【作者】 周莉；

【导师】 黄熙；

【作者基本信息】 南京中医药大学 ， 中西医结合基础， 2023， 硕士

【摘要】 背景及目的:21世纪以来,颅脑损伤(Traumatic brain injury,TBI)导致的人类死亡率仅次于癌症、心脑血管疾病。它作为一种多靶点、多病理生理疾病,仅从单病角度研究,容易遭遇瓶颈,出现相关研究发展趋缓的局面。TBI临床治疗中出现的药效慢、副作用明显、药物抵抗率高等问题,也反映出需将颅脑损伤与情绪障碍、心脑血管、胃肠功能紊乱等疾病作为一个整体辩证看待。然而关于传统中药治疗颅脑损伤多病的研究鲜少出现,为充分探究中草药治疗TBI多病的优势,我们课题组前期已测定冠心Ⅱ号及其吸收入血活性成分8ABCs。本文将进一步从在体动物实验及离体细胞药效、网络药理学角度探究冠心Ⅱ号及其吸收成分在TBI大鼠中的药理作用。方法:1.急性颅脑损伤大鼠模型建立后12h,尾静脉注射埃文斯蓝(EB)溶液,测定并计算大脑中EB含量,评估大鼠血脑屏障渗透性水平。2.急性颅脑损伤大鼠模型建立后24h,利用小动物彩色多普勒超声机检测大鼠心脏左冠状动脉血流速度时间积分(VTI),评价冠心Ⅱ号及其代表性成分对大鼠冠脉血流量的影响。3.动物造模、灌胃给药后24h进行抑郁样行为学实验(旷场实验和强迫游泳实验),选用氟西汀为阳性药。造模后灌胃埃文斯蓝溶液检测胃排空、肠推进率,将莫沙必利作为阳性药。4.在大鼠星形胶质细胞上做划伤造模,检测CCK8细胞活力、ROS值及细胞流式凋亡程度,进一步探究冠心Ⅱ号代表性8成分单体药效。5.通过网络药理学方法,联合TCMSP平台,获取冠心Ⅱ号活性成分,筛选成分作用靶点。利用Uniprot数据库取得药物靶标基因,并利用GeneCards,OMIM,PharmGkb,TTD数据库进行颅脑损伤靶基因筛选,得到冠心Ⅱ号治疗颅脑损伤的潜在作用靶点。利用蛋白互作平台数据库构建作用靶点间的互作关系,用Cytoscape 11.0数据库构建靶蛋白互作关系网络(PPI)。最后对冠心Ⅱ号治疗颅脑损伤的作用靶点进行GO富集分析和KEGG通路富集分析。结果:1.在血脑屏障通透性检测实验中,与假手术组相比,模型组显著增加了脑组织的EB含量,血脑屏障通透性增加(p<0.0001)。与模型组相比,冠心Ⅱ号及其吸收性8成分灌胃给药后EB水平显著降低,且冠心Ⅱ号的疗效更显著。阳性药阿托伐他汀可降低TBI大鼠EB水平。冠心Ⅱ号及其吸收性8成分可改善颅脑损伤后血脑屏障功能。2.在大鼠冠脉血流量测定实验中,与假手术组相比,模型组的大鼠舒张期血流速度时间积分(VTI)下降且有统计学意义(P<0.05),造模后给药冠心Ⅱ号及其吸收性8成分均能使冠脉血流积分增加且有统计学意义(P<0.05),Ghrelin拮抗剂能抑制8成分的作用。3.在行为学的强迫游泳实验中,模型组不动时间明显多于假手术组(p<0.0001)。与模型组相比,冠心Ⅱ号组能显著降低大鼠不动时间(p<0.0001);8成分组不动时间也呈减少趋势且统计学差异有意义;氟西汀组改善TBI大鼠不动时间的效果最明显。与模型组相比,15g/kg冠心Ⅱ号及其吸收成分8成分分别降低了大鼠46.49%和39.82%的不动时间。8成分在冠心Ⅱ号中的贡献率为95.45%。大鼠旷场实验结果中,与假手术组比,造模后大鼠爬行总距离显著减少(p<0.0001);冠心Ⅱ及8ABCs治疗后爬行总距离显著增加(p<0.0001;p<0.01);与模型组相比,冠心Ⅱ号及其吸收成分8ABCs分别提高了大鼠149.5%和89.79%的爬行距离。8ABCs在冠心Ⅱ号中的贡献率为76.07%。胃排空实验中,与模型组相比,冠心Ⅱ号组和8成分组有胃排空率的上升趋势,但差异没有统计学意义。肠推进实验中,模型组肠推进率明显降低(p<0.0001);用冠心Ⅱ号或8成分治疗能增加TBI模型的肠推进率(p<0.0001;p<0.01)。4.细胞实验的CCK8细胞活力检测中,造模后细胞活力显著降低。与模型组相比,冠心Ⅱ号组及其代表性吸收的8个成分组均有提升CCK8的活力趋势,在细胞活力提升方面氧化芍药苷贡献了冠心Ⅱ号103.85%的疗效;细胞造模后ROS明显增加(p<0.001),而冠心Ⅱ号组及其代表性吸收的8个成分组可以显著降低R0S水平,这一情况提示冠心Ⅱ号组及其代表性吸收8个成分具有抗氧化的作用。细胞凋亡实验中,模型组凋亡率显著提高(p<0.0001)。冠心Ⅱ号可将细胞凋亡率降低72.65%,8成分中黄芩苷、氧化芍药苷、羟A降低细胞凋亡率的作用较突出。5.基于网络药理学方法筛选出冠心Ⅱ号活性成分共160个,筛选出627个颅脑损伤疾病靶点;冠心Ⅱ号药物活性成分靶点和颅脑损伤靶点的交集基因80个;冠心Ⅱ号中共有105种活性成分与80个治疗颅脑损伤相关药物靶点相关,其中丹参最多,有54种。PTGS2基因是受影响最广的靶点。作用的关键靶点分别是MAPK14、AKT1、FOS、JUN、CTNNB1、MAPK1、RELA、TP53和MYC。主要富集的信号通路包括:流体剪切应力和动脉粥样硬化、脂质和动脉粥样硬化、AGE-RAGE信号通路在糖尿病并发症中的作用、人巨细胞病毒感染、神经退行性多种疾病途径、卡波西肉瘤相关疱疹病毒感染、PI3K-Akt信号通路、癌症中的蛋白多糖、MAPK信号通路、乙型肝炎。结论:活血通络经典方冠心Ⅱ号及其代表性8ABCs对TBI大鼠具有多靶点的脑及心脏保护作用,且能较好发挥抗抑郁、促进胃肠动力疗效。更多还原

【Abstract】 Background and Purpose:Since the 21st century,Traumatic brain injury(TBI)has been the leading cause of human mortality after cancer and cardiovascular and cerebrovascular diseases.As a multi-target and multi-pathophysiological disease,it is easy to encounter bottlenecks when it is studied from the perspective of a single disease,leading to a slowdown in the development of related research.The problems of slow efficacy,obvious side effects,and high drug resistance rate in the clinical treatment of TBI also reflect the need to dialectically treat craniocerebral injury and emotional disorders,cardiovascular and cerebrovascular disorders,gastrointestinal disorders and other diseases as a whole.However,there are few studies on traditional Chinese medicine in the treatment of multiple diseases after TBI.In order to fully explore the advantages of Chinese herbal medicine in the treatment of multiple TBI diseases,our research group has previously determined Guanxin Ⅱ and its active component 8ABCs absorbed into the blood.This article will further explore the pharmacological effects of Guanxin Ⅱ and its absorbed components in TBI rats from the perspective of network pharmacology,in vivo animal experiments and in vitro cell efficacy.Methods:1.Evans blue(EB)solution was injected into the tail vein 12 hours after the establishment of the rat model of acute brain injury.The content of EB in the brain was measured and calculated to evaluate the permeability of the blood-brain barrier.2.Twenty-four hours after the establishment of the rat model of acute craniocerebral injury,the left coronary artery blood flow velocity time integral(VTI)of the rat heart was detected by small animal color Doppler ultrasound machine to evaluate the effects of Guan xin Ⅱ and its representative components on the coronary blood flow in rats.3.The depression-like behavior test(open field and forced swimming)was performed 24 hours after intragastric administration.Fluoxetine was used as the positive drug.After modeling,the gastric emptying and intestinal propulsion rate were detected by gavage of Evans blue solution,and Mosapride was used as a positive drug.4.Rat astrocytes were scratched to establish a model,and CCK8 cell viability,ROS value and flow cytometry apoptosis degree were detected to further explore the effect of representative 8-component monomer of Guan xin Ⅱ.5.The active ingredients of Guan xin Ⅱ were obtained by network pharmacology method combined with TCMSP platform,and their action targets were screened.Uniprot database was used to obtain drug target genes,and GeneCards,OMIM,PharmGkb and TTD databases were used to screen the target genes of craniocerebral injury,and the potential targets of Guan xin Ⅱ in the treatment of craniocerebral injury were obtained.The protein interaction platform database was used to construct the interaction between the targets,and the Cytoscape 11.0 database was used to construct the target protein interaction network(PPI).Finally,GO enrichment analysis and KEGG pathway enrichment analysis were performed on the targets of Guan xin Ⅱ in the treatment of craniocerebral injury.Results:1.In the blood-brain barrier permeability detection experiment,compared with the sham operation group,the model group significantly increased the EB content in brain tissue,and the blood-brain barrier permeability increased.Compared with the model group,the level of EB was significantly reduced after intragastric administration of Guanxin Ⅱ and its absorbable 8 components,and the curative effect of Guanxin Ⅱ was more significant.The positive drug atorvastatin can reduce the EB level in TBI rats.Guanxin Ⅱ and its absorbable 8 components improve blood-brain barrier function after traumatic brain injury.2.In the rat coronary blood flow measurement experiment,compared with the sham operation group,the diastolic blood flow velocity time integral(VTI)of the rats in the model group decreased and had statistical significance(P<0.05).Administration of Guanxin Ⅱ and its absorbable 8 components can increase coronary blood flow integral with statistical significance(P<0.05),and Ghrelin antagonist can inhibit the effect of 8 components.3.In the behavioral forced swimming test,the immobility time of the model group was significantly longer than that of the sham operation group.Compared with the model group,the Guanxin Ⅱ group can significantly reduce the immobility time(p<0.0001);the immobility time of the 8-component group also showed a decreasing trend,and the statistical difference was significant;the fluoxetine group improved the immobility of TBI rats.The effect of time is most pronounced.Compared with the model group,15g/kg Guanxin Ⅱ and its absorbed components 8 components reduced the immobility time of rats by 46.49%and 39.82%,respectively.The contribution rate of 8 components in Guanxin Ⅱ was 95.45%.In the results of the open field test of rats,compared with the blank group,the total crawling distance of the rats after modeling was significantly reduced(p<0.0001);the total crawling distance of the rats after coronary heart Ⅱ or 8ABCs treatment was significantly increased(p<0.0001;p<0.01);Compared with the model group,Guanxin Ⅱ and its absorbed component 8ABCs increased the crawling distance of rats by 149.5%and 89.79%,respectively.The contribution rate of 8ABCs in Guanxin Ⅱ was 76.07%.In the gastric emptying experiment,compared with the model group,the Guanxin Ⅱ group and the 8-component group showed an upward trend in gastric emptying rate,but the difference was not statistically significant.In the intestinal propulsion test,the intestinal propulsion rate of the model group was significantly reduced(p<0.0001);treatment with Guanxin Ⅱ or 8 components could increase the intestinal propulsion rate of the TBI model(p<0.0001;p<0.01).4.In the CCK8 cell viability test of the cell experiment,the cell viability decreased significantly after modeling.Compared with the model group,Guanxin Ⅱ group and its eight representative absorbed components all had a tendency to increase the activity of CCK8,and oxidized paeoniflorin contributed 103.85%of the curative effect of Guanxin Ⅱ in terms of cell viability improvement;The ROS increased significantly after the mold(p<0.001),and the Guanxin Ⅱ group and its eight representative absorbed ingredients could significantly improve this situation,suggesting that the Guanxin Ⅱ group and its eight representative absorbed ingredients have anti-inflammatory effects.The role of oxidation.In the cell apoptosis experiment,the apoptosis rate in the model group was significantly increased.Guanxin Ⅱ can reduce the apoptosis rate of cells by 72.65%.Among the 8 components,baicalin,oxidized paeoniflorin,and hydroxy A are more prominent in reducing the cell apoptosis rate.5.Based on the network pharmacology method,a total of 160 active ingredients of Guanxin II were screened out,and 627 disease targets of craniocerebral injury were screened out;80 intersection genes of active ingredient targets and disease targets of Guanxin Ⅱ were screened;A total of 105 active ingredients in Xin Ⅱ are related to 80 drug targets related to the treatment of craniocerebral injury,among which Danshen has the most,with 54 species.The PTGS2 gene is the most widely affected target.The key targets of action are MAPK14,AKT1,FOS,JUN,CTNNB1,MAPK1,RELA,TP53 and MYC,respectively.The main enriched signaling pathways include:Fluid shear stress and atherosclerosis,Lipid and atherosclerosis,AGE-RAGE signaling pathway in diabetic complications,Human cytomegalovirus infection,Pathways of neurodegeneration-multiple diseases,Kaposi sarcoma-associated herpesvirus infection,PI3K-A,Proteoglycans in cancer,MAPK signaling pathway,Hepatitis B.Conclusion:Guan xin Ⅱ and its representative 8ABCs have multi-target brain and heart protective effects on TBI rats,and can exert antidepressant effects and promote gastrointestinal motility.更多还原