王琦教授新冠肺炎预防方预防新型冠状病毒肺炎机制的网络药理学探讨＊

目的 借助网络药理学的方法，探究两组王琦教授新冠肺炎预防方（简称预防方）预防新型冠状病毒肺炎（COVID-19）的分子靶点和机制。方法 通过TCMSP数据库检索并筛选其活性成分及其作用靶点，通过Uniprot数据库进行蛋白标准化处理。从Genecards数据库中以“Novel coronavirus pneumonia”为关键词搜索获取COVID-19的靶标，构建相交靶点韦恩图。通过cytoscape3.7.2构建PPI蛋白互作网络，寻找富集数目最多的靶点。通过R语言对预防方治疗COVID-19的靶点进行GO和KEGG富集分析，并绘制气泡图。结果 预防方与新冠肺炎相关靶点涉IL-6、TNF、CXCL8、VEGFA、MMP9；GO功能富集分析分别获得53、57条通路；27、29条KEGG相关信号通路。结论 王琦教授新冠肺炎预防方中的主要活性成分为槲皮素、山奈酚、木犀草素、β-谷甾醇、植物甾醇等，可能通过作用于IL-6、TNF、CXCL8、VEGFA、MMP9、CXCL8、IL10、CCL2、IL1B等靶点和介导TNF信号通路、T细胞受体信号通路、Toll样受体信号通路等发挥作用，从而促进免疫反应、炎症反应及细菌防御反应，预防COVID-19。     

Protective effects of quercetin on uterine receptivity markers and blastocyst implantation rate in diabetic pregnant mice

ObjectiveDiabetic women have different reproductive problems. In pregnant diabetic women, high rates of perinatal mortality, spontaneous abortion and congenital anomalies are observed. We hypothesized that quercetin, as an antidiabetic and phytoestrogen, might have protective effects on the embryo implantation in pregnant diabetic mice. We investigated the ameliorative effects of quercetin on the levels of serum estrogen and progesterone, rate of blastocyst implantation, and uterine receptivity markers in diabetic mice.Materials and methodsDiabetic and healthy female mice were treated with quercetin (30 mg/kg/day) four weeks before pregnancy. Plasma sex-steroid levels were determined on day 4 of pregnancy. Also, uteri were harvested for investigation of protein and mRNA expression changes. In another set of our study, implantation rate was determined on day 5 of pregnancy.ResultsOur results indicated that quercetin was significantly reduced blood glucose levels in diabetic mice. The number of implantation sites as well as serum estradiol level was reduced in diabetic mice, and then treatment with quercetin significantly increased both. On the other hand, insulin like growth factor1, integrin αvβ3, and cyclooxygenase2 mRNA expression in the uterus of diabetic mice were significantly reduced, and quercetin treatment augmented the expression level of these genes. Besides, the level of inactive β-catenin protein level in the uterus of diabetic mice was higher than normal group; treatment with quercetin reduced the level of inactive β-catenin protein as compared to diabetic mice.ConclusionWe conclude that administration of quercetin before pregnancy can probably alleviate reproductive problems in diabetic women likely via its estrogenic and antihyperglycemic effects.     

Bioassay-guided isolation of anti-hepatitis B virus flavonoid myricetin-3-O-rhamnoside along with quercetin from Guiera senegalensis leaves

Recently, we have shown in vitro anti-hepatitis B virus (HBV) activity of G. senegalensis J.F. Gmel leaves, and Identified quercetin and other flavonoids by HPTLC. Here we report bioassay-directed fractionation of G. senegalensis leaves using column chromatography and isolation of two flavonoinds from the n-butanol fraction, their structure determination (¹H NMR, ¹³C NMR and 2D-NMR) and assessment of antiviral activities (HBsAg and HBeAg assay) in HBV-reporter HepG2.2.2.15 cells. Further molecular docking was performed against HBV polymerase (Pol/RT) and capsid (Core) proteins as well as host-receptor sodium taurocholate co-transporting polypeptide (NTCP). The two isolated bioactive compounds were identified as quercetin and myricetin-3-O-rhamnoside. Quercetin significantly inhibited synthesis of HBsAg and HBeAg by about 60% and 62%, respectively as compared to myricetin-3-O-rhamnoside by 44% and 35%, respectively. Molecular docking of the two anti-HBV flavonoids revealed their higher binding affinities towards Pol/RT than Core and NTCP. In conclusion, this is the first report on anti-HBV active myricetin-3-O-rhamnoside along with quercetin isolated from G. senegalensis leaves. Their possible mode of anti-HBV activities are suggested through binding with viral Pol/RT and Core as well as host NTCP proteins.     

Quercetin abrogates bisphenol A induced altered neurobehavioral response and oxidative stress in zebrafish by modulating brain antioxidant defence system

Bisphenol A (BPA), a well-recognized anthropogenic xenoestrogen, has been identified as a causative agent responsible for inducing carcinogenicity, cognitive impairment, neurotoxicity, oxidative stress, etc. However, BPA-induced neurotoxicity and its possible amelioration through natural compound intervention remain elusive. The current study was performed to elucidate the neurotoxic potential of BPA in zebrafish (Danio rerio) by waterborne exposure and its possible amelioration by quercetin co-supplementation. Protective effect of quercetin against BPA-induced altered neurobehavioral response, oxidative stress and neuromorphological changes were evaluated in zebrafish brain. The present findings reveal that BPA-induced altered neurobehavioral response was ameliorated by quercetin. Biochemical studies advocate the potential therapeutic efficacy of quercetin against BPA-induced oxidative stress in zebrafish brain. Quercetin also shows neuroprotection against BPA-induced augmented neuronal pyknosis in periventricular grey zone (PGZ) of zebrafish brain. These basic findings indicate that quercetin may act as an effective intervention against BPA-induced neurotoxicity in zebrafish through down-regulation of oxidative stress.     

槲皮素对糖尿病视网膜病变小鼠氧化应激损伤和炎症反应的影响

目的：探讨槲皮素对糖尿病视网膜病变（DR）的保护作用及其抗氧化和抗炎机制。方法： 腹腔注射链脲佐菌素（STZ）建立小鼠糖尿病模型,随机分为正常对照组、造模组、槲皮素治疗组（n = 10）。治疗后定期监测小鼠精神状态、血糖、体重;检测小鼠视网膜结构变化、超氧化物歧化酶（SOD）活性、8-羟基脱氧鸟苷（8-OHdG）、丙二醛（MDA）、肿瘤坏死因子-α（TNF-α）、白介素-6（IL-6）、白介素-β（IL-β）的mRNA等水平。结果：与造模组相比,槲皮素降低小鼠血糖（P ＜ 0.05）,小鼠体重无明显差异（P ＞ 0.05）,视网膜细胞排列整齐,厚度增加;视网膜组织中8-OHdG、MDA、TNF-α mRNA、IL-6 mRNA、IL-β mRNA、Bax及p-p38 MAPK蛋白表达指数显著降低（P ＜ 0.05）,T-SOD活力和Bcl-2蛋白表达水平明显升高（P ＜ 0.05）,但各组p38 MAPK蛋白表达水平无明显差异（P ＞ 0.05）。结论： 槲皮素改善2型糖尿病小鼠视网膜组织氧化应激损伤和炎症反应,其机制可能与p38 MAPK信号通路有关。     

槲皮素对K562细胞生长及血管内皮生长因子分泌的影响

 目的 探讨槲皮素对K562细胞的形态、血管内皮生长因子（VEGF）的表达影响。方法 以K562细胞为研究对象,细胞形态学观察采用Wright染色法;AnnecxinⅤ标记检测细胞凋亡率;VEGF表达采用ELISA法。结果 经槲皮素处理后,细胞形态学上出现凋亡特征性改变;槲皮素处理后K562细胞凋亡率明显增加;槲皮素处理后K562细胞显著降低VEGF的表达。结论 槲皮素在体外能抑制白血病细胞K562生长并诱导其凋亡;槲皮素可抑制白血病细胞分泌VEGF。     

槲皮素对人结肠癌HT-29细胞凋亡的影响

目的 研究槲皮素对人结肠癌HT-29细胞凋亡的影响,并探讨其对肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor factor 6,Traf6)/转化生长因子β激活酶1(transforming growth factor-βactivated kinase-1,TAK1)信号通路的调控作用.方法 将细胞按随机数字表法分为对照组、16μg/ml槲皮素组、Traf6抑制剂组、16μg/ml槲皮素+Traf6抑制剂组,对照组常规培养,其余各组加入16μg/ml槲皮素和/或Traf6抑制剂20 nmol/L进行干预.培养24 h,采用流式细胞仪检测各组细胞凋亡率,采用免疫印迹法检测Traf6/TAK1信号通路蛋白及caspase-3、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)及Bcl-2表达,采用RT-PCR技术检测Traf6 mRNA表达.结果 与16μg/ml槲皮素组比较,16μg/ml槲皮素+Traf6抑制剂组Traf6[(0.59±0.03)比(0.96±0.04)]、p-TAK1[(0.43±0.02)比(0.72±0.04)]、caspase-3[(0.59±0.03)比(0.70±0.04)]、Bax[(0.48±0.03)比(0.67±0.04)]表达降低,Bcl-2[(0.54±0.03)比(0.44±0.02)]表达升高(P<0.05),Traf6 mRNA[(0.38±0.24)比(0.82±0.08)]表达降低(P<0.05).结论 槲皮素可诱导人结肠癌HT-29细胞凋亡,其作用机制可能与激活Traf6/TAK1信号通路有关.     

HPLC法同时测定楚雄臭菜中槲皮素和山奈酚的含量

目的建立HPLC法测定楚雄臭菜中的槲皮素和山奈酚含量的方法。方法采用色谱柱:InertSustain C18(250 mm×4.6 mm,5μm);以乙腈(A)-0.3%磷酸水溶液(B)为流动相进行梯度洗脱;流速:0.8 m L/min;检测波长:360 nm;柱温:30℃。结果槲皮素的进样量在0.034~0.272μg范围内(r=0.999 9)、山奈酚的进样量在0.039~0.317μg范围内(r=0.999 8)线性关系良好,槲皮素、山奈酚的平均回收率分别为99.33%(RSD=1.33%,n=6)、99.48%(RSD=1.85%,n=6)。结论本方法操作简便、快捷,稳定性高,重现性好,为楚雄臭菜药材的质量评价提供依据。     

槲皮素对人肝癌高转移细胞基质粘附能力的影响

目的 研究槲皮素对肝癌高转移细胞基质粘附能力的影响及其机制.方法 研究对象为人高转移肝细胞癌细胞HCCLM6,用细胞基质粘附实验检测槲皮素对细胞外基质粘附能力的影响,免疫荧光和Western blot 检测细胞内E-Cadherin蛋白表达.结果 槲皮素能抑制肝癌细胞对Marigel胶的粘附,免疫荧光和Western blot检测均发现槲皮素处理组肝癌细胞E-Cadherin蛋白表达较对照组增加(P＜0.05).结论 槲皮素能抑制肝癌高转移细胞的基质粘附能力,其机制之一可能在于槲皮素能上调肝癌细胞E-Cadherin蛋白表达.