Hypoglycemic mechanism of Tegillarca granosa polysaccharides on type 2 diabetic mice by altering gut microbiota and regulating the PI3K-akt signaling pathwaye

Type 2 diabetes mellitus(T2DM)is a complex metabolic disease threatening human health.We investigated the effects of Tegillarca granosa polysaccharide(TGP)and determined its potential mechanisms in a mouse model of T2DM established through a high-fat diet and streptozotocin.TGP(5.1×10^(3) Da)was composed of mannose,glucosamine,rhamnose,glucuronic acid,galactosamine,glucose,galactose,xylose,and fucose.It could significantly alleviate weight loss,reduce fasting blood glucose levels,reverse dyslipidemia,reduce liver damage from oxidative stress,and improve insulin sensitivity.RT-PCR and Western blotting indicated that TGP could activate the phosphatidylinositol-3-kinase/protein kinase B signaling pathway to regulate disorders in glucolipid metabolism and improve insulin resistance.TGP increased the abundance of Allobaculum,Akkermansia,and Bifidobacterium,restored the microbiota abundance in the intestinal tracts of mice with T2DM,and promoted short-chain fatty acid production.This study provides new insights into the antidiabetic effects of TGP and highlights its potential as a natural hypoglycemic nutraceutical.

Spi1 regulates the microglial/macrophage inflammatory response via the PI3K/AKT/mTOR signaling pathway after intracerebral hemorrhage

Preclinical and clinical studies have shown that microglia and macrophages participate in a multiphasic brain damage repair process following intracerebral hemorrhage.The E26 transformation-specific sequence-related transcription factor Spi1 regulates microglial/macrophage commitment and maturation.However,the effect of Spi1 on intracerebral hemorrhage remains unclear.In this study,we found that Spi1 may regulate recovery from the neuroinflammation and neurofunctional damage caused by intracerebral hemorrhage by modulating the microglial/macrophage transcriptome.We showed that high Spi1expression in microglia/macrophages after intracerebral hemorrhage is associated with the activation of many pathways that promote phagocytosis,glycolysis,and autophagy,as well as debris clearance and sustained remyelination.Notably,microglia with higher levels of Soil expression were chara cterized by activation of pathways associated with a variety of hemorrhage-related cellular processes,such as complement activation,angiogenesis,and coagulation.In conclusion,our results suggest that Spi1 plays a vital role in the microglial/macrophage inflammatory response following intracerebral hemorrhage.This new insight into the regulation of Spi1 and its target genes may advance our understanding of neuroinflammation in intracerebral hemorrhage and provide therapeutic targets for patients with intracerebral hemorrhage.

MicroRNA (let-7b-5p)-targeted DARS2 regulates lung adenocarcinoma growth by PI3K/AKT signaling pathway

Background:The aberrant intraellular expression of a mitochondrial aspartyl tRNA synthetase 2(DARS2)has been reported in human cancers.Nevertheless its critical role and detailed mechanism in lung adenocarcinoma(LUAD)remain unexplored.Methods:Initially,The Cancer Genome Atlas(TCGA)based Gene Expression Profiling Interactive Analysis(GEPIA)database (http:/gepia.cancer-pku.cn/)was used to analyze the prognostic relevance of DARS2 expression in LUAD.Further,cell counting kit(CCK)8,immunostaining,and transwell invasion assays in LUAD cell lines in vitro,as well as DARS2 silence on LUAD by tumorigenicity experiments in wivo in nude mice,were performed.Besides,we analyzed the expression levels of p-PI3K(phosphorylated Phosphotylinosital3 kinase),PI3K,AKT(Protein Kinase B),p-AKT(phosphorylated Protein Kinase B),PCNA(proliferating cell nudear antigen),cleaved-caspase 3,E cadherin,and N-cadherin proteins using the Westem blot analysis.Results:LUAD tissues showed higher DARS2 expression compared to normal tissues.Upregulation of DARS2 could be related to Tumor-Node-Metastasis(TNM)stage,high lymph node metastasis,and inferior prognosis.DARS2 silence decreased the proliferation,migration,and invasion abilities of LUAD cells.In addition,the DARS2 downregulation decreased the PCNA and N-cadherin expression and increased cleaved:caspase 3 and E cadherin expressions in LUAD cells,coupled with the inactivation of the PI3K/AKT signaling pathway.Moreover,DARS2 silence impaired the tumonigenicity of LUAD in vivo.Interestingly,let:7b-5p could recognize DARS2 through a complementary sequence.Mechanistically,the increased let 7b 5p expression attenuated the promo oncogenic action of DARS2 during LUAD progression,which were inversely correlated to each other in the LUAD tssues Conclusion:In summary,let 7b-5p,downregulated DARS2 expression,regulating the progression of LUAD cells by the PI3K/AKT signaling pathway.

Acalypha australis L.extract inhibits B16 melanoma cell metastasis through PI3K/AKT signaling pathway

Background:Melanoma is a deadly skin tumor resulting from the malignant transformation of melanocytes.It is highly malignant and invasive,with the highest mortality rate among skin cancers.Acalypha australis L.(AAL),a plant with dual medicinal and culinary purposes,is commonly regarded as an edible wild vegetable in southern China.Additionally,AAL has a long history of medicinal use in China,often employed for its hemostatic,anti-diarrheal,and anti-inflammatory properties.Modern pharmacology has demonstrated that AAL possesses functions such as weight loss,antimicrobial activity,antiviral effects,and treatment for ulcerative colitis.However,there is currently no research available regarding its effectiveness and mechanisms of action on melanoma.Methods:In this investigation,we used methyl thiazolyl tetrazolium assay to detect cell viability,transwell assay to detect cell migration and invasion ability,and Western blot assay to detect relevant signaling pathways.Results:The present study reveals that 2 mg/mL AAL effectively suppresses the metastasis of B16 cells,while simultaneously triggering the expression of key apoptosis-related proteins,including Bcl-2,Bax,and cleaved caspased 3.Subsequent investigations demonstrate that AAL exerts this inhibitory effect via the PI3K/AKT signal transduction pathway,as evidenced by the observed deficits in Ras,AKT,p-AKT,and PI3K expression levels.Conclusion:These findings indicated that AAL could be a valuable therapeutic option for reducing the metastatic potential of B16 melanoma cells.

Melatonin improves synapse development by PI3K/Akt signaling in a mouse model of autism spectrum disorder

Autism spectrum disorders are a group of neurodevelopmental disorders involving more than 1100 genes,including Ctnnd2 as a candidate gene.Ctnnd2knockout mice,serving as an animal model of autis m,have been demonstrated to exhibit decreased density of dendritic spines.The role of melatonin,as a neuro hormone capable of effectively alleviating social interaction deficits and regulating the development of dendritic spines,in Ctnnd2 deletion-induced nerve injury remains unclea r.In the present study,we discove red that the deletion of exon 2 of the Ctnnd2 gene was linked to social interaction deficits,spine loss,impaired inhibitory neurons,and suppressed phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt) signal pathway in the prefrontal cortex.Our findings demonstrated that the long-term oral administration of melatonin for 28 days effectively alleviated the aforementioned abnormalities in Ctnnd2 gene-knockout mice.Furthermore,the administration of melatonin in the prefro ntal cortex was found to improve synaptic function and activate the PI3K/Akt signal pathway in this region.The pharmacological blockade of the PI3K/Akt signal pathway with a PI3K/Akt inhibitor,wo rtmannin,and melatonin receptor antagonists,luzindole and 4-phenyl-2-propionamidotetralin,prevented the melatonin-induced enhancement of GABAergic synaptic function.These findings suggest that melatonin treatment can ameliorate GABAe rgic synaptic function by activating the PI3K/Akt signal pathway,which may contribute to the improvement of dendritic spine abnormalities in autism spectrum disorders.

Myricetin induces M2 macrophage polarization to alleviate renal tubulointerstitial fibrosis in diabetic nephropathy via PI3K/Akt pathway

BACKGROUND Development of end-stage renal disease is predominantly attributed to diabetic nephropathy(DN).Previous studies have indicated that myricetin possesses the potential to mitigate the pathological alterations observed in renal tissue.Never-theless,the precise molecular mechanism through which myricetin influences the progression of DN remains uncertain.AIM To investigate the effects of myricetin on DN and explore its potential therapeutic mechanism.METHODS Db/db mice were administered myricetin intragastrically on a daily basis at doses of 50 mg/kg or 100 mg/kg for a duration of 12 wk.Subsequently,blood and urine indexes were assessed,along with examination of renal tissue pathology.Kidney morphology and fibrosis were evaluated using various staining techniques including hematoxylin and eosin,periodic acid–Schiff,Masson’s trichrome,and Sirius-red.Additionally,high-glucose culturing was conducted on the RAW 264.7 cell line,treated with 25 mM myricetin or co-administered with the PI3K/Akt inhibitor LY294002 for a period of 24 h.In both in vivo and in vitro settings,quantification of inflammation factor levels was conducted using western blotting,real-time qPCR and ELISA.RESULTS In db/db mice,administration of myricetin led to a mitigating effect on DN-induced renal dysfunction and fibrosis.Notably,we observed a significant reduction in expressions of the kidney injury markers kidney injury molecule-1 and neutrophil gelatinase associated lipocalin,along with a decrease in expressions of inflammatory cytokine-related factors.Furthermore,myricetin treatment effectively inhibited the up-regulation of tumor necrosis factor-alpha,interleukin-6,and interluekin-1βinduced by high glucose in RAW 264.7 cells.Additionally,myricetin modulated the M1-type polarization of the RAW 264.7 cells.Molecular docking and bioinformatic analyses revealed Akt as the target of myricetin.The protective effect of myricetin was nullified upon blocking the polarization of RAW 264.7 via inhibition of PI3K/Akt activation using LY294002.CONCLUSION This study demonstrated that myricetin effectively mitigates kidney injury in DN mice through the regulation of macrophage polarization via the PI3K/Akt signaling pathway.

Downregulation of Serum PTEN Expression in Mercury-Exposed Population and PI3K/AKT Pathway-Induced Inflammation

Objective This study investigated the impact of occupational mercury(Hg) exposure on human gene transcription and expression, and its potential biological mechanisms.Methods Differentially expressed genes related to Hg exposure were identified and validated using gene expression microarray analysis and extended validation. Hg-exposed cell models and PTEN lowexpression models were established in vitro using 293T cells. PTEN gene expression was assessed using qRT-PCR, and Western blotting was used to measure PTEN, AKT, and PI3K protein levels. IL-6 expression was determined by ELISA.Results Combined findings from gene expression microarray analysis, bioinformatics, and population expansion validation indicated significant downregulation of the PTEN gene in the high-concentration Hg exposure group. In the Hg-exposed cell model(25 and 10 μmol/L), a significant decrease in PTEN expression was observed, accompanied by a significant increase in PI3K, AKT, and IL-6 expression.Similarly, a low-expression cell model demonstrated that PTEN gene knockdown led to a significant decrease in PTEN protein expression and a substantial increase in PI3K, AKT, and IL-6 levels.Conclusion This is the first study to report that Hg exposure downregulates the PTEN gene, activates the PI3K/AKT regulatory pathway, and increases the expression of inflammatory factors, ultimately resulting in kidney inflammation.

Liqi Huoxue dripping pill protects against myocardial ischemia-reperfusion injury via the PI3K/Akt/GSK-3β signaling pathway in rats

Background:Liqi Huoxue dripping pill(LQHXDP),a traditional Chinese drug for coronary heart disease,has a protective effect on the heart of rats with myocardial ischemia-reperfusion injury(MIRI)in previous studies;however,its mechanism of action remains unclear.The purpose of this study was to investigate the protective mechanism of LQHXDP on MIRI in rats and its relationship with the PI3K/Akt signaling pathway.Methods:In this study,Sprague-Dawley rats were pre-infused with LQHXDP(175 mg/kg/d)for 10 days.PI3K inhibitor LY294002(0.3 mg/kg)was intravenously injected 15 minutes before ischemia.The rat model of MIRI was established by ligating the left anterior descending coronary artery.Subsequently,cardiac hemodynamics,serum myocardial injury markers,inflammatory factors,myocardial infarct size,antioxidant indexes,myocardial histopathology,and phosphorylation levels of key proteins of PI3K/Akt signaling pathway were assessed in rats.Results:LQHXDP was found to improve cardiac hemodynamic indexes,reduce serum creatine kinase MB isoenzyme activity and cardiac troponin and heart-type fatty acid binding protein levels,lower serum interleukin-1 beta,interleukin-6 and tumour necrosis factorαlevels,reduce the myocardial infarct size and enhance the antioxidant capacity of myocardial tissue in MIRI rats.Pathological analysis revealed that LQHXDP attenuated the extent of myocardial injury and protected mitochondria from damage in MIRI rats.Immunoblot analysis revealed that LQHXDP increased the expression levels of p-Akt and p-GSK-3βin MIRI rat cardiomyocytes.PI3K inhibitor LY294002 could impair these effects of LQHXDP.Conclusion:LQHXDP attenuated myocardial injury,attenuated oxidative stress injury and reduced inflammatory response in MIRI rats,and its protective effects were mediated by activating of PI3K/Akt/GSK-3βsignaling pathway.

Zuo Gui Wan Promotes Osteogenesis via PI3K/AKT Signaling Pathway:Network Pharmacology Analysis and Experimental Validation

Objective Osteogenesis is vitally important for bone defect repair,and Zuo Gui Wan(ZGW)is a classic prescription in traditional Chinese medicine(TCM)for strengthening bones.However,the specific mechanism by which ZGW regulates osteogenesis is still unclear.The current study is based on a network pharmacology analysis to explore the potential mechanism of ZGW in promoting osteogenesis.Methods A network pharmacology analysis followed by experimental validation was applied to explore the potential mechanisms of ZGW in promoting the osteogenesis of bone marrow mesenchymal stem cells(BMSCs).Results In total,487 no-repeat targets corresponding to the bioactive components of ZGW were screened,and 175 target genes in the intersection of ZGW and osteogenesis were obtained.And 28 core target genes were then obtained from a PPI network analysis.A GO functional enrichment analysis showed that the relevant biological processes mainly involve the cellular response to chemical stress,metal ions,and lipopolysaccharide.Additionally,KEGG pathway enrichment analysis revealed that multiple signaling pathways,including the phosphatidylinositol-3-kinase/protein kinase B(PI3K/AKT)signaling pathway,were associated with ZGW-promoted osteogensis.Further experimental validation showed that ZGW could increase alkaline phosphatase(ALP)activity as well as the mRNA and protein levels of ALP,osteocalcin(OCN),and runt related transcription factor 2(Runx 2).What’s more,Western blot analysis results showed that ZGW significantly increased the protein levels of p-PI3K and p-AKT,and the increases of these protein levels significantly receded after the addition of the PI3K inhibitor LY294002.Finally,the upregulated osteogenic-related indicators were also suppressed by the addition of LY294002.Conclusion ZGW promotes the osteogenesis of BMSCs via PI3K/AKT signaling pathway.

FGF2 promotes the chemotherapy resistance in colon cancer cells through activating PI3K/Akt signaling pathway

Background:To investigate the role of fibroblast growth factor 2(FGF2)in chemotherapy resistance of colon cancer.Methods:An HCT116/5-fluorouracil(5-FU)-resistant cell line was established,and FGF2 levels were detected in a sensitive cell group(HCT116)and a resistant cell group(HCT1116-R)using different methods.Fibroblast growth factor 2 levels in the medium were determined by enzyme-linked immunoassay.The protein expressions of FGF2,fibroblast growth factor receptor 1(FGFR1),and phospho-FGFR1 were assessed by Western blotting,and FGF2 mRNA levels were detected by quantitative real-time polymerase chain reaction.Fibroblast growth factor 2 recombinant protein was added to sensitive cells,and FGFR inhibitor AZD4547 was added to resistant cells,and the cell survival rate was determined using the cell counting kit-8 method and the protein expressions of PI3K(phosphatidylinositol 3 kinase),p-PI3K(phospho-PI3K),Akt(protein kinase B),p-Akt(phospho-Akt),mammalian target of rapamycin(mTOR),p-mTOR(phospho-mTOR),Bad(Bcl-xL/Bcl-2-associated death promoter),NF-κB(nuclear factorκB),GSK-3(glycogen synthase kinase-3),FKHR(forkhead box protein O1),and PTEN(phosphatase and tensin homolog deleted on chromosome ten)were detected by Western blotting.Results:Fibroblast growth factor 2 protein and mRNA expression levels in the HCT116-R group were significantly higher than those in the HCT116 group.Fibroblast growth factor 2 increased the survival rate of HCT116 cells;improved tolerance to 5-FU;upregulated p-PI3K,p-Akt,and p-mTOR;and downregulated Bad.The FGFR inhibitor AZD4547 decreased cell survival rate and tolerance to 5-FU;downregulated p-PI3K,p-Akt,and p-mTOR expression;and upregulated Bad.Conclusions:Fibroblast growth factor 2 promotes chemotherapy tolerance in colon cancer cells by activating the Akt/mTOR and Akt/Bad signaling pathways downstream of PI3K.

Alleviatory effect of isoquercetin on benign prostatic hyperplasia via IGF-1/PI3K/Akt/mTOR pathway

We evaluated the effect of isoquercetin(quercetin-O-3-glucoside-quercetin,IQ)as a functional component of Abeliophyllum disistichum Nakai ethanol extract(ADLE)on prostate cell proliferation and apoptosis and its effects on the IGF-1/PI3K/Akt/mTOR pathway in benign prostatic hyperplasia(BPH).Metabolites in ADLE were analyzed using UHPLC-qTOF-MS and HPLC.IQ was orally administered(1 or 10 mg/kg)to a testosterone propionate-induced BPH rat model,and its effects on the prostate weight were evaluated.The effect of IQ on androgen receptor(AR)signaling was analyzed in LNCaP cells.Whether IGF-1 and IQ affect the IGF-1/PI3K/Akt/mTOR pathway in BPH-1 cells was also examined.The metabolites in ADLE were identified and quantified,which confirmed that ADLE contained abundant IQ(20.88 mg/g).IQ significantly reduced the prostate size in a concentration-dependent manner in a BPH rat model,and significantly decreased the expression of AR signaling factors in the rat prostate tissue and LNCaP cells in a concentration-dependent manner.IQ also inhibited the PI3K/AKT/mTOR pathway activated by IGF-1 treatment in BPH-1 cells.In BPH-1 cells,IQ led to G0/G1 arrest and suppressed the expression of proliferation factors while inducing apoptosis.Thus,IQ shows potential for use as a pharmaceutical and nutraceutical for BPH.

Accelerated fracture healing by osteogenic Ti45Nb implants through the PI3K–Akt signaling pathway

The key to managing fracture is to achieve stable internal fixation,and currently,biologically and mechanically appropriate internal fixation devices are urgently needed.With excellent biocompatibility and corrosion resistance,titanium–niobium alloys have the potential to become a new generation of internal fixation materials for fractures.However,the role and mechanism of titanium–niobium alloys on promoting fracture healing are still undefined.Therefore,in this study,we systematically evaluated the bone-enabling properties of Ti45Nb via in vivo and in vitro experiments.In vitro,we found that Ti45Nb has an excellent ability to promote MC3T3-E1 cell adhesion and proliferation without obvious cytotoxicity.Alkaline phosphatase(ALP)activity and alizarin red staining and semiquantitative analysis showed that Ti45Nb enhanced the osteogenic differentiation of MC3T3-E1 cells compared to the Ti6Al4V control.In the polymerase chain reaction experiment,the expression of osteogenic genes in the Ti45Nb group,such as ALP,osteopontin(OPN),osteocalcin(OCN),type 1 collagen(Col-1)and runt-related transcription factor-2(Runx2),was significantly higher than that in the control group.Meanwhile,in the western blot experiment,the expression of osteogenic-related proteins in the Ti45Nb group was significantly increased,and the expression of PI3K–Akt-related proteins was also higher,which indicated that Ti45Nb might promote fracture healing by activating the PI3K–Akt signaling pathway.In vivo,we found that Ti45Nb implants accelerated fracture healing compared to Ti6Al4V,and the biosafety of Ti45Nb was confirmed by histological evaluation.Furthermore,immunohistochemical staining confirmed that Ti45Nb may promote osteogenesis by upregulating the PI3K/Akt signaling pathway.Our study demonstrated that Ti45Nb exerts an excellent ability to promote fracture healing as well as enhance osteoblast differentiation by activating the PI3K/Akt signaling pathway,and its good biosafety has been confirmed,which indicates its clinical translation potential.

XB130 inhibits healing of diabetic skin ulcers through the PI3K/Akt signalling pathway

BACKGROUND Diabetic skin ulcers,a significant global healthcare burden,are mainly caused by the inhibition of cell proliferation and impaired angiogenesis.XB130 is an adaptor protein that regulates cell proliferation and migration.However,the role of XB130 in the development of diabetic skin ulcers remains unclear.AIM To investigate whether XB130 can regulate the inhibition of proliferation and vascular damage induced by high glucose.Additionally,we aim to determine whether XB130 is involved in the healing process of diabetic skin ulcers,along with its molecular mechanisms.METHODS We conducted RNA-sequencing analysis to identify the key genes involved in diabetic skin ulcers.We investigated the effects of XB130 on wound healing using histological analyses.In addition,we used reverse transcription-quantitative polymerase chain reaction,Western blot,terminal deoxynucleotidyl transferasemediated dUTP nick end labeling staining,immunofluorescence,wound healing,and tubule formation experiments to investigate their effects on cellular processes in human umbilical vein endothelial cells(HUVECs)stimulated with high glucose.Finally,we performed functional analysis to elucidate the molecular mechanisms underlying diabetic skin ulcers.RESULTS RNA-sequencing analysis showed that the expression of XB130 was up-regulated in the tissues of diabetic skin ulcers.Knockdown of XB130 promoted the healing of skin wounds in mice,leading to an accelerated wound healing process and shortened wound healing time.At the cellular level,knockdown of XB130 alleviated high glucose-induced inhibition of cell proliferation and angiogenic impairment in HUVECs.Inhibition of the PI3K/Akt pathway removed the proliferative effects and endothelial protection mediated by XB130.CONCLUSION The findings of this study indicated that the expression of XB130 is up-regulated in high glucose-stimulated diabetic skin ulcers and HUVECs.Knockdown of XB130 promotes cell proliferation and angiogenesis via the PI3K/Akt signalling pathway,which accelerates the healing of diabetic skin ulcers.

Simiao Wan alleviates obesity-associated insulin resistance via PKCε/IRS-1/PI3K/Akt signaling pathway based on network pharmacology analysis and experimental validation

Background:The purpose of the study was to investigatethe active ingredients and potential biochemicalmechanisms of Simiao Wan(SMW)in obesity-associated insulin resistance.Methods:An integrated network pharmacology method to screen the active compoundsand candidate targets,construct the protein-protein-interaction network,and ingredients-targets-pathways network was constructed for topological analysis to identify core targets and main ingredients.To find the possible signaling pathways,enrichment analysis was performed.Further,a model of insulin resistance in HL-7702 cells was established to verify the impact of SMW and the regulatory processes.Results:An overall of 63 active components and 151 candidate targets were obtained,in which flavonoids were the main ingredients.Enrichment analysis indicated that the PI3K-Akt signaling pathway was the potential pathway regulated by SMW in obesity-associated insulin resistance treatment.The result showed that SMW could significantly ameliorate insulin sensitivity,increase glucose synthesis and glucose utilization and reduce intracellular lipids accumulation in hepatocytes.Also,SMW inhibited diacylglycerols accumulation-induced PKCεactivity and decreased its translocation to the membrane.Conclusion:SMW ameliorated obesity-associated insulin resistance through PKCε/IRS-1/PI3K/Akt signaling axis in hepatocytes,providing a new strategy for metabolic disease treatment.

The role and research progress of PI3K/AKT signaling pathway in non-traumatic osteonecrosis of the femoral head

Non-traumatic osteonecrosis of the femoral head(NONFH)is one of the most common orthopedic diseases,influenced by multiple signaling pathways and inflammatory factors.The PI3K/AKT signaling pathway is closely related to various biological processes such as apoptosis,autophagy,and metabolism in cells.Increasing evidence suggests that it plays an important role in the development of femoral head necrosis.This paper aims to explore the mechanism of the PI3K/AKT signaling pathway in the pathogenesis of NONFH by analyzing its regulation of lipid metabolism,cell apoptosis and autophagy,and intravascular coagulation.This study provides new insights for the research of NONFH.

Baicalin attenuates blood-spinal cord barrier disruption and apoptosis through PI3K/Akt signaling pathway after spinal cord injury

Baicalin is a natural active ingredient isolated from Scutellariae Radix that can cross the blood-brain barrier and exhibits neuroprotective effects on multiple central nervous system diseases.However,the mechanism behind the neuroprotective effects remains unclear.In this study,rat models of spinal cord injury were established using a modified Allen's impact method and then treated with intraperitoneal injection of Baicalin.The results revealed that Baicalin greatly increased the Basso,Beattie,Bresnahan Locomotor Rating Scale score,reduced blood-spinal cord barrier permeability,decreased the expression of Bax,Caspase-3,and nuclear factorκB,increased the expression of Bcl-2,and reduced neuronal apoptosis and pathological spinal cord injury.SH-SY5 Y cell models of excitotoxicity were established by application of 10 m M glutamate for 12 hours and then treated with 40μM Baicalin for 48 hours to investigate the mechanism of action of Baicalin.The results showed that Baicalin reversed tight junction protein expression tendencies(occludin and ZO-1)and apoptosis-related protein expression(Bax,Bcl-2,Caspase-3,and nuclear factor-κB),and also led to up-regulation of PI3 K and Akt phosphorylation.These effects on Bax,Bcl-2,and Caspase-3 were blocked by pretreatment with the PI3 K inhibitor LY294002.These findings suggest that Baicalin can inhibit bloodspinal cord barrier permeability after spinal cord injury and reduce neuronal apoptosis,possibly by activating the PI3 K/Akt signaling pathway.This study was approved by Animal Ethics Committee of Xi'an Jiaotong University on March 6,2014.

PI3K/AKT/mTOR signaling pathway inhibitors in proliferation of retinal pigment epithelial cells

AIM: To determine whether the PI3K/AKT/mTOR pathway is activated in proliferative vitreoretinopathy (PVR) in homo-sapiens. METHODS: The retina of controls and patients with PVR were collected and their levels of PI3K, phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP-1 were determined by Western blot. The cultured human retinal pigment epithelial cell line D407 was treated with a specific mTOR inhibitor, rapamycin (RAPA) or a PI3K inhibitor, LY294002, of various concentrations and durations. Cell morphology was observed by phase contrast microscopy and the proliferation and apoptosis of treated cells were determined by MTT assay and flow cytometry. RESULTS: Levels of PI3K, phospho-AKT, phospho-mTOR, phospho-P70S6K and phospho-4EBP1 was increased in the retina in PVR (P <0.05). In D407 cells, both RAPA and LY294002 significantly inhibited cell proliferation and cell cycle progression, and promoted apoptosis (P <0.05); morphologically, the cells became smaller. Both RAPA and LY294002 reduced levels of phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP1 expression (P <0.05). RAPA, but not LY294002, had no significant effect on PI3K expression. CONCLUSION: PI3K/AKT/mTOR signaling pathway is highly activated in the retinal pigment epithelial cells of PVR. The inhibitors of PI3K/AKT/mTOR signaling pathway, RAPA and LY294002, could inhibited the PI3K/AKT/mTOR signaling pathway by reducing the levels of phosphorylation of mTOR pathway components.

shRNA-interfering LSD1 inhibits proliferation and invasion of gastric cancer cells via VEGF-C/PI3K/AKT signaling pathway

BACKGROUND Histone Lysine Specific Demethylase 1(LSD1)is the first histone demethylase to be discovered,which regulates various biological functions by making lysine of histone H3K4,H3K9 and non-histone substrates demethylated.Abnormal regulation of LSD1 is closely related to the occurrence and development of gastric cancer.The change of LSD1 expression level plays an important role in the proliferation and metastasis of gastric cancer cells.The study of its function and mechanism may provide a theoretical basis for early diagnosis and targeted therapy of gastric cancer.AIM To investigate the effect of downregulation of lysine-specific demethylase 1(LSD1)expression on proliferation and invasion of gastric cancer cells and the possible regulatory mechanisms of the VEGF-C/PI3K/AKT signaling pathway.METHODS The LSD1-specific short hairpin RNA(shRNA)interference plasmid was transiently transfected,and expression of LSD1 was downregulated.The cell proliferation ability of LSD1 was observed by CCK-8 assay after downregulating expression of LSD1.Transwell invasion assay was used to observe the change of cell invasion ability after downregulating expression of LSD1.Expression of phosphorylated phosphoinositide 3-kinase(p-PI3K),PI3K,p-AKT,AKT,vascular endothelial growth factor receptor(VEGFR)-3,matrix metalloproteinase(MMP)-2 and MMP-9 in each group was detected by Western blotting.RESULTS The cell proliferation ability of transiently transfected LSD1-shRNA interference plasmid group was significantly lower than that of the control group(P<0.05).Transwell invasion assay showed that the number of cells across the membrane of the LSD1-shRNA transfection group(238.451±5.216)was significantly lower than that of the control group(49.268±6.984)(P<0.01).Western blotting showed that expression level of VEGF-C,p-PI3K,PI3K,p-AKT,AKT,VEGFR-3,MMP-2 and MMP-9 in the LSD1-shRNA group was significantly lower than that in the control group(P<0.05).CONCLUSION Downregulation of LSD1 expression inhibits metastatic potential of gastric cancer cells,and VEGF-C-mediated activation of PI3K/AKT signaling pathway,which may be an important mechanism for inhibiting lymph node metastasis in gastric cancer cells.

Scoparone inhibits pancreatic cancer through PI3K/Akt signaling pathway

BACKGROUND Pancreatic cancer is a highly malignant tumor of the gastrointestinal system whose emerging resistance to chemotherapy has necessitated the development of novel antitumor treatments.Scoparone,a traditional Chinese medicine monomer with a wide range of pharmacological properties,has attracted considerable attention for its antitumor activity.AIM To explore the potential antitumor effect of scoparone on pancreatic cancer and the possible molecular mechanism of action.METHODS The target genes of scoparone were determined using both the bioinformatics and multiplatform analyses.The effect of scoparone on pancreatic cancer cell proliferation,migration,invasion,cell cycle,and apoptosis was detected in vitro.The expression of hub genes was tested using quantitative reverse transcription polymerase chain reaction(qRT-PCR),and the molecular mechanism was analyzed using Western blot.The in vivo effect of scoparone on pancreatic cancer cell proliferation was detected using a xenograft tumor model in nude mice as well as immunohistochemistry.RESULTS The hub genes involved in the suppression of pancreatic cancer by scoparone were obtained by network bioinformatics analyses using publicly available databases and platforms,including SwissTargetPrediction,STITCH,GeneCards,CTD,STRING,WebGestalt,Cytoscape,and Gepia;AKT1 was confirmed using qRT-PCR to be the hub gene.Cell Counting Kit-8 assay revealed that the viability of Capan-2 and SW1990 cells was significantly reduced by scoparone treatment exhibiting IC50 values of 225.2μmol/L and 209.1μmol/L,respectively.Wound healing and transwell assays showed that scoparone inhibited the migration and invasion of pancreatic cancer cells.Additionally,flow cytometry confirmed that scoparone caused cell cycle arrest and induced apoptosis.Scoparone also increased the expression levels of Bax and cleaved caspase-3,decreased the levels of MMP9 and Bcl-2,and suppressed the phosphorylation of Akt without affecting total PI3K and Akt.Moreover,compared with the control group,xenograft tumors,in the 200μmol/L scoparone treatment group,were smaller in volume and lighter in weight,and the percentages of Ki65-and PCNA-positive cells were decreased.CONCLUSION Our findings indicate that scoparone inhibits pancreatic cancer cell proliferation in vitro and in vivo,inhibits migration and invasion,and induces cycle arrest and apoptosis in vitro through the PI3K/Akt signaling pathway.

Echinoside A from Pearsonothuria graeffei Exert the Cytotoxicity to MDA-MB-231 Cells via Mitochondrial Membrane and Modulation of PI3K/Akt/mTOR Pathway

A kind of triterpene glycosides echinoside A(EA)was extracted from sea cucumber Pearsonothuria graeffei,and its yield was about 0.78%.The purity of EA was 99.0%,and its molecular weight was 1206 Da.EA was a linear tetrasaccharide attached to a pentacyclic triterpene aglycon.It inhibited the growth of MDA-MB-231 cells in vitro.The antitumor effect was related to elevate ROS level,decrease mitochondrial membrane potential,enhance caspase-3 expression,induce cells apoptosis and arrest cell cycle at G2/M phase.EA also dose-dependently suppressed the expressions of phophorylation proteins p-PI3K,p-Akt,and p-mTOR as analyzed by western blotting.These results suggested that EA caused MDA-MB-231 cells apoptosis via intrinsic mitochondrial and PI3K/Akt/mTOR pathway.EA can be a potential anti-breast cancer agent to enhance the clinical efficacy.