目的:通过网络药理学和分子对接技术探讨中药八宝丹中唯一已知植物成分三七治疗胃癌的潜在药效活性成分及其作用机制.方法:通过TCMSP等数据库筛选三七的有效活性成分及相关靶点蛋白,将GeneCards和DisGeNET等数据库挖掘的胃癌相关靶点和药物靶点蛋白交集;采用Cytoscape3.7.0对药效成分进行分析,建立有效的成分-靶点-疾病的关系模型.运用STRING数据库构建交集靶点的PPI蛋白互作图.运用David数据库进行KEGG通路和GO分类的富集分析;利用AutoDock Tools 1.5.6软件对三七关键活性成分和核心靶点进行分子对接验证,运用PyMol 2.5软件绘制对接模式图.结果:筛选出三七的有效活性成分99个,映射273个药靶基因.经GeneCards数据库搜集得到胃癌疾病基因15118个,三七与胃癌的交集靶点234个,分析得到165条KEGG富集通路,195条GO功能富集条目,主要包括癌症的途径、PI3K-Akt通路、MAPK通路、Ras通路,以及RNA聚合酶启动子转录的正向调节、信号转导、基因表达的正调控、正调节细胞增殖、负调节细胞凋亡等生物过程;其中高Hub值的化合物成分豆甾醇、人参皂苷rh2与胃癌基因白细胞介素-6(interleukin-6,IL-6)、TP53、肿瘤坏死因子(tumor necrosis factor,TNF)存在较强的分子对接关系.结论:三七治疗胃癌的机制涉及多成分、多靶点和多途径,三七可能是通过调节癌症的途径、PI3K/Akt通路、MAPK通路、Ras信号通路等在胃癌治疗中发挥作用,其中豆甾醇、人参皂苷rh2可能是三七药效的关键活性成分之一.

Based on the perspective of "industry-university-research" in-depth integration and collaborative innovation,this paper discusses the current industry-university-research integration and the practical problems of the development of traditional Chinese medicine, aiming to explore the new ideas of benign organic integration, collaborative development, and mutual learning between traditional Chinese medicine and industry, schools, and scientific research, and further improve the international influence of traditional Chinese medicine and promote the great progress of traditional Chinese medicine health.

目的 探讨八宝丹对胃癌细胞移植瘤生长及细胞上皮-间质转化(EMT)的影响及其作用机制,为八宝丹的临床应用提供实验依据.方法 构建MGC80-3裸鼠皮下移植瘤模型,分为对照组和八宝丹组各10只,八宝丹组按0.25 mg/kg予八宝丹药液灌胃,对照组予等容积的生理盐水灌胃,每周给药6 d.隔天测量裸鼠瘤体体积和体质量,Western Blot法检测瘤体组织中E-cadherin、Vimentin、MMP2、MMP9、TGF-β1、Smad2/3、p-Smad2/3蛋白表达,HE染色法观察裸鼠的肝和肾组织形态.结果 给药第7天开始,八宝丹组裸鼠移植瘤的体积明显低于对照组(P<0.05),2组裸鼠体质量差异无统计学意义(P>0.05).HE染色结果显示八宝丹对裸鼠无肝、肾毒性.与对照组比较,八宝丹组裸鼠瘤体组织中E-cadherin蛋白表达明显提高(P<0.05),Vimentin、MMP2、MMP9、TGF-β1、p-Smad2/3蛋白表达明显降低(P<0.05),而Smad2/3总蛋白差异无统计学意义(P>0.05).结论 八宝丹具有抑制胃癌细胞移植瘤生长及EMT的作用,下调MMP2、MMP9的表达及TGF-β/Smad信号通路的活化可能是其重要的调节机制.

目的:探讨八宝丹(babao dan,BBD)体外抑制淋巴管生成的作用机制.方法:将人淋巴内皮细胞(human lymphatic endothelial cells,HLECs)分为不同浓度的 BBD 组,即0 g·L-1 BBD 组、0.25 g·L-1 BBD 组、0.5 g·L-1 BBD 组及0.75 g·L-1 BBD组.MTT法检测细胞增殖情况;Hoechs33258染色实验观察细胞凋亡情况;细胞划痕实验考察细胞损伤修复能力;Transwell实验考察细胞的迁移能力;管腔形成实验分析细胞淋巴管生成能力;Western Blot检测血管内皮生长因子-C(vascular endothelial growth factor-C,VEGF-C)、血管内皮生长因子受体-3(vascular endothelial growth factor receptor-3,VEGFR-3)、基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)和 MMP-9蛋白表达水平.结果:BBD可显著降低HLECs增殖活力及迁移率(P＜0.01);显著升高HLECs凋亡率(P＜0.01);显著抑制HLECs损伤修复能力;显著降低HLECs管腔生成数量及淋巴管生成相关蛋白VEGF-C、VEGFR-3、MMP-2及MMP-9蛋白表达水平(P＜0.05).结论:BBD可促进HLECs凋亡,抑制其增殖、迁移及淋巴管生成,作用可能与下调VEGF-C、VEGFR-3、MMP-2和MMP-9蛋白表达水平有关.

目的:探讨黄芩素对AngⅡ诱导高血压小鼠血压及血清中炎症相关因子表达的影响,并基于网络药理学进一步分析黄芩素治疗高血压的潜在作用机制.方法:利用AngⅡ皮下埋泵灌注的方式建立高血压小鼠模型,将24只C57BL/6小鼠随机分为对照组、AngⅡ组、AngⅡ+黄芩素组.黄芩素每天灌胃剂量为5 mg/(kg·d),对照组和AngⅡ组给予等体积生理盐水灌胃,连续干预4周.利用鼠尾无创血压仪每周监测各组小鼠血压;利用Bio-plex试剂盒检测各组小鼠血清中炎症相关因子的表达.同时,运用ETCM、TCMSP数据库筛选黄芩素的作用靶点基因;再运用Disgenet、OMIM等数据库筛选高血压疾病相关的靶点基因,取两者交集,并利用KEGG进行通路富集分析.结果:AngⅡ组第1～4周的收缩压、舒张压及平均动脉压均显著高于对照组(P<0.05);黄芩素干预2周后,能显著抑制AngⅡ诱导高血压小鼠收缩压、舒张压及平均动脉压的升高(P<0.05);与对照组比较,AngⅡ组小鼠血清中的白介素-6(IL-6)、单核细胞趋化蛋白-1(MCP-1)、肿瘤坏死因子-α(TNF-α)、白介素-1α(IL-1α)、单核细胞趋化蛋白-1α(MIP-1α)及T细胞激活分泌调节因子(RANTES)的含量均显著升高(P<0.05),而白介素-10(IL-10)的含量则显著降低(P<0.05);黄芩素干预后,则能显著降低AngⅡ诱导的高血压小鼠血清中IL-6、MCP-1的含量(P<0.05),升高IL-10的含量(P<0.05),但对TNF-α、IL-1α、MIP-1α及RANTES的血清含量没有影响(P>0.05).同时,挖掘出的黄芩素潜在作用靶点基因共有72个、高血压相关靶点基因共9032个,两者的交集靶点基因为58个.基于KEGG通路富集分析提示,其与代谢途径、氮代谢、JAK-STAT信号通路、PI3K-AKT信号通路、趋化因子信号通路等密切相关.结论:黄芩素能显著抑制AngⅡ诱导高血压小鼠的血压升高,调控血清中炎症相关因子的表达,其可能的作用机制与调控代谢途径、氮代谢、JAK-STAT信号通路、PI3K-AKT信号通路、趋化因子信号通路等有关.

Objective To further explore the mechanism of Babao Dan (BBD) combined with oxaliplatin (L-OHP) in treating colorectal cancer (CRC) through a network pharmacology analysis. Methods The analysis involved the following steps: screen the chemical components of BBD through literature review of Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Chemistry Database, PubChem, and other databases; obtain L-OHP-related targets through GeneCards database; and search CRC-related targets through OMIM, GeneMap, TTD, DisGeNET, CTD, GeneCards, and other databases. After the intersection and mapping of drugs and disease, the protein-protein interaction (PPI) network and core targets were obtained using STRING database and CytoScape software. MetaScape database was used to analyze the core targets to obtain GO biological processes and KEGG pathways. Results BBD contained 495 chemical components with 204 active components screened out through the Swiss ADME database and 770 targets were obtained through the Swiss Target Prediction database. After the intersection of BBD and 775 targets of L-OHP with the CRC targets, it resulted in 74 potential targets. Twenty-four core targets were determined from the 74 intersection targets, which were related to the positive regulation of kinase activity, the positive regulation of cell migration, and peptidyl-serine modification in GO biological process. The KEGG pathway analysis showed that the core targets were related to pathway in cancer, proteoglycan in cancer, endocrine resistance, and microRNA in cancer, TNF signaling pathway, platinum resistance, and other pathways. Molecular docking showed that the core targets could bind to the most examined compounds. Conclusion Quercetin-7-olate, cyclo (L-tyrosyl-L-phenylalanyl), panaxadiol, and other compounds in BBD may play an anti-colorectal cancer effect in multiple pathways, including EGFR, AKT1, mTOR, and other targets in synergy with L-OHP.

目的 探讨八宝丹对SGC7901/DDP胃癌耐药细胞生长及转移的作用机制.方法 MTT检测SGC7901/DDP细胞的耐药性及不同浓度的八宝丹干预后SGC7901/DDP细胞的生长情况;Transwell检测八宝丹对SGC7901/DDP细胞迁移和侵袭能力的影响;细胞粘附实验检测八宝丹对SGC7901/DDP细胞粘附能力的影响;Western blot检测八宝丹对TGF-β/Smad信号通路及转移的相关蛋白的影响.结果 SGC7901/DDP细胞对顺铂的耐药指数为1.94＞1.50,具有耐药性;MTT、Transwell及粘附实验结果显示,八宝丹能明显抑制SGC7901/DDP胃癌耐药细胞的生长、迁移、侵袭及粘附,且具有剂量依赖作用.Western blot蛋白检测结果显示,八宝丹下调 TGF-β、Vimentin、MMP2、MMP9、p-Smad2/3、N-cadherin、Smad4蛋白表达及上调Smad2/3、E-cadherin蛋白表达.结论 八宝丹能够抑制胃癌耐药细胞SGC7901/DDP细胞生长和转移,机制可能与其调控TGF-β/Smad通路有关.

目的 探讨齐墩果酸(OA)对人结肠癌细胞(HT-29)增殖与凋亡的影响及其分子机制.方法 体外培养HT-29细胞,用40、80、120μmol·L-1 OA进行干预后,用白介素-6(IL-6)刺激,MTT法和集落形成实验检测细胞增殖情况,倒置显微镜观察细胞密度变化,流式细胞术检测细胞周期,DAPI染色和Annexin V/PI流式细胞仪检测细胞凋亡,Caspase-3活力测定试剂盒测定Caspase-3活力,反转录-聚合酶链反应和Western blot实验检测IL-6/STAT3信号通路活化及其通路相关基因和蛋白的表达.结果 OA可显著抑制HT-29的增殖能力(P<0.01),并诱导其凋亡(P<0.01),显著抑制IL-6/STAT3信号通路的活化.结论 OA通过抑制IL-6/STAT3信号通路的活化,抑制HT-29增殖和促进其凋亡.

目的 研究片仔癀对人结肠癌细胞HCT-116裸鼠皮下移植瘤的抑制作用.方法 取20只BALB/c雄性裸鼠,建立人结肠癌细胞HCT-116裸鼠皮下移植瘤模型后随机分为对照组和片仔癀组,每组各10只.片仔癀组给予25 mg/mL片仔癀溶液0.2 mL/(kg·d)连续灌胃14 d,每日1次;对照组给予等体积生理盐水灌胃14 d,每日1次.从给药开始,隔天监测裸鼠肿瘤体积和体质量;给药结束后,用电子天平称取肝脏、脾脏、肾脏质量;通过免疫组化染色检测肿瘤组织中Ki-67的蛋白表达;采用TUNEL法检测肿瘤组织中细胞凋亡情况;利用RT-qPCR检测肿瘤组织中的Cyclin D1、CDK4、Bcl-2、Bax mRNA相对表达量,并计算Bax/Bcl-2比值;通过Western blot检测肿瘤组织Cyclin D1、CDK4、Bcl-2、Bax蛋白表达水平.结果 与对照组比较,片仔癀组裸鼠在第11、13、15天的肿瘤体积明显降低(P均<0.05),而2组裸鼠体质量无明显差别(P均>0.05);第15天时,2组裸鼠肝脏、脾脏、肾脏质量无明显差别(P均>0.05);与对照组比较,片仔癀组裸鼠肿瘤组织中Ki-67蛋白表达明显减少,细胞凋亡明显增多(P均<0.05);与对照组比较,片仔癀组裸鼠肿瘤组织中Cyclin D1、CDK4 mRNA相对表达量明显减少(P均<0.05),Bcl-2 mRNA相对表达量无明显差别(P>0.05),但Bax mRNA相对表达量和Bax/Bcl-2比值明显升高(P均<0.05);与对照组比较,片仔癀组裸鼠肿瘤组织中Cyclin D1、CDK4和Bcl-2蛋白表达明显下调(P均<0.05),Bax蛋白表达明显上调(P<0.05).结论 片仔癀对人结肠癌HCT-116皮下移植瘤有明显抑制作用,其机制与调控Bcl-2、Bax、Cyclin D1、CDK4表达有关.

Objective:To investigate the effects and mechanisms of Qingjie Fuzheng Granules (QFG) on colorectal cancer SW620 cells in proliferation, apoptosis and migration.Methods:Colorectal cancer SW620 cells in vitro were treated with 0,0.5,1 and 2 mg/mL QFG. The inhibitory effect of QFG on cell proliferation was detected by MTT assay after 24 h and 48 h. The morphology of SW620 cells was observed with inverted microscope after 24 h. The cell cycle after 24 h was measured by flow cytometry. Apoptosis of SW620 cells after 24 h was detected by Hoechst 33258 staining test. The migration ability of cells after 0 h,6 h,12 h and 24 h was detected by scratching test. The expressions of Ki-67, MMP-2 and Cleaved-Caspase-3 proteins were detected by Western blot assay after 24 h.Results:(1) In the concentration range of 0,0.5,1,2 mg/mL, QFG significantly reduced the cell proliferation in a time and dose-dependent manner. The differences were statistically significant when compared with the control group in the same time (P<0.05). (2) After treatment with 0.5,1,2 mg/mL QFG for 24 hours, the morphology of the cells changed significantly. With the increase of the concentration, the cell density decreased gradually, the suspended cells increased, and some cells got shrank and rounded. (3) The result of flow cytometry showed that the G1 phase of the QFG group in the 0.5,1,2 mg/mL concentration was decreased, but the S phase was increased and the G2 phase was not significantly changed. The percentage of each period was significantly different from that of the control group (P<0.01), but not dose dependent. (4) The Hoechst 33258 staining results showed that the cell morphological manifestations of apoptosis were more obvious with the increase of QFG concentration. (5) The scratch test showed that QFG in the 0.5,1,2 mg/mL concentration could reduce the migration ability of SW620 cells, and the scratch healing rate decreased gradually with the increase of intervention time and QFG concentration, showing a time and dose-dependent effect. Compared with the control group, the difference of 12 h and 24 h was statistically significant (P<0.01). (6) In addition, compared with the control group, QFG in the 0.5,1,2 mg/mL concentration significantly reduced the protein expression of Ki-67 and MMP-2, increased the protein expression of Cleaved-Caspase-3, and showed a dose-dependent, the difference was statistically significant (P<0.01).Conclusion:QFG can inhibit the proliferation, induce apoptosis and inhibit migration of colorectal cancer SW620 cells, and its mechanism may be related to down-regulation of Ki-67 and MMP-2 protein expression and up-regulation of Cleaved-Caspase-3 protein expression.

目的:探讨清解扶正颗粒(Qingjiefuzheng Granules,QFG)对体外人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cells,HUVEC)新生的影响及可能的作用机制.方法:以HUVEC为研究对象,给予不同浓度QFG后观察细胞形态变化,MTT检测细胞活力,划痕实验检测细胞迁移能力,管腔形成实验检测细胞成血管能力,蛋白免疫印迹检测促血管新生因子、血管内皮生长因子A、血管内皮生长因子受体2、基质金属蛋白酶2及基质金属蛋白酶9的蛋白表达.结果:QFG对HUVEC形态与细胞生长没有明显影响,但可显著抑制HUVEC的活力、迁移及体外成血管能力;QFG显著抑制血管内皮生长因子A、基质金属蛋白酶2、基质金属蛋白酶9等蛋白表达.结论:QFG在体外具有抑制血管新生的作用,其作用机制与其抑制血管内皮生长因子A、基质金属蛋白酶2及基质金属蛋白酶的表达有关.

目的 研究栝楼桂枝颗粒(Gualou Guizhi granule,GLGZG)是否通过调控PI3K/AKT通路抑制脑缺血再灌注损伤大鼠神经元凋亡而达到抗脑缺血再灌注损伤的作用.方法 SD大鼠36只,♂,分为假手术组、大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)组、GLGZG组,每组12只.其中MCAO组、GLGZG组采用线栓法致MCAO建立大鼠脑缺血再灌注损伤模型,GLGZG灌胃给药,连续给药7d.采用改良的神经功能缺损评分法对大鼠神经功能损伤进行评分,MRI观察大鼠脑梗死体积,Real-time PCR检测脑组织中PI3K、Akt mRNA的表达,Western blot法检测大鼠缺血侧脑组织PI3K(p85)、Akt、p-Akt、PDKl、Bcl-2、Bcl-xL、cleaved-caspase-3、Bad、Bax蛋白表达.结果 与MCAO组比较,GLGZG组神经行为学评分降低,第5、7天神经评分显著低于MCAO组(P＜0.05);与MCAO组比较,MRI观察发现GLGZG组大鼠脑梗死体积显著减小(P＜0.05);PI3K(p85)、p-Akt、PDKl、Bcl-2、Bcl-xL蛋白的表达上调(P＜0.01),cleaved-caspase-3、Bad、Bax蛋白的表达下调(P＜0.01),对Akt的表达没有影响.结论 GLGZG能够提高MCAO大鼠神经功能,抑制神经细胞凋亡,其作用机制可能是通过激活PI3K/AKT信号通路抑制MCAO大鼠神经元凋亡.

厦门中药厂生产的八宝丹(Babao Dan,BBD)是国家中药保护品种,其成分包含天然牛黄、天然麝香、蛇胆、羚羊角、珍珠、三七和其他两种尚未公开的组分,是国家食品药品监督管理总局(CFDA)规定允许使用天然麝香入药的8个中成药之一,具有清利湿热、活血解毒、去黄止痛之效.目前八宝丹被广泛应用于肿瘤、肝胆疾病、炎症疾病、免疫系统疾病、血液病及中医热证等方面的治疗.近年来,八宝丹的基础和临床研究取得了一定的成果,除自身所具有的疗效之外,其作为辅助用药减少化疗不良反应,提升术后患者体能状况,促进患者术后康复等优点也逐渐被发现,药效机制也部分被揭示.现对八宝丹在基础及临床应用中的研究进展综述如下.

Objective To establish a method for the content determination of paeoniflorin and paeonolide in rat plasma by HPLC-MS/MS; To study its characteristics of pharmacokinetics.Methods HPLC-MS/MS method was adopted. The column was Ultimate RXB-C18 (2.1 mm×100 mm, 3 μm) with mobile phase consisted of acetonitrile- 0.1％ formic acid water solution. Flow rate was 0.3 mL/min; column temperature was 30℃; the volume was 5 μL; detection wavelength was 230 nm. Quantitative ions were m/z 525→120.9 (paeoniflorin and paeonolide), m/z 433→224.8 (geniposide). 8 SD male rats were collected blood 0.5 mL from orbital vein 0, 5, 10, 20, 30, 45, 60, 90, 120, 180, 360, 540, 720 min after intragastric administrationBaishaoDecoction 6 g (medicinal materials)/kg to determine the blood concentration of medicine. DAS V2.1.1 software was employed to calculate pharmacokinetic parameters. Results Good linearity was found in the range of 40–8000 ng/mL and 40–10000 ng/mL for paeoniflorin and paeonolide (r=0.9996,r=0.9995). The intra-day and inter-day RSD were less than 6％. The extraction recoveries were 92.33％–95.43％ and 88.65％–95.62％. The matrix effect values were 99.29％–107.86％ and 93.79％–104.05％.All items of method validation were in line with the requirements. The main pharmacokinetic parameters of paeoniflorin and paeonolide were showed as follows: Cmax of (8.50±2.01) and (4.13±0.72) μg/mL; tmax of (10.00± 1.73) and (20.00±2.11) min; t1/2 of (142.98±30.11) and (127.68±35.74) min.Conclusion The HPLC-MS/MS method established in this study is simple, feasible, accurate, reliable, highly sensitive, and specific, which can be applied to study the pharmacokinetic characteristics of paeoniflorin and paeonolide in rats in vivo.

目的 观察八宝丹(BBD)对人脐静脉内皮细胞(HUVEC)增殖和迁移能力的影响. 方法 体外培养HUVEC,使用不同浓度BBD进行干预,MTT法检测HUVEC增殖情况,倒置显微镜观察细胞形态、划痕损伤,Transwell实验观察细胞损伤修复及迁移能力. 结果 BBD能显著降低HUVEC的活力,抑制HUVEC增殖,抑制HUVEC的损伤修复和迁移能力,并呈现剂量依赖性. 结论 BBD具有抑制HUVEC增殖扣迁移的作用.

To study the inhibitory effect of tryptanthrin on drug resistance to cisplatin chemotherapy for esophageal carcinoma and its mechanism,different treatments (blank control,cisplatin treatment,tryptanthrin treatment and cisplatin combined tryptanthrin treatment)of esophageal cancer Eca109 cells and cisplatin-resistant Eca109 cells (Eca109/cDDP)were performed on the basis of the purpose of experiments.Real-time quantitative PCR was used to detect the mRNA levels of multidrug resistance gene 1(MDR1)and glutathione-s-transferase-pi gene (GST-pi)in Eca109 and Eca109/cDDP cells.Western blot and/or immunofluorescence were used to detect the protein levels of MDR1 and GST-pi.The proliferation of cells was tested by cell counting kit (CCK-8).The results showed that tryptanthrin suppressed cell proliferation by inhibition of mRNA and protein levels of MDR1 and GST-pi in Eca109/cDDP cells. In summary,tryptanthrin can reverse the cisplatin resistance in Eca109/cDDP by its inhibition on MDR1 and GST-pi expression.It could be a potential adj uvant agent for chemotherapy.

近年来,我国医学行业不断发展,药理学人才需求逐渐增加,而复合型人才的需求更是供不应求.在药理学学科中最重要的组成就是实验教学,实验不仅仅需要建立在相关基础上,而是需要学生学会自主实验设计,对实验流程进行不断创新,才能更好为药理学服务.药理学实验教学改革成为教学的主要任务,对传统教学模式进行完善,才能真正的提高学生的创新能力和创新意识,提升解决问题的能力,真正的实现高质量教学.