Loss of monopolar spindle-binding protein 3B expression promotes colorectal cancer malignant behaviors by activation of target of rapamycin kinase/autophagy signaling

BACKGROUND Monopolar spindle-binding protein 3B(MOB3B)functions as a signal transducer and altered MOB3B expression is associated with the development of human cancers.AIM To investigate the role of MOB3B in colorectal cancer(CRC).METHODS This study collected 102 CRC tissue samples for immunohistochemical detection of MOB3B expression for association with CRC prognosis.After overexpression and knockdown of MOB3B expression were induced in CRC cell lines,changes in cell viability,migration,invasion,and gene expression were assayed.Tumor cell autophagy was detected using transmission electron microscopy,while nude mouse xenograft experiments were performed to confirm the in-vitro results.RESULTS MOB3B expression was reduced in CRC vs normal tissues and loss of MOB3B expression was associated with poor CRC prognosis.Overexpression of MOB3B protein in vitro attenuated the cell viability as well as the migration and invasion capacities of CRC cells,whereas knockdown of MOB3B expression had the opposite effects in CRC cells.At the molecular level,microtubule-associated protein light chain 3 II/I expression was elevated,whereas the expression of matrix metalloproteinase(MMP)2,MMP9,sequestosome 1,and phosphorylated mechanistic target of rapamycin kinase(mTOR)was downregulated in MOB3B-overexpressing RKO cells.In contrast,the opposite results were observed in tumor cells with MOB3B knockdown.The nude mouse data confirmed these in-vitro findings,i.e.,MOB3B expression suppressed CRC cell xenograft growth,whereas knockdown of MOB3B expression promoted the growth of CRC cell xenografts.CONCLUSION Loss of MOB3B expression promotes CRC development and malignant behaviors,suggesting a potential tumor suppressive role of MOB3B in CRC by inhibition of mTOR/autophagy signaling.

淫羊藿苷调控mTOR/Akt/CREB通路对高糖诱导的足细胞自噬及凋亡的影响

目的 探讨淫羊藿苷对高糖诱导的足细胞自噬、凋亡及哺乳动物雷帕霉素靶蛋白(mTOR)/丝氨酸苏氨酸蛋白激酶(Akt)/环磷酸腺苷反应元件结合蛋白(CREB)通路的影响。方法 将小鼠足细胞MPC5分为5组:正常对照组(5.5 mmol·L^(-1)葡萄糖)、高糖组(30 mmol·L^(-1)葡萄糖)、淫羊藿苷组(30 mmol·L^(-1)葡萄糖+5μmol·L^(-1)淫羊藿苷)、GDC-0349组(30 mmol·L^(-1)葡萄糖+50μmol·L^(-1)GDC-0349)、淫羊藿苷+GDC-0349组(30 mmol·L^(-1)葡萄糖+5μmol·L^(-1)淫羊藿苷+50μmol·L^(-1)GDC-0349)。培养48 h后,噻唑蓝法检测MPC5细胞活力;吖啶橙染色观察MPC5细胞自噬情况;流式细胞术检测MPC5细胞凋亡;蛋白印迹法检测MPC5细胞自噬[微管相关蛋白1轻链3(LC3)Ⅱ、LC3Ⅰ、自噬相关蛋白(Beclin-1)]、凋亡[Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)]和mTOR/Akt/CREB通路相关蛋白的表达。结果 与正常对照组比较,高糖组MPC5细胞活力、Bcl-2、磷酸化mTOR(p-mTOR)/mTOR、磷酸化Akt(p-Akt)/Akt、磷酸化CREB(p-CREB)/CREB蛋白表达水平显著降低(P<0.05),自噬能力增强,自噬体表现出橙色荧光,细胞凋亡率、LC3Ⅱ/LC3Ⅰ、Beclin-1、Bax蛋白表达水平显著升高(P<0.05)。与高糖组比较,淫羊藿苷组MPC5细胞活力、LC3Ⅱ/LC3Ⅰ、Beclin-1、Bcl-2、p-mTOR/mTOR、p-Akt/Akt、p-CREB/CREB蛋白表达水平显著升高,自噬能力进一步增强,自噬体数量增多,自噬体呈现出砖红色荧光(P<0.05),细胞凋亡率、Bax蛋白表达水平显著降低(P<0.05);GDC-0349组MPC5细胞活力、LC3Ⅱ/LC3Ⅰ、Beclin-1、Bcl-2、p-mTOR/mTOR、p-Akt/Akt、p-CREB/CREB蛋白表达水平显著降低,自噬能力减弱,自噬体数量减少,自噬体表现出橙色荧光(P<0.05),细胞凋亡率、Bax蛋白表达水平显著升高(P<0.05);淫羊藿苷+GDC-0349可逆转淫羊藿苷对高糖诱导MPC5细胞的作用效果(P<0.05)。结论 淫羊藿苷通过激活mTOR/Akt/CREB通路促进高糖诱导的足细胞自噬抑制细胞凋亡。

Suppressing high mobility group box-1 release alleviates morphine tolerance via the adenosine5'-monophosphate-activated protein kinase/heme oxygenase-1 pathway

Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory pain,but its role in morphine tolerance is unclear.In this study,we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days.We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1.HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4receptor expression in microglia,thereby inducing morphine tolerance.Glycyrrhizin,an HMGB1 inhibito r,markedly attenuated chronic morphine tole rance in the mouse model.Finally,compound C(adenosine 5’-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin(heme oxygenase-1 inhibitor)alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tole rance,and alleviated morphine tolerance in the mouse model.These findings suggest that morphine induces HMGB1 release via the adenosine 5’-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway,and that inhibiting this signaling pathway can effectively reduce morphine tole rance.

Osteopontin promotes gastric cancer progression via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effects of OPN on the proliferation,invasion and migration of GC cells and its possible mechanism.METHODS The mRNA and protein expression of OPN in the GC cells were analyzed by realtime quantitative-reverse transcription polymerase chain reaction and western blotting,and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC.Next,the effects of OPN knockdown on GC cells migration and invasion were examined.The short hairpin RNA(shRNA)and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer’s instructions.Non transfected cells were classified as control in the identical transfecting process.24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay,and cell invasiveness and migration were detected by Trans well assay.Meanwhile,the expression of protein kinase B(AKT),matrix metalloproteinase 2(MMP-2)and vascular endothelial growth factor(VEGF)in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting.RESULTS The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells.OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation,invasion and migration of SGC-7901 cells.Moreover,in the experiments of investigating the underlying mechanism,results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF,it also decreased the phosphorylation of AKT.Meanwhile,the protein expression levels of MMP-2,VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002).CONCLUSION These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to upregulate MMP-2 and VEGF expression,which contribute SGC-7901 cells to proliferation,invasion and migration.Thus,our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.

下调HMGB2表达对肝癌LM3细胞上皮-间质转化的抑制作用及其AKT/mTOR信号通路机制

目的:探讨下调肝癌细胞中高迁移率族框蛋白2 (HMGB2)表达对肝癌细胞生物学行为及上皮-间质转化(EMT)进程的影响,并阐明其作用机制。方法:对数生长期的人肝癌LM3细胞分为阴性对照组和HMGB2 RNA干扰组(HMGB2 siRNA组),分别以Lipofectamin 2000为载体转染无关序列的RNA寡核苷酸(RNA oligo)和敲除HMGB2序列的RNA oligo。采用实时荧光定量PCR(RT-qPCR)法和Western blotting法检测2组细胞中HMGB2 mRNA和蛋白表达水平,分别采用细胞划痕实验和Transwell小室实验检测2组细胞的迁移和侵袭能力,采用Western blotting法检测2组细胞中E-钙黏蛋白(E-cadherin)、 N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)和蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白表达水平。结果:与阴性对照组比较,HMGB2 siRNA组细胞中HMGB2 mRNA和蛋白表达水平均明显降低(P<0.05),HMGB2 siRNA组细胞划痕愈合率明显降低(P<0.01),侵袭细胞数明显减少(P<0.01),细胞中E-cadherin蛋白表达水平明显升高(P<0.01),N-cadherin、Vimentin、mTOR、AKT和磷酸化AKT (p-AKT)蛋白表达水平明显降低(P<0.05或P<0.01)。结论:下调HMGB2的表达可降低肝癌LM3细胞迁移和侵袭能力并抑制EMT,其作用机制可能与参与调节AKT/mTOR通路相关蛋白表达有关。

黄芪阳和汤调控PI3K/AKT/NF-κB信号通路促进糖尿病足溃疡大鼠创面愈合

目的基于磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/核因子-κB(NF-κB)信号通路探究黄芪阳和汤对糖尿病足溃疡(DFU)大鼠创面愈合的影响。方法构建DFU大鼠模型,将建模成功的48只大鼠随机分为模型组,黄芪阳和汤低(8.5 g/kg)、高(17 g/kg)剂量组,黄芪阳和汤高剂量(17 g/kg)+LY294002(PI3K/AKT通路抑制剂,0.3 mg/kg)组;每组12只;另取12只大鼠为对照组。各组大鼠给予对应药物干预,连续4周。第14、28天给药后,观察大鼠一般状态及创面变化,计算创面愈合率,检测大鼠空腹血糖(FBG)水平和大鼠创面周围组织经皮氧分压(TcpO2);酶联免疫吸附试验检测大鼠血清血管内皮生长因子(VEGF)、缺氧诱导因子-1α(HIF-1α)、C反应蛋白(CRP)、白细胞介素(IL)-6水平;苏木素-伊红染色观察大鼠创面组织病理学变化;免疫组织化学染色测定大鼠创面组织微血管密度;蛋白免疫印迹法检测大鼠创面组织中PI3K、磷酸化PI3K(p-PI3K)、AKT、磷酸化AKT(p-AKT)、NF-κB p65、磷酸化NF-κB p65(p-NF-κB p65)、NF-κB抑制蛋白α(IκB-α)蛋白表达。结果对照组大鼠毛色光滑,饮食、饮水、排泄均正常,较活跃,创面愈合快,创面组织炎症反应较轻,新生血管较多,肉芽组织中成纤维细胞及胶原基质丰富;模型组大鼠毛色暗淡无光泽,活动减少,且出现多饮、多食、多尿症状,创面颜色较深,且周围组织出现水肿、溃疡,创面组织可见大量炎性细胞浸润,伴组织坏死、渗出,新生血管及成纤维细胞较少,创面愈合率、创面周围组织TcpO2、血清VEGF、HIF-1α、创面组织微血管密度、p-PI3K、p-AKT、IκB-α蛋白表达水平降低,FBG、血清CRP、IL-6、创面组织p-NF-κB p65蛋白表达升高(P<0.05);与模型组相比,黄芪阳和汤低、高剂量组大鼠状态逐渐改善,创面组织病变程度依次减轻,创面愈合率、创面周围组织TcpO2、血清VEGF、HIF-1α、创面组织微血管密度、p-PI3K、p-AKT、IκB-α蛋白表达水平依次升高,FBG、血清CRP、IL-6、创面组织p-NF-κB p65蛋白表达依次降低(P<0.05);LY294002能部分逆转高剂量黄芪阳和汤对DFU大鼠的治疗作用(P<0.05)。结论黄芪阳和汤能调控PI3K/AKT/NF-κB信号通路,抑制DFU大鼠炎症反应,促进血管新生,从而促进创面愈合。

Fenofibrate Pre-treatment Suppressed Inflammation by Activating Phosphoinositide 3 Kinase/Protein Kinase B(PI3K/Akt) Signaling in Renal Ischemia-Reperfusion Injury

The aim of this study was to investigate the possible beneficial effects of Fenofibrate on renal ischemia-reperfusion injury（IRI） in mice and its potential mechanism. IRI was induced by bilateral renal ischemia for 60 min followed by reperfusion for 24 h. Eighteen male C57BL/6 mice were randomly divided into three groups： sham-operated group（sham）, IRI＋saline group（IRI group）, IRI＋Fenofibrate（FEN） group. Normal saline or Fenofibrate（3 mg/kg） was intravenously injected 60 min before renal ischemia in IRI group and FEN group, respectively. Blood samples and renal tissues were collected at the end of reperfusion. The renal function, histopathologic changes, and the expression levels of pro-inflammatory cytokines [interleukin-8（IL-8）, tumor necrosis factor alpha（TNF-α） and IL-6] in serum and renal tissue homogenate were assessed. Moreover, the effects of Fenofibrate on activating phosphoinositide 3 kinase/protein kinase B（PI3K/Akt） signaling and peroxisome proliferator-activated receptor-α（PPAR-α） were also measured in renal IRI. The results showed that plasma levels of blood urea nitrogen and creatinine, histopathologic scores and the expression levels of TNF-α, IL-8 and IL-6 were significantly lower in FEN group than in IRI group. Moreover, Fenofibrate pretreatment could further induce PI3K/Akt signal pathway and PPAR-α activation following renal IRI. These findings indicated PPAR-α activation by Fenofibrate exerts protective effects on renal IRI in mice by suppressing inflammation via PI3K/Akt activation. Thus, Fenofibrate could be a novel therapeutic alternative in renal IRI.

Response of Subcutaneous Xenografts of Endometrial Cancer in Nude Mice to Inhibitors of Phosphatidylinositol 3-Kinase/Akt and Mitogen-Activated Protein Kinase (MAPK) Pathways: An Effective Therapeutic Strategy for Endometrial Cancer

Objective: This study was designed to explore whether inhibition of the extracellular-regulated kinase (ERK) and phosphatidylinositol-3-kinase (PI3K) signaling pathways can inhibit the growth of xenografts of endometrial cancer cell lines with different estrogen receptors (ER) profiles in vivo and to provide preliminary laboratory basis for the probability of endometrial adenocarcinoma treatment with blockage of the two pathways, especially to endometrial cancer with low ER status. Methods: Human endometrial cancer Ishikawa bearing ER and HEC-1Awith low ER status cells were subcutaneously injected into BALB/c nude mice to establish endometrial cancer xenograft tumor models. The effects of PI3K/Akt inhibitor LY294002, MAPK/ERK1/2 inhibitor PD-98059 and their combinations on the growth of the xenograft tumors and apoptotic state of Ishikawa and HEC-1Acells were tested in vivo using the inhibitory rate, the terminal deoxynucleotidyl transferase-mediated nick-end labeling assay, H/E-stain. Western blot analysis was used to detect the alterations of activated ERK (P-ERK) and AKT (P-AKT) during this process. Results: LY294002, a PI3K/Akt pathway inhibitor, induced significant suppression in the growth of both Ishikawa and HEC-1Acell xenograft tumors, concomitant with increased apoptosis in xenografts as evidenced by TUNEL. A similar effect was also observed when the MAPK/ERK1/2 signaling pathway was inhibited by PD98059. Concurrent inhibition of the PI3K/Akt and MAPK/ERK1/2 pathways showed enhanced anti-tumor effects in vivo as indicated by increased apoptosis. At the same time, the levels of P-ERK and P-AKT in both xenograft tumors decreased, and their levels in combination group was the lowest. Conclusions: PD98059, LY294002 and their combinations showed remarkable inhibitory effects on xenograft tumors of endometrial carcinoma cell lines with different expression status of ER in vivo through blockage of PI3K/Akt and MAPK/ERK1/2 signaling pathways. This suggests that targeting these pathways may be an effective therapeutic strategy against endometrial carcinomas, especially for ER-negative cancers which show poor response to endocrinal therapy.

基于UBA2/PTEN/PI3K/Akt通路探讨蔓荆子黄素对结直肠癌细胞增殖、迁移和侵袭的影响

目的 基于泛素样修饰激活酶2(UBA2)/磷酸酶及张力蛋白同源物(PTEN)/磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)通路探究蔓荆子黄素对结直肠癌SW480细胞增殖、迁移和侵袭的影响。方法 取对数生长期的SW480细胞,对照组细胞常规培养,蔓荆子黄素组细胞加入10μmol/L蔓荆子黄素培养,UBA2抑制剂组细胞加入0.5μmol/L UBA2抑制剂培养,蔓荆子黄素+UBA2抑制剂组细胞加入10μmol/L蔓荆子黄素和0.5μmol/L UBA2抑制剂共培养。CCK-8实验检测细胞增殖情况,克隆形成实验观察细胞的单克隆形成能力,划痕实验观察细胞的迁移能力,Transwell实验观察细胞的侵袭能力,Western blot法检测细胞中UBA2/PTEN/PI3K/Akt通路相关蛋白表达情况。结果 CCK-8实验和克隆形成实验显示,UBA2抑制剂组和蔓荆子黄素+UBA2抑制剂组培养72 h后的细胞增殖吸光度OD值明显低于蔓荆子黄素组(P均<0.05),细胞克隆形成数量均明显少于蔓荆子黄素组(P均<0.05),UBA2抑制剂组和蔓荆子黄素+UBA2抑制剂组培养不同时间的细胞增殖吸光度OD值和细胞克隆形成数量比较差异均无统计学意义(P均>0.05)。划痕实验和Transwell实验显示,UBA2抑制剂组和蔓荆子黄素+UBA2抑制剂组划痕间距均明显宽于蔓荆子黄素组(P均<0.05),穿膜细胞数量均明显少于蔓荆子黄素组(P均<0.05),UBA2抑制剂组和蔓荆子黄素+UBA2抑制剂组比较差异均无统计学意义(P均>0.05)。蔓荆子黄素组、UBA2抑制剂组和蔓荆子黄素+UBA2抑制剂组细胞中PTEN蛋白相对表达量均明显高于对照组(P均<0.05),UBA2、p-PI3K、p-Akt蛋白相对表达量均明显低于对照组(P均<0.05);UBA2抑制剂组和蔓荆子黄素+UBA2抑制剂组细胞中PTEN蛋白相对表达量均明显高于蔓荆子黄素组(P均<0.05),UBA2、p-PI3K、p-Akt蛋白相对表达量均明显低于蔓荆子黄素组(P均<0.05),UBA2抑制剂组和蔓荆子黄素+UBA2抑制剂组UBA2、PTEN、p-PI3K、p-Akt蛋白相对表达量比较差异均无统计学意义(P均>0.05)。结论 蔓荆子黄素可能通过抑制UBA2/PTEN/PI3K/Akt信号通路发挥抗结直肠癌SW480细胞增殖、迁移和侵袭的能力。

薯蓣皂苷经PI3K/AKT通路对肝癌Bel-7402细胞增殖和凋亡的影响

目的分析薯蓣皂苷对肝癌Bel-7402细胞增殖和凋亡的影响并探讨其机制。方法肝癌Bel-7402细胞分为空白组和薯蓣皂苷低、中、高剂量组(给予1、2、8μmol/L的薯蓣皂苷)及薯蓣皂苷+抑制剂组(给予8μmol/L的薯蓣皂苷+10μmol/L的磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)信号通路抑制剂LY294002),于处理后12、24、36、48及72 h时采用四甲基偶氮唑盐(MTT)比色法测定细胞活力,于24 h时采用流式细胞术检测细胞凋亡情况、采用蛋白免疫印迹法(Western blot)检测p-PI3K和p-AKT的表达。结果与空白组相比较,薯蓣皂苷低、中、高剂量组细胞活力及p-PI3K、p-AKT表达均下降,凋亡率升高(P<0.05),且各剂量组间两两比较,上述指标水平差异均有统计学意义(P<0.05);与薯蓣皂苷高剂量组比较,薯蓣皂苷+抑制剂组细胞活力及p-PI3K、p-AKT表达下降,凋亡率升高(P<0.05)。结论薯蓣皂苷可能通过抑制PI3K/AKT通路抑制肝癌Bel-7402细胞增殖,诱导Bel-7402细胞凋亡。

茯苓酸调节PI3K/AKT/NF-κB信号通路对大鼠幽门螺旋杆菌相关性胃炎的治疗作用

目的 探讨茯苓酸(PA)对大鼠幽门螺旋杆菌(Hp)相关性胃炎的治疗效果及作用机制。方法 建立Hp相关性胃炎大鼠模型;所有大鼠分为对照组(CT组)、模型组(M组)、PA低剂量组(PA L组)和PA高剂量组(PA H组)、PA H+磷脂酰肌醇3-激酶(PI3K)激活剂(740 Y-P)组;评估各组大鼠胃黏膜损伤指数(UI),透射电子显微镜观察胃黏膜细胞形态学,HE染色评价胃黏膜病理学特征,ELISA检测胃组织白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、IL-10、诱导型一氧化氮合酶(iNOS)、超氧化物歧化酶(SOD)的水平,Western blot法检测PI3K、磷酸化-PI3K(p-PI3K)、蛋白激酶B(AKT)、p-AKT、核因子(NF)-κB p65、p-NF-κB p65蛋白表达。结果 与CT组比较,M组大鼠胃黏膜糜烂,上皮水肿、充血、溃疡严重,上皮细胞固缩,炎性细胞浸润,UI、IL-6、TNF-α、iNOS以及p-PI3K/PI3K、p-AKT/AKT、p-NF-κB p65/NF-κB p65蛋白表达水平升高,IL-10和SOD水平降低(P<0.05);与M组比较,PA L组、PA H组大鼠胃黏膜损伤改善,炎性细胞浸润减少,UI、IL-6、TNF-α、iNOS以及p-PI3K/PI3K、p-AKT/AKT、p-NF-κB p65/NF-κB p65蛋白表达水平降低,IL-10和SOD水平升高(P<0.05);与PA H组比较,PA H+740 Y-P组大鼠胃黏膜病理损伤加重,上皮细胞固缩,UI、IL-6、TNF-α、iNOS以及p-PI3K/PI3K、p-AKT/AKT、p-NF-κB p65/NF-κB p65蛋白表达水平升高,IL-10和SOD水平降低(P<0.05)。结论 PA可能通过抑制PI3K/AKT/NF-κB信号通路发挥对大鼠Hp相关性胃炎的治疗作用。

miR-19b对冠心病大鼠血管内皮细胞损伤的影响及PTEN/PI3K/AKT信号通路的作用

目的探讨微小RNA(miR)-19b对冠心病大鼠血管内皮细胞损伤的影响及磷酸酶及张力蛋白同源物/磷脂酰肌醇3-激酶/蛋白激酶B(PTEN/PI3K/AKT)信号通路的作用。方法选取72只大鼠随机分为正常组、模型组、模拟对照物(NC)组、miR-19b组、miR-19b+质粒互补DNA(pcDNA)3.1组、miR-19b+PTEN组。采用高脂饲料+垂体后叶素建立冠心病模型,尾静脉注射miR-19b mimic、pcDNA3.1-PTEN慢病毒液构建miR-19b、PTEN过表达大鼠。采用荧光定量聚合酶链反应检测冠状动脉组织miR-19b和PTEN信使RNA(mRNA)水平,测定大鼠心功能,观察大鼠冠状动脉病理变化,检测心肌组织细胞凋亡情况、血管内皮功能、冠状动脉中PTEN/PI3K/AKT信号通路、含半胱氨酸的天冬氨酸蛋白水解酶(cleaved Caspase)-3、cleaved Caspase-9水平。分离培养冠心病大鼠冠状动脉内皮细胞,根据转染方式分为野生型质粒(WT)+NC组、WT+miR-19b mimic组、突变型质粒(MUT)+NC组、MUT+miR-19b mimic组,采用双荧光素酶报告基因实验验证miR-19b与PTEN的靶向关系。将冠心病大鼠冠状动脉内皮细胞分为正常细胞组、NC细胞组和miR-19b细胞组,检测PTEN mRNA和PTEN蛋白水平。结果miR-19b组左心室射血分数(LVEF)、miR-19b、血清血管内皮生长因子(VEGF)、一氧化氮(NO)、磷酸化磷脂酰肌醇-3激酶(p-PI3K)/PI3K蛋白、磷酸化蛋白激酶B(p-AKT)/AKT蛋白水平高于模型组,左心室收缩末期容积(LVESV)、舒张末期容积(LVEDV)、细胞凋亡率、cleaved Caspase-3和cleaved Caspase-9、PTEN mRNA、内皮素-1(ET-1)、PTEN蛋白水平低于模型组,差异均有统计学意义(P<0.05)。miR-19b+PTEN组LVESV、LVEDV、细胞凋亡率、PTEN mRNA、cleaved Caspase-3、cleaved Caspase-9、ET-1、VEGF、PTEN蛋白水平高于miR-19b组,LVEF、NO、p-PI3K/PI3K蛋白和p-AKT/AKT蛋白水平低于miR-19b组,差异均有统计学意义(P<0.05)。模型组LVEF、miR-19b、VEGF、NO、p-PI3K/PI3K蛋白、p-AKT/AKT蛋白水平低于正常组,LVESV和LVEDV、细胞凋亡率、PTEN mRNA、cleaved Caspase-3、cleaved Caspase-9、ET-1、PTEN蛋白水平高于正常组,差异均有统计学意义(P<0.05)。与正常组比较,模型组大鼠冠状动脉内皮细胞排列紊乱,并伴有明显的水肿。与模型组比较,miR-19b组冠状动脉组织病理损伤明显减轻。WT+miR-19b mimic组细胞中荧光素酶相对活性低于WT+NC组、MUT+NC组、MUT+miR-19b mimic组,差异均有统计学意义(P<0.05)。miR-19b mimic细胞组PTEN mRNA和PTEN蛋白水平低于正常细胞组,差异均有统计学意义(P<0.05)。结论miR-19b靶向抑制PTEN,激活PI3K/AKT信号通路,保护冠心病大鼠心功能和血管内皮功能,减少心肌细胞凋亡。

肠病药方调控PI3K/AKT/NF-κB信号通路改善溃疡性结肠炎的研究

目的探讨肠病药方调控磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/核因子kappa B(NF-κB)信号通路对抗溃疡性结肠炎(UC)的作用机制。方法42只C57BL/6小鼠随机分为正常对照组(n=6,Control组,自由饮去离子水)、模型组(n=12,DSS组)、美沙拉嗪组(n=12,MES组)及肠病药方组(n=12,CBD组),后3组小鼠自由饮用3%葡聚糖硫酸钠(DSS)溶液7 d诱导UC模型,后两组同期灌胃美沙拉嗪或肠病药方,前两组灌胃等体积去离子水;观察实验期间小鼠体质量改变,评测小鼠疾病活动指数(DAI);造模结束后麻醉处死小鼠,测量结肠长度、观察结肠内容物及黏膜,采用苏木素-伊红(HE)染色观察各组小鼠结肠病理组织学变化,酶联免疫吸附试验(ELISA)检测各组小鼠结肠组织匀浆白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平,蛋白免疫印迹法(WB)检测各组小鼠结肠组织中磷酸化PI3K(p-PI3K)-p85、磷酸化AKT1(p-AKT1)、磷酸化NF-κB(p-NF-κB)-p65蛋白表达,实时荧光定量PCR技术(qRT-PCT)检测各组小鼠结肠组织PI3K、AKT、NF-κB信使RNA(mRNA)表达水平。结果与DSS组比较,CBD组治疗后,有效缓解UC小鼠症状,粪便性状及便血情况改善,结肠黏膜炎症浸润减少,体质量下降有所缓解,DAI评分降低及结肠长度接近正常,结肠组织匀浆中IL-6、TNF-α表达降低(P<0.05),p-PI3K-p85、p-AKT1、p-NF-κB-p65蛋白及mRNA表达明显减少(P<0.05)。结论肠病药方可缓解UC小鼠的症状、减轻结肠黏膜损伤,其机制可能与调解结肠组织中PI3K/AKT/NF-κB信号通路中相关蛋白质及mRNA表达、降低结肠组织炎症因子表达有关。

黄芩素调节PI3K/Akt/NF-κB信号通路及对细菌性脑膜炎大鼠血脑屏障的影响

目的:探讨黄芩素调控磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/核因子κB(NF-κB)信号通路对细菌性脑膜炎(BM)大鼠血脑屏障的影响。方法:制备大鼠BM模型,将大鼠分为对照组、模型组、黄芩素组(腹腔注射30 mg/kg黄芩素)、黄芩素+抑制剂组(腹腔注射30 mg/kg黄芩素+7.5 mg/kg LY294002),对大鼠进行Loeffler神经行为评分,H-E染色观察大鼠脑组织病理学变化,伊文思蓝染色检测血脑屏障通透性,分别检测脑脊液IL-1β、IL-6水平及白细胞计数(WBC)、脑组织含水量,水通道蛋白4(AQP4)、闭锁蛋白-5(claudin-5)、PI3K/Akt/NF-κB通路相关蛋白表达。结果:对照组大鼠脑组织结构正常,细胞排列整齐,模型组大鼠脑组织细胞排列紊乱,大量炎症细胞浸润。与对照组比较,模型组Loeffler评分及脑组织AQP4、claudin-5、p-PI3K、p-Akt表达水平显著降低,脑脊液IL-1β、IL-6水平及WBC、脑组织EB含量及含水量、p-NF-κB p65表达水平显著升高;黄芩素组较模型组病理损伤减轻,炎症细胞减少。与模型组比较,黄芩素组Loeffler评分及脑组织AQP4、claudin-5、p-PI3K、p-Akt表达水平显著升高,脑脊液IL-1β、IL-6水平及WBC、脑组织EB含量及含水量、p-NF-κB p65表达水平显著降低;LY294002可部分逆转黄芩素对BM大鼠血脑屏障通透性的改善作用。结论:黄芩素可改善BM大鼠脑水肿及血脑屏障通透性,可能与调控PI3K/Akt/NF-κB信号通路有关。

山姜素调控PI3K/Akt/NF-κB信号通路对冠心病大鼠心肌损伤的影响

目的:探讨山姜素调控磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/核转录因子-κB(NF-κB)信号通路对冠心病(CHD)大鼠心肌损伤的影响。方法:选取健康成年SPF级SD雄性大鼠60只,随机分为空白组、模型组、山姜素小剂量组、山姜素中剂量组、山姜素高剂量组各12只,采用Western blot法检测大鼠PI3K/Akt/NF-κB信号通路蛋白表达,采用ELISA法检测大鼠炎症因子指标[肿瘤坏死因子α(TNF-α)、白介素-1β(IL-1β)、白介素-18(IL-18)]和氧化应激指标[活性氧(ROS)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)],观察并记录大鼠心肌损伤情况。结果:与空白组比较,模型组的心肌缺血和心肌梗死面积更大(P<0.05),与模型组比较,山姜素小、中、高剂量组的心肌缺血和心肌梗死面积均缩小(P<0.05),且高剂量组的心肌缺血和心肌梗死面积小于山姜素中、小剂量组,中剂量组心肌缺血和心肌梗死面积小于山姜素小剂量组(P<0.05)。与空白组比较,模型组的PI3K、Akt、NF-κB更高(P<0.05),与模型组比较,山姜素小、中、高剂量组的PI3K、Akt、NF-κB均升高(P<0.05),且高剂量组的PI3K、Akt、NF-κB高于山姜素中、小剂量组,中剂量组PI3K、Akt、NF-κB高于山姜素小剂量组(P<0.05)。与空白组比较,模型组的TNF-α、IL-1β、IL-18更高(P<0.05),与模型组比较,山姜素小、中、高剂量组的TNF-α、IL-1β、IL-18均降低(P<0.05),且山姜素高剂量组的TNF-α、IL-1β、IL-18低于中、小剂量组,山姜素中剂量组的TNF-α、IL-1β、IL-18低于山姜素小剂量组(P<0.05)。与空白组比较,模型组的ROS、GSH-Px、MDA更高(P<0.05),与模型组比较,山姜素小、中、高剂量组的ROS、GSH-Px、MDA均降低(P<0.05),且山姜素高剂量组ROS、GSH-Px、MDA低于山姜素中、小剂量组,山姜素中剂量组的ROS、GSH-Px、MDA低于山姜素小剂量组(P<0.05)。结论:山姜素可通过调控PI3K/Akt/NF-κB信号通路改善CHD大鼠心肌炎症和氧化应激状况,进而发挥心肌保护作用。

Xuebijing improves intestinal microcirculation dysfunction in septic rats by regulating the VEGF-A/PI3K/Akt signaling pathway

BACKGROUND:This study aims to explore whether Xuebijing(XBJ) can improve intestinal microcirculation dysfunction in sepsis and its mechanism.METHODS:A rat model of sepsis was established by cecal ligation and puncture(CLP).A total of 30 male SD rats were divided into four groups:sham group,CLP group,XBJ + axitinib group,and XBJ group.XBJ was intraperitoneally injected 2 h before CLP.Hemodynamic data(blood pressure and heart rate) were recorded.The intestinal microcirculation data of the rats were analyzed via microcirculation imaging.Enzyme-linked immunosorbent assay(ELISA) kits were used to detect the serum levels of interleukin-6(IL-6),C-reactive protein(CRP),and tumor necrosis factor-α(TNF-α) in the rats.Histological analysis and transmission electron microscopy were used to analyze the injury of small intestinal microvascular endothelial cells and small intestinal mucosa in rats.The expression of vascular endothelial growth factor A(VEGF-A),phosphoinositide 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(Akt),and phosphorylated Akt(p-Akt) in the small intestine was analyzed via Western blotting.RESULTS:XBJ improved intestinal microcirculation dysfunction in septic rats,alleviated the injury of small intestinal microvascular endothelial cells and small intestinal mucosa,and reduced the systemic inflammatory response.Moreover,XBJ upregulated the expression of VEGF-A,p-PI3K/total PI3K,and p-Akt/total Akt in the rat small intestine.CONCLUSION:XBJ may improve intestinal microcirculation dysfunction in septic rats possibly through the VEGF-A/PI3K/Akt signaling pathway.

Inhibition of EGFR attenuates EGF-induced activation of retinal pigment epithelium cell via EGFR/AKT signaling pathway

AIM:To explore the effect of epidermal growth factor receptor(EGFR)inhibition by erlotinib and EGFR siRNA on epidermal growth factor(EGF)-induced activation of retinal pigment epithelium(RPE)cells.METHODS:Human RPE cell line(ARPE-19 cells)was activated by 100 ng/mL EGF.Erlotinib and EGFR siRNA were used to intervene EGF treatment.Cellular viability,proliferation,and migration were detected by methyl thiazolyl tetrazolium(MTT)assay,bromodeoxyuridine(BrdU)staining assay and wound healing assay,respectively.EGFR/protein kinase B(AKT)pathway proteins and N-cadherin,α-smooth muscle actin(α-SMA),and vimentin were tested by Western blot assay.EGFR was also determined by immunofluorescence staining.RESULTS:EGF treatment for 24h induced a significant increase of ARPE-19 cells’viability,proliferation and migration,phosphorylation of EGFR/AKT proteins,and decreased total EGFR expression.Erlotinib suppressed ARPE-19 cells’viability,proliferation and migration through down regulating total EGFR and AKT protein expressions.Erlotinib also inhibited EGF-induced an increase of proliferative and migrative ability in ARPE-19 cells and clearly suppressed EGF-induced EGFR/AKT proteins phosphorylation and decreased expression of N-cadherin,α-SMA,and vimentin proteins.Similarly,EGFR inhibition by EGFR siRNA significantly affected EGF-induced an increase of cell proliferation,viability,and migration,phosphorylation of EGFR/AKT proteins,and up-regulation of N-cadherin,α-SMA,and vimentin proteins.CONCLUSION:Erlotinib and EGFR-knockdown suppress EGF-induced cell viability,proliferation,and migration via EGFR/AKT pathway in RPE cells.EGFR inhibition may be a possible therapeutic approach for proliferative vitreoretinopathy(PVR).

许氏平鲉蛋白激酶B(SsAkt)基因的克隆及其mRNA在细菌胁迫后的表达规律

为研究蛋白激酶B(Akt)在许氏平鲉(Sebastes schlegelii)应答细菌胁迫过程中的作用,本研究应用PCR技术对许氏平鲉Akt基因(SsAkt)的编码区序列进行了克隆和特征分析,并应用实时荧光定量PCR技术检测了健康许氏平鲉各组织和细菌胁迫后肾脏、血液和肝脏中SsAktm RNA的表达规律。结果显示,SsAkt的开放阅读框(ORF)的长度为1440 bp,编码479个氨基酸,预测其蛋白的相对分子质量为55.80 k Da,理论等电点(p I)为5.64。SMART分析显示,Ss Akt蛋白含有丝氨酸/苏氨酸蛋白激酶家族的3个特征性保守结构域和2个磷酸化位点。同源比对发现,Ss Akt与鱼类的同源性较高,与尖嘴鲈(Lates calcarifer)的相似度最高,为99.37%。组织表达分析显示,SsAkt在许氏平鲉健康组织(血液、鳃、肝脏、肌肉、肾脏、脾脏、肠、脑和心脏)中均有表达,在肾脏、脑和血液中的相对表达量较高。许氏平鲉响应细菌胁迫的表达结果显示,藤黄微球菌(Micrococcus luteus)感染后,SsAkt在3种组织中的表达明显上调,呈先上升后下降的趋势;鳗弧菌(Vibrio anguillarum)感染后,SsAkt在3种组织中呈现不同的表达趋势:在肾脏中,SsAkt的表达趋势为先上升后下降又上升再下降,而在血液和肝脏中的表达趋势为先上升后下降。以上研究表明,SsAkt响应了外源微生物对许氏平鲉的胁迫,在抵御外源微生物免疫应答过程中发挥重要作用。本研究结果为许氏平鲉的免疫机理研究奠定了基础。

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.

Yinchenhao decoction attenuates obstructive jaundice-induced liver injury and hepatocyte apoptosis by suppressing protein kinase RNA-like endoplasmic reticulum kinase-induced pathway

BACKGROUND Chronic biliary obstruction results in ischemia and hypoxia of hepatocytes,and leads to apoptosis.Apoptosis is very important in regulating the homeostasis of the hepatobiliary system.Endoplasmic reticulum(ER)stress is one of the signaling pathways that induce apoptosis.Moreover,the protein kinase RNA-like endoplasmic reticulum kinase(PERK)-induced apoptotic pathway is the main way;but its role in liver injury remains unclear.Yinchenhao decoction(YCHD)is a traditional Chinese medicine formula that alleviates liver injury and apoptosis,yet its mechanism is unknown.We undertook this study to investigate the effects of YCHD on the expression of ER stress proteins and hepatocyte apoptosis in rats with obstructive jaundice(OJ).AIM To investigate whether YCHD can attenuate OJ-induced liver injury and hepatocyte apoptosis by inhibiting the PERK-CCAAT/enhancer-binding protein homologous protein(CHOP)-growth arrest and DNA damage-inducible protein 34(GADD34)pathway and B cell lymphoma/leukemia-2 related X protein(Bax)/B cell lymphoma/leukemia-2(Bcl-2)ratio.METHODS For in vivo experiments,30 rats were divided into three groups:control group,OJ model group,and YCHD-treated group.Blood was collected to detect the indicators of liver function,and liver tissues were used for histological analysis.For in vitro experiments,30 rats were divided into three groups:G1,G2,and G3.The rats in group G1 had their bile duct exposed without ligation,the rats in group G2 underwent total bile duct ligation,and the rats in group G3 were given a gavage of YCHD.According to the serum pharmacology,serum was extracted and centrifuged from the rat blood to cultivate the BRL-3A cells.Terminal deoxynucleotidyl transferase mediated dUTP nick end-labelling(TUNEL)assay was used to detect BRL-3A hepatocyte apoptosis.Alanine aminotransferase(ALT)and aspartate transaminase(AST)levels in the medium were detected.Western blot and quantitative real-time polymerase chain reaction(qRT-PCR)analyses were used to detect protein and gene expression levels of PERK,CHOP,GADD34,Bax,and Bcl-2 in the liver tissues and BRL-3A cells.RESULTS Biochemical assays and haematoxylin and eosin staining suggested severe liver function injury and liver tissue structure damage in the OJ model group.The TUNEL assay showed that massive BRL-3A rat hepatocyte apoptosis was induced by OJ.Elevated ALT and AST levels in the medium also demonstrated that hepatocytes could be destroyed by OJ.Western blot or qRT-PCR analyses showed that the protein and mRNA expression levels of PERK,CHOP,and GADD34 were significantly increased both in the rat liver tissue and BRL-3A rat hepatocytes by OJ.The Bax and Bcl-2 levels were increased,and the Bax/Bcl-2 ratio was also increased.When YCHD was used,the PERK,CHOP,GADD34,and Bax levels quickly decreased,while the Bcl-2 levels increased,and the Bax/Bcl-2 ratio decreased.CONCLUSION OJ-induced liver injury and hepatocyte apoptosis are associated with the activation of the PERK-CHOP-GADD34 pathway and increased Bax/Bcl-2 ratio.YCHD can attenuate these changes.