Screening of Anti-inflammatory Active Ingredients from Gancao Qinlian Extract and Study of Its Efficacy

[Objectives]To establish the chromatographic fingerprint of Gancao Qinlian Extracts(GQE)and reveal the possible material basis of the anti-inflammatory effect of GQE by the correlation analysis between the fingerprint chromatographic peaks of different components of GQE and its anti-inflammatory activity.[Methods]Ultra-performance liquid chromatography(UPLC)was used to detect the different ingredients of GQE to establish its chromatographic fingerprint and analyze the differences among the three medicine components;LPS stimulated RAW264.7 cells to construct an inflammatory cell model.The NO secretion of cells was detected by the Griess method.ELISA was used to detect the changes in TNF-αand IL-10 contents.RT-qPCR tested the mRNA expression levels of TNF-αand IL-10.Grey relational analysis was carried out by combining fingerprint chromatographic peak data and anti-inflammatory activity data.[Results]The GQE fingerprint was established,34 fingerprint characteristic peaks were calibrated,and 33 related chromatographic peaks were screened out.The corresponding chromatographic peaks in the three components were obtained,and the content of the components was calculated;the anti-inflammatory results showed that the content of NO,TNF-α,and the expression of TNF-αmRNA in the high and medium-dose groups of GQE were significantly lower than those in the blank group(P<0.01).The NO content and TNF-αmRNA expression in the high-dose group of GQE I was considerably lower than those in the blank group(P<0.01).The secretion of NO,TNF-α,and the expression of TNF-αmRNA in the high,medium,and low dose groups of GQE II were significantly lower than those in the blank group(P<0.01);the results of grey relational analysis showed that the correlation degree of the three components was GQE II>GQE>GQE I,and the characteristic fingerprint peaks 12,15,22,23,28,31,33 may be closely related to the anti-inflammatory effect.[Conclusions]The best component of the anti-inflammatory effect in GQE is water-soluble component,and its main components are flavonoids and alkaloids.These components can alleviate cellular inflammatory damage by inhibiting the excessive secretion of NO and reducing the expression of TNF-αmRNA.

Therapeutic targets and signal transduction mechanisms of medicinal plant formula Gancao Xiexin decoction against ulcerative colitis:A network pharmacological study

Background:Ulcerative colitis(UC)is a chronic disease that often presents with abdominal pain,diarrhea,hematochezia,and significant morbidity.Gancao Xiexin decoction(GXD),a traditional Chinese medicine,has been applied for the clinical treatment of UC,while its action mechanisms are unclear.Methods:The active ingredients and their targets of GXD,and UC-related targets,were derived from public databases.Protein-protein interaction,Gene Ontology(GO),and the Kyoto Encyclopedia of Genes and Genomes(KEGG)were used to analyze the important active compounds,key targets,and signaling pathways.Then,molecular docking and animal experiments were performed to verify the findings.A total of 213 active compounds and 89 common targets of GXD for UC were obtained.Results:The hub gene network showed ALB,AKT1,IL6,TNF,VEGFA,TP53,CXCL8,MAPK1,PTGS2,and IL1βmay be potential targets of GXD against UC.GO and KEGG pathway enrichment analyses suggested that the action of GXD against UC was mainly related to response to oxygen levels,lipopolysaccharide,and molecule of bacterial origin,etc.,and achieved by advanced glycation endproducts/receptors for advanced glycation endproducts signaling pathway in diabetic complications,hypoxia-inducible factor(HIF)-1 signaling pathway,interleukin-17/HIF-1 signaling pathway,TNF signaling pathway,etc.Molecular docking results showed that the GXD had good potency of action with the hub target.In vivo experiments showed that GXD significantly alleviated the symptoms of UC and down-regulated the expression of inflammatory factors,nuclear factor-κB and signal transducer and activator of transcription 3.Conclusions:The anti-UC action of GXD is mainly attributed to its anti-oxidative stress,antiinflammatory,and immunomodulatory functions.

Anti-inflammatory Effect of Wumen Zhike Gancao Decoction on Rats with Lumbar Disc Herniation Associated with Lipid Metabolic Disorder

[Objectives] To study the anti-inflammatory effect of Wumen Zhike Gancao Decoction (ZKGC) on rats with lumbar disc herniation (LDH) associated with lipid metabolic disorder.[Methods] A rat model of LDH was established by implantation of the autologous nucleus pulposus from the coccygeal vertebra of each rat tail, and histopathology, immunohistochemistry and biochemistry assays were employed to evaluate the treatment effects of ZKGC. In addition, the metabolic characteristics of LDH and ZKGC treatment were investigated with a liquid-chromatography with time-of-flight mass spectrometer (LC/Q-TOF-MS)-based metabolomics study. Nucleus pulpous tissues from rat models were collected and analyzed by metabolomics.[Results] By metabolism network analysis, lipid metabolism was up-regulated in LDH rat models and the treatment with ZKGC significantly reversed the abnormal up-regulated lipid metabolism. Meanwhile, the treatment of ZKGC also regulated the markers of neuron autophagy and inflammatory response in serum.[Conclusions] These results indicated that a complex mechanism, including abnormal lipid metabolism, associates with the progress of LDH, and multiple pathways might be involved in ZKGC s therapeutic effects on LDH.

Network pharmacology study on the mechanism of Shaoyao Gancao decoction in treating cancer pain

Objective:This paper aims to explore the key targets and mechanism of action of Shaoyao Gancao Decoction in the treatment of cancer pain.Methods:TCMSP database was adopted to screen the active components and potential targets of Shaoyao Gancao Decoction.Genecards and OMIM databases were used to collect disease targets for cancer pain.Cytoscape software was used to construct the drug-component-target-disease network diagram.STRING database was used to draw PPI network.Finally,gene ontology(GO)enrichment analysis and KEGG pathway enrichment analysis were performed on key targets.Results:There were 98 potential targets for the treatment of cancer pain in Shaoyao Gancao Decoction.The protein interaction network suggested that IL-6,VEGFA,CASP3,EGFR and MAPK8 may be the core targets for the treatment of cancer pain in Shaoyao Gancao Decoction.GO enrichment analysis showed 127 cellular biological processes,and KEGG pathway enrichment analysis showed 116 related signaling pathways,including MPAK,AGE-RAGE,TNF,ErbB and so on.Conclusions:The treatment of cancer pain by Shaoyao Gancao Decoction may be multi-target,multi-channel and multi-level.This consequence may provide ideas and basis for further research.

Effects of Gancao Nourish-Yin Decoction on Liver Metabolic Profiles in hTNF-αTransgenic Arthritic Model Mice

Objective Gancao Nourish-Yin Decoction(GNYD)has been applied to clinical rheumatoid arthritis(RA)patients,and it had shown effectiveness not only in disease activity controlling but also in improving patients'physical status.However,its mechanism of function has not been investigated.Metabolic perturbations have been associated with RA,and targeting the metabolic profile is one of the ways to manage the disease.The aim of this study is to observe the effect of GNYD on metabolic changes of human tumor necrosis factorα(hTNF-α)transgenic arthritic model mice.Methods hTNF-αtransgenic arthritic model mice were divided into the control group and the GNYD group with six mice in each group.After 8 weeks of treatment,liver tissues of mice in both groups were obtained for liquid chromatography-mass spectrometry analysis.Significantly regulated metabolites by GNYD treatment were first identified,followed by Kyoto Encyclopedia of Genes and Genomes pathway and network analysis.Results A total of 126 metabolites were detected in the liver.Compared with the control group,17 metabolites in the GNYD group were significantly altered.Specifically,thiamine,gamma-L-glutamyl-L-valine,pantothenic acid,pyridoxal(vitamin B6),succinic acid,uridine 5′-diphospho-glucuronic acid,uridine,allantoic acid,N-acetyl-D-glucosamine,nicotinamide ribotide,and N2,N2-dimethylguanosine were down-regulated by GNYD treatment,whereas isobutyrylglycine,N-acetylcadaverine,N-carbamoyl-L-aspartic acid,L-anserine,creatinine,and cis-4-hydroxy-D-proline were up-regulated.Six metabolic pathways were significantly altered including the alanine,aspartate,and glutamate metabolism;pyrimidine metabolism;thiamine metabolism;amino sugar and nucleotide sugar metabolism;pantothenate and CoA biosynthesis;and citrate cycle.Integrative metabolic network analysis suggested the possibility of GNYD having both positive and negative effects on RA through the suppression of angiogenesis and the promotion of leukocyte extravasation into the synovium,respectively.Conclusions GNYD can modulate the hepatic metabolism of hTNF-αtransgenic arthritic model mice.Further optimization of this decoction may lead to better therapeutic effects on RA patients.

Exploration of the Potential Mechanism of Gancao Yangyin Decoction on Aging Based on Network Pharmacology

Objective:To explore the targels and molecular mechanism of Gancao Yangyin Decoction(甘草养阴汤,GCYYD)based on network pharmacology.Methods:The effective chemical components of 7 kinds of Chinese materia medica in GCYYD and their relevant targets were obtai ned through the traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP)and the encyelopedia of traditional Chinese medicine(ETCM).The aging-related targets were obtained through GeneCards database.The targets related to the effective chemical components were mapped with the aging-related targets,and the gene targets of GCY YD for intervening in aging were obtained.The protein interaction network diagram of GCY YD interfering with aging was drawn through String database and Cytoscape 3.7.1 software,and the core target genes were screened.The potential targets obtained were analyzed by gene ontology(GO)biological function enrichment analysis and kyoto encyelopedia of genes and genomes(KEGG)pathway enrichment analysis.Results:Totally 130 effective chemical components of the 7 kinds of Chinese materia medica of GCYYD and 276 related targets were obtained from TCMSP and ETCM databases.Totally 216 aging-related targets were obtained through GeneCards database.There were 63 target genes intervening in aging in GCYYD,with core target genes ALB,AKTI,TNF,L 6,MMP-3,VEGFA,CASP5,etc.Through biological function and signaling pathway enrichment analyses for the target genes with R software,147 KEGG signaling pathways were found,mainly related to age-RAGE signaling pathway in diabetic complications,proteoglycans in cancer,fluid shear stress and atherosclero-sis,HIF-1 signaling pathway,human cytomegalovirus infection,celluar senescence,prostate cancer,bladder cancer,elte.Conclusion:GCYYD can intervene in aging through"multicomponents-mulitargets-multipath-ways",which lays foundation for further experimental research.

Therapeutic Effect of Shaoyao Jiawei Gancao Decoction(芍药加味甘草汤)with Acupoint Application on Chronic Lumbar Muscle Strain and the Recovery of Lumbar Strength

Objective:To study the therapeutic effect of Shaoyao Jiawei Gancao Decoction(芍药加味甘草汤)with acupoint application on chronic lumbar muscle strain and the recovery of lumbar strength.Methods:From August 2018 to August 2019,100 patients with chronic lumbar muscle strain admitted to our hospital were selected and randomly divided into an acupoint application group and a combined group of 50 cases each.The acupoint application group was treated by acupoint application,and the combined group was treated with Shaoyao Jiawei Gancao Decoction(芍药加味甘草汤)based on acupoint application.The indexes scores,VAS scores,ADL scores,ODI scores,lumbar endurance,TCM syndrome scores and the treatment effects of the two groups before and after treatment were statistically analyzed,and TNF-αand TXB2 levels were detected.Results:After treatment,the scores of indexes,VAS,ODI,TCM syndrome,TNF-αand TXB2 in the combined group were lower than those in the acupoint application group,and the ADL score,lumbar endurance and treatment efficiency were higher than those in the acupoint application group,with a statistical difference(P<0.05).Conclusion:Shaoyao Jiawei Gancao Decoction(芍药加味甘草汤)with acupoint application can effectively reduce pain,improve lumbar function,restore lumbar strength,effectively relieve inflammation,with significant effects.

Neuroprotective effects of Shaoyao Gancao decoction against excitatory damage in PC12 cells based on the Src-NR2-nNOS pathway

Objective:To explore the neuroprotective effects of the Shaoyao Gancao decoction(SGD)against excitatory damage in PC12 cells and the role of the Src-NR2-nNOS pathway mediation by SGD in regulatingγ-aminobutyric acid(GABA)-glutamate(Glu)homeostasis.Methods: N-Methyl-d-aspartic acid(NMDA)was used to establish a PC12 cell excitability injury model.To investigate the neuroprotective effect of SGD,a cell counting kit-8(CCK-8)assay was used to determine PC12 cell viability,Annexin V/Propidium Iodide(Annexin V/PI)double staining was used to determine PC12 cell apoptosis,and Ca^(2+)concentration was observed using laser confocal microscopy.GABA receptor agonists and antagonists were used to analyze the neuroprotective interactions betweenγ-aminobutyric acid(GABA)and NMDA receptors.Additionally,molecular biology techniques were used to determine mRNA and protein expression in the Src-NR2-nNOS pathway.We analyzed the correlations between the regulatory sites of GABA and NMDA interactions,excitatory neurotoxicity,and brain damage at the molecular level.Results: NMDA excitotoxic injury manifested as a significant decrease in cell activity,increased apoptosis and caspase-3 protein expression,and a significant increase in intracellular Ca^(2+)concentration.Administration of SGD,a GABAA receptor agonist(muscimol),or a GABAB receptor agonist(baclofen)decreased intracellular Ca^(2+)concentrations,attenuated apoptosis,and reversed NMDA-induced upregulation of caspase-3,Src,NMDAR2A,NMDAR2B,and nNOS.Unexpectedly,a GABA_(A)receptor antagonist(bicuculline)and a GABA_(B)receptor antagonist(saclofen)failed to significantly increase excitatory neurotoxicity.Conclusions: Taken together,these results not only provide an experimental basis for SGD administration in the clinical treatment of central nervous system injury diseases,but also suggest that the Src-NR2A-nNOS pathway may be a valuable target in excitotoxicity treatment.

芍药甘草汤加味联合针灸对骨质疏松性椎体压缩性骨折术后Oswestry功能障碍指数的影响

【目的】探析芍药甘草汤加味联合针灸治疗对骨质疏松性椎体压缩性骨折(OVCF)患者术后Oswestry功能障碍指数(ODI)的影响。【方法】将94例肝肾不足、阴血亏虚型OVCF患者随机分为观察组和对照组,每组各47例。2组患者均接受手术治疗(包括经皮穿刺球囊椎体后凸成形术或经皮椎体成形术),术后对照组接受针灸治疗,观察组接受芍药甘草汤加味联合针灸治疗,连续治疗12周(共3个月)。观察2组患者术后不同观察时点的疼痛程度视觉模拟量表(VAS)评分、ODI评分、骨密度[腰椎第2~4节椎体(L2~4)、股骨颈、Wards三角、全髋]、骨代谢指标[人Ⅰ型胶原C端肽(CTX-1)、骨碱性磷酸酶(BALP)、血钙]血清水平的变化情况,并评价2组患者的临床疗效。【结果】(1)疗效方面,治疗3个月后,观察组的总有效率为100.00%(47/47),对照组为87.23%(41/47),组间比较(Fisher精确检验),观察组的临床疗效明显优于对照组(P<0.01)。(2)疼痛程度及胸腰椎功能评分方面,术后第7天及1个月和3个月,2组患者的疼痛程度VAS评分及胸腰椎ODI评分均较前一观察时点明显下降(P<0.05),且观察组在术后第7天及1个月和3个月对疼痛程度VAS评分及胸腰椎ODI评分的下降幅度均明显优于对照组(P<0.05或P<0.01)。(3)骨密度方面,治疗后(术后1个月),2组患者的L2~4、股骨颈、Wards三角、全髋骨密度值均较治疗前明显升高(P<0.05),且观察组对L2~4、股骨颈、Wards三角、全髋骨密度值的升高幅度均明显优于对照组(P<0.01)。(4)骨代谢指标方面,治疗后(术后1个月),2组患者的血清CTX-1、BALP和血钙水平均较治疗前明显下降(P<0.05),且观察组对血清CTX-1、BALP和血钙水平的下降幅度均明显优于对照组(P<0.01)。【结论】芍药甘草汤加味联合针灸治疗肝肾不足、阴血亏虚型OVCF患者疗效确切,能显著减轻患者术后疼痛症状,改善患者骨密度和骨代谢,促进胸腰椎功能恢复,有效提升其整体治疗效果。

甘草抗炎活性物质基础及其作用机制研究进展

炎症是机体对于刺激所产生的一种防御反应,随着炎症的发展可能导致组织的变异和增生,进一步加剧疾病的进程。甘草作为“众药之王”“国之药老”,早在《神农本草经》中就记载其清热作用较强,能“主五脏六腑寒热邪气”。现代研究表明,甘草提取物对肾小球肾炎、结肠炎症状改善明显,甘草及其制剂在治疗肝炎、胃炎、支气管炎、心肌炎、强直性脊柱炎等炎症性疾病方面亦具有一定的独特优势和发展潜力。该文旨在通过对近期国内外甘草抗炎活性物质基础及其作用机制方面的相关文献进行收集和整理,探索甘草发挥抗炎作用的主要活性成分及靶点通路,以期为甘草抗炎作用的进一步研究及抗炎药物的研发提供思路和参考。

《方剂学》教材商榷5则

《方剂学》教材中存在一些值得商榷的问题,如安神药服药时间、麻黄杏仁甘草石膏汤证是否兼有外邪、四逆散证有无外邪的方义解释、牡蛎散是否治疗盗汗、大秦艽汤的分类归属等。这些问题不单涉及到教学,更影响临床应用,对此作一探讨,以有利于中医教学与临床。

基于网络药理学方法和分子对接技术探讨芍药甘草汤合桃红四物汤治疗糖尿病周围神经病变的作用机制

目的:运用网络药理学方法及分子对接技术探讨芍药甘草汤合桃红四物汤治疗糖尿病周围神经病变(DPN)的作用机制。方法:利用中药系统药理学数据库与分析平台(TCMSP)数据库筛选芍药甘草汤合桃红四物汤的活性成分及相关靶点信息;从DrugBank、PharmGKB、GeneCards、OMIM和TTD数据库中获取DPN的靶点基因。运用Cytoscape软件、STRING数据库构建芍药甘草汤合桃红四物汤活性成分-DPN共同靶点基因关系网络及共同靶点基因的蛋白质相互作用(PPI)网络。利用R语言软件进行基因本体论(GO)功能富集分析和京都基因与基因组百科全书(KEGG)通路富集分析。采用AutoDock Vina软件进行分子对接。结果:共获得161个芍药甘草汤合桃红四物汤活性成分,184个芍药甘草汤合桃红四物汤活性成分-DPN共同靶点基因。核心活性成分为槲皮素、木犀草素、山奈酚、柚皮素、7-甲氧基-2-甲基异黄酮,核心靶点基因为信号传导及转录激活蛋白3(STAT3)、丝裂原活化蛋白激酶3(MAPK3)、热休克蛋白90AA1(HSP90AA1)、丝裂原活化蛋白激酶1(MAPK1)、转录因子p65(RELA)。GO功能富集分析得到2940条GO条目,KEGG通路富集分析得到182条信号通路。分子对接结果显示,木犀草素与HSP90AA1的结合活性最好。结论:芍药甘草汤合桃红四物汤具有多成分、多靶点、多通路的效应特点,其作用机制可能是槲皮素、木犀草素、山奈酚、柚皮素、7-甲氧基-2-甲基异黄酮等活性成分作用于STAT3、MAPK3、HSP90AA1、MAPK1、RELA等靶点基因,调控磷脂酰肌醇3-激酶/蛋白激酶B(PI3K-Akt)、晚期糖基化终末产物-晚期糖基化终末产物受体(AGE-RAGE)、白细胞介素-17(IL-17)、肿瘤坏死因子(TNF)、缺氧诱导因子1(HIF-1)等多条信号通路,通过抗氧化应激、抑制炎症反应、保护神经细胞、改善胰岛素抵抗发挥治疗DPN的作用。

雷火灸联合加味芍药甘草汤对单开门颈椎管扩大椎板成形术后轴性症状的临床疗效

目的:探讨雷火灸联合加味芍药甘草汤治疗单开门颈椎管扩大椎板成形术后轴性症状的临床效果,探索治疗术后轴性症状新途径。方法:选取2021年10月—2023年10月于赣州市人民医院脊柱外科就诊的单开门颈椎管扩大椎板成形术后轴性症状且中医辨证属于气血不足症型的70例患者,通过随机数字表法将其分为试验组和对照组,各35例。试验组采用雷火灸联合加味芍药甘草汤内服的治疗方案,对照组采用常规西药治疗。采用视觉模拟评分法(visual analogue scale,VAS)、颈椎日本骨科协会(Japanese orthopaedic association,JOA)评分、C反应蛋白和红细胞沉降率评估治疗效果。结果:治疗前,两组VAS评分、颈椎JOA评分及C反应蛋白和红细胞沉降率比较,差异均无统计学意义(P>0.05);治疗后,试验组C反应蛋白和红细胞沉降率均低于对照组;治疗后2、4周,试验组VAS评分均低于对照组;治疗后4周,颈椎JOA评分高于对照组,差异有统计学意义(P<0.05)。结论:雷火灸配合加味芍药甘草汤治疗颈椎单开门椎管扩大成形术后轴性症状的效果较好,能够有效缓解疼痛,改善脊髓功能障碍,缓解轴性症状。

刘小斌运用麻附细辛汤合甘草干姜汤加减治疗先天性肌无力综合征经验

先天性肌无力综合征(congenital myasthenia syndrome,CMS)为临床罕见的遗传性肌病。CMS患者虽与重症肌无力患者的症状相似,均有肌无力、易疲劳等症,但CMS为先天禀赋不足疾病,以肾元亏损为本,而重症肌无力为后天失养疾病,故在辨治侧重上有所区别。刘小斌教授认为,CMS的中医病机特点为“先天不足,肾元亏损”“脾胃虚损,五脏相关”,属于中医“痿病”范畴;根据患者不易出汗、舌淡红苔白、脉细的病症表现,可辨为脾肾阳虚兼寒邪束表证(少阴太阴兼太阳证);治疗可采用温阳补肾、健脾益损法,常以麻附细辛汤合甘草干姜汤加减,可酌加桑螵蛸、紫河车等益精养血之品,可获显效。

甘草泻心口腔护理液改善脑卒中患者口腔状态的临床研究

目的探讨甘草泻心口腔护理液(甘草泻心口护液)改善脑卒中患者口腔状态的效果。方法采用随机对照试验,选取2021年12月至2023年10月在河南中医药大学第一附属医院神经外科治疗的80例脑卒中患者作为研究对象,以随机数表法分为对照组和观察组,各40例。对照组男25例、女15例,年龄(52.20±4.95)岁,病程(1.84±0.53)个月,予以生理盐水进行口腔护理,2次/d,20 ml/次,治疗10 d;观察组男26例、女14例,年龄(52.20±6.37)岁,病程(1.84±0.53)个月,采用甘草泻心口护液进行口腔护理,2次/d,50 ml/次,治疗10 d。对比两组患者治疗前后牙龈指数、菌斑指数、口腔pH值、口唇湿润度、口腔异味、口腔黏膜炎及不良反应发生情况。统计学方法采用t检验、χ^(2)检验。结果治疗前,两组患者牙龈指数、菌斑指数、口腔pH值、口唇湿润度、口腔异味发生情况比较,差异均无统计学意义(均P>0.05)。治疗5 d、10 d,观察组牙龈指数、菌斑指数、口唇湿润度及口腔异味情况均低于对照组[(1.23±0.42)分比(1.98±0.16)分、(0.65±0.48)分比(1.10±0.30)分、(1.44±0.50)分比(1.93±0.27)分、(0.78±0.48)分比(1.20±0.52)分、(0.65±0.53)分比(0.98±0.42)分、(0.15±0.36)分比(0.65±0.53)分、(2.28±0.45)分比(2.50±0.51)分、(1.45±0.60)分比(1.90±0.38)分],口腔pH值均高于对照组[(6.53±0.08)比(6.42±0.11)、(6.98±0.10)比(6.62±0.14)],差异均有统计学意义(t=10.506、4.987、5.416、3.814、3.086、4.936、2.096、4.007、5.115、13.234,均P<0.05)。治疗后,观察组口腔黏膜炎发生率为5.0%(2/40),低于对照组的15.0%(6/40),差异有统计学意义(χ^(2)=6.486,P<0.05)。两组患者治疗期间均无不良反应情况发生。结论甘草泻心口护液能有效改善脑卒中患者口腔状态,操作简便安全,患者依从性高,值得临床进一步推广应用。

芍药甘草汤通过JAK2/STAT3信号通路对神经病理性疼痛大鼠的镇痛及对免疫调节机制影响

目的探寻芍药甘草汤对神经病理性疼痛(NP)的改善作用及其机制。方法采用坐骨神经分支选择性损伤(SNI)法建立NP大鼠模型,模型制备成功后予以芍药甘草汤灌胃治疗14 d。治疗前后监测大鼠疼痛行为学指标变化,TUNEL染色检测脊髓背角细胞凋亡情况,免疫荧光组织化学法测定p-STAT3在脊髓中与Iba1的共定位情况,RT-PCR检测CD68、SOCS3、Arg-1的mRNA表达,ELISA检测白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)表达,Western blotting检测JAK2/STAT3通路蛋白表达。结果与假手术组比较,模型组大鼠机械缩足阈值(MWT)、热缩足潜伏期(TWL)明显降低(P<0.01),脊髓背角中凋亡细胞显著增加(P<0.05),IL-1β、1L-6和TNF-α的表达显著增加(P<0.01),JAK2、p-JAK2、STAT3、p-STAT3的表达显著增加(P<0.01),术后7、14 d的CD68、SOCS3 mRNA表达显著增加(P<0.01),Arg-1 mRNA表达显著降低(P<0.01)。与模型组比较,芍药甘草汤组大鼠治疗后MWT、TWL明显上升(P<0.01),脊髓背角中凋亡细胞减少,IL-1β、IL-6、TNF-α的水平显著减少(P<0.01),JAK2、p-JAK2、STAT3、p-STAT3蛋白表达水平明显降低(P<0.01),CD68、SOCS3 mRNA表达显著减少(P<0.01),Arg-1 mRNA表达水平显著增加(P<0.01)。结论芍药甘草汤可能通过调控JAK2/STAT3通路调控小胶质细胞极化状态进而发挥改善NP的作用。

基于药材原产地芍药甘草汤质量评价体系研究

目的采用高效液相色谱法(HPLC)同时测定芍药甘草汤中没食子酸、芍药苷、甘草苷、甘草素和甘草酸5种指标成分的含量并建立芍药甘草汤指纹图谱,探究芍药甘草汤中间体关键质量属性,为其药材基源筛选和质量控制提供参考。方法制备来自不同产地不同批次的18批芍药甘草汤冻干粉作为中间体样品;采用Agilent ZORBAX Eclipse Plus C_(18)(4.6 mm×250 mm,5μm)色谱柱,以乙腈-0.2%磷酸水溶液为流动相,梯度洗脱,柱温35℃,流速0.8 mL/min,进样量10μL,对指标成分的含量和指纹图谱进行检测,评价多批次芍药甘草汤中间体质量属性差异,为其质量标准的建立提供参考。结果5种成分线性关系(R^(2)>0.999)、精密度(RSD≤0.89%,n=6)、重复性(RSD≤1.35%,n=6)、稳定性(RSD≤1.87%,72 h)均良好,加样回收率为(98.24±1.85)%~(104.88±1.38)%(n=6)。含量测定结果显示在不同批次芍药甘草汤中间体中仅没食子酸的含量波动范围较小(4.74~5.92 mg/g),芍药苷、甘草苷、甘草素和甘草酸在不同产地不同批次中的含量均有较大差异,含量范围分别为29.58~38.24、7.21~12.12、0.18~0.89、12.14~42.83 mg/g;建立芍药甘草汤指纹图谱,确定了14个共有峰,并对各峰进行药材归属,其中1、4、5、10号峰来自白芍,2、3、6、7、8、9、11、12、13、14号峰来自炙甘草,炙甘草对共有峰的贡献最大。其中13批芍药甘草汤中间体的HPLC指纹图谱与对照指纹图谱的相似度均大于0.98,第S4、S8、S16、S17、S18批次的相似度大于0.91,说明不同产地批次的药材质量差异较大。结论该方法准确可靠、灵敏度高,具有良好的重复性,可用于测定芍药甘草汤中5种成分的含量及建立芍药甘草汤的指纹图谱,可为经典名方芍药甘草汤的质量标准建立及评价提供依据。

“止痛之祖方”芍药甘草汤治疗颈椎病网络药理学机制研究

目的:探究颈椎病中医常用方芍药甘草汤的网络药理学机制。方法:依据BATMAN-TCM数据库和文献查询选出芍药甘草汤(芍药、甘草)的主要活性成分并经Uniprot蛋白质数据库对照后获得其靶点,同时从疾病数据库OMIM、GeneCards等和相关文献获得颈椎病靶点。整理收集芍药甘草汤和颈椎病交集,获得共有靶点。通过蛋白质相互作用网络数据库STRING构建已获得的共有靶点蛋白质-蛋白质相互作用网络图,再用生物信息数据库DAVID平台进行基因本体论(GO)和京都基因与基因组百科全书(KEGG)富集通路分析芍药甘草汤治疗颈椎病过程中可能相关的生物过程和信号通路。结果:(1)筛选出芍药甘草汤的活性成分79个,潜在作用靶点442个;疾病靶点765个,药物、疾病共有靶点45个;(2) GO分析显示芍药甘草汤治疗颈椎病可能是通过调节丝氨酸/苏氨酸蛋白激酶1(Akt Serine/Threonine Kinase 1,AKT1)、白细胞介素-6(Interleukin-6,IL-6)、表皮生长因子受体(Epidermal Growth Factor Receptor,EGFR)、雌激素受体1(Estrogen Receptor 1,ESR1)等靶点,并与蛋白质和DNA结合酶相关,还涉及脂多糖反应、细胞凋亡和分化的负调节、细胞对有机环状化合物的反应等多个生物过程;(3) KEGG富集分析显示芍药甘草汤治疗颈椎病主要与丝裂原活化蛋白激酶(Mitogen-Activated Protein Kinase,MAPK)、核因子-κB(Nuclear Factor-κB,NF-κB)、肿瘤坏死因子(Tumor Necrosis Factor,TNF)等多条信号通路相关。结论:推测芍药甘草汤主要通过调节调节AKT1、IL-6、EGFR、ESR1等靶点以调控MAPK、NF-κB、TNF等信号通路,进而干预颈椎病抗凋亡、黏附、髓核细胞外基质降解、血管的生长、炎症和分解代谢介质水平。

基于整合药理学探讨“茯苓-甘草”协同配伍对肺腺癌的作用机制及验证研究

目的:基于网络药理学和生物信息学探讨“茯苓-甘草”协同配伍对肺腺癌的作用机制及靶点,并通过实验验证“茯苓-甘草”主要成分槲皮素对肺腺癌的抑制作用。方法:对肺腺癌相关基因进行差异分析,从TCMSP数据库下载“茯苓-甘草”有效成分及靶标,获取药物-疾病共同靶标,进行网络构建、富集分析、生存分析等。通过对“茯苓-甘草”主要成分与肺腺癌关键靶点进行分子对接,选出构效关系良好的有效成分进行后续实验验证。通过细胞增殖、划痕及侵袭实验检测药物对肺腺癌细胞的干预作用,实时荧光定量法检测药物对关键靶点的作用。结果:肺腺癌8 109个差异表达基因和102个药物靶基因取交集共获得32个共同基因,富集分析显示靶基因主要参与p53信号通路、细胞衰老等癌症相关通路,分子对接结果显示槲皮素具有良好的构效关系,因此选择槲皮素进行后续实验验证。有效成分槲皮素可以抑制肺腺癌A549及H1299细胞的增殖、迁移及侵袭能力,并且可以显著下调关键靶点含杆状病毒IAP重复序列蛋白5(BIRC5)、细胞周期蛋白B1(CCNB1)、检查点蛋白激酶1(CHEK1)的表达。结论“:茯苓-甘草”有效成分槲皮素可以有效抑制肺腺癌恶性表型,其机制可能与靶向调节关键基因BIRC5、CCNB1、CHEK1的表达有关。

基于UPLC-TOF-MS结合网络药理学探究大黄甘草汤通便止呕作用机制

目的探究大黄甘草汤(DG)通便止呕的作用机制。方法运用UPLC-TOF-MS对DG化学成分进行定性分析,结合网络药理学探讨DG发挥通便止呕功效的潜在作用机制和物质基础,并运用分子对接技术进行验证。结果UPLC-TOF-MS法采用正负离子扫描模式,根据数据库比对和文献数据从DG水煎液中共鉴定27个化合物。网络药理学分析表明DG通便止呕功效的关键成分可能为木犀草素、圣草酚、甘草次酸、大黄素等;关键靶点可能为AKT1、IL6、IL1B、TP53、TNF、EGFR;关键通路可能为胃癌、结直肠癌、IL-17、TNF、HIF-1等信号通路。有效成分与核心靶点分子对接结果良好。结论该研究较全面地阐明了DG水煎液的化学成分,明确了DG通过减轻炎症状态、促进肠道运动等作用发挥通便止呕的功效,可为DG的进一步质量评价和临床研究提供参考。