丹酚酸B通过线粒体凋亡途径抑制口腔白斑癌变的机制研究

目的:探讨丹酚酸B通过线粒体凋亡途径抑制口腔白斑癌变的分子机制。方法:雄性叙利亚金黄地鼠随机分为正常组(15只)、丹酚酸B对照组(15只)、模型组(35只)和丹酚酸B治疗组(35只)。正常组灌胃给予1 ml蒸馏水,不给予造模剂和药物干预;丹酚酸B对照组灌胃给予10 mg/kg丹酚酸B溶液,不涂布0.5%DMBA丙酮溶液;模型组金黄地鼠左侧颊囊涂布0.5%DMBA丙酮溶液,但不给予药物干预;丹酚酸B治疗组金黄地鼠左侧颊囊涂布0.5%DMBA丙酮溶液,同时灌胃给予10 mg/kg丹酚酸B溶液。各组的干预时间均为9周,其中丹酚酸B每日给药1次,0.5%DMBA丙酮溶液每周涂布3次。分别于第4、5、6、7、9周末分批处理动物,采集颊囊黏膜组织样本。HE染色后光镜下比较各组病理损伤的程度;Western blot检测线粒体凋亡途径相关蛋白Caspase 3、cleaved Caspase 3、Caspase 9、cleaved Caspase 9、Bax、Bcl-2、Cyto C以及AKT-mTOR信号通路相关蛋白AKT、p-AKT、mTOR、p-mTOR的表达。结果:①HE染色观察显示,模型组和丹酚酸B治疗组均出现颊囊黏膜异常增生和肿瘤,丹酚酸B对照组未出现异常增生、肿瘤等病变。②干预第4周起,模型组和丹酚酸B治疗组均出现不同程度的黏膜异常增生,至第9周末出现鳞癌病变,模型组肿瘤发生率为50%,丹酚酸B治疗组发生率为8%;丹酚酸B治疗组第6周和第9周的病理损伤程度较模型组明显减轻(P0.05)。③与正常组相比,模型组金黄地鼠颊囊黏膜肿瘤和异常增生组织中cleaved Caspase 9、Bax、Cyto C蛋白表达水平均显著降低(P0.05,P0.01),而Bcl-2蛋白表达水平明显升高(P0.05,P0.01);与模型组相比,丹酚酸B治疗组cleaved Caspase 3、cleaved Caspase 9、Bax、Cyto C蛋白表达水平显著上调(P0.05,P0.01),Bcl-2蛋白表达水平明显下降(P0.05),且Bcl-2/Bax比值下降。④与正常组相比,模型组金黄地鼠颊囊黏膜肿瘤和异常增生组织中的AKT、p-AKT和p-mTOR蛋白表达水平均显著升高(P0.01);与模型组相比,丹酚酸B治疗组AKT、p-AKT和p-mTOR蛋白表达水平显著降低(P0.05,P0.01)。结论:丹酚酸B可有效抑制金黄地鼠口腔白斑癌变,其作用机制可能是通过抑制AKT-mTOR通路激活线粒体凋亡途径。     

Metformin and temozolomide act synergistically to inhibit growth of glioma cells and glioma stem cells in vitro and in vivo

Glioblastoma (GBM) is the most frequent and aggressive brain tumor in adults. In spite of advances in diagnosis and therapy, the prognosis of patients with GBM has remained dismal. The fast recurrence and multi-drug resistance are some of the key challenges in combating brain tumors. Glioma stem cells (GSCs) which are considered the source of relapse and chemoresistance, the need for more effective therapeutic options is overwhelming. In our present work, we found that combined treatment with temozolomide (TMZ) and metformin (MET) synergistically inhibited proliferation and induced apoptosis in both glioma cells and GSCs. Combination of TMZ and MET significantly reduced the secondary gliosphere formation and expansion of GSCs. We first demonstrated that MET effectively inhibited the AKT activation induced by TMZ, and a combination of both drugs led to enhanced reduction of mTOR, 4EBP1 and S6K phosphorylation. In addition, the combination of the two drugs was accompanied with a powerful AMP-activated protein kinase (AMPK) activation, while this pathway is not determinant. Xenografts performed in nude mice demonstrate in vivo demonstrated that combined treatment significantly reduced tumor growth rates and prolonged median survival of tumor-bearing mice. In conclusion, TMZ in combination with MET synergistically inhibits the GSCs proliferation through downregulation of AKT-mTOR signaling pathway. The combined treatment of two drugs inhibits GSCs self-renewal capability and partly eliminates GSCs in vitro and in vivo. This combined treatment could be a promising option for patients with advanced GBM.     

龙葵碱对Panc-1细胞增殖和血管形成能力的影响及对AKT-mTOR通路的调节作用

目的 探讨龙葵碱对胰腺癌细胞Panc-1的增殖和血管形成能力的影响及其相关机制。方法 实验分为对照组和药物组,药物组分别采用浓度为3.5、7.0 和10.5 μmol/L的龙葵碱对细胞进行干预,软琼脂克隆试验观察龙葵碱对胰腺癌细胞Panc-1非贴壁依赖性增殖能力的影响;脉管形成实验观察龙葵碱对胰腺癌细胞Panc-1血管形成能力的影响;蛋白质免疫印迹（Western blotting）法检测Panc-1细胞总蛋白中蛋白激酶B（protein kinase B,AKT）、雷帕霉素靶蛋白（target of rapamycin,mTOR）和血管内皮生长因子 （vascular endothelial growth factor,VEGF）的表达变化。结果 龙葵碱可明显抑制Panc-1细胞非贴壁依赖性增殖能力,且呈剂量依赖性[100% vs （42.1±9.6）%,（24.3±8.5）%,（14.4±1.7）%;P ＜0.05];龙葵碱亦可抑制VEGF蛋白表达[100% vs （74.9±5.5）%,（31.9±6.8）%,（16.5±7.5）%,P ＜0.05],并且抑制脉管形成[100% vs （82.3±9.5）%,（76.9±8.9）%,（56.0±12.1）%,P ＜0.05];龙葵碱可下调Panc-1细胞AKT、mTOR磷酸化蛋白表达[100% vs （72.4±0.8）%,（59.4±1.3）%,（40.7±2.9）%;100% vs （96.7±0.4）%,（77.5±3.4）%,（34.1±7.6）%,P ＜0.05]。结论 龙葵碱可能通过抑制AKT-mTOR细胞信号通路而抑制Panc-1细胞的增殖及血管形成能力。     

Impact of intracellular innate immune receptors on immunometabolism

Immunometabolism, which is the metabolic reprogramming of anaerobic glycolysis, oxidative phosphorylation, and metabolite synthesis upon immune cell activation, has gained importance as a regulator of the homeostasis, activation, proliferation, and differentiation of innate and adaptive immune cell subsets that function as key factors in immunity. Metabolic changes in epithelial and other stromal cells in response to different stimulatory signals are also crucial in infection, inflammation, cancer, autoimmune diseases, and metabolic disorders. The crosstalk between the PI3K–AKT–mTOR and LKB1–AMPK signaling pathways is critical for modulating both immune and nonimmune cell metabolism. The bidirectional interaction between immune cells and metabolism is a topic of intense study. Toll-like receptors (TLRs), cytokine receptors, and T and B cell receptors have been shown to activate multiple downstream metabolic pathways. However, how intracellular innate immune sensors/receptors intersect with metabolic pathways is less well understood. The goal of this review is to examine the link between immunometabolism and the functions of several intracellular innate immune sensors or receptors, such as nucleotide-binding and leucine-rich repeat-containing receptors (NLRs, or NOD-like receptors), absent in melanoma 2 (AIM2)-like receptors (ALRs), and the cyclic dinucleotide receptor stimulator of interferon genes (STING). We will focus on recent advances and describe the impact of these intracellular innate immune receptors on multiple metabolic pathways. Whenever appropriate, this review will provide a brief contextual connection to pathogenic infections, autoimmune diseases, cancers, metabolic disorders, and/or inflammatory bowel diseases.     

马钱苷元对人胰腺癌细胞BXPC3的抗肿瘤作用及机制研究

目的 研究山茱萸提取物马钱苷元（Loganetin）对人胰腺癌细胞株BXPC3的体外抗肿瘤作用,并探讨其可能的抗肿瘤机制。方法 本研究共设置两组实验,即对照组和马钱苷元组。采用CCK8（Cell Counting Kit-8）实验检测细胞存活率的改变,流式细胞仪检测马钱苷元对细胞周期的影响。同时利用划痕实验及基质胶Transwell小室实验分别检测马钱苷元对人胰腺癌细胞株BXPC3迁移和侵袭的影响。最后通过Western blot检测AKT-mTOR信号通路相关蛋白AKT、mTOR和S6磷酸化水平的改变。结果 与对照组相比,马钱苷元对人胰腺癌细胞株BXPC3的增殖具有抑制作用,且呈明显的时间和浓度依赖性,100μM马钱苷元作用于BXPC3细胞24、48h的抑制率分别为（21.49±0.01）%、（29.25±0.03）%;马钱苷元能够使细胞周期阻滞于S期并抑制细胞株BXPC3的迁移（p=0.0002）和侵袭(p＜0.0001),下调AKT、mTOR和S6的磷酸化水平。结论 马钱苷元可能通过抑制AKT-mTOR信号通路发挥其抗肿瘤活性。     

独一味单体抑制脊髓背角内AKT-mTOR信号通路缓解大鼠神经病理性痛

摘 要 目的：观察鞘内给予独一味单体8-O-乙酰山栀苷甲酯（8 O acetyl SM,8 OaS）对慢性神经病理性痛大鼠脊髓背角内丝苏氨酸蛋白激酶B（protein kinase B,AKT） 哺乳动物雷帕霉素靶点（the mammalian target of rapamycin,mTOR）信号转导通路表达的影响。方法: 采用大鼠腰5脊神经结扎（spinal nerve ligation,SNL）构建神经病理性痛模型,使用Von Frey 细丝连续观察造模后大鼠术侧后足的痛阈改变;应用Westernblot法定量分析大鼠腰膨大节段AKT和mTOR的磷酸化水平;采用免疫荧光组织化学染色观察pAKT和磷酸化mTOR(pmTOR)在脊髓背角内的细胞定位。结果: 由行为学数据可见,SNL模型大鼠机械性痛阈明显降低（P＜0.01）。术后1～7 d鞘内连续给予8 OaS进行人为干预,与给药前对比差异有统计学意义（P<0.05）;免疫荧光双重标记记实验结果显示,脊髓背角内pAKT与星形胶质细胞标记物胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP）存在大量共表达,pmTOR在星形胶质细胞和神经元上均观察到大量阳性染色。Western blot实验结果提示,8 OaS能显著下调脊髓背角内pAKT和pmTOR蛋白的表达水平。结论: 鞘内给予8 OaS可有效缓解由SNL诱导的机械性痛阈,其机制可能是8 OaS通过下调脊髓背角内AKT-mTOR信号通路的表达水平,进而抑制了星形胶质细胞的活化,从而达到缓解神经病理性痛的效果。     

黄芪甲苷通过AKT-mTOR信号通路促进脑缺血的自噬改善脑缺血再灌注损伤

目的研究黄芪甲苷对脑缺血/再灌注(I/R)损伤的保护作用。方法将72只健康SD大鼠随机分成假手术组、I/R组、雷帕霉素组和黄芪甲苷不同剂量(20 mg·kg~(-1)、50 mg·kg~(-1)、100 mg·kg~(-1))组。除假手术组外,各组分别采用大脑中动脉闭塞法建立大鼠I/R模型。观察脑梗死面积、细胞形态,免疫组化及Western blot检测海马神经元凋亡和自噬水平。结果 I/R组、雷帕霉素组和黄芪甲苷不同剂量组Bederson评分与假手术组比较,均有差异有统计学意义(P0.05);雷帕霉素组和黄芪甲苷不同剂量组Bederson评分均低于I/R组(P0.05)。雷帕霉素组和黄芪甲苷不同剂量组大鼠的脑梗死面积明显小于I/R组。雷帕霉素组和黄芪甲苷不同剂量组的海马凋亡阳性细胞数减少、凋亡率降低(P0.05)。与I/R组比较,雷帕霉素组和黄芪甲苷不同剂量组的神经细胞核双层膜结构尚完整,细胞器结构破坏较少。I/R组、雷帕霉素和黄芪甲苷不同剂量组与假手术组比较,caspase-3和P62水平均升高,Bcl-2、LC3Ⅱ、Beclinl、ATG5和LAMP2蛋白相对表达量均降低;I/R组与雷帕霉素组和黄芪甲苷不同剂量组比较,caspase-3和P62水平显著升高(P0.05),Bcl-2、LC3Ⅱ、Beclinl、ATG5和LAMP2蛋白相对表达量均降低。结论黄芪甲苷可减轻细胞结构损伤,减少脑梗死面积,减轻细胞坏死及凋亡,增加自噬水平,降低凋亡水平。黄芪甲苷通过AKT-mTOR信号通路促进脑缺血的自噬改善脑I/R损伤。     

Exendin-4通过促进自噬减少非酒精性脂肪性肝病细胞模型的脂质沉积

目的 探究Exendin-4改善肝细胞脂质沉积的作用机制,讨论Exendin-4治疗非酒精性脂肪肝病的可能机制。方法 对照组（人正常肝细胞LO2和肝癌细胞HepG2,不做任何处理）;棕榈酸钠（PA）组（LO2与HepG2分别进行PA处理,以构建肝细胞脂肪变性模型）;PA+Exendin-4组（LO2与HepG2分别进行PA与Exendin-4共处理）;PA+Exendin-4+3BDO组（LO2与HepG2分别进行Exendin-4与3BDO共处理）。通过油红O染色实验、CCK8实验,检测对照组、PA组、PA+Exendin-4组的脂质沉积程度与细胞增殖能力,并通过Western blot检测信号通路关键分子p-mTOR、m-TOR、p-AKT、AKT,以及自噬相关蛋白LC3-I/II和p62的表达;通过 Western blot 与免疫荧光实验,检测 LO2 与 HepG2 细胞中 GLP-1R 表达情况;通过 Western blot,检测对照组、PA+Exendin-4组、PA+Exendin-4+3BDO组中LC3-I/II和p62的表达。结果 油红O染色实验表明,PA诱导后细胞中脂质明显增加,PA+Exendin-4组脂质沉积程度较PA组降低;细胞增殖实验表明,PA组细胞增殖水平较对照组明显受抑制（P<0.01）;PA+Exendin-4组细胞增殖水平较PA组升高（P<0.05）;免疫荧光实验表明,人正常肝细胞LO2和肝癌细胞HepG2细胞株中均有GLP-1R表达;Western blot表明,PA+Exendin-4组p-mTOR的表达较PA组下调,p-AKT的表达较PA组上调。Exendin-4可下调自噬相关蛋白p62,而上调LC3-II的表达,而这一结果可被3BDO逆转。结论 Exendin-4可能通过直接作用于细胞上GLP-1R,激活AKT-mTOR信号通路,进而促进自噬;同时,Exendin-4有助于缓解PA作用下肝细胞的脂质沉积过程,并促进细胞增殖。提示Exendin-4在PA作用下肝细胞的脂质沉积过程中具有重要作用。     

Overexpression of TYRO3 indicates poor prognosis and induces gastric cancer progression via AKT-mTOR pathway

Gastric cancer (GC) is among of the leading causes of cancer mortality worldwide. This is because many patients are diagnosed with advanced GC and postoperative radiotherapy and chemotherapy have also exhibited limited effects on GC. TYRO3 has been considered carcinogenic and a potential therapeutic target for GC. However, TYRO3 function and mechanism in GC remains elusive. The study results indicated that TYRO3 was aberrantly elevated in GC tissues and predicted poor prognosis. TYRO3 is closely associated with clinicopathological indicators in GC tissues such as lymph node metastasis, venous invasion, neural invasion, and the tumor-node-metastasis stage. In addition, TYRO3 expression levels are closely related to the AKT-mTOR pathway in GC tissues. Moreover, the oncogenic role of TYRO3 was determined through in vitro and in vivo functional assays, and knockdown of the TYRO3 expression level in GC cell lines can effectively suppress the AKT-mTOR pathway and inhibit tumor cell proliferation and migration. In conclusion, this study provides a theoretical basis for establishing the potential association and regulatory mechanism between TYRO3 and AKT-mTOR and offers a new strategy for GC-targeted therapy.