基于网络药理学和细胞实验探讨和厚朴酚对 PC12细胞HIF-1α-VEGF通路的调节作用

基于网络药理学和细胞实验探讨和厚朴酚对PC12细胞HIF-1α-VEGF通路的调节作用。通过TCMSP数据库和Swiss Target Prediction数据库筛选出127个药物靶点;采用DisGeNET数据库收集到287个疾病靶点;通过String数据库构建PPI网络;采用DAVID数据库进行GO和KEGG富集分析。GO富集分析得到173个条目。KEGG通路筛选出前20条信号通路,包括HIF-1通路、VEGF通路、PI3K-Akt通路等。分子对接结果表明和厚朴酚与HIF-1α降解酶PHDs、VHL有较好的结合力。CCK-8法发现和厚朴酚能有效增加PC12细胞的存活率。正常环境下,和厚朴酚给药后HIF-1α、VEGF、PSD 95、SYN 1蛋白表达升高(P<0.05,P<0.01)。缺氧环境下,和厚朴酚给药后HIF-1α和VEGF蛋白表达显著降低(P<0.05,P<0.01),而PSD 95和SYN 1蛋白表达显著升高(P<0.01,P<0.001)。综上,正常和缺氧环境下和厚朴酚对PC12细胞HIF-1α-VEGF通路有相反的调节作用,为和厚朴酚抗脑缺血、抗抑郁和抗肿瘤机制研究提供可靠的理论和实验支持。

Zinc oxide nanoparticles inhibit osteosarcoma metastasis by downregulatingβ-catenin via HIF-1α/BNIP3/LC3B-mediated mitophagy pathway

Osteosarcoma(OS)therapy faces many challenges,especially the poor survival rate once metastasis occurs.Therefore,it is crucial to explore new OS treatment strategies that can efficiently inhibit OS metastasis.Bioactive nanoparticles such as zinc oxide nanoparticles(ZnO NPs)can efficiently inhibit OS growth,however,the effect and mechanisms of them on tumor metastasis are still not clear.In this study,we firstly prepared well-dispersed ZnO NPs and proved that ZnO NPs can inhibit OS metastasis-related malignant behaviors including migration,invasion,and epithelial-mesenchymal transition(EMT).RNA-Seqs found that differentially expressed genes(DEGs)in ZnO NP-treated OS cells were enriched in wingless/integrated(Wnt)and hypoxia-inducible factor-1(HIF-1)signaling pathway.We further proved that Zn^(2+)released from ZnO NPs induced downregulation ofβ-catenin expression via HIF-1α/BNIP3/LC3B-mediated mitophagy pathway.ZnO NPs combined with ICG-001,aβ-catenin inhibitor,showed a synergistic inhibitory effect on OS lung metastasis and a longer survival time.In addition,tissue microarray(TMA)of OS patients also detected much higherβ-catenin expression which indicated the role ofβ-catenin in OS development.In summary,our current study not only proved that ZnO NPs can inhibit OS metastasis by degradingβ-catenin in HIF-1α/BNIP3/LC3B-mediated mitophagy pathway,but also provided a far-reaching potential of ZnO NPs in clinical OS treatment with metastasis.

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.

黄芪百合颗粒通过HIF-1α-VEGF信号通路对常压缺氧模型小鼠脑损伤的保护作用

目的观察黄芪百合颗粒通过HIF-1α-VEGF信号通路对常压缺氧环境下小鼠大脑损伤的保护作用。方法72只SPF级昆明种雄性小鼠随机分为空白组、模型组、阳性药组、黄芪百合颗粒低、中、高剂量组。空白组和模型组小鼠给予蒸馏水灌胃,黄芪百合颗粒低、中、高剂量组分别给与(1.75、3.5、7mg·kg^(-1))黄芪百合颗粒灌胃,阳性药组给与0.5mg(kg·d)枸杞红景天胶囊溶液灌胃,干预30d。灌胃的第5天开始,进行小鼠常压耐氧实验。分光光度法检测脑组织总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)、丙二醛(MDA)含量、HE染色观察脑组织的病理形态学改变。Western blot检测各组小鼠HIF-1α、VEGF蛋白的表达水平。RT-PCR检测各组小鼠HIF-1α、VEGF mRNA的表达水平;结果与空白组相比,模型组小鼠病理切片可见细胞水肿明显出现多灶性神经元细胞坏死,周围有淋巴细胞等的炎性浸润,大脑指数明显升高,脑丙二醛(MDA)含量、HIF-1α、VEGF蛋白和mRNA含量明显升高(P<0.01),脑总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)活性明显降低(P<0.01);与模型组相比,黄芪百合颗粒高剂量组神经元细胞水肿、坏死明显减少,其大小、形态未发生明显变化,局部炎性浸润减少。黄芪百合颗粒低、中、高剂量组小鼠大脑指数、脑脑总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)活性明显升高(P<0.01或P<0.05),丙二醛(MDA)含量、VEGF蛋白和mRNA含量均明显降低(P<0.01或P<0.05)。结论黄芪百合颗粒可通过HIF-1α-VEGF信号通路对常压缺氧损伤的小鼠脑组织发挥良好的保护作用。

麦冬皂苷D对氧糖剥夺/复氧后新生鼠大脑皮层离体星形胶质细胞HIF-1 α-VEGF通路的作用

目的:探讨麦冬皂苷D对氧糖剥夺/复氧(OGD/R)后星形胶质细胞HIF-1α-VEGF通路的作用。方法:分离培养和鉴定新生大鼠星形胶质细胞,建立星形胶质细胞OGD/R模型;设正常组、模型组、麦冬皂苷D给药组;在OGD4h/R2h、4h、6h后用ELISA检测星形胶质细胞细胞培养液中VEGF蛋白表达量,Western Blot检测星形胶质细胞胞核内HIF-1α蛋白的含量,免疫荧光检测星形胶质细胞及其在OGD/R后细胞的光密度、面密度和阳性细胞数量。结果:模型组VEGF、HIF-1α蛋白含量高于正常组(P<0.05,P<0.01),麦冬皂苷D给药组高于模型组(P<0.05,P<0.01),且随再灌注时间延长,VEGF、HIF-1α蛋白含量呈增加趋势。模型组星形胶质细胞的面密度、光密度、阳性细胞个数较正常组均明显降低(P<0.01),麦冬皂苷D给药组星形胶质细胞的面密度、光密度、阳性细胞个数较模型组均显著升高(P<0.05,P<0.01)。结论:麦冬皂苷D能够促进OGD/R后新生鼠离体星形胶质细胞的增殖;麦冬皂苷D能够明显上调OGD/R后新生鼠离体星形胶质细胞VEGF、HIF-1α的蛋白含量;麦冬皂苷对OGD/R后神经损伤的保护及修复作用可能是通过星形胶质细胞HIF-1α-VEGF通路实现的。

Network pharmacology and experimental validation of Maxing Shigan decoction in the treatment of influenza virus-inducedferroptosis

Influenza is an acute viral respiratory infection that has caused high morbidity and mortality worldwide.Influenza A virus(IAV)has been found to activate multiple programmed cell death pathways,including ferroptosis.Ferroptosis is a novel form of programmed cell death in which the accumulation of intracellular iron promotes lipid peroxidation,leading to cell death.However,little is known about how influenza viruses induce ferroptosis in the host cells.In this study,based on network pharmacology,we predicted the mechanism of action of Maxing Shigan decoction(MXSGD)in IAV-induced ferroptosis,and found that this process was related to biological processes,cellular components,molecular function and multiple signaling pathways,where the hypoxia inducible factor-1(HIF-1)signaling pathway plays a significant role.Subsequently,we constructed the mouse lung epithelial(MLE-12)cell model by IAV-infected in vitro cell experiments,and revealed that IAV infection induced cellular ferroptosis that was characterized by mitochondrial damage,increased reactive oxygen species(ROS)release,increased total iron and iron ion contents,decreased expression of ferroptosis marker gene recombinant glutathione peroxidase 4(GPX4),increased expression of acyl-CoA synthetase long chain family member 4(ACSL4),and enhanced activation of hypoxia inducible factor-1α(HIF-1α),induced nitric oxide synthase(iNOS)and vascular endothelial growth factor(VEGF)in the HIF-1 signaling pathway.Treatment with MXSGD effectively reduced intracellular viral load,while reducing ROS,total iron and ferrous ion contents,repairing mitochondrial results and inhibiting the expression of cellular ferroptosis and the HIF-1 signaling pathway.Finally,based on animal experiments,it was found that MXSGD effectively alleviated pulmonary congestion,edema and inflammation in IAV-infected mice,and inhibited the expression of ferroptosis-related protein and the HIF-1 signaling pathway in lung tissues.

Self-oxygenated co-assembled biomimetic nanoplatform for enhanced photodynamic therapy in hypoxic tumor

Photodynamic therapy(PDT)has shown great application potential in cancer treatment and the important manifestation of PDT in the inhibition of tumors is the activation of immunogenic cell death(ICD)effects.However,the strategy is limited in the innate hypoxic tumor microenvironment.There are two key elements for the realization of enhanced PDT:specific cellular uptake and release of the photosensitizer in the tumor,and a sufficient amount of oxygen to ensure photodynamic efficiency.Herein,self-oxygenated biomimetic nanoparticles(CS@M NPs)co-assembled by photosensitizer prodrug(Ce6-S-S-LA)and squalene(SQ)were engineered.In the treatment of triple negative breast cancer(TNBC),the oxygen carried by SQ can be converted to reactive oxygen species(ROS).Meanwhile,glutathione(GSH)consumption during transformation from Ce6-S-S-LA to chlorin e6(Ce6)avoided the depletion of ROS.The co-assembled(CS NPs)were encapsulated by homologous tumor cell membrane to improve the tumor targeting.The results showed that the ICD effect of CS@M NPs was confirmed by the significant release of calreticulin(CRT)and high mobility group protein B1(HMGB1),and it significantly activated the immune system by inhibiting the hypoxia inducible factor-1alpha(HIF-1α)-CD39-CD73-adenosine a2a receptor(A2AR)pathway,which not only promoted the maturation of dendritic cells(DC)and the presentation of tumor specific antigens,but also induced effective immune infiltration of tumors.Overall,the integrated nanoplatform implements the concept of multiple advantages of tumor targeting,reactive drug release,and synergistic photodynamic therapy-immunotherapy,which can achieve nearly 90%tumor suppression rate in orthotopic TNBC models.

Extracts of Celastrus Orbiculatus Inhibit Cancer Metastasis by Down-regulating Epithelial-Mesenchymal Transition in Hypoxia-Induced Human Hepatocellular Carcinoma Cells

Objective: To evaluate the effects of Celastrus Orbiculatus extracts(COE) on metastasis in hypoxiainduced hepatocellular carcinoma cells(Hep G2) and to explore the underlying molecular mechanisms. Methods:The effect of COE(160, 200 and 240 μg/mL) on cell viability, scratch-wound, invasion and migration were studied by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide(MTT), scratch-wound and transwell assays, respectively. Co Cl2 was used to establish a hypoxia model in vitro. Effects of COE on the expressions of E-cadherin, vimentin and N-cadherin were investigated with Western blot and immuno?uorescence analysis,respectively. Results: COE inhibited proliferation and metastasis of hypoxia-induced hepatocellular carcinoma cells in a dose-dependent manner(P<0.01). Furthermore, the expression of epithelial-mesenchymal transition(EMT) related markers were also remarkably suppressed in a dose-dependent manner(P<0.01). In addition, the upstream signaling pathways, including the hypoxia-inducible factor 1α(Hif-1α) and Twist1 were suppressed by COE. Additionally, the Hif-1α inhibitor 3-5'-hydroxymethyl-2'-furyl)-1-benzylindazole(YC-1), potently suppressed cell invasion and migration as well as expression of EMT in hypoxia-induced Hep G2 cells. Similarly, the combined treatment with COE and YC-1 showed a synergistic effect(P<0.01) compared with the treatment with COE or YC-1 alone in hypoxia-induced Hep G2 cells. Conclusions: COE signi?cantly inhibited the tumor metastasis and EMT by suppressing Hif-1α/Twist1 signaling pathway in hypoxia-induced Hep G2 cell. Thus, COE might have potential effect to inhibit the progression of Hep G2 in the context of tumor hypoxia.