Mammalian Target Of Rapamycin Phosphorylation Research Articles

Beta-carotene ameliorates diabetic nephropathy in rats: involvement of AMPK/SIRT1/autophagy pathway

Objective This study aimed to demonstrate the protective effect of beta-carotene against STZ-induced DN in rats and explore the possible underlying mechanisms that may have mediated such condition. Material and Methods Wistar rats were allocated into four groups. Normal group received distilled water for 3 weeks. The other three groups were rendered diabetic by an intraperitoneal dose of STZ (50 mg/kg), 48 h later, group 2: received the vehicle and served as control, groups (3 &4) received orally beta-carotene in doses of 10 and 20 mg/kg, respectively for 3 weeks. Then serum and renal tissue were collected for biochemical, molecular, immunohistopathological, and histopathological examination. Results Beta-carotene ameliorated the reduction in body weight, reduced blood glucose, elevated serum insulin, reduced blood urea nitrogen, and serum creatinine levels. Beta-carotene elevated phosphorylated 5′ adenosine monophosphate-activated protein kinase (p-AMPK)/AMPK, alleviated phosphorylated mammalian target of rapamycin (p-mTOR)/mTOR, reduced interleukin 1 beta (IL-1β), increased Beclin 1, LC3II/LC3I, and reduced p62 renal contents. Moreover, it elevated renal SIRT1 gene expression and reduced renal tumor necrosis factor-alpha (TNF-α) and caspase-3 protein expressions. Conclusion Beta-carotene exerted renoprotective effect against STZ-induced DN and histopathological alterations through alleviating hyperglycemia, attenuating inflammation, activating AMPK/SIRT1/autophagy pathway, and combating apoptosis.

Resistance Training in the Heat: Mechanisms of Hypertrophy and Performance Enhancement.

Pryor, JL, Sweet, D, Rosbrook, P, Qiao, J, Hess, HW, and Looney, DP. Resistance training in the heat: Mechanisms of hypertrophy and performance enhancement. J Strength Cond Res 38(7): 1350-1357, 2024-The addition of heat stress to resistance exercise or heated resistance exercise (HRE) is growing in popularity as emerging evidence indicates altered neuromuscular function and an amplification of several mechanistic targets of protein synthesis. Studies demonstrating increased protein synthesis activity have shown temperature-dependent mammalian target of rapamycin phosphorylation, supplemental calcium release, augmented heat shock protein expression, and altered immune and hormone activity. These intriguing observations have largely stemmed from myotube, isolated muscle fiber, or rodent models using passive heating alone or in combination with immobilization or injury models. A growing number of translational studies in humans show comparable results employing local tissue or whole-body heat with and without resistance exercise. While few, these translational studies are immensely valuable as they are most applicable to sport and exercise. As such, this brief narrative review aims to discuss evidence primarily from human HRE studies detailing the neuromuscular, hormonal, and molecular responses to HRE and subsequent strength and hypertrophy adaptations. Much remains unknown in this exciting new area of inquiry from both a mechanistic and functional perspective warranting continued research.

Inhibition of cc chemokine receptor 10 ameliorates osteoarthritis via inhibition of the phosphoinositide-3-kinase/Akt/mammalian target of rapamycin pathway

BackgroundOsteoarthritis (OA) is a joint disease characterized by inflammation and progressive cartilage degradation. Chondrocyte apoptosis is the most common pathological feature of OA. Interleukin-1β (IL-1β), a major inflammatory cytokine that promotes cartilage degradation in OA, often stimulates primary human chondrocytes in vitro to establish an in vitro OA model. Moreover, IL-1β is involved in OA pathogenesis by stimulating the phosphoinositide-3-kinase (PI3K)/Akt and mitogen-activated protein kinases pathways. The G-protein-coupled receptor, cc chemokine receptor 10 (CCR10), plays a vital role in the occurrence and development of various malignant tumors. However, the mechanism underlying the role of CCR10 in the pathogenesis of OA remains unclear. We aimed to evaluate the protective effect of CCR10 on IL-1β-stimulated CHON-001 cells and elucidate the underlying mechanism.MethodsThe CHON-001 cells were transfected with a control small interfering RNA (siRNA) or CCR10-siRNA for 24 h, and stimulated with 10 ng/mL IL-1β for 12 h to construct an OA model in vitro. The levels of CCR10, cleaved-caspase-3, MMP-3, MMP-13, Collagen II, Aggrecan, p-PI3K, PI3K, p-Akt, Akt, phosphorylated-mammalian target of rapamycin (p-mTOR), and mTOR were detected using quantitative reverse transcription polymerase chain reaction and western blotting. Viability, cytotoxicity, and apoptosis of CHON-001 cells were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, lactate dehydrogenase assay (LDH), and flow cytometry analysis, respectively. Inflammatory cytokines (TNF-α, IL-6, and IL-8) were assessed using enzyme-linked immunosorbent assay.ResultsLevel of CCR10 was substantially higher in the IL-1β-stimulated CHON-001 cells than that in the control group, whereas CCR10 was down-regulated in the CCR10-siRNA transfected CHON-001 cells compared to that in the control-siRNA group. Notably, CCR10 inhibition alleviated IL-1β-induced inflammatory injury in the CHON-001 cells, as verified by enhanced cell viability, inhibited LDH release, reduced apoptotic cells, and cleaved-caspase-3 expression. Meanwhile, IL-1β induced the release of tumor necrosis factor alpha, IL-6, and IL-8, increase of MMP-3 and MMP-13, and decrease of Collagen II and Aggrecan in the CHON-001 cells, which were reversed by CCR10-siRNA. However, these effects were reversed upon PI3K agonist 740Y-P treatment. Further, IL-1β-induced PI3K/Akt/mTOR signaling pathway activation was inhibited by CCR10-siRNA, which was increased by 740Y-P treatment.ConclusionInhibition of CCR10 alleviates IL-1β-induced chondrocytes injury via PI3K/Akt/mTOR pathway inhibition, suggesting that CCR10 might be a promising target for novel OA therapeutic strategies.

Identification of Reduced mTOR T1262 Phosphorylation as a Novel Mechanism and Therapeutic Target of Apoptosis in Senescent Cardiomyocytes: Aging and Cardiomyocyte Apoptosis.

The mechanisms through which aging increases heart injury remain partially understood. Protein phosphorylation plays a critical regulatory role in cell survival and death. Using an unbiased phosphoproteomics approach, we aimed to identify the proteins whose phosphorylation could be causatively related to aging-related cardiomyocyte apoptosis and elucidate the underlying mechanisms. Comparative phosphoproteomics was conducted on cardiac tissues obtained from young (8 weeks) and aged (24 months) mice. Our findings revealed that the Mammalian Target of Rapamycin phosphorylation at T1262 (mTORT1262) was reduced in the aging heart. Immunohistochemical and Western blot analyses confirmed these findings in aging myocardia and D-galactose-induced senescent AC16 cardiomyocytes. In hypoxia/reoxygenation cardiomyocytes, mTORT1262 phosphorylation deficiency (mTORT1262A, lentivirus-mediated transfection) inhibited AKT1, suppressed NF-κB, activated FOXO1/3a signaling, and ultimately exacerbated apoptosis. Conversely, mTORT1262 pseudophosphorylation (mTORT1262E) exhibited opposite effects. Through bioinformatics and CO-IP, purinergic receptor P2X4 (P2X4R) was found to be the possible receptor responsible for mTORT1262 phosphorylation. Knockdown of P2X4R increased apoptosis, whereas its overexpression decreased it. In senescent cardiomyocytes, P2X4R expression and mTORT1262 and AKT1S473 phosphorylation were reduced, NF-κB signaling was suppressed, and FOXO1/3a signaling was activated. We demonstrated that P2X4R downregulation and the subsequent reduction of mTORT1262 phosphorylation is a novel mechanism contributing to cardiomyocyte apoptosis in aging hearts. The P2X4R-mTOR-AKT1 signaling pathway represents a potential therapeutic target against accelerated cardiac injury in aging.

Melittin induces autophagy to alleviate chronic renal failure in 5/6-nephrectomized rats and angiotensin II-induced damage in podocytes.

Chronic renal failure (CRF) is a complex pathological condition that lacks a cure. Certain Chinese medicines, such as melittin, a major component in bee venom, have shown efficacy in treating CRF patients. On the other hand, the mechanisms underlying the therapeutic effects of melittin are unclear. A 5/6 nephrectomy model (5/6 Nx) of renal failure was established on rats for in vivo assays, and mouse podocyte clone 5 (MPC5) mouse podocyte cells were treated with angiotensin II (AngII) to establish an in vitro podocyte damage model. The 24-h urine protein, serum creatinine, and blood urea nitrogen levels were evaluated after one, 2, and 4 weeks. Hematoxylin and eosin staining, Masson staining, and periodic acid-Schiff staining were used to examine the pathological changes in kidney tissues. A cell counting kit 8 assay was used to assess the cell viability. Reverse transcription polymerase chain reaction and Western blot were used to assess the mRNA and protein levels in the cells, respectively. In the rat 5/6 Nx, melittin reduced the 24-h urinary protein excretion and the serum creatinine and blood urea nitrogen levels. Furthermore, the renal pathology was improved in the melittin-treated 5/6 Nx rats. Melittin promoted podocin, nephrin, Beclin 1, and the LC3II/LC3I ratio and inhibited phosphorylated mammalian target of rapamycin (mTOR)/mTOR in 5/6 Nx-induced rats and AngII-induced MPC5 mouse podocyte cells. Moreover, inhibiting autophagy with 3-MA weakened the effects of melittin on podocin, nephrin, and the LC3II/LC3I ratio in podocytes. Melittin may offer protection against kidney injury, probably by regulating podocyte autophagy. These results provide the theoretical basis for applying melittin in CRF therapy.

SKI-349, a Sphingosine Kinases 1/2 Inhibitor, Suppresses Cell Viability, Invasion, and AKT/mTOR Signaling Pathway, and Shows Synergistic Cytotoxic Effects with Sorafenib in Hepatocellular Carcinoma.

SKI-349 is a novel sphingosine kinases (SPHK) inhibitor with anti-tumor effects. This study aimed to assess the effect of SKI-349 on cell biological behaviors, downstream pathways, and its synergistic effect with sorafenib in hepatocellular carcinoma (HCC). HCC cell lines (Huh7 and Hep3B) were treated with SKI-349 at concentrations of 1, 2, 4, or 8 μM. Then, SPHK1/2 activity, cell viability, proliferation, apoptosis, invasion, and protein expressions of phosphorylated-protein kinase B (p-AKT), AKT, phosphorylated-mammalian target of rapamycin (p-mTOR) and mTOR were detected. Combination index values of SKI-349 (0, 1, 2, 4, or 8 μM) and sorafenib (0, 2.5, 5, 10, or 20 μM) were calculated. SKI-349 decreased the relative SPHK1 and SPHK2 activity compared with blank control in a dose-dependent manner in the Huh7 and Hep3B cell lines. Meanwhile, SKI-349 reduced cell viability, 5-ethynyl-2'-deoxyuridine (EdU) positive cells, and invasive cells, while it increased apoptotic cells compared to blank control in a dose-dependent manner in Huh7 and Hep3B cell lines. Based on the western blot assay, SKI-349 decreased the ratio of p-AKT to AKT and that of p-mTOR to mTOR compared with blank control in a dose-dependent manner in the Huh7 and Hep3B cell lines. Additionally, SKI-349 combined with sorafenib declined cell viability with concentration gradient effects compared to SKI-349 sole treatment, and they had synergistic cytotoxic effects in Huh7 and Hep3B cell lines. SKI-349 suppresses SPHK1 and SPHK2 activity, cell viability, invasion, and AKT/mTOR signaling pathway, as well as exhibits a synergistic cytotoxic effect with sorafenib in HCC.

Silibinin Alleviates Lipopolysaccharide Induced Inflammation in Porcine Mammary Epithelial Cells via mTOR/NF-κB Signaling Pathway.

Inflammatory responses reduce milk production in lactating sow. Silymarin (Silibinin is main component) reduces the inflammatory reaction and increases milk yield in lactating sow in the previous study. In the present study, silibinin may be a previously unrecognized nutrients in inflammatory resolution in porcine mammary epithelial cells (PMECs) is hypothesized. PMECs are treated with or without lipopolysaccharide (LPS) in the absence or presence of silibinin to test cell viability, cell cycle, cell apoptosis, cellular inflammatory factors, and signaling protein phosphorylation and expression. Silibinin promotes the proliferation of PMEC independent of the estrogen pathway. In LPS-induced damage of PMECs, silibinin protects cell proliferation, as well as reduced cell apoptosis. Silibinin reverses the LPS-induced increase in tumor necrosis factor-alpha (TNF-α) expression compared with control. In addition, silibinin accentuates the LPS-induced decrease in the key proteins phosphorylated-ribosomal protein S6 (p-S6) and phosphorylated-mammalian target of rapamycin (p-mTOR) of the mammalian target of rapamycin (mTOR) signaling pathway. Furthermore, silibinin reverses the increase in phosphorylated-nuclear factor-kappa B p65 (p-NF-κB p65), phosphorylated-Ikappab-alpha (p-IκB-α), and phosphorylated-Mitogen-activated protein kinase p38 (p-MAPK p38) expression in LPS-induced damage in PMECs. This study highlights silibinin-mTOR/NF-κB axis plays an important role in the control of inflammation in PMECs, and suggests that silibinin may be an effective dietary strategy to alleviate the inflammatory response in lactating sow.

Effect of lentivirus-mediated peroxiredoxins 6 gene silencing on the phenotype of human gastric cancer BGC-823 cells.

Peroxiredoxins (PRDX6) regulates the occurrence and progression of cancer. The aim of this study is to investigate the effect of PRDX6 knockdown on the biological behavior of human gastric cancer cell line BGC-823 cells. Research article. The differential expression of PRDX6 in gastric cancer and normal gastric tissues was tested by immunohistochemistry. Ribonucleic acid plasmid of PRDX6 gene was packaged using a lentivirus, and BGC-823 cells were transfected with the lentivirus to obtain a BGC-823 cell line in which the expression of PRDX6 was stably silenced. The proliferation activity of BGC-823 cells was detected using the cell counting kit-8 method. The effect of PRDX6 on the migration and invasion of BGC-823 cells was evaluated using the scratch test and Transwell assay, and the expression of related proteins was detected by western blot. The expression of PRDX6 in gastric cancer was significantly increased (P < 0.05). Compared with those in the untransfected and negative control groups. The proliferation, migration, and invasion of gastric cancer BGC-823 cells were significantly inhibited, and the apoptotic rates were significantly increased in the lentivirus-transfected (short hairpin-PRDX6) group. Western blot analysis showed that the expression of Bax protein increased, whereas that of proliferating cell nuclear antigen, Bcl-2, PI3K, phospho (p-Akt), and phosphorylated-mammalian target of rapamycin (mTOR) decreased significantly compared with that in WT and vector groups (P < 0.05). The knockdown of PRDX6 gene expression in BGC-823 cells can inhibit the proliferation, migration, and invasion of gastric cancer cells and promote apoptosis, thereby affecting gastric cancer cells.

Autophagy protects against high uric acid-induced hepatic insulin resistance

Uric acid (UA), the end-product of purine metabolism, is closely related to hepatic insulin resistance (IR). Autophagy is a conserved intracellular degradation process maintaining cellular homeostasis. Autophagy plays a protective role in obesity-related hepatic IR, but whether it occurs in high uric acid (HUA)-induced hepatic IR is unclear. In this study, spontaneously elevated UA level induced hepatic IR and facilitated hepatic autophagy degradation in uricase knockout (Uox-/-) mice. In vitro, HepG2 cells stimulated with HUA medium showed decreased glucose uptake and inhibition of insulin signaling pathways, concomitant with activation of autophagy, as manifested by increased conversion of LC3B–I to –II. Rapamycin, the autophagy activator, alleviated but the autophagy inhibitor trimethyl adenine (3-MA) aggravated HUA-induced IR in HepG2 cells. Similarly, rapamycin ameliorated and 3-MA worsened HUA-induced blood glucose level and hepatic IR in Uox-/- mice. Mechanistically, HUA enhanced AMPKα phosphorylation (p-AMPKα) and inhibited mammalian target of rapamycin phosphorylation (p-mTOR) in HepG2 cells. The levels of p-AMPKα and LC3B-II/I were downregulated in HepG2 cells transfected with small interfering RNA (siRNA) against AMPKα, which suggests that the AMPKα–mTOR pathway was involved in HUA-induced autophagy. Antioxidant N-acetyl-L-cysteine reversed elevated reactive oxygen species levels induced by HUA in HepG2 cells, and AMPKα level was also inhibited, which suggests that AMPKα activation may be derived from reactive oxygen species. Collectively, these findings demonstrate that HUA increased hepatic autophagy, and autophagy activation plays a protective role in hepatic IR, which may suggest a potential therapeutic target for hepatic IR derived from HUA.

High L-Type Amino Acid Transporter 1 Levels Are Associated with Chemotherapeutic Resistance in Gastric Cancer Patients

Introduction: We investigated whether the expression of L-type amino acid transporter 1 (LAT-1) in clinical gastric cancer (GC) patients could predict patient therapeutic response to postoperative adjuvant chemotherapy. Methods: Immunohistochemistry was used to investigate LAT-1, CD98, and phosphorylated-mammalian target of rapamycin (p-mTOR) expression in 111 GC patients. To clarify whether LAT-1 influences the therapeutic effects of chemotherapy, the correlation between disease-free survival rates and LAT-1 was determined in 2 groups: 59 patients who did not undergo postoperative adjuvant chemotherapy and 52 patients who did undergo postoperative adjuvant chemotherapy. Results: LAT-1 was significantly correlated with CD98 and p-mTOR expressions. We did not find any statistically significant correlation between LAT-1 and recurrence in the nontreated group. In contrast, a significant association was found between LAT-1 expression and disease-free survival in the chemotherapy group. Moreover, multivariate regression analysis demonstrated that LAT-1 was an independent predictor of disease-free survival in the postoperative adjuvant chemotherapy group (p = 0.012). Conclusion: Our findings demonstrate that LAT-1 is a useful predictive marker for a successful postoperative adjuvant chemotherapy treatment.

R (−)-methoxetamine exerts rapid and sustained antidepressant effects and fewer behavioral side effects relative to S (+)-methoxetamine

The newfound antidepressant efficacy of ketamine has provided opportunities for the development of new-generation, rapid-acting, glutamate-based antidepressants. We previously identified that methoxetamine (MXE), a ketamine analog, and an N-Methyl-d-aspartate (NMDA) receptor antagonist, produced rapid and sustained antidepressant effects in mice. MXE (R, S (±)-MXE) is a racemic mixture containing equal parts of S (+)-MXE and R (−)-MXE. However, studies have yet to investigate the antidepressant effects of its enantiomers. Here, we examined the potential antidepressant properties and behavioral side effects of S- and R-MXE in mice. Both S- and R-MXE showed significant NMDA receptor affinity and appreciable inhibitory activity on serotonin transporter. Also, S- and R-MXE (10 mg kg−1) exerted antidepressant effects and increased gamma waves (electroencephalography) but were inhibited by NBQX (an AMPA receptor antagonist). Subsequently, they increased mammalian target of rapamycin phosphorylation and AMPA receptor subunits GluA1 and GluA2 protein levels in the hippocampus or prefrontal cortex. Furthermore, they increased 5HT2a and 5HT2c receptor mRNA levels in the prefrontal cortex, with their antidepressant effects inhibited by ketanserin (a 5HT2a/c receptor antagonist). Taken together, S-MXE and R-MXE elicit antidepressant effects that are probably mediated via glutamatergic and serotonergic mechanisms. Unlike S-MXE, R-MXE did not induce prepulse inhibition deficits, hyperlocomotion, conditioned place preference, and locomotor sensitization, although it acutely altered motor coordination. This suggests that R-MXE induces fewer behavioral side effects and is a safer antidepressant than S-MXE. Overall, this study provides significant implications for future research on the next generation of rapid-acting, glutamate-based antidepressant drugs.

Maternal yeast-based nucleotide supplementation decreased stillbirth by regulating nutrient metabolism.

As an enzymatic product of yeast, yeast-based nucleotide (YN) is rich in nucleotides. To test the effects of maternal dietary supplementation with YN during late pregnancy on placental nutrient transport and nutrient metabolism in neonatal piglets, 64 pregnant sows (day 85 ± 3) were assigned into two groups: (i) control (CON) and (ii) treatment (YN; 4g kg-1 ). Blood, placenta and liver samples of neonates during delivery were collected. The results showed that maternal YN supplementation decreased stillbirth rate and intra-uterine growth restriction rate (P < 0.05). In addition, maternal YN supplementation increased total serum protein, albumin and total cholesterol (P < 0.05). Furthermore, in neonatal piglets in the YN group, both serum amino acidand nucleotide profiles were affected, as well as liver amino acid, and fatty acid profiles were regulated (P < 0.05). Moreover, maternal YN supplementation increased liver mRNA expression of SLC28A3, SLC29A1, SLC29A2, PC, PCK1, FBP1, SREBP1c, HSL and CYP7a1 of neonatal piglets (P < 0.05). Meanwhile, there was a decrease in placental gene expression of EAAT2, EAAT3, LAT1 and PAT1, as well as lower protein expression of peroxisome proliferator-activated receptor (PPAR)γ, AKT, phosphorylated-AKT, phosphorylated-mammalian target of rapamycin (mTOR) and Raptor, in the YN group (P < 0.05). Taken together, these results indicate that maternal YN supplementation regulates placental nutrient transport by regulating the mTOR complex 1-PPAR pathway, and affects the liver metabolism of nucleotides, amino acids and fatty acids in neonatal piglets, thereby improving the reproductive performance of sow to a certain extent. © 2020 Society of Chemical Industry.

Arbutin suppresses osteosarcoma progression via miR-338-3p/MTHFD1L and inactivation of the AKT/mTOR pathway.

Arbutin, a glycoside extracted from the plant Arctostaphylosuva-ursi, has been previously reported to possess antioxidant, anti-inflammatory and anticancer effects. Here, we investigated whether arbutin affects the proliferation of the cells of the osteosarcoma (OS) cell lines MG-63 and SW1353. Arbutin suppressed OS cell viability in a dose- and time-dependent manner, as shown by Cell Counting Kit-8 assay. Furthermore, arbutin exposure decreased the protein levels of MTHFD1L, CCND1 and phosphorylated-protein kinase B (AKT)/phosphorylated-mammalian target of rapamycin (mTOR). Potential upstream miRNAs of MTHFD1L were predicted using TargetScan, PICTAR5, miRanda and miRWalk. We performed luciferase activity assays to show that miR-338-3p directly targets and negatively regulates the expression of MTHFD1L. Knockdown of miR-338-3p promoted cell invasion, migration and proliferation in arbutin-treated OS cells via MTHFD1L. In summary, our data suggest that arbutin inhibits OS cell proliferation, migration and invasion via miR-338-3p/MTHFD1L and by inactivating the AKT/mTOR pathway.

Atorvastatin Enhances Foam Cell Lipophagy and Promotes Cholesterol Efflux Through the AMP-Activated Protein Kinase/Mammalian Target of Rapamycin Pathway.

Foam cells are the main pathological components of atherosclerosis. Therapies reducing foam cell formation can effectively prevent atherosclerotic diseases and cardiovascular events. Beyond lowering plasma cholesterol levels, the pleiotropic functions of statins in atherosclerosis have not been fully elucidated. In the present study, atorvastatin reduced cholesterol content and increased cholesterol efflux from foam cells in a concentration-dependent manner. Atorvastatin (10 μM) inhibited foam cell formation within 48 hours. Furthermore, we found that atorvastatin inhibited foam cell formation by promoting lipophagy, which was manifested by increased autophagy-related gene 5 (Atg5) expression, elevated ratio of microtubule-associated protein1 light chain 3 (LC3) II to LC3I, reduced p62 expression, and increased LC3 and lipid droplets colocalization in foam cells treated with atorvastatin. The autophagy inducer, rapamycin (Rap), did not increase the lipophagy enhancement effect of atorvastatin, but the autophagy inhibitor, 3-methyladenine, suppressed the effect of atorvastatin on Atg5 expression and the LC3II/LC3I ratio, as well as the increased p62 expression, suppressed lipophagy, attenuated cholesterol efflux and increased cholesterol content in foam cells. Further analysis revealed that atorvastatin promoted lipophagy by upregulating adenosine 5'-monophosphate-activated protein kinase (AMPK) phosphorylation, and downregulating mammalian target of rapamycin phosphorylation, whereas the AMPK inhibiter, compound C, attenuated these effects. In conclusion, atorvastatin reduced lipid accumulation and promoted cholesterol efflux by enhancing lipophagy in foam cells and thereby inhibited foam cell formation. The enhanced lipophagy of foam cells was exerted through the AMPK/mammalian target of rapamycin signaling pathway.

Effects of hypoxia/reoxygenation on apoptosis, autophagy and pyroptosis in H9C2 cardiomyocytes

Objective To investigate the effects of hypoxia/reoxygenation (HR) on apoptosis, autophagy and pyroptosis in H9C2 cardiomyocytes. Methods The cultured H9C2 cardiomyocytes were divided into two groups according to the random number table method: a normal control (Ctrl) group and an HR group. The H9C2 cardiomyocytes in the HR group were deprived of oxygen and glucose for 8 h in a hypoxic incubator, followed by reoxygenation for 12 h. Cell damage was assessed through detection of lactate dehy-drogenase (LDH) content in culture medium and determination of cell viability by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) method (n=6). Apoptosis related proteins [cysteinyl aspartate-specific proteinase (caspase)-3, B-cell lymphoma/leuke-mia 2 (Bcl-2) and Bcl-associate x protein (Bax)], autophagy related proteins [(light chain 3 (LC3) Ⅱ/Ⅰ , p62, Beclin-1, and phosphory-lated mammalian target of rapamycin (p-mTOR)]and pyroptosis related proteins [(nod-like receptor pyrin domain 3 (NLRP3), apoptosis associated speck-like protein (ASC), caspase-1p20, interleukin (IL)-1β, and IL-18]were detected by Western blot (n=9). Cell apopto-sis, autophagy and pyroptosis were further assessed by immunofluorescence staining (n=6) Results Compared with the Ctrl group, the HR group presented reduced viability of H9C2 cardiomyocytes (P<0.05), increased LDH release (P<0.05), and up-regulated expres-sion of activated caspase-3 and Bax (P<0.05), as well as decreased expression of Bcl-2 (P<0.05). TUNEL staining showed that apoptosis significantly was enhanced in the cells after HR (P<0.05). These results indicated that apoptosis and cell damage were enhanced after HR. In contrast, the expression of p-mTOR and p62 increased (P<0.05), but the expression of LC3 Ⅱ/Ⅰ and Beclin-1 protein decreased(P<0.05). The immunofluorescence staining of LC3 Ⅱ/Ⅰ showed that the number of autophagosome within the cells increased after HR(P<0.05), indicating that autophagy in H9C2 myocardial cells was significantly inhibited. Meanwhile, the expression of NLRP3 inflamma-some, ASC and caspase-1p20 protein was remarkably up-regulated (P<0.05), and the expression of activated inflammatory cellular factors IL-1β and IL-18 increased (P<0.05), suggesting that NLRP3 inflammasomes were activated and pyroptosis enhanced after HR. Conclusions Autophagy is inhibited in H9C2 cardiomyocytes during HR injury, but pyroptosis and apoptosis are enhanced. Key words: Autophagy; Apoptosis; Pyroptosis; Cardiomyocytes; Hypoxia-reoxygenation injury

Annexin V expression on CD4+ T cells with regulatory function

Regulatory T (Treg) cells induce immunologic tolerance by suppressing effector functions of conventional lymphocytes in the periphery. On the other hand, immune silencing is mediated by recognition of phosphatidylserine (PS) on apoptotic cells by phagocytes. Here we describe expression of the PS-binding protein Annexin V (ANXA5) in CD4+ CD25hi Treg cells at the mRNA and protein levels. CD4+ ANXA5+ T cells constitute about 0·1%-0·6% of peripheral blood CD3+ T cells, exhibit co-expression of several Treg markers, such as Forkhead box P3, programmed cell death protein-1, cytotoxic T-lymphocyte antigen-4 and CD38. In vitro, ANXA5+ Treg cells showed enhanced adhesion to PS+ endothelial cells. Stimulated by anti-CD3 and PS+ syngeneic antigen-presenting cells CD4+ ANXA5+ T cells expanded in the absence of exogenous interleukin-2. CD4+ ANXA5+ T cells suppressed CD4+ ANXA5- T-cell proliferation and mammalian target of rapamycin phosphorylation, partially dependent on cell contact. CD4+ ANXA5+ T-cell-mediated suppression was allo-specific and accompanied by an increased production of anti-inflammatory mediators. In vivo, using a model of delayed type hypersensitivity, murine CD4+ ANXA5+ T cells inhibited T helper type 1 responses. In conclusion, we report for the first time expression of ANXA5 on a subset of Treg cells that might bridge classical regulatory Treg function with immune silencing.

Soluble epoxide hydrolase inhibitor, TUPS, attenuates isoproterenol/angiotensin II-induced cardiac hypertrophy through mammalian target of rapamycin-mediated autophagy inhibition.

To investigate the potential role and mechanism of TUPS, a soluble epoxide hydrolase inhibitor, in cardiac hypertrophy. Rat and H9C2 cell models of cardiac hypertrophy were induced by isoproterenol and angiotensin II, respectively, followed by TUPS treatment. The expression of hypertrophic markers, ANP and BNP, was determined by quantitative real-time PCR. The abundance of Beclin-1, LC3, p-AMPK and phosphorylated-mammalian target of rapamycin (p-mTOR) proteins was analysed by Western blot and immunohistocytology. Cell morphology and viability were evaluated by F-actin staining and MTS. H9C2 cells were transfected with GFP-LC3 to evaluate autophagy flux. TUPS significantly inhibited rat heart size, heart weight-to-body weight ratio, heart wall thickness, hypertrophic H9C2 cell swelling and viability suppression as well as the expression of ANP and BNP genes in hypertrophic models. In addition, autophagic markers Beclin-1 and LC3 were elevated in both cellular and animal models, which were suppressed by TUPS, with corresponding changes of autophagy flux. The abundance of p-AMPK was increased, while p-mTOR was decreased in hypertrophic cells, which were abolished by TUPS. Rapamycin decreased p-mTOR level, increased Beclin-1 and LC3 expression and induced cell size enlargement and cell viability inhibition in hypertrophic H9C2 cells treated with TUPS. TUPS inhibits cardiac hypertrophy by regulating mTOR/autophagy axis.

Metformin Enhances Autophagy and Provides Cardioprotection in δ-Sarcoglycan Deficiency-Induced Dilated Cardiomyopathy.

Metformin is a popular antidiabetic agent that is also used to treat heart failure patients with type 2 diabetes mellitus. Several reports suggest that metformin may also have cardioprotective effects in patients without diabetes mellitus. In the present study, we investigated the possible therapeutic effect of metformin in heart failure and its underlying molecular mechanisms using a δ-sarcoglycan-deficient mouse model of dilated cardiomyopathy. Thirty-two-week-old δ-sarcoglycan-deficient mice exhibiting established cardiomyopathy with extensive left ventricular dilatation and dysfunction were administered saline or metformin (200 mg/kg per day) for 4 weeks using osmotic mini-pumps. Metformin partially reversed the left ventricular dilatation (reverse remodeling) and significantly improved cardiac function. The hearts of metformin-treated mice showed less fibrosis, less cardiomyocyte hypertrophy, and fewer degenerative subcellular changes than saline-treated mice. These effects were accompanied by restored expression of the sarcomeric proteins myosin heavy chain and troponin I, and their transcription factor, GATA-4. Autophagy was enhanced in the hearts from metformin-treated mice, as indicated by increase of myocardial microtubule-associated protein-1 LC-3 (light chain 3)-II levels and LC3-II/-I ratios as well as levels of cathepsin D and ATP. In addition, increased numbers of autophagic vacuoles and lysosomes were accompanied increased AMP-activated protein kinase activity and suppression of mammalian target of rapamycin phosphorylation. Finally, autophagic flux assays using short-term chloroquine treatment revealed that autophagy was activated in δ-sarcoglycan-deficient hearts and was further augmented by metformin treatment. Metformin is a beneficial pharmacological tool that mitigates heart failure caused by δ-sarcoglycan deficiency in association with enhanced autophagy.

Anticancer activities of chalcone flavokawain B from Alpinia pricei Hayata in human lung adenocarcinoma (A549) cells via induction of reactive oxygen species-mediated apoptotic and autophagic cell death.

Chalcones found in fruits and vegetables have promising cancer chemopreventive properties. This study attempts to identify the anticancer efficacies of chalcone flavokawain B (FKB) in the rhizomes of Alpinia pricei Hayata by examining key molecular events in non-small-cell lung cancer (A549) cells. Our results indicated that in human A549 cells, FKB (0-15 μg/ml) decreases cell viability and colony formation, dysregulates the Bax:B-cell lymphoma 2 ratio and increases apoptotic DNA fragmentation. Mitochondrial (caspase-9/-3 and poly ADP ribose polymerase [PARP]) signaling was found to be involved in FKB-induced apoptosis. In addition, FKB-induced reactive oxygen species (ROS) generation, and N-acetylcysteine attenuated FKB-induced apoptotic cell death. Moreover, FKB triggered autophagy, as evidenced by the improved acidic vesicular organelle formation, lipidated light chain 3 (microtubule-related light chain 3) accumulation, and ATG7 expression and the decreasedmammalian target of rapamycin phosphorylation. Furthermore, FKB suppressed ROS-mediated ATG4B expression. Inhibiting autophagy using 3-methyladenine/chloroquine diminished FKB-induced cell death, indicating that autophagy is triggered as a death mechanism by FKB. In summary, FKB has a crucial role in the execution and propagation of ROS-mediated apoptotic and autophagic cell death of lung adenocarcinoma cells.

Effects of α-lipoic acid on chronic cerebrovascular hypoperfusion in an animal model of vascular dementia.

Given the aging population, the treatment of vascular dementia (VaD) is becoming increasingly important. The antioxidant α-lipoic acid (α-LA) protects against neurodegeneration in VaD, but the underlying mechanisms remain unclear. Hence, we aimed to identify the effects of α-LA on cognitive function following chronic cerebrovascular hypoperfusion in a VaD animal model. Mice were categorized into the sham, bilateral common carotid artery stenosis (BCAS), or BCAS + α-LA group. The BCAS + α-LA group was intraperitoneally injected (100 mg/kg) once daily with α-LA for 4 weeks after BCAS surgery, while the BCAS and sham groups were injected with saline. After the last injection, we examined cognitive function and exploration behavior using the Morris water maze. Mice brains were then harvested for Western blot analyses. The BCAS group, but not the BCAS + α-LA group, showed cognitive dysfunction in the Morris water maze. Apoptosis pathways involving poly (ADP-ribose) polymerase (PARP) cleavage, phosphorylated-mammalian target of rapamycin (p-mTOR), phosphorylated-3-phosphoinositide-dependent protein kinase-1, and phosphorylated-protein kinase B (p-AKT) were enhanced in the BCAS group than the α-LA group. The BCAS + α-LA group demonstrated less PARP cleavage and p-mTOR function than did the BCAS group. The activity of autophagy pathways involving LC3B was higher in the BCAS and BCAS + α-LA groups than the sham group, but there were no significant differences between the BCAS and BCAS + α-LA groups. In the BCAS rodent model, cognitive dysfunction and apoptosis mediated by the phosphatase and tensin homolog/AKT/mTOR pathway were observed in the hippocampus. However, acting on the mTOR pathway, α-LA improved cognitive function and led to hippocampal cell survival. Thus, α-LA may be useful for treating VaD.