Spi1 regulates the microglial/macrophage inflammatory response via the PI3K/AKT/mTOR signaling pathway after intracerebral hemorrhage

Preclinical and clinical studies have shown that microglia and macrophages participate in a multiphasic brain damage repair process following intracerebral hemorrhage.The E26 transformation-specific sequence-related transcription factor Spi1 regulates microglial/macrophage commitment and maturation.However,the effect of Spi1 on intracerebral hemorrhage remains unclear.In this study,we found that Spi1 may regulate recovery from the neuroinflammation and neurofunctional damage caused by intracerebral hemorrhage by modulating the microglial/macrophage transcriptome.We showed that high Spi1expression in microglia/macrophages after intracerebral hemorrhage is associated with the activation of many pathways that promote phagocytosis,glycolysis,and autophagy,as well as debris clearance and sustained remyelination.Notably,microglia with higher levels of Soil expression were chara cterized by activation of pathways associated with a variety of hemorrhage-related cellular processes,such as complement activation,angiogenesis,and coagulation.In conclusion,our results suggest that Spi1 plays a vital role in the microglial/macrophage inflammatory response following intracerebral hemorrhage.This new insight into the regulation of Spi1 and its target genes may advance our understanding of neuroinflammation in intracerebral hemorrhage and provide therapeutic targets for patients with intracerebral hemorrhage.

MiR-146a-5p targeting SMAD4 and TRAF6 inhibits adipogenensis through TGF-β and AKT/mTORC1 signal pathways in porcine intramuscular preadipocytes

Background: Intramuscular fat(IMF) content is a vital parameter for assessing pork quality. Increasing evidence has shown that microRNAs(miRNAs) play an important role in regulating porcine IMF deposition. Here, a novel miRNA implicated in porcine IMF adipogenesis was found, and its effect and regulatory mechanism were further explored with respect to intramuscular preadipocyte proliferation and differentiation.Results: By porcine adipose tissue miRNA sequencing analysis, we found that miR-146a-5p is a potential regulator of porcine IMF adipogenesis. Further studies showed that miR-146a-5p mimics inhibited porcine intramuscular preadipocyte proliferation and differentiation, while the miR-146a-5p inhibitor promoted cell proliferation and adipogenic differentiation. Mechanistically, miR-146a-5p suppressed cell proliferation by directly targeting SMAD family member 4(SMAD4) to attenuate TGF-β signaling. Moreover, miR-146a-5p inhibited the differentiation of intramuscular preadipocytes by targeting TNF receptor-associated factor 6(TRAF6) to weaken the AKT/mTORC1 signaling downstream of the TRAF6 pathway.Conclusions: MiR-146a-5p targets SMAD4 and TRAF6 to inhibit porcine intramuscular adipogenesis by attenuating TGF-β and AKT/mTORC1 signaling, respectively. These findings provide a novel miRNA biomarker for regulating intramuscular adipogenesis to promote pork quality.

shRNA-interfering LSD1 inhibits proliferation and invasion of gastric cancer cells via VEGF-C/PI3K/AKT signaling pathway

BACKGROUND Histone Lysine Specific Demethylase 1(LSD1)is the first histone demethylase to be discovered,which regulates various biological functions by making lysine of histone H3K4,H3K9 and non-histone substrates demethylated.Abnormal regulation of LSD1 is closely related to the occurrence and development of gastric cancer.The change of LSD1 expression level plays an important role in the proliferation and metastasis of gastric cancer cells.The study of its function and mechanism may provide a theoretical basis for early diagnosis and targeted therapy of gastric cancer.AIM To investigate the effect of downregulation of lysine-specific demethylase 1(LSD1)expression on proliferation and invasion of gastric cancer cells and the possible regulatory mechanisms of the VEGF-C/PI3K/AKT signaling pathway.METHODS The LSD1-specific short hairpin RNA(shRNA)interference plasmid was transiently transfected,and expression of LSD1 was downregulated.The cell proliferation ability of LSD1 was observed by CCK-8 assay after downregulating expression of LSD1.Transwell invasion assay was used to observe the change of cell invasion ability after downregulating expression of LSD1.Expression of phosphorylated phosphoinositide 3-kinase(p-PI3K),PI3K,p-AKT,AKT,vascular endothelial growth factor receptor(VEGFR)-3,matrix metalloproteinase(MMP)-2 and MMP-9 in each group was detected by Western blotting.RESULTS The cell proliferation ability of transiently transfected LSD1-shRNA interference plasmid group was significantly lower than that of the control group(P<0.05).Transwell invasion assay showed that the number of cells across the membrane of the LSD1-shRNA transfection group(238.451±5.216)was significantly lower than that of the control group(49.268±6.984)(P<0.01).Western blotting showed that expression level of VEGF-C,p-PI3K,PI3K,p-AKT,AKT,VEGFR-3,MMP-2 and MMP-9 in the LSD1-shRNA group was significantly lower than that in the control group(P<0.05).CONCLUSION Downregulation of LSD1 expression inhibits metastatic potential of gastric cancer cells,and VEGF-C-mediated activation of PI3K/AKT signaling pathway,which may be an important mechanism for inhibiting lymph node metastasis in gastric cancer cells.

Simiao Wan alleviates obesity-associated insulin resistance via PKCε/IRS-1/PI3K/Akt signaling pathway based on network pharmacology analysis and experimental validation

Background:The purpose of the study was to investigatethe active ingredients and potential biochemicalmechanisms of Simiao Wan(SMW)in obesity-associated insulin resistance.Methods:An integrated network pharmacology method to screen the active compoundsand candidate targets,construct the protein-protein-interaction network,and ingredients-targets-pathways network was constructed for topological analysis to identify core targets and main ingredients.To find the possible signaling pathways,enrichment analysis was performed.Further,a model of insulin resistance in HL-7702 cells was established to verify the impact of SMW and the regulatory processes.Results:An overall of 63 active components and 151 candidate targets were obtained,in which flavonoids were the main ingredients.Enrichment analysis indicated that the PI3K-Akt signaling pathway was the potential pathway regulated by SMW in obesity-associated insulin resistance treatment.The result showed that SMW could significantly ameliorate insulin sensitivity,increase glucose synthesis and glucose utilization and reduce intracellular lipids accumulation in hepatocytes.Also,SMW inhibited diacylglycerols accumulation-induced PKCεactivity and decreased its translocation to the membrane.Conclusion:SMW ameliorated obesity-associated insulin resistance through PKCε/IRS-1/PI3K/Akt signaling axis in hepatocytes,providing a new strategy for metabolic disease treatment.

Fibroblast-derived CXCL12/SDF-1α promotes CXCL6 secretion and co-operatively enhances metastatic potential through the PI3K/Akt/m TOR pathway in colon cancer

AIM To investigate the underlying mechanism by which CXCL12 and CXCL6 influences the metastatic potential of colon cancer and internal relation of colon cancer and stromal cells. METHODS Western blotting was used to detect the expression of CXCL12 and CXCL6 in colon cancer cells and stromal cells. The co-operative effects of CXCL12 and CXCL6 on proliferation and invasion of colon cancer cells and human umbilical vein endothelial cells(HUVECs) were determined by enzyme-linked immunosorbent assay,and proliferation and invasion assays. The angiogenesis of HUVECs through interaction with cancer cells and stromal cells was examined by angiogenesis assay. We eventually investigated activation of PI3K/Akt/m TOR signaling by CXCL12 involved in the metastatic process of colon cancer.RESULTS CXCL12 was expressed in DLD-1 cancer cells and fibroblasts. The secretion level of CXCL6 by colon cancer cells and HUVECs were significantly promoted by fibroblasts derived from CXCL12. CXCL6 and CXCL2 could significantly enhance HUVEC proliferation and migration(P < 0.01). CXCL6 and CXCL2 enhanced angiogenesis by HUVECs when cultured with fibroblast cells and colon cancer cells(P < 0.01). CXCL12 also enhanced the invasion of colon cancer cells. Stromal cell-derived CXCL12 promoted the secretion level of CXCL6 and co-operatively promoted metastasis of colon carcinoma through activation of the PI3K/Akt/m TOR pathway.CONCLUSION Fibroblast-derived CXCL12 enhanced the CXCL6 secretion of colon cancer cells,and both CXCL12 and CXCL6 co-operatively regulated the metastasis via the PI3K/Akt/m TOR signaling pathway. Blocking this pathway may be a potential anti-metastatic therapeutic target for patients with colon cancer.

Eukaryotic elongation factor-1α 2 knockdown inhibits hepatocarcinogenesis by suppressing PI3K/Akt/NF-κB signaling

AIM: To assess the impact of eukaryotic elongation factor 1 alpha 2 (eEF1A2) on hepatocellular carcinoma (HCC) cell proliferation, apoptosis, migration and invasion, and determine the underlying mechanisms.METHODS: eEF1A2 levels were detected in 62 HCC tissue samples and paired pericarcinomatous specimens, and the human HCC cell lines SK-HEP-1, HepG2 and BEF-7402, by real-time PCR and immunohistochemistry. Experimental groups included eEF1A2 silencing in BEL-7402 cells with lentivirus eEF1A2-shRNA (KD group) and eEF1A2 overexpression in SK-HEP-1 cells with eEF1A2 plasmid (OE group). Non-transfected cells (control group) and lentivirus-based empty vector transfected cells (NC group) were considered control groups. Cell proliferation (MTT and colony formation assays), apoptosis (Annexin V-APC assay), cell cycle (DNA ploidy assay), and migration and invasion (Transwell assays) were assessed. Protein levels of PI3K/Akt/NF-&#x003ba;B signaling effectors were evaluated by Western blot.RESULTS: eEF1A2 mRNA and protein levels were significantly higher in HCC cancer tissue samples than in paired pericarcinomatous and normal specimens. SK-HEP-1 cells showed lower eEF1A2 mRNA levels; HepG2 and BEL-7402 cells showed higher eEF1A2 mRNA levels, with BEL-7402 cells displaying the highest amount. Efficient eEF1A2 silencing resulted in reduced cell proliferation, migration and invasion, increased apoptosis, and induced cell cycle arrest. The PI3K/Akt/NF-&#x003ba;B signaling pathway was notably inhibited. Inversely, eEF1A2 overexpression resulted in promoted cell proliferation, migration and invasion.CONCLUSION: eEF1A2, highly expressed in HCC, is a potential oncogene. Its silencing significantly decreases HCC tumorigenesis, likely by inhibiting PI3K/Akt/NF-&#x003ba;B signaling.

Scoparone inhibits pancreatic cancer through PI3K/Akt signaling pathway

BACKGROUND Pancreatic cancer is a highly malignant tumor of the gastrointestinal system whose emerging resistance to chemotherapy has necessitated the development of novel antitumor treatments.Scoparone,a traditional Chinese medicine monomer with a wide range of pharmacological properties,has attracted considerable attention for its antitumor activity.AIM To explore the potential antitumor effect of scoparone on pancreatic cancer and the possible molecular mechanism of action.METHODS The target genes of scoparone were determined using both the bioinformatics and multiplatform analyses.The effect of scoparone on pancreatic cancer cell proliferation,migration,invasion,cell cycle,and apoptosis was detected in vitro.The expression of hub genes was tested using quantitative reverse transcription polymerase chain reaction(qRT-PCR),and the molecular mechanism was analyzed using Western blot.The in vivo effect of scoparone on pancreatic cancer cell proliferation was detected using a xenograft tumor model in nude mice as well as immunohistochemistry.RESULTS The hub genes involved in the suppression of pancreatic cancer by scoparone were obtained by network bioinformatics analyses using publicly available databases and platforms,including SwissTargetPrediction,STITCH,GeneCards,CTD,STRING,WebGestalt,Cytoscape,and Gepia;AKT1 was confirmed using qRT-PCR to be the hub gene.Cell Counting Kit-8 assay revealed that the viability of Capan-2 and SW1990 cells was significantly reduced by scoparone treatment exhibiting IC50 values of 225.2μmol/L and 209.1μmol/L,respectively.Wound healing and transwell assays showed that scoparone inhibited the migration and invasion of pancreatic cancer cells.Additionally,flow cytometry confirmed that scoparone caused cell cycle arrest and induced apoptosis.Scoparone also increased the expression levels of Bax and cleaved caspase-3,decreased the levels of MMP9 and Bcl-2,and suppressed the phosphorylation of Akt without affecting total PI3K and Akt.Moreover,compared with the control group,xenograft tumors,in the 200μmol/L scoparone treatment group,were smaller in volume and lighter in weight,and the percentages of Ki65-and PCNA-positive cells were decreased.CONCLUSION Our findings indicate that scoparone inhibits pancreatic cancer cell proliferation in vitro and in vivo,inhibits migration and invasion,and induces cycle arrest and apoptosis in vitro through the PI3K/Akt signaling pathway.

Baicalin protects neonatal rat brains against hypoxicischemic injury by upregulating glutamate transporter 1 via the phosphoinositide 3-kinase/protein kinase B signaling pathway

Baicalin is a flavonoid compound extracted from Scutellaria baicalensis root.Recent evidence indicates that baicalin is neuroprotective in models of ischemic stroke.Here,we investigate the neuroprotective effect of baicalin in a neonatal rat model of hypoxic-ischemic encephalopathy.Seven-day-old pups underwent left common carotid artery ligation followed by hypoxia（8% oxygen at 37°C） for 2 hours,before being injected with baicalin（120 mg/kg intraperitoneally） and examined 24 hours later.Baicalin effectively reduced cerebral infarct volume and neuronal loss,inhibited apoptosis,and upregulated the expression of p-Akt and glutamate transporter 1.Intracerebroventricular injection of the phosphoinositide 3-kinase/protein kinase B（PI3 K/Akt） inhibitor LY294002 30 minutes before injury blocked the effect of baicalin on p-Akt and glutamate transporter 1,and weakened the associated neuroprotective effect.Our findings provide the first evidence,to our knowledge that baicalin can protect neonatal rat brains against hypoxic-ischemic injury by upregulating glutamate transporter 1 via the PI3 K/Akt signaling pathway.

AKt/mTOR信号通路中p70S6K1、4E-BPs在促进肿瘤发生作用中的研究进展

当受细胞外生长因子等刺激作用后,可激活PI3K/AKt/m TOR信号通路,参与控制细胞的生长、增殖、生存、凋亡。AKt/m TOR信号通路在肿瘤的发生发展过程中起着很重要的促进作用。AKt/m TOR信号通路在肿瘤细胞中能够通过p70S6K1和4E-BPs的作用抑制细胞的凋亡,促进核糖体以及蛋白质的合成、促进肿瘤细胞的侵袭和转移、促进细胞周期进展、促进肿瘤血管的生成,进而促进肿瘤的发生发展。本文就AKt/m TOR信号通路通过p70S6K1和4E-BPs促进肿瘤发生的机制进行综述。

S6K1 C端自抑制假底物结构域磷酸化与Akt去磷酸化协同调节SP600125诱导巨核细胞系多倍体化

目的:探讨JNK抑制剂SP600125诱导巨核细胞白血病系多倍体化的调控机制。方法:用SP600125和3种抑制剂(PD184352、U0126和LY294002)处理Dami和CMK细胞,用点突变技术对核糖体蛋白S6激酶1(S6K1)C端自抑制假底物结构域和疏水基序突变构建的S6K1质粒转染细胞,流式细胞术分析细胞的DNA倍性,Western印迹检测S6K1、MAPK和Akt蛋白的表达及磷酸化修饰位点的变化。结果:当单独使用3种抑制剂时,对Dami和CMK细胞的倍性无影响。当SP600125与3种抑制剂联合时,尽管PD184352下调了p44/42 MAPK在Thr202/Tyr204位点的磷酸化,但其并不抑制SP600125诱导的Dami和CMK细胞的多倍体化;相反,U0126抑制SP600125诱导的Dami和CMK细胞的多倍体化,但并不下调p44/42 MAPK的磷酸化;而LY294002增加Akt的磷酸化,并阻断SP600125诱导的Dami和CMK细胞的多倍体化。这3种抑制剂均在SP600125诱导的多倍体Dami和CMK细胞中部分抑制S6K1的Thr421/Ser424磷酸化,但并不增加S6K1的Thr389磷酸化。转染S6K1突变质粒的Dami细胞,并没有对SP600125诱导的多倍体化产生影响,同时,无论是模拟磷酸化或去磷酸化的Thr389突变均对SP600125诱导的多倍体化无影响,且未显现出与LY294002的协同作用。C端自抑制假底物结构域突变质粒(S6K1-D3E)可以进一步阻断SP600125诱导的已经被LY294002部分阻断的Dami细胞多倍体化。结论:S6K1的C端自抑制假底物结构域磷酸化与Akt去磷酸化协同调节SP600125诱导巨核细胞白血病系的多倍体化。

Rosmarinic acid elicits neuroprotection in ischemic stroke via Nrf2 and heme oxygenase 1 signaling

Rosmarinic acid（RA） can elicit a neuroprotective effect against ischemic stroke, but the precise molecular mechanism remains poorly understood. In this study, an experimental ischemic stroke model was established in CD-1 mice（Beijing Vital River Laboratory Animal Technology, Beijing, China） by occluding the right middle cerebral artery for 1 hour and allowing reperfusion for 24 hours. After intraperitoneally injecting model mice with 10, 20, or 40 mg/kg RA, functional neurological deficits were evaluated using modified Longa scores. Subsequently, cerebral infarct volume was measured using TTC staining and ischemic brain tissue was examined for cell apoptosis with TUNEL staining. Superoxide dismutase activity and malondialdehyde levels were measured by spectrophometry. Expression of heme oxygenase-1（HO-1）, nuclear factor erythroid 2-related factor 2（Nrf2）, Bcl-2, Bax, Akt, and phospho-Ser473 Akt proteins in ischemic brain tissue was detected by western blot, while mRNA levels of Nrf2, HO-1, Bcl-2, and Bax were analyzed using real time quantitative PCR. In addition, HO-1 enzyme activity was measured spectrophotometrically. RA（20 and 40 mg/kg） greatly improved neurological function, reduced infarct volume, decreased cell apoptosis, upregulated Bcl-2 protein and mRNA expression, downregulated Bax protein and mRNA expression, increased HO-1 and Nrf2 protein and mRNA expression, increased superoxide dismutase activity, and decreased malondialdehyde levels in ischemic brain tissue of model mice. However, intraperitoneal injection of a HO-1 inhibitor（10 mg/kg zinc protoporphyrin IX） reversed the neuroprotective effects of RA on HO-1 enzyme activity and Bcl-2 and Bax protein expression. The PI3 K/Akt signaling pathway inhibitor LY294002（10 mM） inhibited Akt phosphorylation, as well as Nrf2 and HO-1 expression. Our findings suggest that RA has anti-oxidative and anti-apoptotic properties that protect against ischemic stroke by a mechanism involving upregulation of Nrf2 and HO-1 expression via the PI3 K/Akt signaling pathway.

PC-1解除S6K对AKT信号通路的负反馈抑制

目的:通过建立过表达PC-1的前列腺癌LNCaP细胞系及敲低PC-1表达的C4-2细胞系,探究PC-1激活AKT信号通路的分子机制。方法:将PC-1基因及针对PC-1的siRNA序列,分别克隆至慢病毒表达载体pCDH-EF1-Myc-MCS-T2A-Puro及干扰载体pSIH1-H1-Puro,包装成慢病毒后分别感染前列腺癌LNCaP及C4-2细胞,通过Western印迹鉴定PC-1过表达及敲低效果,并检测PI3K/AKT/mTOR信号通路相关蛋白S6K、AKT的磷酸化水平。结果:PC-1过表达时,S6K磷酸化水平下降,而AKT的磷酸化水平上升。结论:PC-1可以通过抑制S6K激酶活性,解除其对AKT的负反馈抑制作用,从而激活AKT激酶的活性。

长链非编码RNA CBR3-AS1通过PI3K/AKT/mTOR/S6K通路介导白血病细胞对阿糖胞苷的耐药机制

目的探讨长链非编码RNA(lncRNA)CBR3-AS1通过PI3K/AKT/mTOR/S6K通路介导急性髓细胞白血病(AML)细胞对阿糖胞苷耐药的可能机制。方法在2个AML细胞株K562和HL-60中建立耐药模型,分别为K562-R和HL-60-R。逆转录-实时定量聚合酶链反应(RT-qPCR)法检测lncRNA CBR3-AS1在K562-R和HL-60-R中的表达情况。在K562和HL-60细胞株中,利用质粒过表达lncRNA CBR3-AS1;在K562-R和HL-60-R细胞株中,利用siRNA敲低lncRNA CBR3-AS1表达,并计算阿糖胞苷的半数抑制浓度(IC_(50))。蛋白印迹法检测PI3K/AKT/mTOR/S6K通路激活情况。结果相较于K562和HL-60细胞株,K562-R和HL-60-R细胞株中lncRNA CBR3-AS1表达升高,差异有统计学意义(P<0.05)。过表达lncRNA CBR3-AS1可提高阿糖胞苷IC_(50)超过1000μmol/L(P<0.05)。敲低lncRNA CBR3-AS1后,阿糖胞苷在耐药细胞株K562-R和HL-60-R中的IC_(50)分别降低到21.27μmol/L和12.10μmol/L,差异有统计学意义(P<0.05)。过表达lncRNA CBR3-AS1显著提高磷酸肌醇3激酶(PI3K)、AKT、哺乳动物雷帕霉素靶蛋白(mTOR)、S6K蛋白的磷酸化水平(P<0.05);敲低lncRNA CBR3-AS1显著降低PI3K、AKT、mTOR、S6K蛋白的磷酸化水平(P<0.05)。结论lncRNA CBR3-AS1可能通过激活PI3K/AKT/mTOR/S6K通路介导AML细胞对阿糖胞苷的耐药。

Effect of acupotomy on chondrocyte proliferation and expression of CyclinD1,CDK4 and CDK6 in rabbits with knee osteoarthritis

Objective:The aim of this study was to investigate the mechanism of acupotomology(Apo)in the prevention of articular cartilage destruction via the promotion of chondrocyte proliferation and chondrocyte expression of cell cycle regulators,CyclinD1,CDK4 and CDK6 in a rabbit knee osteoarthritis(KOA)model.Methods:Twenty-eight rabbits were randomly divided into a control group,an OA(osteoarthritis)model group,an Apo(acupotomology)group and EA(electro-acupuncture)group(n Z 7).Improved Videman’s method was used to induce a rabbit model of KOA over 6 weeks.One week later,acupotomy and electro-acupuncture therapy was applied to animals in the respective groups and treatment lasted 4 weeks.Following these treatments,quantitative real-time PCR,immunohistochemical staining and western blotting were performed to assess the mRNA and protein levels of cell cycle regulators CyclinD1(Cell cycle protein D1),CDK4(Cyclin-dependent kinase 4)and CDK6(Cyclin-dependent kinase 6).Ethology measures and knee morphology were also compared among groups.Results:The Lequesne MG index score of morphology was increased(P<.01),and the passive range of motion(PROM)and the mRNA and protein levels of CyclinD1,CDK4,and CDK6 were significantly decreased(P<.01)in the OA model compared with the control group.The Lequesne MG index score and the morphology score were decreased in the Apo and EA group compared with the OA model group(P<.05 or P<.01),while the mRNA levels of CyclinD1,CDK4,and CDK6,and the protein levels of CDK4 were increased in the Apo and EA groups compared with the OA model group(P<.05 or P<.01).The PROM,and the protein levels of CyclinD1 and CDK6 were increased(P<.05)in the Apo group compared with the OA model group,while the PROM and the protein levels of CyclinD1 and CDK6 in the EA group were not significantly different(P>.05).Compared with the EA group,the morphology score was decreased in Apo group(P<.05).Conclusions:The mRNA levels of CyclinD1 and CDK4,and the protein level of CDK4 in chondrocytes are regulate by both Apo and EA.Apo is more effective than EA in regulating the protein levels of CyclinD1 and CDK6.According to the observed changes in morphology and cytokine levels,acupotomy can promote chondrocyte proliferation and can alleviate the destruction of articular cartilage in a model of KOA.

Solanine Interferes with AKT/p-AKT and PI3K/p-PI3K Pathway to Inhibit HIF and Destroy Cell Energy Metabolism

The purpose of this study was to explore the mechanism of Solanine disrupting energy metabolism in human renal cancer ACHN cells and to clarify its target. The specific method was to culture human renal cancer ACHN cell lines, and to intervene with Solanine of high, medium and low concentrations. The content of ATP in cells was measured by ELISA method. The expression of HIF-1α protein and the expression of PI3K, AKT, p-PI3K, p-AKT in PI3K/AKT pathway were detected by Western blotting. The results showed that compared with the control group, the relative expression of p-PI3K and p-AKT showed a downward trend with the increase of Solanine concentration (P < 0.05), while the relative expression of PI3K and AKT showed no significant change (P > 0.05). In addition, the relative expression of HIF-1α also showed a downward trend (P < 0.05). According to the above results, it is suggested that Solanine can significantly inhibit the energy metabolism of renal cancer cells, the main mechanism of which is the down-regulation of HI-1αf downstream of the PI3K/Akt pathway by inhibiting the phosphorylation process of PI3K/p-PI3K and Akt/p-Akt.

抑制mTOR信号通路对幼鼠肺损伤时p-AKT1分子的影响及意义

目的:探讨抑制哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路在高体积分数氧(高氧)致SD幼鼠肺损伤时对磷酸化AKT1(p-AKT1)分子的影响和意义。方法:72只SD幼鼠(3周龄)随机分为空气+生理盐水组、高氧+生理盐水组、高氧+OSI-027组及高氧+雷帕霉素组(n=18),分别构建动物模型。高氧选择90%氧气持续干预,生理盐水、OSI-027和雷帕霉素干预分别在观察期第1、3、6、8、10和13天时经腹腔注射给药。在造模第3、7和14天时取各组幼鼠进行体重测量、肺湿干重比(wet/drg weight ratio,W/D)计算、肺组织病理学检查、肺泡间隔宽度测定和肺损伤评分,肺组织免疫组化和Western blot检测磷酸化S6K1(p-S6K1)和p-AKT1的分布与水平。结果:与空气组比较,高氧组幼鼠体重明显下降(P<0.05),肺损伤急性期肺W/D增高(P<0.05),肺泡间隔宽度及肺损伤评分明显增加(P<0.05),肺组织p-S6K1阳性细胞增多(P<0.05),肺组织p-AKT1阳性细胞减少(P<0.05),p-S6K1蛋白显著升高(P<0.01),p-AKT1蛋白明显减低(P<0.01);与高氧组比较,高氧+OSI-027组的肺组织损伤减轻,肺组织p-S6K1阳性细胞减少(P<0.05),p-AKT1阳性细胞增多(P<0.05),p-S6K1蛋白水平显著降低(P<0.05),p-AKT1蛋白水平增加(P<0.05);高氧+雷帕霉素组的肺损伤进一步加重(P<0.05),p-S6K1阳性细胞减少(P<0.05),p-AKT1阳性细胞增加(P<0.05),p-S6K1蛋白水平显著降低(P<0.05),p-AKT1蛋白水平显著增加(P<0.05)。与高氧+雷帕霉素组比较,高氧+OSI-027组的肺组织损伤减轻(P<0.05),肺组织p-AKT1阳性细胞减少(P<0.05),p-AKT1蛋白水平降低(P<0.05)。结论:p-AKT1参与了高氧肺损伤的发生发展,其调控机制可能与抑制mTOR信号通路的活化有关。高氧肺损伤时,p-AKT1蛋白水平下降,mTOR抑制剂能增加p-AKT1蛋白水平,但只有mTORC1/2双重抑制剂OSI-027能减轻高氧所致SD幼鼠的肺损伤及纤维化。

Expanding mTOR signaling

The mammalian target of rapamycin （mTOR） has drawn growth control and its involvement in human tumorigenesis much attention recently because of its essential role in cell Great endeavors have been made to elucidate the functions and regulation of mTOR in the past decade. The current prevailing view is that mTOR regulates many fundamental biological processes, such as cell growth and survival, by integrating both intracellular and extracellular signals, including growth factors, nutrients, energy levels, and cellular stress. The significance of roTOR has been highlighted most recently by the identification of mTOR-associated proteins. Amazingly, when bound to different proteins, mTOR forms distinctive complexes with very different physiological functions. These findings not only expand the roles that mTOR plays in cells but also further complicate the regulation network. Thus, it is now even more critical that we precisely understand the underlying molecular mechanisms in order to directly guide the development and usage of anti-cancer drugs targeting the mTOR signaling pathway. In this review, we will discuss different mTOR-associated proteins, the regulation of mTOR complexes, and the consequences of mTOR dysregulation under pathophysiological conditions.

Phosphorylation of Protein Kinase Akt by Mtorc2 in Peripheral Blood Mononuclear Cells of Patients with Cancer and Diabetes

Akt/mTOR/p70S6K1 signaling pathway plays an important role in the pathogenesis of cancer and diabetes.Macrophages and lymphocytes are involved in the pathogenesis of diabetes,diabetic atherosclerosis,formation of insulin resistance as well as immune response to cancer and tumor maintenance.The aim of the study was to determine the Akt activation by mTORC2 in peripheral blood mononuclear cell(PBMC)of patients with type 2 diabetes and cancer.The following groups were studied:control group,patients with type 2 diabetes,cancer patients and patients with both cancer and diabetes.The amounts of phospho-Akt(р-S473)and phospho-p70S6K1(p-T389)were determined using ELISA kits.The amount of phosphorylated Akt significantly increases in PBMC of patients with cancer.There was no effect in PBMC from patients with type 2 diabetes and significant decrease in the amount of phospho-Akt in PBMC of the patients group both with cancer and diabetes.p70S6K1 activation was observed in PBMC of the groups 2 and 3 patients.Thus,chronic diseases such as type 2 diabetes and cancer can affect the signaling mechanisms in blood cells.The state of Akt phosphorylation in leukocytes can indicate the activity of mTORC1 and its substrates,which may be important for the evaluation of the pathological process and the efficacy of the drugs.

强精煎介导PI3K/Akt/mTOR通路调控少精子症大鼠精子发生的实验研究

目的:探索强精煎通过介导PI3K/Akt/mTOR通路及其下游靶因子4EBP1、p70S6K,调控环磷酰胺(CTX)诱导的少精子症模型大鼠精子发生的作用机制。方法:将75只SD雄性大鼠随机分为空白对照组、模型组、mTOR通路抑制剂组(雷帕霉素组)、强精煎+雷帕霉素组和强精煎组,每组15只。采用腹腔注射CTX法诱导少精子症大鼠模型。连续药物干预38 d后,剪开各组大鼠腹腔摘取睾丸,采用免疫组化法检测PI3K/Akt/mTOR通路蛋白及其下游通路蛋白(4EBP1、p70S6 K)、细胞增殖标记蛋白Ki-67表达;采用RT-PCT法检测睾丸组织PI3K/Akt/mTOR基因及其下游靶基因(4EBP1、p70S6K)表达。结果:与空白对照组比较,模型组、雷帕霉素组睾丸组织PI3K、Akt、mTOR、4EBP1、p70S6 K、Ki-67蛋白表达水平均显著降低(P<0.05),雷帕霉素组降低尤为明显。与模型组、雷帕霉素组相比,强精煎组睾丸组织PI3K、Akt、mTOR、4EBP1、p70S6K、Ki-67蛋白表达水平明显上调(P<0.05)。与空白对照组比较,模型组、雷帕霉素组睾丸组织PI3K、Akt、mTOR、4EBP1、p70S6K基因表达水平均显著降低(P<0.05)。强精煎组睾丸组织PI3K、Akt、mTOR、4EBP1、p70S6K基因表达水平明显上调(P<0.05)。结论:强精煎可能通过激活PI3K/Akt//mTOR/4EBP1/p70S6K蛋白/基因表达、激活Ki-67蛋白表达等多途径调控少精子症大鼠生精细胞的增殖,促进精子发生,提高精子数量。

环孢素对耐力运动大鼠骨骼肌Akt及其下游信号分子影响

目的本研究旨在探讨耐力运动时骨骼肌Akt/mTOR/S6k1/GSK-3/FoxO1信号通路变化及钙调神经磷酸酶(CaN)抑制剂环孢素(CSA)是否影响这些信号分子。方法雄性SD大鼠随机分成对照组、运动组和CSA+运动组,6周实验结束取比目鱼肌(SOL)和趾长伸肌(EDL),用W estern b lotting法测基础P-Akt(Ser473)、p-mTOR(Ser2448)、P-S6K1(Thr389)和GSK-3、FoxO1蛋白含量,并用放射性核素法测GSK-3活性。结果 CSA不影响耐力运动大鼠SOL和EDL基础胞质P-Akt(Ser473)、p-mTOR(Ser2448)、P-S6K1(Thr389)含量;耐力运动增加EDL胞质GSK-3蛋白含量,但降低其活性;CSA不影响耐力运动诱导的EDL GSK-3蛋白含量和活性变化。耐力运动增加SOL FoxO1蛋白含量,CSA进一步增加其胞质含量,但CSA不影响SOL、EDL胞核FoxO1含量。结论耐力运动可影响骨骼肌静息GSK-3蛋白含量和活性以及FoxO1蛋白含量,CSA进一步增加耐力运动时大鼠SOL胞质FoxO1蛋白。