中药复方益糖康干预代谢综合征的疗效评价及其代谢组学研究
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摘要
目的:
代谢综合征(metabolic syndrome，MS)是一组以肥胖、高血糖（糖尿病或糖调节受损）、血脂异常（指高甘油三酯血症和/或低高密度脂蛋白胆固醇血症）以及高血压等聚集发病，严重影响机体健康的临床征候群，是一组在代谢上相互关联的危险因素的组合，这些因素直接促进了动脉粥样硬化性心血管疾病的发生，也增加了发生2型糖尿病的风险。本研究以前瞻性的研究方法，选择并有糖调节受损的代谢综合征患者作为研究对象，并符合中医辨证诊断者。通过随机、双盲、平行对照、多中心临床试验的方法，以血糖、血脂、血压、糖化血红蛋白和体重指数等作为主要考察指标，观察中药复方益糖康干预代谢综合征的有效性及生活方式干预+中药复方益糖康治疗方案的优效性。代谢组学着重研究的是生物整体、器官或组织的内源性代谢物质的代谢途径及其所受内在和外在因素影响下代谢整体的变化轨迹来反映某种病理生理过程中所发生的一系列生物事件。这种整体的研究模式可全面了解代谢物质在疾病发生、发展过程中的变化规律，对发病机制研究以及疾病的诊断、治疗和疗效评估具有重要意义。本研究拟通过复制脾虚证代谢综合征大鼠模型，对模型血浆标本的代谢组学研究，寻找脾虚证代谢综合征病变的特征性代谢产物，观察代谢物变化与生理病理相关的改变关系，探寻脾虚证代谢综合征病变的分子机制，从而进一步揭示脾虚证代谢综合征本质。同时运用代谢组学的技术与方法，从机体内源性代谢物的角度阐释中药复方益糖康的干预机制及作用靶点，为中药治疗代谢综合征作用机理的系统研究提供科学依据。
材料与方法：
1.临床研究
采用随机、双盲、平行对照、多中心临床试验方法，收集符合中医辨证诊断标准的代谢综合征患者165名。随机分配到两个组中。治疗组：生活方式干预（饮食控制，运动）+中药。对照组：生活方式干预（饮食控制，运动）+中药安慰剂。在整个的临床试验周期内，对全部病例进行基线（包括流行病学资料、安全性指标、有效性指标等）分析，观测在试验前治疗组与对照组的各项数据和指标有无显著性差异,是否具有可比性。对全部病例的各种有效性指标，包括主要效应指标（血糖、血脂、血压、糖化血红蛋白）和次要效应指标（体重、腰围、体重指数、中医证型指标、中医证候指标、生存质量积分等）进行治疗前后及组间的比较，并确立达标率判断标准，观察中药复方益糖康及中西医结合治疗方案的临床疗效。分别对治疗组和对照组全部病例的安全性指标进行治疗前后的比较，涉及的内容有血、尿、便常规，肝功，肾功等，观察本试验用药中药复方益糖康安全性。
2.实验研究
2.1动物疾病模型的复制
健康雄性SD大鼠30只，体重（180士20）g，适应性饲养1天后按体重随机分成两组：空白对照组（n=10）和模型组(n=30)。模型组单日喂食甘蓝15～20g/只，自由饮水，游泳至耐力极限；双日高糖高脂膳食持续喂养，共计12周，复制脾气虚证代谢综合征模型。
2.2代谢组学研究
将复制成功的脾气虚证代谢综合征大鼠按体重随机分为两组：模型组（n=6）和模型+中药组（n=6）。运用超高压液相色谱-四极杆-时间飞行串联质谱仪（ultra highperformance liquid chromatography–quadrupole–time of flight mass spectrometry，UPLC/Q-TOF-MS）技术检测模型组血浆中的小分子代谢产物，建立UPLC/Q-TOF-MS大鼠血样总离子流色谱图，对所得到的代谢指纹图谱进行主成分分析和偏最小二乘法-判别分析，找出具有差异的特征代谢物，从而辨识生物标志物并追溯其来源和代谢途径。根据临床患者一日中药用量折算成大鼠的等效剂量为每日0.4g/100g体重，模型+中药组大鼠每天一次灌胃，连续4周。通过UPLC/Q-TOF-MS技术检测，观察脾气虚证代谢综合征模型经中药复方益糖康干预后，血浆小分子代谢物组的差异性变化谱。
结果:
1.临床试验：
1.1基线分析：人口学资料基线比较，包括年龄、性别分布。两组患者间无显著性差异（P＞0.05）。病程基线比较，两组患者间无显著性差异（P＞0.05）。主要疗效指标基线比较，包括收缩压、舒张压、空腹血糖、餐后2小时血糖、糖化血红蛋白。两组患者间无显著性差异（P＞0.05）。次要疗效指标基线比较，包括体重、腰围、臀围、体重指数、中医证候总计分、生存量表计分。两组患者间无显著性差异（P＞0.05）。
1.2有效性分析：生活方式干预可以显著降低餐后2小时血糖、血压和体重指数。中药复方益糖康干预后，不仅可以改善患者餐后2小时血糖、血压和体重指数，还能显著降低MS患者的甘油三酯和总胆固醇。同时，对于空腹血糖、低密度脂蛋白和高密度脂蛋白也有改善作用，并可提高空腹血糖、餐后两小时血糖、高密度脂蛋白、血压和体重指数的达标率。还能够有效地改善MS患者的主要临床症状及全身状况，同时提高患者的生存质量。
1.3安全性分析：两组安全性指标在治疗前后均无统计学差异（P＞0.05），说明中药复方益糖康安全无毒性。
2.动物实验：
2.1动物疾病模型复制部分：模型组大鼠造模开始后，出现了游泳耐力逐日下降，食少懒动，大便溏薄，精神萎靡不振，体毛光亮度减退，从第4周后体重开始显著增加，第6周后空腹血糖增高，甘油三酯和高密度脂蛋白分别从第2周后表现出异常，造模结束后模型组出现高胰岛素血症、胰岛素抵抗，符合本研究拟建立的脾气虚证、代谢综合征的评估标准。
2.2代谢组学研究部分：将UPLC/Q-TOF-MS获得的原始质谱数据导入MarkerLynx软件包，进行峰识别、峰对齐和基线矫正等前处理，最后输出三维矩阵，即由精确质核比与保留时间组成的变量、样品名称和峰强度/面积。将处理后的数据矩阵导入Simca-P软件（版本11.5）分别进行主成分分析（PCA），偏最小二乘方判别分析（PLS-DA）和正交偏最小二乘方-判别分析（OPLS-DA），对两组样本之间的差异进行模型分析，各分组血样代谢谱存在差异，找到了与脾气虚证代谢综合征大鼠模型相关的潜在生物标记物，主要有N-乙酰-D-葡萄糖胺（N-acetyl-D-glucosamine）、溶血磷脂酰胆碱（lysophosphatidylcholine，LPC）、鞘磷脂(sphingomyelin，SM)、5-甲基胞嘧啶（5-methylcytosine）、促黑素抑制素（Melanostatin）、三羟基异黄酮（Genistein）、前列腺素A2（Prostaglandin A2）、同型半胱氨酸(homocysteine, HCY)、L-丝氨酸（L-Homoserine）等，上述物质的集合共同构成了脾虚证代谢综合征大鼠模型的代谢组学特征，而这些物质正是体内糖类、脂类、蛋白质等代谢紊乱的结果。经中药复方益糖康治疗4周后，模型组中具有重要生物活性意义的LPC、SM、HCY水平下降，而且出现一些新的生物活性物质，如甘油二酯(diacylglycerol，DAG)、6-酮-前列腺素F1α（6-Keto-prostaglandin F1a）、苯丙氨酸（phenylalanine）、半乳糖基鞘氨醇（galactosylsphingosine）、生物胞素（Biocytin）等代谢产物，呈现代谢网络修复的结果，整个代谢谱有向正常范围回归的趋势。
结论:
1.生活方式干预（保持适当的体重、适当运动、改变饮食结构以减少热量摄入，尽量避免吸烟和适度减少饮酒等）代谢综合征能减轻餐后2小时血糖、血压和体重指数。
2.中药复方益糖康不仅能降低血糖、血压，调节血脂代谢紊乱减轻代谢综合征危险因素，也能改善代谢综合征患者的主要临床症状和中医证候，同时提高患者的生存质量，且安全无毒性。
3.生活方式干预+中药复方益糖康治疗方案临床疗效优于单纯生活方式干预，可延缓脾气虚证代谢综合征病情发展，值得推广应用。
4.通过劳倦加饮食不节（高糖高脂膳食）造模方法可成功复制出脾气虚证代谢综合征动物模型。
5.发现了N-乙酰-D-葡萄糖胺、溶血磷脂酰胆碱、鞘磷脂、5-甲基胞嘧啶、促黑素抑制素、三羟基异黄酮、前列腺素A2、同型半胱氨酸、L-丝氨酸等潜在脾气虚证代谢综合征动物模型生物标志物，揭示脾气虚证代谢综合征的发生与糖类、脂类、蛋白质代谢紊乱有关，这些体内的小分子化合物可能是代谢综合征中医证候的物质基础。
6.中药复方益糖康的作用显示出多途径、多靶点、双向调节特点。不仅能降低血浆中溶血磷脂酰胆碱、鞘磷脂、同型半胱氨酸等代谢物质的含量，并产生一些新的生物活性物质如甘油二酯、6-酮-前列腺素F1α、苯丙氨酸等，修复了糖类、脂类、蛋白质代谢网络紊乱，使整个代谢谱向正常范围回归。
7.代谢组学方法是研究代谢综合征和相关中药机理的良好平台。
Purpose: Metabolic Syndrome is a combination of risk factors including obesity,high blood sugar(diabetes or impaired glucose regulation),dyslipidemia(hypertriglyceridemia and/or low high-density lipoproteincholesterol), hypertension,which can seriously affect the health of the body.These factors are directly contributed to the occurrence of atheroscleroticcardiovascular disease and increase the risk of type2diabetes. In thisprospective study, the object of study is the metabolic syndrome and impairedglucose regulation,and in line with the TCM diagnosis.Randomized,double-blind, parallel-group, multicenter clinical trial methods are used asresearch methods, the main inspection targets include glucose, lipids, bloodpressure, glycosylated hemoglobin and body mass index. We observed theeffectiveness of traditional Chinese medicine Yi Tangkang treatmentingmetabolic syndrome and the lifestyle intervention+traditional Chinese medicineYi Tangkang. Metabolomics focuses on the metabolic pathways of metabolism ofendogenous substances including whole-organ or tissue and metabolism of theoverall change in trajectory suffered internal and external factors, reflecta series of biological events that occured in certain pathophysiological process.In this study, by copying the spleen deficiency syndrome metabolic syndrome ratmodel, we researched model plasma specimens by metabolomics and looked for spleendeficiency syndrome metabolic syndrome lesions characteristic metabolites,observed metabolite changes related to physiological and pathological, exploredthe molecular mechanism of spleen deficiency syndrome metabolic syndrome lesionsand further revealled the essence of spleen deficiency syndrome metabolicsyndrome. We explained traditional Chinese medicine Yi Tangkang interventionmechanism and targets of interpretation from the point of view of endogenousmetabolites by using metabolomics technology and methods,
Material and method:
1. Clinical research
Double-blind, parallel-group, multicenter clinical trial methods are used asresearch methods.We Collected165metabolic syndrome in line with the TCMdiagnostic criteria. Patients were randomly assigned to two groups. Treatmentgroups: lifestyle intervention (diet, exercise)+traditional Chinese medicine.Control group: Lifestyle intervention (diet, exercise)+Chinese medicineplacebo. In the entire clinical trial period,we analyzed all cases ofbaseline(epidemiological data, safety indicators, indicators of effectiveness).We observed the data and indicators of treatment and control groups before thetest and the existence of comparable. In all cases a variety of indicators ofeffectiveness,including the main effect indicators (blood glucose, blood lipids,blood pressure,glycosylated hemoglobin)and indicators of secondary effects(weight, waist circumference, BMI, and TCM syndromeindicators, traditionalChinese medicine syndrome indicators, quality of life points, etc.) have beenanalyzed.We compared the data and established a compliance rate criterion. Wehave observed that the traditional Chinese medicine Yi Tangkang and Chinese andWestern medicine combined with the clinical efficacy of the treatment program.We compared the safety index concerned with the blood, urine, stool routine,liver function, renal function, etc and observe the security of trial medicationChinese herbal compound Yi Tangkang.
2. Experimental study
2.1Replication of animal disease models
30healthy male SD rats, weighing (180±20) g, were randomly divided intotwo groups after adaptive feeding days. Blank control group (n=10) and modelgroup (n=30). The model group were fed cabbage15~20g/a rat in a singleday and allowed to drink water freely, swimm to the endurance limit. The modelgroup were fed high sugar, high fat diet feeding in double day, a total of12weeks. W looked forward to Copy the model of spleen deficiency syndrome andmetabolic syndrome.
2.2metabolomics studies
Spleen deficiency syndrome and metabolic syndrome rats were dividedrandomly into two groups: model group (n=6) and model+TCM group (n=6).Weused ultra high performance liquid chromatography–quadrupole–time offlight mass spectrometry(UPLC/Q-TOF-MS)technology and detected smallmolecule metabolites in plasma. We builted a UPLC/Q-TOF-MS-MS blood totalion chromatogram and getted a metabolic fingerprint which were Were analyzedusing principal Component Analysis and Partial Least Squares-DiscriminantAnalysis.We identified the differences in characteristics of metabolites andFound some biomarkers, traced their sources and metabolic pathways. Accordingto the clinical patient day of Chinese medicine dosage were converted into theratequivalent dose, once a day orally, for4weeks. After the end of the UPLC/Q-TOF-MS-MS detection,we observed the group differences in changes of plasmasmall-molecule metabolites spectrum of the spleen deficiency syndrome andmetabolic syndrome model.
Results:
1.Clinical trials:
1.1Baseline analysis: Demographic data baseline: age, sex distribution,no significant difference between the two groups（P＞0.05）. Duration baseline:systolic blood pressure, diastolic blood pressure, fasting glucose,2-hourpostprandialblood glucose, glycosylated hemoglobin,no significantdifference between the two groups（P＞0.05）. The primary efficacy endpointbaseline:no significant difference between the two groups （P＞0.05）Secondary outcome measures baseline: Weight, waist circumference, hipcircumference, BMI,, TCM syndromes total points, the survival scale scoring,no significant difference between the two groups（P＞0.05）.
1.2Analysis of efectiveness: lifestyle intervention can significantlyreduce the2-hour postprandial blood glucose,blood pressure and BMI.After the intervention of Chinese herbal compound Yi Tangkang,2hours postprandial blood sugar, blood pressure and BMI had been improved, triglycerides and total cholesterol declined, improved fasting blood glucose,LDL and HDL, improve the compliance rate of fasting plasma glucose, twohours postprandial blood sugar, high-density lipoprotein, blood pressure andBMI. Chinese herbal compound Yi Tangkang can improve effectively the mainclinical symptoms and general condition of MS patients, whileimproving patient quality of life.
1.3Security analysis: the safety index of the two groups before and aftertreatment were not statistically different (P>0.05). Chinese herbalcompound Yi Tang Kang is safe.
2.Animal experiments:
2.1Animal disease model experiment: rats showed swimming endurance decreased,eat less lazy, loose stools thin, spiritual malaise, body Mao Guangliang degreediminished.The weight began to increase significantly after the the four weeks,fasting blood glucose increased after the the sixr weeks,triglycerides and highdensity lipoprotein showed abnormal after the the two weeks. Rats showedhyperinsulinemia, insulin resistance Lining with spleen deficiency syndrome andmetabolic syndrome evaluation criteria.
2.2Metabolomics studies: The original mass spectrometry data wered importedinto MarkerLynx package and completed the pre-treatment of the peakidentification, peak alignment and baseline correction,outputedthree-dimensional matrix last. The processed data matrix wered imported Simca-Psoftware (version11.5)to analyze PCA, PLS-DA, OPLS-DA. there were significantdifferences in each grouping blood metabolic spectrum. We found a number ofpotential biomarkers related to the metabolic syndrome rat modelof spleendeficiency syndrome: N-acetyl-D-glucosamine, lysophosphatidylcholine,sphingomyelin,5-methylcytosine, Melanostatin, Genistein, Prostaglandin A2,homocysteine, L-Homoserine. The collection of these substances togetherconstitute the Spleen Deficiency Syndromemetabolic syndrome rat model ofmetabolic group characteristics and are the results of the carbohydrates, lipids,proteins and other metabolic disorders.4weeks of treatment by traditional Chinese medicine Yi Tangkang, LPC、SM、HCY decreased, some new biologically activesubstances appeared: diacylglycerol,6-Keto-prostaglandin F1a, phenylalanine,galactosylsphingosine, Biocytin,which showing metabolic network repairment,the whole metabolic spectrum returned to the normal range.
Conclusions:
1. Lifestyle intervention in metabolic syndrome (to maintain properweight, proper exercise, changes in diet to reduce calorieintake, avoid smoking and a modest reduction indrinking, etc.) canreduce the2-hour postprandial blood glucose, blood pressure andBMI.
2. Yi Tangkang, a Chinese herbal formula, can not only lower blood sugar, bloodpressure, regulation of lipid metabolism disorders to reduce the risk factorsof metabolic syndrome, can also improve the clinical symptoms of the metabolicsyndrome and TCM syndromes, while improving the quality of life of patients andsafe non-toxic.
3. Lifestyle intervention+Chinese medicine treatment options in clinicalefficacy is superior to the simple lifestyle interventions,which can delay thedevelopment of spleen deficiency syndrome and metabolic syndrome condition andshould be widely applied.
4. Weary plus improper diet (high sugar, high fat diet) modeling method can besuccessfully copied spleen deficiency syndrome and metabolic syndrome in animalmodels.
5.N-acetyl-D-glucosamine, lysophosphatidylcholine, sphingomyelin,5-methylcytosine, Melanostatin, Genistein, Prostaglandin A2, homocysteine,L-Homoserine. The collection of these substances together constitute the SpleenDeficiency Syndromemetabolic syndrome rat model of metabolic groupcharacteristics and are the results of the carbohydrates, lipids, proteins andother metabolic disorders.
6. Chinese herbal compound Yi Tangkang shows the multi-channel, multi-target,two-way adjustment features. LPC、SM、HCY decreased, some new biologically active substances appeared: diacylglycerol,6-Keto-prostaglandin F1a, phenylalanine,galactosylsphingosine, Biocytin,which showing metabolic network repairment,the whole metabolic spectrum returned to the normal range.
7. Metabonomics is a good platform to study the mechanism of metabolic syndromeand traditional Chinese medicine.
代谢综合征(metabolic syndrome，MS)是一组以肥胖、高血糖（糖尿病或糖调节受损）、血脂异常（指高甘油三酯血症和/或低高密度脂蛋白胆固醇血症）以及高血压等聚集发病，严重影响机体健康的临床征候群，是一组在代谢上相互关联的危险因素的组合，这些因素直接促进了动脉粥样硬化性心血管疾病的发生，也增加了发生2型糖尿病的风险。本研究以前瞻性的研究方法，选择并有糖调节受损的代谢综合征患者作为研究对象，并符合中医辨证诊断者。通过随机、双盲、平行对照、多中心临床试验的方法，以血糖、血脂、血压、糖化血红蛋白和体重指数等作为主要考察指标，观察中药复方益糖康干预代谢综合征的有效性及生活方式干预+中药复方益糖康治疗方案的优效性。代谢组学着重研究的是生物整体、器官或组织的内源性代谢物质的代谢途径及其所受内在和外在因素影响下代谢整体的变化轨迹来反映某种病理生理过程中所发生的一系列生物事件。这种整体的研究模式可全面了解代谢物质在疾病发生、发展过程中的变化规律，对发病机制研究以及疾病的诊断、治疗和疗效评估具有重要意义。本研究拟通过复制脾虚证代谢综合征大鼠模型，对模型血浆标本的代谢组学研究，寻找脾虚证代谢综合征病变的特征性代谢产物，观察代谢物变化与生理病理相关的改变关系，探寻脾虚证代谢综合征病变的分子机制，从而进一步揭示脾虚证代谢综合征本质。同时运用代谢组学的技术与方法，从机体内源性代谢物的角度阐释中药复方益糖康的干预机制及作用靶点，为中药治疗代谢综合征作用机理的系统研究提供科学依据。
材料与方法：
1.临床研究
采用随机、双盲、平行对照、多中心临床试验方法，收集符合中医辨证诊断标准的代谢综合征患者165名。随机分配到两个组中。治疗组：生活方式干预（饮食控制，运动）+中药。对照组：生活方式干预（饮食控制，运动）+中药安慰剂。在整个的临床试验周期内，对全部病例进行基线（包括流行病学资料、安全性指标、有效性指标等）分析，观测在试验前治疗组与对照组的各项数据和指标有无显著性差异,是否具有可比性。对全部病例的各种有效性指标，包括主要效应指标（血糖、血脂、血压、糖化血红蛋白）和次要效应指标（体重、腰围、体重指数、中医证型指标、中医证候指标、生存质量积分等）进行治疗前后及组间的比较，并确立达标率判断标准，观察中药复方益糖康及中西医结合治疗方案的临床疗效。分别对治疗组和对照组全部病例的安全性指标进行治疗前后的比较，涉及的内容有血、尿、便常规，肝功，肾功等，观察本试验用药中药复方益糖康安全性。
2.实验研究
2.1动物疾病模型的复制
健康雄性SD大鼠30只，体重（180士20）g，适应性饲养1天后按体重随机分成两组：空白对照组（n=10）和模型组(n=30)。模型组单日喂食甘蓝15～20g/只，自由饮水，游泳至耐力极限；双日高糖高脂膳食持续喂养，共计12周，复制脾气虚证代谢综合征模型。
2.2代谢组学研究
将复制成功的脾气虚证代谢综合征大鼠按体重随机分为两组：模型组（n=6）和模型+中药组（n=6）。运用超高压液相色谱-四极杆-时间飞行串联质谱仪（ultra highperformance liquid chromatography–quadrupole–time of flight mass spectrometry，UPLC/Q-TOF-MS）技术检测模型组血浆中的小分子代谢产物，建立UPLC/Q-TOF-MS大鼠血样总离子流色谱图，对所得到的代谢指纹图谱进行主成分分析和偏最小二乘法-判别分析，找出具有差异的特征代谢物，从而辨识生物标志物并追溯其来源和代谢途径。根据临床患者一日中药用量折算成大鼠的等效剂量为每日0.4g/100g体重，模型+中药组大鼠每天一次灌胃，连续4周。通过UPLC/Q-TOF-MS技术检测，观察脾气虚证代谢综合征模型经中药复方益糖康干预后，血浆小分子代谢物组的差异性变化谱。
结果:
1.临床试验：
1.1基线分析：人口学资料基线比较，包括年龄、性别分布。两组患者间无显著性差异（P＞0.05）。病程基线比较，两组患者间无显著性差异（P＞0.05）。主要疗效指标基线比较，包括收缩压、舒张压、空腹血糖、餐后2小时血糖、糖化血红蛋白。两组患者间无显著性差异（P＞0.05）。次要疗效指标基线比较，包括体重、腰围、臀围、体重指数、中医证候总计分、生存量表计分。两组患者间无显著性差异（P＞0.05）。
1.2有效性分析：生活方式干预可以显著降低餐后2小时血糖、血压和体重指数。中药复方益糖康干预后，不仅可以改善患者餐后2小时血糖、血压和体重指数，还能显著降低MS患者的甘油三酯和总胆固醇。同时，对于空腹血糖、低密度脂蛋白和高密度脂蛋白也有改善作用，并可提高空腹血糖、餐后两小时血糖、高密度脂蛋白、血压和体重指数的达标率。还能够有效地改善MS患者的主要临床症状及全身状况，同时提高患者的生存质量。
1.3安全性分析：两组安全性指标在治疗前后均无统计学差异（P＞0.05），说明中药复方益糖康安全无毒性。
2.动物实验：
2.1动物疾病模型复制部分：模型组大鼠造模开始后，出现了游泳耐力逐日下降，食少懒动，大便溏薄，精神萎靡不振，体毛光亮度减退，从第4周后体重开始显著增加，第6周后空腹血糖增高，甘油三酯和高密度脂蛋白分别从第2周后表现出异常，造模结束后模型组出现高胰岛素血症、胰岛素抵抗，符合本研究拟建立的脾气虚证、代谢综合征的评估标准。
2.2代谢组学研究部分：将UPLC/Q-TOF-MS获得的原始质谱数据导入MarkerLynx软件包，进行峰识别、峰对齐和基线矫正等前处理，最后输出三维矩阵，即由精确质核比与保留时间组成的变量、样品名称和峰强度/面积。将处理后的数据矩阵导入Simca-P软件（版本11.5）分别进行主成分分析（PCA），偏最小二乘方判别分析（PLS-DA）和正交偏最小二乘方-判别分析（OPLS-DA），对两组样本之间的差异进行模型分析，各分组血样代谢谱存在差异，找到了与脾气虚证代谢综合征大鼠模型相关的潜在生物标记物，主要有N-乙酰-D-葡萄糖胺（N-acetyl-D-glucosamine）、溶血磷脂酰胆碱（lysophosphatidylcholine，LPC）、鞘磷脂(sphingomyelin，SM)、5-甲基胞嘧啶（5-methylcytosine）、促黑素抑制素（Melanostatin）、三羟基异黄酮（Genistein）、前列腺素A2（Prostaglandin A2）、同型半胱氨酸(homocysteine, HCY)、L-丝氨酸（L-Homoserine）等，上述物质的集合共同构成了脾虚证代谢综合征大鼠模型的代谢组学特征，而这些物质正是体内糖类、脂类、蛋白质等代谢紊乱的结果。经中药复方益糖康治疗4周后，模型组中具有重要生物活性意义的LPC、SM、HCY水平下降，而且出现一些新的生物活性物质，如甘油二酯(diacylglycerol，DAG)、6-酮-前列腺素F1α（6-Keto-prostaglandin F1a）、苯丙氨酸（phenylalanine）、半乳糖基鞘氨醇（galactosylsphingosine）、生物胞素（Biocytin）等代谢产物，呈现代谢网络修复的结果，整个代谢谱有向正常范围回归的趋势。
结论:
1.生活方式干预（保持适当的体重、适当运动、改变饮食结构以减少热量摄入，尽量避免吸烟和适度减少饮酒等）代谢综合征能减轻餐后2小时血糖、血压和体重指数。
2.中药复方益糖康不仅能降低血糖、血压，调节血脂代谢紊乱减轻代谢综合征危险因素，也能改善代谢综合征患者的主要临床症状和中医证候，同时提高患者的生存质量，且安全无毒性。
3.生活方式干预+中药复方益糖康治疗方案临床疗效优于单纯生活方式干预，可延缓脾气虚证代谢综合征病情发展，值得推广应用。
4.通过劳倦加饮食不节（高糖高脂膳食）造模方法可成功复制出脾气虚证代谢综合征动物模型。
5.发现了N-乙酰-D-葡萄糖胺、溶血磷脂酰胆碱、鞘磷脂、5-甲基胞嘧啶、促黑素抑制素、三羟基异黄酮、前列腺素A2、同型半胱氨酸、L-丝氨酸等潜在脾气虚证代谢综合征动物模型生物标志物，揭示脾气虚证代谢综合征的发生与糖类、脂类、蛋白质代谢紊乱有关，这些体内的小分子化合物可能是代谢综合征中医证候的物质基础。
6.中药复方益糖康的作用显示出多途径、多靶点、双向调节特点。不仅能降低血浆中溶血磷脂酰胆碱、鞘磷脂、同型半胱氨酸等代谢物质的含量，并产生一些新的生物活性物质如甘油二酯、6-酮-前列腺素F1α、苯丙氨酸等，修复了糖类、脂类、蛋白质代谢网络紊乱，使整个代谢谱向正常范围回归。
7.代谢组学方法是研究代谢综合征和相关中药机理的良好平台。
Purpose: Metabolic Syndrome is a combination of risk factors including obesity,high blood sugar(diabetes or impaired glucose regulation),dyslipidemia(hypertriglyceridemia and/or low high-density lipoproteincholesterol), hypertension,which can seriously affect the health of the body.These factors are directly contributed to the occurrence of atheroscleroticcardiovascular disease and increase the risk of type2diabetes. In thisprospective study, the object of study is the metabolic syndrome and impairedglucose regulation,and in line with the TCM diagnosis.Randomized,double-blind, parallel-group, multicenter clinical trial methods are used asresearch methods, the main inspection targets include glucose, lipids, bloodpressure, glycosylated hemoglobin and body mass index. We observed theeffectiveness of traditional Chinese medicine Yi Tangkang treatmentingmetabolic syndrome and the lifestyle intervention+traditional Chinese medicineYi Tangkang. Metabolomics focuses on the metabolic pathways of metabolism ofendogenous substances including whole-organ or tissue and metabolism of theoverall change in trajectory suffered internal and external factors, reflecta series of biological events that occured in certain pathophysiological process.In this study, by copying the spleen deficiency syndrome metabolic syndrome ratmodel, we researched model plasma specimens by metabolomics and looked for spleendeficiency syndrome metabolic syndrome lesions characteristic metabolites,observed metabolite changes related to physiological and pathological, exploredthe molecular mechanism of spleen deficiency syndrome metabolic syndrome lesionsand further revealled the essence of spleen deficiency syndrome metabolicsyndrome. We explained traditional Chinese medicine Yi Tangkang interventionmechanism and targets of interpretation from the point of view of endogenousmetabolites by using metabolomics technology and methods,
Material and method:
1. Clinical research
Double-blind, parallel-group, multicenter clinical trial methods are used asresearch methods.We Collected165metabolic syndrome in line with the TCMdiagnostic criteria. Patients were randomly assigned to two groups. Treatmentgroups: lifestyle intervention (diet, exercise)+traditional Chinese medicine.Control group: Lifestyle intervention (diet, exercise)+Chinese medicineplacebo. In the entire clinical trial period,we analyzed all cases ofbaseline(epidemiological data, safety indicators, indicators of effectiveness).We observed the data and indicators of treatment and control groups before thetest and the existence of comparable. In all cases a variety of indicators ofeffectiveness,including the main effect indicators (blood glucose, blood lipids,blood pressure,glycosylated hemoglobin)and indicators of secondary effects(weight, waist circumference, BMI, and TCM syndromeindicators, traditionalChinese medicine syndrome indicators, quality of life points, etc.) have beenanalyzed.We compared the data and established a compliance rate criterion. Wehave observed that the traditional Chinese medicine Yi Tangkang and Chinese andWestern medicine combined with the clinical efficacy of the treatment program.We compared the safety index concerned with the blood, urine, stool routine,liver function, renal function, etc and observe the security of trial medicationChinese herbal compound Yi Tangkang.
2. Experimental study
2.1Replication of animal disease models
30healthy male SD rats, weighing (180±20) g, were randomly divided intotwo groups after adaptive feeding days. Blank control group (n=10) and modelgroup (n=30). The model group were fed cabbage15~20g/a rat in a singleday and allowed to drink water freely, swimm to the endurance limit. The modelgroup were fed high sugar, high fat diet feeding in double day, a total of12weeks. W looked forward to Copy the model of spleen deficiency syndrome andmetabolic syndrome.
2.2metabolomics studies
Spleen deficiency syndrome and metabolic syndrome rats were dividedrandomly into two groups: model group (n=6) and model+TCM group (n=6).Weused ultra high performance liquid chromatography–quadrupole–time offlight mass spectrometry(UPLC/Q-TOF-MS)technology and detected smallmolecule metabolites in plasma. We builted a UPLC/Q-TOF-MS-MS blood totalion chromatogram and getted a metabolic fingerprint which were Were analyzedusing principal Component Analysis and Partial Least Squares-DiscriminantAnalysis.We identified the differences in characteristics of metabolites andFound some biomarkers, traced their sources and metabolic pathways. Accordingto the clinical patient day of Chinese medicine dosage were converted into theratequivalent dose, once a day orally, for4weeks. After the end of the UPLC/Q-TOF-MS-MS detection,we observed the group differences in changes of plasmasmall-molecule metabolites spectrum of the spleen deficiency syndrome andmetabolic syndrome model.
Results:
1.Clinical trials:
1.1Baseline analysis: Demographic data baseline: age, sex distribution,no significant difference between the two groups（P＞0.05）. Duration baseline:systolic blood pressure, diastolic blood pressure, fasting glucose,2-hourpostprandialblood glucose, glycosylated hemoglobin,no significantdifference between the two groups（P＞0.05）. The primary efficacy endpointbaseline:no significant difference between the two groups （P＞0.05）Secondary outcome measures baseline: Weight, waist circumference, hipcircumference, BMI,, TCM syndromes total points, the survival scale scoring,no significant difference between the two groups（P＞0.05）.
1.2Analysis of efectiveness: lifestyle intervention can significantlyreduce the2-hour postprandial blood glucose,blood pressure and BMI.After the intervention of Chinese herbal compound Yi Tangkang,2hours postprandial blood sugar, blood pressure and BMI had been improved, triglycerides and total cholesterol declined, improved fasting blood glucose,LDL and HDL, improve the compliance rate of fasting plasma glucose, twohours postprandial blood sugar, high-density lipoprotein, blood pressure andBMI. Chinese herbal compound Yi Tangkang can improve effectively the mainclinical symptoms and general condition of MS patients, whileimproving patient quality of life.
1.3Security analysis: the safety index of the two groups before and aftertreatment were not statistically different (P>0.05). Chinese herbalcompound Yi Tang Kang is safe.
2.Animal experiments:
2.1Animal disease model experiment: rats showed swimming endurance decreased,eat less lazy, loose stools thin, spiritual malaise, body Mao Guangliang degreediminished.The weight began to increase significantly after the the four weeks,fasting blood glucose increased after the the sixr weeks,triglycerides and highdensity lipoprotein showed abnormal after the the two weeks. Rats showedhyperinsulinemia, insulin resistance Lining with spleen deficiency syndrome andmetabolic syndrome evaluation criteria.
2.2Metabolomics studies: The original mass spectrometry data wered importedinto MarkerLynx package and completed the pre-treatment of the peakidentification, peak alignment and baseline correction,outputedthree-dimensional matrix last. The processed data matrix wered imported Simca-Psoftware (version11.5)to analyze PCA, PLS-DA, OPLS-DA. there were significantdifferences in each grouping blood metabolic spectrum. We found a number ofpotential biomarkers related to the metabolic syndrome rat modelof spleendeficiency syndrome: N-acetyl-D-glucosamine, lysophosphatidylcholine,sphingomyelin,5-methylcytosine, Melanostatin, Genistein, Prostaglandin A2,homocysteine, L-Homoserine. The collection of these substances togetherconstitute the Spleen Deficiency Syndromemetabolic syndrome rat model ofmetabolic group characteristics and are the results of the carbohydrates, lipids,proteins and other metabolic disorders.4weeks of treatment by traditional Chinese medicine Yi Tangkang, LPC、SM、HCY decreased, some new biologically activesubstances appeared: diacylglycerol,6-Keto-prostaglandin F1a, phenylalanine,galactosylsphingosine, Biocytin,which showing metabolic network repairment,the whole metabolic spectrum returned to the normal range.
Conclusions:
1. Lifestyle intervention in metabolic syndrome (to maintain properweight, proper exercise, changes in diet to reduce calorieintake, avoid smoking and a modest reduction indrinking, etc.) canreduce the2-hour postprandial blood glucose, blood pressure andBMI.
2. Yi Tangkang, a Chinese herbal formula, can not only lower blood sugar, bloodpressure, regulation of lipid metabolism disorders to reduce the risk factorsof metabolic syndrome, can also improve the clinical symptoms of the metabolicsyndrome and TCM syndromes, while improving the quality of life of patients andsafe non-toxic.
3. Lifestyle intervention+Chinese medicine treatment options in clinicalefficacy is superior to the simple lifestyle interventions,which can delay thedevelopment of spleen deficiency syndrome and metabolic syndrome condition andshould be widely applied.
4. Weary plus improper diet (high sugar, high fat diet) modeling method can besuccessfully copied spleen deficiency syndrome and metabolic syndrome in animalmodels.
5.N-acetyl-D-glucosamine, lysophosphatidylcholine, sphingomyelin,5-methylcytosine, Melanostatin, Genistein, Prostaglandin A2, homocysteine,L-Homoserine. The collection of these substances together constitute the SpleenDeficiency Syndromemetabolic syndrome rat model of metabolic groupcharacteristics and are the results of the carbohydrates, lipids, proteins andother metabolic disorders.
6. Chinese herbal compound Yi Tangkang shows the multi-channel, multi-target,two-way adjustment features. LPC、SM、HCY decreased, some new biologically active substances appeared: diacylglycerol,6-Keto-prostaglandin F1a, phenylalanine,galactosylsphingosine, Biocytin,which showing metabolic network repairment,the whole metabolic spectrum returned to the normal range.
7. Metabonomics is a good platform to study the mechanism of metabolic syndromeand traditional Chinese medicine.
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