摘要
1相关的文献研究
高脂血症是当今一种很常见的慢性疾病,其发病的原因是由于机体的脂质代谢紊乱,导致机体代谢失去平衡,有关的物质大量积累,引发脂肪肝、糖尿病、动脉粥样硬化、高血压及心血管疾病等,极大地威胁着人们的生命健康。高脂血症的临床表现为血清TC、TG的浓度较高,HDL-C的浓度较低。
目前有较多的中药及复方用于高脂血症的治疗,并取得了较好的疗效。泽泻汤是出自东汉医圣张仲景《金匮要略》里的经典验方,该药方由泽泻、白术两位中药材配伍组成,泽泻的作用重在利水下行、祛湿功效,可以使已停之饮从小便而去；白术的作用则重在健脾制水功效,可以使水湿既化而不复聚。该方最早用于治疗昏冒眩晕,后来也用于治疗高脂血症,实际上是对泽泻汤应用范围的进一步扩大,用药作用目的及机理基本相同。并经过长期的临床验证,证明该方有较好的降血脂作用。但是目前对泽泻汤降血脂机理方面的研究文献不多,且作用机理没有得到较好地阐明,还处在初期的探索阶段。普遍认为泽泻汤降血脂的作用机理有,泽泻汤能影响外源性胆固醇的吸收,或者是泽泻汤能干预机体对内源性胆固醇的分解代谢,同时泽泻汤的降血脂作用还可能与其能提高机体的抗氧化能力,降低机体的氧化应激性有关。也有学者认为,一些中药降血脂的作用机理还可能与药物能对机体的肝,脾、肾的保护作用有关,调节这些器官功能的平衡,有助于维持机体正常的生理功能,调节脂质代谢紊乱等作用有关。用分子生物学手段研究泽泻汤的进行作用机制也处在探索的阶段。对肝脏中调节胆固醇合成的基因以及影响影响胆固醇合成、促进胆固醇分解的关键酶等(如Hmgcr、Srebf2、Fasn、Cyp7a1等)的研究,是目前降血脂药物机理研究的重点之一,对调节高脂血症的关键基因进行研究可能是从根本上阐明药物降血脂作用的有效方法。对泽泻汤降血脂作用的机理的阐明一方面可以指导临床用药安全,另一方面也是寻找及开发新型降血脂药物的有效途径,具有重要意义。
2实验部分
2.1泽泻汤降血脂作用机理初步研究
目的：通过泽泻汤对高脂血症小鼠血脂的影响,初步探讨泽泻汤降血脂的作用机理。方法：采用高脂饲料喂养法建立小鼠实验性高脂血症模型,将高脂血症小鼠分成6个组,分别为高脂血症模型组,正常对照组,阳性组,泽泻汤水提取物高、中、低三个剂量组。用泽泻汤水提物高、中、低三个剂量组连续给药4w,观察并记录各组小鼠的行为及生活状况。摘眼球取血,分离血清,测定高脂血症小鼠血清TC、TG、HDL-C、SOD、 MDA、ALT、AST等生化指标,观察各组织器官的病理变化及取部分肝脏用10%的福尔马林固定,石蜡包埋切片,HE染色后进行光镜检查。其余的脏器置于-80℃冰箱保存备用。结果：给药4w后,高脂血症小鼠血清TC、TG显著降低,HDL-C、SOD显著升高,ALT、AST也显著降低。结论：中药泽泻汤水提取物对高脂血症小鼠有较好的降血脂作用,且高剂量组效果最好。其降血脂作用机理可能是对肝、肾、脾等脏器的保护,调节机体代谢平衡,以及改善机体抗氧化能力,减小氧化应激性等。
2.2泽泻汤的LC-MS/MS指纹图谱的建立
目的：通过建立泽泻汤水提取物的LC-MS/MS指纹图谱,标准化泽泻汤降血脂的有效成分,为泽泻汤降血脂作用机理的研究奠定基础。方法：采用LC-MS/MS联用技术,以实验一中使用的泽泻汤水提取物为样本,建立泽泻汤降血脂有效成分的指纹图谱。结果：建立了泽泻汤降血脂有效成分的指纹图谱。结论：该方法建立的泽泻汤指纹图谱方便、可行,能有效地对泽泻汤降血脂有效成分进行控制,标准化了泽泻汤的各成分,对研究泽泻汤降血脂作用以及降血脂作用机制的研究提供了实验依据。
2.3泽泻汤降血脂作用机理研究
目的：通过对泽泻汤进行拆方与组方研究泽泻汤的降血脂作用,探讨泽泻汤降血脂作用的机理。方法：对泽泻汤进行拆分,先分别制备泽泻,白术,以及复方泽泻汤的水提取物。采用高脂饲料喂养法建立小鼠实验性高脂血症模型,将高脂血症小鼠分成6个组,分别为高脂血症模型组,正常对照组,阳性组,泽泻汤水提取物组、泽泻水提取物组、白术水提取物组。用泽泻汤水提取物、泽泻水提取物、白术水提取物连续给药4w,观察小鼠的行为及生活状况。摘眼球取血,分离血清,测定高脂血症小鼠血清TC、TG、HDL-C、SOD、MDA、ALT、AST等生化指标,观察各组织器官的病理变化及取部分肝脏用10%的福尔马林固定,石蜡包埋切片HE染色后进行光镜检查,各组织置于-80℃冰箱保存备用。研究复方泽泻汤水提取物、单一的泽泻水提取物、白术水提取物对高脂血症小鼠的血脂影响,并采用现代分子生物学技术,研究泽泻汤等对调血脂的关键基因(Hmgcr、Srebf2、Fasn、Cyp7a1)表达的影响。结果：泽泻汤水提取物组仍然有较好的降血脂作用,泽泻水提取物组也有较弱的降血脂作用,而白术水提物组基本没有降血脂作用。泽泻汤对高脂血症小鼠肝脏中Hmgcr、Srebf2、Fasn的表达有较显著地抑制作用,而对Cyp7a1的表达无显著作用。结论：泽泻汤有较好的降血脂作用,其降血脂作用与泽泻和白术的配伍协同作用有关,其降血脂作用机理还与泽泻汤能调节胆固醇的合成与代谢基因表达有关,泽泻汤通过抑制Hmgcr、Srebf2、Fasn的表达,抑制胆固醇的合成,从而降低血清TC、TG等生化指标,其到降血脂的作用。
Part One:Related Literature Review
Hyperlipidemia is a common chronic disease, the incidence of which is due to the disorder of body lipid metabolism, and the accumulation of a large number of related substances, causing fatty liver, diabetes, arterial atherosclerosis, hypertension and cardiovascular diseases, and threatening people's lives and health. The clinical manifestations of hyperlipidemia is higher concentrations of serum TC and TG and lower concentrations of HDL-C.
There are many Chinese medicine and compound for the treatment of hyperlipidemia, and have achieved good effects. Alismatis Rhizoma Decoction is a classical Traditional Chinese Medicine formula for treatment of vertigo with its long history of efficacious and successful clinical effect. AD is composed of two component Chinese herbal medicine which are Alismatis Rhizoma and Atractylodis Macrocephalae Rhizoma. Since vertigo is a symptom of hyperlipidemia, the present study aimed at evaluating the hypolipidemic effect of ARD in hypolipidemic mice induced by high fat diet and investigated the rationality of formula combination of AR and AMR. Compared with mice in control group, hypolipidemic mice in AR and AD groups displayed a reduction of following parameters:body weight, liver and serum total cholesterol, triglyceride concentration, liver coefficient, activities of serum aspartate aminotransferase AST and alanine aminotransferase ALT; whereas the concentrations of serum HDL-cholesterol were significantly elevated in both AR and AD groups. AR and AD treatments significantly down regulated the expressions of3-hydroxy-3-methylglutharyl-coenzyme A reductase HMG-CoA reductase and sterol regulatory element binding factor-2SREBF-2.hese findings clearly provided evidences that molecular mechanisms of the hypolipidemic effects of AD and AR in hypolipidemic mice are due to their suppression on biosynthesis of cholesterol in liver. Since no significantly hypolipidemic effect of AMR was observed in hypolipidemic mice, the more prominent effect of AD than that of AR indicated a synergistic effect of AR and AMR, and confirmed the rationality of AD formula. The Alismatis Rhizoma Decoction is a classic prescription from the Synopsis of the Golden Chamber written by medical saint Zhang Zhongjing in Eastern Han dynasty. The prescription were composed by Alisma and atractylodes. Alisma's role focuses on Lishuixiaxing, banishing dampness effect; Atractylodes's role focuses on strengthening spleen and making water effects. The prescription for the treatment of faint and dizziness earliest, and later also for the treatment of hyperlipidemia, is actually a further expansion of Alismatis Rhizoma Decoction in applications, and the purpose and mechanism of drug were basically the same. long-term clinical validation proved the prescription has a good effect on hyperlipidemia. However, the research literature of Alismatis Rhizoma Decoction's mechanism in hypolipidemic is few, and the mechanism is not clear and was still in the early exploratory stage. It is commonly believed that Alismatis Rhizoma Decoction can affect the absorption of exogenous cholesterol and Alismatis Rhizoma Decoction can interfere the endogenous cholesterol catabolism, and the hypolipidemic effect of Alismatis Rhizoma Decoction may be related to improving the body's antioxidant capacity and reducing the body's oxidative stress. Some scholars believe that the mechanism of some traditional Chinese medicine hypolipidemic drug may also related to the protective effect of liver, spleen, kidney and regulating the organ balance which is helpful for the maintainance of body's normal physiological function, and the mechanism may also related to the regulation of lipidmetabolic disorders. The research of Alismatis Rhizoma Decoction's mechanism by molecular biology is in the exploration stage. The study of genes in the liver that regulate cholesterol synthesis and the key enzymes(such as Hmgcr, Srebf2Fasn, Cyp7al, etc.) that affect the synthesis and decomposition of cholestero is one of the major mechanism of lipid-lowering drugs. The study in key gene that regulating the lipemia may be fundamental to clarify the drug effect on lipid. To clarify the mechanism of Alismatis Rhizoma Decoction hypolipidemic effect not only can guide clinical medication safety, but also meaningful for the development of new effective lipid-lowering drug.
Part two:Experimental research
Experiment one:The preliminary mechanism study of Alismatis Rhizoma Decoction in hypolip-idemic
Objective:observe the Alismatis Rhizoma Decoctions's effect of blood lipids in hyperlipidemic mice and study the preliminary hypolipidemic mechanism of Alismatis Rhizoma Decoction. Methods: use high fat diet to establish hyperlipidemia model, Sixty male Kunming mice were randomly divided into normal control, high-fat control, positive control group, Alismatis Rhizoma Decoction extract high dose group, medium dose group and low dose group, each group consisting of10animals. In addition to normal group which was fed with normal diet, other four groups were fed with high-fat diet for4weeks to establish the experimental hyperlipemia model. Then the normal control and high-fat control were given distilled water orally, positive control group was given simvastatin, others were given Alismatis Rhizoma Decoction extract high, medium and low dose, continuous administration for4w, determinate hyperlipidemia mice in serum TC, TG HDL-C, SOD, MDA, ALT, and AST and other biochemical parameters, to observe the pathological changes in tissues and organs, and the liver biopsy stained with Hematoxylin-eosin (HE) were observed under light microscope. Results:After4w administration, hyperlipidemic mice serum TC, TG was significantly lower, and HDL-C, SOD significantly increased, ALT, AST also significantly reduced. Conclusion:Alismatis Rhizoma Decoction extract have a good effect in hyperlipidemic mice especially the high dose group. Its lipid-lowering mechanism may be the protection of the liver, kidney, spleen and other organs, and the balance regulating of body metabolism, thus to improve the antioxidant capacity, and reduce oxidative stress.
Experiment two:LC-MS/MS Fingerprinting of the Alismatis Rhizoma Decoction
Objective:To establish LC-MS/MS fingerprint of Alismatis Rhizoma Decoction extract, and standardized the active lowering lipid ingredients of Alismatis Rhizoma Decoction, lay the foundation of Alismatis Rhizoma Decoction's lipid-lowering mechanism. Methods:use LC-MS/MS techniques, the Alismatis Rhizoma Decoction extract as samples, to establish the active ingredients of the soup hypolipidemic fingerprint. Results:establish fingerprint of the active ingredients of the Alismatis Rhizoma Decoction. Conclusion:The method to establish the Alismatis Rhizoma Decoction fingerprint is convenient, feasible, has effective control in lipid-lowering active ingredients of Alismatis Rhizoma Decoction, and provides an experimental basis for the study of Alismatis Rhizoma Decoction hypolipidemic effect and the mechanism of lipid-lowering effect.
Experiment three:Study on the mechanism for the hypolipidemic effect of Alismatis Rhizoma Decoction
Objective:To explore the Alismatis Rhizoma Decoction hypolipidemic effect mechanism by the partly prescription and combinated prescription Methods:Study compound Alismatis Rhizoma Decoction extract, a single the Alisma water extract, Sixty male Kunming mice were randomly divided into normal control, high-fat control, positive control group, Alismatis Rhizoma Decoction extract group, Alisma water extract group and Atractylodes water extract group, each group consisting of10animals. In addition to normal group which was fed with normal diet, other four groups were fed with high-fat diet for4weeks to establish the experimental hyperlipemia model. Then the normal control and high-fat control were given distilled water orally, positive control group was given simvastatin, others were given Alismatis Rhizoma Decoction extract, Alisma water extract and Atractylodes water extrac, continuous administration for4w. Determinate hyperlipidemia mice in serum TC, TG HDL-C, SOD, MDA, ALT, and AST and other biochemical parameters, to observe the pathological changes in tissues and organs, and the liver biopsy stained with Hematoxylin-eosin (HE) were observed under light microscope. The Atractylodes water extract of blood lipids in hyperlipidemic mice, and using modern molecular biology techniques to study the effect of Alismatis Rhizoma Decoction in the expression of key genes (Hmgcr, Srebf2, Fasn, Cyp7al) that regulating lipid. Results:Alismatis Rhizoma Decoction extract group still have a good effect on lowering the lipid, and Alisma water extract group also had a weak effect in lowering blood pressure, while the Atractylodes water extract group had basically no effect on lowering lipid. Alismatis Rhizoma Decoction more could inhibit the expression of Hmgcr, Srebf2, Fasn in the liver of hyperlipidemic mice significantly, but had no significant effect on the expression of Cyp7al. Conclusion:Alismatis Rhizoma Decoction has a good effect on lowering hypolipidemic effect and its lipid-lowering effect may be related to the combination with Atractylodes, mechanism of action with the Alisma Tang can regulate cholesterol synthesis and metabolism gene expression related to Chak spilled soup inhibition Hmgcr, SrebfZ, Fasn expression, inhibition of cholesterol synthesis, thereby reducing serum TC TG, and other biochemical markers, to the hypolipidemic effect.
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