灯盏花MYB基因克隆及其荧光表达载体的构建

目的克隆灯盏花MYB基因的全长序列,为解析灯盏花MYB基因的功能奠定基础。方法根据灯盏花转录组的相关信息,利用RACE方法从灯盏花中克隆到一个可能参与灯盏花乙素合成的MYB基因,在对其cDNA序列、核苷酸序列的相似性、理化性质、疏水性、跨膜结构、二级结构及三级结构进行分析预测的基础上,对其进行多序列比对并构建系统树。同时,还构建了该基因与绿色荧光蛋白的融合表达载体,并进行了初步转化研究。结果克隆获得灯盏花MYB基因,命名为ebMYB06,其开放阅读框为783 bp,编码260个氨基酸残基,相对分子质量为63 800,理论等电点(p I)为5.18,属稳定蛋白。其蛋白二级结构主要由无规卷曲、α-螺旋和β-折叠构成。根据灯盏花MYB与拟南芥MYB(AtMYB)的系统树比对分析结果,发现ebMYB基因与拟南芥中的AtMYB4、7、32、6、8和AtMYB11、12、111 2亚群的基因聚类,推测所克隆基因在结构或功能上,可能与这两组具有共同性。实验还表明所构建的表达可用于灯盏花的高效转化。结论首次从灯盏花中克隆到可能参与其苯丙烷代谢或环境响应调控的MYB基因。     

灯盏花制剂治疗急性脑梗死的网状Meta分析

目的采用网状Meta分析的方法比较不同灯盏花制剂治疗急性脑梗死的疗效。方法计算机检索中国生物医学文献数据库(CBM)、中国知网(CNKI)、万方全文数据库(Wanfang),以及PubMed、Embase、the Cochrane Library,查找与灯盏花制剂相关的系统评价,检索时限为建库至2019年2月,对合格系统评价中包含的随机对照试验(RCT)进行筛选、数据提取及偏倚风险评价,并采用Stata 13.0软件进行网状Meta分析。结果共纳入74项RCT,包括8803名患者,涉及12种干预措施。网状Meta分析结果显示,在治疗急性脑梗死的总有效率方面,当疗程小于15 d时,不同的灯盏花制剂之间无统计学差异(P>0.05);当疗程大于20 d时,灯盏花素注射液治疗疗效优于灯盏细辛注射液[RR=1.16,95%CI(1.02,1.33),P<0.05],其余不同的灯盏花制剂之间互相比较无统计学差异(P>0.05),累积排序曲线下面积(SUCRA)由高到低排序为灯盏花素注射液、灯盏生脉胶囊、灯盏细辛注射液。在神经功能缺损评分方面,灯盏生脉胶囊组的治疗前后的美国国立卫生研究院卒中量表(NIHSS)变化值大于灯盏细辛注射液组[MD=1.95,95%CI(0.31,1.60),P<0.05],其余不同的灯盏花制剂之间互相比较无统计学差异(P>0.05)。SUCRA由高到低排序为灯盏生脉胶囊、灯盏花素注射液、灯盏细辛注射液。灯盏生脉胶囊的不良事件较其他灯盏花制剂少。结论灯盏花制剂均可在一定程度上提高急性脑梗死患者的有效率及改善神经功能缺损,而不同制剂的优势与疗程长短、结局指标差异有关。综合平衡疗效与安全性,灯盏生脉胶囊对于急性脑梗死的优势最为明显。     

Extraction (DSX) from Erigeron breviscapus modulates outward potassium currents in rat retinal ganglion cells

AIM: To investigate the effect of DSX, an active component extracted from Erigeron breviscapus, on the voltage-gated outward K+ channel currents in rat retinal ganglion cells (RGCs) by using electrophysiological method, and to explore the possible mechanisms of DSX on optic nerve protection. METHODS: Outward K+ currents were recorded by using whole-cell patch-clamp techniques on acutely isolated rat RGCs. Outward K+ currents were induced by a series of depolarizing voltage pulses from a holding potential of -70 mV to +20 mV in an increment of 10 mV. RESULTS: Extracellular application of DSX voltage-dependently suppressed both the steady-state and peak current amplitudes of outward K+ currents in rat RGCs. Furthermore, DSX reversibly and dose-dependently inhibited the amplitudes of outward K+ currents of the cells. At +20 mV membrane potential DSX at the concentrations of 0.02 g/L and 0.05 g/L showed no significant effects on the currents. In contrast, DSX at higher concentrations (0.1 g/L, 0.2 g/L and 0.5 g/L) significantly suppressed the current amplitudes. CONCLUSION: These results suggest that DSX reversibly and dose-dependently suppress outward K+ channel currents in rat RGCs, which may be one of the possible mechanisms underlying Erigeron breviscapus prevents vision loss and RGC damage caused by glaucoma.     

A New Procedure for Separation and Purification of Scutellarin from Erigeron multiradiatus

Objective To establish a new procedure for isolating scutellarin from Erigeron multiradiatus. Methods A proposed method was developed by combining macroporous resins with ODS column. Firstly, E. multiradiatus was extracted by ultrasound with 80% methanol. Preliminary separation was performed on macroporous resin column. The performance and adsorption characteristics of three macroporous resins, D140, D141, and D605, were compared and the enrichment procedure was optimized. Further purification was carried out by medium pressure liquid chromatography (MPLC) with ODS column. Results It was demonstrated that D141 had better extractive effects on scutellarin. The MPLC conditions were optimized as follows: 15% ethanol aqueous as mobile phase with flow rate at 2.5 mL/min. The yield and purity of the isolated scutellarin were 1.20 mg/g and 96.5%, respectively. Conclusion The overall procedure is efficient and low-cost, which is considered suitable for the separation and purification of scutellarin from E. multiradiatus. The results provide the scientific basis for developing and using scutellarin in clinic.     

灯盏细辛与赤芍配伍对永久性脑缺血损伤大鼠的保护作用

目的探讨灯盏细辛与赤芍配伍药对脑缺血性损伤大鼠的保护作用及可能机制。方法方法 48只成年SD雄性大鼠随机分为4组:假手术组、脑缺血组、配伍药物治疗组、阻滞剂组,每组12只。采用线栓法制作大脑中动脉闭塞模型。于缺血后24 h后行神经功能缺损评分,断头取脑行TTC染色检测脑梗死面积,实时荧光定量PCR法、蛋白质免疫印记法测缺血区脑皮质SHH、Patched-1、Gli-1mRNA及蛋白的表达。结果配伍药物组缺损评分、脑组织梗死面积显著低于脑缺血组和阻滞剂组。脑组织shh、Patched-1、Gli-1 mRNA及蛋白表达量显著高于脑缺血组,阻滞剂组Gli-1mRNA及蛋白表达量显著低于配伍药物组。结论灯盏乙素与芍药苷配伍对大鼠局灶性脑缺血损伤具有一定的保护作用,其机制与上调Sonic Hedgehog(Shh)通路关键蛋白分子表达量有关。     

灯盏细辛治疗眼压已控制的青光眼的临床研究

目的：研究灯盏细辛治疗眼压已控制的青光眼的临床疗效。方法：将86例患者采用双盲随机对照法分为2组,A组口服含灯盏细辛的A药片,B组口服安慰剂B药片,随访观察4个月。结果：A组治疗2月及4月后视野计分点平均值较治疗前均有显著性提高（P〈0.05）,B组治疗前后无显著性差异（P〉0.05）;A组治疗后视力进步率（38.46%）明显高于B组（23.53%）;A、B两组治疗前、后2月及4月的眼压、血压、心率、血液流变学指标均无显著性差异（P〉0.05）;所有患者服药后未出现不良反应。结论：灯盏细辛在治疗眼压已控制的青光眼性视神经病变中具有一定的疗效,且未发现毒副作用,其视神经保护机制还有待我们长期的临床观察和进一步的基础研究。     

Comparative Study of Anti-oxidative Effects of Tibetan Folk Medicine Erigeron multiradiatus during Plant Growth

Objective To explore the effects of a potential anti-oxidative plant, Erigeron multiradiatus (Asteraceae), plant materials from naturally distributed high-altitude populations at different stages of life cycle were collected. Methods Fifteen extracts obtained from the Ganzi region (Sichuan, China) were studied to assess their radical- scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals and reducing power ability. Moreover, considering that anti-oxidants and free radical scavengers can also exert protective effect on endothelial cells from oxidative injury, these extracts were also evaluated for their anti-oxidative activity against cellular injury in the cultured human endothelial cell line (ECV304) induced by hydrogen peroxide (H2O2). Results All the extracts had radical-scavenging and/or reducing power ability, and the most active extract was found during flowering whereas the lowest appeared during vegetative growth period. The accumulation of anti-oxidative compounds was found to be affected by the altitude of growth environment. Total flavonoid content assay was also performed to support this outcome. Furthermore, these extracts also exhibited different effects on attenuating H2O2-induced cytotoxicity and inhibiting lipid peroxidation and LDH leakage from endothelial cells. Conclusion E. multiradiatus may be an important natural anti-oxidant and this property may contribute to verifying the utilization of this plant in Tibet folk medicine.     

灯盏花提取物对青光眼视神经保护作用的临床观察

目的 评价灯盏花提取物对眼压已控制的青光眼视神经保护作用.方法 采用随机、双盲与安慰剂对照临床试验方法.对2009年10月至2009年在解放军第五医院眼科就诊符合纳入标准的青光眼患者共34例(42只眼),分为治疗组和对照组,分别给予口服益脉康胶囊和安慰剂,每两月观察安全(血压、肝功、肾功)及疗效指标(视力、视野、HRT),疗程6月,评价疗效.结果 治疗组与对照组两组治疗前及治疗后2月平均光敏感度组间比较P＞0.05,差异无统计学意义,而治疗4、6月后两组组间比较的P＜0.05,差异有统计学意义;两组治疗后2月视网膜平均光敏感度改善率组间比较P＞0.05,差异无统计学意义,而治疗4月后两组组间比较P＜0.05,差异有统计学意义.治疗6月后两组组间比较的P＜0.01,差异显著有统计学意义.结论 益脉康胶囊能提高视网膜平均光敏感度,随治疗时间延长,其改善率明显提高,表明灯盏花提取物对眼压已控制的青光眼有一定的视神经保护作用.

Abstract：

Objective To evaluate the action for optic nerve protection of engeron extracts in treating glaucoma with control of intraocular pressure. Methods Thiny-four patients (42 eyes) with glaucoma who met the inclusion criteria, in randomized, double-blind and placebo-controlled clinical trial were divided into treatment group and control group were given oral Yi Mai Kang capsules and placebo, observed every two months to test security (blood pressure, liver function, renal function) and outcome measures (visual acuity, visual field, HRT), and were evaluated after 6 months. Results The mean sensitivity of treatment group and control group, the difference was not statistically significant both before treatment and 2 months after treatment, (P＞0.05), but the difference of the two groups,4, 6 months after treatment were statistically significant (P ＜0.05). The average rate of retinal light sensitivity of the 2 months, the difFerence between two groups was not statistically significant (P ＞0.05), but the difference of the two groups, 4 months at/er treatment was statistically significant (P ＜0.05). There was statistically sipiificant difference 6 months after treatment between the two groups (P ＜0.01). Conclusions Yi Mai Kang Capsule can improve retinal mean sensitivity, with longer treatment time, the improvement rate is significantly increased. It indicates that the extract of Erigeron has certain protective effect of optic nerve on glaucoma with control of intraocular pressure.     

中草药灯盏花对兔正畸牙移动过程中牙周组织血管内皮生长因子表达的影响

目的 检测中草药灯盏花对正畸牙牙周组织中血管内皮生长因子（VEGF）表达的影响,推测其加速正畸牙移动的可能作用机理。方法 45只兔分为A、B、C组,均以双侧下颌第一磨牙为实验牙。A组右侧第一磨牙离子导入灯盏花（A-R组）,左侧导入生理盐水作为对照（A-L组）。B组安装使双侧下颌第一磨牙近中移动的正畸加力装置,同时右侧离子导入灯盏花（B-R组）,左侧导入生理盐水作为对照（B-L组）。C组为空白对照组。A、B组分别于实验的1、3、7、14 d处死,每次处死5只兔。取所有动物的双侧下颌骨标本,B组测量牙移动距离后,所有标本制成第一磨牙的牙周组织切片,免疫组化法检测VEGF的表达。结果 B组第一磨牙移动距离在加力1、3、7、14 d时依次递增,B-R组在3、7、14 d的移动距离较B-L组大（P<0.01）。免疫组化染色表明C组和A-L组VEGF表达阴性,
A-R组、B-L组和B-R组表达较强,其中B-R组表达最强;A-R组和B-R组表达高峰在实验第3天,而B-L组表达高峰出现于第7天。结论 灯盏花可能通过上调正畸牙加力初期牙周组织中VEGF的表达而加速其移动。     

氮、磷和钾肥对灯盏花生长和有效成分积累的影响

目的 探讨N、P和K肥在植物生物量和黄酮类次生代谢产物积累中的影响作用,揭示药用植物生物量积累与次生代谢有效成分积累的关系,为建立合理的施用技术提供理论基础.方法 以尿素、磷酸和氯化钾3种N、P和K肥按正交设计组成3因素3水平的9个处理组合.用盆栽法比较灯盏花开花植株的生物量、总咖啡酸酯和灯盏乙素的量.结果 生物量适宜高N、P和K施肥量组合,有效成分量则偏向低施肥量组合.3肥素中,N作用最强,对生物量积累具有显著的促进作用,对有效成分积累有显著的抑制作用,对有效成分产量的作用不显著.P对有效成分量的作用高于K,但对生物量的影响低于K.K对有效成分产量的作用最大.结论 N、P和K施肥提高灯盏花药材生物产量时将会导致黄酮等次生代谢有效成分量下降,但可提高其产量.