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.

IGF-1对Rh1肉瘤细胞PI3K/Akt/mTOR信号通路的影响

目的探讨胰岛素样生长因子(IGF)1对Rh1肉瘤细胞生长活性和PI3K/Akt/mTOR信号通路的背景变化。方法常规细胞培养,用无血清培养基消除内源性因子影响27 h,再用IGF-1(终浓度为10 ng/ml)刺激72 h,流式细胞仪检测细胞生长活性;另外Western印迹方法观察IGF-1刺激细胞5、10、20、30和60 min后Akt(s473)、S6的动态变化。结果与对照组相比,IGF-1可促进Rh1细胞存活。IGF-1刺激不同时间后S6磷酸化则随着时间的延长逐渐增强;IGF-1亦导致Akt(s473)位点的磷酸化,随时间的延长,磷酸化Akt在5 min时达高峰,此后逐渐减弱。结论Akt、S6等是PI3K/Akt/mTOR信号通路中的重要信号分子,对Rh1细胞而言,在IGF-1刺激下S6有逐渐增强的变化,Akt(s473)位点磷酸化则有减弱的动态变化。

更年汤对肾阳虚围绝经期模型大鼠卵巢PI3K/AKT/mTOR蛋白表达的影响

目的:探讨肾阳虚围绝经期模型大鼠在自拟更年汤不同剂量干预下对卵巢PI3K/AKT/mTOR蛋白表达的影响。方法:选24只4~6月龄的青年大鼠作为对照组,构建肾阳虚模型。另采集阴道脱落细胞将10~12月龄雌性SD大鼠筛选出围绝经期模型,并将该模型大鼠随机分为模型组、利维爱组、更年汤低、中及高剂量共5组,每组24只。以上6组均给予相应药物灌胃,末次给药24 h后,检测卵巢组织中IGF-1、IGF-1R及PI3K/AKT/mTOR蛋白表达。结果:更年汤高剂量组、西药组IGF-1、IGF-1R及PI3K/AKT/mTOR蛋白表达均较高,与模型组比较均有统计学差异(P<0.05)。结论:更年汤能改善肾阳虚型围绝经期模型大鼠卵巢组织中PI3K/AKT/mTOR蛋白表达,可能与能调节IGF-1及其受体的表达量有关。

CD26 upregulates proliferation and invasion in keloid fibroblasts through an IGF-1-induced PI3K/AKT/mTOR pathway

Background:Keloid is a fibrotic dermal disease characterized by an abnormal increase in fibroblast proliferation and invasion.These pathological behaviours may be related to the heterogeneity of keloid fibroblasts(KFs);however,because of a lack of effective biomarkers for KFs it is difficult to study the underlying mechanism.Our previous studies revealed that the expansion of CD26+KFs was responsible for increased keloid proliferation and invasion capabilities;the intrinsic relationship and mechanism between CD26 and keloid is therefore worthy of further investigation.The aim of this studywas to explore molecular mechanisms in the process of CD26 upregulated KFs proliferation and invasion abilities,and provide more evidence for CD26 as an effective biomarker of keloid and a new clinical therapeutic target.Methods:Flow cytometry was performed to isolate CD26+/CD26−fibroblasts from KFs and normal fibroblasts.To generate stably silenced KFs for CD26 and insulin-like growth factor-1 receptor(IGF-1R),lentiviral particles encoding shRNA targeting CD26 and IGF-1R were used for transfection.Cell proliferations were analysed by cell counting kit-8 assay and 5-ethynyl-2-deoxyuridine(EdU)incorporation assay.Scratching assay and transwell assay were used to assess cell migration and invasion abilities.To further quantify the regulatory role of CD26 expression in the relevant signalling pathway,RT-qPCR,western blot,ELISA,PI3K activity assay and immunofluorescence were used.Results:Aberrant expression of CD26 in KFs was proven to be associated with increased proliferation and invasion of KFs.Furthermore,the role of the IGF-1/IGF-1 receptor axis was also studied in CD26 and was found to upregulate KF proliferation and invasion.The PI3K/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway was shown to affect CD26-regulated KF proliferation and invasion by increasing phosphorylation levels of S6 kinase and 4E-binding protein.Conclusions:CD26 can be the effective biomarker for KFs,and its expression is closely related to proliferation and invasion in keloids through the IGF-1-induced PI3K/AKT/mTOR pathway.This work provides a novel perspective on the pathological mechanisms affecting KFs and therapeutic strategies against keloids.

巴戟天提取物对地塞米松诱导小鼠肌少症的治疗效果及作用机制研究

目的:旨在探究巴戟天提取物(MoE)在地塞米松(Dex)诱导的肌少症小鼠中的治疗效果及可能作用机制。方法:首先利用网络药理学方法研究巴戟天的药效成分、重要靶点并富集通路,以预测MoE的作用机制。在动物实验中,50只KM小鼠随机分为5组(control、sarcopenia、MoE-H、MoE-M、MoE-L)。除control组,其他组皮下注射Dex 45 d诱导形成肌少症,3个治疗组同时经口给予不同剂量的MoE。实验过程中定期检测动物体重、运动能力(跑台和拉力计)和体成分。处死动物后计算后肢肌肉系数,进行组织学及相关基因表达检测。结果:网络药理学结果表明MoE可能通过上调IGF-1/AKT/mTOR信号通路来治疗肌少症。动物实验结果显示MoE显著逆转Dex导致的小鼠体重、肌肉系数的下降,提升运动能力,改善组织学病变,上调了IRS-1、mTOR、MFN2、OPA1、MYOD、MYOG等基因的表达,下调了Atrogin-1、MuRF-1、MSTN等基因的表达。结论:MoE对Dex引起的肌肉萎缩具有治疗作用,可改善肌肉组织学形态,其作用机制可能是激活IGF-1/AKT/mTOR通路。

IGF-1/PI3K/Akt/mTOR信号通路失衡与孤独症关系研究进展

IGF-1/PI3K/Akt/mTOR信号通路在调节细胞生长、增殖、分化、能动性、生存、代谢和蛋白质合成过程中起重要作用。孤独症是广泛性发育障碍疾病,病因机制复杂,其发展与其分子机制改变是近年研究的热点。IGF-1/PI3K/Akt/mTOR信号通路失调与孤独症发生有关,该信号通路在孤独症的诊断、治疗中可能有重要作用。本文就IGF-1/PI3K/Akt/mTOR信号通路失衡与孤独症关系的研究进展作一综述。

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.

Wide Pulse Width Electroacupuncture Ameliorates Denervation-Induced Skeletal Muscle Atrophy in Rats via IGF-1/PI3K/Akt Pathway

Objective:To evaluate the effect of the pulse width of electroacupuncture(EA)in the treatment of denervation-induced skeletal muscle atrophy in rats and examine the role of insulin-like growth factor 1(IGF-1)/phosphatidylinositol 3-kinase(PI3 K)/Akt signaling pathway during EA.Methods:Sciatic nerve functional index(SFI),muscle wet weight and the cross-sectional area(CSA)of the gastrocnemius muscle were analyzed after treatment in model rats with EA of various pulse widths(0.5,50,100 and 200 ms).The apoptosis index(AI)and paired box(PAX)3 and PAX7 protein expression were also determined.Further,the mRNA and protein expressions of components of IGF-1/PI3 K/Akt pathway and their downstream targets were determined,along with the inhibiting effect of the pathway with a PI3-specific inhibitor.Results:EA with a pulse width of 200 ms was found to have the best effect with regard to increasing SFI,CSA and muscle weight,decreasing AI,and increasing the expression of PAX3 and PAX7.The IGF-1/PI3 K/Akt pathway was found to be activated by denervation,although the downstream forkhead box O(FoxO)pathway was not suppressed by its activation.The PI3 K/Akt pathway and its downstream molecule mammalian target of rapamycin(mTOR)were up-regulated further by EA to promote muscle protein synthesis.Meanwhile,the expressions of downstream FoxO and F-box protein 32(ATROGIN-1)were down-regulated to reduce protein degradation.Conclusions:EA with 200-ms pulse width was found to have a more significant effect than 0.5-ms EA.The positive effects of EA disappeared after inhibition of the PI3 K/Akt pathway.

CircMAN1A2 promotes vasculogenic mimicry of nasopharyngeal carcinoma cells through upregulating ERBB2 via sponging miR-940

Nasopharyngeal carcinoma(NPC)is the most prevalent human primary malignancy of the head and neck,and the presence of vasculogenic mimicry(VM)renders anti-angiogenic therapy ineffective and poorly prognostic.However,the underlying mechanisms are unclear.In the present study,we used miR-940 silencing and overexpression for in vitro NPC cell EdU staining,wound healing assay and 3D cell culture assay,and in vivo xenograft mouse model and VM formation to assess miR-940 function.We found that ectopic miR-940 expression reduced NPC cell proliferation,migration and VM,as well as tumorigenesis in vivo.By bioinformatic analysis,circMAN1A2 was identified as a circRNA that binds to miR-940.Mechanistically,we confirmed that circMAN1A2 acts as a sponge for miR-940,impairs the inhibitory effect of miR-940 on target ERBB2,and then activates the PI3K/AKT/mTOR signaling pathway using RNA-FISH,dual luciferase reporter gene and rescue analysis assays.In addition,upregulation of ERBB2 expression is associated with clinical staging and poor prognosis of NPC.Taken together,the present findings suggest that circMAN1A2 promotes VM formation and progression of NPC through miR-940/ERBB2 axis and further activates the PI3K/AKT/mTOR pathway.Therefore,circMAN1A2 may become a biomarker and therapeutic target for anti-angiogenic therapy in patients with nasopharyngeal carcinoma.

Association of a genetic variant in AKT1 gene with features of the metabolic syndrome

Metabolic syndrome(MetS)is a clustering of metabolic abnormalities that is associated with increased risk of developing cardiovascular disease and type 2 diabetes.There is growing body of data showing the associations of genetic variants of the genes involved in the PI3K/AKT/mTOR pathway with diabetes and obesity.We aimed to investigate the association between MetS and its components with the genetic polymorphism in AKT1,rs1130233(T>C).Total of 618 participants,recruited from Mashhad stroke and heart atherosclerosis disorder cohort(MASHAD study).Patients with MetS were defined by using international diabetes federation(IDF)criteria(n Z 326)and those without MetS(n Z 261)were recruited.Anthropometric and biochemical parameters were measured in all subjects.Genetic analysis for the rs1130233 polymorphism was performed,using the ABI-StepOne instruments with SDS version-2.0 software.Individuals with MetS had a significantly higher levels of BMI,waistcircumference,total cholesterol,triglyceride,high sensitivity-c reactive protein(hs-CRP)and blood-pressure,and lower concentrations of high density lipoprotein(HDL-C),compared to non-MetS individuals(P<0.05).The association between the rs1130233 and MetS was not significant.Subjects with a CC or CT genotypes had a significantly higher serum hs-CRP-level(OR:1.5;95%CI(1.05e2.1),P Z 0.02).Additionally,subjects who carried the TC genotype had a higher BMI compared to the CC genotype(p value Z 0.045).Our findings demonstrated that AKT1,rs1130233(T>C)polymorphism was associated with major components of MetS such as hs-CRP,and BMI,indicating further investigation in a multi-center setting to explore its value as an emerging biomarker of risk stratification marker.

WJH 6^(th) Anniversary Special Issues(2): Hepatocellular carcinoma Mammalian target of rapamycin inhibition in hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is one of the leading causes of cancer-related death worldwide. It is associated with a poor prognosis and has limited treatment options. Sorafenib, a multi-targeted kinase inhibitor, is the only available systemic agent for treatment of HCC that improves overall survival for patients with advanced stage disease; unfortunately, an effective second-line agent for the treatment of progressive or sorafenib-resistant HCC has yet to be identified. This review focuses on components of the mammalian target of rapamycin(mTOR) pathway, its role in HCC pathogenesis, and dual mTOR inhibition as a therapeutic option with potential efficacy in advanced HCC. There are several important upstream and downstream signals in the mTOR pathway, and alternative tumor-promoting pathways are known to exist beyond mTORC1 inhibition in HCC. This review analyzes the relationships of the upstream and downstream regulators of mTORC1 and mTORC2 signaling; it also provides a comprehensive global picture of the interaction between mTORC1 and mTORC2 which demonstrates the pre-clinical relevance of the mTOR pathway in HCC pathogenesis and progression. Finally, it provides scientific rationale for dual mTORC1 and mTORC2 inhibition in the treatment of HCC. Clinical trials utilizing mTORC1 inhibitors and dual mTOR inhibitors in HCC are discussed as well. The mTOR pathway is comprised of two main components, mTORC1 and mTORC2; each has a unique role in the pathogenesis and progression of HCC. In phase Ⅲ studies, mTORC1 inhibitors demonstrate anti-tumor ac-tivity in advanced HCC, but dual mTOR(mTORC1 and mTORC2) inhibition has greater therapeutic potential in HCC treatment which warrants further clinical investigation.

Zebrafish ppp1r21 mutant as a model for the study of primary biliary cholangitis

Primary biliary cholangitis (PBC) is an autoimmune cholestatic liver disease that progresses to fibrosis and cirrhosis, resulting from the gradual destruction of intrahepatic bile ducts. Exploring genetic variants associated with PBC is essential to understand the pathogenesis of PBC. Here we identify a zebrafish balloon dog (blg) mutant with intrahepatic bile duct branching defects, exhibiting several key pathological PBC-like features, including immunodominant autoantigen PDC-E2 production, cholangiocyte apoptosis, immune cell infiltration, inflammatory activation, and liver fibrosis. blg encodes the protein phosphatase 1 regulatory subunit 21 (Ppp1r21), which is enriched in the liver and its peripheral tissues and plays a vital role in the early intrahepatic bile duct formation stage. Further studies show an excessive activation of the PI3K/AKT/mTOR pathway in the hepatic tissues in the mutant, while treatment with the pathway inhibitor LY294002 and rapamycin partially rescues intrahepatic bile duct branching defects and alleviates the PBC-like symptoms. These findings implicate the potential role of the Ppp1r21-mediated PI3K/AKT/mTOR pathway in the pathophysiology of PBC.

四物汤在卵巢衰老大鼠骨骼肌中的雌激素样作用机制研究

目的:探讨四物汤通过胰岛素样生长因子-1(Insulin-ike growth factors-1,IGF-1)/磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/雷帕霉素靶蛋白(Mammalian target of rapamycin,mTOR)mTOR信号通路发挥对4-乙烯基环己烯二环氧化合物(4-Vinylcyclohexene Diepoxide,VCD)诱导的卵巢衰老大鼠骨骼肌的保护作用及其分子机制。方法:选用28日龄雌性F-344大鼠,随机选取6只作为空白对照组,剩余大鼠连续腹腔注射VCD溶液(160 mg/kg/d)20天,每天阴道涂片检测动情周期,连续观察12d无角化细胞或仅有少量角化细胞即符合"卵巢衰老"表现,经动情周期筛选出24只造模成功的大鼠,分为模型组(Model)、阳性(戊酸雌二醇,E2)对照组、四物汤高剂量(SWT-H)组和四物汤低剂量(SWT-L)组,每组6只,灌胃持续3周后取材。取大鼠骨骼肌组织进行HE染色观察病理组织结构;MASSON染色骨骼肌纤维形态变化;RT-PCR检测骨骼肌组织中各基因的mRNA水平。结果:与空白组比较,模型组骨骼肌肌纤维排列疏松,分布不均且有断点,胞浆不均,细胞核增多,出现增生的结缔组织,胶原纤维逐渐增多且肌纤维间距变宽;与模型组相比,E2组、SWT-H组与SWT-L组肌纤维排列相对整齐,胞浆较为均匀。肌肉形态较规则,纤维间距缩短,胶原纤维减少。RT-PCR结果显示,与空白组相比,模型组IGF-1、PI3K、AKT、mTOR基因的mRNA表达量明显下调,E2组、SWT-H组与SWT-L组的IGF-1、PI3K、mTOR的表达明显上升,SWT-H组AKT表达无明显变化,无统计学意义,SWT-L组AKT表达相对降低。与模型组相比,E2组、SWT-H组与SWT-L组的IGF-1、AKT、mTOR的表达明显增加,且具有剂量依赖性,PI3K表达增加,但无明显剂量依赖性。结论:四物汤可以明显改善VCD诱导的卵巢衰老大鼠的肌肉减少情况,其机制可能是通过IGF-1-PI3K-AKT-mTOR信号通路来发挥其抑制肌肉流失的作用。

曲妥珠单抗治疗HER2阳性乳腺癌的耐药机制及新疗法探索

研究表明大约有20%的乳腺癌患者存在HER2过表达现象。HER2的异常表达及异常信号通路与乳腺癌的侵袭转移、治疗抵抗及不良预后密切相关。在临床上,对于HER2阳性的初期乳腺癌患者常联合曲妥珠单抗及化学药物治疗,但部分患者对曲妥珠单抗产生耐药。因此,研究其耐药机制对于HER2阳性乳腺癌患者的治疗、预后及新疗法的探索具有重要的临床意义。目前引起曲妥珠单抗抵抗的主要机制有:p95-HER2累积、PI3K/AKT/mTOR信号异常激活、HER家族受体和IGF-1R信号增加、非受体酪氨酸激酶c-SRC活性增加等。将对上述机制及治疗HER2阳性乳腺癌的新疗法进行综述。