杏仁核注射红藻氨酸诱导的边缘叶发作癫痫大鼠海马 Bad去磷酸化和Bcl-2表达上调

应用红藻氨酸(kainic acid,KA)诱导的人鼠边缘叶发作癫痫模型,检测Bad(Bcl-2-associated death protein)、14-3-3、磷酸化Bad、Bcl-XL和Bcl-2在癫痫人鼠海码神经元的表达。单侧杏仁核内注射KA诱导癫痫发作,持续记录脑电和局部脑血流(regional cerebral blood flow,r-CBF),发作1h后静脉注射30mg／kg安定终止发作,然后分别用cresyl violet染色和TUNEL染色观察海马神经元存活和凋亡的变化;用免疫荧光、Western blot和免疫沉淀俭测海马Bad、14-3-3、磷酸化Bad、Bcl-XL和Bcl-2的表达。结果表明,发作终止8h时出现TUNEL阳忡细胞,24h时达高峰;发作诱导Bad去磷酸化,去磷酸化的Bad与分了伴侣蛋F114-3-3解离,然后Bad与Bcl-XL结合：磷酸化Bad表达减少而Bcl-2表达增加;发作前后r-CBF无明显变化。以上结果提示,癫痫发作诱导Bad的去磷酸化和Bcl-2表达上调,Bad的上磷酸化可能具有损伤作用,而Bcl-2的表达上调则对癫痫神经元损伤具有保护作用,但与脑缺血无关。     

Odorant Stimulation Promotes Survival of Rodent Olfactory Receptor Neurons via PI3K/Akt Activation and Bcl-2 Expression

Olfactory stimulation activates multiple signaling cascades in order to mediate activity-driven changes in gene expression that promote neuronal survival. To date, the mechanisms involved in activity-dependent olfactory neuronal survival have yet to be fully elucidated. In the current study, we observed that olfactory sensory stimulation, which caused neuronal activation, promoted activation of the phosphatidylinositol 3′-kinase (PI3K)/Akt pathway and the expression of Bcl-2, which were responsible for olfactory receptor neuron (ORN) survival. We demonstrated that Bcl-2 expression increased after odorant stimulation both in vivo and in vitro. We also showed that odorant stimulation activated Akt, and that Akt activation was completely blocked by incubation with both a PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) and Akt1 small interfering RNA. Moreover, blocking the PI3K/Akt pathway diminished the odorant-induced Bcl-2 expression, as well as the effects on odorant-induced ORN survival. A temporal difference was noted between the activation of Akt1 and the expression of Bcl-2 following odorant stimulation. Blocking the PI3K/Akt pathway did not affect ORN survival in the time range prior to the increase in Bcl-2 expression, implying that these two events, activation of the PI3K pathway and Bcl-2 induction, were tightly connected to promote post-translational ORN survival. Collectively, our results indicated that olfactory activity activated PI3K/Akt, induced Bcl-2, and promoted long term ORN survival as a result.     

Mechanism of salidroside relieving the acute hypoxia-induced myocardial injury through the PI3K/Akt pathway

ObjectiveThe objective was to investigate the anti-inflammatory effects of salidroside through the PI3K/Akt signaling pathway and its protective effects on acute hypoxia-induced myocardial injury in rats.MethodsA total of 24 healthy Sprague-Dawley male rats were selected as the experimental subjects. All rats were divided into 4 groups by using the random number table method, with 6 rats in each group. The groups included the normal control group, the salidroside group, the hypobaric hypoxia group, and the hypobaric hypoxia + salidroside group. Rats in the salidroside group were fed in the original animal laboratory and were intragastrically administered with salidroside every morning at a dosage of 35 mg/kg. Rats in the normal control group were intragastrically administered with an equal dosage of saline. Rats in the hypobaric hypoxia + salidroside group were intragastrically administered with salidroside every morning at a dosage of 35 mg/kg, who were fed in the hypoxic experiment module for animals. The altitude was increased to 4000 m, and the rats were kept in the module for 24 h. Rats in the hypobaric hypoxia group were intragastrically administered with an equal dosage of saline in the same environment, and the altitude was increased to 4000 m after administration. Parameters of blood gas analysis, histopathological changes in cardiac tissues, cardiac indexes, and inflammatory factors IL-6 and TNF-α levels of rats in groups were compared.Results1. The cardiac indexes of rats in groups were compared. The differences between the hypobaric hypoxia group and the hypobaric hypoxia + salidroside group were statistically significant (P < 0.05). 2. The results of blood gas analysis of rats in groups were compared. The differences between the hypobaric hypoxia group and the hypobaric hypoxia + salidroside group were significantly different (P < 0.05). 3. In the hypobaric hypoxia group, the myocardial cells of rats were arranged disorderly and shaped differently, with cases such as edema, degeneration, necrosis, nucleus pyknosis, and massive infiltration of inflammatory cells. In the hypobaric hypoxia + salidroside group, the above-mentioned pathological changes in myocardial cells were relieved. 4. Compared with the hypobaric hypoxia group, in the hypobaric hypoxia + salidroside group, the concentrations of IL-6 and TNF-α in rats decreased apparently, and the differences were statistically significant (P < 0.05).ConclusionSalidroside had the repairing and protective effects on the hypobaric hypoxia-induced myocardial injuries in rats. The application of salidroside could reduce the inflammatory responses of rats with hypobaric hypoxia-induced myocardial injuries through PI3K/Akt signaling pathway, thereby protecting the myocardial cells.     

PESV 对K562 细胞BCR/ABL 融合基因及凋亡调控因子Bcl-2、Bad 表达的影响

目的:探讨PESV对K562细胞BCR/ABL融合基因及凋亡调控因子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,荧光定量RT-PCR检测BCR/ABL、Bcl-2、Bad mRNA水平变化.结果:与对照组相比,PESV处理后K562细胞,凋亡率增加,BCR/ABL融合基因表达降低,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能降低降低K562细胞BCR/ABL融合基因的表达,可能通过调节Bcl-2和Bad表达,抑制K562细胞增殖,促进其凋亡.     

NEAT1通过PI3K/AKT/Bcl-2信号通路抑制骨质疏松

为探讨NEAT1在骨质疏松症中的作用以及可能的病理机制,本研究通过建立卵巢去势和鼠尾悬挂2种骨质疏松的小鼠模型,将C57BL/6分为假手术组(Sham组)、OVX组和TS组;经过PCR测定小鼠NEAT1的表达;Elisa法检测小鼠E2、ALP和TRACP水平;Western blotting检测细胞凋亡因子PI3K/AKT/Bcl-2的蛋白水平。结果显示,建模4周后,3组小鼠体重没有显著变化;与Sham组相比,OVX组和TS组小鼠的骨密度值显著降低,骨生化指标ALP和TRACR水平明显升高;OVX组小鼠的E2水平与Sham组相比明显降低;与Sham组相比,OVX组和TS组小鼠的NEAT1表达显著下调;与Sham组相比,OVX组和TS组小鼠p-AKT和Bcl-2蛋白水平明显降低。本研究结果表明,NEAT1可能通过抑制PI3K/AKT/Bcl-2细胞凋亡途径诱导骨质疏松。     

人参皂苷Rb1对H9N2流感病毒诱导急性肺损伤小鼠抗氧化酶的影响

目的探讨人参皂苷Rb1(G-Rb1)对肺损伤小鼠抗氧化酶活力的影响。方法将195只6~8周龄BALB/c小鼠随机分为对照组、肺损伤模型组(ALI组)、人参皂苷Rb1组(G-Rb1组),每组65只。ALI与G-Rb1组采用100μL SI A/swine/HeBei/012/2008/猪流感病毒(H9N2 SIV)经鼻腔接种建立急性肺损伤模型,同时G-Rb1组腹腔注射人参皂苷Rb1液0.1 mL,剂量为10 mg/(kg·bw),连续7d;对照组鼻腔接种相同剂量生理盐水稀释的正常鸡胚尿囊液。观察临床症状、肺病理组织学变化,计算小鼠肺湿干重比、肺系数,检测小鼠肺组织T-SOD、MPO、CAT、GSH-PX活力。结果从第2天末开始ALI组大部分小鼠出现高度的精神沉郁,呼吸极度困难,采食量明显减少,体重下降。肺部明显水肿、淤血和出血,炎性细胞渗出,对照组小鼠各器官未见异常。肺系数及肺干湿重比逐渐升高,第8天开始下降,第14天趋于正常。G-Rb1组症状明显轻于攻毒组,症状出现较缓,症状较轻,死亡时间延迟,死亡率降低。在第4、6、8天,与对照组比G-Rb1组和ALI组T-SOD及CAT活力显著降低(P0.01),组间比,G-Rb1组明显高于ALI组(P0.05);在各时间点,与对照组比,ALI组GSH-PX活力显著降低(P0.01),而GRb1组则显著升高(P0.01),实验组组间差异显著(P0.01)。结论 G-Rb1在一定浓度范围内,具有提高小鼠抗氧化酶活力作用,一定程度上改善H9N2猪流感病毒对肺组织的氧化损伤。     

Integrin beta1 mediates the effect of telocytes on mesenchymal stem cell proliferation and migration in the treatment of acute lung injury

Co-transplantation of mesenchymal stem cells (MSCs) with telocytes (TCs) was found to have therapeutic effects, although the mechanism of intercellular communication is still unknown. Our current studies aim at exploring the potential molecular mechanisms of TCs interaction and communication with MSCs with a focus on integrin beta1 (ITGB1) in TCs. We found that the co-culture of MSCs with ITGB1-deleted TCs (TC^ITGB1-ko) changed the proliferation, differentiation and growth dynamics ability of MSC in responses to LPS or PI3K inhibitor. Changes of MSC proliferation and apoptosis were accompanied with the dysregulation of cytokine mRNA expression in MSCs co-cultured with TC^ITGB1-ko during the exposure of PI3Kα/δ/β inhibitor, of which IL-1β, IL-6 and TNF-α increased, while IFN-γ, IL-4 and IL-10 decreased. The responses of PI3K p85, PI3K p110 and pAKT of MSCs co-cultured with TC^ITGB1-ko to LPS or PI3K inhibitor were opposite to those with ITGB1-presented TCs. The intraperitoneal injection of TC^ITGB1-ko, TC^vector or MSCs alone, as well as the combination of MSCs with TC^ITGB1-ko or TC^vector exhibited therapeutic effects on LPS-induced acute lung injury. Thus, our data indicate that telocyte ITGB1 contributes to the interaction and intercellular communication between MSCs and TCs, responsible for influencing other cell phenomes and functions.     

RhoGDI2与PI3K/Akt/mTOR信号通路在肺癌细胞中的相关性研究

目的探讨肿瘤转移相关因子RhoGDI2与PI3K/Akt/mTOR信号通路在肺癌侵袭转移过程中的作用及相关机制。方法利用PI3K/Akt/mTOR信号通路上特异性的抑制剂,采用MTT法,伤口愈合实验及侵袭实验观察不同浓度药物对肺癌95D细胞生长侵袭转移能力的影响,通过Western Blot方法观察RhoGDI2蛋白水平的变化。结果PI3K抑制剂LY294002及mTOR抑制剂Rapamycin都能抑制肺癌细胞95D的侵袭转移能力,联合应用抑制作用更强。PI3K抑制剂LY294002处理组RhoGDI2蛋白的表达量增加,且随浓度增加RhoGDI2蛋白表达也增加。mTOR抑制剂Rapamycin组,在低浓度时增加RhoGDI2蛋白的表达,但增大Rapamycin的浓度,RhoGDI2蛋白的表达反而降低。低浓度LY294002组和Rapa-mycin组联合应用可以明显增加RhoGDI2蛋白的表达。结论PI3K/Akt/mTOR信号通路中Akt的活化与RhoGDI2密切相关,RhoGDI2可能直接或间接通过与Akt的相互作用参与调节肺癌的侵袭转移的过程。     

1,7-Bis(4-hydroxyphenyl)-1,4-heptadien-3-one induces lung cancer cell apoptosis via the PI3K/Akt and ERK1/2 pathways

1,7-Bis(4-hydroxyphenyl)-1,4-heptadien-3-one (EB30) is a diarylheptanoid-like compound isolated from Viscum coloratum. This curcumin analog exhibits significant cytotoxic activity against HeLa, SGC-7901, and MCF-7 cells. However, little is known about the anticancer effects and mechanisms of EB30 in human lung cancer. The current study reports that EB30 significantly reduced the cell viability of A549 and NCI-H292 human lung cancer cells. Further examination revealed that EB30 not only induced cell cycle arrest and promoted the generation of reactive oxygen species (ROS) but also induced cell apoptosis through the intrinsic and extrinsic signaling pathways. Furthermore, EB30 upregulated the expression levels of p-ERK1/2 and p-P90RSK, whereas downregulating the phosphorylation of Akt and P70RSK. Cell viability was further inhibited by the combination of EB30 with LY294002 (a specific PI3K inhibitor) or U0126 (a MEK inhibitor). The current study indicates that EB30 is a potential anticancer agent that induces cell apoptosis via suppression of the PI3K/Akt pathway and activation of the ERK1/2 pathway.     

Endogenously produced ganglioside GM3 endows etoposide and doxorubicin resistance by up-regulating Bcl-2 expression in 3LL Lewis lung carcinoma cells

The ganglioside patterns have been shown to dramatically change during cell proliferation and differentiation and in certain cell-cycle phases, brain development, and cancer malignancy. To investigate the significance of the ganglioside GM3 in cancer malignancy, we established GM3-reconstituted cells by transfecting the cDNA of GM3 synthase into a GM3-deficient subclone of the 3LL Lewis lung carcinoma cell line (Uemura, S. (2003) Glycobiology, 13, 207-216). The GM3-reconstituted cells were resistant to apoptosis induced by etoposide and doxorubicin. There were no changes in the expression levels of topoisomerase IIalpha or P-glycoprotein, or in the uptake of doxorubicin between the GM3-reconstituted cells and the mock-transfected cells. To understand the mechanism of the etoposide-resistant phenotype acquired in the GM3-reconstituted cells, we investigated their apoptotic signaling. Although no difference was observed in the phosphorylation of p53 at serine-15-residue site by etoposide between the GM3-reconstituted cells and mock-transfected cells, the activation of both caspase-3 and caspase-9 was specifically inhibited in the former. We found that the anti-apoptotic protein B-cell leukemia/lymphoma 2 (Bcl-2) was increased in the GM3-reconstituted cells. Moreover, wild-type 3LL Lewis lung carcinoma cells, which have an abundance of GM3, exhibited no DNA fragmentation following etoposide treatment and expressed higher levels of the Bcl-2 protein compared with the J5 subclone. Thus, these results support the conclusion that endogenously produced GM3 is involved in malignant phenotypes, including anticancer drug resistance through up-regulating the Bcl-2 protein in this lung cancer cell line.     

Toll-like receptor 4 mediates lipopolysaccharide-induced collagen secretion by phosphoinositide3-kinase-akt pathway in fibroblasts during acute lung injury

Gram-negative bacillus infection is an important risk factor of acute lung injury (ALI). Previous experiments have revealed that lipopolysaccharide (LPS), a primary component of endotoxin of gram-negative bacilli, stimulated the inflammatory reactions that contribute to ALI and pulmonary interstitial fibrosis, but the mechanisms were not well understood. We reported that LPS was able to directly induce secretion of collagen in mouse lung fibroblasts via activation of phosphoinositide3-kinase-Akt (PI3K-Akt) pathway through toll-like receptor 4 (TLR4) in vitro. We found that overexpression of TLR4, type I procollagen, alpha smooth muscle actin (alpha-SMA), and p-AKT in primary cultured mouse lung fibroblast stimulated by LPS were detected by real-time PCR or Western blots, and the contents of C-terminal propeptide of type I procollagen (PICP) in cell culture supernatants were increased simultaneously. The activation of TLR4 stimulated by LPS could also up-regulate the expression of integrin beta1 and TLR4 in mouse lung fibroblast, which could accelerate ALI and pulmonary interstitial fibrosis processes. All these changes could be inhabited by transfection of Lentivirus-TLR4-siRNA or application of PI3K inhibitor LY294002. Therefore, we infer that besides pulmonary macrophage, lung fibroblasts are also important target cells directly influenced by LPS, which may play an important role in ALI and pulmonary interstitial fibrosis.     

复方清下汤对脓毒症大鼠肺组织细胞因子白介素-1及白介素-6基因表达的影响

目的 探讨复方清下汤对脓毒症大鼠肺组织白介素-1(IL-1)及白介素-6(IL-6)基因表达的影响,进一步探讨其减轻肺损伤机制.方法 将健康SD大鼠随机分为4组,每组10只:假手术组(SHAM组),脓毒症肺损伤组(模型组),盲肠结扎穿孔+复方清下汤组,以及盲肠结扎穿孔+头孢哌酮舒巴坦(舒普深)组,造模24 h后收集标本.应用免疫组织化学和Westernblotting法检测肺组织中IL-1、IL-6的表达,RT-PCR检测肺组织上述蛋白mRNA表达.结果 与SHAM组比较,模型组IL-1、IL-6的mRNA转录水平和蛋白水平表达均显著升高(P＜0.01);抗生素及中药处理组与模型组比较,IL-1、IL-6的表达明显降低(P＜0.01),抗生素及中药处理组两组检测数据相近.结论 脓毒症大鼠肺损伤时细胞因子IL-1、IL-6过度表达可能是造成脓毒症肺损伤的重要原因;复方清下汤处理的动物模型肺损伤减轻的同时IL-1、IL-6表达变化,提示它可能通过调控IL-1、IL-6表达起作用.     

BMP9对人肺腺癌AS49细胞侵袭、迁移的影响及机制的研究

BMP9属于TGF-β超家族的成员,参与多种细胞的增殖、分化、凋亡、侵袭、转移过程。以人肺腺癌细胞A549作为目的细胞,采用腺病毒体外感染方式,外源性高表达BMP9。RT-PCR及Westernblot检测重组细胞中BMP9的表达,通过细胞划痕实验、Transwell侵袭实验检测AdBMP9细胞侵袭及迁移改变,RT-PCR及Westernblot检测感染BMP9腺病毒后IL-6的mRNA和蛋白表达：Westemblotg检测NPI3K／Akt信号通路中总Akt和磷酸化Akt蛋白的表达。结果显示：与对照组细胞相比,感染BMP9腺病毒后,A549中BMP9mRNA和蛋白表达明显升高;实验组划痕愈合率由对照的（85．4±2．11％与（86．5±3．4）％上升至（97．4±2．6）％（P〈0．05）;实验组穿膜细胞数由对照的（115．5±13．1）个与（123．3±14．9）个上升至（224．3±24．6）个（P〈0．05）;与对照组细胞相比,AdBMP9组IL-6的表达上调,磷酸化Akt蛋白表达上调。该研究表明,BMP9可能通过上调IL-6的表达,激活P13K／Akt信号通路,促进人肺腺癌A549的侵袭、迁移。     

Matrine attenuates high‐fat diet‐induced in vivo and ox‐LDL‐induced in vitro vascular injury by regulating the PKCα/eNOS and PI3K/Akt/eNOS pathways

Lipid metabolism disorders lead to vascular endothelial injury. Matrine is an alkaloid that has been used to improve obesity and diabetes and for the treatment of hepatitis B. However, its effect on lipid metabolism disorders and vascular injury is unclear. Here, we investigated the effect of matrine on high‐fat diet fed mice and oxidized low‐density lipoprotein (ox‐LDL)‐induced human umbilical vein endothelial cells (HUVECs). Computational virtual docking analyses, phosphoinositide 3‐kinase (PI3K) and protein kinase C‐α (PKCα) inhibitors were used to localize matrine in vascular injuries. The results showed that matrine‐treated mice were more resistant to abnormal lipid metabolism and inflammation than vehicle‐treated mice and exhibited significantly alleviated ox‐LDL‐stimulated dysfunction of HUVECs, restored diminished nitric oxide release, decreased reactive oxygen species generation and increased expression phosphorylation of AKT‐Ser473 and endothelial nitric oxide synthase (eNOS)‐Ser1177. Matrine not only up‐regulates eNOS‐Ser1177 but also down‐regulates eNOS‐Thr495, a PKCα‐controlled negative regulator of eNOS. Using computational virtual docking analyses and biochemical assays, matrine was also shown to influence eNOS/NO via PKCα inhibition. Moreover, the protective effects of matrine were significantly abolished by the simultaneous application of PKCα and the PI3K inhibitor. Matrine may thus be potentially employed as a novel therapeutic strategy against high‐fat diet‐induced vascular injury.     

人参皂甙 Rb1对大鼠缺血再灌注心肌细胞Bcl-2、Bax、Bad、Fas基因表达的影响

目的观察人参皂甙Rb1对缺血再灌注心肌细胞Bcl-2、Bax、Bad、Fas基因表达的影响.方法结扎/松解Wistar大鼠左冠状动脉前降支,建立大鼠缺血再灌注动物模型,免疫组化法检测Bcl-2、Bax、Bad、Fas基因的蛋白表达,并利用图象分析系统测量蛋白阳性表达区域平均光密度值,进行定量分析.结果缺血再灌注组及Rb1治疗组Bcl-2、Bax、Bad、Fas基因的表达较假手术组明显增加(P<0.05), Rb1治疗组Bcl-2的表达与缺血再灌注组比较无明显差异(P>0.05),而Bax、Bad、Fas的表达明显下降(P<0.05),人参皂甙Rb1治疗组Bcl-2/Bax、Bcl-2/Bad以及Bcl-2/Fas比值均较假手术组与缺血再灌注组明显增加.结论人参皂甙Rb1治疗可以抑制缺血再灌注心肌细胞中促凋亡基因Bax、Bad、Fas的表达,并使Bcl-2/Bax、Bcl-2/Bad以及Bcl-2/Fas比值增加.     

褪黑素对油酸致大鼠急性肺损伤的防护作用及其机制研究

目的：研究油酸（OA）致大鼠急性肺损伤（ALI）时,P-选择素（Ps）、细胞间粘附分子-1（ICAM-1）和核因子-κB（NF-KB）在肺组织中的表达及褪黑素（MT）对肺组织的保护作用及其机制。方法：将48只SD大鼠随机分为4组（n=12）,对照组（Control）、油酸组（OA）、MT＋OA组,SB203580＋OA组。采用尾静脉注射油酸的方法建立大鼠Au的模型,测定肺系数,光镜下观察大鼠肺组织形态学改变,并通过免疫组织化学染色技术观察肺组织中Ps、ICAM-1和NF-κB的表达变化。结果：与control组相比,OA组大鼠肺系数明显升高（P〈0．05）;肺组织损伤严重,肺泡间隔明显增宽,肺泡腔及肺间质弥漫性炎细胞浸润;Ps、ICAM-1和NF-κB的阳性表达信号明显增强（P〈0．05）;应用MT和SB203580均显著缓解上述变化（P〈0．05）。结论：MT对ALI时的肺组织起明显的保护作用,其保护机制可能与抑制Ps、ICAM-1和NF-κB的表达有关。     

ST2 protein induced by inflammatory stimuli can modulate acute lung inflammation

We have investigated gene and protein expression of ST2/ST2L in a murine alveolar macrophage (AM) cell line, MH-S, reacting to inflammatory stimuli in vitro and in the lung tissue of an acute lung injury model in vivo. We have also analyzed the effect of soluble ST2 protein on inflammatory response of MH-S cells. Lipopolysaccharide (LPS) and proinflammatory cytokines such as IL-1beta, IL-6, and TNF-alpha induced ST2 mRNA expression in MH-S cells. In an acute lung injury model, protein and mRNA expression levels of ST2 increased to the maximal level at 24-72h after the LPS challenge. Furthermore, pretreatment with ST2 protein significantly reduced the protein production and gene expression of IL-1alpha, IL-6, and TNF-alpha in LPS-stimulated MH-S cells in vitro. These results suggest that increases in endogenous ST2 protein in AM, which is induced by inflammatory stimuli, such as LPS and proinflammatory cytokines, may modulate acute lung inflammation.