Upregulation Of Akt Phosphorylation Research Articles

Inhibition of Selenoprotein I promotes ferroptosis and reverses resistance to platinum chemotherapy by impairing Akt phosphorylation in ovarian cancer.

Ovarian cancer (OV) ranks among the deadliest gynecological cancer, known for its high risk of relapse and metastasis, and a general resistance to conventional platinum-based chemotherapy. Selenoprotein I (SELENOI) is a crucial mediator implicated in human hereditary spastic paraplegia. However, its role in human tumors remains poorly elucidated. Here, we comprehensively analyzed SELENOI expression patterns, functions, and clinical implications across various malignancies through the integration of bulk transcriptomics, cancer databases, and in vitro and in vivo experiments. Pan-cancer analysis indicated upregulated SELENOI expression across various cancers, correlating with augmented malignancy, suppressed tumor immunity and poor prognosis. Knockdown of SELENOI caused G0/G1-phase cell cycle arrest and diminished aggressive cancer phenotypes in OV cells. Moreover, SELENOI inhibition augments ferroptosis and reverses the cisplatin resistance in OV cells by modulating Akt phosphorylation. Conversely, overexpression of SELENOI in OV cells enhanced therapeutic sensitivity to cisplatin by upregulating Akt phosphorylation. Importantly, in vivo studies demonstrated that SELENOI inhibition suppressed ovarian tumor growth and enhanced cisplatin's anticancer effects. These findings highlight the significant role of SELENOI in OV by modulating ferroptosis and chemotherapy resistance. Targeting SELENOI represents a promising therapeutic approach to promote the efficacy of platinum-based chemotherapy in OV, particularly in cases of resistance.

Bioinformatics and validation reveal the potential target of curcumin in the treatment of diabetic peripheral neuropathy

Diabetic peripheral neuropathy (DPN) is a common nerve-damaging complication of diabetes mellitus. Effective treatments are needed to alleviate and reverse diabetes-associated damage to the peripheral nerves. Curcumin is an effective neuroprotectant that plays a protective role in DPN promoted by Schwann cells (SCs) lesions. However, the potential molecular mechanism of curcumin remains unclear. Therefore, our aim is to study the detailed molecular mechanism of curcumin-mediated SCs repair in order to improve the efficacy of curcumin in the clinical treatment of DPN. First, candidate target genes of curcumin in rat SC line RSC96 cells stimulated by high glucose were identified by RNA sequencing and bioinformatic analyses. Enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) was carried out by Metascape, followed by 8 algorithms on Cytoscape to determine 4 hub genes, namly Hmox1, Pten, Vegfa and Myc. Next, gene set enrichment analysis (GSEA) and Pearson function showed that Hmox1 was significantly correlated with apoptosis. Subsequently, qRT-PCR, MTT assay, flow cytometry, caspase-3 activity detection and westernblot showed that curcumin treatment increased RSC96 cell viability, reduced cell apoptosis, increased Hmox1, Pten, Vegfa and Myc expression, and up-regulated Akt phosphorylation level under high glucose environment. Finally, molecular docking predicted the binding site of curcumin to Hmox1. These results suggest that curcumin can reduce the apoptosis of SCs induced by high glucose, and Hmox1 is a potential target for curcumin. Our findings provide new insights about the mechanism of action of curcumin on SC as a potential treatment in DPN.

Metformin ameliorates mitochondrial damage induced by C9orf72 poly(GR) via upregulating AKT phosphorylation.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are devastating neurodegenerative diseases with no effective cure. GGGGCC repeat expansion in C9orf72 is the most common genetic cause of both ALS and FTD. A key pathological feature of C9orf72 related ALS/FTD is the presence of abnormal dipeptide repeat proteins translated from GGGGCC repeat expansion, including poly Glycine-Arginine (GR). In this study, we observed that (GR)50 conferred significant mitochondria damage and cytotoxicity. Metformin, the most widely used clinical drug, successfully relieved (GR)50 induced mitochondrial damage and inhibited (GR)50 related cytotoxicity. Further research revealed metformin effectively restored mitochondrial function by upregulating AKT phosphorylation in (GR)50 expressed cells. Taken together, our results indicated restoring mitochondrial function with metformin may be a rational therapeutic strategy to reduce poly(GR) toxicity in C9orf72 ALS/FTD patients.

Interaction between neutrophil extracellular traps and cardiomyocytes contributes to atrial fibrillation progression

Atrial fibrillation (AF) is a frequent arrhythmia associated with cardiovascular morbidity and mortality. Neutrophil extracellular traps (NETs) are DNA fragments with cytoplasm proteins released from neutrophils, which are involved in various cardiovascular diseases. To elucidate the role of NETs in AF, we investigated the effect of NETs on AF progression and the secretion of NETs in AF. Results showed that: NETs induced the autophagic apoptosis of cardiomyocytes, and NETs also led to mitochondrial injury by promoting mitochondrial depolarization and ROS production. Ongoing tachy-pacing led to the structural loss of cardiomyocytes and provided potent stimuli to induce NETs secretion from neutrophils. In the meanwhile, increased Ang II in AF facilitated NETs formation through the upregulation of AKT phosphorylation, while it could not directly initiate NETosis as the autophagy was not induced. In vivo, DNase I was administrated to abrogate NETs formation, and AF-related fibrosis was ameliorated as expected. Correspondingly, the duration of the induced AF was reduced. Our study addresses the formation mechanism of NETs in AF and demonstrates the lethal effects of NETs on cardiomyocytes through the induction of mitochondrial injury and autophagic cell death, which comprehensively describes the positive feedback comprised of NETs and stimuli secreted by cardiomyocytes that sustains the progression of AF and AF related fibrosis.

TRIM31 promotes the progression of oral squamous cell carcinoma through upregulating AKT phosphorylation and subsequent cellular glycolysis.

The regulation of protein kinase B (AKT) phosphorylation by Tripartite motif-containing protein 31 (TRIM31) is implicated as an essential mechanism in the progression of many malignant tumors. Nevertheless, the function of the TRIM31/AKT pathway in oral squamous cell carcinoma (OSCC) remains elusive. Here, immunohistochemistry analysis of human OSCC tissue microarrays indicated significantly higher levels of TRIM31 and phosphorylated AKT (p-AKT) in OSCC tumors than in adjacent tissue samples. Also, we detected a positive association between TRIM31 expression and clinical OSCC development. In in vitro studies, TRIM31 knockdown severely impaired OSCC cell growth, invasion, and migration. By contrast, TRIM31 overexpression improved these cell behaviors, while subsequent AKT inhibition abrogated the effect. In vivo tumorigenesis experiments using nude mice also validated the effects of TRIM31/AKT signaling in tumor growth. Furthermore, TRIM31 upregulation facilitated glucose uptake, as well as lactate and adenosine triphosphate production of OSCC cells, while such positive effects on glycolysis and malignant cell phenotypes were reversed by treatment with AKT or glycolysis inhibitors. In conclusion, TRIM31 may improve OSCC progression by enhancing AKT phosphorylation and subsequent glycolysis. Hence, TRIM31 has the potential as a treatment target in OSCC.

Role of PI3K/Akt-Mediated Nrf2/HO-1 Signaling Pathway in Resveratrol Alleviation of Zearalenone-Induced Oxidative Stress and Apoptosis in TM4 Cells.

Zearalenone (ZEA) is a common mycotoxin that induces oxidative stress (OS) and affects the male reproductive system in animals. Resveratrol (RSV) has good antioxidant activity and can activate nuclear factor erythroid 2-related factor (Nrf2) to protect cells through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. The objective of this study was to investigate the protective effect and the mechanism of RSV on OS and apoptosis in TM4 cells induced by ZEA. Prior to being exposed to ZEA, TM4 cells were pretreated with RSV or the PI3K/Akt inhibitor LY294002. Cell viability was measured by Cell Counting Kit-8 (CCK-8) assays. Flow cytometry was used to determine the level of apoptosis and intracellular reactive oxygen species (ROS). The expression of poly ADP-ribose polymerase (PARP), caspase-3, BCL2-associated X (Bax)/B-cell lymphoma-2 (Bcl-2), and PI3K/Akt-mediated Nrf2/heme oxygenase 1 (HO-1) signaling pathway-related proteins was evaluated by Western blotting. Nrf2 siRNA transfection and LY294002 treatment were used to investigate the role of the Nrf2/HO-1 and PI3K/Akt signaling pathways in RSV alleviation of ZEA-induced OS. The results showed that pretreatment with RSV significantly reduced the expression of apoptosis-related proteins and increased cell viability. Catalase (CAT) activity and glutathione (GSH) levels were also increased, whereas malondialdehyde (MDA) and ROS levels decreased (p < 0.05). RSV also upregulated Akt phosphorylation, Nrf2 nuclear translocation, and HO-1 expression under conditions of OS (p < 0.05). Transfection with Nrf2 siRNA abolished the protective effects of RSV against ZEA-induced cytotoxicity (p < 0.05), ROS accumulation (p < 0.05), and apoptosis (p < 0.05). LY294002 completely blocked the RSV-mediated increase in Nrf2 nuclear translocation (p < 0.05), HO-1 expression (p < 0.05), and cytoprotective activity (p < 0.05). Collectively, the above findings indicate that RSV can protect against ZEA-induced OS and apoptosis in TM4 cells by PI3K/Akt-mediated activation of the Nrf2/HO-1 signaling pathway.

EPAC Regulates Melanoma Growth by Stimulating mTORC1 Signaling and Loss of EPAC Signaling Dependence Correlates with Melanoma Progression.

This study establishes loss of dependency on EPAC-mTORC1 signaling as hallmark of primary melanoma evolution and targeting this escape mechanism is a promising strategy for metastatic melanoma.

Kai-Xin-San Inhibits Tau Pathology and Neuronal Apoptosis in Aged SAMP8 Mice

Alzheimer’s disease (AD) is an age-related neurological disorder. Currently, there is no effective cure for AD due to its complexity in pathogenesis. In light of the complex pathogenesis of AD, the traditional Chinese medicine (TCM) formula Kai-Xin-San (KXS), which was used for amnesia treatment, has been proved to improve cognitive function in AD animal models. However, the active ingredients and the mechanism of KXS have not yet been clearly elucidated. In this study, network pharmacology analysis predicts that KXS yields 168 candidate compounds acting on 863 potential targets, 30 of which are associated with AD. Enrichment analysis revealed that the therapeutic mechanisms of KXS for AD are associated with the inhibition of Tau protein hyperphosphorylation, inflammation, and apoptosis. Therefore, we chose 7-month-old senescence-accelerated mouse prone 8 (SAMP8) mice as AD mouse model, which harbors the behavioral and pathological hallmarks of AD. Subsequently, the potential underlying action mechanisms of KXS on AD predicted by the network pharmacology analyses were experimentally validated in SAMP8 mice after intragastric administration of KXS for 3 months. We observed that KXS upregulated AKT phosphorylation, suppressed GSK3β and CDK5 activation, and inhibited the TLR4/MyD88/NF-κB signaling pathway to attenuate Tau hyperphosphorylation and neuroinflammation, thus suppressing neuronal apoptosis and improving the cognitive impairment of aged SAMP8 mice. Taken together, our findings reveal a multi-component and multi-target therapeutic mechanism of KXS for attenuating the progression of AD, contributing to the future development of TCM modernization, including KXS, and broader clinical application.Graphical abstract

Substance P modulates BMSCs migration for tissue repair through NK-1R/CXCR4/p-Akt signal activation.

The migration of bone marrow-derived mesenchymal stem cells (BMSCs) to the wound site played an important role in tissue repair. Substance P (SP) has been studied and reported to be involved in tissue repair by promoting the growth of endothelial cells and the migration of BMSCs. However, the complicated process and the molecular mechanisms were not fully understood. Thus, we aimed to investigate the effect of SP-induced BMSCs migration on tissue repair and its possible mechanism. Western blot and q-PCR assay revealed that SP could induce the BMSCs migration through overexpression of CXCR4 and upregulation of Akt phosphorylation. And the upregulation was related to the activation of neurokinin-1 receptor (NK-1R). Besides, we found that the increased phosphorylation Akt caused by SP could be canceled by the inhibition of CXCR4 both in vitro and in vivo. Furthermore, a skin-injury animal model was established and used to observe the tissue repair process. Results showed that SP could accelerate wound closure, gain more granulation tissue accumulation, and more collagen deposition through the promotion of angiogenesis and induction of the BMSCs migration to the wound site. And these effects could be impaired by inhibition of CXCR4 and p-Akt. Our results suggested that SP promoted tissue repair through BMSCs migration via upregulation of CXCR4 and p-Akt. The expression of CXCR4 and p-Akt were regulated by NK-1R activation. These findings add more evidence in understanding the mechanisms of SP-induced BMSCs migration and highlight the potential for clinical implementation of SP in tissue repair.

Phytochemical‑rich herbal formula ATG‑125 protects against sucrose‑induced gastrocnemius muscle atrophy by rescuing Akt signaling and improving mitochondrial dysfunction in young adult mice.

The antioxidant capability of herbal remedies has attracted widespread attention, but their molecular mechanisms in a muscle atrophy model have not been explored. The aim of the present study was to compare the bioactivity of sucrose challenged mice following treatment with ATG-125. Here, through a combination of transcriptomic and biomedical analysis, herbal formula ATG-125, a phytochemical-rich formula, was identified as a protective factor against muscle atrophy in sucrose challenged mice. Gene ontology (GO) identified differentially expressed genes that were primarily enriched in the ‘negative regulation of proteolysis’, ‘cellular amino acid metabolic process’, ‘lipoprotein particle’ and ‘cell cycle’, all of which were associated with the ATG-125-mediated prevention of muscle atrophy, particularly with regard to mitochondrial biogenesis. In skeletal muscle, a set of mitochondrial-related genes, including angiopoietin-like 4, nicotinamide riboside kinase 2 (Nmrk2), pyruvate dehydrogenase lipoamide kinase isozyme 4, Asc-type amino acid transporter 1 and mitochondrial uncoupling protein 3 (Ucp3) were markedly upregulated following ATG-125 intervention. An increase in Nmrk2 and Ucp3 expression were noted after ATG-125 treatment, in parallel with upregulation of the ‘nicotinate and nicotinamide metabolism’ pathway, as determined using the Kyoto Encyclopedia of Genes and Genomes (KEGG). Furthermore, KEGG pathway analysis revealed the downregulation of ‘complement and coagulation cascades’, ‘cholesterol metabolism’, ‘biosynthesis of amino acids’ and ‘PPAR signaling pathway’, which were associated with the downregulation of serine (or cysteine) peptidase inhibitor clade A member (Serpina)3, Serpina1b, Serpina1d, Serpina1e, apolipoprotein (Apo)a1 and Apoa2, all of which were cardiovascular and diabetes-associated risk factors and were regulated by ATG-125. In addition, ATG-125 treatment resulted in downregulated mRNA expression levels of ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2, troponin-I1, troponin-C1 and troponin-T1 in young adult gastrocnemius muscle compared with the sucrose group. Nuclear factor-κB-hypoxia inducible factor-1α-TGFβ receptor type-II-vascular endothelial growth factor staining indicated that ATG-125 decreased sucrose-induced chronic inflammation. ATG-125 was sufficient to prevent muscle atrophy, and this protective effect may be mediated through upregulation of AKT phosphorylation, upregulating the insulin growth factor-1R-insulin receptor substrate-PI3K-AKT pathway, which in turn resulted in a forkhead box O-dependent decrease in protein degradation pathways, including regulation of atrogin1 and E3 ubiquitin-protein ligase TRIM63. Peroxisome-proliferator activated receptor γ coactivator 1α (PGC1α) was decreased in young adult mice challenged with sucrose. ATG-125 treatment significantly increased PGC1α and significantly increased UCP-1,2,3 expression levels, which suggested ATG-125 poised the mitochondria for uncoupling of respiration. This effect is consistent with the increased SIRT1 levels and may explain an increase in mitochondria biogenesis. Taken together, the present study showed that ATG-125, as an integrator of protein synthesis and degradative pathways, prevented muscle wasting.

MEK1/2 is a bottleneck that induces cancer stem cells to activate the PI3K/AKT pathway

Cancer stem cells (CSCs) are responsible for cancer initiation, drug resistance, and aggressive tumor phenotypes. Our lab has established a novel method to induce CSCs from induced pluripotent stem (iPS) cells in a microenvironment mimicking chronic inflammation. The converted cells acquired CSC characteristics and developed malignant tumors. Recently, we demonstrated that nonmutagenic chemical inhibitors accelerated the conversion of mouse iPS (miPS) cells into CSCs. Here, we investigated the effects of AZD-6244, a MEK1/2-specific inhibitor, on the conversion of iPS cells into CSCs. The miPS cells were cultured for one week in the presence of the conditioned medium (CM) of Lewis lung carcinoma (LLC) cells and AZD-6244, PD0325901, a pan-MEK inhibitor, or GDC-0879, a B-Raf inhibitor. As a result, AZD-6244 enhanced the conversion of iPS cells into CSCs and upregulated AKT phosphorylation as same as GDC-0879 and PD0325901. The converted cells maintained their self-renewal ability and stemness gene expression. The expression of the CSC markers CD24, CD44 and CD133 was higher in the cells cultured with MAPK inhibitors than in those cultured without MAPK inhibitors. Moreover, converted cells gained migration and invasion abilities assessed by in vitro assays. Therefore, the inhibition of MEK1/2 was found to be critical for the conversion of normal stem cells into CSCs in the tumor-inducing microenvironment.

Protective Effect of Chrysanthemum morifolium cv. Fubaiju Hot-Water Extracts Against ARPE-19 Cell Oxidative Damage by Activating PI3K/Akt-Mediated Nrf2/HO-1 Signaling Pathway

Chrysanthemum morifolium cv. Fubaiju is a kind of widely consumed herb tea with multiple health benefits. The present study was aimed to evaluate the protective capacity of C. morifolium cv. Fubaiju hot-water extracts (CMs) against ARPE-19 cell oxidative damage. The results showed that pretreatment with 100 μg/mL CM could significantly reduce cell oxidative damage and apoptosis. Proapoptotic protein expression such as Bax, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP) was significantly decreased after CM addition, while the expression level of antioxidant enzymes including catalase, glutamate-cysteine ligase catalytic subunit (GCLc), superoxide dismutase 2 (SOD2), and NAD(P)H:quinone oxidoreductase 1 (NQO-1) was significantly promoted. Meanwhile, CM treatment upregulated Akt phosphorylation, nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation, and the expression level of antioxidant gene heme oxygenase-1 (HO-1) in a dose-dependent manner under oxidative stress. Knockdown of Nrf2 by targeted small interfering RNA (siRNA) alleviated CM-mediated HO-1 transcription and almost abolished CM-mediated protection against hydrogen peroxide (H2O2)-induced cell damage. Correspondingly, the protective effect of CM was dramatically blocked after interference with phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor LY294002, indicating that the protective effect of CM on cell oxidative damage was attributed to PI3K/Akt-mediated Nrf2/HO-1 signaling pathway.

Effects of Phthalate Esters on Human Myometrial and Fibroid Cells: Cell Culture and NOD-SCID Mouse Data.

Evidence is growing that phthalate esters play an important role in the pathogenesis of estrogen-dependent gynecologic diseases, especially uterine fibroids. We aimed to investigate whether in vitro treatment with di-(2-ethylhexyl)-phthalate (DEHP) affects angiogenesis, proliferation, and apoptosis in uterine fibroids. To ascertain this, we evaluated vascular endothelial growth factor (VEGF) expression and AKT/ERT phosphorylation and compared the fibroid volume between nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice fed with and without DEHP. VEGF expression was measured using enzyme-linked immunosorbent assay, and AKT/ERK phosphorylation was analyzed by western blot analysis in human myometrial and fibroid cells. The volume of the fibroid tissues implanted to NOD/SCID mice was measured, and the expression of collagen type I protein, Ki-67, proliferating cell nuclear antigen, and B cell lymphoma 2 were analyzed using immunohistochemistry. We could see significant increases in VEGF expression and AKT phosphorylation in human myometrial and fibroid cells treated with DEHP. The volume of the fibroid tissues was significantly increased in NOD/SCID mice fed with DEHP, which was accompanied by increased expression of collagen type I and AKT phosphorylation. Taken together, these results suggest that exposure to phthalate esters may influence uterine fibroid pathogenesis by increasing VEGF and collagen expression and upregulating AKT phosphorylation.

Scratching damages tight junctions through the Akt–claudin 1 axis in atopic dermatitis

Atopic dermatitis (AD) is a common, chronic, severely pruritic, eczematous skin disease that seriously deteriorates the quality of life of patients. Scratching is a cardinal symptom of AD. Although the vicious itch-scratch cycle continues and aggravates skin barrier dysfunction in AD, how scratching induces skin barrier dysfunction through tight junctions remains unclear. To study the effect of scratching on tight junctions in the itch-scratch cycle. Scratching behaviour and skin barrier dysfunction on the neck and back in an AD mouse model were assessed. The expression of tight junction proteins was compared between the neck and back mice, and the mechanisms underlying the involvement of Akt/CLDN1 pathways in this process were explored. We used oxazolone to induce AD on the neck or back of mice. There was significantly more scratching behaviour and more pronounced skin barrier dysfunction with the neck than with the back. Downregulation of claudin-1 (CLDN1) and upregulation of Akt phosphorylation in skin were well correlated with scratching behaviour in this AD model. Furthermore, SC79, an agonist of Akt phosphorylation, could downregulate CLDN1 expression in HaCaT cells. An antagonist of Akt phosphorylation (LY294002) was used to treat the AD mice; this treatment rescued CLDN1 expression through inhibiting Akt phosphorylation in skin, and importantly, also inhibited the scratching behaviour induced by AD. The results reveal the underlying mechanism of tight junction damage promoted by scratching in the itch-scratch cycle of AD, and opens a new avenue to pruritus management in AD, through Akt antagonists.

Scutellarin, a modulator of mTOR, attenuates hepatic insulin resistance by regulating hepatocyte lipid metabolism via SREBP-1c suppression.

High levels of consumption of saturated lipids have been largely associated with the increasing prevalence of metabolic diseases. In particular, saturated fatty acids such as palmitic acid (PA) have been implicated in the development of insulin resistance (IR). Scutellarin (Scu) is one of the effective traditional Chinese medicines considered beneficial for liver diseases and diabetes. In this study, we investigated the effect of Scu on IR and lipid metabolism disorders in vitro and in high fat diet (HFD)-fed mice. In vitro, we found that Scu decreased insulin-dependent lipid accumulation and the mRNA expression of CD36, Fasn, and ACC in PA-treated HepG2 cells. Additionally, Scu upregulated Akt phosphorylation and improved the insulin signalling pathway. Moreover, Scu downregulated mammalian target of rapamycin (mTOR) phosphorylation and the n-SREBP-1c protein level and also reduced lipid accumulation via the mTOR-dependent pathway, as confirmed by the molecular docking of Scu to mTOR. In HFD-fed C57BL/6 mice, Scu improved oral glucose tolerance, pyruvate tolerance and the IR index and also increased the Akt phosphorylation level. Moreover, Scu reduced hepatocyte steatosis, decreased lipid accumulation and triglyceride levels, inhibited mTOR phosphorylation, and decreased the SREBP-1c level in the liver. Taken together, these findings suggest that Scu ameliorates hepatic IR by regulating hepatocyte lipid metabolism via the mTOR-dependent pathway through SREBP-1c suppression.

Resveratrol Attenuates Oxidative Stress-Induced Intestinal Barrier Injury through PI3K/Akt-Mediated Nrf2 Signaling Pathway.

Oxidative stress is implicated in a wide range of intestinal disorders and closely associated with their pathological processes. Resveratrol (RSV), a plant extract, plays a vital role in protecting various organs in vitro and in vivo. However, the benefits of RSV are controversial, and underlying mechanisms for its antioxidant effects on intestinal epithelial cells remain unclear. In this study, we evaluated the effects of RSV on oxidative stress induced by H2O2 in IPEC-J2 cells. We found that pretreatment with RSV significantly increased cell viability; increased expression levels of tight junction (TJ) proteins (claudin-1, occludin, and ZO-1); improved activities of superoxide dismutase-1 (SOD-1), catalase (CAT), and glutathione peroxidase (GSH-Px); and decreased intracellular reactive oxygen species (ROS) levels and apoptosis induced by H2O2 (P < 0.05). In addition, RSV upregulated Akt phosphorylation, Nrf2 phosphorylation, and expression levels of antioxidant genes HO-1, SOD-1, and CAT in a dose-dependent manner (P < 0.05) under oxidative stress. Knockdown of Nrf2 by short-hairpin RNA (shRNA) abrogated RSV-mediated protection against H2O2-induced apoptosis, RSV-induced increase of TJ protein levels, and antioxidant gene expression (SOD-1, CAT, and GSH-Px) (P < 0.05). Consistent with Nrf2 knockdown, the PI3K/Akt inhibitor LY294002 significantly suppressed RSV-induced Nrf2 phosphorylation and RSV-induced increase of TJ protein levels and antioxidant gene expression under H2O2 treatment (P < 0.05). Collectively, these results demonstrate that RSV can directly protect IPEC-J2 cells against oxidative stress through the PI3K/Akt-mediated Nrf2 signaling pathway, suggesting that RSV may be an effective feed additive against intestinal damage in livestock production.

17-DMAG, an Hsp90 inhibitor, ameliorates ovariectomy-induced obesity in rats

AimsObesity is not only associated with metabolic diseases but is also a symptom of menopause in women. To date, there are no effective drugs for the management of obesity, and it is important to find new agents with fewer side effects, for the treatment of obesity. This study aimed to determine the anti-obesity effect of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a heat shock protein 90 (Hsp90) inhibitor, and its underlying mechanism in rats with ovariectomy-induced obesity. Main methodsOvariectomy (Ovx) rats were treated with 17-DMAG (1 mg kg−1, intraperitoneally) for eight weeks from one week after surgery. The body weight, food intake, locomotor activity, adipogenic- and autophagy-related protein expression in white adipose tissue (WAT) and plasma triglyceride (TG) levels were measured in sham and Ovx rats. Key findingsCompared with sham rats, Ovx rats showed increased weight gain, food intake, WAT mass, TG levels, adipogenic protein expression, and decreased locomotor activity. Furthermore, autophagy-related proteins and Foxo3a of WAT were significantly increased in Ovx rats. However, with the exclusion of increased food intake, the changes induced by Ovx were all reversed in 17-DMAG-treated Ovx rats. In addition, the expression of Hsp70 and phosphorylation of Akt increased in 17-DMAG-treated Ovx rats. SignificanceThese results suggest that 17-DMAG significantly ameliorated obesity induced by Ovx, and this phenomenon is accompanied by the downregulation of adipogenic-related and autophagy-related proteins as well as the upregulation of Akt-phosphorylation and Hsp70 expression. Therefore, 17-DMAG may be a potential agent for preventing or treating obesity in postmenopausal women.

Identification of a Calcium-sensing Receptor in Human Dental Pulp Cells That Regulates Mineral Trioxide Aggregate–induced Mineralization

IntroductionThe purpose of this study was to verify the expression of the calcium-sensing receptor (CaSR) and its role in mineral trioxide aggregate (MTA)-induced odontoblastic differentiation and mineralization in human dental pulp cells (hDPCs). MethodsThe expression of CaSR in human dental pulp tissue and hDPCs was detected using immunohistochemical and immunofluorescent assays. Then, hDPCs were cultured in specific medium supplemented with defined concentrations of MTA dilute alone or in combination with calcimimetic R-568 (a positive allosteric modulator of CaSR [Tocris Bioscience, Bristol, UK]), and cell viability was monitored by Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) analysis. Alkaline phosphatase activity, alizarin red S staining, quantitative real-time polymerase chain reaction, and Western blot were used to investigate the gene/protein expression of odontoblastic-associated markers and CaSR in medium supplemented with different combinations of diluted MTA, R-568, and calcilytic Calhex 231 (a negative allosteric modulator of CaSR [Sigma-Aldrich, St Louis, MO]). ResultsCaSR was slightly expressed in the central pulp tissue, whereas it was strongly expressed in the odontoblast layer, plasma membrane, and cytoplasm of hDPCs. Cell Counting Kit-8 assay indicated maximum cell viability in cultures treated with 1:8 diluted MTA additives. Compared with undifferentiated controls, the cells at the early stage of odontoblastic differentiation exhibited lower CaSR protein expression. The combination of 1:8 diluted MTA with 0.1 and 1.0 μmol/L R-568 led to significantly increased cell vitality but decreased alkaline phosphatase activity and mineralized deposit formation, and this negative effect could be attenuated by 1.0 μmol/L Calhex 231 supplementation. Quantitative polymerase chain reaction results showed a significant up-regulation of RUNX2, DSPP, DMP-1, and OCN gene expression in the 1 μmol/L R-568–treated hDPCs. Western blot analysis indicated that the treatment by MTA and R-568 alone or their combination gave no clear trend on the protein levels of CaSR and dentin sialophosphoprotein, whereas Calhex 231 can increase their expressions. In addition, the up-regulation of Akt phosphorylation was observed in R-568– and Calhex 231–treated hDPCs. ConclusionsOur data indicated that CaSR is expressed in human dental pulp and hDPCs and that it can negatively or positively regulate MTA-induced mineralization of hDPCs via the phosphoinositide 3-kinase/Akt pathway in a ligand-dependent manner, suggesting a therapeutic target for modulating reparative dentin formation.

MT1‐MMP downregulation via the PI3K/Akt signaling pathway is required for the mechanical stretching‐inhibited invasion of bone‐marrow‐derived mesenchymal stem cells

Mobilization from the bone marrow and the migration of bone-marrow-derived mesenchymal stem cells (BMSCs) through the peripheral circulation to injured tissue sites are regulated by multiple mechanical and chemical factors. We previously demonstrated that mechanical stretching promotes the migration but inhibits the invasion of BMSCs. However, the involved mechanisms, especially the mechanism of stretching-inhibited BMSC invasion, have not been thoroughly elucidated to date. In this study, we found that mechanical stretching with a 10% amplitude at a 1-Hz frequency for 8 hr significantly reduces BMSC invasion and downregulates the expression of membrane type-1 matrix metalloproteinases (MT1-MMP) at both the messenger RNA and protein levels. The overexpression of MT1-MMP restores mechanical stretching-reduced BMSC invasion. Moreover, phosphatidylinositol 3-kinase (PI3K)-dependent Akt phosphorylation in BMSCs was found to be inactivated by mechanical stretching. Pharmacological inhibitors of PI3K/Akt signaling (LY294002 or A443654) reduced the expression of MT1-MMP and impaired BMSC invasion. In addition, the upregulation of Akt phosphorylation by a pharmacological activator (SC79) increased MT1-MMP expression and suppressed mechanical stretching-reduced BMSC invasion. Taken together, our results suggest that mechanical stretching inhibits BMSC invasion by downregulating MT1-MMP expression by suppressing the PI3K/Akt signaling pathway.

MiR‑214 reduces cisplatin resistance by targeting netrin‑1 in bladder cancer cells.

miR‑214 has been reported to be downregulated in several cancer types, such as bladder cancer. However, its involvement in apoptosis and chemoresistance has not been investigated. The present study aimed to clarify the biological function of miR‑214 and potential mechanisms in chemoresistance of bladder cancer cells. Reverse transcription‑quantitative polymerase chain reaction demonstrated that miR‑214 was downregulated in bladder cancer tissues compared with the level in normal tissues. miR‑214 was downregulated in bladder cancer cell lines compared with the level in the normal cell line SV‑HUC‑1. miR‑214 mimics were transfected into T24 and J82 cell lines to restore its expression. The results indicated that miR‑214 mimic inhibited proliferation and invasion in these cell lines. In addition, miR‑214 mimic reduced cisplatin resistance in T24 and J82 cells, indicated by the inhibition of cell viability and upregulation of cell apoptosis. Western blotting demonstrated that miR‑214 mimic was able to upregulate cleaved caspase‑3 and cleaved poly (ADP‑ribose) polymerase (PARP), while downregulate caspase‑3 and PARP expression, and AKT phosphorylation. Using prediction software, it was revealed that the netrin‑1 oncoprotein is on the target list of miR‑214. miR‑214 also downregulated netrin‑1 protein and mRNA expression levels in the T24 and J82 cell lines. Luciferase reporter assays demonstrated that netrin‑1 acted as a direct target of miR‑214. A negative correlation between netrin‑1 and miR‑214 expression in bladder cancer tissues was also observed. In addition, cisplatin treatment could induce netrin‑1 protein expression in bladder cancer cells and miR‑214 mimic partly blocked this phenomenon. Netrin‑1 plasmid transfection inhibited cisplatin‑induced apoptosis, upregulated AKT phosphorylation, and downregulated caspase‑3 and PARP cleavage. Netrin‑1 was restored in cells transfected with miR‑214 mimic using plasmid transfection. Netrin‑1 transfection restored AKT phosphorylation and blocked caspase/PARP cleavage in the T24 and J82 cell lines. In conclusion, the present study demonstrated that miR‑214 is downregulated in bladder cancer tissues and cell lines. miR‑214 reduces chemoresistance by targeting netrin‑1 in bladder cancer cell lines.