中药黄芪(Astragali Radix)抗阿霉素所致心肌细胞毒性的作用机制尚未完全阐明.本文基于阿霉素心肌细胞毒性转录组学数据,以网络药理学方法探讨黄芪抗阿霉素心肌细胞毒性的物质基础与作用机制,并在细胞水平进行验证.网络药理学研究结果显示,黄芪中7-O-甲基异丁香酚、山柰酚、槲皮素、刺芒柄花素、异鼠李素等21个有效成分主要通过TP53、STAT3、AKT1、MAPK1等12个核心靶点作用于PI3K-AKt等信号通路发挥抗阿霉素心肌细胞毒性作用.细胞实验结果显示,给予黄芪处理能显著降低阿霉素导致的H9C2心肌细胞凋亡,增加G1期细胞,减少G2期细胞;同时阿霉素上调核心靶点TP53、STAT3、MAPK1的mRNA表达,下调AKT1的mRNA表达,黄芪处理能剂量依赖降低核心靶点TP53、STAT3、MAPK1的mRNA表达并上调AKT1的mRNA表达.本研究表明黄芪可能主要通过PI3K-Akt信号通路中的TP53、STAT3、AKT1、MAPK1对阿霉素引起的心肌细胞毒性发挥保护作用.

The normal development of follicles determines the reproductive performance of females. Granulosa cells (GC) play crucial roles in follicular maturation. Numerous studies have shown that miRNAs are involved in the regulation of GC. According to our previous sequencing data, gga-miR-146b-3p was differentially expressed in normal and atretic chicken follicles. In this study, we verified that gga-miR-146b-3p attenuated proliferation and autophagy but promoted apoptosis in chicken GC. Threonine kinase1 (AKT1), a key member of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, was predicted to be a target gene of gga-miR-146b-3p via bioinformatic analysis. Dual-luciferase reporter gene assays were used to determine target relationships. Moreover, knockout of AKT1 decelerated proliferation and autophagy while accelerating the apoptosis of GC. However, overexpression of AKT1 reversed these results. In summary, our results demonstrated that gga-miR-146b-3p repressed the proliferation and autophagy of chicken GC while up-regulating apoptosis by targeting AKT1 through the PI3K/AKT signaling pathway. These findings may provide great insights for further exploration of the molecular regulation of gga-miR-146b-3p and AKT1 on the functions of GC during folliculogenesis.

目的 建立基于亲水相互作用色谱-高效液相色谱法测定潞党参中游离果糖、蔗糖含量的方法,并考察不同温度干燥温度处理对潞党参药材中果糖、蔗糖以及多糖含量的影响.方法 建立了亲水相互作用色谱-高效液相色谱-蒸发光散射检测器(HILIC-ELSD)的方法测定不同温度干燥的潞党参中果糖、蔗糖含量,用苯酚-硫酸法测定不同温度干燥的潞党参中多糖含量.结果 果糖、蔗糖色谱峰能很好分离;果糖含量高低顺序:常温阴干≈ 40℃烘干＞60℃烘干;蔗糖含量高低顺序:常温阴干＞40℃烘干＞60℃烘干;多糖含量高低顺序:60℃烘干＞40℃烘干≈常温阴干.结论 该HILIC-ELSD法操作简便,重复性、分离度好,可用于党参中果糖、蔗糖含量测定;不同温度干燥的潞党参中果糖、蔗糖及多糖含量存在差异,且显现出不同变化趋势,该研究结果可为潞党参药材质量评价提供科学参考.

基于网络药理学及分子对接探讨黄芪抗肝癌的活性成分与分子作用机制.通过TCMSP数据库获取黄芪活性成分,Swiss Target Prediction预测成分靶点,采用Genecards数据库与OMIM数据库搜集肝癌靶点,Venny相映射黄芪抗肝癌的作用靶点,String数据库结合Cytoscape 3.7.2软件绘制肝癌靶点的蛋白相互作用网络(PPI)及"黄芪-成分-通路-肝癌"相互作用网络,DAVID数据库对核心靶点基因功能富集和通路富集分析.Surflex-Dock软件对黄芪关键成分与核心靶点进行分子对接验证.MTT法检测槲皮素、毛蕊异黄酮、山奈酚、芒柄花素、异鼠李素及华良姜素对肝癌细胞(HepG2)的影响.RT-qPCR法验证华良姜素对TP53、MAPK1、AKT1、IL6、MAPK8与VEGFA基因表达相对水平.本研究筛选出20个黄芪活性成分,涉及202个作用靶点及其100条KEGG信号通路,GO分析为487条生物功能.网络药理学分析黄芪可能是通过TP53、MAPK1、AKT1、IL6、MAPK8与VEGFA等关键靶点起到抗肝癌作用.分子对接表明靶点与成分有一定的结合性.MTT表明华良姜素对肝癌细胞(HepG2)的抑制作用较强于其他五个成分.RT-qPCR验证不同浓度的华良姜素对6个基因的表达量均为上调趋势,与KEGG通路分析所涉及基因一致.本研究初步探讨黄芪治疗肝癌具有多靶点、多通路的潜在作用机制,为后续验证黄芪抗肝癌的分子机制提供了依据.

为了更好地了解实验室基本技术和中医学综合实验指导课程的教学情况,采用问卷的方式对修完本课程的研究生进行了调查,力求探讨一种更符合中医药院校研究生需求的实验课程教学模式.问卷的统计和分析结果 显示,学生对课程内容、教师教学、考核、教学效果等方面都较为满意,并对课程提出了中肯的建议.这为该课程的发展、改革和探索提供了一定的依据.

目的 优化黄芪蛋白水解液工艺条件,并考察其体外抗氧化活性.方法 在单因素试验的基础上,选择温度、pH、酶用量、酶解时间为影响因素,以1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除率为指标,采用中心组合Box-Benhnken法建立数学模型,进行响应面分析.结果 黄芪水解蛋白的最佳酶解条件为酶解温度50℃、pH值3、酶用量800 U/g、酶解时间8 h,该条件下水解度为52.96％,DPPH自由基清除率为80.64％,O2-·清除率为98.93％,·OH清除率为74.83％,还原能力为2.3.结论 黄芪蛋白水解液具有较强的抗氧化活性,可作为一种天然食品抗氧化剂或抗衰老、抗氧化饮品.

To investigate the inhibitory effects of two xanthone compounds, 1-hydroxy-2,3,4,8-4 methoxy xanthone(here in after referred to as Fr15) and 1-hydroxy-2,3,4,6-4 methoxy xanthone(here in after referred to as Fr17), on the proliferation of hepatocellular carcinoma cells HepG2, and to further investigate their mechanism in combination with transcriptomics. Cell counting was used to detect the effects of two kinds of xanthone compounds Fr15 and Fr17(0, 0.03, 0.15, 0.3 mmoL·L~(-1)) on the proliferation of HepG2 cells; the effects of the two compounds Fr15 and Fr17 on HepG2 cell cycle were detected by flow cytometry; the changes of autophagosomes count in cells were observed under fluorescence microscope; the expression of autophagy marker proteins autophagy marker proteins SQSTM 1(p62) and microtubule associated protein 1 light chain 3 Ⅰ/Ⅱ(LC3 Ⅰ/Ⅱ) in the cells was detected by Western blot; the differentially expressed genes between the control group and the experimental group were analyzed by RNA-seq transcriptome sequencing; qRT-PCR was used to verify the differentially expressed genes in sequencing. The results showed that compounds Fr15 and Fr17 inhibited the proliferation of HepG2 cells with the increase of drug concentration and time. Flow cytometry showed that compounds Fr15 and Fr17 had little effect on HepG2 cell cycle. Fluorescence microscopy results showed that the number of autophagosomes in cells increased with the increase of drug concentration. Western blot showed that the expression of p62 protein was decreased and the expression of LC3-Ⅱ protein was significantly increased after drug addition. The results of RNA sequencing showed that 26 102 and 52 351 differentially expressed genes were obtained in Fr15 and Fr17 respectively. Analysis of KEGG showed that drug treatment had a great effect on autophagy pathway. qRT-PCR verified that 6 up-regulated genes were related to autophagy, and their trend was consis-tent with sequencing results, where all 6 genes showed an up-regulated trend. Two xanthone compounds Fr15 and Fr17 may inhibit proliferation of HepG2 cells by inducing autophagy.

本文考察了黄芪蛋白对肝癌细胞HepG2的增殖抑制作用,结合转录组学探讨黄芪蛋白抗肿瘤作用机制.黄芪干燥根部经硫酸铵沉淀,得到分子质量大小不一的黄芪蛋白(Huang Qi protein,HQP).通过血球计数法检测黄芪蛋白对肿瘤细胞HepG2的影响及其毒性作用;结合流式细胞术和Hoechst/propidium iodide (PI)双染测定细胞死亡情况;Western blot测定坏死标志蛋白受体相互作用的丝氨酸/苏氨酸激酶l(RIPl);将对照组与加药组RNA进行转录组测序,对RNA测序(RNA-seq)结果进行差异表达基因分析;qRT-PCR验证候选基因mRNA相对表达量.结果 表明,随着HQP浓度增加,对肝癌细胞HepG2增殖抑制作用愈加明显,当HQP质量浓度为100 μtg·mL-1时,细胞坏死率增加到18.78％,同时在显微镜下观察到PI单染的红色坏死细胞增多,Western blot结果显示RIP1蛋白水平增加.RNA-seq结果分析得到2.6万个相关基因受HQP调控,其中979个基因受调控较明显.KEGG分析发现部分差异表达基因与p53信号通路相关,qRT-PCR验证测序结果可靠.黄芪蛋白使HepG2细胞发生程序性坏死可能与p53信号通路有关.

目的 观察黄芪糖蛋白(Huang Qi Glycoprotein,HQGP)对肺纤维化小鼠肺组织病理形态、细胞因子及α-平滑肌肌动蛋白(α-SMA)蛋白表达的影响.方法 将80只雌性ICR小鼠随机分为空白组、模型组、地塞米松组、HQGP组,每组20只.空白组鼻腔滴注生理盐水,其余3组鼻腔滴注博来霉素(15 mg/kg)诱导肺纤维化,造模后第1天起,各组小鼠给予相应药物腹腔注射,连续治疗14 d.给药后的第7、28天分两批取材,用HE染色和ELISA测定肺组织TNF-α和IL-1β含量来观察对肺泡炎程度的影响;Masson染色评价对肺纤维化程度的影响;采用免疫组化和Western Blot法分别检测肺组织中α-SMA蛋白表达.结果 与模型组比较,第7、28天HQGP组小鼠的肺泡炎、肺纤维化程度明显减轻;第7、28天HQGP组小鼠肺组织中α-SMA蛋白表达显著降低(P＜0.01).结论 HQGP对肺纤维化的干预机制可能与抑制肺组织中α-SMA的表达有关.

目的 按古籍方法制备沙参麦冬汤物质基准,建立特征图谱及指标成分检测方法,完善其质量控制体系.方法 采用HPLC建立15批沙参麦冬汤物质基准的特征图谱,使用《中药色谱指纹图谱相似度评价系统》(2012A版)计算相似度,按2015年版《中华人民共和国药典》"甘草"项下方法测定15批物质基准中甘草苷的含量和转移率.结果 以甘草苷为参照峰,沙参麦冬汤特征图谱共标定20个共有峰,指认出2个共有峰,15批物质基准相似度均大于0.9.15批物质基准甘草苷含量为0.053％～0.478％,转移率为38.92％～60.98％.结论 本研究建立的方法所得数据真实可靠,可为沙参麦冬汤物质基准的质量控制奠定基础.

如何使中医药类硕士研究生熟练掌握细胞培养技术,并能灵活应用于中医药研究中是中医药高等院校开展细胞培养实验技术课程的首要任务.针对山西中医药大学开展细胞培养实验技术课程的教学现状,我们从优化实验内容,调整教学模式;强调细节,夯实基本功;优化教学资源,注重教师队伍建设;重视教学效果评价,构建合理的考核评定体系等4个方面进行了教学改革探索,以期提升课程的教学质量,增强中医药类硕士研究生的科研创新能力.

目的:以玉米根尖为材料,研究叠氮化钠对玉米根尖细胞遗传毒性的影响.方法:用不同浓度的叠氮化钠溶液(0.002 mmol/L、0.02 mmol/L、0.2 mmol/L)作为化学诱变剂,分别对玉米根尖进行8h、16 h、24h的染毒处理.采用常规染色体压片技术,在光学显微镜下观察玉米根尖细胞染色体结构变异情况,并测定玉米根尖细胞的有丝分裂指数,染色体畸变率和微核率.结果:叠氮化钠能影响玉米根尖细胞染色体的结构,出现了不同的畸变现象,包括多极、成环、桥(单桥和多桥)、滞后、不均等分裂、断片、微核等,并随着叠氮化钠溶液浓度升高和染毒时间加长,有丝分裂指数逐渐下降,染色体畸变率和微核率逐渐上升.结论:叠氮化钠对玉米根尖染色体的结构有影响,对玉米根尖有毒害作用,所以我们要合理地使用以叠氮化钠为原料的除草剂.

目的 观察黄芪糖蛋白(HQGP)干预肺纤维化小鼠的病理学改变及并分析机制.方法 将60只健康ICR小鼠随机分为对照组、模型组及治疗组,每组20只.采用鼻腔滴入博莱霉素建立小鼠肺纤维化模型,造模次日起治疗组每天腹腔注射HQGP,模型组及对照组腹腔注射等体积的生理盐水,连续14 d;造模后第7、14、28天取材,取肺组织进行HE、Masson染色,观察肺泡炎及纤维化程度;免疫组化法检测肺组织转化生长因子(TGF)-β1表达.结果 与模型组比较,第7、14、28天治疗组肺泡炎、肺纤维化程度明显减轻;肺组织TGF-β1蛋白表达明显降低(P<0.05).结论 HQGP抑制小鼠肺纤维化的发展,其作用机制可能与降低肺组织中TGF-β1表达有关.

Previous studies have shown that Huangqi glycoprotein (HQGP) has an anti-inflammatory effect in vitro, and suppressed experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis; however, the mechanism underlying its effect is largely unknown. In this manuscript we investigated the mechanisms by which HQGP protect mice from EAE. HQGP was extracted from Astragalus membranaceus and purified by anion-exchange and gel filtration chromatography. HQGP delayed disease onset, reduced disease severity and alleviated inflammation and demyelination in the central nervous system (CNS). Moreover, HQGP reduced the infiltration of pathogenic immune cells and increased the expression of microtubule-associated protein 2 (MAP-2) and neuronal nuclei (NeuN) in the CNS. HQGP treatment also reduced the expression of chemokines such as CCL2 and CCL5 and the production of tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6, but increased the level of IL-10. These results demonstrate that HQGP suppressed EAE development by modulating the immune system and the infiltration of leukocytes to the CNS as well as promoting axon and neural repair.

Triptolide is a major active ingredient isolated from the traditional Chinese herb Tripterygium wilfordii Hook F. However, its use in clinical practice is limited due to its severe hepatotoxicity. Autophagy, a highly conserved intracellular process, is essential for maintaining cytoplasmic homeostasis. Considering that abnormalities in autophagy are closely associated with drug-mediated hepatotoxicity, we applied human normal liver HL7702 cells to elucidate the roles of autophagy in triptolide-induced hepatotoxicity. Our study revealed that triptolide was cytotoxic to HL7702 cells. It markedly increased autophagosome formation and expression of autophagy-related proteins, namely Beclin1 and microtubule-associated protein 1 light chain 3II, and induced oxidative stress. These proautophagic effects were counteracted by pretreatment with N-acetylcysteine, a reactive oxygen species scavenger. Moreover, the pharmacological suppression of autophagy further exacerbated triptolide-elicited decrease in cell viability, increase in lactate dehydrogenase leakage, and activation of apoptosis proteases (caspase 3 and caspase 9). Our findings suggest that triptolide-induced oxidative stress consequently enhances autophagic activity, and autophagy is a cytoprotective mechanism against triptolide-induced cytotoxicity in HL7702 cells.

目的 观察黄芪糖蛋白(HQGP)对C57BL/6小鼠实验性自身免疫性脑脊髓炎(EAE)血脑屏障(BBB)的影响,探讨HQGP治疗EAE的机制.方法 选择C57BL/6雌性小鼠40只,用髓鞘少突胶质细胞糖蛋白35-55(MOG35-55)诱导建立EAE模型,并随机分为HQGP组和EAE组.HQGP组于免疫后第3天腹腔注射HQGP(1 mg/kg),EAE组每天给予等量生理盐水,直至免疫后第18天处死.观察小鼠的发病情况;检测小鼠脑组织含水量和伊文思蓝(EB)含量;免疫组织化学法检测小鼠脊髓组织中CD4+T细胞和CD68+巨噬细胞的浸润;Western blotting检测脊髓组织BBB紧密连接蛋白Occludin、ZO-1的表达.结果 与EAE组比较,HQGP组发病延缓,临床症状轻,脑组织含水量及EB含量降低.HQGP能降低小鼠脊髓组织中CD4+T细胞和CD68+巨噬细胞的浸润,显著增加脊髓组织中Occludin及ZO-1的表达.结论 HQGP可以有效保护EAE小鼠的BBB,进而减轻EAE的临床症状.

目的 观察黄芪糖蛋白(Huangqi Glycoprotein,HQGP)对实验性自身免疫性脑脊髓膜炎(Experimental Autoimmune Encephalomyelitis,EAE)小鼠的干预作用,并探讨其可能的保护机制.方法 复制MOG35-55多肽诱导的C57BL/6雌性小鼠EAE模型,随机分为EAE对照组和HQGP治疗组.通过5级临床症状评分和小鼠体重变化观察HQGP对EAE小鼠的保护效应;取小鼠脊髓进行HE染色和髓鞘固蓝染色观察HQGP的神经保护作用;免疫荧光组织化学染色检测小鼠脊髓相关免疫细胞数量的变化;ELISA法检测脾脏单个核细胞(Mononuclear Cells,MNCs)培养上清液中相关细胞因子的水平.结果 HQGP有效缓解了EAE小鼠的临床症状,推迟发病时间,减轻中枢神经系统的炎性反应和髓鞘脱失;HQGP能减少小鼠脊髓CD4+T细胞、CD68+巨噬细胞和CDllb+细胞的数量,并能明显抑制小鼠IL-17、TNF-α、IL-12的分泌,促进IL-4、IL-10、IFN-γ的分泌.结论 HQGP具有治疗EAE的潜在作用,其作用机制与抗炎和免疫调节有关.

目的:观察黄芪糖蛋白(Huang Qi Glycoprotein,HQGP)对肺纤维化小鼠的治疗作用,并初步探讨其作用机制.方法:选取60只健康ICR小鼠,随机分为对照组、模型组及观察组,每组20只.采用鼻腔滴入博莱霉素建立小鼠肺纤维化模型,造模次日起观察组每天每只小鼠腹腔注射HQGP,连续14 d,模型组及对照组腹腔注射等体积的生理盐水;给药后第7、28天每组分别处死5只动物,取肺组织,评价HQGP对肺组织病理形态、肺系数、肺组织羟脯氨酸(Hyp)、超氧化物歧化酶(SOD)、丙二醛(MDA)及转化生长因子-β1(TGF-β1)表达的影响.结果:与模型组比较,第7、28天HQGP观察组小鼠的肺纤维化程度减轻,肺组织的TGF-β1、Hyp、MDA水平显著降低,差异有统计学意义(P<0.05),肺组织SOD水平及肺系数显著升高,差异有统计学意义(P<0.05).结论:HQGP可能通过降低胶原合成,提高机体的抗氧化能力,对肺纤维化的发生发展起到治疗作用.

As a major active component extracted from traditional Chinese herb Tripterygium wilfordii Hook F, triptolide exhibits multiple pharmacological effects. Autophagy is an evolutionary conserved intracellular catabolic process involved in cytoplasmic materials degradation. Autophagic dysfunction contributes to the pathologies of many human diseases, which makes it a promising therapeutic target. Recent studies have shown that triptolide exerts neuroprotection, anti-tumor activities, organ toxicity, and podocyte protection by modulating autophagy. This article highlights the current information on triptolide-modulated autophagy, analyzes the possible pathways involved, and describes the crosstalk between autophagy and apoptosis modulated by triptolide, in hope of providing implications for the roles of autophagy in pharmacological effects of triptolide and expanding its novel usage as an autophagy modulator.

目的 探讨外周血吲哚胺2,3-双加氧酶和白介素-6水平与乳腺癌的关系.方法 ELISA法测定乳腺癌患者外周血吲哚胺2,3-双加氧酶和白介素-6的水平.结果 乳腺癌组外周血吲哚胺2,3-双加氧酶和白介素-6水平高于乳腺良性肿瘤组(P<0.05).乳腺良性肿瘤组外周血IDO和IL-6水平与对照组无明显差异(P>0.05).淋巴结转移阳性患者血清IDO和IL-6水平明显高于淋巴结转移阴性患者(P<0.05).结论 外周血吲哚胺2,3-双加氧酶和白介素-6与乳腺癌之间有着密切的联系,IDO和IL-6在癌症进展和化学疗效中可能起重要作用.