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.

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.

基于IGF-1R/PI3K/AKT的益气固表丸对慢性阻塞性肺疾病模型大鼠的影响及作用机制研究

目的:益气固表丸对慢性阻塞性肺疾病(COPD)有明显的治疗作用,但具体作用机制尚不清楚。本研究旨在探讨益气固表丸对脂多糖(LPS)联合烟雾暴露构建慢性阻塞性肺病模型大鼠的治疗作用机制。方法:通过大鼠气管内灌注LPS和吸入香烟烟雾构建慢性阻塞性肺病模型,同时给予模型大鼠低、中、高剂量益气固表丸治疗14 d。观察呼吸参数及肺组织病理变化。采用酶联免疫吸附试验测定支气管肺泡灌洗液(BALF)及血清胰岛素样生长因子1型受体(IGF-1R)、白细胞介素-1β(IL-1β)、IL-10水平。采用蛋白质印迹法检测大鼠肺组织样品中胰岛素样生长因子1受体(IGF-1R)/PI3K/AKT信号通路组分的磷酸化状态。结果:与对照组和低剂量益气组比较,大剂量益气固表丸治疗可显著改善COPD大鼠呼吸参数,减轻肺损伤和炎症,降低BALF和血清炎症介质水平。此外,高剂量益气固表丸干预的COPD大鼠肺组织标本中磷酸化IGF-1R、p-PI3K、p-AKT、p-GSK3、p-mTOR蛋白水平显著降低。结论:益气固表丸对COPD有明显的治疗作用,可能与靶向IGF-1R/PI3K/AKT信号通路有关。

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.

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.

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.

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.

ASF1 regulates asexual and sexual reproduction in Stemphylium eturmiunum by DJ-1 stimulation of the PI3K/AKT signaling pathway

Most fungi display a mixed mating system with both asexual and sexual reproduction.The timing of the two modes of reproduction must be carefully coordinated through signal perception and coordination in the cell along with chromatin modification.Here,we investigated coordination of reproductive output by investigating the function of the histone chaper-one anti-silencing factor 1(ASF1)in a fungal species amenable to characterization of both asexual and sexual reproduction.We used knockout approach to show that SeASF1 influenced asexual and sexual reproduction in Stemphylium eturmiunum.SeASF1-deleted strains failed to produce pseudothecia,but produce abnormal conidia and showed an irregular distribution of nuclei in mycelium.Transcriptome sequencing was then used to identify genes with altered expression in the SeASF1-deleted strains.The transcriptional expression of the identified SeDJ-1 was strongly regulated by SeASF1.The interaction of SeDJ-1 and SeASF1 was confirmed using Y2H,Co-IP,and pull-down.Due to some components of phosphatidylinositol 3-kinase/protein kinase B(PI3K/AKT)signaling pathway were known to interact with DJ-1 in mammals,we verified SePI3K,an element of PI3K/AKT signaling pathway in S.eturmiunum,was directly linked to SeDJ-1 and then these two proteins were defined as a coordinator of reproduction.However,knockout of SeDJ-1 or SePI3K altered the asexual and sexual repro-duction,but SePI3K recovered the asexual and sexual development of∆Sedj-1.The SeDJ-1-M6 segment of SeDJ-1 was essential for its interaction with SePI3K and played a critical role in restoring sexual reproduction in the∆Sepi3k,providing a deep understanding of the regulatory mechanism of SeDJ-1 in S.eturmiunum development.Summarily,SeASF1 is able to trigger SeDJ-1 and SeDJ-1can also activate SePI3K,which is orchestrally involved in asexual and sexual reproduction in S.eturmiunum.All these results reveal that SeASF1 manipulates asexual and sexual reproduction in S.eturmiunum by SeDJ-1 perception of PI3K/AKT signaling pathway.These data highlight the deep similarities in coordinating asexual and sexual processes in both fungi and eukaryotes in general.

更年汤对肾阳虚围绝经期模型大鼠卵巢PI3K/AKT/mTOR蛋白表达的影响

目的:探讨肾阳虚围绝经期模型大鼠在自拟更年汤不同剂量干预下对卵巢PI3K/AKT/mTOR蛋白表达的影响。方法:选24只4~6月龄的青年大鼠作为对照组,构建肾阳虚模型。另采集阴道脱落细胞将10~12月龄雌性SD大鼠筛选出围绝经期模型,并将该模型大鼠随机分为模型组、利维爱组、更年汤低、中及高剂量共5组,每组24只。以上6组均给予相应药物灌胃,末次给药24 h后,检测卵巢组织中IGF-1、IGF-1R及PI3K/AKT/mTOR蛋白表达。结果:更年汤高剂量组、西药组IGF-1、IGF-1R及PI3K/AKT/mTOR蛋白表达均较高,与模型组比较均有统计学差异(P<0.05)。结论:更年汤能改善肾阳虚型围绝经期模型大鼠卵巢组织中PI3K/AKT/mTOR蛋白表达,可能与能调节IGF-1及其受体的表达量有关。

DDR1抑制剂与IGF-1R抑制剂联合应用及机制

【目的】研究盘状结构域受体(DDR1)的抑制剂3-(2-(吡唑并[1,5-a]嘧啶-6-基)乙炔基)苯甲酰胺7RH对鼻咽癌细胞的体外抗肿瘤作用及相关机制,为该药用于鼻咽癌的临床治疗提供实验依据。【方法】采用MTT法绘制细胞生长曲线,计算7RH对鼻咽癌细胞的半数抑制浓度(IC50值);Western Blot检测7RH引起鼻咽癌细胞中信号通路的改变,并检测Si RNA瞬时转染方法干扰DDR1后phospho-IGF-1R的改变。【结果】7RH在体外明显抑制鼻咽癌细胞的增殖并且呈浓度依赖性;7RH作用鼻咽癌细胞株CNE2和HK1的IC50值分别为2.60μmol/L与6.33μmol/L;应用7RH处理或者使用SiRNA瞬时转染方法干扰DDR1后,CNE2的IGF-1R的磷酸化水平明显增强;联用DDR1抑制剂和IGF-IR抑制剂能更有效地抑制肿瘤细胞的增殖,两者联用能更显著地抑制AKT活性。【结论】DDR1的抑制剂3-(2-(吡唑并[1,5-a]嘧啶-6-基)乙炔基)苯甲酰胺7RH能在体外有效地抑制鼻咽癌细胞的增殖。联用7RH和IGF-IR抑制剂能通过抑制PI3K/AKT通路更有效抑制鼻咽癌细胞的增殖。

miRNA-1对胃癌细胞体外EGFR-TKI敏感性影响的实验研究

目的探讨上调或沉默miRNA-1(miR-1)表达对胃癌细胞体外表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)敏感性影响的研究。方法选择人胃癌细胞系MKN-45作为受试对象,慢病毒转染miR-1或miR-1 shRNA,分别作为miR-1组和miR-1 shRNA组,并采用实时荧光定量PCR(qPCR)法检测GES-1和MKN-45细胞中miR-1表达。另外设置空白对照组和阴性对照组。采用MTT法检测MKN-45细胞对不同浓度AZD9291(奥西替尼)的敏感性;流式细胞术检测1μmol/L AZD9291作用48 h后MKN-45细胞凋亡活力和细胞周期。采用Western blotting法检测各组细胞中PI3K/AKT通路相关蛋白的表达。结果qPCR证实miR-1在MKN-45细胞系中低表达。MTT法检测结果显示,与空白对照组比较,miR-1组细胞对AZD9291敏感性明显增加,而miR-1 shRNA组细胞对AZD9291敏感性明显降低。Annexin V/PI双染法和BrdU法检测,miR-1组细胞凋亡率为(44.65±5.18)%,高于空白对照组的(15.40±2.13)%和阴性对照组的(17.92±3.14)%,差异有统计学意义(P<0.05)。miR-1 shRNA组细胞凋亡率为(11.20±2.48)%,低于空白对照组和阴性对照组,差异有统计学意义(P<0.05)。与空白对照组和阴性对照组比较,miR-1组细胞IGF-1R、p-IGF-1R、p-AKT、p-p70S6K蛋白表达量明显降低,miR-1 shRNA组细胞IGF-1R、p-IGF-1R、p-AKT、p-p70S6K蛋白表达量明显升高,差异有统计学意义(P<0.05)。结论上调miR-1表达可增加MKN-45胃癌细胞系对AZD9291的敏感性,其作用机制可能与抑制IGF-1R/PI3K/AKT信号通路的激活有关。

CD26 upregulates proliferation and invasion in keloid fibroblasts through an IGF-1-induced PI3K/AKT/mTOR pathway

Background:Keloid is a fibrotic dermal disease characterized by an abnormal increase in fibroblast proliferation and invasion.These pathological behaviours may be related to the heterogeneity of keloid fibroblasts(KFs);however,because of a lack of effective biomarkers for KFs it is difficult to study the underlying mechanism.Our previous studies revealed that the expansion of CD26+KFs was responsible for increased keloid proliferation and invasion capabilities;the intrinsic relationship and mechanism between CD26 and keloid is therefore worthy of further investigation.The aim of this studywas to explore molecular mechanisms in the process of CD26 upregulated KFs proliferation and invasion abilities,and provide more evidence for CD26 as an effective biomarker of keloid and a new clinical therapeutic target.Methods:Flow cytometry was performed to isolate CD26+/CD26−fibroblasts from KFs and normal fibroblasts.To generate stably silenced KFs for CD26 and insulin-like growth factor-1 receptor(IGF-1R),lentiviral particles encoding shRNA targeting CD26 and IGF-1R were used for transfection.Cell proliferations were analysed by cell counting kit-8 assay and 5-ethynyl-2-deoxyuridine(EdU)incorporation assay.Scratching assay and transwell assay were used to assess cell migration and invasion abilities.To further quantify the regulatory role of CD26 expression in the relevant signalling pathway,RT-qPCR,western blot,ELISA,PI3K activity assay and immunofluorescence were used.Results:Aberrant expression of CD26 in KFs was proven to be associated with increased proliferation and invasion of KFs.Furthermore,the role of the IGF-1/IGF-1 receptor axis was also studied in CD26 and was found to upregulate KF proliferation and invasion.The PI3K/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway was shown to affect CD26-regulated KF proliferation and invasion by increasing phosphorylation levels of S6 kinase and 4E-binding protein.Conclusions:CD26 can be the effective biomarker for KFs,and its expression is closely related to proliferation and invasion in keloids through the IGF-1-induced PI3K/AKT/mTOR pathway.This work provides a novel perspective on the pathological mechanisms affecting KFs and therapeutic strategies against keloids.

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.

Wide Pulse Width Electroacupuncture Ameliorates Denervation-Induced Skeletal Muscle Atrophy in Rats via IGF-1/PI3K/Akt Pathway

Objective:To evaluate the effect of the pulse width of electroacupuncture(EA)in the treatment of denervation-induced skeletal muscle atrophy in rats and examine the role of insulin-like growth factor 1(IGF-1)/phosphatidylinositol 3-kinase(PI3 K)/Akt signaling pathway during EA.Methods:Sciatic nerve functional index(SFI),muscle wet weight and the cross-sectional area(CSA)of the gastrocnemius muscle were analyzed after treatment in model rats with EA of various pulse widths(0.5,50,100 and 200 ms).The apoptosis index(AI)and paired box(PAX)3 and PAX7 protein expression were also determined.Further,the mRNA and protein expressions of components of IGF-1/PI3 K/Akt pathway and their downstream targets were determined,along with the inhibiting effect of the pathway with a PI3-specific inhibitor.Results:EA with a pulse width of 200 ms was found to have the best effect with regard to increasing SFI,CSA and muscle weight,decreasing AI,and increasing the expression of PAX3 and PAX7.The IGF-1/PI3 K/Akt pathway was found to be activated by denervation,although the downstream forkhead box O(FoxO)pathway was not suppressed by its activation.The PI3 K/Akt pathway and its downstream molecule mammalian target of rapamycin(mTOR)were up-regulated further by EA to promote muscle protein synthesis.Meanwhile,the expressions of downstream FoxO and F-box protein 32(ATROGIN-1)were down-regulated to reduce protein degradation.Conclusions:EA with 200-ms pulse width was found to have a more significant effect than 0.5-ms EA.The positive effects of EA disappeared after inhibition of the PI3 K/Akt pathway.

Silencing novel long non-coding RNA FKBP9P1 represses malignant progression and inhibits PI3K/AKT signaling of head and neck squamous cell carcinoma in vitro

Background:Long non-coding RNAs(lncRNAs)play key roles in human cancers.In our previous study,we demonstrated that lncRNA FKBP prolyl isomerase 9 pseudogene 1(FKBP9P1)was highly expressed in head and neck squamous cell cancer(HNSCC)tissues.However,its functional significance remains poorly understood.In the present study,we identify the role and potential molecular biologic mechanisms of FKBP9P1 in HNSCC.Methods:Quantitative real-time polymerase chain reaction was used to detect the expression of FKBP9P1 in HNSCC tissues,matched adjacent normal tissues,human HNSCC cells(FaDu,Cal-27,SCC4,and SCC9),and human immortalized keratinocytes cell HaCaT(normal control).Cal-27 and SCC9 cells were transfected with sh-FKBP9P1-1,sh-FKBP9P1-2,and normal control(sh-NC)lentivirus.Cell counting kit-8 assay,colony formation assay,wound healing assay,and trans-well assay were used to explore the biologic function of FKBP9P1 in HNSCC cells.Furthermore,western blotting was used to determine the mechanism of FKBP9P1 in HNSCC progression.Chi-squared test was performed to assess the clinical significance among FKBP9P1 high-expression and low-expression groups.Survival analyses were performed using the Kaplan-Meier method and assessed using the log-rank test.The comparison between two groups was analyzed by Student t test,and comparisons among multiple samples were performed by one-way analysis of variance and a Bonferroni post hoc test.Results:FKBP9P1 expression was significantly up-regulated in HNSCC tissues(tumor vs.normal,1.914 vs.0.957,t=7.746,P<0.001)and cell lines(P<0.01 in all HNSCC cell lines).Besides,the median FKBP9P1 expression of HNSCC tissues(1.677)was considered as the threshold.High FKBP9P1 level was correlated with advanced T stage(P=0.022),advanced N stage(P=0.036),advanced clinical stage(P=0.018),and poor prognosis of HNSCC patients(overall survival,P=0.002 and disease-free survival,P<0.001).Knockdown of FKBP9P1 led to marked repression in proliferation,migration,and invasion of HNSCC cells in vitro(P all<0.01).Mechanistically,silencing FKBP9P1 was observed to restrain the PI3K/AKT signaling pathway.Conclusions:Silencing lncRNA FKBP9P1 represses HNSCC progression and inhibits PI3K/AKT(phosphatidylinositol 3 kinase/AKT Serine/Threonine Kinase)signaling in vitro.Therefore,FKBP9P1 could be a potential new target for the diagnosis and treatment of HNSCC patients.

逍遥散正丁醇部位基于IGF-1Rβ/PI3K/Akt信号通路的抗抑郁作用

目的:观察逍遥散正丁醇部位对慢性不可预知应激(CUMS)模型小鼠抑郁样行为的干预作用,及其作用发挥与调控类胰岛素生长因子-1受体β(IGF-1Rβ)/磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)通路的关系。方法:首先基于小鼠行为绝望模型初步获得逍遥散正丁醇部位发挥抗抑郁作用的有效剂量。再将雄性C57BL/6小鼠随机分为正常组、模型组、氟西汀组、逍遥散组、逍遥散正丁醇部位20,40 g·kg^(-1)组,采用CUMS法建立抑郁症模型小鼠研究其抗抑郁作用及机制,以糖水偏好实验检测造模效果。模型成功后,各给药组连续灌胃小鼠5周,正常组与模型组给予等体积溶媒。第5周末,开展小鼠糖水偏好实验、悬尾实验及新奇环境抑制进食实验;酶联免疫吸附测定法(ELISA)测定小鼠血清、海马区类胰岛素生长因子-1(IGF-1)含量,甲苯胺蓝染色法检测海马CA3区神经元尼氏小体平均光密度,免疫荧光法标记海马齿状回(DG)5-溴脱氧尿嘧啶核苷(Brdu)和双肾上腺皮质激素(DCX)阳性表达神经元,蛋白免疫印迹法(Western blot)检测海马区IGF-1Rβ,PI3K,磷酸化磷脂酰肌醇3-激酶(p-PI3K),Akt,磷酸化蛋白激酶B(p-Akt),半胱氨酸天冬氨酸蛋白水解酶3(Caspase-3),剪切后的半胱氨酸天冬氨酸蛋白水解酶(cleaved Caspase-3)蛋白表达水平。结果:小鼠强迫游泳和悬尾实验结果表明,逍遥散正丁醇部位生药量9.1,40 g·kg^(-1)均能明显缩短小鼠不动时间(P<0.05,P<0.01),表明其在9.1~40 g·kg^(-1)剂量之间具有有效性。CUMS模型中,与模型组比较,逍遥散正丁醇部位(生药量20,40 g·kg^(-1))能明显提高小鼠糖水偏好率(P<0.05,P<0.01),缩短悬尾不动时间(P<0.01)及新奇环境摄食潜伏期(P<0.01);并逆转模型小鼠血清、海马区下调的IGF-1含量(P<0.01),增加小鼠海马CA3区神经元尼氏小体数目(P<0.01),促进海马DG区Brdu和DCX表达(P<0.05,P<0.01),下调模型小鼠海马IGF-1Rβ(P<0.05,P<0.01),p-PI3K/PI3K(P<0.05,P<0.01),p-Akt/Akt(P<0.05),cleaved Caspase-3/Caspase-3蛋白表达水平。结论:逍遥散正丁醇部位具有与逍遥散全方相当的抗抑郁作用,可改善CUSM模型小鼠的抑郁样行为,诱导模型小鼠海马CA3区神经元合成尼氏小体,增加DG区神经元增殖、分化,促进神经发生;其作用发挥可能与抑制过度激活的IGF-1Rβ/PI3K/Akt通路、减少神经元凋亡有关。

Garcinia xanthochymus extract protects PC12 cells from H2O2-induced apoptosis through modulation of PI3K/AKT and NRF2/HO-1 pathways

The aim of the present study was to investigate the protective effects and underlying mechanisms of Garcinia xanthochymus, a perennial medicinal plant native to Yunnan, China, against H2 O2-induced oxidative damage in rat pheochromacytoma PC12 cells. Preincubation of PC12 cells with fruit Et OAc fraction(fruit-EFr., 12.5–50 μmol·L^(-1)) of G. xanthochymus for 24 h prior to H_2O_2 exposure markedly improved cell viability and increased the activities of antioxidant enzymes(superoxide dismutase, catalase, and heme oxygenase-1 [HO-1]), prevented lactate dehydrogenase release and lipid peroxidation malondialdehyde production, attenuated the decrease of matrix metalloproteinases(MMP), and scavenged reactive oxygen species(ROS). Fruit-EFr. also reduced BAX and cytochrome C expression and improved BCL-2 expression, thereby decreasing the ratio of BAX to BCL-2. Fruit-EFr. activated the nuclear translocation of NRF2 to increase HO-1 and induced the phosphorylation of AKT. Its cytoprotective effect was abolished by LY294002, a specific inhibitor of PI3 K. Taken together, the above findings suggested that fruit-EFr.of G. xanthochymus could enhance cellular antioxidant defense capacity, at least in part, through upregulating HO-1 expression and activating the PI3 K/AKT pathway and that it could suppress H_2O_2-induced oxidative damage via PI3 K/AKT and NRF2/HO-1 signaling pathways.

CircMAN1A2 promotes vasculogenic mimicry of nasopharyngeal carcinoma cells through upregulating ERBB2 via sponging miR-940

Nasopharyngeal carcinoma(NPC)is the most prevalent human primary malignancy of the head and neck,and the presence of vasculogenic mimicry(VM)renders anti-angiogenic therapy ineffective and poorly prognostic.However,the underlying mechanisms are unclear.In the present study,we used miR-940 silencing and overexpression for in vitro NPC cell EdU staining,wound healing assay and 3D cell culture assay,and in vivo xenograft mouse model and VM formation to assess miR-940 function.We found that ectopic miR-940 expression reduced NPC cell proliferation,migration and VM,as well as tumorigenesis in vivo.By bioinformatic analysis,circMAN1A2 was identified as a circRNA that binds to miR-940.Mechanistically,we confirmed that circMAN1A2 acts as a sponge for miR-940,impairs the inhibitory effect of miR-940 on target ERBB2,and then activates the PI3K/AKT/mTOR signaling pathway using RNA-FISH,dual luciferase reporter gene and rescue analysis assays.In addition,upregulation of ERBB2 expression is associated with clinical staging and poor prognosis of NPC.Taken together,the present findings suggest that circMAN1A2 promotes VM formation and progression of NPC through miR-940/ERBB2 axis and further activates the PI3K/AKT/mTOR pathway.Therefore,circMAN1A2 may become a biomarker and therapeutic target for anti-angiogenic therapy in patients with nasopharyngeal carcinoma.

Uncovering the mechanism of Sophora flavescens in the treatment of glioma based on network pharmacology

Glioma is a common primary intracranial tumor with high mortality and postoperative recurrence.Developing efficient therapies with lower toxicity is urgently needed.Sophora flavescens(SF)is a common Chinese medicine used to treat eczema,wet ulcers and itchy skin.Modern pharmacological studies have showed that SF has anti-glioma effects,but the mechanism of action remains unclear.This study aims to reveal the pharmacological mechanism of SF in treating glioma.The active components and related targets of SF were obtained from TCMSP.Genecard and Online Mendelian Inheritance in Man(OMIM)databases were used to explore the therapeutic targets for glioma.By making Venn diagram,we obtained 132 common targets of compounds and diseases.STRING databases and Cytoscape were used to construct diagrams of Protein-Protein Interaction(PPI)networks.Through the construction of PPI network,potential targets with degree value greater than the median were taken as core targets for further analysis.A total of 66 core targets were screened out.The degree values of TP53,HSP90AA1,MAPK1,and AKT1 were higher,indicating that these genes played important roles in this network.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were used to further discover the mechanism of active components in SF.Through enrichment analysis,it was found that the core targets were mainly enriched in PI3K/AKT signaling pathway,cell senescence related signaling pathway and IL-17 signaling pathway.In order to further explore the binding between active components and selected target,molecular docking was carried out.Finally,SwissADME was used to determine whether the compound could cross the blood-brain barrier.Based on network pharmacology,we speculated that matrine and formononetin in SF might inhibit PI3K/AKT signaling pathway and cellular senescence related signaling pathway by targeting AKT1,TP53,MAPK1 and other key targets.In summary,this study preliminarily explored the target and mode of action of SF in the treatment of glioma,laying the foundation for further research on its mechanism.