A Network Pharmacology Approach to Uncover the Molecular Targets and Associated Potential Pathways of Lycii Fructus for the Treatment of Retinitis Pigmentosa

Objective To explore the molecular targets and associated potential pathways of Lycii Fructus(LF,Gou Qi Zi,枸杞子)in the treatment of retinitis pigmentosa(RP)by the approaches of network pharmacology and bioinformatics.Methods The potential blood-entry active ingredients and targets of LF were retrieved by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).RP-related gene targets were retrieved through disease comprehensive databases.Protein-protein interaction(PPI)network of LF component-targets and RP disease-targets was constructed by STRING,and the intersection of the 2 networks was extracted.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis of theintersection network were conducted by Database for Annotation,Visualization and Integrated Discovery(DAVID).CytoHubba was used to screen the key targets.Results A total of 188 chemical constituents related to LF was retrieved from TCMSP database.45 active ingredients were screened according to pharmacokinetic parameters oral bioavailability(OB)and drug similarity(DL).36 active ingredients were further screened and 201 targets related to these constituents were obtained.206 target genes directly related to RP were obtained from the disease comprehensive databases,and 89 genes were obtained from the intersection of componenttarget and disease-target PPI network.These genes were mainly involved in intracellular signal transduction,GTPase activity regulation,cell morphology regulation,and other biological processes.Molecular functions were mainly related to Rho guanine nucleotide exchange factor activity,GTPase activator activity,receptor signal protein serine/threonine kinase activity and so on.They were enriched in the cytoplasm,cell membrane,Golgi apparatus,and other regions.The mechanism was related to cell cycle pathways,neurotrophin signaling pathways,Ras signaling pathways,and so on.10 key gene targets of LF in the treatment of RP were screened.Conclusions The material basis for LF to exert its pharmacodynamic effect is 36 active ingredients such as cycloartenol,mandenol,and so on.The key targets of LF in the treatment of RP include 10 genes,such as Rho,PAK,and so on.The main mechanism is related to the regulation of the Ras signaling pathway,neurotrophin signaling pathway,cell cycle related pathway,and other signaling networks.

Effects of Lycii Fructus and Salviae Miltiorrhizae on the Syndrome of Deficiency with Blood Stasis in RCS(rdy-/-, p-/-) Rats with Retinitis Pigmentosa: An Intervention Study

Objective To investigate the effects of Lycii Fructus(LF,Gou Qi Zi,枸杞子)and Salviae Miltiorrhizae Radix Ex Rhizoma(SM,Dan Shen,丹参)on the syndrome of deficiency with blood stasis in the RCS(rdy-/-,p-/-)rats with retinitis pigmentosa(RP).Methods A total of 32 RCS(rdy-/-,p-/-)rats were divided into 4 groups(equal amounts of female and male rats in each group):model group treated with 0.9%normal saline,LF group treated with LF formula granules,SM group treated with SM formula granules,and LF and SM(L·S)group treated with LF and SM formula granules.Eight RCS(rdy+/+,p+/+)rats(4 males and 4 females)were treated with 0.9%normal saline to serve as blank group.The contents of E2,PG,P-Selectin,plasma viscosity,whole blood relative index of the high shear rate and fibrinogen content in plasma,and the content of cAMP and cGMP in retinal homogenate were detected.The retina was evaluated by hematoxylin-eosin staining.Results The contents of E2,PG,P-Selectin,plasma viscosity,whole blood relative index of the high shear rate,and fibrinogen content in the plasma of L·S group significantly differed from those of model group(P<0.01),but were similar to those of blank group.The contents of cAMP and cGMP in the retinal homogenate of L·S group significantly differed from those in model group(P<0.01)but were similar to those in blank group(P>0.05).Conclusions LF and SM can effectively treat retinitis pigmentosa by ameliorating the syndrome of deficiency with blood stasis.

枸杞子-丹参诱导穆勒细胞重编程治疗视网膜色素变性的实验研究

探讨滋阴活血明目药对枸杞子-丹参(Lycii Fructus-Salviae Miltiorrhizae Radix, LFSMR)抑制穆勒细胞(Müller cell,MC)胶质化,促进MC重编程转分化为视网膜神经细胞治疗视网膜色素变性(retinitis pigmentosa, RP)的效应及机制。将12只C57小鼠作为正常对照组,48只转基因RP(rd10)小鼠随机分为模型组、阳性对照组和LFSMR低、高剂量组,每组12只。HE染色检测视网膜病理改变,视网膜电图检测视网膜功能,眼底光学相干断层扫描检测视网膜厚度并进行眼底照相,激光散斑灌注成像检测视网膜局部血流,数字PCR检测视网膜神经细胞相关基因表达,免疫荧光法检测视网膜神经细胞相关蛋白表达。LFSMR可明显改善rd10小鼠视网膜病理改变;LFSMR可明显增加小鼠视网膜a波和b波振幅;LFSMR可显著增加小鼠视网膜厚度;LFSMR可明显恢复RP病变过程小鼠眼底损伤;LFSMR可显著提升RP病变过程小鼠视网膜血流量;LFSMR可显著抑制RP发病过程胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)mRNA表达,上调性别决定域Y链蛋白2(sex determining region Y box protein 2, SOX2)、双链复合蛋白6(paired box protein 6, Pax6)、视紫红质(rhodopsin)、蛋白激酶C-α(protein kinase C-α, PKCα)、突触融合蛋白(syntaxin)、胸腺细胞抗原1.1(thymic cell antigen 1.1, Thy1.1)mRNA表达;LFSMR可显著抑制GFAP蛋白表达,提升SOX2、Pax6、rhodopsin、PKCα、syntaxin、Thy1.1蛋白表达。LFSMR可逆转rd10小鼠视网膜病理改变,改善视网膜功能和眼底表现,增加视网膜厚度,提升视网膜局部血流量,发挥治疗RP的效应。LFSMR的作用机制可能与抑制穆勒细胞胶质化,促进穆勒细胞重编程转分化为各类视网膜神经细胞有关。

丹参对视网膜色素变性病理过程Müller细胞特征性基因变化及关键蛋白表达的影响

目的基于网络药理学和生物信息学方法探讨丹参(SM)通过干预Müller细胞(MC)特征性基因和关键蛋白表达治疗视网膜色素变性(RP)的分子机制。方法运用中药系统药理学数据库和分析平台(TCMSP)检索、筛选SM的入血活性成分和作用靶点;通过GEO数据库获取正常和RP小鼠MC差异表达基因;通过疾病数据库检索RP相关的基因靶点;采用Cytoscape构建MC差异表达基因和疾病靶点、成分靶点的蛋白质-蛋白质交互作用网络并提取交集;运用DAVID数据库对特征性基因进行基因本体和KEGG通路分析;采用cytoHubba分析筛选关键蛋白靶点。结果检索得到与SM相关的化学成分202个,依据ADME参数筛得活性成分65个,其中入血活性成分13个,进一步检索配对得到这些成分可能作用的靶点117个;从芯片数据中分析得到正常和RP小鼠MC中差异表达基因242个;从疾病数据库获得与RP密切相关的靶点206个;提取交集得到85个SM影响RP病理过程MC的特征性基因;这些基因主要涉及转录调控、凋亡信号通路调控、DNA核内复制调节等生物学过程,分子功能主要包括转录辅激活子活性、蛋白激酶活性、核心启动子结合等,富集于细胞核、核质、转录因子复合物、Rb-E2F复合体等区域,主要与剪接体信号通路、肌动蛋白细胞骨架调控信号途径、细胞周期调控通路等有关;分析筛选出SM干预RP病理过程MC中的8个关键蛋白靶点。结论 SM药效作用的物质基础为隐丹参酮、木犀草素、丹参酮ⅡA等13个化学成分;其所干预的RP病理过程MC特征性基因与剪接体信号通路、肌动蛋白细胞骨架调控信号途径、细胞周期调控通路等机制相关,关键靶点包括RB1、E2F1、TFDP1等8个蛋白。

益气养阴活血利水复方联合玻璃体内注射曲安奈德对非增生性糖尿病视网膜病变黄斑水肿患者TNF-α、sICAM-1的影响

目的:观察益气养阴活血利水复方联合玻璃体内注射曲安奈德(TA)对非增生性糖尿病视网膜病变黄斑水肿(DME)患者血清和玻璃体液中TNF-α、sICAM-1含量和临床疗效的影响,从而探索其治疗的分子作用机制。方法:将60例(71眼)非增生性糖尿病视网膜病变DME患者随机分为治疗组(30例,36眼)和对照组(30例,35眼)。两组均予以玻璃体内注射TA,每月注射1次,共注射3次,治疗组同时予以益气养阴活血利水中药口服,服用2个月。观察比较两组患者治疗前和治疗1、2个月后血清和玻璃体液中TNF-α、sICAM-1含量差异,观察比较两组患者治疗前和治疗1、2、5个月后视力、视网膜厚度、黄斑区渗漏及治疗随访期间并发症发生情况。结果:治疗1、2个月后,治疗组血清、玻璃体液中TNF-α、sICAM-1含量均显著低于对照组(P<0.05,P<0.01)。治疗组患者患眼在治疗1、2、5个月后视力、视网膜厚度情况均显著优于对照组(P<0.05,P<0.01),在治疗2、5个月后黄斑区渗漏情况少于对照组(P<0.05,P<0.01)。治疗及随访期间,治疗组眼压升高眼数少于对照组(P<0.05)。结论:益气养阴活血利水复方联合玻璃体内注射TA能够更好、更持久地减轻非增生性糖尿病视网膜病变DME患者黄斑区水肿和渗漏,提高患者的视功能可能与其能够明显持续降低患者血清与玻璃体液中TNF-α、sICAM-1等炎性因子质量浓度密切相关。

密蒙花滴眼液对去势兔干眼模型泪腺细胞凋亡相关因子Caspase-3、Caspase-8的干预作用

目的通过建立去势兔干眼模型,观察密蒙花滴眼液对其泪腺细胞凋亡相关因子Caspase-3、Caspase-8的干预作用。方法将35只新西兰白兔随机分为正常组(A组)、模型组(B组)、0.3、0.5、1.0 mg/mL、1.5、3.0 mg/mL密蒙花滴眼液(C、D、E、F、G组)。A、B组不予处理,C、D、E、F、G组分别予以相应密蒙花滴眼液局部滴眼,3次/日。疗程为4周。造模前、用药前及用药后检测各组基础泪液分泌试验(Schirmer′s test I,SIT)水平,免疫组化检测各组泪腺组织中Caspase-3、Caspase-8的表达情况,Real-time PCR法检测各组泪腺组织中Caspase-3、Caspase-8 mRNA的相对表达情况。结果与A比较,用药前及用药后B组SIT值降低(P<0.01);与B组比较,各治疗组用药后SIT值升高(P<0.01);各治疗组用药后与本组用药前比较,SIT值升高(P<0.01)。与A组比较,B组Caspase-3、Caspase-8表达及mRNA相对表达量升高(P<0.01)。与B组比较,D、F、G组Caspase-3及C、D、E、F、G组Caspase-8表达降低(P<0.01);C、D、E、F、G组Caspase-3及D、E、F、G组Caspase-8 mRNA相对表达量降低(P<0.01)。与C组比较,Caspase-3表达降低(P<0.05);D、E、F、G组Caspase-8 mRNA相对表达量降低(P<0.01)。除D与G组比较外,其他各浓度组间Caspase-3 mRNA相对表达量比较均有统计学意义(P<0.01)。结论密蒙花滴眼液可以通过下调去势兔干眼模型泪腺细胞凋亡因子Caspase-3和Caspase-8的表达,达到维持泪腺基础分泌量和保护泪腺的作用。

彭清华辨治干眼经验

介绍彭清华教授多年治疗干眼的经验,分为肺阴不足证、肝经郁热证、气阴两虚证、邪热留恋证4种证型,治疗上常用柴胡、密蒙花、枸杞子等药物,重视阳虚与干眼的关系,强调治未病。临床上取得了较好的疗效,列举3例,以供大家学习参考。