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.

IGF1/IGF-1R通过Akt/Bad通路促进肝癌进展

目的为了探索影响肝癌进展的新的分子机制。方法通过免疫荧光技术、免疫组织化学技术、Western blot技术检测肝癌细胞与正常肝细胞中IGF-1R的表达水平,并且通过克隆实验、JC-1实验检测IGF-1R对肝癌细胞的增殖、凋亡的影响,同时运用Western blot实验检测IGF-1R下游信号分子Akt/Bad的蛋白磷酸化水平。结果验证了IGF-1R在肝癌细胞上的表达水平明显升高,并且促进肝癌增殖、抑制凋亡。进一步研究发现IGF-1R上调Akt、Bad分子磷酸化水平影响肝癌进展。结论IGF-1R在肝癌细胞中呈明显上调表达,并且可能通过活化Akt/Bad通路促进肝癌进展。

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.

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.

芹菜素对人肺癌H1975细胞增殖、凋亡、转移及IGF-1R/Akt信号通路的影响

目的:探讨芹菜素对人肺癌H1975细胞增殖、凋亡和转移及胰岛素样生长因子1受体(IGF-1R)/蛋白激酶B(Akt)信号通路的影响。方法:设H1975细胞组、氟尿嘧啶组(100μg·ml-1)、芹菜素低(100μg·ml-1)、高(200μg·ml-1)剂量组,以上各组每孔设6个平行样,培养72h。MTT法、结晶紫染色测定细胞增殖、克隆形成数目,Transwell细胞培养室评估细胞迁移,流式细胞仪测定凋亡水平,实时荧光定量逆转录-聚合酶链反应(RT-PCR)法及蛋白免疫印迹(WEST-BLOT)法检测IGF-1R、Akt基因和蛋白表达水平。结果:与H1975细胞组比较,氟尿嘧啶组、芹菜素低、高剂量组的光密度(OD)值、存活率、细胞克隆形成数目、穿膜数、IGF-1R、Akt mRNA和蛋白降低,凋亡率升高(P<0.05)。与氟尿嘧啶组比较,芹菜素低剂量组的光密度(OD)值、存活率、细胞克隆形成数目、穿膜数、IGF-1R、Akt mRNA和蛋白升高,凋亡率降低(P<0.05);芹菜素高剂量组OD值、存活率、细胞克隆形成数目、穿膜数、IGF-1R、Akt mRNA和蛋白降低,凋亡率升高(P<0.05)。与芹菜素低剂量组比较,芹菜素高剂量组的OD值、存活率、细胞克隆形成数目、穿膜数、IGF-1R、Akt mRNA和蛋白降低,凋亡率升高(P<0.05)。结论:芹菜素能明显抑制人肺癌H1975细胞增殖、凋亡和转移;其机制与芹菜素抑制IGF-1R、Akt mRNA和蛋白的表达,进而抑制了IGF-1R/Akt信号通路的激活有关。

基于IGF-1R/Akt/PGC-1α通路探讨芍药苷对H2O2诱导PC12细胞氧化损伤的保护作用

目的研究芍药苷(Paeoniflorin,PF)对过氧化氢(H2O2)诱导高分化大鼠肾上腺嗜铬瘤细胞(PC12细胞)氧化损伤模型的保护作用及其机制。方法采用无血清H2O2体外诱导PC12细胞氧化损伤,并以不同浓度PF(100、200、300μmol·L-1)进行干预。采用噻唑蓝(MTT)法检测细胞存活率;倒置生物显微镜观察细胞形态;Annexin-V/PI双染法检测细胞凋亡率;ELISA法检测细胞内超氧化物歧化酶(SOD)活力、丙二醛(MDA)含量和总抗氧化能力(T-AOC);Western Blot法检测胰岛素样生长因子-1受体(IGF-1R)、丝氨酸/苏氨酸激酶(Akt)、磷酸化Akt(p-Akt)、过氧化物酶体增殖物激活受体γ辅助激活因子-1α(PGC-1α)蛋白的表达水平。结果选择8μmol·L-1 H2O2作为模型复制浓度。与正常组比较,H2O2模型组细胞形态异常;细胞存活率明显降低、凋亡率显著升高(P<0.01);MDA含量增高(P<0.01),SOD活力及T-AOC降低(P<0.01);IGF-1R、Akt、pAkt及PGC-1α蛋白表达水平均明显下调(P<0.01)。与模型组比较,PF不同浓度组均能改善细胞形态,显著提高细胞存活率和降低细胞凋亡率(P<0.05,P<0.01);显著提高SOD酶活力和T-AOC(P<0.05,P<0.01),降低MDA含量(P<0.01,200、300μmol·L-1 PF组);明显上调IGF-1R、Akt及PGC-1α蛋白表达水平(P<0.05,P<0.01),提高Akt蛋白的磷酸化(p-Akt)水平(P<0.05,P<0.01)。结论PF可能是通过提高IGF-1R的表达,激活Akt/PGC-1α信号转导通路,增强细胞抗氧化损伤能力,抑制细胞凋亡,从而发挥保护H2O2损伤的PC12细胞的作用。

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.

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。

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.

African swine fever virus MGF505-3R inhibits cGAS-STING-mediated IFN-βpathway activation by degrading TBK1

African swine fever virus(ASFV)is an important pathogen causing acute infectious disease in domestic pigs and wild boars that seriously endangers the global swine industry.As ASFV is structurally complex and encodes a large number of functional proteins,no effective vaccine has been developed to date.Thus,dissecting the mechanisms of immune escape induced by ASFV proteins is crucial.A previous study showed that the ASFV-encoded protein is an important factor in host immunity.In this study,we identified a negative regulator,MGF505-3R,that significantly downregulated cGAS/STING-and poly(dG:dC)-mediated IFN-βand interferon stimulation response element(ISRE)reporter activity and suppressed IFNB1 and IFIT2 mRNA levels.In addition,TBK1,IRF3 and IκBαphosphorylation levels were also inhibited.Mechanistically,MGF505-3R interacted with cGAS/TBK1/IRF3 and targeted TBK1 for degradation,thereby disrupting the cGAS-STING-mediated IFN-βsignaling pathway,which appears to be highly correlated with autophagy.Knockdown MGF505-3R expression enhanced IFN-βand IL-1βproduction.Taken together,our study revealed a negative regulatory mechanism involving the MGF505-3R-cGAS-STING axis and provided insights into an evasion strategy employed by ASFV that involves autophagy and innate signaling pathways.

Novel phenanthrene/bibenzyl trimers from the tubers of Bletilla striata attenuate neuroinflammation via inhibition of NF-κB signaling pathway

Five novel(9,10-dihydro)phenanthrene and bibenzyl trimers,as well as two previously identified biphenanthrenes and bibenzyls,were isolated from the tubers of Bletilla striata.Their structures were elucidated through comprehensive analyses of NMR and HRESIMS spectroscopic data.The absolute configurations of these compounds were determined by calculating rotational energy barriers and comparison of experimental and calculated ECD curves.Compounds 5b and 6 exhibited inhibitory effects on LPS-induced NO production in BV-2 cells,with IC_(50) values of 12.59±0.40 and 15.59±0.83μmol·L^(-1),respectively.A mechanistic study suggested that these compounds may attenuate neuroinflammation by reducing the activation of the AKT/IκB/NF-κB signaling pathway.Additionally,compounds 3a,6,and 7 demonstrated significant PTP1B inhibitory activities,with IC_(50) values of 1.52±0.34,1.39±0.11,and 1.78±0.01μmol·L^(-1),respectively.Further investigation revealed that compound 3a might inhibit LPS-induced PTP1B overexpression and NF-κB activation,thereby mitigating the neuroinflammatory response in BV-2 cells.

Network pharmacology and molecular docking analysis reveal insights into the molecular mechanism of Gualou Qumai Wan in clear cell renal cell carcinoma

Background:To initially clarify the potential therapeutic targets and pharmacological mechanism regarding Gualou Qumai Wan(GQW),a kind of traditional Chinese medicine(TCM),in clear cell renal cell carcinoma(ccRCC)by virtue of the network pharmacology analysis and molecular docking analysis.Methods:The screening of bioactive components and targets of GQW was based on the Traditional Chinese Medicine System Pharmacology(TCMSP)and the UniProt platform served for standardizing their targets.Online Mendelian Inheritance in Man(OMIM),PharmGkb,TTD,DrugBank and GeneCards databases were searched to collect the disease targets of ccRCC.Cytoscape assisted in constructing herb-compound-target(H-C-T)networks.The STRING database was searched for constructing the target protein-protein interaction(PPI)networks,while the R programming language served for analyzing GO functional terms and the KEGG pathways related to potential targets.Analyses of core genes related to survival and tumor microenvironment(TME)were conducted respectively based on the GEPIA2 database and TIMER 2.0 database.Human Protein Atlas(HPA)and The Cancer Genome Atlas(TCGA)helped to obtain core genes’protein expression as well as transcriptome expression level.Autodock Vina software validated the molecular docking regarding GQW components and pivotal targets.Results:The constructed H-C-T networks mainly had 33 compounds and 65 targets.A topological analysis of the PPI network identified that ESR1,AKT1,HIF1A,PTGS2,TP53 and VEGFA serve as core targets in the way GQW affects ccRCC.According to the GO and KEGG pathway enrichment analyses,the effects of GQW are mediated by genes related to hypoxia and oxidative stress as well as the Chemical carcinogenesis-receptor activation and PI3K-Akt signaling pathways.AKT1 shows a close relation to the recruitment of various immune cells and can remarkably affect disease prognosis according to reports.Molecular docking and molecular dynamics simulations showed that diosgenin has higher affinity with core targets.Conclusion:The study makes a comprehensive explanation of the biological activity,potential targets,as well as molecular mechanism regarding GQW against ccRCC,which promisingly assists in revealing the action mechanism of TCM formulae in disease treatment and the respective and scientific basis.

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.

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。

The effects of claudin 14 during early Wallerian degeneration after sciatic nerve injury

Claudin 14 has been shown to promote nerve repair and regeneration in the early stages of Wallerian degeneration （0-4 days） in rats with sciatic nerve injury, but the mechanism underlying this process remains poorly understood. This study reported the effects of claudin 14 on nerve degeneration and regeneration during early Wallerian degeneration. Claudin 14 expression was up-regulated in sciatic nerve 4 days after Wallerian degeneration. The altered expression of claudin 14 in Schwann cells resulted in expression changes of cytokines in vitro. Expression of claudin 14 affected c-Jun, but not Akt anal ERK1/2 patl^ways, l^urther studies reve^ed that enhanced expression of claudin 14 could promote Schwann cell proliferation and migration. Silencing of claudin 14 expression resulted in Schwann cell apoptosis and reduction in Schwann cell proliferation. Our data revealed the role of claudin 14 in early Wallerian degeneration, which may provide new insights into the molecular mechanisms of Wallerian degeneration.

GRK2 overexpression inhibits IGF1-induced proliferation and migration of human hepatocellular carcinoma cells by down-regulating EGR1

OBJECTIVE To investigate the role and mechanism of G protein-coupled receptor kinase 2(GRK2)involving in hepatocel ular carcinoma(HCC)progression.METHODS Cel Counting Kit 8 and tumor colony formation assay were designed to detect HCC cell proliferation,wound healing assay was to detect HCC migration.The correlation between GRK2 and early growth response-1(EGR1)were detected by RT-PCR and real-time PCR assays.Co-immunoprecipitation and Western blot assay were adopted to detect the relationship between GRK2and insulin-like growth factor 1 receptor(IGF-1R)signaling pathway.RESULTS In this study we find that GRK2plays an inhibition role in IGF1-induced HCC cell proliferation and migration.Overexpression of GRK2 causes a decrease in EGR1 expression,while knockdown of GRK2 leads to the dramatically increase in EGR1 expression in the treatment of IGF1.Through co-immunoprecipitation and Western blot assay,we confirm that GRK2can interact with IGF-1R and inhibiting IGF1-induced activation of IGF1R signaling pathway.Silencing EGR1attenuates GRK2 overexpression-caused inhibition of cell proliferation,tumor colony number and migrationactivity,while overexpressing of EGR1 restores the antiproliferative and migratory effect by GRK2 overexpression in HCCLM3 cells.CONCLUSION Taken together,these results suggest that GRK2 may inhibit IGF1-induced HCC cell growth and migration through down-regulation of EGR1 and indicate that enforced GRK2 may offer a potential therapeutic approach against HCC.