calcium uptake, which lead to increased mitochondrial membrane potential (JC-1 and mitochondria tracker green detection) and specific proteins (complex I), raised anti-apoptosis Messenger Ribonucleic Acid level (B-cell lymphoma 2 and Bcl-xL), and increased endothelial junction proteins (VE-cadherin and occludin). Reversely, mTOR inhibitor rapamycin and STAT3 inhibitor S3I-201 could raise oxidative stress and mitochondria-dependent apoptosis even with HGF treatment in LPS-induced endothelial cells. Similarly, mTORC1 as well as mTORC2 have the same protective effects in mitochondria damage and apoptosis. In in vivo experiments of ALI mouse, HGF also increased mitochondria structural integrity via the mTOR/STAT3 pathway.@*CONCLUSION@#In all, these reveal that mTOR/STAT3 signaling mediates the HGF suppression effects to oxidative level, mitochondria-dependent apoptosis, and endothelial junction protein in ARDS, contributing to the pulmonary endothelial survival and barrier integrity.

Down-regulation of SIK2 expression alleviates myocardial ischemia-reperfusion injury in rats by inhibiting autophagy through the mTOR-ULK1 signaling pathway / 南方医科大学学报

OBJECTIVE@#To explore the role of salt-inducible kinase 2 (SIK2) in myocardial ischemia-reperfusion (IR) injury in rats.@*METHODS@#Fifteen male SD rats were randomized equally into sham operation group, myocardial IR model group, and SIK2 inhibitor group (in which the rats were treated with intravenous injection of 10 mg/kg bosutinib via the left femoral vein 24 h before modeling). Ultrasound was used to detect the cardiac function of the rats, and myocardial pathologies were observed with HE staining. Transmission electron microscopy was used to observe autophagy of myocardial cells, and Western blotting was performed to detect the contents of the autophagy-related proteins SIK2, LC3B, Beclin-1, p62 and the expressions of p-mTOR, mTOR, p-ULK1, and ULK1 in myocardial tissue.@*RESULTS@#Myocardial IR injury significantly increased the number of autophagosomes (P < 0.05) and the expression of SIK2 protein (P < 0.01) in the myocardial tissues. Treatment with bosutinib before modeling obviously lowered the expression of SIK2 protein (P < 0.01), alleviated myocardial pathologies, and reduced the number of autophagosomes (P < 0.05) in the myocardial tissue. The rats with myocardial IR injury showed obviously lowered LVEF and FS values (P < 0.001), which were significantly improved by bosutinib treatment (P < 0.05); no significant difference was detected in IVSDd or LVPWDd among the 3 groups (P > 0.05). Myocardial IR injury obviously increased the expressions of LC3-II/LC3-I and Beclin-1 proteins and lowered the expression of p62 protein (P < 0.01), and these changes were significantly rescued by bosutinib treatment (P < 0.05). The rat models of myocardial IR injury showed significantly increased expression of p-ULK1 (Ser757) (P < 0.01) and lowered expression of p-mTOR protein (P < 0.0001) in the myocardium, and these changes were obviously reversed by bosutinib (P < 0.01 or 0.05); there was no significant difference in mTOR and ULK1 expressions among the 3 groups (P > 0.05).@*CONCLUSION@#SIK2 may promote autophagy through the mTOR/ULK1 signaling pathway, and inhibiting SIK2 can reduce abnormal autophagy and alleviate myocardial IR injury in rats.

Mibefradil improves skeletal muscle mass, function and structure in obese mice / 南方医科大学学报

OBJECTIVE@#To observe the effect of mibefradil on skeletal muscle mass, function and structure in obese mice.@*METHODS@#Fifteen 6-week-old C57BL/6 mice were randomized equally into normal diet group (control group), high-fat diet (HFD) group and high-fat diet +mibefradil intervention group (HFD +Mibe group). The grip strength of the mice was measured using an electronic grip strength meter, and the muscle content of the hindlimb was analyzed by X-ray absorptiometry (DXA). Triglyceride (TG) and total cholesterol (TC) levels of the mice were measured with GPO-PAP method. The cross-sectional area of the muscle fibers was observed with HE staining. The changes in the level of autophagy in the muscles were detected by Western blotting and immunofluorescence assay, and the activation of the Akt/mTOR signaling pathway was detected with Western blotting.@*RESULTS@#Compared with those in the control group, the mice in HFD group had a significantly greater body weight, lower relative grip strength, smaller average cross sectional area of the muscle fibers, and a lower hindlimb muscle ratio (P < 0.05). Immunofluorescence assay revealed a homogenous distribution of LC3 emitting light red fluorescence in the cytoplasm in the muscle cells in HFD group and HFD+Mibe group, while bright spots of red fluorescence were detected in HFD group. In HFD group, the muscular tissues of the mice showed an increased expression level of LC3 II protein with lowered expressions of p62 protein and phosphorylated AKT and mTOR (P < 0.05). Mibefradil treatment significantly reduced body weight of the mice, lowered the expression level of p62 protein, and increased forelimb grip strength, hindlimb muscle ratio, cross-sectional area of the muscle fibers, and the expression levels of LC3 II protein and phosphorylated AKT and mTOR (P < 0.05).@*CONCLUSION@#Mibefradil treatment can moderate high-fat diet-induced weight gain and improve muscle mass and function in obese mice possibly by activating AKT/mTOR signal pathway to improve lipid metabolism and inhibit obesityinduced autophagy.

Inhibition effect of active fraction from clove on PI3K/Akt/mTOR signaling pathway to induce apoptosis of human colon cancer HCT116 cells / 中国中药杂志

To screen the sensitive cell lines of active fraction from clove(AFC) on human colon cancer cells, investigate the effects of AFC on the cells proliferation and apoptosis as well as PI3 K/Akt/mTOR(phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin) signaling pathways involved, and reveal the mechanism of AFC for inducing apoptosis of human colorectal carcinoma cells. Cell counting kit-8(CCK-8) assay was used to detect the cytotoxic effect of different concentrations of AFC. AFC-induced apoptosis was detected by Hoechst 33258 fluorescence staining and Annexin V-FITC/PI double staining. HCT116 cells were treated with AFC with or without pretreatment with insulin-like growth factor-Ⅰ(IGF-Ⅰ), and then the protein expression levels of caspase-3, caspase-9, poly ADP-ribose polymerase(PARP), PI3 K, p-PI3 K, Akt, p-Akt, mTOR and p-mTOR in PI3 K/Akt/mTOR signaling pathway were detected by Western blot. RESULTS:: showed that the most obvious inhibitory effect of AFC was on human colon cancer HCT116 cells, and the optimal AFC treatment time was 48 hours. After AFC treatment, typical apoptotic features such as nuclear chromatin concentration, nuclear fragmentation and apoptotic bodies appeared in a dose-dependent manner. Annexin V-FITC/PI double staining showed that as compared with the control group, 50 and 100 μg·mL~(-1) AFC groups increased the apoptosis rate of HCT116 cells significantly(P<0.001); AFC activated caspase-9, cleaved caspase-3 and cleaved PARP in a concentration-dependent manner. The protein expression levels of cleaved caspase-3/procaspase-3, cleaved PARP/PARP and caspase-9/β-actin after treatment of AFC(100 μg·mL~(-1)) were significantly different from those in the control group(P<0.001). The relative protein expression of p-PI3 K, p-Akt and p-mTOR decreased in a concentration dependent manner, while Akt and mTOR showed no significant differences among groups. The ratios of p-PI3 K/PI3 K, p-Akt/Akt and p-mTOR/mTOR in the AFC groups(50 and 100 μg·mL~(-1)) were significantly lower than those in the control group(P<0.01). Its combination with IGF-Ⅰ weakened the effect of AFC in inhibiting PI3 K/Akt/mTOR signaling pathway. The ratios of p-Akt/Akt and p-mTOR/mTOR in the AFC+IGF-Ⅰ group were significantly enhanced as compared with the AFC group(P<0.05). Apoptosis-related protein expression levels(cleaved caspase-3 and cleaved PARP) in HCT116 cells treated with AFC+IGF-Ⅰ were also down regulated. As compared with the AFC group, the ratios of cleaved caspase-3/procaspase-3 and cleaved PARP/PARP in the AFC+IGF-Ⅰ group were significantly decreased(P<0.01). In summary, AFC activated caspase-mediated cascades and induced HCT116 cells apoptosis in a dose-dependent manner, which may be associated with the inhibition of the PI3 K/Akt/mTOR signaling pathway.

ubtor Mutation Causes Motor Hyperactivity by Activating mTOR Signaling in Zebrafish / 神经科学通报·英文版

Mechanistic target of rapamycin (mTOR) signaling governs important physiological and pathological processes key to cellular life. Loss of mTOR negative regulators and subsequent over-activation of mTOR signaling are major causes underlying epileptic encephalopathy. Our previous studies showed that UBTOR/KIAA1024/MINAR1 acts as a negative regulator of mTOR signaling, but whether UBTOR plays a role in neurological diseases remains largely unknown. We therefore examined a zebrafish model and found that ubtor disruption caused increased spontaneous embryonic movement and neuronal activity in spinal interneurons, as well as the expected hyperactivation of mTOR signaling in early zebrafish embryos. In addition, mutant ubtor larvae showed increased sensitivity to the convulsant pentylenetetrazol, and both the motor activity and the neuronal activity were up-regulated. These phenotypic abnormalities in zebrafish embryos and larvae were rescued by treatment with the mTORC1 inhibitor rapamycin. Taken together, our findings show that ubtor regulates motor hyperactivity and epilepsy-like behaviors by elevating neuronal activity and activating mTOR signaling.

Pinocembrin Promotes OPC Differentiation and Remyelination via the mTOR Signaling Pathway / 神经科学通报·英文版

The exacerbation of progressive multiple sclerosis (MS) is closely associated with obstruction of the differentiation of oligodendrocyte progenitor cells (OPCs). To discover novel therapeutic compounds for enhancing remyelination by endogenous OPCs, we screened for myelin basic protein expression using cultured rat OPCs and a library of small-molecule compounds. One of the most effective drugs was pinocembrin, which remarkably promoted OPC differentiation and maturation without affecting cell proliferation and survival. Based on these in vitro effects, we further assessed the therapeutic effects of pinocembrin in animal models of demyelinating diseases. We demonstrated that pinocembrin significantly ameliorated the progression of experimental autoimmune encephalomyelitis (EAE) and enhanced the repair of demyelination in lysolectin-induced lesions. Further studies indicated that pinocembrin increased the phosphorylation level of mammalian target of rapamycin (mTOR). Taken together, our results demonstrated that pinocembrin promotes OPC differentiation and remyelination through the phosphorylated mTOR pathway, and suggest a novel therapeutic prospect for this natural flavonoid product in treating demyelinating diseases.