Construction of Rat Model of Hepatic Fibrosis with Blood Stasis Syndrome Integrated with Traditional Chinese Medicine(TCM)Syndrome and Western Medicine Disease

[ Objective ] According to the theory of blood stasis in traditional Chinese medicine （ TCM）, the animal modeling method of hepatic fibrosis integrated with pathology and symptoms was explored and improved, to construct a new type of rat model of hepatic fibrosis with blood stasis syndrome integrated with tradition- al Chinese and westem medicine. [ Method] The hepatic fibrosis model of blood stasis with blood stasis syndrome was constructed by jointing multi factors, inclu- ding intragastric administration of ethanol, high fat and low protein feeding, joint injection of dimethylnitrosamine （DMN）, bovine serum albumin （BSA） and nor- epinephrine （NE）. The modeling method was further compared with traditional CC14 single-factor modeling method from the aspects of mortality, blood stasis syn- drome of TCM, syndrome grading, general morphology of liver pathology, liver function changes, as well as expression levels of three kinds of collagen （type I and type III collagen, a-SMA） determined by immunohistochemical staining method, and four items of rat hepatic fibrosis （HA, P3NP, LN, CIV content） determined by radio enzyme immunoassay. [ Result ] In blood stasis group, （ 1 ） the mortality of rats was 20% ; （2） model rats appeared typical blood stasis syndromes of TCM such as ecchymosis, dark purple tongue, loose stool, and blood stasis syndrome grading was high; （3） fibrosis changes of liver such as dark white surface, dense gray nodules and brittle texture were observed in general morphological examination; （4） serological liver function tests found that ALT and AST of model rats, as well as TBIL, DBIL and IBIL contents increased significantly; （5） immunohistochemical staining demonstrated that the expression levels of three kinds of collagen （type I and type III collagen, ot-SMA） increased significantly; （6） four items of rat serum hepatic fibrosis, HA, P3NP, LN, CIV content, increased significantly; （7） the above results in blood stasis syndrome model group （except morality and liver function） were higher than those in CC14 modeling group, and the difference was statistically significant （P 〈 0.05 ）. [ Conclusion ] The new improved modeling method effectively reduces high mortality in traditional CC 14 modeling method. In addition to low mortality, the model animal has dual characteristics of disease in western medicine and syndrome in TCM. It is consistent with the pathological characteristics of hepatic fibrosis in western medicine when according with the basic characteristics of blood stasis syndrome in TCM.

Comparative pharmacokinetics of five saponins after intravenous administration of TSFS injection and TSFS injection plus TFFG in rats under different physiological states

Sanqi is a popular traditional Chinese medicine and commonly used for promoting blood circulation and removing blood stasis. Notoginsenoside R1, ginsenoside Rg1, Re, Rb1 and Rd are the major active constituents of Sanqi. The purpose of the study was to investigate the pharmacokinetic behavior of the five active constituents from total saponin from Sanqi when it was used in the blood stasis animals or in combination with Gegen. The concentrations of the five active constituents in rat plasma were determined by an ultra-HPLC-ESI-MS/MS method. The main pharmacokinetic parameters were calculated and statistically analyzed using the unpaired student＆#39;s t-test. It was found that the pharmacokinetic parameters of notoginsenoside R1, ginsenoside Rg1 and Rb1 represented a statistically significant difference （Po0.05） between the normal rats and the blood stasis rats after administration of total saponin from Sanqi （TSFS）. And there were statistically significant differences （Po0.05） in the pharmacokinetic parameters of all the five constituents between administration of TSFS alone and combined with total flavonoid from Gegen （TFFG） in blood stasis rats. It suggested that the pharmacokinetic behavior of the active constituents from TSFS could be changed when it was used in blood stasis animals or in combination with TFFG.

冠心病心血瘀阻证大鼠心肌组织代谢组学研究

目的研究冠心病心血瘀阻证大鼠心肌组织代谢产物谱,探讨其病证生物学基础。方法SD大鼠随机分为假手术组和模型组,采用冠状动脉左前降支结扎法制备冠心病心血瘀阻证大鼠模型,观察大鼠一般状态,检测舌象色度、心电图、心功能,HE染色、透射电镜观察心肌组织形态及超微结构,用超高效液相色谱-质谱技术检测心肌组织差异代谢物,并对代谢通路进行富集分析。结果与假手术组比较,模型组大鼠舌象色度R、G、B值均显著降低(P<0.05),心电图心率、ST段抬高幅度显著上升(P<0.05),左室射血分数、左室短轴缩短率显著下降(P<0.05),左室收缩末期内径、左室舒张末期内径显著上升(P<0.05);心肌组织结构模糊,有炎性细胞浸润,线粒体肿胀、破裂、溶解,嵴结构断裂减少。代谢组学共鉴定出29个假手术组与模型组差异显著的潜在生物标志物(7个上调、22个下调),主要富集于硫胺素代谢,精氨酸生物合成,嘌呤代谢,氨酰基-tRNA生物合成,丙氨酸、天冬氨酸和谷氨酸代谢,戊糖和葡萄糖醛酸相互转化,糖酵解/糖异生,缬氨酸、亮氨酸和异亮氨酸降解,三羧酸循环,丙酮酸代谢10条代谢通路。结论冠状动脉左前降支结扎法可较好地模拟冠心病心血瘀证病理过程,其病理机制涉及葡萄糖代谢、线粒体能量代谢、氨基酸代谢、蛋白质生物合成、嘌呤代谢等多层次代谢网络紊乱。

大鼠急性血瘀证模型造模方法的网状Meta分析

目的:采用网状Meta分析评价不同急性血瘀证模型造模方法的效果,为中医实验研究急性血瘀证动物模型的制备提供循证依据。方法:检索中国期刊全文数据库(CNKI)、中国学术期刊数据库(CSPD)、中文科技期刊数据库(CCD)中有关急性血瘀证模型的建模方法,应用Stata软件进行网状Meta分析。结果:最终纳入32项研究,共纳入9种造模方案。各项指标最优前3位概率排序分别为:1)全血黏度:1 S^(-)1切变率右旋糖酐>高脂>氢化可的松+肾上腺素,10 S^(-)1切变率冰水浴>肾上腺素>高脂+力竭游泳,30 S^(-)1切变率多因素刺激>右旋糖酐>肾上腺素,60 S^(-)1切变率多因素刺激>右旋糖酐>肾上腺素,150 S^(-)1切变率肾上腺素>冰水浴>高脂+力竭游泳,200 S^(-)1切变率右旋糖酐>氢化可的松+肾上腺素>角叉莱胶+内毒素;2)血浆黏度:右旋糖酐>多因素刺激>肾上腺素;3)红细胞聚集指数:冰水浴>多因素刺激>高脂+力竭游泳。受纳入研究质量和数量的限制,所得结论仍待进一步验证。结论:右旋糖酐、冰水浴、多因素刺激、肾上腺素造模效果更显著。

益心泰颗粒对慢性心衰(心气虚兼血瘀水停证)大鼠AMPK、PGC-1α的影响

目的观察益心泰颗粒对慢性心力衰竭(CHF)(心气虚兼血瘀水停证)大鼠AMPK、PGC-1α的影响。方法采用丙硫氧嘧啶灌胃及阿霉素腹腔注射复制CHF(心气虚兼血瘀水停证)模型大鼠,将造模成功大鼠分为模型组与益心泰低、中、高剂量组和曲美他嗪组;另设正常对照组。灌胃4周后观察心肌组织病理结构,检测LVEF、血清NT-proBNP及心肌组织FFA、ATP/AMP、LAC、pAMPK、AMPK、PGC-1α水平。结果与模型组比较,益心泰颗粒低、中、高剂量组及曲美他嗪组LVEF、心肌组织ATP/AMP值、心肌组织p-AMPK、AMPK、PGC-1α相对灰度值均明显升高(P<0.05或P<0.01),血清NT-proBNP及心肌组织FFA、LAC明显降低(P<0.05或P<0.01)。结论益心泰颗粒能促进慢性心力衰竭(心气虚兼血瘀水停证)大鼠AMPK、PGC-1α表达,改善心肌能量代谢。

Metabonomic phenotype and identification of “heart blood stasis obstruction pattern” and “qi and yin deficiency pattern” of myocardial ischemia rat models

The traditional Chinese medicine concepts of "Xinxueyuzuzheng (heart blood stasis obstruction pattern)" and "Qiyinliangxuzheng (qi and yin deficiency pattern)" for myocardial ischemia rat models were constructed in the present study. Endogenous metabolites in rat plasma were analyzed using the GC/TOF-MS-based metabonomic method. Significant metabolic differences were observed between the control and two model groups, and the three groups were distinguished clearly by pattern recognition. Compared with those of the control, the levels of hydroxyproline, threonic acid, glutamine and citric acid were strikingly up or down-regulated in model rats. The metabolites contributing most to the classification between the two "pattern" rats were identified, such as valine, serine, threonine, ornithine, hydroxyproline, lysine, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, galactofuranose and inositol. These compounds were indicated as the potential biomarkers. The results suggested that the two "patterns" are involved in dysfunction in oxidative stress, energy metabolism and amino acid metabolism. These findings also provided the substantial foundation for exploring the scientific connotation of these two "Zhengxing (pattern types)" of myocardial ischemia, and "Bianzheng (pattern identification)".

Improvement and Application of Acute Blood Stasis Rat Model Aligned with the 3Rs (Reduction, Refinement and Replacement) of Humane Animal Experimentation

Objective:To establish a novel cardiocentesis method for withdrawing venous blood from the right atrium,and to improve an acute blood stasis rat model using an ice bath and epinephrine hydrochloride(Epi)while considering the 3Rs(reduction,refinement,and replacement)of humane animal experimentation.Methods:An acute blood stasis model was established in male Sprague-Dawley rats by subcutaneous injection(s.c.)Epi(1.2mg/kg)administration at 0h,followed by a 5-min exposure to an ice-bath at 2h and s.c.Epi administration at 4h.Control rats received physiological saline.Rats were fasted overnight and treated with Angelicae Sinensis Lateralis Radix(ASLR)and Pheretima the following day.Venous blood was collected using our novel cardiocentesis method and used to test whole blood viscosity(WBV),prothrombin time(PT),activated partial thromboplastin time(APTT),and fibrinogen(FIB)8ntent.Results:The rats survived the novel cardiocentesis technique;WBV value returned to normal while hematological parameters such as hemoglobin level and red blood cell count were restored to>94%of the corresponding values in normal rats following a 14-day recovery.Epi(1.2 mg/kg,s.c.)combined with a 5-min exposure to the ice bath replicated the acute blood stasis rat model and was associated with the highest WBV value.In rats showing acute blood stasis,ASLR treatment[4g/(kg-d)for 8 days]decreased WBV by 9.98%,11.09%,9.34%,9.00%,7.66%,and 7.03%(P<0.05),while Pheretima treatment[2.6g/(kg?d),for 8 days]decreased WBV by 25.49%,25.94%,16.28%,17.76%,11.07%,and 7.89%(P<0.01)at shear rates of 1,3,10,30,100,and 180 s'1,respectively.Furthermore,Pheretima treatment increased APTT significantly(P<0.01).Conclusions:We presented a stable,reproducible,and improved acute blood stasis rat model,which could be applied to screen drugs for promoting blood circulation and eliminating blood stasis.

优化溃结方对溃疡性结肠炎气滞血瘀模型大鼠的影响及其作用机制研究

背景经验方优化溃结方能有效提高溃疡性结肠炎(UC)患者生活质量,促进肠黏膜愈合和临床缓解,但其作用机制尚不完全明确。目的探讨优化溃结方对UC气滞血瘀模型大鼠的影响及其作用机制。方法2023年9—10月,选取SPF级雄性SD大鼠70只并采用随机数字表法将其随机分为正常组、模型组、柳氮磺胺吡啶组、低剂量组、标准剂量组、益气组、活血组,每组10只。采用三硝基苯磺酸(TNBS)/乙醇二次致炎法结合束缚法建立UC气滞血瘀模型。正常组大鼠在造模时使用0.9%氯化钠溶液灌肠,并与其他组大鼠进行同步抓取、固定,在造模成功后给予等体积水灌胃,1次/d,共灌胃14次;模型组大鼠在造模成功后给予等体积水灌胃,1次/d,共灌胃14次;柳氮磺胺吡啶组、低剂量组、标准剂量组、益气组、活血组大鼠分别在造模成功后给予柳氮磺胺吡啶药液0.54 g/kg灌胃、低剂量优化溃结方药液0.837 g/kg、标准剂量优化溃结方药液1.674 g/kg灌胃、黄芪药液1.8 g/kg、红花药液0.9 g/kg,均为1次/d,均灌胃14次。采用高精度透射电镜观察7组大鼠干预后结肠组织超微结构变化,并比较7组大鼠干预后结肠组织趋化因子受体4(CXCR4)、血管内皮生长因子A(VEGFA)、转化生长因子激酶1(TAK1)表达水平。结果低剂量组、标准剂量组大鼠干预后结肠组织超微结构趋于正常。模型组大鼠干预后结肠组织CXCR4、VEGFA、TAK1蛋白灰度比值、mRNA相对表达量高于正常;柳氮磺胺吡啶组、低剂量组、标准剂量组、益气组、活血组大鼠干预后结肠组织CXCR4、VEGFA、TAK1蛋白灰度比值、mRNA相对表达量低于模型组(P<0.05);标准剂量组大鼠干预后结肠组织CXCR4、VEGFA、TAK1蛋白灰度比值低于柳氮磺胺吡啶组,活血组大鼠干预后结肠组织VEGFA、TAK1 mRNA相对表达量低于柳氮磺胺吡啶组(P<0.05)。结论优化溃结方及其益气、活血组分可有效降低UC气滞血瘀模型大鼠结肠组织CXCR4、VEGFA、TAK1表达水平,其治疗作用可能是益气组分、活血组分通过协同调节结肠组织中CXCR4、VEGFA、TAK1表达水平而实现。

基于病证结合的冠心病动物模型制备研究进展与思考

冠心病的临床研究和动物实验研究一直是心血管领域的常见病。病证结合动物模型体现了中西医结合的理念,不仅有西医的疾病特点,而且借助中医思维用不同实验方法来表现独特的中医证候特点,是中医药研究冠心病预防和治疗的有效工具。文章对常用冠心病中医证候的动物模型建立方法进行总结,并以冠心病痰瘀互结证动物模型为例提出思考。

气虚血瘀证小鼠模型拓展望诊指标的应用与评价

目的 以气虚血瘀证为代表,探索拓展望诊指标,为开展模型动物证候诊断的可行性提供依据。方法 将30只小鼠分为空白对照组、气虚血瘀组、中药反证组,每组10只。气虚血瘀组与中药反证组小鼠采用饥饿、疲劳、寒湿法进行造模,持续20 d;中药反证组自造模第3天开始每日予中药黄芪-莪术浓煎液灌胃,空白对照组和气虚血瘀组同期每日予相同容积蒸馏水灌胃,连续18 d。持续观测小鼠一般状态和体质量变化;于造模结束后检测粪便含水率、觅食欲,并通过避风实验、机械痛阈、旷场实验观察各组小鼠行为特点,拍照并利用图片分析软件对小鼠舌色进行分析。结果 与空白对照组比较,气虚血瘀组与中药反证组体质量低(P<0.01);避风实验中,2、3区域停留时间短、跨域次数少(P<0.01);旷场实验中,总路程短、平均速度低、跨格次数少、静止时间长(P<0.01);舌色R、G、B值均低(P<0.01)。与气虚血瘀组比较,中药反证组体质量高(P<0.01);避风实验中,2、3区域停留时间长、跨域次数多(P<0.01);旷场实验中,总路程长、平均速度高、跨格次数多、静止时间短(P<0.01);舌色R、G、B值均高(P<0.05)。气虚血瘀组、中药反证组机械痛阈均低于空白对照组(P<0.01),2组间差异无统计学意义(P>0.05);气虚血瘀组、中药反证组粪便颗粒稀软、易夹断,含水率高于空白对照组(P<0.01),2组间差异有统计学意义(P<0.05)。结论 避风实验和机械痛阈指标,以及旷场实验、粪便含水率和舌色检测指标,可作为诊断小鼠气虚血瘀证的拓展望诊指标,而觅食欲不可作为其有效指标。

Pharmacokinetic Comparison of Four Major Bio-Active Components of Naoxintong Capsule in Normal and Acute Blood Stasis Rats Using Ultra-Performance Liquid Chromatography Coupled with Triple-Quadrupole Mass Spectrometry

Objective: To compare the pharmacokinetic differences of the main components of Naoxintong capsule(NXTC) in normal and acute blood stasis rats. Materials and Methods: Rats were subcutaneously injected with adrenaline hydrochloride twice;during the two subcutaneous injections, the rats were placed in ice water for 4 min to reproduce the model rat of acute blood stasis. The normal and acute blood stasis rats were administrated a 5.04 g/kg dose of NXTC suspension. Then, blood samples were collected from the posterior retinal venous plexus at different time points. Plasma concentrations of four major bio-active components including caffeic acid, ferulic acid, formononetin, and tanshinone IIA in NXTC were measured using ultra-performance liquid chromatography coupled with triple-quadrupole mass spectrometry. Phoenix Win Nonlin v6.2 software was used to calculate the pharmacokinetic parameters. Results: Compared with the normal rats, the acute blood stasis rats showed a significant decrease in C_(max) of ferulic acid and formononetin, AUC_(all) of caffeic acid and ferulic acid, and AUC_(INF_obs) of ferulic acid. Conversely, an increase in the Vz_F_obs and MRT_(last) of ferulic acid and caffeic acid was observed. These findings demonstrate that the absorption of the four NXTC components was weakened in the acute blood stasis rats and that the elimination time was prolonged. Conclusions: The significant difference in some parameters of the four NXTC components between the normal and acute blood stasis rats might be caused by an increase in blood viscosity and the subsequent slowing down of blood flow in the acute blood stasis rats. The pharmacokinetic study conducted in pathological state can provide important information and scientific basis for further rational clinical application of NXTC.

基于LASSO回归建立与验证中风痰瘀滞络证诊断模型的探索

[目的]探索并建立一个中风痰瘀滞络证的临床预测诊断模型,可应用于互联网医疗等现代大环境。[方法]对2021年6月至2022年2月上海市浦东新区浦南医院中医科收治的新发脑卒中有偏瘫后遗症的患者305例进行病史采集,给予中医证型诊断。将病例以7:3的比例区分为训练集及验证集,运用R语言进行LASSO回归筛选疾病研究因子,以二元Logistic回归建立模型,并予区分度、校准度验证,以列线图形式展示模型。[结果]30个研究因子经过筛选后留下9个因子建立模型,模型区分度:训练集曲线下面积(area under curve,AUC)0.942,95%可信区间(0.906,0.979);验证集AUC 0.951,95%可信区间(0.895,1.000)。校准度:Hosmer-Lemeshow指数(H-L)训练集P=0.47,验证集P=0.39。模型以列线图进行可视化展示。[结论]该诊断模型有较好的诊断效能,可在多种无法进行望诊、切诊的情况下给予辨证参考。

基于蛋白质组学的冠心病痰瘀互结证生物标志物及诊断模型研究

目的痰瘀互结证是当代冠心病的主要证候之一,而目前尚缺乏可靠的定量蛋白质组学检测结果。基于数据非依赖性采集模式(Data independent acquisition,DIA)对冠心病痰瘀互结证患者的血浆蛋白质组进行研究分析,探索冠心病痰瘀互结证的蛋白标志物及关键生物学过程,并构建证候的诊断模型。方法选取2020年8月—2020年11月期间中国中医科学院广安门医院心血管科收治的5例冠心病痰瘀互结证患者作为疾病观察组,同时选取2020年10月—2020年11月期间在读研究生的健康成人5人作为对照组,采用DIA蛋白质组学技术对受试者的血浆进行检测,进行差异蛋白表达分析及GO富集分析、KEGG富集分析、蛋白质互作分析。根据生物信息学结果筛选冠心病痰瘀互结证的关键蛋白,对其进行酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA),并根据ELISA结果开展logistic回归分析。结果在差异倍数为1.5倍时,疾病观察组相比于健康对照组存在9个蛋白质表达上调,19个蛋白质表达下调。生物信息学分析表明,血管性血友病因子(VWF)、补体因子H是参与凝血和补体反应的关键蛋白。进一步ELISA结果表明,冠心病痰瘀互结证患者的血浆VWF水平高于健康受试者,差异有统计学意义(P<0.05),CFH水平低于健康受试者,差异有统计学意义(P<0.05),与蛋白质组学的结果相一致。VWF的AUC=0.73,CFH的AUC=0.66。基于两指标的Logistic回归模型测试集的准确率、灵敏度、特异度和AUC分别为73%、74%、70%和0.78。结论VWF相较于CFH具有更好的诊断冠心病痰瘀互结证的潜力。基于CFH和VWF两种蛋白质分子的证候诊断模型,具有一定的诊断效能。

冠状动脉微血管疾病气虚血瘀证大鼠模型的建立及评价

目的冠状动脉微血管疾病(coronary microvascular disease,CMVD)发病率逐年增高,中医认为CMVD属中医学“络脉”的范畴,气虚血瘀证是CMVD主要证型,然而目前尚无气虚血瘀证CMVD动物模型的报道,因此本研究探索建立冠状动脉微血管疾病气虚血瘀证大鼠模型并进行评价。方法雄性SD大鼠45只,随机分为假手术组、冠脉微血管疾病组和冠脉微血管疾病气虚血瘀证组,每组15只。冠脉微血管疾病气虚血瘀证组动物每天随机14~16 h睡眠剥夺,持续6周,复制气虚证大鼠模型;假手术组及冠脉微血管疾病组动物正常饲养6周。实验6周后,冠脉微血管疾病组和冠脉微血管疾病气虚血瘀证组大鼠麻醉后开胸,向左心室内注射栓塞微球(40~120μm),假手术组动物行开胸手术但不注射栓塞微球。术后7 d,各组测定相关检测指标。结果冠脉微血管疾病组与假手术组相比,左室射血分数、左室缩短率明显降低,肌酸激酶MB同工酶(CK-MB)以及乳酸脱氢酶(LDH)活性明显增加。冠脉微血管疾病气虚血瘀证组与假手术组相比,心功能下降、心肌损伤标志物活性显著升高、血浆及全血粘度明显升高。结论睡眠剥夺复合心室内注射栓塞微球可成功建立冠脉微血管疾病气虚血瘀证大鼠模型,比较符合临床发病特征适合于发病与中药作用机制研究。

痰瘀互结证小动物模型类人证候评价量表理论思路及类人证候测量方法构建

证候辨识是中医辨证论治的起点,中医证候评价量表的研究一直在中医证候研究中备受关注。该文以中医传统理论为基础,以“类人证候”思想为指导,集合多种动物行为学及机能学实验,构建痰瘀互结证小动物模型类人证候评价量表理论思路,并对痰瘀互结证包括舌象、脉象、精神状况、疼痛、异常分泌物、麻木、痿废、眩晕、肿块等证候的测量方式进行探索,以期为中医证候动物模型评价建立新标准,为中医辨证的发展提供帮助。

冠心病心阳虚痰瘀互结证大鼠模型的代谢组学特征分析

目的 构建冠心病心阳虚痰瘀互结证大鼠模型,阐释其代谢组学特征。方法 采用丙基硫氧嘧啶灌胃联合高脂饮食、腹腔注射维生素D3和皮下注射异丙肾上腺素构建冠心病心阳虚痰瘀互结证模型,通过观察大鼠行为学改变,测定心功能、心电图、耳温、尾静脉脉波、舌象与足底RGB(red,green,blue)值、心肌组织三磷酸腺苷(adenosine triphosphate,ATP)含量、血脂含量、血液流变学指标、炎症因子白细胞介素(interleukin,IL)-6与肿瘤坏死因子(tumor necrosis factor,TNF)-α含量及甲状腺功能指标,结合主动脉弓组织、心肌组织病理改变对该模型进行评价;应用代谢组学技术筛选出冠心病心阳虚痰瘀互结证大鼠差异代谢物,并进行通路富集分析。结果 冠心病心阳虚痰瘀互结证大鼠表现出精神萎靡、行动迟缓、蜷缩扎堆、耳温降低、毛发缺少光泽,舌象、足底色泽偏淡暗伴RGB值升高,左心室射血分数(ejection fraction, EF)、左心室短轴缩短率(fraction shortening, FS)降低,心电图ST段抬高、脉波增宽;血清中总胆固醇(total cholesterol, TC)、低密度脂蛋白胆固醇(low density lipoprotein cholesterol, LDL-C)、IL-6、TNF-α含量升高, T3、T4及心肌组织中ATP含量降低;主动脉弓平滑肌细胞坏死钙化、心肌组织纤维化坏死溶解。冠心病心阳虚痰瘀互结证组大鼠与空白组大鼠之间共有160个差异代谢物,主要涉及ABC转运器、花生四烯酸代谢、嘧啶代谢、不饱和脂肪酸的生物合成和氨基酸的生物合成等代谢通路。结论 本研究构建了冠心病心阳虚痰瘀互结证大鼠模型,并初步揭示了其代谢特征。

中医气虚血瘀证斑马鱼模型的构建与评价

目的:对中医气虚血瘀证斑马鱼模型进行构建与评价。方法:随机选取90尾受精后4 d(4 dpf)野生型AB品系斑马鱼于6孔板中,每孔均处理30尾斑马鱼。分为正常对照组、气虚血瘀证组和药物验证组。采用无水亚硫酸钠和异丙肾上腺素盐酸盐联合刺激建立气虚血瘀证斑马鱼模型。药物验证组斑马鱼在造模基础上给予水溶脑心通胶囊。每组随机选取10尾斑马鱼,使用行为学分析仪分析斑马鱼的总运动距离;采用乳酸和ATP试剂盒测定斑马鱼体内乳酸和ATP水平;置于荧光显微镜下拍照,图像处理软件分析斑马鱼心脏凋亡细胞心肌细胞凋亡荧光强度;采用心跳血流分析系统分析斑马鱼血流速度和心输出量。结果:与正常对照组比较,气虚血瘀证组斑马鱼总运动距离和ATP水平均明显降低(P<0.05,P<0.001),气虚血瘀证组斑马鱼体内乳酸水平和心肌细胞凋亡荧光强度明显高于正常对照组(P<0.001,P<0.01),而血流速度和心输出量明显低于正常对照组(P<0.01,P<0.05)。与气虚血瘀证组比较,药物验证组斑马鱼总运动距离、体内ATP水平、血流速度和心输出量明显升高(P<0.05,P<0.001),斑马鱼心肌细胞凋亡荧光强度和体内乳酸水平明显降低(P<0.001)。结论:无水亚硫酸钠和异丙肾上腺素盐酸盐联合干预可成功建立气虚血瘀证斑马鱼模型。

基于病证结合探讨动脉粥样硬化瘀毒证的模型构建与评价

动脉粥样硬化(atherosclerosis,AS)是心脑血管疾病的共同病理基础,建立具有中医瘀毒证候特色的动脉粥样硬化动物模型及客观规范的评价体系是现代心脑血管疾病研究的重要内容,对中医药防治心脑血管疾病基础研发及药效机制评价具有重要意义。本文通过对动脉粥样硬化瘀毒证的动物模型构建及评价进行了多方面的总结和归纳,同时分析了当前模型构建及评价存在的问题,以期为建立规范化的动物模型提供参考。

基于机器学习的特发性肺纤维化气阴两虚兼血瘀证诊断模型研究

目的应用机器学习理论3种方法构建诊断模型,比较各模型对特发性肺纤维化气阴两虚兼血瘀证的临床诊断价值。方法收集2015—2020年于辽宁中医药大学附属医院住院部及门诊部病例库中特发性肺纤维化患者348例,采用机器学习理论之决策树(CHAID、QUEST、CRT、C5.0)、神经网络、支持向量机方法建立特发性肺纤维化气阴两虚兼血瘀证的诊断模型,比较各模型的判别准确率、特异度、敏感度、可用度。结果决策树CHAID、QUEST、CRT、C5.0构建的诊断模型判别准确率分别为87.20%、89.10%、89.30%、90.53%,C5.0决策树模型优于其他,神经网络模型判别准确率92.63%,支持向量机模型准确率91.58%。神经网络模型的准确率最高,特异度、敏感度、可用度与其他模型无显著差异,故神经网络模型为研究的最优诊断模型。结论舌质黯在特发性肺纤维化气阴两虚兼血瘀证的临床诊断中贡献率较大,并结合中医四诊,共同形成符合特发性肺纤维化气阴两虚兼血瘀证的诊断判别模式,为该病的诊断提供客观、规范的参考依据。

小鼠膝骨关节炎阳虚血瘀证模型的构建与评价

目的:探讨构建小鼠膝骨关节炎(knee osteoarthritis,KOA)阳虚血瘀证模型的有效方法。方法:将12只10周龄雄性无特定病原C57BL/6J小鼠采用内侧半月板失稳(destabilized medial meniscus,DMM)法构建KOA模型。KOA造模成功后,将12只KOA模型小鼠随机分为KOA阳虚血瘀证组和KOA对照组,每组6只。KOA阳虚血瘀证组小鼠于浸在冰水混合物中的塑料鼠箱中饲养,并以冰水混合物为饮用水;KOA对照组小鼠于22℃干燥环境中正常饲养。饲养10周后,评价小鼠阳虚血瘀证表现,分析小鼠步态,处死小鼠后进行小鼠膝关节MicroCT分析、膝关节组织病理学观察及膝关节软骨Ⅱ型胶原蛋白α1链表达检测。结果:①小鼠阳虚血瘀证评价结果。KOA阳虚血瘀证组小鼠的阳虚证评分和血瘀证评分均高于KOA对照组[(13.33±1.37)分,(3.50±1.23)分,t=13.120,P=0.000;(4.83±0.75)分,(0.83±0.75)分,t=9.200,P=0.000]。②小鼠步态分析结果。2组各收集到5只小鼠完整的步态分析数据。KOA阳虚血瘀证组小鼠右后肢的爪面积和步幅均小于KOA对照组[(0.610±0.094)cm2,(0.758±0.042)cm2,t=-3.211,P=0.012;(1.780±0.045)cm,(2.040±0.152)cm,t=-3.677,P=0.006];2组小鼠右后肢的摆动期时间和站立期时间比较,组间差异均无统计学意义[(0.060±0.004)s,(0.060±0.009)s,t=-0.096,P=0.963;(0.062±0.009)s,(0.076±0.014)s,t=-1.810,P=0.137]。③小鼠膝关节MicroCT分析结果。KOA阳虚血瘀证组小鼠的骨体积分数低于KOA对照组[(55.582±2.810)%,(69.248±1.884)%,t=-6.997,P=0.002],骨小梁分离度大于KOA对照组[(0.114±0.009)mm,(0.077±0.010)mm,t=4.883,P=0.008];2组小鼠的骨小梁厚度、骨小梁数量比较,组间差异均无统计学意义[(0.148±0.005)mm,(0.210±0.036)mm,t=-2.939,P=0.094;(3.767±0.100)个·mm^(-1),(3.368±0.547)个·mm^(-1),t=1.243,P=0.282]。④小鼠膝关节组织病理学观察结果。2组小鼠膝关节软骨均有缺失,且KOA阳虚血瘀证组小鼠膝关节软骨缺失较KOA对照组更严重。⑤小鼠膝关节软骨Ⅱ型胶原蛋白α1链表达检测结果。KOA阳虚血瘀证组小鼠膝关节软骨Ⅱ型胶原蛋白α1链阳性表达面积比和平均光密度均低于KOA对照组[(0.144±0.003)%,(0.163±0.003)%,t=-6.580,P=0.003;(0.051±0.002)lux,(0.095±0.004)lux,t=-15.878,P=0.001]。结论:DMM法联合低温诱导法是构建小鼠KOA阳虚血瘀证模型的有效方法。