Puerarin protects rat brain against ischemia/reperfusion injury by suppressing autophagy via the AMPK-mT OR-ULK1 signaling pathway

Puerarin suppresses autophagy to alleviate cerebral ischemia/reperfusion injury, and accumulating evidence indicates that the AMPKm TOR signaling pathway regulates the activation of the autophagy pathway through the coordinated phosphorylation of ULK1. In this study, we investigated the mechanisms underlying the neuroprotective effect of puerarin and its role in modulating autophagy via the AMPK-m TOR-ULK1 signaling pathway in the rat middle cerebral artery occlusion model of cerebral ischemia/reperfusion injury. Rats were intraperitoneally injected with puerarin, 50 or 100 mg/kg, daily for 7 days. Then, 30 minutes after the final administration, rats were subjected to transient middle cerebral artery occlusion for 90 minutes. Then, after 24 hours of reperfusion, the Longa score and infarct volume were evaluated in each group. Autophagosome formation was observed by transmission electron microscopy. LC3, Beclin-1 p62, AMPK, m TOR and ULK1 protein expression levels were examined by immunofluorescence and western blot assay. Puerarin substantially reduced the Longa score and infarct volume, and it lessened autophagosome formation in the hippocampal CA1 area following cerebral ischemia/reperfusion injury in a dose-dependent manner. Pretreatment with puerarin（50 or 100 mg/kg） reduced Beclin-1 expression and the LC3-II/LC3-I ratio, as well as p-AMPK and p S317-ULK1 levels. In comparison, it increased p62 expression. Furthermore, puerarin at 100 mg/kg dramatically increased the levels of p-m TOR and p S757-ULK1 in the hippocampus on the ischemic side. Our findings suggest that puerarin alleviates autophagy by activating the APMK-m TOR-ULK1 signaling pathway. Thus, puerarin might have therapeutic potential for treating cerebral ischemia/reperfusion injury.

Forkhead box protein O1(FoxO1)regulates lipids metabolism and cell proliferation mediated by insulin and PI3K-Akt-mTOR pathway in goose primary hepatocytes

In order to explore the role of forkhead box protein O1(FoxO1)in the lipid metabolism and cell proliferation,goose primary hepatocytes were isolated and incubated with insulin or PI3K-Akt-mTOR pathway dual inhibitor NVPBEZ235,and then transfected with FoxO1 interference plasmid.The related parameters of lipid metabolism and cell proliferation were measured.The results firstly showed that FoxO1 interference increased the intracellular TG and lipids concentration(P<0.05);and increased the proliferative index(PI),cell DNA synthesis,protein expression of Cyclin D1 in goose primary hepatocytes(P<0.05).Secondly,the co-treatment of insulin and FoxO1 interference increased the mRNA level and protein content of Cyclin D1(P<0.05);however,there was no significant difference between the insulin treatment and the co-treatment of insulin and miR-FoxO1 interference in the intracellular TG and lipids concentration and PI(P>0.05).Lastly,the decrease of intracellular TG and lipids concentration and PI induced by NVP-BEZ235 was up-regulated by FoxO1 interference significantly(P<0.05).In summary,FoxO1 could regulate the lipids metabolism and cell proliferation mediated by PI3K-Akt-mTOR signaling pathway in goose primary hepatocytes.Further investigations are required to highlight the potential role of FoxO1 in the lipid metabolism and cell proliferation mediated by insulin in goose primary hepatocyte.

Regulatory effects of antitumor agent matrine on FOXO and PI3K-AKT pathway in castration-resistant prostate cancer cells

We previously demonstrated that matrine could inhibit the proliferating, migrating, as well as invading processes of both PC-3 and DU145 cells. However, the underlying molecular mechanisms have not yet been clearly defined. In this study, using various techniques such as high throughput sequencing technology, bioinformatics, quantitative real-time PCR, and immunoblot analysis,we aimed to understand whether matrine serves as a novel regulator of FOXO and PI3K-AKT signaling pathway. DU145 and PC-3 cell lines were cultured for 24 h in vitro. Cells were treated with either matrine or control serum for 48 h, followed by extraction of total RNA. The RNA was sequenced using HiSeq 2500 high-throughput sequencing platform (Illumina). A gene library was established and quality analysis of read data carried out. Integrated database from the website DAVID was used to analyze Gene Ontology (GO), and Kyoto encyclopedia of genes and genomes (KEGG) pathway of differential genes was used for pathway analysis, screening for fold differences of more than two times. The FOXO and PI3K-AKT signaling pathways were screened, and expression levels of mRNA and core protein detected by real-time PCR and immunoblotting, respectively. High throughput sequencing and GO analysis revealed that differentially expressed genes before and after treatment played an important role in cell metabolic process, growth process, anatomical structure formation, cellular component organization, and biological regulation. KEGG signal pathway analysis revealed that FOXO and PI3K-AKT signal pathways had a significant difference between before and after matrine-treated androgen-independent prostate cancer cells PC-3 and DU145. Real-time PCR showed that matrine treatment led to a significant increase in the expression levels of FOXO1A, FOXO3A, FOXO4, and FOXO6 in DU145 and PC-3 cells (P<0.01 or P<0.05), whereas the PI3K expression levels decreased (P<0.01). Similarly, immunoblotting revealed a significant increase (P<0.05) in the expression levels of FOXO1A FOXO3A, FOXO4, and FOXO6 in both PC-3 and DU145 cells, whereas PI3K expression levels decreased (P<0.05). Matrine had a broad regulating effect on the mRNA expression profiles of both PC-3 and DU145 cells. Matrine may inhibit cell proliferation, migration, as well as invasion, and induce apoptosis in both PC-3 and DU145 cells through FOXO and PI3K-AKT signaling pathways. Matrine could therefore be used as a complementary drug to present chemotherapeutic agents, for treating androgen-independent prostate cancer.

Anti-hyperglycemic effects of dihydromyricetin in streptozotocin-induced diabetic rats

Dihydromyricetin(DHM),as a bioactive flavanonol compound,is mainly found in“Tengcha”(Ampelopsis grossedentata)cultivated in south of China.This study aimed to investigate the anti-hyperglycemic and antidyslipidemic activities of DHM using type 2 diabetes mellitus(T2D)rats,which was induced by feeding with high fat and fructose diet for 42 days and intraperitoneal administration of streptozocin.Forty-eight freshlyweaned rats were randomly assigned into the negative control(Blank),low dose(100 mg/kg),medium dose(200 mg/kg),high dose(400 mg/kg),and positive(40 mg/kg,met)groups.Fasting blood glucose and body weight were measured at weekly interval.Oral glucose tolerance tests were performed on days 42.The results revealed that DHM possessed significant antihyperglycaemic and antihyperinsulinemic effects.Moreover,after the DHM treatment,p-Akt and p-AMPK expression was upregulated,and glycogen synthase kinase-3β(GSK-3β)expression was downregulated,indicating that the potential anti-diabetic mechanism of DHM might be due to the regulation of the AMPK/Akt/GSK-3βsignaling pathway.

Novel nervous and multi-system regenerative therapeutic strategies for diabetes mellitus with mTOR

Throughout the globe,diabetes mellitus（DM） is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder.DM impacts multiple organs and affects all components of the central and peripheral nervous systems that can range from dementia to diabetic neuropathy.The mechanistic target of rapamycin（m TOR） is a promising agent for the development of novel regenerative strategies for the treatment of DM.m TOR and its related signaling pathways impact multiple metabolic parameters that include cellular metabolic homeostasis,insulin resistance,insulin secretion,stem cell proliferation and differentiation,pancreatic β-cell function,and programmed cell death with apoptosis and autophagy.m TOR is central element for the protein complexes m TOR Complex 1（m TORC1） and m TOR Complex 2（m TORC2） and is a critical component for a number of signaling pathways that involve phosphoinositide 3-kinase（PI 3-K）,protein kinase B（Akt）,AMP activated protein kinase（AMPK）,silent mating type information regulation 2 homolog 1（Saccharomyces cerevisiae）（SIRT1）,Wnt1 inducible signaling pathway protein 1（WISP1）,and growth factors.As a result,m TOR represents an exciting target to offer new clinical avenues for the treatment of DM and the complications of this disease.Future studies directed to elucidate the delicate balance m TOR holds over cellular metabolism and the impact of its broad signaling pathways should foster the translation of these targets into effective clinical regimens for DM.