The human adenovirus PI3K-Akt activator E4orf1 is targeted by the tumor suppressor p53.

Human adenoviruses (HAdV) are classified as DNA tumor viruses due to their potential to mediate oncogenic transformation in non-permissive mammalian cells and certain human stem cells. To achieve transformation, the viral early proteins of the E1 and E4 regions must block apoptosis and activate proliferation: the former predominantly through modulating the cellular tumor suppressor p53 and the latter by activating cellular pro-survival and pro-metabolism protein cascades, such as the phosphoinositide 3-kinase (PI3K-Akt) pathway, which is activated by HAdV E4orf1. Focusing on HAdV-C5, we show that E4orf1 is necessary and sufficient to stimulate Akt activation through phosphorylation in H1299 cells, which is not only hindered but repressed during HAdV-C5 infection with a loss of E4orf1 function in p53-positive A549 cells. Contrary to other research, E4orf1 localized not only in the common, cytoplasmic PI3K-Akt-containing compartment, but also in distinct nuclear aggregates. We identified a novel inhibitory mechanism, where p53 selectively targeted E4orf1 to destabilize it, also stalling E4orf1-dependent Akt phosphorylation. Co-IP and immunofluorescence studies showed that p53 and E4orf1 interact, and since p53 is bound by the HAdV-C5 E3 ubiquitin ligase complex, we also identified E4orf1 as a novel factor interacting with E1B-55K and E4orf6 during infection; overexpression of E4orf1 led to less-efficient E3 ubiquitin ligase-mediated proteasomal degradation of p53. We hypothesize that p53 specifically subverts the pro-survival function of E4orf1-mediated PI3K-Akt activation to protect the cell from metabolic hyper-activation or even transformation.IMPORTANCEHuman adenoviruses (HAdV) are nearly ubiquitous pathogens comprising numerous subtypes that infect various tissues and organs. Among many encoded proteins that facilitate viral replication and subversion of host cellular processes, the viral E4orf1 protein has emerged as an intriguing yet under-investigated player in the complex interplay between the virus and its host. Nonetheless, E4orf1 has gained attention as a metabolism activator and oncogenic agent, while recent research is showing that E4orf1 may play a more important role in modulating the cellular pathways such as phosphoinositide 3-kinase-Akt-mTOR. Our study reveals a novel and general impact of E4orf1 on host mechanisms, providing a novel basis for innovative antiviral strategies in future therapeutic settings. Ongoing investigations of the cellular pathways modulated by HAdV are of great interest, particularly since adenovirus-based vectors actually serve as vaccine or gene vectors. HAdV constitute an ideal model system to analyze the underlying molecular principles of virus-induced tumorigenesis.

Regulation of the voltage-dependent sodium channel NaV1.1 by AKT1.

The voltage-sensitive sodium channel NaV1.1 plays a critical role in regulating excitability of GABAergic neurons and mutations in the corresponding gene are associated to Dravet syndrome and other forms of epilepsy. The activity of this channel is regulated by several protein kinases. To identify novel regulatory kinases we screened a library of activated kinases and we found that AKT1 was able to directly phosphorylate NaV1.1. In vitro kinase assays revealed that the phosphorylation site was located in the C-terminal part of the large intracellular loop connecting domains I and II of NaV1.1, a region that is known to be targeted by other kinases like PKA and PKC. Electrophysiological recordings revealed that activated AKT1 strongly reduced peak Na+ currents and displaced the inactivation curve to more negative potentials in HEK-293 cell stably expressing NaV1.1. These alterations in current amplitude and steady-state inactivation were mimicked by SC79, a specific activator of AKT1, and largely reverted by triciribine, a selective inhibitor. Neurons expressing endogenous NaV1.1 in primary cultures were identified by expressing a fluorescent protein under the NaV1.1 promoter. There, we also observed a strong decrease in the current amplitude after addition of SC79, but small effects on the inactivation parameters. Altogether, we propose a novel mechanism that might regulate the excitability of neural networks in response to AKT1, a kinase that plays a pivotal role under physiological and pathological conditions, including epileptogenesis.

Role of PI3K/AKT Signaling Pathway in Nucleus Pulposus Cells.

OBJECTIVE: To investigate the role of PI3K/AKT signaling pathway in nucleus pulposus (NP) cells. METHODS: Nucleus pulposus (NP) cells were isolated from SD rat, and thereafter, passage three (P3) NP cells were divided into the following experimental groups: control, PI3K/AKT agonist IGF-1 (25 ng/ml, 50 ng/ml, and 100 ng/ml), and PI3K/AKT inhibitor LY294002 (5 µM, 10 µM, and 20 µM). Flow cytometry and BrdU cell proliferation assays were performed to assess apoptosis and the proliferation rate of NP cells. Western blot analysis was performed to examine the protein expression level of Col II, Col X, Aggrecan, and MMP13. RESULTS: PI3K/AKT inhibitor LY294002 increased the rate of apoptosis in NP cells when compared to the control and decreased the proliferation rate when compared to control. Moreover, LY294002 decreased the protein expression level of Col-II and Aggrecan in NP cells. At the same time, LY294002 increased the protein expression level of MMP13 and Col-X in NP cells. Through activating PI3K/AKT, IGF-1 increased the proliferation rate when compared to control and decreased the rate of apoptosis when compared to control. Additionally, IGF-1 decreased the protein expression level of MMP13 and Col-X and increased Col-II and Aggrecan in NP cells. CONCLUSION: The inhibition of PI3K/AKT signaling pathway accelerated the apoptosis of NP cells and facilitated the extracellular matrix degradation. However, the activation of PI3K/AKT pathway partly prevented the NP cell from apoptosis and promoted their proliferation. Meanwhile, its activation also delayed the loss of extracellular matrix.

Pterostilbene Improves Insulin Resistance Caused by Advanced Glycation End Products (AGEs) in Hepatocytes and Mice.

SCOPE: Increased consumption of modern processed foods rich in AGEs is drawing worldwide concerns because they are related with rising diabetes prevalence. This study aimed to investigate if pterostilbene (PTE) regulates glucose metabolism and insulin signaling, as well as its potential mechanism in the context of AGEs exposure. METHODS AND RESULTS: In vitro, Lo2 and HepG2 cells are treated with vehicle, AGEs with or without PTE. AGEs exposure directly impair insulin action as evidenced by assays of insulin-stimulated glucose uptake, consumption, and output. However, PTE efficiently rescue the AGE-induced phenotypes in both cell lines, and enhance IRS-1/PI3K/AKT insulin signaling in a dose-dependent manner. In vivo, C57BL6 mice are fed with regular, high AGEs diet and high AGEs plus PTE. PTE administration effectively improves hyperglycemia, glucose tolerance, and impaired hepatic insulin signaling induced by AGEs, consistent with the in vitro experiments. Moreover, PTE reduce AGEs accumulation in liver and serum. RNA-seq data indicate that PTE counteracts several AGEs-induced dysfunctions including diabetes related process, glucose metabolic process, immune response, and so on. CONCLUSION: PTE treatment prominently reduced AGEs accumulation and alleviated AGEs-associated diabetes symptoms. PTE could be used as a promising glucose-sensitizing agent for nutritional intervention.

Knockdown of TRIM26 inhibits the proliferation, migration and invasion of bladder cancer cells through the Akt/GSK3ß/ß-catenin pathway.

Tripartite motif-containing protein 26 (TRIM26) is a member of the TRIM protein family and has been demonstrated to play crucial roles in several types of cancers. However, the biological role of TRIM26 in bladder cancer and the mechanism have not been studied. In this study, we investigated the expression of TRIM26 in bladder cancer tissues and their adjacent non-tumor tissues by Western blot and qRT-PCR. In vitro investigations were performed to assess the roles of TRIM26 in bladder cancer using TRIM26-silencing and TRIM26-overexpressing bladder cancer cell lines. MTT and EdU assays were performed to evaluate cell proliferation. Cell migration and invasion were determined by transwell assays. Western blot analysis was performed to detect the expression levels of p-Akt, Akt, p-GSK3ß, GSK3ß, ß-catenin and c-Myc. Our results showed that TRIM26 expression was upregulated in human bladder cancer tissues and cell lines at both mRNA and protein levels. Knockdown of TRIM26 significantly inhibited the proliferation, migration and invasion of bladder cancer cells. In contrast, TRIM26 overexpression promoted bladder cancer cell proliferation, cell migration and invasion. Furthermore, knockdown of TRIM26 significantly decreased the levels of p-Akt, p-GSK3ß, ß-catenin and c-Myc in bladder cancer cells. Additionally, induction of Akt by SC79 treatment reversed the inhibitory effects of TRIM26 knockdown on the cellular behaviors of bladder cancer cells, while inhibition of ß-catenin reversed the effects of TRIM26 overexpression on the behaviors. Finally, knockdown of TRIM26 attenuated the growth of tumor xenografts in nude mice. In conclusion, these findings demonstrated that TRIM26 exerted an oncogenic role in bladder cancer through regulation of cell proliferation, migration and invasion via the Akt/GSK3ß/ß-catenin pathway.

Oridonin inhibits epithelial-mesenchymal transition of human nasopharyngeal carcinoma cells by negatively regulating AKT/STAT3 signaling pathway.

Oridonin, derived from Rabdosia rubescens, has exhibited anticancer activity in a variety of cancers. However, few studies have explored the effect of oridonin (ORI) on migration, invasion and epithelial-mesenchymal transition (EMT) in nasopharyngeal carcinoma. In our study, the results demonstrated that oridonin significantly inhibited migration and invasion of human nasopharyngeal carcinoma CNE-2Z and HNE-1 cell lines, as depicted by wound healing and Transwell assays. In addition, oridonin increased the expression of E-Cadherin while decreased the expressions of vimentin and twist1 at the mRNA and protein levels in a dose-dependent manner. Interestingly, oridonin also decreased cell mobility in nasopharyngeal carcinoma. The subsequent results of western blotting uncovered that the phosphorylation levels of AKT and signal transducer and activator of transcription 3 (STAT3) were decreased upon oridonin treatment. Furthermore, co-treatment with the AKT activator SC-79 attenuated the anti-metastatic effect of oridonin on nasopharyngeal carcinoma and partially abolished the high expression of E-cadherin and the low expression of twist1 mediated by oridonin. In conclusion, the results revealed that oridonin could repress metastatic phenotype and reverse epithelial-mesenchymal transition (EMT) in nasopharyngeal carcinoma by negatively regulating AKT/STAT3 signaling pathway, suggesting that AKT/STAT3 signaling may be the potential therapeutic target of oridonin against nasopharyngeal carcinoma.

The gut microbiota metabolite propionate ameliorates intestinal epithelial barrier dysfunction-mediated Parkinson's disease via the AKT signaling pathway.

OBJECTIVE: Parkinson's disease is a common neurodegenerative disease. Here, we investigated the protective effect and potential mechanisms of propionate on the intestinal epithelial barrier in mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease. METHODS: Gas chromatography was used to determine short-chain fatty acids (SCFA) concentrations in the fecal samples of Parkinson's disease patients and healthy controls. The stepping test was used to analyze forelimb akinesia, whisker test was used to analyze sensorimotor injury, cylinder test was used to analyze sensorimotor function, and Western blotting was used to analyze protein expression. RESULTS: The concentrations of SCFAs, including acetate, butyrate and propionate, were significantly downregulated in the fecal samples of Parkinson's disease patients, and among the SCFAs, propionate decreased the most. Propionate administration improved the stepping test score, whisker test score and cylinder test score of MPTP-induced Parkinson's disease mice. Additionally, propionate administration increased the protein expression of zonula occludens-1 and occludin. Moreover, the effects of propionate on motor behavior and the intestinal epithelial barrier were dependent on the proteirrserinc-threonine kinases (AKT) signaling pathway. More importantly, treatment with SC79, a specific AKT agonist, abolished the effects of propionate on the intestinal epithelial barrier and motor behavior. CONCLUSION: Our results demonstrated that propionate, which was decreased in the fecal samples of Parkinson's disease patients, exerted beneficial effects on intestinal epithelial barrier function and improved motor behavior in MPTP-induced Parkinson's disease mice through the AKT signaling pathway.

Kcnh2 mediates FAK/AKT-FOXO3A pathway to attenuate sepsis-induced cardiac dysfunction.

OBJECTIVES: Myocardial dysfunction is a significant manifestation in sepsis, which results in high mortality. Even Kcnh2 has been hinted to associate with the pathological process, its involved signalling is still elusive. MATERIALS AND METHODS: The caecal ligation puncture (CLP) surgery or lipopolysaccharide (LPS) injection was performed to induce septic cardiac dysfunction. Western blotting was used to determine KCNH2 expression. Cardiac function was examined by echocardiography 6 hours after CLP and LPS injection in Kcnh2 knockout (Kcnh2+/- ) and NS1643 injection rats (n ≥ 6/group). Survival was monitored following CLP-induced sepsis (n ≥ 8/group). RESULTS: Sepsis could downregulate KCNH2 level in the rat heart, as well as in LPS-stimulated cardiomyocytes but not cardiac fibroblast. Defect of Kcnh2 (Kcnh2+/- ) significantly aggravated septic cardiac dysfunction, exacerbated tissue damage and increased apoptosis under LPS challenge. Fractional shortening and ejection fraction values were significantly decreased in Kcnh2+/- group than Kcnh2+/+ group. Survival outcome in Kcnh2+/- septic rats was markedly deteriorated, compared with Kcnh2+/+ rats. Activated Kcnh2 with NS1643, however, resulted in opposite effects. Lack of Kcnh2 caused inhibition of FAK/AKT signalling, reflecting in an upregulation for FOXO3A and its downstream targets, which eventually induced cardiomyocyte apoptosis and heart tissue damage. Either activation of AKT by activator or knockdown of FOXO3A with si-RNA remarkably attenuated the pathological manifestations that Kcnh2 defect mediated. CONCLUSION: Kcnh2 plays a protection role in sepsis-induced cardiac dysfunction (SCID) via regulating FAK/AKT-FOXO3A to block LPS-induced myocardium apoptosis, indicating a potential effect of the potassium channels in pathophysiology of SCID.

Downregulation of Cypher induces apoptosis in cardiomyocytes via Akt/p38 MAPK signaling pathway.

Background: Dilated cardiomyopathy (DCM) is considered as the most common form of non-ischemic cardiomyopathy with a high mortality worldwide. Cytoskeleton protein Cypher plays an important role in maintaining cardiac function. Genetic studies in human and animal models revealed that Cypher is involved in the development of DCM. However, the underlying molecular mechanism is not fully understood. Accumulating evidences suggest that apoptosis in myocytes may contribute to DCM. Thus, the purpose of this study is to define whether lack of Cypher in cardiomyocytes can elevate apoptosis signaling and lead to DCM eventually. Methods and Results: Cypher-siRNA sufficiently inhibited Cypher expression in cardiomyocytes. TUNEL-positive cardiomyocytes were increased in both Cypher knockdown neonatal rat cardiomyocytes and Cypher knockout mice hearts, which were rare in the control group. Flow cytometry further confirmed that downregulation of Cypher significantly increased myocytes apoptosis in vitro. Cell counting kit-8 assay revealed that Cypher knockdown in H9c2 cells significantly reduced cell viability. Cypher knockdown was found to increase cleaved caspase-3 expression and suppress p21, ratio of bcl-2 to Bax. Cypher-deficiency induced apoptosis was linked to downregulation of Akt activation and elevated p-p38 MAPK accumulation. Pharmacological activation of Akt with SC79 attenuated apoptosis with enhanced phosphorylation of Akt and reduced p-p38 MAPK and Bax expression. Conclusions: Downregulation of Cypher participates in the promotion of cardiomyocytes apoptosis through inhibiting Akt dependent pathway and enhancing p38 MAPK phosphorylation. These findings may provide a new potential therapeutic strategy for the treatment of DCM.

Triptolide prevents bone loss via suppressing osteoclastogenesis through inhibiting PI3K-AKT-NFATc1 pathway.

Bone loss (osteopenia) is a common complication in human solid tumour. In addition, after surgical treatment of gynaecological tumour, osteoporosis often occurs due to the withdrawal of oestrogen. The major characteristic of osteoporosis is the low bone mass with micro-architectural deteriorated bone tissue. And the main cause is the overactivation of osteoclastogenesis, which is one of the most important therapeutic targets. Inflammation could induce the interaction of RANKL/RANK, which is the promoter of osteoclastogenesis. Triptolide is derived from the traditional Chinese herb lei gong teng, presented multiple biological effects, including anti-cancer, anti-inflammation and immunosuppression. We hypothesized that triptolide could inhibits osteoclastogenesis by suppressing inflammation activation. In this study, we confirmed that triptolide could suppress RANKL-induced osteoclastogenesis in bone marrow mononuclear cells (BMMCs) and RAW264.7 cells and inhibited the osteoclast bone resorption functions. PI3K-AKT-NFATc1 pathway is one of the most important downstream pathways of RANKL-induced osteogenesis. The experiments in vitro indicated that triptolide suppresses the activation of PI3K-AKT-NFATc1 pathway and the target point located at the upstream of AKT because both NFATc1 overexpression and AKT phosphorylation could ameliorate the triptolide suppression effects. The expression of MDM2 was elevated, which demonstrated the MDM-p53-induced cell death might contribute to the osteoclastogenesis suppression. Ovariectomy-induced bone loss and inflammation activation were also found to be ameliorated in the experiments in vivo. In summary, the new effect of anti-cancer drug triptolide was demonstrated to be anti-osteoclastogenesis, and we demonstrated triptolide might be a promising therapy for bone loss caused by tumour.

Lgr4 Deletion Delays the Hair Cycle and Inhibits the Activation of Hair Follicle Stem Cells.

It is known that LGR4 plays an important role in hair follicle (HF) development, but the impact of LGR4 on the hair cycle is still unclear. In this study, we have found that K14-Cre-mediated skin epithelia-specific deletion of Lgr4 results in delayed anagen entry during the physiological hair cycle and compromised HF regeneration upon transplantation. We show that, although Lgr4 deletion does not appear to affect the number of quiescent HF stem cells, it leads to reduced numbers of LGR5+ and actively proliferating stem cells in the HFs. Moreover, LGR4-deficient HFs show molecular changes consistent with decreased mTOR and Wnt signaling but upregulated BMP signaling. Importantly, the reactivation of the protein kinase B pathway by injecting the protein kinase B activator SC79 in Lgr4-/- mice can effectively reverse the hair cycle delay. Together, these data suggest that LGR4 promotes the normal hair cycle by activating HF stem cells and by influencing the activities of multiple signaling pathways that are known to regulate HF stem cells. Our study also implicates LGR4 as a potential target for treating hair disorder in the future.

Metformin promotes osteogenic differentiation and protects against oxidative stress-induced damage in periodontal ligament stem cells via activation of the Akt/Nrf2 signaling pathway.

Periodontal ligament stem cell (PDLSC)-based tissue engineering is an important method for regenerating lost bone in periodontitis. Maintaining or enhancing the osteogenic differentiation of PDLSCs, as well as enhancing the resistance of PDLSCs to oxidative stress, is necessary in this process. As a common hypoglycemic drug, metformin has been reported to have multiple effects on cell functions. This study found that low concentrations of metformin did not affect cell proliferation but did inhibit adipogenic differentiation and promote osteogenic differentiation of PDLSCs. This positive effect was associated with activation of Akt signaling by metformin. Moreover, applying metformin as either a pretreatment or co-treatment could reduce the amount of reactive oxygen species, enhance antioxidant capacity, and rescue the cell viability and osteogenic differentiation that were negatively affected by H2O2-induced oxidative stress in PDLSCs. In addition, metformin was found to activate the Nrf2 signaling pathway in PDLSCs, and knockdown of Nrf2 by siRNA impaired the protective effect of metformin. Taken together, these results indicate that metformin not only promotes osteogenic differentiation of PDLSCs, but also protects PDLSCs against oxidative stress-induced damage, suggesting that metformin could be potentially useful in promoting PDLSC-based bone regeneration in the treatment of periodontitis.

Lithium ameliorates tubule-interstitial injury through activation of the mTORC2/protein kinase B pathway.

Tubule-interstitial injury (TII) is a critical step in the progression of renal disease. It has been proposed that changes in proximal tubule (PT) albumin endocytosis plays an important role in the development of TII. Some reports have shown protective effects of lithium on kidney injury animal models that was correlated to proteinuria. We tested the hypothesis that lithium treatment ameliorates the development of TII due to changes in albumin endocytosis. Two experimental models were used: (1) TII induced by albumin overload in an animal model; (2) LLC-PK1 cells, a PT cell line. Lithium treatment ameliorates TII induced by albumin overload measured by (1) proteinuria; (2) collagen deposition; (3) area of tubule-interstitial space, and (4) macrophage infiltration. Lithium treatment increased mTORC2 activity leading to the phosphorylation of protein kinase B (PKB) at Ser473 and its activation. This mechanism enhanced albumin endocytosis in PT cells, which decreased the proteinuria observed in TII induced by albumin overload. This effect did not involve changes in the expression of megalin, a PT albumin receptor. In addition, activation of this pathway decreased apoptosis in LLC-PK1 cells, a PT cell line, induced by higher albumin concentration, similar to that found in pathophysiologic conditions. Our results indicate that the protective role of lithium treatment on TII induced by albumin overload involves an increase in PT albumin endocytosis due to activation of the mTORC2/PKB pathway. These results open new possibilities in understanding the effects of lithium on the progression of renal disease.

C-type lectin domain family 5, member A (CLEC5A, MDL-1) promotes brain glioblastoma tumorigenesis by regulating PI3K/Akt signalling.

OBJECTIVES: Glioblastoma is the most common malignant glioma of all brain tumours. It is difficult to treat because of its poor response to chemotherapy and radiotherapy and high recurrence rate after treatment. The aetiology of glioblastoma is a result of disorders of multiple factors. Depending on cell signal transduction, these glioblastoma-associated factors lead to cell proliferation, differentiation and apoptosis. Therefore, investigation of the potential factors which involved in the development of glioblastoma could provide a new target for the treatment of glioblastoma. MATERIALS AND METHODS: We analysed the transcript expression of CLEC5A in glioblastoma by accessing The Cancer Genome Atlas (TCGA). qRT-PCR was performed to detect the RNA expression of genes in cells and tissues, and Western blot was used to measure the protein levels (Cyclin D1, Bcl-2, BAX, PCNA, MMP2, MMP9, Akt and Akt phosphorylation) in tissues and cells. Cell proliferation, migration, invasion, cycle and apoptosis were measured by CCK-8, transwell and flow cytometry assays, respectively. Ki67 level and lung metastasis were determined by immunochemistry and H&E staining. RESULTS: In this study, we found that CLEC5A was highly upregulated in glioblastoma compared to normal brain tissues, which had an opposite relation with the overall patient survival. Downregulation of CLEC5A could inhibit cell proliferation, migration and invasion via promoting apoptosis and G1 arrest. In contrast, overexpression of CLEC5A stimulated cell proliferation, migration and invasion. In addition, we found that CLEC5A level was positively correlated with Akt phosphorylation level. Akt inhibitor or agonist could reverse the modulation effects of CLEC5A in glioblastoma. Moreover, In vivo results suggested that inhibition of CLEC5A significantly reduced tumour size, weight, cell proliferation ability and lung metastasis via inhibition of phosphorylation Akt. CONCLUSION: Both in vitro and in vivo evidences supported that CLEC5A was involved in glioblastoma pathogenesis via regulation of PI3K/Akt pathway. Thus, CLEC5A might serve as a potential therapeutic target in the treatment of glioblastoma in the future.

Neutrophils Derived from Genetically Modified Human Induced Pluripotent Stem Cells Circulate and Phagocytose Bacteria In Vivo.

Bacterial and fungal infections are a major cause of morbidity and mortality in neutropenic patients. Donor-derived neutrophil transfusions have been used for prophylaxis or treatment for infection in neutropenic patients. However, the short half-life and the limited availability of large numbers of donor-derived neutrophils for transfusion remain a significant hurdle in the implementation of neutrophil transfusion therapy. Here, we investigate the in vitro and in vivo activity of neutrophils generated from human induced pluripotent stem cells (iPSC), a potentially unlimited resource to produce neutrophils for transfusion. Phenotypic analysis of iPSC-derived neutrophils reveal reactive oxygen species production at similar or slightly higher than normal peripheral blood neutrophils, but have an ∼50%-70% reduced Escherichia coli phagocytosis and phorbol 12-myristate 13-acetate induced formation of neutrophil extracellular traps (NET). Signaling of granulocytic precursors identified impaired AKT activation, but not ERK or STAT3, in agonist-stimulated iPSC-derived neutrophils. Expression of a constitutively activated AKT in iPSC-derived neutrophils restores most phagocytic activity and NET formation. In a model of bacterial induced peritonitis in immunodeficient mice, iPSC-derived neutrophils, with or without corrected AKT activation, migrate similarly to the peritoneal fluid as peripheral blood neutrophils, whereas the expression of activated AKT significantly improves their phagocytic activity in vivo. Stem Cells Translational Medicine 2019;8:557-567.

A pilot study on the effect of lactoferrin on Alzheimer's disease pathological sequelae: Impact of the p-Akt/PTEN pathway.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases in which the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (PKB or Akt) pathway is deregulated in response to phosphatase and tensin homolog (PTEN) overexpression. Lactoferrin (LF), a multifunctional iron-binding glycoprotein, is involved in AD pathology; however, direct evidence of its impact upon AD remains unclear. To elucidate LF's role in AD, the possible protective mechanism post-LF administration for 3 months was investigated in AD patients by observing changes in the p-Akt/PTEN pathway. AD patients showed decreased serum acetylcholine (ACh), serotonin (5-HT), antioxidant and anti-inflammatory markers, and decreased expression of Akt in peripheral blood lymphocytes (PBL), as well as PI3K, and p-Akt levels in PBL lysate; all these parameters were significantly improved after daily LF administration for 3 months. Similarly, elevated serum amyloid ß (Aß) 42, cholesterol, oxidative stress markers, IL-6, heat shock protein (HSP) 90, caspase-3, and p-tau, as well as increased expression of tau, MAPK1 and PTEN in AD patients, were significantly reduced upon LF intake. Improvement in the aforementioned AD surrogate markers post-LF treatment was reflected in enhanced cognitive function assessed by the Mini-Mental State Examination (MMSE) and Alzheimer's Disease Assessment Scale-Cognitive Subscale 11-item (ADAS-COG 11) questionnaires as clinical endpoints. These results provide a basis for a possible protective mechanism of LF in AD through its ability to alleviate the AD pathological cascade and cognitive decline via modulation of the p-Akt/PTEN pathway, which affects the key players of inflammation and oxidative stress that are involved in AD pathology.

Piceatannol pretreatment alleviates acute cardiac injury via regulating PI3K-Akt-eNOS signaling in H9c2 cells.

Piceatannol (3,3',4,5'-trans-trihydroxystilbene) is a natural polyphenols compound that occurs hydroxylated analogue of resveratrol showing widely biological activities. Previous studies have demonstrated its functions on anti-cancer, neuroprotection and cardioprotection. However, few studies have clarified the benefits of piceatannol on cardiomyocytes except its anti-oxidative effect based on the original property of polyphenols. Here we apply H9c2 cardiomyocytes to study the cardioprotective mechanisms of piceatannol in vitro. We firstly verify its anti-peroxidation effect by using H2O2-induced in vitro model. Then, flow cytometry results show piceatannol reduce cellular apoptosis by enhancing Bcl-2 expressions in immunoblot analysis. Meantime, piceatannol decreases H2O2-induced excessive ROS and calcium overloading, and prevents mitochondrial depolarization. Most importantly, piceatannol pretreatment can regulate PI3K-Akt-eNOS signaling pathway to alleviate peroxidative injury. Immunoblot analysis of PI3K, Akt, p-Akt and eNOS shows H2O2 significantly reduces expressions of these proteins. Pretreatment of piceatannol evidently increases their expressions and decreases iNOS expression, implying piceatannol can upregulate PI3K-Akt-eNOS signaling to protect cardiomyocytes from peroxidative injury.

Side-chain cleaved phytoecdysteroid metabolites as activators of protein kinase B.

Phytoecdysteroids exert their non-hormonal anabolic and adaptogenic effects in mammals, including humans, through a partially revealed mechanism of action involving the activation of protein kinase B (Akt). We have recently found that poststerone, a side-chain cleaved in vivo metabolite of 20-hydroxyecdysone, exerts potent anabolic activity in rats. Here we report the semi-synthetic preparation of a series of side-chain cleaved ecdysteroids and their activity on the Akt phosphorylation in murine skeletal muscle cells. Twelve C-21 ecdysteroids including 8 new compounds were obtained through the oxidative side-chain cleavage of various phytoecdysteroids, or through the base-catalyzed autoxidation of poststerone. The complete 1H and 13C NMR spectroscopic assignments of the new compounds are presented. Among the tested compounds, 9 could activate Akt stronger than poststerone revealing that side-chain cleaved derivatives of phytoecdysteroids other than 20-hydroxyecdysone are valuable bioactive metabolites. Thus, our results suggest that the expectable in vivo formation of such compounds should contribute to the bioactivity of herbal preparations containing ecdysteroid mixtures.

Effects of fluvoxamine on nerve growth factor-induced neurite outgrowth inhibition by dexamethasone in PC12 cells.

In the present study, we examined the effects of fluvoxamine on nerve growth factor (NGF)-induced neurite outgrowth inhibition by dexamethasone (DEX) in PC12 cells. Fluvoxamine increased NGF-induced neurite outgrowth. Compared with co-treatment with NGF and fluvoxamine, p-Akt levels were higher than the values without fluvoxamine. The phosphorylated extracellular regulated kinase 1/2 levels were slightly increased by co-treatment with NGF and fluvoxamine. Fluvoxamine concentration-dependently improved NGF-induced neurite outgrowth inhibition by DEX. Fluvoxamine also improved the decrease in the NGF-induced p-Akt level caused by DEX. Interestingly, the sigma-1 receptor antagonist NE-100 blocked the improvement effects of fluvoxamine on NGF-induced neurite outgrowth inhibition by DEX. The selective sigma-1 receptor agonist PRE-084 also improved NGF-induced neurite outgrowth inhibition by DEX, which is blocked by NE-100. These results indicate that the improvement effects of fluvoxamine on NGF-induced neurite outgrowth inhibition by DEX may be attributable to the phosphorylation of Akt and the sigma-1 receptor.

Histamine H3 receptor antagonist Clobenpropit protects propofol-induced apoptosis of hippocampal neurons through PI3K/AKT pathway.

OBJECTIVE: The aim of the study was to explore whether histamine H3 receptor antagonist Clobenpropit could protect propofol-induced neurotoxicity in hippocampal neurons. MATERIALS AND METHODS: Hippocampal neurons were extracted from neonatal rats and induced with propofol. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed to detect apoptotic rate of neurons. Western blot was conducted to detect protein levels of cleaved-caspase-3 and Bax/Bcl2. After LY294002 treatment, the PI3K pathway antagonist was applied in neurons, protein levels of cleaved-caspase-3 and Bax/Bcl2 were detected by Western blot as well. RESULTS: Propofol treatment remarkably induced neuronal apoptosis. Clobenpropit alleviated cell apoptosis induced by propofol. Protein expressions of cleaved-caspase-3 and Bax/Bcl2 were remarkably downregulated in neurons treated with Clobenpropit. LY294002 induction remarkably reverses the protective role of Clobenpropit in neuronal apoptosis, manifesting as downregulated PI3K and p-AKT after LY294002 treatment. CONCLUSIONS: Clobenpropit protects propofol-induced neuronal apoptosis through activating PI3K/AKT pathway.