Micro RNA-21 promotes phosphatase gene and protein kinase B/phosphatidylinositol 3-kinase expression in colorectal cancer

AIM: To explore the regulatory mechanism of the target gene of micro RNA-21(mi R-21), phosphatase gene(p TEN), and its downstream proteins, protein kinase B(AKT) and phosphatidylinositol 3-kinase(p I3K), in colorectal cancer(CRC) cells. METHODS: Quantitative real-time p CR(q RT-p CR) and Western blot were used to detect the expression levels of mi R-21 and p TEN in HCT116, HT29, Colo32 and SW480 CRC cell lines. Also, the expression levels of p TEN m RNA and its downstream proteins AKT and p I3 K in HCT116 cells after downregulating mi R-21 were investigated. RESULTS: Comparing the mi R-21 expression in CRC cells, the expression levels of mi R-21 were highest in HCT116 cells, and the expression levels of mi R-21 were lowest in SW480 cells. In comparing mi R-21 and p TEN expression in CRC cells, we found that the protein expression levels of mi R-21 and p TEN were inversely correlated(p < 0.05); when mi R-21 expression was reduced, m RNA expression levels of p TEN did not significantly change(p > 0.05), but the expression levels of its protein significantly increased(p < 0.05). In comparing the levels of p TEN protein and downstream AKT and p I3 K in HCT116 cells after downregulation of mi R-21 expression, the levels of AKT and p I3 K protein expression significantly decreased(p < 0.05). CONCLUSION: p TEN is one of the direct target genesof mi R-21. Thus, phosphatase gene and its downstream AKT and p I3 K expression levels can be regulated by regulating the expression levels of mi R-21, which in turn regulates the development of CRC.

Salvianolic acid B promotes the invasion and migration of H_(2)O_(2)-induced HTR-8/Svneo trophoblast cells by upregulating matrix metalloproteinase-9 via the phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B pathway

OBJECTIVE:To elucidate the regulatory effects of salvianolic acid B(Sal B)on trophoblast cells in preeclampsia(PE).METHODS:3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide(MTT)assays were used to detect the viability of human extravillous trophoblast HTR-8/Svneo cells induced by H_(2)O_(2)following treatment with different concentrations of Sal B.The levels of oxidative stressrelated molecules,including superoxide dismutase,glutathione-Px and malondialdehyde were detected using corresponding kits.Cell apoptosis was detected using a Terminal deoxynucleotidyl transferase(Td T)d UTP NickEnd Labeling(TUNEL)assay,and the expression of apoptosis-related proteins was detected using western blot analysis.In the present study,wound healing and Transwell assays were performed to measure the levels of cell invasion and migration.Western blot analysis was also used to detect the expression levels of epithelialmesenchymal transition-related proteins.The mechanisms underlying Sal B were further investigated using reverse transcription-quantitative real-time polymerase chain reaction(RT-q PCR)and western blot analysis,to determine the expression levels of matrix metallopeptidase 9(MMP-9)and phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/protein kinase B(Akt).RESULTS:Sal B increased the activity of HTR-8/Svneo cells,inhibited oxidative damage and promoted the invasion and migration of trophoblast cells induced by H_(2)O_(2).Furthermore,the expression levels of MMP-9 and members of the PI3K/Akt signaling pathway were significantly decreased.The pathway agonist,LY294002,and MMP-9 inhibitor,GM6001,reversed the effects of Sal B on H_(2)O_(2)-induced cells.CONCLUSIONS:Sal B promoted the invasion and migration of H_(2)O_(2)-induced HTR-8/Svneo trophoblast cells by upregulating MMP-9 via the PI3K/Akt signaling pathway.

Bornyl acetate extracted from Sharen(Fructus Amomi)inhibits proliferation,invasion and induces apoptosis by suppressing phosphatidylinositol-3-kinase/protein kinase B signaling in colorectal cancer

OBJECTIVE:To investigate the antitumor effects of bornyl acetate(BA)isolated from Sharen(Fructus Amomi)in colorectal cancer(CRC)and the underlying mechanisms.METHODS:SW480 and HT29 cells were treated with increasing doses of BA in order to determine its antitumor effects in vitro.Cell viability,colony formation,cell cycle,and apoptosis as well as migration and invasion were assessed using various assays.In addition,the in vivo antitumor effects of BA were assessed using a xenograft mouse model.We then assessed the mechanism of action of BA by conducting pathway activator-mediated rescue experiments and assessed the protein levels by Western blot analysis.RESULTS:BA showed anti-CRC tumor activities in vitro by suppressing cell proliferation and colony formation,inducing apoptosis,blocking cell cycle,and inhibiting migration and invasion.These effects were mediated via suppression of the phosphatidylinositol-3-kinase/protein kinase B(PI3K/AKT)pathway.In the tumor xenograft experiment,BA was found to repress tumor growth in vivo with low toxicity.CONCLUSIONS:The results demonstrated that BA exerts antitumor effects by suppressing the PI3K/AKT pathway,with low toxicity.Thus,BA might be a potential novel therapeutic agent for CRC.

右美托咪定下调PI3K/AKT信号通路改善脂多糖诱导的结肠炎结肠上皮细胞损伤

为研究右美托咪定对脂多糖(LPS)诱导的人结肠上皮NCM-460细胞的炎症、增殖和凋亡的影响及磷脂酰肌醇-3-激酶/丝氨酸-苏氨酸激酶(PI3K/AKT)信号通路的调控作用,本研究体外培养人结肠上皮NCM-460细胞,将其分为对照组、LPS组(1μg/mL LPS)和不同质量浓度右美托咪定组(1μg/mL LPS+1.25、2.50、5.00和10.00μg/mL右美托咪定),干预24 h,筛选出合适的右美托咪定作用质量浓度用于后续试验。随后,将人结肠上皮NCM-460细胞分为对照组、LPS组、右美托咪定组(1μg/mL LPS+5.00μg/mL右美托咪定)、LY294002组(1μg/mL LPS+10μmol/L PI3K/AKT通路抑制剂LY294002)、抑制剂组(1μg/mL LPS+5.00μg/mL右美托咪定+10μmol/L LY294002)和激活剂组(1μg/mL LPS+5.00μg/mL右美托咪定+10μmol/L PI3K/AKT通路激动剂SC79),干预24 h。用细胞计数试剂盒-8(CCK-8)检测细胞活力;通过酶联免疫吸附试验(ELISA)检测肿瘤坏死因子-α(TNF-α)和白细胞介素-8(IL-8)的表达水平;用5-乙炔基-2'脱氧尿嘧啶核苷(EdU)测定细胞增殖率;用Hoechst 33258染色法测定细胞凋亡率;用蛋白免疫印迹(WB)法测定细胞周期蛋白D1(Cyclin D1)、剪切的半胱氨酸蛋白酶-3(cleaved Caspase 3)和PI3K/AKT信号通路关键蛋白的表达水平。根据细胞活力和炎症因子TNF-α的表达水平选择5.00μg/mL右美托咪定用于后续试验。结果显示,与对照组相比,LPS组细胞增殖率和Cyclin D1的表达水平显著降低(P<0.05),细胞凋亡率、TNF-α、IL-8、cleaved Caspase 3的表达水平、p-PI3K/PI3K及p-AKT/AKT的比值显著升高(P<0.05);右美托咪定组和LY294002组中的右美托咪定和LY294002扭转了LPS对人结肠上皮NCM-460细胞的上述作用(P<0.05);与右美托咪定组相比,抑制剂组中的LY294002增强了右美托咪定对LPS诱导的人结肠上皮NCM-460细胞的作用(P<0.05),激活剂组中的SC79则削弱了右美托咪定对LPS诱导的人结肠上皮NCM-460细胞的作用(P<0.05)。研究表明,右美托咪定能促进LPS诱导的人结肠上皮NCM-460细胞增殖,抑制其炎症和凋亡,其作用机制可能与阻滞PI3K/PI3K信号通路信号转导有关。本研究为结肠炎的治疗提供了新的方向。

中风活心软胶囊通过PPARγ受体上调STAT3磷酸化激活PI3K/AKT通路抑制脑缺血再灌注损伤内质网应激的作用机制研究

目的探讨中风活心软胶囊介导过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptorγ,PPARγ)受体上调信号传导转录激活因子3(signal transducer and activator of transcription 3,STAT3)磷酸化诱导磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinase,PI3K)/丝氨酸/苏氨酸蛋白激酶(BAKT serine/threonine kinase,AKT)信号通路参与脑缺血再灌注损伤内质网(endoplasmic reticulum,ER)应激和神经保护的作用机制研究,阐明中风活心软胶囊对脑缺血后神经损伤修复的调控机制。方法选取30只SPF级雄性SD大鼠,随机分为对照组、模型组和治疗组,采用Zea Longa法建立右侧大脑中动脉缺血(middle cerebral artery ischemia,MCAI)大鼠模型,采用改良神经功能损伤评分(modified neurological severity score,mNSS)评估大鼠神经功能缺损情况,以mNSS评分2~18分为造模成功。仅治疗组给予中风活心软胶囊。Western blot法分别检测缺血半暗区皮质PPARγ受体蛋白、STAT3磷酸化蛋白、PI3K/AKT信号通路蛋白的表达,检测ER应激标记分子葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)及真核翻译起始因子2α激酶3(PKR-like endoplasmic reticulum kinase,PERK)-激活转录因子4(activating transcription factor 4,ATF4)-C/EBP同源蛋白(C/EBP-homologous protein,CHOP)通路蛋白表达水平。结果模型组大鼠神经功能缺损情况,透射电镜观察缺血半暗区皮质神经元显著减少,提示右侧MCAI大鼠模型构建成功。模型组、治疗组mNSS评分均高于对照组,但治疗组低于模型组,差异有统计学意义(P<0.05)。模型组大鼠缺血区PPARγ受体蛋白、STAT3磷酸化蛋白、PI3K及AKT蛋白表达均低于对照组与治疗组,差异有统计学意义(P<0.05);治疗组与对照组各指标比较差异无统计学意义。治疗组、模型组大鼠缺血区GRP78、PERK、ATF4和CHOP蛋白表达均高于对照组,但治疗组均低于模型组,差异有统计学意义(P<0.05)。结论中风活心软胶囊可显著提高脑缺血大鼠缺血半暗区皮质PPARγ受体蛋白、STAT3磷酸化蛋白、PI3K/AKT信号通路蛋白表达,其作用机制可能是通过介导PPARγ受体上调STAT3磷酸化诱导PI3K/AKT信号通路参与脑缺血再灌注损伤ER应激和神经保护,从而发挥脑保护效应。

基于PI3K/Akt通路探讨醒脑静注射液改善颅脑创伤患者神经损伤及预后的效果

目的基于磷酸肌醇-3-激酶/丝氨酸/苏氨酸蛋白激酶(PI3K/Akt)通路探讨醒脑静注射液治疗颅脑创伤患者的效果,分析其对神经损伤、预后的影响。方法选取2020-06—2023-06武汉市第一医院收治的96例颅脑创伤患者,分为对照组48例(常规治疗)和观察组48例(常规治疗+醒脑静注射液治疗)。比较2组治疗效果、治疗前后PI3K/Akt通路分子[外周血单个核细胞(PMBC)中PI3K、Akt、哺乳动物雷帕霉素靶蛋白(mTOR)mRNA]、神经损伤因子、神经损伤程度[中国脑卒中临床神经功能缺损程度评分量表(CSS)、美国国立卫生研究院卒中量表(NIHSS)、格拉斯哥昏迷量表(GCS)评分],以及并发症发生率、预后情况。结果与对照组比较,观察组总有效率升高,并发症发生率降低(P<0.05)。观察组治疗7 d、14 d后PI3K、Akt、mTOR mRNA相对表达量升高(P<0.05),血清神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)、S-100β蛋白(S-100β)、髓鞘碱性蛋白(MBP)水平降低(P<0.05),CSS、NIHSS评分降低,GCS评分升高(P<0.05)。治疗后3个月观察组预后良好率升高(P<0.05)。结论醒脑静注射液治疗颅脑创伤患者的疗效确切,可促进神经功能恢复,减少并发症,并可改善患者预后,其作用机制可能与激活PI3K/Akt通路有关。

艾司氯胺酮联合丙泊酚全麻诱导对于腹腔镜胆囊手术中的老年患者PI3K/AKT信号通路的影响研究

目的 探究艾司氯胺酮联合丙泊酚全麻诱导对接受腹腔镜胆囊手术的老年患者血清磷脂酰肌醇-3-羟基酶(P13K)/丝氨酸-苏氨酸蛋白激酶(AKT)信号通路的影响。方法 前瞻性选取2021年1月至2023年12月在安徽省公共卫生临床中心行腹腔镜胆囊手术的80例老年患者为研究对象,按照随机数字表法分为对照组和研究组,每组各40例。对照组实施丙泊酚全麻诱导方案,研究组在对照组麻醉方案中加用艾司氯胺酮。比较两组患者的临床指标(苏醒时间、拔管时间及定向力恢复时间),麻醉诱导前5 min(T0)、诱导完成(T1)、插管(T2)、拔管(T3)患者的心率及平均动脉压,术后30 min、术后2 h、6 h、12 h、24 h的Ramsay评分,术后30 min、2 h、6 h、12 h、24 h时的视觉模拟评分法(VAS)评分,以及术前、术后即刻、术后1 d及术后3 d时的PI3K、AKT水平,并观察两组患者的不良反应发生情况。结果 研究组患者的苏醒时间、拔管时间及定向力恢复时间均短于对照组,差异均有统计学意义(P<0.05)。T0时,两组间平均动脉压、心率比较,差异均无统计意义(P>0.05);研究组T1时平均动脉压、心率均高于对照组,T2、T3时平均动脉压、心率均低于对照组,差异均有统计学意义(P<0.05)。术后30 min和术后2 h时,研究组Ramsay评分高于对照组,差异均有统计学意义(P<0.05);但在术后6、12、24 h时的Ramsay评分比较,差异均无统计学意义(P>0.05)。术后30 min、2 h、6 h、12 h、24 h时,研究组患者的VAS评分均低于对照组,差异均有统计学意义(P<0.05)。术后即刻、术后1 d及术后3 d时,研究组血清PI3K、AKT水平均低于对照组,差异均有统计学意义(P<0.05)。两组总不良反应发生率比较,差异无统计学意义(P>0.05)。结论 艾司氯胺酮联合丙泊酚全麻诱导在腹腔镜胆囊手术中的应用对老年患者的生命体征、麻醉恢复过程和术后疼痛控制均有积极影响,其对疼痛的控制与调节PI3K/AKT信号通路有关,可为临床麻醉管理提供潜在的有效策略。

辛开苦降法对多囊卵巢综合征胰岛素抵抗患者卵巢功能、子宫内膜容受性及磷脂酰肌醇3激酶/丝苏氨酸蛋白激酶通路的影响

目的 观察辛开苦降法对多囊卵巢综合征(Polycystic ovarian syndrome,PCOS)胰岛素抵抗(Insulin resistance,IR)患者卵巢功能、子宫内膜容受性及磷脂酰肌醇3激酶/丝苏氨酸蛋白激酶(Phosphatidylinositol 3 kinase/serine threonine protein kinase,PI3K/AKT)通路的影响。方法 选取2018年11月—2020年1月期间四川中医药高等专科学校附属绵阳市中医医院收治的PCOS-IR患者83例,按随机数字表法分为对照组41例和治疗组42例。对照组采用炔雌醇环丙孕酮联合二甲双胍治疗,治疗组采用中医辛开苦降法(中药),均连续治疗12周。观察比较两组患者治疗前后体质量指数(Body mass index,BMI)、卵巢功能[卵泡刺激素(Follicle stimulating hormone,FSH)、黄体生成素(Luteinizing hormone,LH)、LH/FSH与抗缪勒管激素(Anti-mullerian hormone,AMH)]、血糖[空腹胰岛素(Fasting insulin,FIN)、空腹血糖(Fasting blood glucose,FPG)、餐后2 h血糖(2 hours postprandial blood glucose,2 h PBG)与胰岛素抵抗指数(Homeostasis model assessmentinsulin resistance,HOMA-IR)]、子宫内膜容受性[收缩期峰值流速(Vmax)、搏动指数(Pulse index,PI)、阻力指数(Resistance index,RI)、血流参数血管化指数(Vascularity index,VI)、血流指数(Flow index,FI)与血管化血流指数(Vascularity flow index,VFI)]、外周血PI3K/AKT通路(PI3K、AKT mNRA),治疗期间不良反应情况。结果 治疗后两组患者BMI、FPG、FIN、2 h GLU、HOMA-IR水平均较治疗前明显降低,差异有统计学意义(P<0.05);且治疗组BMI、FPG、FIN、2 h GLU、HOMA-IR水平均明显低于对照组,差异有统计学意义(P<0.05)。治疗后两组患者AMH、LH、LH/FSH水平均较治疗前降低,差异有统计学意义(P<0.05);FSH较治疗前无显著变化,差异无统计意义(P>0.05);且治疗组AMH、LH、LH/FSH明显低于对照组,差异有统计学意义(P<0.05)。治疗后两组患者子宫内膜厚度、子宫容积、Vmax、VI、FI、VFI均较治疗前明显升高,PI、RI均较治疗前降低,差异有统计学意义(P<0.05);且治疗组子宫内膜厚度、子宫容积、Vmax、VI、FI、VFI均明显高于对照组,PI、RI均明显低于对照组,差异有统计学意义(P<0.05)。治疗后两组患者PI3K、AktmRN表达均较治疗前升高,差异有统计学意义(P<0.05);且治疗组PI3K、AktmRN表达均明显高于对照组,差异有统计学意义(P<0.05)。治疗期间,两组患者均未发生严重不良反应。结论 中医辛开苦降法不仅能改善PCOS-IR患者胰岛素抵抗,还可改善其卵巢功能、子宫内膜容受性,激活PI3K/AKT通路,安全性高。

熊果苷调节PI3K/Akt/GLUT1信号通路对胃癌细胞恶性进展的影响

目的 探讨熊果苷(Arb)调节磷脂酰肌醇-3激酶B/蛋白激酶B/葡萄糖转运蛋白1(PI3K/Akt/GLUT1)信号通路对胃癌细胞恶性进展的影响。方法 使用不同浓度(12.5、25、50、100、200、400 mol/L)Arb处理SNU-601细胞,检测细胞活性,筛选最佳浓度;将细胞分为对照组(Control组)、熊果苷低、中、高浓度组(L-Arb组、M-Arb组、H-Arb组)、熊果苷高浓度+PI3K/Akt激活剂组(H-Arb+740 Y-P组),分别检测细胞凋亡率、细胞周期、葡萄糖摄取、乳酸生成、ATP生成、细胞迁移数和细胞侵袭数;Western blot检测Bax、半胱氨酸天冬氨酸蛋白水解酶3(cleaved-caspase3)、B细胞淋巴瘤-2(Bcl-2)、p-PI3K、PI3K、p-Akt、Akt、GLUT1、HKⅡ蛋白表达。结果 12.5~400 mol/L的Arb可显著抑制SNU-601细胞增殖,选择50、100、200 mol/L的Arb进行后续实验。与Control组比较,L-Arb组、M-Arb组、H-Arb组细胞活性、葡萄糖摄取、乳酸生成、ATP生成、细胞迁移数、细胞侵袭数及Bcl-2、p-PI3K/PI3K、p-Akt/Akt、GLUT1、HKⅡ蛋白表达降低,细胞凋亡率和Bax、cleaved-caspase3蛋白表达增加(P<0.05);与H-Arb组比较,H-Arb+740 Y-P组细胞活性、葡萄糖摄取、乳酸生成、ATP生成、细胞迁移数、细胞侵袭数及Bcl-2、p-PI3K/PI3K、p-Akt/Akt、GLUT1、HKⅡ蛋白表达增加,细胞凋亡率和Bax、cleaved-caspase3蛋白表达降低(P<0.05)。结论 Arb通过抑制PI3K/Akt/GLUT1信号通路抑制胃癌细胞恶性进展。

紫杉叶素调控PI3K/AKT/mTOR通路对糖尿病模型大鼠氧化应激和胰岛功能的机制研究

目的:探索紫杉叶素(TAX)调控磷脂酰肌醇3-激酶(PI3K)/丝苏氨酸蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)通路对糖尿病模型大鼠氧化应激和胰岛功能的影响。方法:102只无特定病原体(SPF)级SD大鼠高脂高糖饮食饲养4周,建立2型糖尿病大鼠模型,造模成功后随机分为Model组、TAX低剂量组(5 mg/kg)、TAX中剂量组(10 mg/kg)、TAX高剂量组(20 mg/kg)、PI3K激活剂组(0.02 mg/kg PI3K激活剂740Y-P+20 mg/kg TAX)、mTOR激活剂组(10 mg/kg mTOR激活剂MHY1485+20 mg/kg TAX),每组12只。另外取12只正常大鼠作为Control组,造模成功后,给予相应药物,每日1次,连续干预5周。测量各组大鼠体质量;血糖仪检测空腹血糖(FBG);对各组大鼠进行葡萄糖耐量试验(OGTT);酶联免疫吸附测定法(ELISA)测定大鼠血清空腹胰岛素(FINS)水平,计算胰岛素抵抗指数(HOMA-IR)、胰岛β细胞功能指数(HOMA-β)、胰岛素敏感指数(ISI);苏木精-伊红(HE)染色观察大鼠胰腺组织损伤情况,计算胰岛个数;全自动生化分析仪检测大鼠血清总胆固醇(TC)、三酰甘油(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、游离脂肪酸(FFA)水平;试剂盒检测大鼠血清丙二醛(MDA)、活性氧(ROS)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)水平;蛋白免疫印迹法(Western Blot)检测大鼠胰腺组织中PI3K/AKT/mTOR通路蛋白表达。结果:与Control组比较,Model组大鼠体质量、FBG、OGTT(0.5、1.0、2.0 h)血糖值、FINS、HOMA-IR、TC、TG、LDL-C、FFA、ROS、MDA、p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR增加,ISI、HOMA-β、HDL-C、GSH-Px、SOD、胰岛数量降低(P<0.05);与Model组比较,TAX低剂量组、TAX中剂量组、TAX高剂量组大鼠体质量、FBG、OGTT(0.5、1.0、2.0 h)血糖值、FINS、HOMA-IR、TC、TG、LDL-C、FFA、ROS、MDA、p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR水平降低,ISI、HOMA-β、HDL-C、GSH-Px、SOD、胰岛数量增加(P<0.05);PI3K、mTOR激活剂均减弱了高剂量TAX对糖尿病模型大鼠的改善作用(P<0.05)。结论:TAX通过抑制PI3K/AKT/mTOR信号通路,抑制STZ诱导的糖尿病模型大鼠氧化应激,改善胰岛功能。

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.

PI3K-like Kinases Restrain Pim Gene Expression in Endothelial Cells

Pim kinases contribute to tumor formation and development of lymphoma,which shows enhanced DNA replication,DNA recombination and repair.Endothelial cells (ECs) express all the three members of Pim kinase gene family.We hypothesized that DNA repair gene would regulate Pim ex-pression in ECs.Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in M199 culture medium.The cellular distribution of Pim-3 in ECs was determined by immunofluorescent staining.The siRNA fragments were synthesized and transfected by using Lipofectamine LTX.The total cellular RNA was extracted from the cells by using Trizol reagent.cDNAs were quantified by semi-quantity PCR.The effects of LY294002 and wortmannin on RNA stability in ECs were also ex-amined.Our data showed that LY294002 and wortmannin,phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase inhibitors,increased Pim mRNA expression in ECs without altering the mRNA stabil-ity.RNA interference (RNAi) targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) increased mRNA expression of Pim-3 and Pim-1,respectively.Silencing of Akt decreased Pim-1 instead of Pm-2 and Pim-3 gene expression in ECs.But etoposide,a nucleoside analogue,which could activate DNA-PKcs and ATM,increased Pim expression in ECs.Our study indicates that the expression of Pim kinases is physiologically related to DNA-PKcs and ATM in ECs.

汉黄芩素调节磷脂酰肌醇3激酶/丝氨酸苏氨酸蛋白激酶/核因子κB信号通路对慢性阻塞性肺疾病大鼠辅助性T细胞17/调节性T细胞平衡的影响

目的探讨汉黄芩素(Wog)调节磷脂酰肌醇3激酶(PI3K)/丝氨酸苏氨酸蛋白激酶(Akt)/核因子κB(NF-κB)信号通路对慢性阻塞性肺疾病(COPD)大鼠辅助性T细胞17(Th17)/调节性T细胞(Treg)平衡的影响。方法2022年8-12月,大鼠采用随机数字表法分为Model组、低剂量Wog组(Wog-L组,50 mg/kg)、高剂量Wog组(Wog-H组,100 mg/kg)、阳性药物氨茶碱组(Ami组,2.3 mg/kg)、IGF-1(PI3K激活剂)组(1.33 mg/kg)、Wog-H+IGF-1组(100 mg/kg+1.33 mg/kg)、对照组(CK组),每组12只。除CK组外,其他组大鼠均需利用烟熏法联合气管滴注脂多糖(LPS)的方法构建COPD模型,建模成功24 h后,进行给药处理,每天1次给药,持续4周。检测呼气峰流量(PEF)、每分钟通气量(MV)、吸气峰流量(PIF);流式细胞术检测外周血中Th17/Treg;HE染色检测肺组织病理;酶联免疫吸附法检测大鼠肺组织中白细胞介素(IL)-17、IL-10水平;蛋白质印迹法检测肺组织中维甲酸相关孤核受体γt(RORγt)、叉头框蛋白P3(Foxp3)、磷酸化PI3K(p-PI3K)、磷酸化Akt(p-Akt)、磷酸化NF-κB p65(p-NF-κB p65)蛋白。结果与CK组比较,Model组大鼠PEF(12.56±0.47比8.72±0.39)、PIF(9.35±0.32比7.24±0.17)、MV(132.26±5.78比96.63±3.28)、Treg(31.18±2.62比15.52±1.01)比例、IL-10(23.35±1.16比8.85±0.27)明显降低(均P<0.05);Th17(3.14±0.13比18.86±1.67)比例、Th17/Treg(0.10±0.01比1.22±0.11)、肺泡间隔(33.36±1.48比49.78±1.73)、气道炎症评分(0比4.56±0.23)及IL-17(75.83±3.60比185.56±8.62)水平明显升高(均P<0.05)。与Model组比较,Wog-L组、Wog-H组PEF(9.66±0.40,11.49±0.51)、PIF(8.28±0.19,9.03±0.22)、MV(105.54±4.11,126.67±5.72)、Treg(19.93±1.18,27.73±2.05)比例、IL-10(11.56±0.33,20.72±0.59)水平明显升高(均P<0.05);Th17(3.14±0.13比18.86±1.67)比例、Th17/Treg(0.10±0.01比1.22±0.11)、肺泡间隔(43.45±1.26,35.78±1.12)、气道炎症评分(3.75±0.17,0.86±0.07)、IL-17(162.27±7.14,103.35±4.33)水平明显降低(均P<0.05)。与CK组比较,Model组大鼠RORγt(0.15±0.01比1.34±0.11)、p-PI3K(0.22±0.01比0.86±0.07)、p-Akt(0.18±0.01比0.75±0.06)、p-NF-κB p65(0.11±0.01比0.69±0.06)蛋白表达升高,Foxp3(1.45±0.27比0.35±0.02)蛋白表达降低(均P<0.05)。与Model组相比,Wog-L组、Wog-H组RORγt(1.08±0.10,0.36±0.02)、p-PI3K(0.71±0.06,0.35±0.03)、p-Akt(0.62±0.06,0.28±0.02)、p-NF-κB p65(0.52±0.05,0.26±0.02)蛋白表达明显降低,Foxp3(0.57±0.04,1.13±0.09)蛋白表达明显升高(均P<0.05)。Wog-H组与Ami组大鼠上述各指标水平近似,均差异无统计学意义(P>0.05)。IGF-1逆转了高剂量Wog对COPD大鼠Th17/Treg的影响。结论Wog促进COPD大鼠Th17/Treg平衡的机制可能与下调PI3K/Akt/NF-κB通路有关。

TopoisomeraseⅡalpha promotes gallbladder cancer proliferation and metastasis through activating phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

Background:TopoisomeraseⅡalpha(TOP2A)has been reported to play a crucial role in the tumorigenesis of various cancer types.However,the biological role of TOP2A in gallbladder cancer(GBC)remains unknown.The current study aimed to explore the function and potential mechanism of TOP2A in GBC.Methods:Based on Gene Expression Profiling Interactive Analysis data,we found TOP2A was significantly up-regulated in GBC tissues and resulting in shorter overall survival.Quantitative real-time polymerase chain reaction and immunohistochemistry were conducted to detect the expression of TOP2A in 45 pairs of GBC tissues and adjacent non-tumor tissues.In vitro,cell proliferation,migration,and invasion ability were examined by cell counting kit-8 and transwell assay,respectively.Epithelial-mesenchymal transition(EMT)related and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway-related markers were measured by Western blotting.Xenograft model assay was performed to evaluate the effect of TOP2A in vivo.Results:TOP2A was found up-regulated in GBC(tumor vs.normal,12.62 vs.0.34)and correlated with the late tumor node metastasis stage(P=0.0032),present of lymph node metastasis(P=0.0273),and poor prognosis in GBC patients(log-rank P=0.028).In vitro and in vivo assays showed that knockdown of TOP2A notably inhibited cell proliferation,migration,invasion,EMT process,and tumor growth in GBC.In addition,TOP2A down-regulation significantly decreased the protein levels of phosphor(p)-PI3K,p-Akt,and p-mTOR.Conclusion:Our study demonstrates that TOP2A was overexpressed in GBC and associated with poor prognosis in GBC patients.TOP2A promotes GBC cell proliferation,migration,invasion,EMT process,and tumor growth through activating PI3K/Akt/mTOR signaling pathway,and may serve as a novel prognostic biomarker and therapeutic target for GBC.

Efficacy of self-made Gengnian decoction on phosphatidylinositol 3-kinases/protein kinase B/mammalian target of rapamycin signaling pathway in perimenopausal rats

OBJECTIVE:To investigate the efficacy of self-made Gengnian decoction on expressions of phosphatidylinositol 3-kinase(PI3 K),protein kinase B(Akt)and mammalian target of rapamycin(m TOR)in ovarian tissues of perimenopausal rats.They were identified with symptom pattern of kidney-Yang deficiency in terms of Traditional Chinese Medicine.METHODS:Female Sprague-Dawley rats aged10-12 months were selected.Estrous cycle was observed by vaginal smears of keratinocytes to screen the perimenopausal model rats.The chosen rats were randomly divided into five groups,including perimenopausal model of kidney-Yang deficiency group(24 rats),self-made Gengnian decoction of high-dose group(24 rats),self-made Gengnian decoction of middle-dose group(24 rats),self-made Gengnian decoction of low dose group(24 rats)and tibolone control group(24 rats).In addition,rats aged 4-6 months were selected as young control group.The perimenopausal model rats of kidney-Yang deficiency were prepared by alternative intramuscular injection of hydrocortisone 5 mg·kg^-1·d^-1The successfully prepared models in self-made Gengnian decoction of high-dose,middle-dose and low-dose groups and tibolone control group were given self-made Gengnian decoction 26.4,13.2 and 6.6 mg·kg^-1·d^-1,and tibolone tablets solvent 0.22 mg·kg^-1·d^-1,respectively,through intragastric administration.Models group and young control group were given the same dose of normal saline,1 time a day for 15 consecutive days.24 h after the last administration,blood and ovarian tissues were collected after anesthesia with 20%ethyl carbamate.The follicles of different levels in ovarian tissue were observed and counted by histopathological hematoxylin-eosin staining.Enzyme linked immunosorbent assay was applied to test insulin-like growth factor-1(IGF-1)level in the serum of experimental rats.The expression levels of PI3 K,phosphorylated-Akt(p-Akt)and phosphorylated-m TOR(p-m TOR)m RNA in ovarian tissue were detected by quantitative real-time polymerase chain reaction.RESULTS:The total follicle counts of perimenopausal model rats with kidney-Yang deficiency were significantly reduced,and the number of follicles(mainly increased in preantral follicles and antral follicles)in perimenopausal model rats with kidney-Yang deficiency was significantly increased after intervention of high and middle doses of Gengnian decoction and tibolone(P<0.05).Compared with normal rats in young control group,the levels of IGF-1 in serum of perimenopausal rats with kidney-Yang deficiency were significantly decreased(P<0.01),and those intervened by high dose of Gengnian decoction and tibolone were significantly up-regulated.The relative expression levels of PI3 K,p-Akt,p-m TOR m RNA in ovarian tissues of perimenopausal rats with kidney-Yang deficiency were significantly lower than those of young rats(P<0.01),and those intervened by high dose of Gengnian decoction and tibolone were significantly up-regulated(P<0.05).CONCLUSION:Self-made Gengnian decoction can increase the levels of IGF-1,PI3 K,Akt and m TOR m RNA expression in serum.

正畸牙移动中脂联素调控PI3K/AKt信号通路对牙周组织改建的影响

目的探究正畸牙移动中脂联素(adiponectin,APN)调控磷脂酰肌醇3激酶(Phosphatidy linositol 3 kinase,PI3K)/蛋白质丝氨酸-苏氨酸激酶(Protein serine threonine kinase,AKt)信号通路对牙周组织改建的影响。方法选取60只大鼠构建正畸牙移动模型并随机分为空白组、PBS组(注射PBS)、APN组(注射APN),以及APN+胰岛素生长因子1(Insulin growth factor 1,IGF-1)组(注射APN,同时给予PI3K/AKt通路激活剂IGF-1),每组15只。观察加力后1,7,14 d第1磨牙移动距离及破骨细胞数量,观察14 d时牙周组织形态,比较各组牙周组织PI3K、AKt mRNA表达及PI3K、p-PI3K、AKt、p-AKt蛋白表达。结果加力后7 d,14 d,APN组第1磨牙移动距离明显小于空白组(P<0.05),APN组新骨形成量及新骨矿化程度高于空白组、PBS组,APN组破骨细胞数量明显少于空白组(P<0.05)。APN组牙周组织PI3K、AKt mRNA及p-PI3K、p-AKt蛋白表达均明显低于空白组(P<0.05)。APN+IGF-1组PI3K、AKt mRNA及p-PI3K、p-AKt蛋白表达均明显高于APN组(P<0.05)。结论APN可降低正畸牙移动大鼠模型压力侧破骨细胞数量及牙移动速度,在延缓正畸牙移动牙周组织改建过程中具有重要作用,其机制可能是通过抑制PI3K/AKt信号通路实现。

黄连温胆汤通过PI3K/Akt通路对缺氧复氧诱导心肌细胞损伤作用机制

目的:探讨黄连温胆汤通过磷脂酰肌醇3激酶(PI3K)/丝氨酸苏氨酸蛋白激酶(Akt)通路对缺氧复氧诱导心肌细胞损伤的作用机制。方法:构建缺氧复氧诱导的大鼠心肌细胞H9C2损伤模型,分为对照组、缺氧复氧组、黄连温胆汤低剂量组、黄连温胆汤中剂量组、黄连温胆汤高剂量组。CCK-8法检测H9C2细胞增殖。流式细胞术检测H9C2细胞凋亡。检测细胞内活性氧(ROS)、细胞上清中超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量。Western blotting检测细胞中Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)、p-PI3K、p-Akt蛋白表达。结果:缺氧复氧组H9C2细胞OD 450值、SOD活性、Bcl-2、p-PI3K、p-Akt蛋白表达均低于对照组,而细胞凋亡率、ROS平均荧光强度、MDA含量、Bax蛋白表达高于对照组,差异有统计学意义(均P<0.05)。黄连温胆汤低剂量组、黄连温胆汤中剂量组、黄连温胆汤高剂量组H9C2细胞OD 450值、SOD活性、Bcl-2、p-PI3K、p-Akt蛋白表达高于缺氧复氧组,而细胞凋亡率、ROS平均荧光强度、MDA含量、Bax蛋白表达低于缺氧复氧组,差异有统计学意义(均P<0.05)。结论:黄连温胆汤通过激活PI3K/Akt信号通路减轻缺氧复氧诱导的H9C2细胞损伤。

抑制PI3K/Akt/mTOR信号通路对MPP+处理的SH-SY5Y细胞自噬、凋亡及PD特征蛋白表达的影响

目的探讨抑制磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对1-甲基-4-苯基吡啶离子(MPP+)处理的SH-SY5Y细胞自噬、凋亡及帕金森病(PD)特征蛋白α-突触核蛋白(α-syn)、酪氨酸羟化酶(TH)表达的影响。方法以MPP+、PI3K/Akt激活剂胰岛素样生长因子-1(IGF-1)、PI3K/Akt抑制剂LY294002作用于人神经母细胞瘤SH-SY5Y细胞,CCK-8检测细胞存活率;流式细胞术检测细胞凋亡率;吖啶橙(AO)染色检测自噬空泡;Western blot检测α-syn、TH、p62、微管相关蛋白1轻链3B(LC3B)、p-mTOR、mTOR、p-PI3K、PI3K、p-Akt、Akt蛋白水平。结果MPP+干预显著降低SH-SY5Y细胞存活率,且呈现剂量依赖性(P<0.05)。MPP+和IGF-1处理后SH-SY5Y细胞存活率降低,凋亡率增高(P<0.05);细胞中自噬空泡减少,LC3BⅡ/Ⅰ降低,p62蛋白水平增高(P<0.05);TH蛋白水平降低,α-syn蛋白水平增高(P<0.05);p-PI3K/PI3K、p-Akt/Akt与p-mTOR/mTOR增高(P<0.05)。LY294002对SH-SY5Y细胞的影响与MPP+和IGF-1相反,且LY294002可在一定程度上逆转MPP+对SH-SY5Y细胞的影响(P<0.05)。结论抑制PI3K/Akt/mTOR信号通路可能对MPP+诱导的SH-SY5Y细胞毒性具有保护作用。

桃核承气汤对多囊卵巢综合征大鼠卵巢组织PI3K/AKT/mTOR信号通路的影响

目的探究桃核承气汤对多囊卵巢综合征(PCOS)大鼠卵巢组织磷脂酰肌醇3-激酶(PI3K)/丝氨酸/苏氨酸蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路的影响。方法将96只雌性SD大鼠随机分为对照组,PCOS组,二甲双胍组,桃核承气汤低、中、高剂量组,每组16只;除对照组外,其余各组大鼠皮下注射脱氢表雄酮构建PCOS模型。造模成功后二甲双胍组大鼠给予0.1 g/kg二甲双胍灌胃,桃核承气汤低、中、高剂量组分别给予1.89、3.78、7.56 g/kg桃核承气汤灌胃,对照组和PCOS组灌胃等量生理盐水,1次/d,共28 d。血糖仪检测空腹血糖(FPG)水平,酶联免疫吸附试验检测血清卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E_(2))、睾酮(T)、空腹胰岛素(FINS)水平,计算胰岛素抵抗指数(HOMA-IR);苏木素伊红染色观察大鼠卵巢组织病理学情况;荧光定量PCR和蛋白免疫印迹实验分别检测大鼠卵巢组织PI3K/AKT/mTOR通路相关分子mRNA和蛋白表达。结果与对照组相比,PCOS组大鼠卵泡体积增大,含卵泡液,且细胞间隙增大、黄体减少、泡膜细胞层增生,血清FSH、E_(2),卵巢组织中PI3K/AKT/mTOR通路mRNA和蛋白表达降低,LH、T、FPG、FINS及HOMA-IR水平升高(P<0.05);与PCOS组相比,二甲双胍组及桃核承气汤低、中、高剂量组大鼠卵泡体积减小、黄体数目增加、泡膜细胞层增生现象减轻,血清FSH、E_(2),卵巢组织中PI3K/AKT/mTOR通路mRNA和蛋白表达升高,LH、T、FPG、FINS及HOMA-IR水平降低(P<0.05);桃核承气汤低、中、高剂量组上述指标变化呈剂量依赖性,低、中剂量组与二甲双胍组差异有统计学意义,高剂量组与二甲双胍组差异无统计学意义。结论桃核承气汤可促进PCOS模型大鼠性激素恢复正常水平,抑制胰岛素抵抗,这可能与其促进PI3K/AKT/mTOR信号通路激活有关。

基于PI3K/AKT信号通路探讨益气养阴活血利水方抑制早期糖尿病视网膜病变大鼠微血管周细胞凋亡的作用机制

目的观察益气养阴活血利水方对早期糖尿病视网膜病变(diabetic retinopathy,DR)大鼠视网膜微血管周细胞中磷酸肌醇3-激酶蛋白(phosphatidylinositol 3-kiases,PI3K)/丝氨酸/苏氨酸蛋白激酶(phospho-alpha serine/threonine-protein kinase,AKT)信号通路相关因子表达的影响,探讨益气养阴活血利水方抑制早期DR视网膜微血管周细胞凋亡的作用机制。方法138只雄性SPF级SD大鼠随机分成空白组(等体积蒸馏水),模型组,羟苯磺酸钙组[150 mg/(kg·d)],益气养阴活血利水方低剂量组[6.2 g/(kg·d)]、中剂量组[12.4 g/(kg·d)]、高剂量组[24.8 g/(kg·d)],每组23只。除空白组外,各组均采用链佐脲菌素腹腔注射并维持10周高血糖状态,诱导早期DR大鼠模型。造模成功后给药4周,取大鼠眼球进行检测。采用HE染色观察视网膜病理变化,采用TUNEL染色检测大鼠视网膜微血管周细胞凋亡,采用免疫组织化学法及RT-PCR法检测PI3K、AKT、Bcl-2相关X蛋白(Bcl2-associated X protein,Bax)、胱天蛋白酶-8(cysteine aspartic acid specific protease-8,Caspase-8)、胱天蛋白酶-3(cysteine aspartic acid specific protease-3,Caspase-3)、细胞凋亡死亡激动剂BID蛋白(apoptotic death agonist BID protein,Bid)在视网膜微血管周细胞中的蛋白和mRNA表达水平。结果与空白组比较,模型组大鼠血糖显著升高(P<0.01),体质量显著降低(P<0.01),视网膜层次结构不清,各层细胞排列疏松,可见大量凋亡细胞,视网膜中PI3K、AKT mRNA表达水平下降(P<0.01),视网膜细胞凋亡数及Bax、Caspase-8、Caspase-3、Bid mRNA表达水平明显升高(P<0.05)。与模型组比较,益气养阴活血利水方中剂量、高剂量组大鼠血糖均降低(P<0.01);羟苯磺酸钙组、益气养阴活血利水方各剂量组大鼠体质量均升高(P<0.01);益气养阴活血利水方各剂量组大鼠视网膜组织层次结构较清楚,细胞排列相对紧密,视网膜微血管周细胞凋亡数下降(P<0.05);益气养阴活血利水方高剂量组大鼠视网膜微血管周细胞中PI3K、AKT蛋白和mRNA表达水平均明显升高(P<0.01),Bax、Caspase-8、Caspase-3、Bid蛋白和mRNA表达水平均明显下降(P<0.05)。结论益气养阴活血利水方可能通过调控PI3K/AKT信号通路,上调PI3K、AKT,下调Bax、Caspase-8、Caspase-3、Bid的表达,抑制早期DR大鼠视网膜微血管周细胞的凋亡,从而发挥其对早期DR视网膜微血管周细胞凋亡的保护作用。