绞股蓝复方降糖、降脂作用的实验研究
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摘要
目的：高脂血症是一种以血浆中脂质成分异常增高为特征的代谢紊乱综合病症。随着生活水平的提高和生活习惯的改变，其发病率有逐年上升的趋势，同时，它也是引起脂肪肝、糖尿病、肥胖症、动脉粥样硬化和心脑血管疾病的罪魁祸首。糖尿病是由各种致病因子作用于机体导致胰岛功能减退、胰岛素分泌不足和胰岛素抵抗等而引发的以高血糖为主要特征的代谢紊乱综合征。糖尿病在全球已经成为继心脑血管疾病、肿瘤之后严重危害人类健康的第三大慢性病。目前在临床上，这两种疾病的治疗均以西药为主，虽然西药疗效确切，但作用途径单一，有的具有严重的不良反应，不能从根本上控制疾病的发展和缓解疾病的并发症。中医药理论在预防和治疗高脂血症和糖尿病的过程中，能够通过多途径和多靶点的优势发挥广泛作用，疗效稳定，毒性和不良反应小，作用温和持久。本课题旨在通过对绞股蓝复方及其两味君药绞股蓝和苦丁茶降糖降脂作用的动物实验研究，检测动物模型血糖、总胆固醇、甘油三酯等血清指标的变化，确定其有效活性部位；并通过对氧化活性检测、胆固醇代谢和糖代谢相关基因表达的变化，探讨其降糖调脂作用的可能机制，为开发出具有确切调脂降糖疗效的复方绞股蓝提供有效的理论支撑和科学依据。
方法：选取雄性昆明小鼠，用不同的造模方法，建立糖尿病动物模型和高脂血症动物模型；绞股蓝水提、醇沉提取并经大孔树脂分离得到三个组分，苦丁茶水提、醇沉提取并经硅胶柱层析得到七个组分；不同组分的绞股蓝和苦丁茶及绞股蓝复方分别作用于高脂血症和糖尿病动物模型，观察小鼠体重及饮水量变化，并通过血液各项生化指标的测量来分析各组分发挥的降糖调脂作用；通过肝脏病理切片观察了不同药物组分作用后肝脏组织的病理形态学变化；通过RT-PCR的方法检测肝脏中与脂代谢相关基因载脂蛋白AI（ApoAI）、胆固醇7α-羟化酶（Cyp7α1）、羟甲基戊二酸单酰辅酶A还原酶（Hmgcr）及固醇调控元件结合转录因子2（Srebf2）mRNA的相对表达量和糖代谢相关基因葡萄糖-6-磷酸酶催化酶（G6pc）、葡萄糖激酶（Gck）和胰岛素受体（Insr）mRNA的相对表达量来推断绞股蓝、苦丁茶及复方降糖调脂作用的可能机制。
结果：①与模型组比较，绞股蓝总提物和组分3均能降低糖尿病小鼠的GLU，NEFA，TC，TG和MDA值，升高糖尿病小鼠的血清SOD值，缓解糖尿病小鼠“三多一少”的症状，确定绞股蓝组分3为降血糖的有效部位。②与模型组对比，苦丁茶组分1和组分2能显著降低高脂血症动物模型的血脂代谢指标TC和TG，显著降低心脏收缩压，对心率没有影响；病理学研究表明，经过组分1和组分2治疗后的两组高脂血症动物模型肝脏内脂肪沉积样变性和水肿样变性减少；此外，ApoAI和Cyp7α1基因mRNA的表达明显上调，而Hmgcr和Srebf2基因mRNA的表达下调；研究确定了苦丁茶组分1和组分2是降脂作用的的有效部位。③与模型组对比，苦丁茶组分1和组分2能显著降低糖尿病小鼠动物模型的血糖和血脂水平，有效改善OGTT，但体重、心率和脏器系数没有明显的改变；病理学研究表明，经过组分1和组分2治疗后的两组糖尿病动物模型肝脏内脂肪沉积样变性和水肿样变性减少；此外，Gck和Insr基因mRNA的表达明显上调，而G6pc基因mRNA的表达下调；研究确定了苦丁茶组分1和组分2是降糖作用的的有效部位。④与高脂模型组相比较，复方给药组小鼠血清TC、TG含量显著降低，HDL-C含量显著升高，小鼠肝脏病变情况较高脂模型组显著好转；同时小鼠肝脏中Cyp7α1和ApoAI基因mRNA相对表达量显著上调，Srebf2及Hmgcr基因mRNA的相对表达量显著下调。
结论：本研究通过对绞股蓝和苦丁茶两味植物药主要有效成分降糖、降脂作用的实验研究，找到了能够降低糖尿病小鼠的血糖值，改善糖尿病小鼠的症状，并可调节其血脂代谢的绞股蓝组分，证实了绞股蓝治疗糖尿病的药效，并找到了主要药效部位；证实了苦丁茶活性成分能够有效降低TC、TG，调节脂质代谢，减轻肝脏脂肪病变，有效缓解高脂血症；证实了苦丁茶活性成分能够有效发挥胰岛素增敏剂的作用，有效降低小鼠空腹血糖，改善OGTT，有效降低血液中的胆固醇，增加肝脏的抗氧化状态，缓解糖尿病引起的主动脉和肝脏病变；证实了绞股蓝软胶囊能够有效降低小鼠模型的TC、TG、LDL，升高HDL，改善肝脏脂肪病变；探讨了以绞股蓝和苦丁茶为主要成分的复方药降糖、降脂的作用机制，有效证明了中药复方能够通过多作用靶点，治疗高脂血症，为阐明绞股蓝等调脂降糖中药的作用机理进行了初步探讨，为进一步开发新型调脂降糖药物提供了一定的实验依据。在药物作用机制方面，通过对苦丁茶活性成分和绞股蓝复方降脂作用的研究，发现其作用机制与促进小鼠肝脏中Cyp7α1和ApoAI基因表达，抑制Srebf2、Hmgcr基因表达有关，并因此基因表达的上调和下调，抑制体内胆固醇的合成、促进胆固醇分解等作用达到调节脂质代谢的作用；证实苦丁茶活性成分和绞股蓝活性成分能够通过上调Insr，缓解胰岛素抵抗；通过上调G6pc和下调Gck，影响糖酵解和糖异生，影响体内葡萄糖代谢，有效降低糖尿病小鼠的血糖。通过机制的研究，为下一步深入研究绞股蓝及其复方降糖的机制和信号传导通路奠定了良好的基础。
Objective： Hyperlipidemia is a kind of metabolic disorder syndromewhich is characterized by abnormally increasing of plasma lipidcomposition.With the improvement of living standards and changing of livinghabits, the incidence rate of Hyperlipidemia is increasing year by year. It alsocan cause the fatty liver, diabetes, obesity, atherosclerosis, cardiovascular andcerebrovascular diseases. Diabetes is another kind of metabolic disordersyndrome which is caused by various pathogenic factors acting on the bodylead to islet dysfunction, insulin secretion and insulin resistance characterizedby abnormally increasing of glucose. Diabetes in the world becomes the thirdchronic diseases seriously harm to human health after tumor andcardiovascular and cerebrovascular diseases. At present, clinical doctorsusually use chmical medicine as conventional treatment of these two diseases,chemical medicine has curative effect, but the ways of effect is not wideenough, some have serious adverse reactions, some can not control thedevelopment of the disease and alleviate the complications of the diseasefundamentally. The theory of Chinese Traditional Medicine in the preventionand treatment of hyperlipidemia and diabetes can play an extensive rolethrough multiple ways of effect and multiple target points.Chtnese TraditionalMedicine has stable efficacy, little toxicity and adverse reaction. This research aims to study on the effect of hypoglycemic and lipid lowering ofGynostemma pentaphyllum compound and two kinds of main drugs by animalexperiments. Find the activity fraction by change detection of total cholesterol,triglyceride and blood glucose in animal model. And explore the possiblemechanism of its hypoglycemic and lipid-lowering effects, by detection ofgene expression of oxidative activity, and the gene expression of cholesterolmetabolism and sugar metabolism. Provide effective theoretical support andscientific basis for the development of compound Gynostemma which hasexact curative effect of hypoglycemic and lipid-lowering.
Methods：Select the male Kunming mice, by diffferent molding method,to establish animal model of diabetes and hyperlipidemia. Gynostemmapentaphyllum extracted by water and alcohol, and three fractions wereobtained by macroporous resin separation. Kudingcha extracted by water andalcohol, and silica gel column chromatography yielded seven fractions.Different components of Gynostemma pentaphyllum, Kudingcha and thecompound respectively act on hyperlipidemia and diabetes animal models, toobserve the mice weight and water volume changes, to analysis thehypoglycemic and lipid-lowering effects of different groups by measuring theblood biochemical index, to observe the pathological changes of liver tissue ofdifferent drug groups.To inference the possible mechanism of hypoglycemicand lipid-lowering effects through detecting the expression of genes associatedwith lipid metabolism, such as apolipoprotein AI(ApoAI), cholesterol7α-hydroxylase(Cyp7α1),3-hydroxy-3-ethylglutaryl coenzyme A(Hmgcr),sterol regulatory element-binding transcription factor-2(Srebf2), andexpression of genes associated with glucose metabolism, such asglucokinase(Gck), glucose-6-phosphatase catalytic enzyme(G6pc),insulinreceptor(Insr),by RT-PCR method.
Results：①Compared with the model group, groups treated by totalextract and third fraction of Gynostemma pentaphyllum can reduce the valueof GLU, NEFA, TC, TG and MDA of diabetes mice, and increase the value ofserum SOD of diabetes mice. The experiment confirm that fractions ofGynostemma pentaphyllum can alleviate the symptoms of diabetes mice, andidentify the third fraction of gynostemma is the active part of falling bloodglucose.②Compared with the model group, groups treated by fractions1and2of Kudingcha can significantly reduce index of lipid metabolism such as TCand TG in hyperlipidemia animal model, and significant lower systolic bloodpressure, but has no effect on heart rate. The pathological study shows that,deposition of fat degeneration and edema degeneration in liver has reduced intwo groups of hyperlipidemia treated by fraction1and2. In addition, theexpression of ApoAI and Cyp7α1gene mRNA increased, and the expressionof Hmgcr and Srebf2gene mRNA decreased. The study identified thefractions1and2of Kudingcha is the active part of lipid-lowering effect.③Compared with the model group, groups treated by fractions1and2ofKudingcha can significantly reduce the value of blood glucose and blood lipidin diabetic mice animal model, Improve the OGTT, but the body weight, heartrate and organ coefficients do not change significantly after treated byfractions of Kudingcha. The pathological study shows that, deposition of fatdegeneration and edema degeneration in liver has reduced in two groups ofdiabetic mice treated by fraction1and2. In addition, the expression of Gckand Insr gene mRNA increased, and the expression of G6pc gene mRNAdecreased. The study identified the fractions1and2of Kudingcha is theactive part of lipid-lowering effect.④Compared with the hyperlipidemiamodel group, groups treated by the CGS could significantly reduce the level ofserum TC and TG, significantly increase the level of serum HDL-C in thehyperlipemia mice. In addition, the expression of ApoAI and Cyp7α1gene mRNA increased, and the expression of Hmgcr and Srebf2gene mRNAdecreased.
Conclusion：Through the experimental study on effects of hypoglycemicand lipid-lowering on main effective components of Gynostemmapentaphyllum and Kudingcha, we found the effective Gynostemma groupswhich can decrease blood glucose values, improve the symptoms of diabeticmice, and regulate their blood lipid metabolism, we confirmed the efficacy ofdiabetes of Gynostemma, and finds out the main pharmacodynamics part. Weconfirmed that the active ingredients of Kudingcha can effectively reduce TC,TG, regulate lipid metabolism, reduce hepatic steatosis, effectively alleviatehyperlipidemia. We confirmed Kudingcha active ingredients can effectivelyplay the role of insulin sensitizing agent, effectively reduce the mice fastingblood glucose, improve OGTT, reduce the cholesterol in the blood, increasingthe antioxidant status of the liver, relieving diabetic aorta and liver lesions. Weconfirmed the Gynostemma pentaphyllum soft capsule can effectively reducethe mouse model of TC, TG, LDL, increase HDL, improve the hepaticsteatosis. In this study, we discussed the interaction mechanism ofGynostemma pentaphyllum and Kudingcha as the main component of thehypoglycemic and lipid-lowering compound drugs, proves that the traditionalChinese medicine compound can Treatment of hyperlipidemia throughmultiple target. It is helpful to clarify the mechanism of regulating function ofblood lipid and reducing effect of blood glucose of Gynostemma, providecertain experimental basis for the further development of new lipid-loweringhypoglycemic drugs.In the aspect of mechanism of drug action, through thestudy of the active components of lipid-lowering effect of Kudingcha, wefound the mechanism is concerned with the promoting of the expression ofCyp7α1and ApoAI genes in mouse liver, and inhibiting the expression ofSrebf2and Hmgcr genes. Kudingcha ingredients are confirmed can relieve insulin resistance through the up regulation of Insr, affect the process ofglycolysis and gluconeogenesis through the upregulation of G6pc anddownregulation of Gck, affect glucose metabolism in vivo, reduce bloodglucose of the small rat of diabetes. All these research will help us to explainthe hypoglycemic mechanism and signal transduction pathway ofGynostemma pentaphyllum and it’s compound.
方法：选取雄性昆明小鼠，用不同的造模方法，建立糖尿病动物模型和高脂血症动物模型；绞股蓝水提、醇沉提取并经大孔树脂分离得到三个组分，苦丁茶水提、醇沉提取并经硅胶柱层析得到七个组分；不同组分的绞股蓝和苦丁茶及绞股蓝复方分别作用于高脂血症和糖尿病动物模型，观察小鼠体重及饮水量变化，并通过血液各项生化指标的测量来分析各组分发挥的降糖调脂作用；通过肝脏病理切片观察了不同药物组分作用后肝脏组织的病理形态学变化；通过RT-PCR的方法检测肝脏中与脂代谢相关基因载脂蛋白AI（ApoAI）、胆固醇7α-羟化酶（Cyp7α1）、羟甲基戊二酸单酰辅酶A还原酶（Hmgcr）及固醇调控元件结合转录因子2（Srebf2）mRNA的相对表达量和糖代谢相关基因葡萄糖-6-磷酸酶催化酶（G6pc）、葡萄糖激酶（Gck）和胰岛素受体（Insr）mRNA的相对表达量来推断绞股蓝、苦丁茶及复方降糖调脂作用的可能机制。
结果：①与模型组比较，绞股蓝总提物和组分3均能降低糖尿病小鼠的GLU，NEFA，TC，TG和MDA值，升高糖尿病小鼠的血清SOD值，缓解糖尿病小鼠“三多一少”的症状，确定绞股蓝组分3为降血糖的有效部位。②与模型组对比，苦丁茶组分1和组分2能显著降低高脂血症动物模型的血脂代谢指标TC和TG，显著降低心脏收缩压，对心率没有影响；病理学研究表明，经过组分1和组分2治疗后的两组高脂血症动物模型肝脏内脂肪沉积样变性和水肿样变性减少；此外，ApoAI和Cyp7α1基因mRNA的表达明显上调，而Hmgcr和Srebf2基因mRNA的表达下调；研究确定了苦丁茶组分1和组分2是降脂作用的的有效部位。③与模型组对比，苦丁茶组分1和组分2能显著降低糖尿病小鼠动物模型的血糖和血脂水平，有效改善OGTT，但体重、心率和脏器系数没有明显的改变；病理学研究表明，经过组分1和组分2治疗后的两组糖尿病动物模型肝脏内脂肪沉积样变性和水肿样变性减少；此外，Gck和Insr基因mRNA的表达明显上调，而G6pc基因mRNA的表达下调；研究确定了苦丁茶组分1和组分2是降糖作用的的有效部位。④与高脂模型组相比较，复方给药组小鼠血清TC、TG含量显著降低，HDL-C含量显著升高，小鼠肝脏病变情况较高脂模型组显著好转；同时小鼠肝脏中Cyp7α1和ApoAI基因mRNA相对表达量显著上调，Srebf2及Hmgcr基因mRNA的相对表达量显著下调。
结论：本研究通过对绞股蓝和苦丁茶两味植物药主要有效成分降糖、降脂作用的实验研究，找到了能够降低糖尿病小鼠的血糖值，改善糖尿病小鼠的症状，并可调节其血脂代谢的绞股蓝组分，证实了绞股蓝治疗糖尿病的药效，并找到了主要药效部位；证实了苦丁茶活性成分能够有效降低TC、TG，调节脂质代谢，减轻肝脏脂肪病变，有效缓解高脂血症；证实了苦丁茶活性成分能够有效发挥胰岛素增敏剂的作用，有效降低小鼠空腹血糖，改善OGTT，有效降低血液中的胆固醇，增加肝脏的抗氧化状态，缓解糖尿病引起的主动脉和肝脏病变；证实了绞股蓝软胶囊能够有效降低小鼠模型的TC、TG、LDL，升高HDL，改善肝脏脂肪病变；探讨了以绞股蓝和苦丁茶为主要成分的复方药降糖、降脂的作用机制，有效证明了中药复方能够通过多作用靶点，治疗高脂血症，为阐明绞股蓝等调脂降糖中药的作用机理进行了初步探讨，为进一步开发新型调脂降糖药物提供了一定的实验依据。在药物作用机制方面，通过对苦丁茶活性成分和绞股蓝复方降脂作用的研究，发现其作用机制与促进小鼠肝脏中Cyp7α1和ApoAI基因表达，抑制Srebf2、Hmgcr基因表达有关，并因此基因表达的上调和下调，抑制体内胆固醇的合成、促进胆固醇分解等作用达到调节脂质代谢的作用；证实苦丁茶活性成分和绞股蓝活性成分能够通过上调Insr，缓解胰岛素抵抗；通过上调G6pc和下调Gck，影响糖酵解和糖异生，影响体内葡萄糖代谢，有效降低糖尿病小鼠的血糖。通过机制的研究，为下一步深入研究绞股蓝及其复方降糖的机制和信号传导通路奠定了良好的基础。
Objective： Hyperlipidemia is a kind of metabolic disorder syndromewhich is characterized by abnormally increasing of plasma lipidcomposition.With the improvement of living standards and changing of livinghabits, the incidence rate of Hyperlipidemia is increasing year by year. It alsocan cause the fatty liver, diabetes, obesity, atherosclerosis, cardiovascular andcerebrovascular diseases. Diabetes is another kind of metabolic disordersyndrome which is caused by various pathogenic factors acting on the bodylead to islet dysfunction, insulin secretion and insulin resistance characterizedby abnormally increasing of glucose. Diabetes in the world becomes the thirdchronic diseases seriously harm to human health after tumor andcardiovascular and cerebrovascular diseases. At present, clinical doctorsusually use chmical medicine as conventional treatment of these two diseases,chemical medicine has curative effect, but the ways of effect is not wideenough, some have serious adverse reactions, some can not control thedevelopment of the disease and alleviate the complications of the diseasefundamentally. The theory of Chinese Traditional Medicine in the preventionand treatment of hyperlipidemia and diabetes can play an extensive rolethrough multiple ways of effect and multiple target points.Chtnese TraditionalMedicine has stable efficacy, little toxicity and adverse reaction. This research aims to study on the effect of hypoglycemic and lipid lowering ofGynostemma pentaphyllum compound and two kinds of main drugs by animalexperiments. Find the activity fraction by change detection of total cholesterol,triglyceride and blood glucose in animal model. And explore the possiblemechanism of its hypoglycemic and lipid-lowering effects, by detection ofgene expression of oxidative activity, and the gene expression of cholesterolmetabolism and sugar metabolism. Provide effective theoretical support andscientific basis for the development of compound Gynostemma which hasexact curative effect of hypoglycemic and lipid-lowering.
Methods：Select the male Kunming mice, by diffferent molding method,to establish animal model of diabetes and hyperlipidemia. Gynostemmapentaphyllum extracted by water and alcohol, and three fractions wereobtained by macroporous resin separation. Kudingcha extracted by water andalcohol, and silica gel column chromatography yielded seven fractions.Different components of Gynostemma pentaphyllum, Kudingcha and thecompound respectively act on hyperlipidemia and diabetes animal models, toobserve the mice weight and water volume changes, to analysis thehypoglycemic and lipid-lowering effects of different groups by measuring theblood biochemical index, to observe the pathological changes of liver tissue ofdifferent drug groups.To inference the possible mechanism of hypoglycemicand lipid-lowering effects through detecting the expression of genes associatedwith lipid metabolism, such as apolipoprotein AI(ApoAI), cholesterol7α-hydroxylase(Cyp7α1),3-hydroxy-3-ethylglutaryl coenzyme A(Hmgcr),sterol regulatory element-binding transcription factor-2(Srebf2), andexpression of genes associated with glucose metabolism, such asglucokinase(Gck), glucose-6-phosphatase catalytic enzyme(G6pc),insulinreceptor(Insr),by RT-PCR method.
Results：①Compared with the model group, groups treated by totalextract and third fraction of Gynostemma pentaphyllum can reduce the valueof GLU, NEFA, TC, TG and MDA of diabetes mice, and increase the value ofserum SOD of diabetes mice. The experiment confirm that fractions ofGynostemma pentaphyllum can alleviate the symptoms of diabetes mice, andidentify the third fraction of gynostemma is the active part of falling bloodglucose.②Compared with the model group, groups treated by fractions1and2of Kudingcha can significantly reduce index of lipid metabolism such as TCand TG in hyperlipidemia animal model, and significant lower systolic bloodpressure, but has no effect on heart rate. The pathological study shows that,deposition of fat degeneration and edema degeneration in liver has reduced intwo groups of hyperlipidemia treated by fraction1and2. In addition, theexpression of ApoAI and Cyp7α1gene mRNA increased, and the expressionof Hmgcr and Srebf2gene mRNA decreased. The study identified thefractions1and2of Kudingcha is the active part of lipid-lowering effect.③Compared with the model group, groups treated by fractions1and2ofKudingcha can significantly reduce the value of blood glucose and blood lipidin diabetic mice animal model, Improve the OGTT, but the body weight, heartrate and organ coefficients do not change significantly after treated byfractions of Kudingcha. The pathological study shows that, deposition of fatdegeneration and edema degeneration in liver has reduced in two groups ofdiabetic mice treated by fraction1and2. In addition, the expression of Gckand Insr gene mRNA increased, and the expression of G6pc gene mRNAdecreased. The study identified the fractions1and2of Kudingcha is theactive part of lipid-lowering effect.④Compared with the hyperlipidemiamodel group, groups treated by the CGS could significantly reduce the level ofserum TC and TG, significantly increase the level of serum HDL-C in thehyperlipemia mice. In addition, the expression of ApoAI and Cyp7α1gene mRNA increased, and the expression of Hmgcr and Srebf2gene mRNAdecreased.
Conclusion：Through the experimental study on effects of hypoglycemicand lipid-lowering on main effective components of Gynostemmapentaphyllum and Kudingcha, we found the effective Gynostemma groupswhich can decrease blood glucose values, improve the symptoms of diabeticmice, and regulate their blood lipid metabolism, we confirmed the efficacy ofdiabetes of Gynostemma, and finds out the main pharmacodynamics part. Weconfirmed that the active ingredients of Kudingcha can effectively reduce TC,TG, regulate lipid metabolism, reduce hepatic steatosis, effectively alleviatehyperlipidemia. We confirmed Kudingcha active ingredients can effectivelyplay the role of insulin sensitizing agent, effectively reduce the mice fastingblood glucose, improve OGTT, reduce the cholesterol in the blood, increasingthe antioxidant status of the liver, relieving diabetic aorta and liver lesions. Weconfirmed the Gynostemma pentaphyllum soft capsule can effectively reducethe mouse model of TC, TG, LDL, increase HDL, improve the hepaticsteatosis. In this study, we discussed the interaction mechanism ofGynostemma pentaphyllum and Kudingcha as the main component of thehypoglycemic and lipid-lowering compound drugs, proves that the traditionalChinese medicine compound can Treatment of hyperlipidemia throughmultiple target. It is helpful to clarify the mechanism of regulating function ofblood lipid and reducing effect of blood glucose of Gynostemma, providecertain experimental basis for the further development of new lipid-loweringhypoglycemic drugs.In the aspect of mechanism of drug action, through thestudy of the active components of lipid-lowering effect of Kudingcha, wefound the mechanism is concerned with the promoting of the expression ofCyp7α1and ApoAI genes in mouse liver, and inhibiting the expression ofSrebf2and Hmgcr genes. Kudingcha ingredients are confirmed can relieve insulin resistance through the up regulation of Insr, affect the process ofglycolysis and gluconeogenesis through the upregulation of G6pc anddownregulation of Gck, affect glucose metabolism in vivo, reduce bloodglucose of the small rat of diabetes. All these research will help us to explainthe hypoglycemic mechanism and signal transduction pathway ofGynostemma pentaphyllum and it’s compound.
引文
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