白藜芦醇通过激活PI3K/Akt通路抑制高糖致肾小管上皮细胞凋亡的研究

目的:白藜芦醇对高糖作用下肾小管上皮细胞(HK-2)氧化应激介导的凋亡影响及作用机制。方法:体外培养肾小管上皮细胞,高糖作用于肾小管上皮细胞诱导其凋亡,设置空白对照组、模型组(30 mmol·L~(-1)高糖处理)、不同浓度(5、10μmol·L~(-1))白藜芦醇组,LY294002(PI3K抑制剂)组(10μmol·L~(-1))。使用MTT法检测各组细胞活力;流式细胞术检测各组细胞凋亡率;JC-1检测线粒体膜电位(MMP);DHE荧光探针检测ROS的变化;特定试剂盒检测乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH-PX)含量;Western-blot法检测各组细胞色素C(CYT-C)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白表达水平以及Akt的磷酸化情况。结果:与空白对照组相比高糖损伤组能够显著降低肾小管上皮细胞的活力,增加细胞中ROS的含量和凋亡率,LDH(细胞外)、MDA含量显著增加,SOD、GSH-PX活性显著下降,同时降低线粒体膜电位,增加了CYT-C、Caspase-3的表达,而p-Akt/Akt表达降低(P0.01)。不同浓度白藜芦醇(起效剂量5μmol·L~(-1),高剂量10μmol·L~(-1))和高糖同时作用肾小管上皮细胞时,细胞存活率逐渐上升,ROS含量及凋亡率逐渐下降,LDH、MDA含量逐渐下降,SOD、GSH-PX活性随之上升,线粒体膜电位提升,CYT-C、Caspase-3表达显著下降,p-Akt/Akt表达增加(P0.01)。LY294002(PI3K抑制剂)组上述指标的检测结果与白藜芦醇组相反。结论:白藜芦醇可能部分通过激活PI3K/Akt通路抑制氧化应激及其诱导的凋亡,从而对肾小管上皮细胞发挥保护作用。     

姜黄素作用PTEN/PI3K/AKT通路诱导膀胱癌EJ细胞凋亡

目的:研究姜黄素抗癌作用的分子机制。方法:以人膀胱癌细胞株EJ为模型,采用MTT法、流式细胞术,检测姜黄素对EJ细胞生长和凋亡的影响,并通过Western Blotting法检测姜黄素对EJ细胞PTEN/PI3K/AKT通路凋亡相关蛋白表达的影响。结果:姜黄素以浓度及时间依赖的方式抑制EJ细胞的增殖;流式细胞仪检测发现姜黄素作用于EJ细胞24 h后凋亡率呈剂量依赖性增加。Western Blotting显示50μM姜黄素作用EJ细胞后,PTEN、GSK-3β、C-raf、Bad、caspase-9、caspase-3的活性增强,PARP的裂解增加和Akt、PDK1的表达水平降低。结论:姜黄素能增加EJ细胞PTEN的表达,进而抑制PI3K/Akt信号通路的激活,继之激活下游GSK-3β,caspase-9和Bad等多种促凋亡分子的表达,诱导EJ细胞发生凋亡。该研究表明PTEN/PI3K/Akt信号通路在姜黄素诱导的EJ膀胱癌细胞凋亡中起了重要作用。     

虎杖苷通过PI3K/AKT/mTOR信号通路诱导人宫颈癌细胞凋亡的初步研究

通过观察虎杖苷对宫颈癌HeLa细胞体外生长的抑制作用,初步探讨其诱导凋亡的可能机制。不同浓度的虎杖苷(50,100,150μmol·L~(-1))处理HeLa细胞后,采用MTT法检测虎杖苷对HeLa细胞增殖的抑制作用,AO/EB染色法荧光显微镜观察HeLa细胞凋亡的形态学变化;Annexin/PI双标记法检测HeLa细胞凋亡率;流式细胞仪分析HeLa细胞周期分布;RTPCR和Western blot法检测HeLa细胞中PI3K,AKT,mTOR,P70S6K的mRNA和蛋白表达。结果显示,虎杖苷显著抑制HeLa细胞增殖,且具有一定剂量依赖性;虎杖苷能够引起HeLa细胞发生S期阻滞,促进细胞凋亡,显著下调HeLa细胞中PI3K,AKT,mTOR,P70S6K的mRNA和蛋白表达。表明虎杖苷具有抑制宫颈癌HeLa细胞增殖及诱导凋亡的作用,其机制可能与抑制PI3K/AKT/mTOR信号通路及其下游基因蛋白表达有关。     

半枝莲总黄酮通过PI3K/AKT/mTOR通路诱导肿瘤细胞自噬的体内实验研究

该研究拟从体内角度研究半枝莲总黄酮(TF-SB)的抗肿瘤作用,并探讨TF-SB对肿瘤细胞自噬的影响及其对PI3K/AKT/mTOR通路的调控作用,为TF-SB的抗肿瘤作用提供实验依据。实验以黑色素瘤荷瘤小鼠模型为研究对象,分为空白对照组、雷帕霉素组(Rap,1.5 mg·kg~(-1))、TF-SB高、中、低剂量组(200,100,50 mg·kg~(-1)),每天给药1次,连续用药11 d,通过测量肿瘤的体积及抑瘤率,来观察TF-SB对黑色素瘤生长的抑制效果;通过TUNEL法检测肿瘤细胞凋亡情况,来进一步验证TF-SB的抗肿瘤活性;通过Western blot法检测LC3-Ⅰ,LC3-Ⅱ的蛋白表达,计算LC3-Ⅱ对LC3-Ⅰ的相对表达量,探讨TF-SB对肿瘤细胞自噬的诱导作用;通过检测PI3K/AKT/mTOR通路标志蛋白的磷酸化水平,研究TF-SB诱导细胞自噬的分子机制。结果表明,TF-SB可有效抑制荷瘤小鼠体内黑色素瘤的生长,减小肿瘤体积、提高抑瘤率,并且可以显著增加肿瘤细胞凋亡指数,增加LC3-Ⅱ/LC3-Ⅰ,抑制p-PI3K,p-AKT及p-mTOR的蛋白表达水平,与对照组比较有显著性差异(P0.05,P0.01或P0.001)。由此可见,半枝莲总黄酮具有抑制体内黑色素瘤生长的作用,其机制可能与通过抑制PI3K/AKT/mTOR通路,诱导肿瘤细胞自噬及凋亡有关。     

中医药干预肝纤维化PI3K/AKT信号通路研究进展

调控HCS凋亡是阻止肝纤维化进程的重要方法之一。针对PI3K/AKT通路在肝纤维化形成机制中举足轻重的地位,利用中药中特殊成分,从信号传导通路调控HSC凋亡,从而发挥抗肝纤维化作用。     

清解扶正颗粒通过PI3K/AKT通路抑制宫颈癌Caski细胞移植瘤的生长

目的 探讨清解扶正颗粒(QFG)对宫颈癌Caski细胞株裸鼠皮下移植瘤生长的抑制作用及其机制.方法 在12只雄性BALB/c裸鼠右前肢腋窝处接种Caski细胞,构建宫颈癌皮下移植瘤模型,待裸鼠右侧腋窝下出现绿豆大小移植瘤,即为模型建立成功.瘤体长至70～100 mm3后,将12只BALB/c裸鼠随机分为对照组和QFG组...     

参麦方通过PI3K/AKT通路改善糖尿病心肌损伤的作用研究

目的 通过糖尿病大鼠模型研究参麦方改善糖尿病心肌损伤的作用机制的研究,为临床应用提供理论基础和实验依据。方法 建立高脂饮食联合链脲佐菌素腹腔注射诱导的大鼠糖尿病模型,通过参麦方进行治疗,检测大鼠空腹血糖(FBG),血清中甘油三酯(TG)、乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)及天门冬氨酸氨基转移酶(AST)水平;HE染色观察心肌病理学变化,Masson染色观察心肌胶原沉积,PAS染色观察心肌糖原沉积;Western blot检测PI3K、p-AKT/AKT和抗凋亡蛋白Bcl-2、促凋亡蛋白Bax、Caspase-3表达水平。结果 参麦方可以降低糖尿病大鼠FBG水平,但作用较小,能显著降低血清中TG、LDH、CK-MB、AST水平(P<0.01或P<0.05)。病理染色结果显示,与正常组比较,模型组动物心肌细胞间隙变大排列不均,存在心肌损伤,心肌胶原纤维增多,糖原沉积增多,经参麦方给药治疗,心肌病理变化有所改善。Western blot检测结果显示,与模型组相比,参麦方给药上调PI3K、p-AKT/AKT、Bcl-2蛋白表达,下调Bax、Caspase-3蛋白表达...     

姜黄素通过PI3K/AKT信号通路调控神经干细胞增殖和凋亡实验研究

目的:观察姜黄素对神经干细胞增殖和凋亡的影响,并探讨其作用机制是否与PI3K/AKT信号通路有关。方法:分离新生24 h昆明小鼠的海马组织,提取原代神经干细胞并培养,并用免疫荧光检测鉴定。收集第3代神经干细胞并随机分为空白组、缺血模型组及不同浓度姜黄素干预组(0、0.1、0.5、2.5、12.5及62.5μmol/L),通过3-(4,5)-2-噻唑-(2,5)-二苯基溴化四氮唑蓝药敏试验(MTT)检测姜黄素干预后神经干细胞的存活情况,5-溴脱氧尿嘧啶核苷(Brdu)增殖试剂盒检测神经干细胞增殖情况。利用免疫蛋白印迹(Western blotting)法检测不同浓度姜黄素干预后神经干细胞pPI3K、AKT蛋白的表达情况。结果:观察到立体感强且折光好的神经球。MTT结果显示:姜黄素可增强神经干细胞增殖能力,并具有浓度和时间依赖性。Brdu检测结果显示:随着姜黄素浓度增加和干预时间延长,神经干细胞凋亡逐渐受抑制。姜黄素可上调PI3K、AKT蛋白的表达,并随着浓度逐渐上调,增强作用更加明显。结论:姜黄素可促进神经干细胞增殖,抑制其凋亡,这可能与激活PI3K/AKT信号通路有关。     

PI3K/AKT信号通路与流感病毒感染相关性研究

PI3K/AKT信号通路是一条关于细胞增殖和存活的信号通路,该信号通路在流感病毒的感染过程中发挥着重要的作用.从PI3K/AKT信号通路的构成、主要功能、通路下游关键分子与流感病毒的相关性及调控PI3K/AKT信号通路的中医药研究等方面出发,通过梳理有关PI3K/AKT信号通路的研究,能够对中医药科学有效防治流行性感冒...     

牛黄−麝香联合应用通过PI3K/AKT/mTOR信号通路抑制肝癌细胞增殖并诱导其凋亡

目的 探讨牛黄?麝香联合使用对人肝癌细胞SMMC-7721及裸鼠肝癌皮下移植瘤的影响,探讨其抗肝癌机制.方法 将牛黄?麝香联合应用于体内、体外两种肝癌模型.体外培养人肝癌SMMC-7721细胞系,实验分为牛黄?麝香处理组和对照组,采用不同浓度(生药量0.625、1.25、2.5、5 mg/mL)牛黄?麝香萃取液进行干预...     

白藜芦醇通过PI3K/Akt信号通路协同5-氟尿嘧啶对结肠癌细胞的抑制作用

[目的] 观察白藜芦醇（RES）对5-氟尿嘧啶（5-FU）化疗结肠癌敏感性及磷脂酰肌醇激酶（PI3K）/蛋白激酶（Akt）信号通路的影响。[方法] 体外培养SW620细胞,CCK8法检测细胞活力,倒置显微镜分析细胞克隆数,流式细胞术分析细胞凋亡率,蛋白免疫印迹法（Western blot）检测凋亡蛋白Cle-caspase 9、Cle-caspase 7和Cle-PARP及PI3K/Akt信号通路相关蛋白表达水平。[结果] 与对照组比较,RES组、5-FU组和RES+5-FU组SW620细胞活力、克隆数及p-p85、p-110β、p-PDK1和p-Akt表达水平明显降低,而细胞凋亡率及Cle-caspase 9、Cle-caspase 7和Cle-PARP表达水平明显增高（P<0.05）;与RES组和5-FU组比较,RES+5-FU组SW620细胞活力、克隆数及p-p85、p-110β和p-PDK1和p-Akt表达水平明显降低,而细胞凋亡率及Cle-caspase 9、Cle-caspase 7和Cle-PARP表达水平明显增高（P<0.05）。PI3K抑制剂LY294002增强RES和5-FU联合处理对SW620细胞生长的抑制作用,而PI3K基因过表达则降低RES和5-FU联合处理对SW620细胞生长的抑制作用。[结论] RES与5-FU协同抑制PI3K/Akt信号通路诱导结肠癌的化疗效果。     

Paeoniflorin ameliorates ANIT‐induced cholestasis by activating Nrf2 through an PI3K/Akt‐dependent pathway in rats

Cholestasis causes hepatic accumulation of bile acids leading to liver injury, fibrosis and liver failure. Paeoniflorin, the major active compound isolated from the roots of Paeonia lactiflora pall and Paeonia veitchii Lynch, is extensively used for liver diseases treatment in China. However, the mechanism of paeoniflorin's hepatoprotective effect on cholestasis has not been investigated yet. In this study, we administered paeoniflorin to rats for 3 days prior to alpha‐naphthylisothiocyanate (ANIT) administration for once, then went on administering paeoniflorin to rats for 3 days. The data demonstrated that paeoniflorin significantly prevented ANIT‐induced change in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphates (ALP), serum total bilirubin (TBIL), direct bilirubin (DBIL), total bile acid (TBA) and gamma‐glutamyl transpeptidase (γ‐GT). Histology examination revealed that paeoniflorin treatment rats relieved more liver injury and bile duct proliferation than ANIT‐administered rats. Moreover, our data indicated that paeoniflorin could restore glutathione (GSH) and its related synthase glutamate‐cysteine ligase catalytic subunit (GCLc) and glutamate‐cysteine ligase modifier subunit (GCLm) in ANIT‐treated group. In addition, the RNA and protein expression of Akt and nuclear factor‐E2‐related factor‐2 (Nrf2) were also activated by paeoniflorin in ANIT‐induced rats. These findings indicated that paeoniflorin protected ANIT‐induced cholestasis and increased GSH synthesis by activating Nrf2 through PI3K/Akt‐dependent pathway. Therefore, paeoniflorin might be a potential therapeutic agent for cholestasis. Copyright © 2015 John Wiley & Sons, Ltd.     

PESV干预K562细胞PI3K/Akt 信号通路凋亡调控的研究

[目的]探讨PESV对K562细胞PI3K/Akt信号蛋白及凋亡调控因子Bcl-2和Bad表达的影响。[方法]将体外培养K562细胞,经PESV处理不同时间后,流式细胞术检测细胞凋亡率,Western blot检测PI3K及p-Akt蛋白水平变化,实时荧光定量RT-PCR检测Bcl-2、Bad mRNA水平变化。[结果]与对照组相比,PESV处理后K562细胞凋亡率显著增加,PI3K及p-Akt表达降低,抗凋亡相关基因Bcl-2 mRNA表达降低,促凋亡基因Bad mRNA表达增加。[结论]PESV可能通过降低PI3K、Akt信号蛋白表达,调节Bcl-2和Bad表达,抑制K562细胞增殖,促进其凋亡。     

Aerobic exercise suppresses hepatocellular carcinoma by downregulating dynamin-related protein 1 through PI3K/AKT pathway

ObjectiveExercise, as a common non-drug intervention, is one of several lifestyle choices known to reduce the risk of cancer. Mitochondrial division has been reported to play a key role in the occurrence and transformation of hepatocellular carcinoma (HCC). This study investigated whether exercise could regulate the occurrence and development of HCC through mitosis.MethodsBioinformatics technology was used to analyze the expression level of dynamin-related protein 1 (DRP1), a key protein of mitochondrial division. The effects of DRP1 and DRP1 inhibitor (mdivi-1) on the proliferation and migration of liver cancer cells BEL-7402 were observed using cell counting kit-8, plate colony formation, transwell cell migration, and scratch experiments. Enzyme-linked immunosorbent assay, Western blot and real-time polymerase chain reaction were used to detect the expression of DRP1 and its downstream phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway. A treadmill exercise intervention was tested in a nude mouse human liver cancer subcutaneous tumor model expressing different levels of DRP1. The size and weight of subcutaneous tumors in mice were detected before and after exercise.ResultsThe expression of DRP1 in liver cancer tissues was significantly upregulated compared with normal liver tissues (P < 0.001). The proliferation rate and the migration of BEL-7402 cells in the DRP1 over-expression group were higher than that in the control group. The mdivi-1 group showed an inhibitory effect on the proliferation and migration of BEL-7402 cells at 50 μmol/L. Aerobic exercise was able to inhibit the expression of DRP1 and decrease the size and weight of subcutaneous tumors. Moreover, the expression of phosphorylated PI3K (p-PI3K) and phosphorylated AKT (p-AKT) decreased in the exercise group. However, exercise could not change p-PI3K and p-AKT levels after knocking down DRP1 or using mdivi-1 on subcutaneous tumor.ConclusionAerobic exercise can suppress the development of tumors partially by regulating DRP1 through PI3K/AKT pathway.     

木犀草素对气道平滑肌细胞PI3 K/AKT通路的影响

目的观察木犀草素对小鼠气道平滑肌细胞增殖的影响及其与PI3 K/AKT通路的关系。方法分离并培养小鼠气道平滑肌细胞,α-actin免疫细胞化学染色鉴定,细胞分4组:对照组、PDGF组、PDGF+LUT组、LUT组,MTT法检测细胞增殖,Western Blot方法观察p-AKT表达的变化。结果 MTT检测发现,PDGF刺激气道平滑肌细胞后A490值较对照组明显增加(P<0.01),PDGF+LUT组A490值较PDGF组明显降低(P<0.05),LUT组A490值较对照组无明显变化;Western Blot发现,对照组p-AKT极低,PDGF组明显升高,PDGF+LUT组p-AKT较PDGF组降低。结论木犀草素能够通过抑制AKT的磷酸化水平,限制体外培养的气道平滑肌细胞的PI3 K/AKT通路激活,从而发挥抑制小鼠气道平滑肌增殖的作用。     

化瘀消癥复方对PI3 K/Akt/mTOR信号通路及输卵管妊娠滋养细胞的作用机制研究

目的 探讨化瘀消癥复方对输卵管妊娠滋养细胞的凋亡、侵袭影响及PI3K/Akt/mTOR信号通路的调控机制.方法 以不同浓度的化瘀消癥复方水提液干预输卵管妊娠滋养细胞,设立空白组、甲氨蝶呤作为西药组、胞磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)抑制剂LY294002与雷帕霉...     

槲皮素对SMMC-7721肝癌细胞PI3K/AKT信号通路影响的探讨

目的:观察槲皮素( quercetin)对人肝癌细胞SMMC-7721增殖与细胞凋亡的影响,探讨其对SMMC-7721细胞PI3K/AKT信号通路的影响.方法:采用MTT法检测槲皮素对SMMC-7721细胞生长的抑制,流式细胞术检测细胞周期变化,Western blot检测槲皮素对SMMC-7721细胞PI3K/AKT信号通路凋亡相关蛋白表达的影响.结果:槲皮素抑制SMMC-7721肝癌细胞增殖作用明显,且呈浓度和时间依赖性.顺铂和槲皮素40,80,160,320 μmol·L-148 h抑制率分别为62.19％,25.47％,27.18％,36.96％,51.28％.流式细胞术结果提示,槲皮素80,160,320μmol ·L -可使SMMC-7721肝癌细胞周期阻滞于G0/G1期.Western blot凋亡相关蛋白表达检测表明,药物组AKT的表达受抑制,PTEN,Caspase-9蛋白的表达率随着药物浓度的增加而增加.结论:槲皮素能诱导SMMC-7721肝癌细胞凋亡,其机制可能是使肝癌细胞SMMC-7721周期阻滞于G0/G1期,PTEN的过表达抑制AKT活化,激活Caspase-9从而促进细胞凋亡.     

The vasoprotective effect of JP05 through the activation of PI3K/Akt-dependent eNOS and MEK/ERK pathways in brain endothelial cells

Aim of the study

Endothelial dysfunction is involved in stroke. Recent therapeutic options for stroke have focused on the combination therapy with a polyherbal mixture. This study was designed to provide insight into the effects of JP05, a water extract of 12 herbs, on the levels of regulators in bEnd.3 mouse brain endothelial cells.

Materials and methods

Production of endothelial nitric oxide synthase (eNOS)-mediated nitric oxide (NO), the expression of vascular endothelial growth factor (VEGF) and the phosphorylations of eNOS, phosphatidylinositol 3-kinase (PI3K)/Akt, extracellular signal-regulated protein kinase (ERK) and cAMP response element binding protein (CREB) in JP05 were assayed in bEnd.3 cells, a mouse brain endothelial line.

Results

JP05 led to increase the levels of eNOS-mediated NO generation and VEGF expression in bEnd.3 cells. JP05 induced the phosphorylation of eNOS, Akt and ERK in bEnd.3 cells. As well, JP05 blocked the inhibition of PI3K/Akt and ERK activities by LY294002 (PI3K/Akt inhibitor) and PD98059 (mitogen-activated protein kinase inhibitor), respectively. JP05 also induced the phosphorylation of CREB, which plays an important role in endothelial cell function and blood vessel development.

Conclusion

Taken together, these results indicate that JP05 can upregulate eNOS-mediated NO generation and VEGF expression through the ERK and/or PI3K/Akt activation, an upstream event of angiogenesis. JP05 with vasoprotective properties has a potential therapy for human brain diseases including stroke.