Synergistic inhibitory effect of wogonin and low-dose paclitaxel on gastric cancer cells and tumor xenografts

Objective：To investigate the synergistic inhibitory effects of wogonin （WOG） and chemotherapeutic drugs on growth of gastric cancer cells and tumor xenografts.Methods：The IC50 values of WOG,cisplatin （CDDP） and paclitaxel （PTX） in four gastric cancer cell lines were determined by MTS assay.Hoechst staining and the median effect method of Chou-Talalay were used to assess the apoptosis of cells and the interaction of two drugs,respectively.BGC-823-derived xenografts in nude mice were established to investigate the effects of WOG combined with chemotherapeutic drugs in vivo.Results：WOG,CDDP and PTX inhibited the growth of BGC-823,MGC-803,MKN-45 and HGC-27 gastric cancer cells in a dose-dependent manner.WOG combined with CDDP or PTX synergistically inhibited the growth of all gastric cancer cell lines in vitro.In BGC-823,MGC-803,HGC-27 and MKN-45 cell lines,synergisms between WOG and PTX were shown when the fraction affected （Fa） values were ＜0.45,＜0.90,＜0.85 and ＜0.60.While WOG and CDDP had a synergistic inhibitory.effect when the Fa values were ＞0,＞0,＞0.65 and ＞0.10.From the results of in vivo experiments using tumor xenografts,WOG and low-dose PTX showed better efficacy than either drug alone.The inhibitory percentages of tumor weight were 61.58％,20.29％,and 22.28％ for the combination,WOG-alone,and low-dose PTX-alone groups,respectively.Notably,WOG combined with CDDP displayed very high toxicity.Conclusions：A synergistic inhibitory effect on growth was observed when WOG was combined with low-dose PTX in gastric cancer cells and tumor xenografts.These findings provide evidence for the design of a clinical trial to test the combination of WOG with low-dose PTX in human gastric cancer.

EFFECTS OF BAICALEIN, WOGONIN, BAICALIN AND Na2MoO_4 ON N-NITROSATION REACTION

The in vitro effects of baicalein,wogonin,baicalin and Na_2MoO_4 on N-nitrosation reaction have been studied. The N-nitrosation reaction has been determined by the amount of dimethylnitrosamine(DMN) and diethylnitrosamine(DEN) produced using the UV-photclysis spectrophotometric and pyrolysis gas chromatography test. Baicalein,wogonin and Na_2MoO_4 showed varing extents of inhibition of the formation of DMN and DEN. Baicalin promoted the formation of DMN and DEN under the condition of simulating gastric Juices. It is also found that E. coli showed a remarkable promoting effect on the formation of DMN and DEN,but this promoting effect could be blocked to some extent by baicalein,wogonin,baicalin and Na_2MoO_4. Besides, Na_2MoO_4 and wogonin have shown synergic effect on the blocking of N-nitrosation reaction.

Identification of novel targets and mechanisms of wogonin on lung cancer,bladder cancer,and colon cancer

Wogonin(WOG)has been demonstrated to have anti-cancer activity,but the mechanisms remain unclear.In this study,new targets of WOG were predicted for lung cancer,bladder cancer,and colon cancer by using bioinformatics methods.wOG might primarily suppress cancers via regulating arachidonic acid,Ras,MAPK,linoleic acid,PI3K Akt,and folate biosynthesis pathways.3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)assay showed that WOG inhibited the proliferation of A549 cells.Real-time quantitative reverse transcription PCR(RT-qPCR)results indicated that anti-lung cancer effect of WOG was achieved by regulating the expression of 18 target genes,including AKRIBI0,AKRIC3,BDNF,CAVI,CXCL2,CYP2B6,CYP4F3,DAO,EGF,ENO3,IL6,PLA2GlB,PLA2G2F,PLA2G4A,PTGES,SLCOIBI,SLCOIB3,and TFAP2A.The Kaplan-Meier survival curves further confirmed that DAO,PLA2G1B,SLCO1B3 and TFAP2A were essential targets via which WOG affected lung cancer survival.Moreover,BDNF,FGF2,and PTGS1 were predicted to be the targets via which WOG alleviated cancer proliferation and invasion in bladder cancer.As for colon cancer,WOG might induce autophagy and inhibit proliferation by down-regulating NTF4 and TH.The study will provide clue for using wOG as a promising antineoplastic agent in basic and translational research,and bring light to the application of herbs containing WOG as food supplements.

黄芩微乳凝胶对小鼠慢性湿疹的疗效研究

目的:拟在前期黄芩自微乳的研究基础上,制备黄芩微乳凝胶,并考察其对慢性湿疹的治疗效果。方法:采用单因素法和响应面法优选凝胶基质、保湿剂等,确定黄芩微乳凝胶的处方工艺;采用Franz扩散池法评价微乳和微乳凝胶的体外透皮性能;建立鼠耳慢性湿疹模型,测定黄芩微乳凝胶对小鼠耳肿胀度、肿胀抑制率、病理改变以及血清中炎症因子TNF-α、IL-1β和IL-6含量,考察对小鼠慢性湿疹的治疗效果。结果:黄芩微乳凝胶理化性质稳定,微乳凝胶相比于微乳具有更好的透皮性能,微乳凝胶中主要成分黄芩素、汉黄芩素的累积透皮量分别为微乳的1.85倍、2.77倍,且微乳凝胶的稳态流量和渗透系数显著升高,滞后时间明显缩短;药效学研究表明,与模型组相比,黄芩微乳凝胶可显著降低小鼠耳廓肿胀度(P<0.05),血清中炎症因子TNF-α、IL-1β和IL-6含量减少37%以上,相较于传统的以黄芩苷为主的水提物和水提物凝胶对慢性湿疹的治疗作用更好。结论:黄芩微乳凝胶制备工艺可行,透皮性强,对慢性湿疹有显著的治疗作用。

Anti-tumor activity of wogonin, an extract from Scutellaria baicalensis, through regulating different signaling pathways

Wogonin is a plant flavonoid compound extracted from Scutellaria baicalensis(Huang-Qin or Chinese skullcap) and has been studied thoroughly by many researchers till date for its anti-viral, anti-oxidant, anti-cancerous and neuro-protective properties. Numerous experiments conducted in vitro and in vivo have demonstrated wogonin's excellent tumor inhibitory properties. The anticancer mechanism of wogonin has been ascribed to modulation of various cell signaling pathways, including serine-threonine kinase Akt(also known as protein kinase B) and AMP-activated protein kinase(AMPK) pathways, p53-dependent/independent apoptosis, and inhibition of telomerase activity. Furthermore, wogonin also decreases DNA adduct formation with a carcinogenic compound 2-Aminofluorene and inhibits growth of drug resistant malignant cells and their migration and metastasis, without any side effects. Recently, newly synthesized wogonin derivatives have been developed with impressive anti-tumor activity. This review is the succinct appraisal of the pertinent articles on the mechanisms of anti-tumor properties of wogonin. We also summarize the potential of wogonin and its derivatives used alone or as an adjunct therapy for cancer treatment. Furthermore, pharmacokinetics and side effects of wogonin and its analogues have also been discussed.

汉黄芩素通过调控miR-451/PI3K/AKT/RXRA/Bcl-2信号通路改善低氧诱导的肺动脉高压

目的:评价汉黄芩素对低氧诱导的肺动脉高压(HPH)的保护作用及其相关分子机制。方法:在体内采用成年雄性野生型C57BL/6小鼠建立HPH模型,将小鼠随机分为3组:对照组(N)、低氧(H+Saline)组、低氧+汉黄芩素(H+w)组。造模3周后,使用压力传感器系统采集小鼠血流动力学指标;肺小动脉管壁直径/管总直径(WT/TT)、肺小动脉管壁面积/管总面积(WA/TA)来评估肺动脉结构重塑;基于网络药理学方法筛选汉黄芩素作用于HPH潜在靶点及分子信号通路。在体外采用小鼠肺动脉平滑肌细胞(MPASMCs),将细胞分为5组:常氧(N)组、低氧(H+PBS)组、低氧+低剂量汉黄芩素(H+40μmol/L w)组、低氧+高剂量汉黄芩素(H+80μmol/L w)组、低氧+高剂量汉黄芩素+740Y-P(H+80μmol/L w+740Y-P)组。N组在常氧(5%CO_(2),21%O_(2),74%N2,37℃)环境中培养48 h,H+PBS、H+40μmol/L w、H+80μmol/L w、H+80μmol/L w+740Y-P组在低氧(5%CO_(2),3%O_(2),92%N2,37℃)环境中培养48 h。通过CCK-8、Ed U法检测评估细胞增殖能力,Transwell和伤口愈合/划痕实验用于检测评估细胞迁移能力,Western blot检测PI3K/AKT/RXRA/Bcl-2信号通路的主要蛋白(P-PI3K、PI3K、P-AKT、AKT、RXRA、Bcl-2)的表达,并利用PI3K通路激活剂740Y-P进行功能回补实验;RT-q PCR检测汉黄芩素对miR-451表达水平的影响,Western blot检测汉黄芩素对PI3K/AKT/RXRA/Bcl-2通路上游调控因子CAB39和MIF表达的影响。结果:与对照组相比,低氧组MPASMCs的增殖、迁移能力以及小鼠右心室压力(RVSP)显著增加(P<0.05),肺动脉结构发生显著重构,经汉黄芩素干预后MPASMCs的增殖、迁移能力以及小鼠RVSP显著下降(P<0.05),肺动脉结构重塑得到显著改善。与对照组相比,低氧组中PI3K/AKT/RXRA/Bcl-2信号通路相关蛋白p-PI3K、p-AKT、RXRA、Bcl-2表达显著升高;与低氧组相比,汉黄芩素干预组中PI3K/AKT/RXRA/Bcl-2信号通路相关的蛋白表达水平显著下调(P<0.05);功能回补实验证实在缺氧条件下,激活PI3K通路能够显著削弱汉黄芩素对MPASMCs增殖抑制效应。与对照组相比,低氧组中CAB39和MIF表达水平显著上升,miR-451表达水平显著减少(P<0.05);与低氧组相比,汉黄芩素干预组中CAB39和MIF表达水平显著下降,miR-451表达水平显著上升(P<0.05);miR-451 inhibitor干预能够部分逆转汉黄芩素对MPASMCs增殖和迁移能力的抑制效应(P<0.05)。结论:汉黄芩素可能通过上调mi R-451的表达靶向调控CAB39和MIF进而抑制PI3K/AKT/RXRA/Bcl-2信号通路,抑制低氧诱导的MPASMCs的增殖和迁移、改善HPH小鼠右心室压力和肺动脉结构重塑,最终缓解肺动脉高压。

汉黄芩素通过调节Hedgehog-YAP信号通路减轻肝硬化大鼠的肝纤维化

目的:探讨汉黄芩素(Wog)对肝纤维化模型大鼠的影响及其机制。方法:将大鼠分为对照组,模型组,Cym组(Hedgehog抑制剂组),Wog低、中、高剂量组。检测各组大鼠血清肝功能指标丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、谷氨酰转肽酶(GGT)水平,肝纤维化指标透明质酸(HA)、Ⅳ型胶原(CⅣ)、层黏蛋白(LN)、Ⅲ型前胶原(PCⅢ)水平;天狼星红染色观察肝组织胶原纤维;RT-qPCR法检测肝组织Ⅰ型胶原(ColⅠ)、α-肌动蛋白(α-SMA)mRNA水平;检测肝组织羟脯氨酸(Hyp)、超氧化物歧化酶(SOD)、丙二醛(MDA)水平;免疫印迹检测Hedgehog-YAP通路相关蛋白SMO、GLI2、SHH、Yes相关蛋白1(YAP)、p-YAP水平。结果:与对照组相比,模型组血清ALT、AST、GGT、HA、CⅣ、LN、PCⅢ水平和肝组织ColⅠ、α-SMA mRNA、Hyp、MDA、SMO、GLI2、SHH、p-YAP/YAP水平均升高,SOD水平降低;与模型组相比,Cym组和Wog低、中、高剂量组血清ALT、AST、GGT、HA、CⅣ、LN、PCⅢ水平和肝组织ColⅠ、α-SMA mRNA、Hyp、MDA、SMO、GLI2、SHH、p-YAP/YAP水平均降低,SOD水平均升高;其中Cym组与Wog高剂量组相比,上述指标无显著差异。对照组肝组织未见增生的纤维组织,模型组肝组织胶原纤维增生明显,胶原染色面积增大;与模型组相比,Cym组和Wog低、中、高剂量组肝组织胶原纤维增生程度均减轻,胶原染色面积减小;其中Cym组与Wog高剂量组相比,胶原纤维增生程度无显著差异。结论:Wog可减轻肝纤维化模型大鼠肝纤维化程度,对肝纤维化模型大鼠具有保护作用,其机制可能与Wog可抑制Hedgehog-YAP信号通路有关。

汉黄芩素对青光眼小鼠视网膜神经节细胞的保护作用及其机制研究

目的观察汉黄芩素对青光眼小鼠视网膜神经节细胞(RGC)的保护作用,并探究其相关作用机制。方法10只(10眼)C57BL/6J小鼠作为对照组;40只(40眼)DBA/2J小鼠用于制备青光眼模型,并分为模型组和实验1、2、3组,每组各10只。对照组、模型组小鼠灌胃生理盐水,实验1、2、3组小鼠分别灌胃50、100、150 mg·kg^(-1)汉黄芩素。以小鼠右眼为入选眼,比较各组小鼠灌胃前和灌胃2周、4周、8周眼压;苏木素-伊红染色并比较各组小鼠RGC数量、视网膜厚度;比色法检测各组小鼠视网膜组织超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、一氧化氮合酶(NOS)水平;酶联免疫吸附法检测各组小鼠视网膜组织白细胞介素(IL)-6、IL-1β、肿瘤坏死因子-α(TNF-α)水平;Western blot检测各组小鼠视网膜组织核苷酸寡聚化结构域样受体家族3(NLRP3)、半胱天冬酶-1(Caspase-1)、凋亡相关斑点样蛋白(ASC)的蛋白表达水平。结果灌胃前,与对照组相比,模型组和实验1、2、3组小鼠眼压均升高,差异均有统计学意义(均为P<0.05)。灌胃2周、4周、8周:与对照组相比,模型组和实验1、2、3组小鼠眼压均增加,差异均有统计学意义(均为P<0.05);与模型组相比,实验1、2、3组小鼠眼压均降低,差异均有统计学意义(均为P<0.05);与实验1组相比,实验2、3组小鼠眼压均降低,差异均有统计学意义(均为P<0.05);与实验2组相比,实验3组小鼠眼压均降低,差异均有统计学意义(均为P<0.05)。与对照组相比,模型组和实验1、2、3组小鼠RGC数量及视网膜厚度、SOD水平、GSH-Px水平均降低,NOS、IL-6、IL-1β、TNF-α水平以及NLRP3蛋白、Caspase-1蛋白、ASC蛋白表达水平均增加,差异均有统计学意义(均为P<0.05);与模型组相比,实验1、2、3组小鼠RGC数量及视网膜厚度、SOD水平、GSH-Px水平均增加,NOS、IL-6、IL-1β、TNF-α水平以及NLRP3蛋白、Caspase-1蛋白、ASC蛋白表达水平均降低,差异均有统计学意义(均为P<0.05)。与实验1组相比,实验2、3组小鼠RGC数量及视网膜厚度、SOD水平、GSH-Px水平均增加,NOS、IL-6、IL-1β、TNF-α水平以及NLRP3蛋白、Caspase-1蛋白、ASC蛋白表达水平均降低,差异均有统计学意义(均为P<0.05);与实验2组相比,实验3组小鼠RGC数量及视网膜厚度、SOD水平、GSH-Px水平均增加,NOS、IL-6、IL-1β、TNF-α水平以及NLRP3蛋白、Caspase-1蛋白、ASC蛋白表达水平均降低,差异均有统计学意义(均为P<0.05)。结论汉黄芩素可改善青光眼小鼠视网膜厚度,缓解视网膜组织氧化损伤和炎症反应,其作用机制可能与抑制NLRP3炎性小体激活有关。

汉黄芩素促进自身免疫性肝炎模型大鼠Th17/Treg细胞平衡

目的探讨汉黄芩素(WG)对自身免疫性肝炎(AIH)大鼠Th17/Treg细胞平衡的影响。方法随机选出10只大鼠作为对照组,其余大鼠通过尾静脉注射伴刀豆球蛋白A(ConA,12.5 mg/kg)溶液构建AIH模型大鼠。将造模成功大鼠随机平分为AIH组、L-WG组(10 mg/kg)、M-WG组(20 mg/kg)、H-WG组(30 mg/kg)、H-WG+VPA组[30 mg/kg WG+300 mg/kg Notch信号通路激活剂丙戊酸(VPA)],每组10只,每天给药1次,持续10 d。ELISA检测血清中炎性因子及肝功能指标;HE染色观察肝脏组织病理形态;流式细胞测量术检测脾脏Th17/Treg细胞水平;Western blot检测脾脏类视黄酸相关孤儿受体γt(RORγt)、叉头框蛋白P3(Foxp3)以及肝组织Notch信号通路蛋白质的表达。结果对照组大鼠肝组织结构正常,染色清晰;AIH组大鼠肝组织细胞出现水肿,细胞体积增大导致肝窦受压变窄,出现大量炎性细胞浸润以及少量坏死现象。AIH组较对照组丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)活性、白介素(IL)-17和IL-23含量、Th17细胞水平、Th17/Treg、RORγt、Notch、hes家族bHLH转录因子1(HES1)、含YRPW基序的bHLH转录因子hes相关家族1(HEY1)蛋白表达均显著上升(P<0.05),IL-10和TNF-β含量、Treg细胞水平、Foxp3蛋白水平均显著下降(P<0.05);与AIH组相比,L-WG组、M-WG组、H-WG组肝损伤情况得到改善,ALT、AST活性、IL-17和IL-23含量、Th17细胞水平、Th17/Treg、RORγt、Notch、HES1、HEY1蛋白表达均显著降低(P<0.05),IL-10和TNF-β含量、Treg细胞水平、Foxp3蛋白水平均显著升高(P<0.05);VPA逆转了H-WG对AIH大鼠的改善效果。结论WG可能通过下调Notch信号通路促进AIH大鼠Th17/Treg细胞平衡。

Apoptotic Mechanism of Human Leukemia K562/A02 Cells Induced by Magnetic Ferroferric Oxide Nanoparticles Loaded with Wogonin

Background： Traditional Chinese medicine wogonin plays an important role in the treatment of leukemia. Recently, the application of drug-coated magnetic nanoparticles （MNPs） to increase water solubility of the drug and to enhance its chemotherapeutic efficiency has attracted much attention. Drugs coated with MNPs are becoming a promising way for better leukemia treatment. This study aimed to assess the possible molecular mechanisms of wogonin-coated MNP-Fe3O4 （Wog-MNPs-Fe3O4） as an antileukemia agent. Methods： After incubated for 48 h, the antiproliferative effects of MNPs, wogonin, or Wog-MNPs-Fe3O4on K562/A02 cells were determined by methyl thiazolyl tetrazolium （MTT） assay. The apoptotic rates of K562/A02 cells treated with either wogonin or Wog-MNPs-Fe3O4were determined by flow cytometer （FCM） assay. The cell cycle arrest in K562/A02 cells was determined by FCM assay. The elementary molecular mechanisms of these phenomena were explored by Western blot and reverse transcriptase polymerase chain reaction （RT-PCR）. Results： With cell viabilities ranging from 98.76% to 101.43%, MNP-Fe3O4was nontoxic to the cell line. Meanwhile, the wogonin and Wog-MNPs-Fe3O4had little effects on normal human embryonic lung fibroblast cells. The cell viabilities of the Wog-MNPs-Fe3O4group （28.64–68.36%） were significantly lower than those of the wogonin group （35.53–97.28%） in a dose-dependent manner in 48 h （P 〈 0.001）. The apoptotic rate of K562/A02 cells was significantly improved in 50 μmol/L Wog-MNPs-Fe3O4group （34.28%） compared with that in 50 μmol/L wogonin group （23.46%; P〈 0.001）. Compared with those of the 25 and 50 μmol/L wogonin groups, the ratios of G0/G1-phase K562/A02 cells were significantly higher in the 25 and 50 μmol/L Wog-MNPs-Fe3O4groups （all P〈 0.001）. The mRNA and protein expression levels of the p21 and p27 in the K562/A02 cells were also significantly higher in the Wog-MNPs-Fe3O4group compared with those of the wogonin group （all P〈 0.001）. Conclusions： This study demonstrated that MNPs were the effective drug delivery vehicles to deliver wogonin to the leukemia cells. Through increasing cells arrested at G0/G1-phase and inducing apoptosis of K562/A02 cells, MNPs could enhance the therapeutic effects of wogonin on leukemia cells. These findings indicated that MNPs loaded with wogonin could provide a promising way for better leukemia treatment.

Study on Plasma Concentration and Bioavailability of Wogonin in Beagle's Dogs

Objective To develop an LC-MS/MS method for determining the concentration of wogonin in dog plasma and investigate the pharmacokinetics and bioavailability by different administrations of wogonin in Beagle’s dogs.Methods LC-MS/MS was employed in determining the concentration of wogonin with the selected ion monitoring model after liquid-liquid extraction with ethyl acetate of dog plasma samples.The lower limit of quantification was 0.105 μg/L.Target ions were at m/z 285.0→270.0 for wogonin and 373.3→305.3 for finasteride.In a randomized,self-control,and cross-over study,six male Beagle’s dogs were treated with different administration methods in three test periods.Pharmacokinetic parameters were calculated with DAS software(Ver.2.0).Results The calibration curve was linear in the range of 0.105-107.36 μg/L for wogonin in dog plasma samples.The main pharmacokinetic parameters of ig administration(native drug of 15 mg/kg and solution preparation of 5 mg/kg) and iv route were as follows:Cmax(2.5 ± 1.1),(7.9 ± 3.3),and(6838.7 ± 1322.1) μg/L,tmax(0.7 ± 0.3) and(0.3 ± 0.2) h for the both former,AUC0-t(7.1 ± 2.0),(21.0 ± 3.2),and(629.7 ± 111.8) μg.h/L.The absolute bioavailability of native and solution of wogonin were(0.59 ± 0.35)% and(3.65 ± 2.00)%,respectively.Conclusion The validated method is convenient,sensitive,and specific,and the improvement of wogonin solubility could remarkably increase the absolute bioavailability.

汉黄芩素调节AMPK/SIRT1信号通路对骨质疏松大鼠骨折愈合的影响

目的 探究汉黄芩素(WOG)调节腺苷酸活化蛋白激酶(AMPK)/沉默信息调节因子1(SIRT1)信号通路对骨质疏松(osteoporosis,OP)大鼠骨折愈合的影响。方法 将60只SPF级SD雌性大鼠随机分为假手术组(Sham组)、骨质疏松性骨折(OPF)模型组(Model组)、低剂量WOG组(WOG-L组,50mg/kg)、高剂量WOG组(WOG-H组,100 mg/kg)和高剂量WOG+AMPK抑制剂Compound C组(WOG-H+CC组,100 mg/kg WOG+0.2 mg/kg Compound C),每组12只。Model组、WOG-L组、WOG-H组、WOG-H+CC组采用双侧卵巢切除术与右侧股骨干骨折髓内固定术构建骨质疏松性骨折(OPF)大鼠模型。放射性检查评估骨折愈合情况。采用双能X线骨密度扫描仪检测大鼠股骨痂骨密度(BMD)。通过三点弯曲实验和压缩实验对大鼠股骨骨生物力学进行检测。比色法检测大鼠血清骨钙素(OC)、I型前胶原羧基端肽(PICP)水平。HE染色观察骨折处骨痂的病理学变化。Western blot法检测股骨组织中AMPK/SIRT1信号通路相关蛋白表达。结果 与Sham组相比,Model组大鼠骨折愈合评分、股骨上、下端骨痂BMD、骨痂最大位移、最大应力和最大负荷、血清OC、PICP水平、股骨组织中AMPK磷酸化水平、SIRT1蛋白表达显著降低(P <0.05),骨折端未见骨痂生长,骨折线清晰,骨小梁较细,数量减少,间隙增大,结构紊乱疏松。与Model组相比,WOG-H组大鼠骨折愈合评分、股骨上、下端骨痂BMD、骨痂最大位移、最大应力和最大负荷、血清OC、PICP水平、股骨组织中AMPK磷酸化水平、SIRT1蛋白表达显著升高(P <0.05),有连续性骨痂包绕贯穿于骨折端,髓腔再通,骨折线模糊不清,骨小梁形态较完整。Compound C减弱了WOG对OPF大鼠骨折愈合的促进作用。结论 WOG可能通过激活AMPK/SIRT1信号通路促进OPF大鼠的骨折愈合。

基于PI3K/AKT信号通路探讨汉黄芩素对宫颈癌HeLa细胞移植瘤抑制作用影响

目的探究汉黄芩素对宫颈癌HeLa细胞移植瘤的抑制作用及机制。方法随机选取2018年1月—2020年12月常熟市中医院的50只Balb/c小鼠进行宫颈癌荷瘤模型造模,随机分为5组:模型对照组、阳性对照组、小剂量组、中剂量组及大剂量组。模型对照组不做特殊处理,阳性对照组腹腔注射25 mg/kg环磷酰胺,小剂量组、中剂量组、大剂量组小鼠灌胃25 mg/kg、50 mg/kg、100 mg/kg汉黄芩素。处死小鼠取出瘤体,比较各组肿瘤质量、体积,计算肿瘤生长抑制率;取出胸腺、脾脏并计算相关脏体指数。将小鼠肿瘤分为两份,一份采用伊红-美兰(HE)染色进行组织学观察;另一份采用免疫印迹法检测各组小鼠PI3K、AKT、p-AKT表达情况。结果阳性对照组的肿瘤生长抑制率为69.63%,大剂量组、中剂量组、小剂量组的肿瘤生长抑制率分别为77.78%、54.81%和31.85%均高于模型对照组,差异有统计学意义(P<0.05)。大剂量组肿瘤生长抑制率为77.78%高于阳性对照组,差异有统计学意义(P<0.05)。免疫印迹法结果显示,与模型对照组比较,大剂量组、中剂量组与小剂量组PI3K、AKT及p-AKT蛋白表达均显著降低,差异有统计学意义(P<0.05);高剂量组与阳性对照组PI3K、AKT和p-AKT表达量接近,差异有统计学意义(P<0.05)。结论汉黄芩素对宫颈癌Hela细胞移植瘤生长具有明显抑制作用,其作用机制可能通过抑制PI3K/AKT通路与诱导细胞凋亡有关。

活血法灌肠治疗溃疡性结肠炎的文献计量学研究及生信分析

目的:整合文献计量学方法探究活血法治疗溃疡性结肠炎的研究脉络、热点和发展趋势,基于网络药理学及分子对接技术共同探究了在活血法治则指导下加减少腹逐瘀汤治疗溃疡性结肠炎的潜在通路与物质基础。方法:检索中国知网(CNKI)数据库中从2002年1月至2022年12月基于活血治疗溃疡性结肠炎的相关文献,通过Citespace软件生成相关文献发文量、作者、研究机构与单位及关键词等的共现图谱,研究的热点,展开分析。以口服生物利用度(OB)≥30%与类药性(DL)≥0.18为筛选条件,在中药系统药理学平台(TCMSP)数据库获取加减少腹逐瘀汤主要活性成分及关键靶点,利用GeneCards、OMIM、PharmGkb、TDD和Drug Bank数据库获得溃疡性结肠炎主要靶点;绘制Venn图,构建主要成分–关键靶点网络图,绘制蛋白互作(PPI)网络;进行基因本体(GO)功能及京都基因与基因组百科全书(KEGG)通路富集分析,最后以分子对接来验证所得靶点。结果:研究纳入文献409篇,涉及主要作者268位,发文量前5名的作者为:张涛、王新月、于玫、安贺军、沈静,发文量居前5名的单位为:北京中医药大学、北京中医药大学东直门医院、成都中医药大学、广西中医药大学附属瑞康医院、南京中医药大学。其中105个关键词被纳入,高频词(≥7)合计8个,具有研究意义的聚类合计9个,近几年的热点主要聚集于分期论治、中医及中药灌肠的研究。通过筛选得到药物靶点3562个,基因靶点2718个,核心基因13个;加减少腹逐瘀汤治疗溃疡性结肠炎的活性成分是槲皮素(quercetin)、山柰酚(Kaempferol)、汉黄芩素(wogonin)等。GO功能富集分析得到生物过程(BP)2193个、细胞组成(CC)65个、分子功能(MF)165个;KEGG通路富集分析获得164条通路。主要活性成分与核心靶点均具有一定的结合活性,其中槲皮素与PTGS2靶点对接活性最好。结论:以可视化图谱的方式分析,基于活血治疗溃疡性结肠炎的发展现状及研究热点,探究中医治法的优势特色,并通过实际工作中以活血为立法作为出发点的加减少腹逐瘀汤进行生信分析,发现其可通过多成分、多靶点和多通路等协同发挥其治疗溃疡性结肠炎的重要作用。

基于代谢组学对延安五倍子蜂蜜特征物筛选的研究

陕西延安地区盛产高质量五倍子蜂蜜,然而常有低价多花蜜冒充五倍子蜂蜜的现象发生,严重损害蜂农和消费者的利益。本研究以延安地区五倍子蜂蜜和多花蜜为主要研究对象,系统分析了两类蜂蜜中的酚类化合物,并结合多元统计分析,筛选出次黄岑素可作为这两类蜂蜜的差异化合物。进一步通过建立UHPLC-MS/MS方法对蜂蜜中的次黄岑素进行准确定量,其在五倍子蜂蜜中的含量为30~100μg/kg,而在百花蜜中的含量低于5μg/kg。本研究为延安地区五倍子蜂蜜的质量控制提供了一定的参考依据。

HPLC法同时测定五虎汤加味中6种成分

目的 建立HPLC法同时测定五虎汤加味中黄芩苷、汉黄芩苷、黄芩素、6-姜辣素、细辛脂素、甘草酸的含量。方法 该药物甲醇提取液的分析采用Ultimate XB-C_(18)色谱柱(4.6 mm×250 mm, 5μm);流动相甲醇-水(含0.1%甲酸),梯度洗脱;体积流量1.0 mL/min;柱温30℃;检测波长250、275、280、285 nm。结果 6种成分在各自范围内线性关系良好(r≥0.999 9),平均加样回收率94.36%~99.98%,RSD 0.16%~1.72%。结论 该方法精密稳定,重复性好,可用于五虎汤加味的质量控制。

基于OATP1B3转运体活性与表达研究黄芩-黄连药对中黄连对黄芩黄酮肝脏分布的影响及其机制

目的探究在连续给药过程中,黄连对黄芩黄酮在大鼠肝组织中分布的影响机制。方法大鼠灌胃给药黄芩、黄连-黄芩药对提取物14 d后,LC-MS/MS分析肝脏中黄芩苷、黄芩素、汉黄芩苷、汉黄芩素的含量;采用稳定转染OATP1B3的HEK293细胞,以黄芩苷和汉黄芩苷为底物,小檗碱作为抑制剂,进行药物转运实验;大鼠灌胃给药黄芩、黄连-黄芩药对提取物14 d后,取肝脏进行Western blot实验,验证Oatp4的蛋白表达水平。结果连续给药后,在肝脏中,黄连仅降低汉黄芩素的含量。小檗碱可抑制OATP1B3对黄芩苷和汉黄芩苷的转运功能;黄连可上调Oatp4的蛋白表达水平。结论在连续给药过程中,黄连可通过影响OATP1B3转运体活性和蛋白水平共同影响黄芩黄酮在肝脏中的转运过程。

基于网络药理学研究汉黄芩素治疗糖尿病视网膜病变的机制

目的基于网络药理学筛选汉黄芩素治疗糖尿病视网膜病变(diabetic retinopathy,DR)的特征靶点和分子通路,并通过细胞实验对关键靶点进行初步验证,阐述汉黄芩素治疗DR的作用机制。方法利用中药系统药理学数据库与分析平台(Traditional Chinese Medicine Systems Pharma-cology Database and Analysis Platform,TCMSP)筛选出汉黄芩素的作用靶点,利用比较毒理学数据库(Comparative Toxicogenomics Database,CTD)、人类基因功能和网络分析数据库(Human Gene Function and Network Analysis Database,GenCLiP)、DisGeNET和GeneCards数据库筛选出DR的相关作用靶点,利用STRING数据库及Cytoscape 3.6.1软件构建蛋白相互作用网络(Protein-Pro-tein-Interaction-Network,PPI),利用注释可视化和集成发现数据库(Database for Annotation Visual-ization and Integrated Discovery,DAVID)对靶点进行基因本体(Gene Ontology,GO)注释和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)信号通路富集分析。最后通过汉黄芩素干预高浓度D-葡萄糖诱导ARPE-19细胞病变模型实验初步验证相关通路中的核心靶点,在光镜下观察细胞形态,用细胞增殖及毒性检测试剂盒(cell counting Kit-8,CCK8)检测细胞存活率,采用实时荧光定量聚合酶链反应(real time quantitative polymerase chain reaction,RT-qPCR)法分析靶点基因的表达量。结果筛选出汉黄芩素的潜在靶点204个,与DR相关的靶点313个,汉黄芩素靶点与DR靶点交集后获得30个核心靶点。GO富集分析显示,汉黄芩素主要涉及渗透胁迫响应、膜筏和胰岛素受体底物结合等生物过程。KEGG靶点分析显示,糖尿病并发症的晚期糖基化终末产物-晚期糖基化终末产物受体(advanced glycation end-advanced glycation end receptor,AGE-RAGE)、内分泌抵抗、表皮生长因子受体(epidermal growth factor receptor,EGFR)酪氨酸激酶抑制剂抵抗和白细胞介素-17(interleukin-17,IL-17)等信号通路基因富集较多。最后,通过体外ARPE-19细胞实验成功验证糖尿病并发症的AGE-RAGE信号通路中蛋白激酶B(protein kinase B,AKT1)、B淋巴细胞瘤-2基因(B-cell lymphoma 2,BCL2)和胱天蛋白酶3(caspase-3,CASP3)是重要靶点,提示汉黄芩素对DR有显著改善作用。结论利用网络药理学揭示了汉黄芩素通过多靶点、多通路的形式治疗DR疾病,通过体外细胞实验成功验证了AGE-RAGE信号通路中的核心靶点,为进一步阐释汉黄芩素治疗DR的作用机制提供了科学依据。

汉黄芩素对慢性阻塞性肺疾病模型大鼠气道炎症的影响及机制

目的研究汉黄芩素(Wog)对慢性阻塞性肺疾病(COPD)模型大鼠气道炎症的影响及可能机制。方法将84只大鼠按随机数字表法分为对照组,模型组,Wog低、高剂量组(灌胃给药,50、100 mg/kg),氨茶碱组(阳性对照,灌胃给药,2.3 mg/kg),重组大鼠受体相互作用蛋白激酶1[rRIPK1,受体相互作用蛋白激酶1(RIPK1)激活剂]组(尾静脉注射给药,8μg/kg),Wog高剂量+rRIPK1组(灌胃100 mg/kg Wog并尾静脉注射8μg/kg rRIPK1),每组12只。除对照组外,其余各组大鼠均利用烟熏联合气管注射脂多糖的方法构建COPD模型。建模24 h后,进行给药处理;每天给药1次,持续4周。末次给药后,测定各组大鼠的吸气峰流量(PIF)、呼气峰流量(PEF)、每分钟通气量(MV)和第1秒用力呼气容积(FEV_(1))/用力肺活量(FVC)比值,测定大鼠血清中白细胞介素1β(IL-1β)、IL-6、肿瘤坏死因子α(TNF-α)水平,观察大鼠肺组织病理形态学变化,测定大鼠肺上皮细胞凋亡率,并测定大鼠肺组织中RIPK1、RIPK3、混合系激酶区域样蛋白(MLKL)mRNA和RIPK1、RIPK3、磷酸化MLKL(p-MLKL)蛋白表达水平。结果与对照组比较,模型组大鼠PIF、PEF、MV、FEV_(1)/FVC比值显著降低(P<0.05),IL-1β、IL-6、TNF-α水平显著升高(P<0.05),肺组织有大量炎症细胞浸润、支气管壁增厚,肺上皮细胞凋亡率及RIPK1、RIPK3、MLKL mRNA和RIPK1、RIPK3、p-MLKL蛋白表达水平也显著升高(P<0.05)。与模型组比较,Wog低、高剂量组大鼠上述指标均显著改善(P<0.05),病理损伤明显减轻;而rRIPK1组大鼠对应指标却显著恶化(P<0.05),病理损伤也进一步加重;并且,rRIPK1可明显减弱高剂量Wog对COPD大鼠气道炎症和RIPK1/RIPK3/MLKL通路的抑制作用(P<0.05)。结论Wog可能通过抑制RIPK1/RIPK3/MLKL信号通路改善COPD大鼠的气道炎症。

汉黄芩素抗非小细胞肺癌细胞的作用研究

目的:探讨汉黄芩素对H1299与A549非小细胞肺癌细胞的作用,以期为汉黄芩素抗肺癌作用研究提供理论依据。方法:采用CCK-8法检测汉黄芩素对H1299和A549细胞活力的影响,确定汉黄芩素后续实验给药剂量;采用平板集落形成实验研究汉黄芩素对H1299和A549细胞增殖的抑制作用;细胞划痕实验考察汉黄芩素对H1299和A549细胞迁移能力的影响;流式细胞术检测汉黄芩素对H1299和A549细胞周期及凋亡的影响。结果:汉黄芩素可抑制H1299和A549细胞的生长,对H1299和A549细胞集落形成有显著抑制作用(p<0.01),表明汉黄芩素能有效抑制H1299和A549细胞的生长和增殖。汉黄芩素能够抑制H1299和A549细胞的迁移能力,且随着给药浓度和给药时间的增加,细胞的迁移率减小。流式细胞术检测发现汉黄芩素能将H1299和A549细胞周期阻滞在G0/G1期且能促进H1299和A549细胞凋亡。结论:汉黄芩素能够抑制H1299和A549细胞的生长、增殖和迁移,阻滞细胞周期在G0/G1期并可促进肺癌细胞凋亡。