The Effects of Six Weeks of Endurance Training and CGRP Inhibition on Nrf2 and AKT Expression in the Hippocampal Tissue of Male Wistar Rats.

Purpose: To study the effects of a six-week endurance training protocol and calcitonin gene-related peptide (CGRP) inhibition on the nuclear factor erythroid 2-related factor 2 (Nrf2) and protein kinase B (PKB) or AKT expression in the hippocampal tissue of male Wistar rats. Main Methods. Building on a controlled experimental design with a posttest, 28 healthy Wistar male rats were randomly assigned to four groups (n = 7 per group), including control, control+CGRP inhibition, endurance training, and endurance training+CGRP inhibition groups. The training groups were trained for six weeks. Rats in the CGRP inhibition group received CGRP receptor antagonist daily (0.25 mg/kg) via intravenous (IV) injection. The Nrf2 and AKT (PKB) expression was measured using the real-time PCR technique. Results: In the endurance training group, Nrf2 expression in the hippocampal tissue was increased significantly more than in other groups (P < 0.05). There was also a significant increase in the AKT expression in the endurance training group compared to the control group (P = 0.048) and in the endurance training+CGRP inhibition compared to the control group (P = 0.012). In addition, there was no significant relationship between AKT (PKB) and Nrf2 (r = -0.27, n = 28, P = 0.16). Conclusion: Endurance training alone has been able to increase Nrf2 and AKT (PKB) mRNA levels in the hippocampal tissue, considering that endurance training had no significant effect on AKT and Nrf2 expression after adding to CGRP inhibition.

Inhibitory Effect of Lotus Leaf-Enriched Flavonoid Extract on the Growth of HT-29 Colon Cancer Cells through the Expression of PI3K-Related Molecules.

Objectives: Lotus leaf is rich in flavonoids, and this study is aimed at examining the inhibitory effect of lotus leaf-enriched flavonoid extract (LLEFE) on HT-29 colon cancer cells through phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) expression regulation. Methods: Lotus leaves were extracted by ethanol and purified using FL-3 macroporous resin to create the LLEFE. HT-29 colon cancer cells were tested using various methods: their proliferation was observed by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, their survival status was observed by fluorescence staining, their oxidative stress level was observed by biochemical kits, and their mRNA expression was determined by quantitative polymerase chain reaction (qPCR) assay. Additionally, the composition of the flavonoids in lotus leaf was determined by HPLC. Results: The results showed that the proliferation of NCM460 normal human colon cells was not affected by 0-500 µg/mL LLEFE but the proliferation of HT-29 human colon cancer cells decreased. LLEFE increased the LDH level in an HT-29 colon cancer cell culture medium; also increased the superoxide dismutase (SOD), catalase (CAT) activities, and glutathione (GSH) level in HT-29 cells; and decreased the malondialdehyde (MDA) level. Further experimental results showed that LLEFE upregulated the expression of SOD1, CAT, and GSH mRNA and downregulated the expression of PI3K, Akt, and mammalian target of rapamycin (mTOR) in HT-29 cells. The high-performance liquid chromatography (HPLC) results showed that kaempferin, hyperoside, astragaloside, phloridzin, and quercetin were the main chemical constituents of lotus leaf. Conclusion: Lotus leaves contain functional flavonoids that inhibit the proliferation of HT-29 colon cancer cells and regulate the expression of PI3K/Akt through five important chemicals.

Propranolol inhibits cell viability and expression of the pro-tumorigenic proteins Akt, NF-ĸB, and VEGF in oral squamous cell carcinoma.

BACKGROUND: Propranolol (PPL) has been suggested as an option for the treatment of various types of cancer. However, data regarding its effectiveness against oral cancer are scarce. Thus, we aimed to evaluate the antitumor potential of PPL in oral squamous cell carcinoma (OSCC) in vitro. METHODS: OSCC cell lines, SCC-9, SCC-25, and Cal27, were treated with PPL at different times and concentrations. OSCC cells were treated with PPL alone or in combination with cisplatin (CDDP) or 5-fluorouracil (5-FU). Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of phosphorylated (p)-Akt, p-S6, p-PTEN, p-P65, and VEGF was verified by immunofluorescence. The migratory activity of OSCC cells was evaluated using a wound-healing assay. RESULTS: PPL reduced OSCC cell viability in a dose- and time-dependent manner. Concentrations above 300 µM, 110 µM, and 100 µM for SCC-9, Cal27, and SCC-25, respectively, significantly eliminated tumor cells. The combination of PPL with CDDP and 5-FU enhanced their antitumor effects. There was a modest difference between the use of the IC30 and IC50 of PPL in the combinatory options. PPL downregulated p-P65 NF-ĸB and VEGF expression in SCC-9 and Cal27 cells but not in SCC-25 cells. PPL inhibited the phosphorylation of Akt and s6 and increased the phosphorylation of PTEN in all OSCC cell lines studied. PPL inhibited OSCC cell migration after 24 h of treatment. CONCLUSION: PPL was effective against oral cancer cells and enhanced standard-of-care. PPL inhibited cell viability and the expression of pAkt, NF-ĸB, and VEGF.

Viral expression of constitutively active AKT3 induces CST axonal sprouting and regeneration, but also promotes seizures.

Increasing the intrinsic growth potential of neurons after injury has repeatedly been shown to promote some level of axonal regeneration in rodent models. One of the most studied pathways involves the activation of the PI3K/AKT/mTOR pathways, primarily by reducing the levels of PTEN, a negative regulator of PI3K. Likewise, activation of signal transducer and activator of transcription 3 (STAT3) has previously been shown to boost axonal regeneration and sprouting within the injured nervous system. Here, we examined the regeneration of the corticospinal tract (CST) after cortical expression of constitutively active (ca) Akt3 and STAT3, both separately and in combination. Overexpression of caAkt3 induced regeneration of CST axons past the injury site independent of caSTAT3 overexpression. STAT3 demonstrated improved axon sprouting compared to controls and contributed to a synergistic improvement in effects when combined with Akt3 but failed to promote axonal regeneration as an individual therapy. Despite showing impressive axonal regeneration, animals expressing Akt3 failed to show any functional improvement and deteriorated with time. During this period, we observed progressive Akt3 dose-dependent increase in behavioral seizures. Histology revealed increased phosphorylation of ribosomal S6 protein within the unilateral cortex, increased neuronal size, microglia activation and hemispheric enlargement (hemimegalencephaly).

Regulation of AKT Signaling in Mouse Uterus.

17ß-estradiol (E2) treatment of ovariectomized adult mice stimulates the uterine PI3K-AKT signaling pathway and epithelial proliferation through estrogen receptor 1 (ESR1). However, epithelial proliferation occurs independently of E2/ESR1 signaling in neonatal uteri. Similarly, estrogen-independent uterine epithelial proliferation is seen in adulthood in mice lacking Ezh2, critical for histone methylation, and in wild-type (WT) mice treated neonatally with estrogen. The role of AKT in estrogen-independent uterine epithelial proliferation was the focus of this study. Expression of the catalytically active phosphorylated form of AKT (p-AKT) and epithelial proliferation were high in estrogen receptor 1 knockout and WT mice at postnatal day 6, when E2 concentrations were low, indicating that neither ESR1 nor E2 are essential for p-AKT expression and epithelial proliferation in these mice. However, p-AKT levels and proliferation remained estrogen responsive in preweaning WT mice. Expression of p-AKT and proliferation were both high in uterine luminal epithelium of mice estrogenized neonatally and ovariectomized during adulthood. Increased expression of phosphorylated (inactive) EZH2 was also observed. Consistent with this, Ezh2 conditional knockout mice show ovary-independent uterine epithelial proliferation and high epithelial p-AKT. Thus, adult p-AKT expression is constitutive and E2/ESR1 independent in both model systems. Finally, E2-induced p-AKT expression and normal uterine proliferation did not occur in mice lacking membrane (m)ESR1, indicating a key role for membrane ESR1 in AKT activation. These findings emphasize the importance of AKT activation in promoting uterine epithelial proliferation even when that proliferation is not E2/ESR1 dependent and further indicate that p-AKT can be uncoupled from E2/ESR1 signaling in several experimental scenarios.

Aerobic exercise and resistance exercise alleviate skeletal muscle atrophy through IGF-1/IGF-1R-PI3K/Akt pathway in mice with myocardial infarction.

Myocardial infarction (MI)-induced heart failure (HF) is commonly accompanied with profound effects on skeletal muscle. With the process of MI-induced HF, perturbations in skeletal muscle contribute to muscle atrophy. Exercise is viewed as a feasible strategy to prevent muscle atrophy. The aims of this study were to investigate whether exercise could alleviate MI-induced skeletal muscle atrophy via insulin-like growth factor 1 (IGF-1) pathway in mice. Male C57/BL6 mice were used to establish the MI model and were divided into three groups: sedentary MI group (MI), MI with aerobic exercise group, and MI with resistance exercise group; sham-operated group was used as control. Exercise-trained animals were subjected to 4 wk of aerobic exercise (AE) or resistance exercise (RE). Cardiac function, muscle weight, myofiber size, levels of IGF-1 signaling and proteins related to myogenesis, protein synthesis, and degradation and apoptosis in gastrocnemius muscle were detected. H2O2-treated C2C12 cells were intervened with recombinant human IGF-1, IGF-1 receptor (IGF-1R) inhibitor NVP-AEW541, and PI3K inhibitor LY294002 to explore the mechanism. Exercises upregulated the IGF-1/IGF-1R-phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling; increased the expressions of Pax7, myogenic regulatory factors (MRFs), and protein synthesis; and reduced protein degradation and cell apoptosis in MI mice. In vitro, IGF-1 upregulated the levels of Pax7, MRFs, mTOR, and P70S6K; reduced MuRF1 and MAFbx; and inhibited cell apoptosis via IGF-1R-PI3K/Akt pathway. AE and RE, safely and effectively, alleviate skeletal muscle atrophy by regulating the levels of myogenesis, protein degradation, and cell apoptosis in mice with MI via activating IGF-1/IGF-1R-PI3K/Akt signaling pathway.

Phosphoinositide-3-Kinase/Akt-Endothelial Nitric Oxide Synthase Signaling Pathway Mediates the Neuroprotective Effect of Sevoflurane Postconditioning in a Rat Model of Hemorrhagic Shock and Resuscitation.

BACKGROUND: Although extensive reports have demonstrated the neuroprotection of sevoflurane postconditioning in cases of focal and global cerebral ischemia/reperfusion, the underlying mechanisms are not completely elucidated. This study investigated whether this effect is related to endothelial nitric oxide synthase (eNOS) and mediated by the phosphoinositide-3-kinase pathway in a rat model of hemorrhagic shock and resuscitation. METHODS: Adult male Sprague Dawley rats were subjected to hemorrhagic shock for 60 minutes and then resuscitation for 30 minutes in experimental groups. Sevoflurane postconditioning was performed at the beginning of resuscitation to completion. At 24 hours after resuscitation, the brain infarct volume was evaluated by 2,3,5-triphenyltetrazolium chloride staining. The neuronal morphological changes and apoptosis were determined by hematoxylin and eosin staining and immunohistochemistry analysis, respectively. The activity of phosphorylated Akt and eNOS was evaluated by Western blot analysis. RESULTS: Brain injuries such as the cerebral infarct volume and pathological neuronal changes as well as cell apoptosis were observed in the hippocampus after hemorrhagic shock and resuscitation. Postconditioning with 2.4% sevoflurane significantly attenuated brain injuries. Wortmannin prevented the improvements of neuronal characteristics elicited by sevoflurane postconditioning as well as the hyperactivity of eNOS and phosphorylated Akt. CONCLUSIONS: Sevoflurane postconditioning could attenuate brain injury induced by hemorrhagic shock and resuscitation, and this neuroprotective effect may be partly by upregulation of eNOS through the phosphoinositide-3-kinase/Akt signaling pathway.

Endogenous regulation of the Akt pathway by the aryl hydrocarbon receptor (AhR) in lung fibroblasts.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known to mediate toxic responses to dioxin. However, the role of the AhR in the regulation of cellular physiology has only recently been appreciated, including its ability to control cell cycle progression and apoptosis by unknown mechanisms. We hypothesized that the AhR enhances the activation of the AKT serine/threonine kinase (Akt) pathway to promote cell survival. Utilizing AhR knock-out (Ahr-/-) and wild-type (Ahr+/+) mouse lung fibroblasts (MLFs), we found that Ahr-/- MLFs have significantly higher basal Akt phosphorylation but that AhR did not affect Akt phosphorylation in MLFs exposed to growth factors or AhR ligands. Basal Akt phosphorylation was dependent on PI3K but was unaffected by changes in intracellular glutathione (GSH) or p85α. There was no significant decrease in cell viability in Ahr-/- MLFs treated with LY294002-a PI3K inhibitor-although LY294002 did attenuate MTT reduction, indicating an affect on mitochondrial function. Using a mass spectrometry (MS)-based approach, we identified several proteins that were differentially phosphorylated in the Ahr-/- MLFs compared to control cells, including proteins involved in the regulation of extracellular matrix (ECM), focal adhesion, cytoskeleton remodeling and mitochondrial function. In conclusion, Ahr ablation increased basal Akt phosphorylation in MLFs. Our results indicate that AhR may modulate the phosphorylation of a variety of novel proteins not previously identified as AhR targets, findings that help advance our understanding of the endogenous functions of AhR.

Tumor necrosis factor-α induced protein 8 (TNFAIP8/TIPE) family is differentially expressed in oral cancer and regulates tumorigenesis through Akt/mTOR/STAT3 signaling cascade.

BACKGROUND: Highest incidence of oral cancer is reported in India with reduced survival rate in the advanced stages due to lack of effective biomarkers. Therefore, it is essential to develop novel biomarkers for the better management of this disease. In the current study, TNFAIP8/TIPE protein family comprising of four proteins is explored for its role in oral cancer. METHODS: IHC analysis of oral cancer TMA and Western blot analysis of tobacco treated oral cancer cells were performed to determine the differential expression of TIPE proteins in oral cancer. Further, CRISPR/Cas9-mediated gene editing was done to generate TIPE proteins' knockouts and MTT, colony formation, wound healing, cell cycle and Western blot analysis were performed to determine the effect of gene knockouts on various cancer hallmarks and the associated molecular targets of TIPE proteins. RESULTS AND DISCUSSION: IHC results revealed that expression of TIPE, TIPE2 and TIPE3 were upregulated and TIPE1 was downregulated in oral cancer tissues compared to normal tissues. Similar results were observed upon treating oral cancer cells with tobacco carcinogens. Furthermore, knockout of TIPE or TIPE2 or TIPE3 significantly reduced the survival, proliferation, colony formation and migration of oral cancer cells whereas knockout of TIPE1 had an opposite effect. Further, TIPE, TIPE2 and TIPE3 knockout-mediated inhibition of proliferation was associated with inhibition of cell cycle progression at S or G2/M phases, and downregulation of proteins involved in cancer progression. We found that TIPE, TIPE1 and TIPE2 proteins regulate oral cancer progression through modulation of Akt/mTOR signaling cascade, whereas TIPE3 acts through an Akt-independent mTOR/STAT3 pathway. CONCLUSION: Collectively, the TIPE proteins were proved to play significant roles in the progression of oral cancer thus warranting research and clinic attention for their therapeutic and prognostic values and raising the importance of specific targeting of TIPE proteins in cancer treatment.

The Effects of Flavonoid Apigenin on Male Reproductive Health: Inhibition of Spermatogonial Proliferation through Downregulation of Prmt7/Akt3 Pathway.

Apigenin, a common dietary flavonoid abundantly present in a variety of fruits and vegetables, has promising anticancer properties. As an effector of apigenin in myoblasts, protein arginine methyltransferase 7 (Prmt7) is required for male germ cell development. However, whether apigenin may influence male reproductive health through Prmt7 is still unclear. To this end, mouse spermatogonia were treated with different concentrations (2.5 to 50 µM) of apigenin for 48 h, which showed that apigenin could cause reduced cell proliferation in conjunction with longer S phase and G2/M phase (with concentrations of 10 and 20 µM, respectively), and increased apoptosis of spermatogonia (with concentration of 20 µM). Reduced Prmt7 expression was found in 20 µM apigenin-treated spermatogonia. Moreover, siRNA-induced Prmt7 knockdown exhibited similar influence on spermatogonia as that of apigenin treatment. In mechanistic terms, transcriptome analysis revealed 287 differentially expressed genes between Prmt7-downregulated and control spermatogonia. Furthermore, rescue experiments suggested that the effects of apigenin on spermatogonia might be mediated through the Prmt7/Akt3 pathway. Overall, our study supports that apigenin can interfere with mouse spermatogonial proliferation by way of the downregulated Prmt7/Akt3 pathway, which demonstrates that the concentration should be taken into account in future applications of apigenin for cancer therapy of men.

Cytoglobin as a Prognostic Factor for Pancreatic Ductal Adenocarcinoma: A Retrospective Analysis of 75 Patients.

OBJECTIVES: The aim was to evaluate the relationship between cytoglobin (Cygb) expression and both clinicopathologic factors and prognosis in patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: Seventy-five patients with PDAC who underwent pancreatectomy between 2009 and 2014 at our department were included. Diagnosis was based on World Health Organization standards, with staging by TNM classification of Union for International Cancer Control. Expressions of Cygb, phosphoinositide-3 kinase, phosphorylated protein kinase B, interleukin-6, and vascular endothelial growth factor were evaluated by immunohistochemical staining of resected surgical specimens and densitometrical analysis. RESULTS: Elevated expression of Cygb was found mainly in carcinoma cells of PDAC. Patients with low expression of Cygb showed significantly shorter disease-free survival and disease-specific survival than those with high expression. There was also a significant negative correlation between Cygb expression and the expressions of phosphoinositide 3-kinase, phosphorylated protein kinase B, interleukin-6, and vascular endothelial growth factor. In univariate analysis, Cygb expression, clinical stage, histologic tumor grade, lymphatic invasion, and vascular invasion were prognostic factors. In multivariate analysis, Cygb expression and the clinical stage were independent prognostic factors. CONCLUSIONS: Loss of Cygb may contribute to tumor recurrence and poor prognosis of PDAC by increases in angiogenic factor.

Selenium inhibits growth of trastuzumab-resistant human breast cancer cells via downregulation of Akt and beclin-1.

The response rate to treatment with trastuzumab (Tz), a recombinant humanized anti-HER2 monoclonal antibody, is only 12-34% despite demonstrated effectiveness on improving the survival of patients with HER2-positive breast cancers. Selenium has an antitumor effect against cancer cells and can play a cytoprotective role on normal cells. This study investigated the effect of selenium on HER2-positive breast cancer cells and the mechanism in relation to the response of the cells to Tz. HER2-positive breast cancer cell lines, SK-BR-3 as trastuzumab-sensitive cells, and JIMT-1 as Tz-resistant cells were treated with Tz and sodium selenite (selenite). Cell survival rates and expression of Her2, Akt, and autophagy-related proteins, including LC3B and beclin 1, in both cell lines 72 h after treatment were evaluated. Significant cell death was induced at different concentrations of selenite in both cell lines. A combined effect of selenite and Tz at 72 h was similar to or significantly greater than each drug alone. The expression of phosphorylated Akt (p-Akt) was decreased in JIMT-1 after combination treatment compared to that after only Tz treatment, while p-Akt expression was increased in SK-BR-3. The expression of beclin1 increased particularly in JIMT-1 after only Tz treatment and was downregulated by combination treatment. These results showed that combination of Tz and selenite had an antitumor effect in Tz-resistant breast cancer cells through downregulation of phosphorylated Akt and beclin1-related autophagy. Selenite might be a potent drug to treat Tz-resistant breast cancer by several mechanisms.

Silencing lncRNA LINC01410 suppresses cell viability yet promotes apoptosis and sensitivity to temozolomide in glioblastoma cells by inactivating PTEN/AKT pathway via targeting miR-370-3p.

BACKGROUND: Long non-coding RNAs (LncRNAs) are involved in glioblastoma (GBM), but the role of long intergenic non-protein coding RNA 01410 (lncRNA LINC01410) is poorly understood. METHODS: The expression of LINC01410 in GBM tissues and cells was analyzed. After transfection or temozolomide (TMZ) treatment, the cell viability and apoptosis were detected using cell counting kit-8 assay and flow cytometry. The targeting relationship between LINC01410 and microRNA (miR)-370-3p was confirmed by dual-luciferase reporter assay. Expressions of LINC01410, miR-370-3p and drug resistance- and Phosphatase and Tensin Homolog (PTEN)/AKT pathway-related factors were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. RESULTS: LINC01410 expression was upregulated in GBM, and silencing of LINC01410 decreased cell viability. A slowed decreased trend in cell viability yet an increased half maximal inhibitory concentration (IC50 for TMZ) value and increased expressions of drug resistance-related factors as well as LINC01410 were found in TMZ-resistant GBM cells. Silencing of LINC01410 also decreased the IC50 value yet promoted the sensitivity and apoptosis in TMZ-resistant cells, while upregulating the expression of PTEN and downregulating the phosphorylation of AKT. MiR-370-3p could competitively bind to LINC01410 and its expression was decreased in both parental and TMZ-resistant GBM cells. Downregulation of miR-370-3p reversed the effects of LINC01410 silencing on cell viability, apoptosis and the expressions of miR-370-3p and PTEN/AKT pathway-related factors. CONCLUSION: Silencing of LINC01410 inhibits cell viability yet enhances apoptosis and sensitivity to TMZ in GBM cells by inactivating PTEN/AKT pathway via targeting miR-370-3p.

Formononetin attenuates H2O2-induced cell death through decreasing ROS level by PI3K/Akt-Nrf2-activated antioxidant gene expression and suppressing MAPK-regulated apoptosis in neuronal SH-SY5Y cells.

Formononetin is an isoflavone, found in herbs like Trifolium pratense, which executes a variety of physiological activities including anti-neurodegenerative effect. However, the molecular mechanism of formononetin-mediated neuroprotection remains unclear. In this study, we investigated the protective effect of formononetin on hydrogen peroxide (H2O2)-induced death of human neuroblastoma SH-SY5Y cells and its underlying molecular mechanism. Formononetin suppressed H2O2-induced cytotoxicity. H2O2-induced increase in the intracellular reactive oxygen species (ROS) levels was decreased by formononetin, together with the enhanced expression of the antioxidant genes. H2O2-induced elevation of the Bax/Bcl-2 ratio and cleaved caspase-3 and caspase-7 levels were lowered by formononetin treatment. Moreover, formononetin repressed H2O2-induced phosphorylation of mitogen-activated protein kinases (MAPKs). Nuclear factor erythroid 2-related factor 2 (Nrf2) siRNA decreased antioxidant gene expression and elevated the H2O2-induced ROS level in the formononetin-treated cells. Furthermore, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling is involved in the activation of the nuclear translocation of Nrf2. These results indicate that the neuroprotective effect of formononetin against H2O2-induced cell death is due to a decrease in the ROS level with the enhanced expression of the antioxidant genes through activation of the PI3K/Akt-Nrf2 signaling. In addition, formononetin suppressed apoptosis through inhibition of phosphorylation of MAPKs in SH-SY5Y cells. Thus, formononetin is a potential therapeutic agent for the treatment of neurodegenerative diseases.

miR-150 and SRPK1 regulate AKT3 expression to participate in LPS-induced inflammatory response.

miR-150 was found to target the 3'-untranslated regions of AKT3, and the AKT pathway was affected by SR protein kinase 1 (SRPK1). However, the expression and significance of miR-150, AKT3 and SRPK1 in acute lung injury (ALI) were not clear. Here, we found that the expression of miR-150 was significantly reduced, while the expression of AKT3 and SRPK1 were markedly increased in LPS-treated A549, THP-1 and RAW 264.7 cells. miR-150 significantly decreased levels of pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α, reduced the expression of AKT3, but had no impact on SRPK1 expression compared with the control group in LPS-treated A549, THP-1 and RAW 264.7 cells. AKT3 silencing only reduced the production of pro-inflammatory cytokines and showed no effect on miR-150 and SRPK1 expression. Finally, we observed that miR-150 mimics and/or silencing of SRPK1 decreased the expression of AKT3 mRNA. Besides, over-expression of miR-150 or silencing of SRPK1 also reduced the expression of AKT3 protein, which exhibited the lowest level in the miR-150 mimics plus si-SRPK1 group. However, si-SRPK1 had no effect on miR-150 level. In conclusion, miR-150 and SRPK1 separately and cooperatively participate into inflammatory responses in ALI through regulating AKT3 pathway. Increased miR-150 and silenced SRPK1 may be a novel potential factor for preventing and treating more inflammatory lung diseases.

Pinocembrin-7-Methylether Protects SH-SY5Y Cells Against 6-Hydroxydopamine-Induced Neurotoxicity via Modulating Nrf2 Induction Through AKT and ERK Pathways.

The present study aimed to evaluate the neuroprotective effects and underlying mechanisms of pinocembrin-7-methylether (PME), a natural bioflavonoid, in 6-hydroxydopamine (6-OHDA)-induced models of Parkinson's disease in vivo and in vitro. First, we found that PME decreased apoptosis in 6-OHDA-intoxicated SH-SY5Y cells. PME also blocked several 6-OHDA-induced mitochondrial apoptotic cascades, including loss of mitochondrial membrane potential, caspase 3 and PARP activation, and a decrease in the Bcl-2/Bax ratio. Also, PME suppressed 6-OHDA-induced oxidative stress while increasing antioxidant enzymatic activity. Further investigations indicated that PME significantly enhanced nuclear accumulation of Nrf2, improved ARE promoter activity, and upregulated HO-1 and NQO1 expression levels. In addition, siRNA-mediated Nrf2 knockdown abolished PME-induced anti-oxidative and anti-apoptotic effects. Interestingly, we found that PME promoted phosphorylation of AKT and ERK, whereas pharmacological inhibition of AKT or ERK pathways diminished PME-induced Nrf2 activation and protective actions. Moreover, PME attenuated 6-OHDA-induced loss of dopaminergic neurons and ameliorated locomotor deficiency in zebrafish, supporting the neuroprotective actions of PME in vivo. In summary, we found that PME conferred neuroprotection against 6-OHDA-induced neurotoxicity in PD models in vivo and in vitro. Taken together, our findings suggest that activation of Nrf2/ARE/HO-1 signaling cascades contributes to PME-induced anti-oxidative and neuroprotective actions, which are at least partially mediated by AKT and ERK pathways.

The effect of young and old ex vivo human serum on cellular protein synthesis and growth in an in vitro model of aging.

In vitro models of muscle aging are useful for understanding mechanisms of age-related muscle loss and aiding the development of targeted therapies. To investigate mechanisms of age-related muscle loss in vitro utilizing ex vivo human serum, fasted blood samples were obtained from four old (72 ± 1 yr) and four young (26 ± 3 yr) men. Older individuals had elevated levels of plasma CRP, IL-6, HOMA-IR, and lower concentric peak torque and work-per-repetition compared with young participants (P < 0.05). C2C12 myotubes were serum and amino acid starved for 1 h and conditioned with human serum (10%) for 4 h or 24 h. After 4 h, C2C12 cells were treated with 5 mM leucine for 30 min. Muscle protein synthesis (MPS) was determined through the surface sensing of translation (SUnSET) technique and regulatory signaling pathways were measured via Western blot. Myotube diameter was significantly reduced in myotubes treated with serum from old, in comparison to young donors (84%, P < 0.001). MPS was reduced in myotubes treated with old donor serum, compared with young serum before leucine treatment (32%, P < 0.01). MPS and the phosphorylation of Akt, p70S6K, and eEF2 were increased in myotubes treated with young serum in response to leucine treatment, with a blunted response identified in cells treated with old serum (P < 0.05). Muscle protein breakdown signaling pathways did not differ between groups. In summary, we show that myotubes conditioned with serum from older individuals had decreased myotube diameter and MPS compared with younger individuals, potentially driven by low-grade systemic inflammation.

Inhibited effect of an RGD peptide hydrogel on the expression of ß1-integrin, FAK, and Akt in Tenon's capsule fibroblasts.

Tenon's capsule fibroblasts are the main cellular components of filtration tract scar that limit the success rate of glaucoma filtration surgery. Scar formation results from infiltration and proliferation of fibroblasts into damaged areas, meanwhile synthesis of extracellular matrix glycoproteins. Integrins are cell surface receptors for extracellular molecules that mediate cell adhesion, spreading, migration, and invasion. They bind their ligands often through recognition of short amino-acid sequences-arginine-glycine-aspartic acid (RGD). Peptides that contain RGD sequence can compete with RGD containing insoluble matrix proteins for binding to the integrin receptor and thus prevent the downstream signaling pathway. Increasing evidence supports that ß1-integrin/focal adhesion kinase (FAK)/Akt signal pathway plays an important role in fibrogenesis and scar formation in different tissues. In consideration of advantages of peptide hydrogel, that is well biocompatibility, gel state, degradability, good drug loading, we designed and fabricated an RGD peptide hydrogel, and hypothesized that it could inhibit the expression of ß1-integrin, FAK, and Akt in Tenon's capsule fibroblasts. Rheology results showed that 1% wt Fmoc-FFGGRGD peptide solution could self-assemble into hydrogel. Western blot analysis revealed that there were statistical differences between control group and 1% wt group in ß1-integrin/ß-actin, FAK/ß-actin, Akt/ß-actin respectively (*p < .05). The relative mRNA expression of ß1-integrin, FAK, Akt in control group and 1% wt group were also statistically different respectively (*p < .05). We proved that 1% wt Fmoc-FFGGRGD self-assembly peptide hydrogel could inhibit the expression of ß1-integrin, FAK and Akt in Tenon's capsule fibroblasts. It is a promising way to solve scar formation of glaucoma filter channel.

HIF-1/AKT Signaling-Activated PFKFB2 Alleviates Cardiac Dysfunction and Cardiomyocyte Apoptosis in Response to Hypoxia.

Myocardial infarction (MI) is the most prevalent disease with severe mortality, and hypoxia-induced cardiac injury and cardiomyocyte apoptosis are the significant and harmful consequences of this disease. The cross talk between hypoxia signaling and glycolysis energy flux plays a critical role in modulating MI-related heart disorder. However, the underlying mechanism remains unclear. Here, we aimed to explore the effect of a key glycolytic enzyme of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 2 (PFKFB2) on cardiac dysfunction and apoptosis in response to hypoxia. Our data demonstrated that the mRNA and protein expression of PFKFB2 were significantly elevated in the MI mice. The MI treatment promoted the activation of PFKFB2 in vivo, as presented by the remarkably increased phosphorylation levels of PFKFB2. PFKFB2 depletion enhanced MI-induced cardiac dysfunction and cardiomyocyte apoptosis in the MI mouse model. Moreover, hypoxia treatment dramatically upregulated the expression and activation of PFKFB2 in a time-dependent manner in cardiomyocytes. Hypoxia-stimulated PFKFB2 relieved hypoxia-induced cardiomyocyte apoptosis in vitro. PFKFB2 activated the fructose-2, 6-bisphosphate (Fru-2, 6-p2) /PFK/anaerobic adenosine triphosphate (ATP) glycolysis energy flux in response to hypoxia in cardiomyocytes. Mechanically, hypoxia-activated PFKFB2 by stimulating the hypoxia-inducible factor 1 (HIF-1) /ATK signaling. Thus, we conclude that HIF-1/AKT axis-activated PFKFB2 alleviates cardiac dysfunction and cardiomyocyte apoptosis in response to hypoxia. Our finding presents a new insight into the mechanism by which HIF-1/AKT/PFKFB2 signaling modulates MI-related heart disorder under the hypoxia condition, providing potential therapeutic targets and strategy for hypoxia-related myocardial injury.

Anti-type I allergic effects of Jing-Fang powder extracts via PI3K/Akt pathway in vitro and in vivo.

Jing-Fang powder (Schizonepeta tenuifolia Briq. and Saposhnikovia divaricata (Turcz.) Schischk.) was used to treat chronic bronchitis, asthma and chronic urticaria. Based on the preliminary results of screening research on the antiallergic effective parts of Jing-Fang powder, its ethyl acetate extract fractions (JFEE) and isolate D (JFEE-D) showed the best anti-allergic effect. RBL-2H3 cell activation degranulation model and mice passive cutaneous anaphylaxis (PCA) reaction model were used to investigate the effects and mechanisms of JFEE and JFEE-D on IgE-mediated type I allergic reactions. LC-MS was utilized to determine the composition of JFEE and JFEE-D. We found that JFEE and JFEE-D significantly reduced ß-HEX, histamine, IL-4, IL-6 levels in cell supernatants, and improved the degree and morphology of cell degranulation. JFEE and JFEE-D significantly inhibited the increase of ear vascular permeability and abnormal increase of serum IgE, TNF-α, IL-6 levels. JFEE and JFEE-D inhibited mRNA expression of PI3K and Akt and down-regulated protein expression of PI3K, Akt, p-Akt, and PLCγ1 in sensitized RBL-2H3 cells. The combined use of JFEE and JFEE-D with pathway inhibitor Wortmannin revealed synergistic down-regulation of PI3K, Akt, and p-Akt protein expression. The combined use of pathway agonist IGF-1, JFEE and JFEE-D down-regulated increase of p-Akt/Akt protein expression. Moreover, JFEE and JFEE-D significantly inhibited protein expression of PI3K, p-Akt and PLCγ1 in PCA model mice. These results show that JFEE and JFEE-D inhibit type I allergic reactions by inhibiting PI3K/Akt signaling pathway.