[Effect of moxibustion on autophagy in mice with Alzheimer's disease based on mTOR/p70S6K signaling pathway].

OBJECTIVE: To investigate the effect of moxibustion on autophagy and amyloid ß-peptide1-42 (Aß1-42) protein expression in amyloid precursor protein/presenilin 1 (APP/PS1) double-transgenic mice with Alzheimer's disease (AD). METHODS: After 2-month adaptive feeding, fifty-six 6-month-old APP/PS1 double transgenic AD mice were randomly divided into a model group, a moxibustion group, a rapamycin group and an inhibitor group, 14 mice in each group. Another 14 C57BL/6J mice with the same age were used as a normal group. The mice in the moxibustion group were treated with monkshood cake-separated moxibustion at "Baihui"(GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14) for 20 min; the mice in the rapamycin group were intraperitoneally injected with rapamycin (2 mg/kg); the mice in the inhibitor group were treated with moxibustion and injection of 1.5 mg/kg 3-methyladenine (3-MA). All the treatments were given once a day for consecutive 2 weeks. The morphology of hippocampal tissue was observed by HE staining; the ultrastructure of hippocampal tissue was observed by transmission electron microscopy; the expression of Aß1-42 protein in frontal cortex and hippocampal tissue was detected by immunohistochemistry; the expressions of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), p70 ribosomal protein S6 kinase (p70S6K) and phosphorylated p70S6K (p-p70S6K) protein in hippocampus were detected by Western blot method. RESULTS: Compared with the normal group, the number of neuron cells was decreased, cells were necrotic and deformed, and autophagy vesicle and lysosome were decreased in the model group. Compared with the model group, the number of neuron cells was increased, cell necrosis was decreased, and autophagy vesicle and lysosome were increased in the moxibustion group and the rapamycin group. Compared with the normal group, the protein expressions of Aß1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the model group were increased (P<0.05); compared with the model group, the protein expressions of Aß1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group, rapamycin group and inhibitor group were decreased (P<0.05); compared with the inhibitor group, the protein expressions of Aß1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group and rapamycin group were decreased (P<0.05); compared with the rapamycin group, the protein expressions of mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group were decreased (P<0.05). CONCLUSION: Moxibustion could enhance autophagy in hippocampal tissue of APP/PS1 double transgenic AD mice and reduce abnormal Aß aggregation in brain tissue, the mechanism may be related to the inhibition of mTOR/p70S6K signaling pathway.

[Protective effect of salidroside on high fat-induced apoptosis in H9c2 cardiomyocytes through AMPK/mTOR/p70S6K pathway].

The study explored the effect of salidroside(SAL) on high fat-induced apoptosis in H9 c2 cardiomyocytes based on AMPK/mTOR/p70 S6 K pathway.H9 c2 cardiomyocytes were cultured in vitro and the lipotoxicity model of H9 c2 cardiomyocytes was constructed by 0.2 mmol·L~(-1) palmitic acid(PA) treatment for 24 hours.The cells were divided into control group, PA group, and SAL group(20 µmol·L~(-1)).Cell proliferation was detected with cell proliferation kit I(MTT) assay after SAL and PA treatment.Dihydroethidium(DHE) probe, Annexin V-FITC/PI kit, and JC-1 probe were used to estimate reactive oxygen species(ROS) level, cell apoptosis, and mitochondrial membrane potential(MMP) change, respectively.The expression levels of p-AMPK/AMPK, p-mTOR/mTOR, p-p70 S6 K/p70 S6 K and apoptosis-related proteins Bax, Bcl-2, and cleaved caspase-3 were investigated with Western blot.The mRNA levels of AMPK, mTOR and p70 S6 K were determined by quantitative reverse transcription-polymerase chain reaction(qRT-PCR).RESULTS:: showed that compared with control group, PA group had decreased cell proliferation ability, MMP, Bcl-2 protein expression and AMPK protein and mRNA expression, while increased ROS level, Bax and cleaved caspase-3 protein expression, and mTOR and p70 S6 K mRNA and protein expression, and the difference was statistically significant(P<0.05, P<0.01).Compared with PA group, SAL improved cell proliferation ability, MMP level, Bcl-2 protein expression, and AMPK mRNA and protein expression, while down-regulated ROS level, cell apoptosis, Bax and cleaved caspase-3 protein expression, and mTOR and p70 S6 K mRNA and protein expression, and the difference was statistically significant(P<0.05, P<0.01).In conclusion, SAL exerted protective effects on high fat-induced lipotoxicity of H9 c2 cardiomyocytes, alleviated the oxidative stress injury and reduced cell apoptosis via regulating AMPK/mTOR/p70 S6 K signaling pathway.

L-Arginine/nitric oxide regulates skeletal muscle development via muscle fibre-specific nitric oxide/mTOR pathway in chickens.

L-Arginine (L-Arg), the precursor of nitric oxide (NO), plays an important role in muscle function. Fast-twitch glycolytic fibres are more susceptible to age-related atrophy than slow-twitch oxidative fibres. The effect of L-Arg/NO on protein metabolism of fast- and slow-twitch muscle fibres was evaluated in chickens. In Exp. 1, 48 chicks at 1 day old were divided into 4 groups of 12 birds and subjected to 4 treatments: basal diet without supplementation or supplemented with 1% L-Arg, and water supplemented with or without L-nitro-arginine methyl ester (L-NAME, 18.5 mM). In Exp. 2, 48 chicks were divided into 4 groups of 12 birds fed with the basal diet and subjected to the following treatments: tap water (control), tap water supplemented with L-NAME (18.5 mM), or molsidomine (MS, 0.1 mM), or 18.5 mM L-NAME + 0.1 mM MS (NAMS). The regulatory effect of L-Arg/NO was further investigated in vitro with myoblasts obtained from chicken embryo pectoralis major (PM) and biceps femoris (BF). In vivo, dietary L-Arg supplementation increased breast (+14.94%, P < 0.05) and thigh muscle mass (+23.40%, P < 0.05); whereas, MS treatment had no detectable influence. However, L-NAME treatment blocked the beneficial influence of L-Arg on muscle development. L-Arg decreased (P < 0.05) protein synthesis rate, phosphorylated mTOR and ribosomal protein S6 kinase beta-1 (p70S6K) levels in breast muscle, which was recovered by L-NAME treatment. In vitro, L-Arg or sodium nitroprusside (SNP) reduced protein synthesis rate, suppressed phosphorylated mTOR/p70S6K and decreased atrogin-1 and muscle RING finger 1 (MuRF1) in myoblasts from PM muscle (P < 0.05). L-NAME abolished the inhibitory effect of L-Arg on protein synthesis and the mTOR/p70S6K pathway. However, myoblasts from BF muscle showed the weak influence. Moreover, blocking the mTOR/p70S6K pathway with rapamycin suppressed protein synthesis of the 2 types of myoblasts; whereas, the protein expression of atrogin-1 and MuRF1 levels were restricted only in myoblasts from PM muscle. In conclusion, L-Arg/NO/mTOR/p70S6K pathway enhances protein accumulation and muscle development in fast-twitch glycolytic muscle in chickens. L-Arg/NO regulates protein turnover in a muscle fibre specific way, which highlights the potential clinical application in fast-twitch glycolytic muscle fibres.

Panax notoginseng Saponins Stimulates Neurogenesis and Neurological Restoration After Microsphere-Induced Cerebral Embolism in Rats Partially Via mTOR Signaling.

P. Notoginseng Saponins (PNS), the main active component of herbal medicine Panax notoginseng, has been widely used to treat cerebrovascular diseases. It has been acknowledged that PNS exerted protection on nerve injuries induced by ischemic stroke, however, the long-term impacts of PNS on the restoration of neurological defects and neuroregeneration after stroke have not been thoroughly studied and the underlying molecular mechanism of stimulating neurogenesis is difficult to precisely clarify, much more in-depth researches are badly needed. In the present study, cerebral ischemia injury was induced by microsphere embolism (ME) in rats. After 14 days, PNS administration relieved cerebral ischemia injury as evidenced by alleviating neurological deficits and reducing hippocampal pathological damage. What's more, PNS stimulated hippocampal neurogenesis by promoting cell proliferation, migration and differentiation activity and modulated synaptic plasticity. Increased number of BrdU/Nestin, BrdU/DCX and NeuroD1-positive cells and upregulated synapse-related GAP43, SYP, and PSD95 expression were observed in the hippocampus. We hypothesized that upregulation of brain-derived neurotrophic factor (BDNF) expression and activation of Akt/mTOR/p70S6K signaling after ME could partially underlie the neuroprotective effects of PNS against cerebral ischemia injury. Our findings offer some new viewpoints into the beneficial roles of PNS against ischemic stroke.

Mechanism of erythropoietin-induced M2 microglia polarization via Akt / Mtor / P70S6k signaling pathway in the treatment of brain injury in premature mice and its effect on biofilm.

We investigated the mechanism of erythropoietin (EPO) in brain injury in premature mice based on Akt/mTOR/p70S6K signaling pathway. The brain injury model group of premature mice was obtained by intraperitoneal injection of lipopolysaccharide during pregnancy. Normal mice were taken as the control group. The model mice were divided into low-dose EPO (1,000 IU/kg, L-EPO), medium-dose EPO (2,500 IU/kg, M-EPO), and high-dose EPO groups (5,000 IU/kg, H-EPO) by intraperitoneal injection. The levels of malondialdehyde (MDA) and total superoxide dismutase (T-SOD) were detected. TUNEL staining and Western blotting were used to detect the differences in neuronal apoptosis index (AI), microglial polarization marker protein, and Akt/mTOR/p70S6K-related protein expression levels in each group. Compared with the control group, the protein levels of AI, MDA, Bax, and iNOS in the model, L-EPO, and M-EPO groups were significantly increased, while the T-SOD level and Bcl-2, ARG1, p-Akt, p-mTOR, and p-70S6K protein levels were significantly decreased (P < 0.05). Compared with the model group, AI, MAD levels and Bax, iNOS protein expression levels in L-EPO, M-EPO, and H-EPO groups were significantly decreased, while T-SOD level and Bcl-2, ARG1, p-Akt, p-mTOR, and p-70S6K protein levels were significantly increased. The changes were dose-dependent. In summary, EPO can activate microglia transformation from M1 to M2 through Akt/mTOR/p70S6K signaling pathway.

Lingguizhugan decoction dynamically regulates MAPKs and AKT signaling pathways to retrogress the pathological progression of cardiac hypertrophy to heart failure.

BACKGROUND: Heart failure (HF) is a grave health concern, with high morbidity and mortality, calling for the urgent need for new and alternative pharmacotherapies. Lingguizhugan decoction (LD) is a classic Chinese formula clinically used to treat HF. However, the underlying mechanisms involved are not fully elucidated. PURPOSE: Based on that, this study aims to investigate the effects and underlying mechanisms of LD on HF. METHODS: After confirming the therapeutic benefits of LD in transverse aortic constriction (TAC)-induced HF mice, network pharmacology and transcriptomic analyzes were utilized to predict the potential molecular targets and pathways of LD treatment in failing hearts, which were evaluated at 3 and 9 w after TAC. UHPLC-QE-MS analysis was utilized to detect bioactive ingredients from LD and plasma of LD-treated rats. RESULTS: Our results showed that LD markedly alleviated cardiac dysfunction via down-regulating CH-related genes and proteins expression in TAC mice. Significantly, cardiac hypertrophy signaling, including AKT and MAPKs signaling pathways, were identified, suggesting the pathways as likely regulatory targets for LD treatment. LD inhibited p38 and ERK phosphorylated expression levels, with the latter effect likely dependent on regulation of AMPK. Interestingly, LD exerted a dual modulatory role in the AKT-GSK3ß/mTOR/P70S6K signaling pathway's regulation, which was characterized by stimulatory activity at 3 w and inhibitory effects at 9 w. Finally, 15 bioactive compounds detected from plasma were predicted as the potential regulators of the AKT-GSK3ß/mTOR and MAPKs signaling pathways. CONCLUSION: Our study shows LD's therapeutic efficacy in failing hearts, signifies LD as HF medication that acts dynamically by balancing AKT-GSK3ß/mTOR/P70S6K and MAPKs pathways, and reveals possible bioactive compounds responsible for LD effects on HF.

Alnustone inhibits the growth of hepatocellular carcinoma via ROS- mediated PI3K/Akt/mTOR/p70S6K axis.

Alnustone, a diarylheptane compound, exhibits potent growth inhibition against hepatocellular carcinoma (HCC) BEL-7402 cells. However, the underlying mechanisms associated with its anticancer activity remain unknown. In the present study, we evaluated the anticancer effect of alnustone against several human cancers focused on HCC and the possible associated mechanisms. The results showed that alnustone significantly inhibited the growth of several cancer cells by CCK-8 assay. Alnustone markedly induced apoptosis and decreased mitochondrial membrane potential in BEL-7402 and HepG2 cells. Alnustone inhibited the expression of proteins related to apoptosis and PI3K/Akt/mTOR/p70S6K pathways and generated ROS production in BEL-7402 and HepG2 cells. Moreover, N-acetyl-L-cysteine (NAC, a ROS inhibitor) could significantly reverse the effects of alnustone on the growth inhibition of BEL-7402 and HepG2 cells and the expression of proteins related to apoptosis and PI3K/Akt/mTOR signaling pathway in HepG2 cells. Furthermore, alnustone significantly inhibited tumor growth of HepG2 xenografts, obviously induced apoptosis in the tumor tissues and improved the pathological condition of liver tissues of mice in vivo. The study provides evidence that alnustone is effective against HCC via ROS-mediated PI3K/Akt/mTOR/p70S6K pathway and the compound has the potential to be developed as a novel anticancer agent for the treatment of HCC clinically.

L-carnitine ameliorates the muscle wasting of cancer cachexia through the AKT/FOXO3a/MaFbx axis.

BACKGROUND: Recent studies suggest potential benefits of applying L-carnitine in the treatment of cancer cachexia, but the precise mechanisms underlying these benefits remain unknown. This study was conducted to determine the mechanism by which L-carnitine reduces cancer cachexia. METHODS: C2C12 cells were differentiated into myotubes by growing them in DMEM for 24 h (hrs) and then changing the media to DMEM supplemented with 2% horse serum. Differentiated myotubes were treated for 2 h with TNF-α to establish a muscle atrophy cell model. After treated with L-carnitine, protein expression of MuRF1, MaFbx, FOXO3, p-FOXO3a, Akt, p-Akt, p70S6K and p-p70S6K was determined by Western blotting. Then siRNA-Akt was used to determine that L-carnitine ameliorated cancer cachexia via the Akt/FOXO3/MaFbx. In vivo, the cancer cachexia model was established by subcutaneously transplanting CT26 cells into the left flanks of the BALB/c nude mice. After treated with L-carnitine, serum levels of IL-1, IL-6 and TNF-α, and the skeletal muscle content of MuRF1, MaFbx, FOXO3, p-FOXO3a, Akt, p-Akt, p70S6K and p-p70S6K were measured. RESULTS: L-carnitine increased the gastrocnemius muscle (GM) weight in the CT26-bearing cachexia mouse model and the cross-sectional fiber area of the GM and myotube diameters of C2C12 cells treated with TNF-α. Additionally, L-carnitine reduced the protein expression of MuRF1, MaFbx and FOXO3a, and increased the p-FOXO3a level in vivo and in vitro. Inhibition of Akt, upstream of FOXO3a, reversed the effects of L-carnitine on the FOXO3a/MaFbx pathway and myotube diameters, without affecting FOXO3a/MuRF-1. In addition to regulating the ubiquitination of muscle proteins, L-carnitine also increased the levels of p-p70S6K and p70S6K, which are involved in protein synthesis. Akt inhibition did not reverse the effects of L-carnitine on p70S6K and p-p70S6K. Hence, L-carnitine ameliorated cancer cachexia via the Akt/FOXO3/MaFbx and p70S6K pathways. Moreover, L-carnitine reduced the serum levels of IL-1 and IL-6, factors known to induce cancer cachexia. However, there were minimal effects on TNF-α, another inducer of cachexia, in the in vivo model. CONCLUSION: These results revealed a novel mechanism by which L-carnitine protects muscle cells and reduces inflammation related to cancer cachexia.

Inhibition of fucoidan on breast cancer cells and potential enhancement of their sensitivity to chemotherapy by regulating autophagy.

Fucoidan is a marine-origin sulfated polysaccharide that has gained attention for its anticancer activities. However, the inhibitory effect of fucoidan on breast cancers by regulating autophagy and its mechanism are not clear, and the chemotherapeutic sensitization of fucoidan is largely unknown. In the present study, the anticancer potential of fucoidan was revealed in MCF-7 and MDA-MB-231 cells. Additionally, we also studied the chemotherapeutic sensitization of fucoidan by combining chemotherapeutic drugs doxorubicin (ADM) and cisplatin (DDP) with fucoidan on breast cancer cells. In the two kinds of human breast cancer cells, cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Apoptosis was examined with flow cytometry. Transfection assay was used to examine autophagy flow. Western blot was used to examine the expressions of related proteins. Results suggested that fucoidan could induce autophagy and might enhance the sensitivity of breast cancer cells to chemotherapeutic drugs. Mechanistically, fucoidan induced autophagy in breast cancer cells by down-regulating m-TOR/p70S6K/TFEB pathway. In conclusion, our research revealed that fucoidan could induce autophagy of breast cancer cells by mediating m-TOR/p70S6K/TFEB pathway, thus inhibiting tumor development. Furthermore, fucoidan might enhance the sensitivity of breast cancer cells to ADM and DDP, and this enhancement was related to autophagy.

Clinical efficacies, underlying mechanisms and molecular targets of Chinese medicines for diabetic nephropathy treatment and management.

Diabetic nephropathy (DN) has been recognized as a severe complication of diabetes mellitus and a dominant pathogeny of end-stage kidney disease, which causes serious health problems and great financial burden to human society worldwide. Conventional strategies, such as renin-angiotensin-aldosterone system blockade, blood glucose level control, and bodyweight reduction, may not achieve satisfactory outcomes in many clinical practices for DN management. Notably, due to the multi-target function, Chinese medicine possesses promising clinical benefits as primary or alternative therapies for DN treatment. Increasing studies have emphasized identifying bioactive compounds and molecular mechanisms of reno-protective effects of Chinese medicines. Signaling pathways involved in glucose/lipid metabolism regulation, antioxidation, anti-inflammation, anti-fibrosis, and podocyte protection have been identified as crucial mechanisms of action. Herein, we summarize the clinical efficacies of Chinese medicines and their bioactive components in treating and managing DN after reviewing the results demonstrated in clinical trials, systematic reviews, and meta-analyses, with a thorough discussion on the relative underlying mechanisms and molecular targets reported in animal and cellular experiments. We aim to provide comprehensive insights into the protective effects of Chinese medicines against DN.

Ma xing shi gan decoction eliminates PM2.5-induced lung injury by reducing pulmonary cell apoptosis through Akt/mTOR/p70S6K pathway in rats.

The present study was designed to investigate the anti-apoptosis effect of Ma xing shi gan decoction (MXD) on PM2.5-induced lung injury via protein kinase B (Akt)/mTOR/p70S6K pathway. A UPLC-MS/MS system was introduced for component analysis of MXD. Rats were instilled with PM2.5 solution suspension intratracheally to induce acute lung injury. The rats were then orally administered with MXD (16, 8, and 4 g/kg) once a day for 7 consecutive days. The therapeutic effects of MXD were evaluated by Hematoxylin and Eosin (HE) staining. The apoptotic cell death was analyzed by terminal-deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay. The alterations in cytochrome c (Cytc) and cleaved-caspase-3 (C-caspase-3) were measured by immunohistochemistry (IHC). The expressions of Bax, B-cell lymphoma 2 (Bcl-2), p-Akt, p-mTOR and p-p70S6K were detected by Western blot. In vitro, PM2.5 exposure model was introduced in A549 cell, followed by incubation with MXD-medicated serum. Hoechst staining was used to determine apoptotic rate. The levels of Bax, Bcl-2, p-Akt, p-mTOR and p-p70S6K were detected by Western blot. Our results in vivo indicated that treatment with MXD decreased histopathological changes score, TUNEL-positive cells rate, expressions of Cytc and C-caspase-3. The in vitro results revealed that incubation with MXD-mediated serum decreased apoptotic rate. Both results in vivo and in vitro demonstrated that MXD inhibited pro-apoptotic protein Bax and promoted anti-apoptotic protein Bcl-2 expression. Likewise, MXD activated Akt/mTOR/p70S6K signal pathway, which was also confirmed by Western immunoblotting. In conclusion, MXD attenuates lung injury and the underlying mechanisms may relate to regulating the apoptosis via Akt/mTOR/p70S6K signaling pathway activation.

A novel role for α-viniferin in suppressing angiogenesis by blocking the VEGFR-2/p70S6K signaling pathway.

Angiogenesis plays important roles in pathological conditions such as cancer and inflammation as well as normal tissue development and homeostasis. Here, we investigated the effects and molecular mechanisms of α-viniferin, an oligostilbene isolated from Caragana sinica, on human umbilical vein endothelial cell responses in vitro and angiogenic sprouting in aortic rings ex vivo. α-viniferin treatment inhibited mitogen-induced HUVEC proliferation by retinoblastoma protein hypophosphorylation. In addition, α-viniferin suppressed mitogen-induced HUVEC adhesion, migration, invasion, and microvessel outgrowth. These anti-angiogenic activities of α-viniferin might be mediated through downregulation of cell cycle-related proteins, vascular endothelial growth factor receptor-2 (VEGFR-2), and matrix metalloproteinase-2. Furthermore, inactivation of VEGFR-2/p70 ribosomal S6 kinase signaling pathway was found to be involved in α-viniferin-mediated modulation of endothelial cell responses. Our results demonstrate the pharmacological functions and molecular mechanisms of α-viniferin in regulating angiogenesis, suggesting the therapeutic potential of α-viniferin to treat and prevent various angiogenesis-related diseases.

The Dephosphorylation of p70S6 (Thr389) Kinase as a Marker of L-Glutamate-Induced Excitotoxicity Related to Diabetes Disturbances-an Unconventional In Vitro Model.

Excitotoxicity is a modern clinical condition included in the pathogenesis of Alzheimer's disease. It is connected with diabetic disturbance, and it is still being analyzed in the context of the participation of the PI3K/mTOR pathway. A very important protein belonging to this pathway is p70S6K, whose activation promotes the pathogenesis of type 2 diabetes by the induction of insulin resistance. The study model was based on a PC12 cell line, derived from the pheochromocytoma of a rat adrenal medulla, cultured in RPMI 1640. The three reagents were used in different concentrations to create the model of excitotoxicity related to diabetes disturbances: L-glutamate (2.5 mM; 10 mM), glucose (150 mM; 200 mM), and insulin (0.093 mM; 0.371 mM). The aim of our study was to examine and evaluate the levels of phosphorylation of proteins involved in signal transduction controlled by MAPK, PI3K/Akt, and mTOR signaling pathways in L-glutamate-induced excitotoxicity with comorbid hyperglycemia and hyperinsulinemia imitating diabetic disturbances in in vitro conditions on PC12 cells. The results we obtained demonstrated the increased phosphorylation of p70S6K in Thr389 residue in almost all combinations of reagents, except for those including the highest concentration of L-glutamate, in which dephosphorylation was confirmed. This confirms the inhibition of mTOR kinase and suggests that p70S6K (Thr389) plays a functional role in the regulation of the signaling pathway in excitotoxicity related to diabetic disturbances.

Superimposed Whole-Body Electrostimulation Augments Strength Adaptations and Type II Myofiber Growth in Soccer Players During a Competitive Season.

BACKGROUND: The improvement of strength and athletic performance during a competitive season in elite soccer players is a demanding task for the coach. AIMS: As whole-body electrostimulation (WB-EMS) training provides a time efficient stimulation potentially capable in exerting skeletal muscle adaptations we aimed to test this approach over 7 weeks in trained male soccer players during a competitive season. HYPOTHESIS: We hypothesized that a superimposed WB-EMS will increase maximal strength and type I and type II myofiber hypertrophy. METHODS: Twenty-eight male field soccer players were assigned in either a WB-EMS group (EG, n = 10), a training group (TG, n = 10), or a control group (CG, n = 8). The regular soccer training consists of two to four sessions and one match per week. In concurrent, the EG performed 3 × 10 squat jumps superimposed with WB-EMS twice per week, TG performed 3 × 10 squat jumps without EMS twice per week, and the CG only performed the regular soccer training. Muscle biopsies were collected and strength tests were performed under resting conditions before (Baseline) and after the intervention period (Posttest). Muscle biopsies were analyzed via western blotting and immunohistochemistry for skeletal muscle adaptive responses. To determine the effect of the training interventions a 2 × 3 (time ∗ group) mixed ANOVA with repeated measures was conducted. RESULTS: Maximal strength in leg press (p = 0.009) and leg curl (p = 0.026) was significantly increased in EG along with a small but significant increase in type II myofiber diameter (p = 0.023). All of these adaptations were not observed in TG and CG. CONCLUSION: WB-EMS can serve as a time efficient training method to augment strength capacities and type II fiber myofiber growth in soccer players when combined with specific resistance training. This combination may therefore be a promising training modification compared to traditional strength training for performance enhancement.

Curcumin induces apoptotic cell death and protective autophagy by inhibiting AKT/mTOR/p70S6K pathway in human ovarian cancer cells.

PURPOSE: Curcumin (Cur), a yellow-colored dietary flavor from the plant (Curcuma longa), has been demonstrated to potentially resist diverse diseases, including ovarian cancer, but drug resistance becomes a major limitation of its success clinically. The key molecule or mechanism associated with curcumin resistance in ovarian cancer still remains unclear. The aim of our study was to investigate the effects of curcumin on autophagy in ovarian cancer cells and elucidate the underlying mechanism. METHODS: In our study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), EdU proliferation assay and colony-forming assay were used to assess cell viability. Apoptosis was detected by western blot and flow cytometric analysis of apoptosis. Autophagy was defined by both electron microscopy and immunofluorescence staining markers such as microtubule-associated protein 1 light chain 3 (LC3). Plasmid construction and shRNA transfection helped us to confirm the function of curcumin. RESULTS: Curcumin reduced cell viability and induced apoptotic cell death by MTT assay in human ovarian cancer cell lines SK-OV-3 and A2780 significantly. Electron microscopy, western blot and immunofluorescence staining proved that curcumin could induce protective autophagy. Moreover, treatment with autophagy-specific inhibitors or stable knockdown of LC3B by shRNA could markedly enhance curcumin-induced apoptosis. Finally, the cells transiently transfected with AKT1 overexpression plasmid demonstrated that autophagy had a direct relationship with the AKT/mTOR/p70S6K pathway. CONCLUSIONS: Curcumin can induce protective autophagy of human ovarian cancer cells by inhibiting the AKT/mTOR/p70S6K pathway, indicating the synergistic effects of curcumin and autophagy inhibition as a possible strategy to overcome the limits of current therapies in the eradication of epithelial ovarian cancer.

Sasanquasaponin ΙΙΙ from Schima crenata Korth induces autophagy through Akt/mTOR/p70S6K pathway and promotes apoptosis in human melanoma A375 cells.

BACKGROUND: Melanoma is a high fatality skin cancer which lacks effective drugs. Sasanquasaponin, an important sort of constituents in theaceae, has been demonstrated to have potent anti-tumor effect in breast cancer and hepatocellular carcinoma. As a sasanquasaponin, we speculate that Sasanquasaponin III (SQS III) isolated from Schima crenata Korth may also have anti-tumor activity. PURPOSE: This study aims to investigate whether SQS III has anti-melanoma activity and examine the underlying mechanisms of SQS III against melanoma. METHODS/STUDY DESIGNS: The anti-proliferative effect of SQS III was assessed by cells viability assay. Annexin V-FITC/PI double staining assay was utilized for detection of apoptosis. Mitochondrial membrane potential and reactive oxygen species (ROS) production were detected using JC-1 and DCFH-DA assay, respectively. Autophagy was monitored using transmission electron microscopy (TEM) and GFP-LC3 transfection fluorescence analysis. Autophagosome-lysosome fusion and lysosomal degradation were determined using a GFP-LC3 & LAMP1 co-localization assay and DQ-BSA staining. Proteins related to apoptosis and autophagy were analyzed by Western blotting. RESULTS: Our results demonstrated that the SQS III exhibited potent anti-cancer activity in A375 cells by inducing both apoptosis and autophagy. In melanoma cells treated with SQS III, caspases were activated and PARP was cleaved, proving the occurrence of apoptosis. Mechanistic studies indicated that the pro-apoptosis activity of SQS III was mediated by death receptor pathway and mitochondrial dysfunction which was induced by ROS accumulation and reversed by the ROS inhibitor N-acetyl-cysteine (NAC). In addition to triggering apoptosis, SQS III may also cause autophagy in melanoma cells. Our results demonstrated that SQS III induced up-regulated expression of GFP-LC3, autophagosome-lysosomal fusion and lysosomal degradation. Additionally, the ROS accumulation was also involved in the activation of autophagy. Meanwhile, it was also found that after SQS III treatment, the expression of LC3-II was up-regulated and the AKT/mTOR signaling pathway was inhibited. The autophagy inhibitor 3-MA converted cytotoxicity and apoptosis of SQS III in A375 cells, which indicated that autophagy promoted the SQS III-induced apoptosis. CONCLUSION: SQS III showed potent anti-cancer activity by inducing apoptosis and autophagy, which provides insights into its possible use as a therapy for melanoma.

Lycium barbarum polysaccharide protects diabetic peripheral neuropathy by enhancing autophagy via mTOR/p70S6K inhibition in Streptozotocin-induced diabetic rats.

Lycium barbarum polysaccharide (LBP), the major active component of Lycium barbarum, has been found to be effective in the management of some diabetic complications. We evaluated the protective effect of LBP in diabetic peripheral neuropathy (DPN) and explored the possible mechanisms. We found that LBP mildly decreased blood glucose levels and partially rescued allodynia and hyperalgesia in the diabetes mellitus (DM) rats. For the electrophysiological function of the sciatic nerve, the decrease in sensory nerve conduction velocity (SNCV) and sensory nerve action potential (SNAP) amplitudes in DM rats were partially rescued. Moreover, DM-induced structural damage to the nerve fiber myelination showed great improvement by 12 weeks of LBP treatment. The decreased expression of the myelin-related proteins, myelin protein zero (P0) and myelin basic protein (MBP), in the DM sciatic nerve was also markedly rescued after 12 weeks of LBP treatment. Furthermore, the possible role of mammalian target of rapamycin (mTOR)-mediated autophagy during these protective processes was examined. The expression of microtubule-associated protein light chain 3-II(LC3-II) and Beclin1 in the sciatic nerve was significantly decreased while the expression of P62 increased in DM rats, demonstrating an decreased activation of autophagy. As expected, the LC3-II and Beclin1 protein levels were markedly increased, and P62 was markedly decreased after LBP treatment. The expression of mTOR, p-mTOR, p70 ribosomal protein S6 kinase (p70S6K) and p-p70S6K in the DM group were markedly increased, while all of these proteins decreased in LBP group. These results demonstrate that LBP exerts protective effects on DPN, which is likely to be mediated through the induction of autophagy by inhibiting the activation of the mTOR/p70S6K pathways.

Inhibition of miR-128-3p by Tongxinluo Protects Human Cardiomyocytes from Ischemia/reperfusion Injury via Upregulation of p70s6k1/p-p70s6k1.

Background and Aims: Tongxinluo (TXL) is a multifunctional traditional Chinese medicine that has been widely used to treat cardiovascular and cerebrovascular diseases. However, no studies have explored whether TXL can protect human cardiomyocytes (HCMs) from ischemia/reperfusion (I/R) injury. Reperfusion Injury Salvage Kinase (RISK) pathway activation was previously demonstrated to protect the hearts against I/R injury and it is generally activated via Akt or (and) Erk 1/2, and their common downstream protein, ribosomal protein S6 kinase (p70s6k). In addition, prior studies proved that TXL treatment of cells promoted secretion of VEGF, which could be stimulated by the increased phosphorylation of one p70s6k subtype, p70s6k1. Consequently, we hypothesized TXL could protect HCMs from I/R injury by activating p70s6k1 and investigated the underlying mechanism. Methods and Results: HCMs were exposed to hypoxia (18 h) and reoxygenation (2 h) (H/R), with or without TXL pretreatment. H/R reduced mitochondrial membrane potential, increased bax/bcl-2 ratios and cytochrome C levels and induced HCM apoptosis. TXL preconditioning reversed these H/R-induced changes in a dose-dependent manner and was most effective at 400 µg/mL. The anti-apoptotic effect of TXL was abrogated by rapamycin, an inhibitor of p70s6k. However, inhibitors of Erk1/2 (U0126) or Akt (LY294002) failed to inhibit the protective effect of TXL. TXL increased p70s6k1 expression and, thus, enhanced its phosphorylation. Furthermore, transfection of cardiomyocytes with siRNA to p70s6k1 abolished the protective effects of TXL. Among the micro-RNAs (miR-145-5p, miR-128-3p and miR-497-5p) previously reported to target p70s6k1, TXL downregulated miR-128-3p in HCMs during H/R, but had no effects on miR-145-5p and miR-497-5p. An in vivo study confirmed the role of the p70s6k1 pathway in the infarct-sparing effect of TXL, demonstrating that TXL decreased miR-128-3p levels in the rat myocardium during I/R. Transfection of HCMs with a hsa-miR-128-3p mimic eliminated the protective effects of TXL. Conclusions: The miR-128-3p/p70s6k1 signaling pathway is involved in protection by TXL against HCM apoptosis during H/R. Overexpression of p70s6k1 is, therefore, a potential new strategy for alleviating myocardial reperfusion injury.

Luteoloside induces G0/G1 arrest and pro-death autophagy through the ROS-mediated AKT/mTOR/p70S6K signalling pathway in human non-small cell lung cancer cell lines.

Autophagy has attracted a great deal of interest in tumour therapy research in recent years. However, the anticancer effect of luteoloside, a naturally occurring flavonoid isolated from the medicinal plant Gentiana macrophylla, on autophagy remains poorly understood in human lung cells. In the present study, we have investigated the anticancer effects of luteoloside on non-small cell lung cancer (NSCLC) cells and demonstrated that luteoloside effectively inhibited cancer cell proliferation, inducing G0/G1 phase arrest associated with reduced expression of CyclinE, CyclinD1 and CDK4; we further found that treatment with luteoloside did not strongly result in apoptotic cell death in NSCLC (A549 and H292) cells. Interestingly, luteoloside induced autophagy in lung cancer cells, which was correlated with the formation of autophagic vacuoles, breakdown of p62, and the overexpression of Beclin-1 and LC3-II, but not in a human bronchial epithelial cell line (BEAS-2B). Notably, pretreatment of cancer cells with 3-MA, an autophagy inhibitor, protected against autophagy and promoted cell viability but not apoptosis. To further clarify whether luteoloside-induced autophagy depended on the PI3K/AKT/mTOR/p70S6K signalling pathway, a major autophagy-suppressive cascade, cells were treated with a combination of AKT inhibitor (LY294002) and mTOR inhibitor (Rap). These results demonstrated that luteoloside induced autophagy in lung cancer cell lines by inhibiting the pathway at p-Akt (Ser473), p-mTOR and p-p70S6K (Thr389). Moreover, we observed that luteoloside-induced cell autophagy was correlated with production of reactive oxygen species (ROS). NAC-mediated protection against ROS clearly implicated ROS in the activation of autophagy and cell death. In addition, the results showed that ROS served as an upstream effector of the PI3K/AKT/mTOR/p70S6K pathway. Taken together, the present study provides new insights into the molecular mechanisms underlying luteoloside-mediated cell death in NSCLC cells and supports luteoloside as a potential anti-cancer agent for targeting NSCLC through the induction of autophagy, inhibition of proliferation and PI3K/AKT/mTOR/p70S6K signalling.

Anti-fibrosis effect for Hirsutella sinensis mycelium based on inhibition of mTOR p70S6K phosphorylation.

Hirsutella sinensis, cultured in vitro, is an attractive substitute for Cordyceps sinensis as health supplement. The aim of this study was to demonstrate whether H. sinensis mycelium (HSM) attenuates murine pulmonary fibrosis induced by bleomycin and to explore the underlying molecular mechanisms. Using lung fibrosis modle induced by intratracheal instillation of bleomycin (BLM; 4 mg/kg), we observed that the administration of HSM reduced HYP, TGF-ß1 and the production of several pro-fibrosis cytokines (α-smooth muscle actin, fibronectin and vimentin) in fibrotic mice lung sections. Histopathological examination of lung tissues also demonstrated that HSM improved BLM-induced pathological damage. Concurrently, HSM supplementation markedly reduced the chemotaxis of alveolar macrophages and potently suppressed the expression of inflammatory cytokines. Also, HSM influenced Th1/Th2 and Th17/Treg imbalance and blocked the phosphorylation of mTOR pathway in vivo. Alveolar epithelial A549 cells acquired a mesenchymal phenotype and an increased expression of myofibroblast markers of differentiation (vimentin and fibronectin) after treatment with TGF-ß1. HSM suppressed these markers and blocked the phosphorylation of mTOR pathway in vitro. The results provide evidence supporting the use of HSM in the intervention of pulmonary fibrosis and suggest that HSM is a potential therapeutic agent for lung fibrosis.