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.

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.

YBX1 inhibits mitochondrial-mediated apoptosis in ischemic heart through the PI3K/AKT signaling pathway

Background:Myocardial infarction(MI)is associated with higher morbidity and mortality in the world,especially in cold weather.YBX1 is an RNA-binding protein that is required for pathological growth of cardiomyocyte by regulating cell growth and protein synthesis.But YBX1,as an individual RNA-binding protein,regulates cardiomyocytes through signaling cascades during myocardial infarction remain largely unexplored.Methods:In vivo,the mouse MI model was induced by ligating the left anterior descending coronary artery(LAD),and randomly divided into sham operation group,MI group,MI+YBX1 knockdown/overexpression group and MI+negative control(NC)group.The protective effect of YBX1 was verified by echocardiography and triphenyltetrazolium chloride staining.In vitro,mitochondrial-dependent apoptosis was investigated by using CCK8,TUNEL staining,reactive oxygen species(ROS)staining and JC-1 staining in hypoxic neonatal mouse cardiomyocytes(NMCMs).Results:YBX1 expression of cardiomyocytes was downregulated in a mouse model and a cellular model on the ischemic condition.Compared to mice induced by MI,YBX1 overexpression mediated by adeno-associated virus serotype 9(AAV9)vector reduced the infarcted size and improved cardiac function.Knockdown of endogenous YBX1 by shRNA partially aggravated ischemia-induced cardiac dysfunction.In hypoxic cardiomyocytes,YBX1 overexpression decreased lactic dehydrogenase(LDH)release,increased cell viability,and inhibited apoptosis by affecting the expression of apoptosis related proteins,while knockdown of endogenous YBX1 by siRNA had the opposite effect.Overexpression of YBX1 restored mitochondrial dysfunction in hypoxic NMCMs by increasing mitochondrial membrane potential and ATP content and decreasing ROS.In hypoxic NMCMs,YBX1 overexpression increased the expression of phosphorylated phosphatidylinositol 3 kinase(PI3K)/AKT,and the anti-apoptosis effect of YBX1 was eliminated t by LY294002,PI3K/AKT inhibitor.Conclusion:YBX1 protected the heart from ischemic damage by inhibiting the mitochondrial-dependent apoptosis through PI3K/AKT pathway.It is anticipated that YBX1 may serve as a novel therapeutic target for MI.

Human neural stem cell-derived extracellular vesicles protect against ischemic stroke by activating the PI3K/AKT/mTOR pathway

Human neural stem cell-derived extracellular vesicles exhibit analogous functions to their parental cells,and can thus be used as substitutes for stem cells in stem cell therapy,thereby mitigating the risks of stem cell therapy and advancing the frontiers of stem cell-derived treatments.This lays a foundation for the development of potentially potent new treatment modalities for ischemic stroke.However,the precise mechanisms underlying the efficacy and safety of human neural stem cell-derived extracellular vesicles remain unclear,presenting challenges for clinical translation.To promote the translation of therapy based on human neural stem cell-derived extracellular vesicles from the bench to the bedside,we conducted a comprehensive preclinical study to evaluate the efficacy and safety of human neural stem cell-derived extracellular vesicles in the treatment of ischemic stroke.We found that administration of human neural stem cell-derived extracellular vesicles to an ischemic stroke rat model reduced the volume of cerebral infarction and promoted functional recovery by alleviating neuronal apoptosis.The human neural stem cell-derived extracellular vesicles reduced neuronal apoptosis by enhancing phosphorylation of phosphoinositide 3-kinase,mammalian target of rapamycin,and protein kinase B,and these effects were reversed by treatment with a phosphoinositide 3-kinase inhibitor.These findings suggest that human neural stem cell-derived extracellular vesicles play a neuroprotective role in ischemic stroke through activation of phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway.Finally,we showed that human neural stem cell-derived extracellular vesicles have a good in vivo safety profile.Therefore,human neural stem cell-derived extracellular vesicles are a promising potential agent for the treatment of ischemic stroke.

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.

Ascophyllum nodosum and Fucus vesiculosus ameliorate restenosis via improving inflammation and regulating the PTEN/PI3K/AKT signaling pathway

Restenosis is a common complication following coronary angioplasty.The traditional use of seaweeds for health benefits has increasingly been explored,however few studies exist reporting its protective effects on the development of restenosis and gut dysbiosis.The aim of this study was to investigate the potential of seaweed extracts(SE) of Ascophyllum nodosum and Fucus vesiculosus in inhibiting intimal hyperplasia in a rat model of restenosis and its underlying mechanisms in macrophages and vascular smooth muscle cells(vSMCs).16S rRNA sequencing was done to investigate the regulatory effect of SE on the gut microbiome of injured rats.As indicated by the results,SE significantly inhibited the progression of intimal hyperplasia in vivo,attenuated inflammation in macrophages and could inhibit the proliferation,dedifferentiation and migration of vSMCs.It was observed through immunoblotting assays that treatment with SE significantly upregulated PTEN expression in macrophages and inhibited the upregulation of PI3K and AKT expression in vSMCs.Meanwhile,according to the 16S rRNA gene sequencing analysis,supplementation with SE modulated gut microbiota composition in injured rats.In conclusion,SE could ameliorate intimal hyperplasia by inhibiting inflammation and vSMCs proliferation through the regulation of the PTEN/PI3K/AKT pathway and modulating the gut microbiome.

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.

Mechanism of stilbene glycosides on apoptosis of SH-SY5Y cells via regulating PI3K/AKT signaling pathway

Objective:To investigate the effects of stilbene glycoside(TSG)on okadaic acid-induced apoptosis in human neuroblastoma cells(SH-SY5Y)via the PI3K/AKT pathway.Methods:The optimal concentration of OA was screened by CCK-8 assay,and SH-SY5Y cells were divided into control group,model group,TSG group,LY294002 group and LY294002+TSG group.The proliferation and apoptosis in each group were detected by CCK-8 and TUNEL assays;Western blotting method and real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of PI3K,P-PI3K(Y607),AKT,P-AKT(Ser473),Bcl-2 and Bax proteins.The relative protein expression was represented by P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax gray ratio.Results:CCK-8 screened the optimal concentration of OA as 40 nmol/L.Compared with the control group,the model group increased relative cell viability,decreased apoptosis rate,the pathway and apoptotic proteins expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were decreased,and the mRNA expression levels of PI3K,AKT and Bcl-2 were decreased.Bax mRNA expression level increased(P<0.05);Compared with model group,TSG group increased relative cell viability,decreased apoptosis rate,increased protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT,Bcl-2/Bax,and increased mRNA expression levels of PI3K,AKT,and Bcl-2.Bax mRNA expression decreased(P<0.05),LY294002 group decreased relative cell viability,increased apoptosis rate,P-PI3K(Y607)/PI3K protein expression levels were significantly decreased(P<0.05),P-AKT(Ser473)/AKT and Bcl-2/Bax protein expression levels were significantly decreased,but there was no statistical significance,PI3K,AKT and Bcl-2 mRNA expression levels were decreased,and Bax mRNA expression levels were increased(all P<0.05);Compared with LY294002 group,LY294002+TSG group increased relative cell viability,decreased apoptosis rate,and the protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were increased.The mRNA expression levels of PI3K,AKT,Bcl-2 were increased,Bax was decreased(all P<0.05).Conclusion:Stilbene glycoside may alleviate okadaic acid-induced apoptosis in SH-SY5Y cells by interfering with the PI3K/AKT signaling pathway,which in turn regulates the expression of apoptotic factors such as Bcl-2 and Bax.

不同强度运动抑制糖尿病大鼠肾脏PI3K/AKT/mTOR信号通路改善自噬的比较

背景:2型糖尿病损害肾功能。研究表明运动干预可以保护肾脏;鸢尾素可以通过抑制磷脂酰肌醇3-激酶/蛋白激酶B/雷帕霉素靶蛋白信号通路恢复自噬,保护糖尿病肾病患者的肾功能。目的:探讨运动能否通过抑制肾脏磷脂酰肌醇3-激酶/蛋白激酶B/雷帕霉素靶蛋白信号通路过度激活来恢复自噬,改善肾损伤,以及分析不同方式运动产生影响的差异。方法:将6周龄的SD大鼠随机分为空白对照组(正常大鼠)和糖尿病组,其中糖尿病组大鼠经过高脂高糖喂养加腹腔注射低剂量1%链脲佐菌素(30 mg/kg)建立2型糖尿病模型。造模成功后再将糖尿病组大鼠随机分成糖尿病模型组、中强度持续运动组和高强度间歇运动组。两个运动组大鼠分别进行8周不同强度运动干预。取材后采用葡萄糖氧化酶法检测大鼠空腹血糖,使用试剂盒检测糖化血红蛋白水平,Elisa法检测血清胰岛素浓度,计算胰岛素抵抗指数,RT-PCR检测肾组织磷脂酰肌醇3-激酶、蛋白激酶B、雷帕霉素靶蛋白、Beclin-1、podocin、nephrin的基因表达量,Western Blot检测肾组织雷帕霉素靶蛋白及自噬标记蛋白LC3-1、LC3-2、Beclin-1的蛋白表达量。结果与结论:①2型糖尿病大鼠空腹血糖和糖化血红蛋白水平极显著性升高,胰岛素抵抗水平显著上升,胰岛素水平显著下降;两种运动均能使2型糖尿病大鼠空腹血糖和糖化血红蛋白水平极显著下降,胰岛素抵抗水平显著下降,胰岛素水平显著上升;与中强度持续运动组相比,高强度间歇运动组胰岛素水平显著上升。②2型糖尿病大鼠podocin、nephrin基因表达量显著降低;两种不同形式运动均能显著提高其表达;与高强度间歇运动组相比,中等强度持续性运动组足细胞相关蛋白基因表达有进一步上升趋势,但无显著性差异。③2型糖尿病大鼠肾组织磷脂酰肌醇3-激酶、蛋白激酶B、mTORC1的mRNA及蛋白的表达量显著增加,自噬标志蛋白Beclin-1、LC3-2表达量以及LC3-2/LC3-1显著降低;两种不同形式运动均能使肾组织磷脂酰肌醇3-激酶、蛋白激酶B、mTORC1的mRNA及雷帕霉素靶蛋白蛋白的表达量显著降低,自噬标志蛋白Beclin-1、LC3-2以及LC3-2/LC3-1显著升高;与中等强度持续性运动组相比,高强度间歇运动的磷脂酰肌醇3-激酶、蛋白激酶B、mTORC1的mRNA及雷帕霉素靶蛋白的蛋白表达量有进一步下降的趋势,Beclin-1、LC3-2以及LC3-2/LC3-1有进一步升高的趋势,但仅Beclin-1有显著性差异。④结果说明2型糖尿病肾脏足细胞损伤,自噬受到抑制,与磷脂酰肌醇3-激酶/蛋白激酶B/mTORC1信号通路被异常激活密切相关。高强度间歇运动和中等强度持续性运动可以保护糖尿病肾脏,减少足细胞损伤,促进自噬恢复,这可能与运动抑制磷脂酰肌醇3-激酶/蛋白激酶B/雷帕霉素靶蛋白信号通路过度激活有关。与中等强度持续性运动相比,高强度间歇运动恢复自噬的效果呈更优趋势,但足细胞蛋白表达稍有下降。

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.

ROR2 promotes invasion and chemoresistance of triple-negative breast cancer cells by activating PI3K/AKT/mTOR signaling

Objective:This study aimed to investigate the role of receptor tyrosine kinase-like orphan receptor 2(ROR2)in triple-negative breast cancer(TNBC).Methods:ROR2 expression in primary TNBC and metastatic TNBC tissues was analyzed by immunohistochemical staining and PCR.ROR2 expression in TNBC cell lines was detected by PCR and Western blot analysis.The migration,invasion and chemosensitivity of TNBC cells with overexpression or knockdown of ROR2 were examined.Results:ROR2 expression was high in metastatic TNBC tissues.ROR2 knockdown suppressed the migration,invasion and chemoresistance of TNBC cells.ROR2 overexpression in MDA-MB-435 cells promoted the migration,invasion,and chemoresistance.Moreover,ROR2 knockdown in HC1599 and MDA-MB-435 adriamycin-resistant cells enhanced chemosensitivity to adriamycin.ROR2 could activate PI3K/AKT/mTOR signaling in TNBC cells.Conclusion:ROR2 is upregulated and promotes metastatic phenotypes of TNBC by activating PI3K/AKT/mTOR signaling.

二甲双胍抑制PI3K/AKT/mTOR信号通路保护骨关节炎模型大鼠关节软骨

背景:研究表明,二甲双胍具有抗炎、抗肿瘤、抗衰老与血管保护作用,可抑制骨关节炎的进展,但其具体的作用机制仍不明确。目的:探讨二甲双胍对骨关节炎模型大鼠软骨保护的作用机制。方法:取40只雄性SD大鼠,采用随机数字表法分4组(n=10):空白组不进行任何手术,假手术组暴露关节腔,模型组、二甲双胍组采用改良Hulth法建立骨关节炎模型;造模后1 d,二甲双胍组大鼠灌胃给予二甲双胍200 mg/(kg·d),模型组、空白组、假手术组灌胃给予生理盐水,连续给药4周。给药结束后,苏木精-伊红、甲苯胺蓝和番红O-固绿染色观察大鼠膝关节软骨病理形态,免疫组化染色与Western blotting检测大鼠软骨组织中SOX9、Ⅱ型胶原、ADAMTS5、Beclin1、P62、p-PI3K、PI3K、p-AKT、AKT、p-mTOR、mTOR的蛋白表达。结果与结论:①苏木精-伊红、甲苯胺蓝和番红O-固绿染色结果显示,空白组、假手术组大鼠膝关节软骨表面光滑,组织形态正常;模型组大鼠膝关节软骨表面不规则,软骨组织出现缺损,软骨细胞数量减少,软骨基质中蛋白多糖含量减少;相较于模型组,二甲双胍组大鼠膝关节软骨结构损伤有明显改善,软骨表面趋于平整,软骨细胞数量与软骨基质中蛋白多糖含量增加;②免疫组化染色与Western blotting检测结果显示,与空白组、假手术组比较,模型组大鼠软骨组织中SOX9、Ⅱ型胶原、Beclin1蛋白表达降低(P<0.05),ADAMTS5、P62及p-PI3K、p-AKT、p-mTOR蛋白表达升高(P<0.05);与模型组比较,二甲双胍组大鼠软骨组织中SOX9、Ⅱ型胶原、Beclin1蛋白表达升高(P<0.05),ADAMTS5、P62及p-PI3K、p-AKT、p-mTOR蛋白表达降低(P<0.05);③结果表明,二甲双胍可通过抑制PI3K/AKT/mTOR信号通路的活化提高骨关节炎模型大鼠软骨细胞自噬活性、减少软骨基质降解,进而发挥关节软骨保护作用。

Indirubin alleviates retinal neurodegeneration through the regulation of PI3K/AKT signaling

Retinal neurodegenerative disease is a leading cause of blindness among the elderly in developed countries,including glaucoma,diabetic retinopathy,traumatic optic neuropathy and optic neuritis,etc.The current clinical treatment is not very effective.We investigated indirubin,one of the main bioactive components of the traditional Chinese medicine Danggui Longhui Pill,in the present study for its role in retinal neurodegeneration.Indirubin exhibited no detectable tissue toxicity in vivo or cytotoxicity in vitro.Moreover,indirubin improved visual function and ameliorated retinal neurodegeneration in mice after optic nerve crush injury in vivo.Furthermore,indirubin reduced the apoptosis of retinal ganglion cells induced by oxidative stress in vitro.In addition,indirubin significantly suppressed the increased production of intracellular reactive oxygen species and the decreased activity of superoxide dismutase induced by oxidative stress.Mechanically,indirubin played a neuroprotective role by regulating the PI3K/AKT/BAD/BCL-2 signaling.In conclusion,indirubin protected retinal ganglion cells from oxidative damage and alleviated retinal neurodegeneration induced by optic nerve crush injury.The present study provides a potential therapeutic medicine for retinal neurodegenerative diseases.

Wedelolactone attenuates sepsis-associated acute liver injury by regulating the macrophage M1/M2 polarization balance through the PI3K/AKT/NF-κB signalling pathway

Background:Liver injury caused by sepsis seriously impairs the normal physiology of the liver.Wedelactone(WED)has an obvious anti-inflammatory effect against liver damage caused by various factors.Nevertheless,further research is needed to determine if WED might mitigate acute liver damage linked to sepsis by influencing macrophage polarization.Methods:We first assessed the effect of WED on lipopolysaccharides-triggered liver injury by biochemistry assay and tissue staining.Inflammatory factors were assessed using the ELISA kits.The expression of Cluster of Differentiation 86(CD86)and Cluster of Differentiation 206(CD206)was measured by immunofluorescence assay.The protein levels of inducible nitric oxide sythase(iNOS),Arginase 1(Arg-1),phosphatidylinositol 3-kinase(PI3K),protein kinase B(AKT),PI3K phosphorylation(p-PI3K),AKT phosphorylation(p-AKT),inhibitor of kappa B kinase(IKK),inhibitor of kappa B(IκB),and nuclear factor kappa-B(NF-κB)p65 were quantified by western blot analysis.Results:WED decreased the level of alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP)and malondialdehyde,and increased the activity of superoxide dismutase(SOD)and glutathione peroxidase(GSH-PX).Moreover,WED exerted effective anti-inflammatory effects by decreasing the level of Tumor necrosis factor-α(TNF-α)and Interleukin 6(IL-6)and increasing the level of Interleukin 10(IL-10)in serum and cells.WED not only decreased CD86 and iNOS expression but also increased CD206 and Arg-1 expression.WED also downregulated the increased expression of PI3K,AKT,p-PI3K,p-AKT,IKK,and NF-κB p65 induced by lipopolysaccharides,while up-regulated the decreased expression of IκB.Besides,LY294002 with WED decreased the expression of protein PI3K,AKT,p-PI3K,p-AKT,IKK and NF-κB p65,and raised the expression of IκBα.Conclusion:Wedelolactone could attenuate sepsis-associated acute liver injury,and its mechanism may be associated with balancing pro-inflammatory and anti-inflammatory by the regulation of M1/M2 macrophage polarization via the PI3K/AKT/NF-κB signaling pathway.

Human-induced pluripotent stem cell-derived neural stem cell exosomes improve blood-brain barrier function after intracerebral hemorrhage by activating astrocytes via PI3K/AKT/MCP-1 axis

Cerebral edema caused by blood-brain barrier injury after intracerebral hemorrhage is an important factor leading to poor prognosis.Human-induced pluripotent stem cell-derived neural stem cell exosomes(hiPSC-NSC-Exos)have shown potential for brain injury repair in central nervous system diseases.In this study,we explored the impact of hiPSC-NSC-Exos on blood-brain barrier preservation and the underlying mechanism.Our results indicated that intranasal delivery of hiPSC-NSC-Exos mitigated neurological deficits,enhanced blood-brain barrier integrity,and reduced leukocyte infiltration in a mouse model of intracerebral hemorrhage.Additionally,hiPSC-NSC-Exos decreased immune cell infiltration,activated astrocytes,and decreased the secretion of inflammatory cytokines like monocyte chemoattractant protein-1,macrophage inflammatory protein-1α,and tumor necrosis factor-αpost-intracerebral hemorrhage,thereby improving the inflammatory microenvironment.RNA sequencing indicated that hiPSC-NSC-Exo activated the PI3K/AKT signaling pathway in astrocytes and decreased monocyte chemoattractant protein-1 secretion,thereby improving blood-brain barrier integrity.Treatment with the PI3K/AKT inhibitor LY294002 or the monocyte chemoattractant protein-1 neutralizing agent C1142 abolished these effects.In summary,our findings suggest that hiPSC-NSC-Exos maintains blood-brain barrier integrity,in part by downregulating monocyte chemoattractant protein-1 secretion through activation of the PI3K/AKT signaling pathway in astrocytes.

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.

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.

Targeting the PI3K/Akt signaling pathway in gastric carcinoma: A reality for personalized medicine?

Frequent activation of phosphatidylinositol-3 kinases(PI3K)/Akt/m TOR signaling pathway in gastric cancer(GC) is gaining immense popularity with identification of mutations and/or amplifications of PIK3 CA gene or loss of function of PTEN,a tumor suppressor protein,to name a few; both playing a crucial role in regulating this pathway. These aberrations result in dysregulation of this pathway eventually leading to gastric oncogenesis,hence,there is a need for targeted therapy for more effective anticancer treatment. Several inhibitors are currently in either preclinical or clinical stages for treatment of solid tumors like GC. With so many inhibitors under development,further studies on predictive biomarkers are needed to measure the specificity of any therapeutic intervention. Herein,we review the common dysregulation of PI3K/Akt/m TOR pathway in GC and the various types of single or dual pathway inhibitors under development that might have a superior role in GC treatment. We also summarize the recent developments in identification of predictive biomarkers and propose use of predictive biomarkers to facilitate more personalized cancer therapy with effective PI3K/Akt/m TOR pathway inhibition.

SHP2 promotes proliferation of breast cancer cells through regulating Cyclin D1 stabilityviathe PI3K/AKT/GSK3β signaling pathway

Objective:The tyrosine phosphatase SHP2 has a dual role in cancer initiation and progression in a tissue type-dependent manner.Several studies have linked SHP2 to the aggressive behavior of breast cancer cells and poorer outcomes in people with cancer.Nevertheless,the mechanistic details of how SHP2 promotes breast cancer progression remain largely undefined.Methods:The relationship between SHP2 expression and the prognosis of patients with breast cancer was investigated by using the TCGA and GEO databases.The expression of SHP2 in breast cancer tissues was analyzed by immunohistochemistry.CRISPR/Cas9 technology was used to generate SHP2-knockout breast cancer cells.Cell-counting kit-8,colony formation,cell cycle,and EdU incorporation assays,as well as a tumor xenograft model were used to examine the function of SHP2 in breast cancer proliferation.Quantitative RT-PCR,western blotting,immunofluorescence staining,and ubiquitination assays were used to explore the molecular mechanism through which SHP2 regulates breast cancer proliferation.Results:High SHP2 expression is correlated with poor prognosis in patients with breast cancer.SHP2 is required for the proliferation of breast cancer cellsin vitro and tumor growthin vivo through regulation of Cyclin D1 abundance,thereby accelerating cell cycle progression.Notably,SHP2 modulates the ubiquitin–proteasome-dependent degradation of Cyclin D1viathe PI3K/AKT/GSK3βsignaling pathway.SHP2 knockout attenuates the activation of PI3K/AKT signaling and causes the dephosphorylation and resultant activation of GSK3β.GSK3βthen mediates phosphorylation of Cyclin D1 at threonine 286,thereby promoting the translocation of Cyclin D1 from the nucleus to the cytoplasm and facilitating Cyclin D1 degradation through the ubiquitin–proteasome system.Conclusions:Our study uncovered the mechanism through which SHP2 regulates breast cancer proliferation.SHP2 may therefore potentially serve as a therapeutic target for breast cancer.