温阳解郁颗粒调节BDNF/TrkB/ERK信号通路保护皮质酮诱导损伤型海马神经细胞的作用机制研究＊

目的 观察温阳解郁颗粒（Wenyang Jieyu granule，WYJY）对皮质酮（Corticosterone，CORT）诱导损伤型小鼠海马神经细胞（TH22 cell）的保护作用，基于脑源性神经营养因子（Brain derived neurotrophic factor， BDNF）/酪氨酸激酶B（Tyrosine kinase B， TrkB）/细胞外信号调节蛋白激酶（Extra cellular regulated protein kinases， ERK）信号通路探讨WYJY保护海马神经细胞的作用机制。方法 体外构建小鼠海马神经细胞皮质酮诱导损伤模型，以不同浓度的WYJY和氟西汀（Fluoxetine，FXT）含药血清作用于模型细胞，细胞增殖-毒性检测（Cell Counting Kit-8， CCK-8）法分析细胞活性，倒置显微镜下观察给药前后细胞形态结构的改变，采用蛋白免疫印迹法（Western Blot）、实时荧光定量PCR（Quantitative real-time PCR， qPCR）法检测神经细胞内凋亡因子（BCL2-Associated X， Bax）、抗凋亡因子（B-cell lymphoma-2， Bcl-2）、BDNF、Trkb、ERK以及丝氨酸/苏氨酸激酶（Phospho-p90RSK， RSK）、环磷腺苷效应元件结合蛋白（cAMP-response element binding protein， CREB）蛋白表达水平以及相关基因的表达水平。结果 在浓度为459.5 μmol·L^-1的CORT作用24 h后，HT22细胞的活性抑制率达到50%，在此条件作用下细胞形态结构损伤明显，凋亡程度严重，细胞上清中BDNF的含量显著减少（P<0.05），细胞内凋亡相关因子Bax/Bcl-2的比值明显升高（P<0.01），BDNF、Trkb、ERK、RSK、CREB磷酸化蛋白表达水平和mRNA表达水平明显降低（P<0.01）；以5%的浓度为2.85 g·kg^-1的WYJY和10%的FXT含药血清作用于受损的HT22细胞后，HT22细胞存活率明显提升（P<0.01），细胞结构的损伤明显改善，细胞凋亡程度减轻，细胞外BDNF的含量显著升高（P<0.05），细胞内Bax/Bcl-2比值显著下调（P<0.01），BDNF、Trkb、ERK、RSK、CREB磷酸化蛋白表达水平和mRNA表达水平显著提升（P<0.05，P<0.01）。结论 温阳解郁颗粒可有效保护高浓度CORT造成的小鼠海马神经细胞损伤。调控BDNF/Trkb/ERK通路，放大CREB信号传导，影响Bcl-2、BDNF水平，可能是其保护海马神经元，发挥抗抑郁疗效的重要机制。     

ArgD of Mycobacterium tuberculosis is a functional N-acetylornithine aminotransferase with moonlighting function as an effective immune modulator

Mycobacterium tuberculosis (M. tuberculosis) encodes an essential enzyme acetyl ornithine aminotransferase ArgD (Rv1655) of arginine biosynthetic pathway which plays crucial role in M. tuberculosis growth and survival. ArgD catalyzes the reversible conversion of N-acetylornithine and 2 oxoglutarate into glutamate-5-semialdehyde and L-glutamate. It also possesses succinyl diaminopimelate aminotransferase activity and can thus carry out the corresponding step in lysine biosynthesis. These essential roles played by ArgD in amino acid biosynthetic pathways highlight it as an important metabolic chokepoint thus an important drug target. We showed that M. tuberculosis ArgD rescues the growth of ΔargD E. coli grown in minimal media validating its functional importance. Phylogenetic analysis of M. tuberculosis ArgD showed homology with proteins in gram positive bacteria, pathogenic and non-pathogenic mycobacteria suggesting the essentiality of this protein. ArgD is a secretory protein that could be utilized by M. tuberculosis to modulate host innate immunity as its moonlighting function. In-silico analysis predicted it to be a highly antigenic protein. The recombinant ArgD protein when exposed to macrophage cells induced enhanced production of pro-inflammatory cytokines TNF, IL6 and IL12 in a dose dependent manner. ArgD also induced the increased production of innate immune effector molecule NOS2 and NO in macrophages. We also demonstrated ArgD mediated activation of the canonical NFkB pathway. Notably, we also show that ArgD is a specific TLR4 agonist involved in the activation of pro-inflammatory signaling for sustained production of effector cytokines. Intriguingly, ArgD protein treatment activated macrophages to acquire the M1 phenotype through the increased surface expression of MHCII and costimulatory molecules CD80 and CD86. ArgD induced robust B-cell response in immunized mice, validating its antigenicity potential as predicted by the in-silico analysis. These properties of M. tuberculosis ArgD signify its functional plasticity that could be exploited as a possible drug target to combat tuberculosis.     

基于细胞因子的人参败毒散治疗新型冠状病毒肺炎(COVID-19)的网络药理学研究

目的从细胞因子角度,探究人参败毒散多成分、多靶点、多途径抗新型冠状病毒肺炎(COVID-19)的作用机制。方法应用中药系统药理分析平台(TCMSP)收集人参败毒散中活性化合物,结合药物靶点数据库收集细胞因子风暴相关靶点,利用Cytoscape构建"药材-化合物-疾病靶点"网络。通过String和DAVID数据库对靶点的互作网络、GO功能和KEGG通路进行分析。结果人参败毒散"药材-活性化合物-疾病靶点"网络包括10种药材,211个活性成分和151个疾病靶点。互作网络发现人参败毒散抑制细胞因子风暴治疗COVID-19的靶点可能包含STAT3、MAPK1、NFκB1、PIK3CA、MAPK3、TNF、CXCR4、VEGFA、IL-6、IL-2等。GO功能分析发现上述靶点在生物功能方面涉及趋化性、类固醇代谢过程等;在分子功能方面涉及血红素结合、铁离子结合、氧结合等;在细胞组成方面涉及细胞表面、细胞膜等。KEGG通路富集发现上述靶点参与查加斯病通路、HIF-1信号通路、肿瘤坏死因子信号通路等的调控。结论人参败毒散可能通过调节趋化性细胞因子,增加血氧饱和度,抑制STAT、MAPK、NFκB、PIK3K、IL-6等炎症相关信号通路,实现多成分-多靶点-多途径抑制细胞因子风暴形成的抗COVID-19作用。     

旋覆代赭汤对反流性食管炎大鼠模型TLR4/NF-κB的影响

目的观察旋覆代赭汤对反流性食管炎(RE)模型大鼠食管黏膜与脂多糖(LPS)、Toll样受体4(TLR4)、核因子κB(NF-κB)表达的影响。方法将60只雄性Wistar大鼠按随机数字表法分为正常对照组、模型组、旋覆代赭汤组(简称中药组,9.89 g/kg)、西药(奥美拉唑+莫沙比利,2.58 mg/kg)组,每组15只。除正常对照组外大鼠采用“4.2 mm幽门夹+2/3胃底结扎术”制备酸碱混合反流RE大鼠模型。术后第7天予相应药物干预,持续干预14天。利用光学显微镜观察大鼠食管下段黏膜组织形态学变化;应用ELISA法检测定外周血中LPS含量;采用Western Blot法与RT-PCR法检测食管黏膜组织中TLR4、NF-κB蛋白和基因表达。结果与正常对照组比较,模型组大鼠食管黏膜镜下损伤最为严重,食管黏膜呈炎性改变,可见急慢性炎细胞浸润,病理积分和外周血中LPS含量升高(P<0.05),食管组织中TLR4、NF-κB蛋白和基因表达升高(P<0.05)。与模型组比较,中药组、西药组病理积分和外周血中LPS含量降低(P<0.05),食管组织中TLR4、NF-κB蛋白和基因表达亦明显降低(P<0.05)。结论旋覆代赭汤能够减轻RE模型大鼠食管黏膜的损伤,抑制TLR4、NF-κB的表达,促进食管黏膜损伤的恢复。     

基于TGF-β1/p38MAPK/FN信号通路的放射性食管炎发病机制的研究

Objective To investigate the pathogenesis mechanism of radiation esophagitis from the perspective of mucosal regeneration and to determine whether it is associated with TGF-β1/p38MAPKs/FN signaling pathway. Methods The pathological analysis of esophageal specimens was performed by HE staining method. The expression of FN and TGF-β1 genes were observed by real time-PCR method, and the expression of tissue proteins TGF-β1, p38 and FN were detected by Western blot. Results The weights, food intakes and water intakes at the first week after the occurrence of radiation esophagitis were significantly decreased (P<0.05) and recovered at the fourth week. The esophageal mucosa was destructed at the first and second weeks, and the regeneration occurred in the fourth weeks; TGF-β1 and p38MAPK protein expression increased first and then decreased, while FN protein expression decreased first and then increased. Conclusion The TGF-β1/p38MAPK/FN signaling pathway may be involved in the process of mucosal repair. © 2020, CHINA RESEARCH ON PREVENTION AND TREATMENT. All rights reserved.     

Biogenic silver,gold and copper nanoparticles - A sustainable green chemistry approach for cancer therapy

The advent of nanotechnology has revolutionized the way clinicians are treating cancers. Treatment for cancer includes surgery, radiotherapy, hormonal therapy, chemotherapy, and now nano therapy, which could be a possible alternative. This new treatment regime can be beneficial since it shows minimum side effects as compared to other treatment methods. Metallic nanoparticles synthesized through green chemistry by using biological entities minimizes the side effects and enhances the properties of the metal against cancer cells. These green nanoparticles are widely used in research and have shown promising cytotoxic activity against various cancer cell lines. Mechanistically, these nanoparticles can enter the cancer cell and cause cell death by the activation of various molecular pathways such as apoptosis, necrosis and autophagy. This review focuses on the metal nanoparticles (silver, gold and copper) synthesized by the green chemistry approach that have been utilized to study the cancer cell death and we are also discussing the underlying molecular pathways.     

基于网络药理学方法探究补肾活血汤治疗激素性股骨头坏死的作用机制

目的:探讨补肾活血汤治疗激素性股骨头坏死(steroid-induced osteonecrosis of the femoral head,SONFH)的作用机制。方法:通过中医药整合药理学网络计算研究平台(integrative pharmacology-based network computational research platform of Traditional Chinese Medicine,TCMIP)v2.0预测、筛选补肾活血汤组方中14味中药的作用靶标,通过GeneCards、CTD和OMIM数据库查询SONFH的疾病靶点。根据获取的药物靶标和疾病靶点,进一步利用TCMIP v2.0中医药关联网络分析模块构建"药物靶标-疾病靶点"相互作用网络,根据网络拓扑特征值筛选补肾活血汤治疗SONFH的核心作用靶点。利用GO和KEGG数据库,采用富集算法挖掘上述方剂核心作用靶点的生物学功能和通路信息。结果:共获得891个补肾活血汤药物靶标和365个SONFH疾病靶点。经"药物靶标-疾病靶点"相互作用网络分析,最终筛选出31个补肾活血汤治疗SONFH核心靶点,GO功能分析富集出生物过程532条、分子功能29条,KEGG信号通路富集分析出相关通路共12条,主要涉及炎症免疫调节(chemokine signaling pathway,Toll-like receptor signaling pathway,NOD-like receptor signaling pathway,leukocyte transendothelial migration,Complement and coagulation cascades,cytokine-cytokine receptor interaction)、血管新生及血液循环调节(VEGF signaling pathway)、神经系统调节(neuroactive ligand-receptor interaction)和细胞功能调节(apoptosis,regulation of actin cytoskeleton)等方面。结论:结合SONFH的病理机制,排除缺乏特异性的信号通路,我们推测补肾活血汤可能通过调节TLR4/NF-κB和VEGF信号通路发挥补肾壮骨、活血化瘀的功效,这可能是其治疗SONFH的作用机制之一。     

黄芩苷对肝星状细胞iNOS/PUMA信号通路的影响

目的研究黄芩苷通过iNOS/PUMA信号通路促进肝星状细胞凋亡的作用。方法MTT检测细胞增殖抑制率,Western Blot检测iNOS、PUMA蛋白表达,real-time PCR检测PUMA mRNA表达,一氧化氮检测试剂盒检测NO含量,iNOS抑制剂L-NAME预处理后检测细胞增殖抑制率和PUMA表达,Hoechst 33342核染色检测细胞凋亡。结果50 mM黄芩苷引起肝星状细胞活化诱导性死亡;黄芩苷促进肝星状细胞PUMA mRNA、蛋白表达;iNOS抑制剂L-NAME预处理后PUMA表达降低、细胞凋亡减少。结论黄芩苷能通过iNOS/PUMA信号通路促进肝星状细胞凋亡。