基于网络药理学和分子对接技术探讨生脉注射液抗新型冠状病毒肺炎的作用机制

目的采用网络药理学与分子对接技术探讨生脉注射液的活性成分和治疗新型冠状病毒肺炎(COVID-19)的潜在作用机制。方法利用TCMSP及BATMAN-TCM数据库筛选生脉注射液的活性化合物,通过TCMSP及Targetnet在线数据库预测作用靶点,通过Cytoscape3.7.1构建活性成分-作用靶点网络图;在GeneCards及OMIM数据库中以"coronavirus pneumonia"为关键词搜索冠状病毒肺炎相关疾病靶点,与生脉注射液化合物靶点进行交集筛选出共同靶点作为研究靶点,将共同靶点导入STRING数据库获取数据后在Cytoscape 3.7.1软件中构建蛋白质-蛋白质相互作用网络图;利用R语言进行GO(gene ontology)功能、KEGG(Kyoto encyclopedia of genes and genomes)通路富集分析,预测其作用机制,并构建"成分-靶点-通路"网络图;通过DiscoveryStudio 2.5软件对关键靶点进行分子对接分析。结果生脉注射液筛选得到22个活性化合物,分别为邻苯二甲酸二辛酯、β-谷甾醇、当归酰基戈米辛O、戈米辛A、戈米辛R、五味子丙素、内南五味子酯乙、长南酸、南五味子内酯、香蒲木脂素B、新杜松烷酸A、新杜松烷酸B、新杜松烷酸C、新南五味子木脂宁、五味子内酯A、五味子内酯E、五味子酸、尿苷、薯蓣皂苷元、鸟嘌呤核苷、N-反式阿魏酰酪胺、豆甾醇。相应作用靶点224个,与COVID-19的共同靶点16个,分别为CASP3、CASP8、PTGS2、BCL2、BAX、PRKCA、PTGS1、PIK3CG、F10、NOS3、DPP4、NOS2、TLR9、ACE、ICAM1、PRKCE,关键靶点涉及CASP3、PTGS2、NOS2、NOS3、ICAM1。GO功能富集分析得到生物过程(BP)条目771个,细胞组成(CC)条目11个,分子功能(MF)条目79个。KEGG通路富集分析筛选得到67条(P0.05)信号通路,主要涉及糖尿病并发症AGE-RAGE信号通路、凋亡通路、P53信号通路、小细胞肺癌通路等。分子对接结果显示与关键靶点对接较好的成分有五味子内酯E、豆甾醇、N-反式阿魏酰酪胺。结论生脉注射液中的活性化合物五味子内酯E、豆甾醇、N-反式阿魏酰酪胺等能作用于CASP3、PTGS2、NOS2、NOS3等靶点调节多条信号通路发挥抗炎、免疫调节、抗休克、增加血氧饱和度等作用,从而可能发挥对COVID-19的治疗作用。     

Amino acid metabolism as drug target in autoimmune diseases

In mammals, amino acid metabolism has evolved to control immune responses. Autoimmune diseases are heterogeneous conditions that involve the breakdown of tolerogenic circuitries and consequent activation of autoreactive immune cells. Therefore, critical enzymes along amino acid degradative pathways may be hijacked to keep in check autoimmunity. We examined here current knowledge of indoleamine 2,3-dioxygenase 1 (IDO1) and arginase 1 (ARG1), the main enzymes catabolizing tryptophan and arginine, respectively, in organ-specific and systemic autoimmune diseases as well as in the development of autoantibodies to therapeutic proteins. At variance with neoplastic contexts, in which it is known to act as a pure immunosuppressive molecule, ARG1 exhibited a protective or pathogenetic profile, depending on the disease. In contrast, in several autoimmune conditions, the bulk of data indicated that drugs capable of potentiating IDO1 expression and activity may represent valuable therapeutic tools and that IDO1-based immunotherapeutic protocols could be more effective if tailored to the genetic profile of individual patients.     

兔下颌骨牵张成骨中VEGF与NOS的相关性研究

目的:研究VEGF、iNOS和eNOS在兔下颌骨牵张成骨中的表达及其相关关系;探讨NOS和VEGF的相互作用及在促进新骨生成中的作用机制。方法:日本大耳白兔20只,随机分为五组,每组4只。分别处死空白对照组、牵张后1天组、1周组、2周组、4周组动物,取牵张处骨组织做成切片,行X线和组织学观察。结果:X线下示4周后牵张处骨缺损消失,组织学示牵张处骨小梁逐渐成熟;VEGF与NOS在形成新骨中均有高水平表达,VEGF同iNOS成正相关,r=0.844,P<0.01;VEGF同eNOS成正相关,r=0.647,P<0.;01;结论:VEGF、NOS可能共同参与促进牵张成骨新骨的形成,二者可能在上诉过程相互作用,促使成骨。     

Study on the effects of mechanical pressure to the ultrastructure and secretion ability of mandibular condylar chondrocytes

During mandibular movement, condyle is subjected to repetitive compression and the mandibular condylar chondrocytes (MCCs) can detect and respond to this biomechanical environment by altering their metabolism. The present study was undertaken to investigate the effects of pressure to the ultrastructure, aggrecan synthesis, nitric oxide (NO) and prostaglandin F(1)alpha(PGF(1)alpha) secretion in MCCs. In vitro cultured rabbit MCCs were incubated and pressed under continuous pressure of 90kPa for 60min and 360min by hydraulic pressure controlled cellular strain unit. The ultrastructure, aggrecan mRNA expression, activity of nitric oxide synthase (NOS) and PGF(1)alpha secretion were investigated. Besides, nitric oxide inhibitor was used together with pressure to investigate the role of NO in mechanical effects. The appearance of MCC on TEM showed that after been pressed under 90kPa for 60min, the cellular processes became elongated and voluminous, together with aggrecan mRNA increasing. Under 90kPa for 360min, some of the cells showed distinct sign of apotosis and the aggrecan mRNA decreased. Pressure of 90kPa could cause increase of NOS activity and decrease of PGF(1)alpha composition. Inhibitor experiments indicated that pressure-induced upregulation of aggrecan mRNA and inhibition of PGF(1)alpha synthesis was partly mediated by NO. Continuous pressure could cause changes on the ultrastructure and function of MCC, as well as up-regulation of aggrecan synthesis, increase of NO secretion and decrease of PGF(1)alpha composition. NO was the upstream molecule, which mediated the response of aggrecan and PGF(1)alpha to mechanical pressure.     

胃癌中一氧化氮合酶表达与幽门螺旋杆菌以及胃癌血管生成之间的关系

目的研究诱导型一氧化氮合酶（iNOS）和内皮型一氧化氮合酶（eNOS）在人胃癌中的表达;探讨两者与人胃癌血管生成和临床病理特征的关系;研究幽门螺旋杆菌（H.pylori）在肿瘤发生机制中对NO的影响。方法应用免疫组织化学方法检测59例胃癌及癌旁组织iNOS和eNOS的表达,快速尿素酶及组织学检测幽门螺旋杆菌。第VⅢ因子相关抗原（F-VⅢRAg）血管内皮细胞特异性染色计数肿瘤微血管密度（MVD）。结果①胃癌组织中iNOS阳性率明显高于癌旁组织,二者之间差异有显著性（P〈0.05）,eNOS的阳性率略高于癌旁组织,二者之间差异无显著性（P〉0.05）。②iNOS、eNOS表达与胃癌分化程度无明显相关。③iNOS表达与胃癌的浸润程度及淋巴结转移呈正相关,eNOS表达与胃癌的浸润程度及淋巴结转移无关。④H.pylori感染与iNOS呈正相关,与eNOS无关。⑤iNOS的表达与胃癌MVD呈正相关,eNOS的表达与胃癌MVD无相关性。结论①iNOS在胃癌的发生发展中起重要作用。②H.pylori在致癌机制中可有iNOS的参与。③iNOS对胃癌血管的生成具有促进作用。     

基因治疗原发性高血压的新途径:内皮型一氧化氮合成酶CDNA腺病毒载体在人胎肾293细胞的表达

研究内皮型一氧化氮合成酶（endothelialnitric oxide synthase,eNOS)CDNA 腺病毒载体 PadRSVeNOSCDNA 导入在人胎肾 293细胞 ( )中的表达。方法:大量制备及纯化质粒 eNOSCDNA ,将纯化eNOSCDNA 载体通过脂质体转染 293细胞48h 后,应用化学比色法测定293细胞提取物NOS 活性。结果:被转染 293细胞 NOS活性显著提高于未转染293细胞 NOS 活性犤( 0.62±0.03),(0.093±0.01)kU /L,P <0.001犦。结论:PA dRSVeNOSCDNA 导入人胎肾 293细胞是一种能使 NOS 活性显著升高的方法,为基因治疗原发性高血压及肾脏血管性疾病研究提供一条新的途径。     

Th1/Th2类细胞因子失衡和一氧化氮合酶在内毒素休克性肺损伤中的作用机制

目的　观察内毒素 (LPS)诱导的急性肺损伤 (ALI)大鼠血Th1/Th2类细胞因子浓度以及肺组织一氧化氮合酶 (NOS)活性和NOS基因表达 ,以探讨它们在ALI中的发病机制。方法　采用颈静脉注入LPS复制大鼠急性肺损伤模型 ,用ELISA法检测其外周血单个核细胞产生IFN -γ、IL - 4水平及IFN -γ/IL - 4比值 ;用放射免疫 (RIA)的方法测定LPS注射后大鼠肺组织NOS活性 ;采用逆转录聚合酶链反应 (RT -PCR)检测LPS注射大鼠肺组织内源型一氧化氮合酶 (eNOS)、诱导型一氧化氮合酶 (iNOS)mRNA表达情况。结果　与正常对照组相比 ,急性肺损伤组外周血PBMC产生IFN -γ水平、IFN -γ/IL - 4比值明显升高 (P <0 0 5 ,P <0 0 0 1) ,而IL - 4水平差异无显著性 (P >0 0 5 ) ,同时伴随NOS活性 [( 0 30± 0 0 5 )U/mgpro]明显高于对照组 [( 0 2 4± 0 0 3) ,P <0 0 5 ]。iNOSmRNA表达( 0 70± 0 2 0 )显著高于对照组 (P <0 0 5 ) ,eNOSmRNA表达未见明显变化 (P >0 0 5 )。肺组织病理切片显示大鼠发生ALI。结论　LPS休克肺损伤时 ,体内存在Th1/Th2类细胞因子水平失衡 ,同时伴有NOS活性增高 ,而NOS的主要来源为iNOS基因表达增强     

Molecular signalling of a novel curcumin derivative versus Tadalafil in erectile dysfunction

The efficacy of a novel curcumin derivative (NCD) versus tadalafil in erectile signalling was assessed. Ten control male rats and 50 diabetic male rats were used and divided into the following: diabetic (DM), curcumin (CURC), NCD, tadalafil and NCD combined with tadalafil rat groups. Cavernous tissue gene expression of heme oxygenase‐1 (HO‐1), Nrf2, NF‐B and p38, enzyme activities of heme oxygenase (HO) and nitric oxide synthase (NOS), cGMP and intracavernosal pressure (ICP)/mean arterial pressure (MAP) were assessed. Results showed that 12 weeks after induction of diabetes, erectile dysfunction (ED) was confirmed by the significant decrease in ICP/MAP, a significant decrease in cGMP, NOS, HO enzyme activities, a significant decrease in HO‐1 gene and a significant increase in NF‐?β, p38 genes. Administration of all therapeutic interventions led to a significant increase in ICP/MAP, cGMP levels, a significant increase in HO‐1 and NOS enzymes, a significant increase in HO‐1, and Nrf2 gene expression, and a significant decrease in NF‐?β, p38 gene expression. NCD or its combination with tadalafil showed significant superiority and more prolonged duration of action. In conclusion, a tendency was observed that CURC and NCD have high efficacy and more prolonged duration of action in enhancing erectile function.     

七氟醚预处理对全身缺氧损伤小鼠脑NO含量和NOS活性的影响

目的观察七氟醚(Sev)预处理后全身缺氧损伤小鼠的生存状况,测定脑内一氧化氮(NO)含量及一氧化氮合酶(NOS)活性,探讨七氟醚预处理的保护作用。方法 6~7周龄健康雄性昆明小鼠120只,随机分为3组:Sev 0.5 h组、Sev 24 h组及对照组(P组),每组40只。Sev 0.5 h组吸入1.0%Sev 30 min,在空气中洗脱30 min后放入体积分数5%O2+95%N2环境中持续20 min;Sev 24 h组每天吸入体积分数1.0%Sev30 min,连续2 d,第3天放入体积分数5%O2+95%N2环境中持续20 min;P组不吸入Sev,直接放入体积分数5%O2+95%N2环境中持续20 min。观察小鼠在体积分数5%O2环境中20 min内的生存时间、生存率,测定脑组织NO含量和NOS活性。结果与P组比较,Sev 0.5 h组小鼠生存率提高、生存时间延长(P<0.05),脑组织NO含量降低,NOS活性增高(P<0.05);而Sev 24 h组与P组比较差异无统计学意义(P>0.05)。Sev 0.5 h组与Sev 24 h组比较,生存率提高,生存时间延长(P<0.05),NO含量和NOS活性差异有统计学意义(P<0.05)。结论 Sev预处理能对全身缺氧损伤小鼠产生保护作用,通过即刻效应提高小鼠在低氧环境中的生存率,延长其生存时间,降低脑组织中的NO含量,提高NOS活性,减轻脑组织缺氧损伤。