摘要
荜茇为胡椒科植物荜茇(Piper longum L.)的近成熟或成熟果穗,是中蒙医常用药材,具有温中散寒、理气止痛功能。临床中不仅用于治疗胃脘痛、呕吐、泄泻、脐腹痛、牙痛、头痛,蒙医还用于治疗高脂血症、冠心病等病。近些年,国内外对荜茇及其有效成分的认识不断深入,其药理作用一直是一些学者的研究热点。经过数年的研究,我们课题组从荜茇中提取得到化学结构明确的单一化合物—荜茇宁,并且首次发现荜茇宁具有显著的降血脂活性。为了深入系统地研究荜茇宁的调脂、抗动脉粥样硬化(AS)作用,并从分子水平探讨其作用机制,本文综合采用了化学、医学、生物和药理等研究方法,主要进行了以下三方面的研究:
第一,采用优化的中试合成工艺,以胡椒碱为起始原料制备荜茇宁,利用高效液相色谱、核磁共振氢谱及核磁共振碳谱的表征,人工合成化合物荜茇宁。第二,采用高脂血症(HLP)模型,研究荜茇宁对大鼠脂代谢紊乱的影响,并进行小鼠急性毒性试验研究,明确荜茇宁的调脂作用和安全性;进一步系统观察荜茇宁对家兔实验性HLP和AS形成的影响,检测兔血清脂质、一氧化氮(NO)、载脂蛋白的含量,以及血清及组织抗氧化酶类活性,并取主动脉、心脏、肝脏进行病理形态学观察,测定肝脏脂质。第三,采用半定量RT-PCR技术检测荜茇宁对HLP大鼠肝脏脂代谢相关的基因低密度脂蛋白受体(LDLR)、载脂蛋白B(ApoB)和3-羟基-3-甲基-戊二酰辅酶A还原酶(HMG-CoAR)mRNA表达的影响;采用实时定量PCR(Real-time PCR)技术,检测荜茇宁对HLP和AS家兔主动脉血凝素样氧化型低密度脂蛋白受体-1(LOX-1)和血管细胞间黏附分子(VCAM-1)基因表达的影响。通过上述研究获得以下研究结果:
1.采用半合成的方法成功地制备了大剂量的化合物荜茇宁,其纯度达到98%以上,不仅充分满足了药理、毒理实验的需要,而且为今后工业化生产荜茇宁提供了依据。
2.动物实验表明荜茇宁可显著降低HLP大鼠血清总胆固醇(TC)、甘油三酯(TG)和低密度脂蛋白胆固醇(LDL-C),升高高密度脂蛋白胆固醇(HDL-C),并呈剂量依赖性,能够有效预防HLP大鼠血脂升高,同时降低动脉硬化指数(AI)。
3.小鼠急性毒性试验显示,一次性经口给予荜茇宁5g·kg~(-1),未见毒性反应。初步表明荜茇宁毒性极低,是一种安全、可靠的调脂化合物,具有进一步深入研究的价值。
4.动态观察了荜茇宁对HLP和AS家兔血清脂质含量的影响,结果显示荜茇宁能够降低兔血清TC、TG、LDL-C、AI,在降低TG方面,优于阳性对照药辛伐他汀:通过光镜、透射电镜等病理组织学观察,荜茇宁能减少高脂饲料诱导的兔主动脉粥样斑块的面积和内膜增厚,减轻主动脉病理损伤程度和冠状动脉狭窄程度,具有较好的抗AS作用。
5.荜茇宁可升高HLP大鼠和AS兔血清载脂蛋白Al(ApoAl),降低ApoB含量及ApoB/ApoAl比值,具有调节脂蛋白代谢的作用。
6.荜茇宁可降低HLP和AS兔肝脏脂质含量,减轻HLP引起兔肝脏脂肪变性的病变程度。提示荜茇宁的降血脂作用,不仅不会引起脂质在肝脏的堆积,而且能降低肝脏TC、TG,初步表明荜茇宁对脂肪肝具有防治作用。
7.荜茇宁能显著提高AS兔血清NO的水平,具有保护血管内皮细胞的功能。
8.荜茇宁能增强HLP和AS动物超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)的活力,降低丙二醛(MDA)的含量,提高机体的抗氧化能力,减少自由基的损伤,减轻脂质过氧化物对血管内皮的损伤。
9.通过RT-PCR技术检测HLP大鼠肝脏LDLR、ApoB、HMG-CoAR基因表达水平,表明荜茇宁可能是通过提高LDLR基因转录水平,降低ApoB mRNA表达,从而起到调节血脂作用,而与HMG-CoAR mRNA的表达无关。
10.荜茇宁能够靶向抑制HLP和AS家兔主动脉LOX-1、VCAM-1基因表达,与模型组比较分别减少42%和51%,这可能是其抗AS的一种分子机制。
综上所述,本研究首次发现荜茇宁具有调节脂代谢紊乱及其抗AS作用,且毒性极低,是一种安全、可靠的调脂、抗AS药用化合物。其作用机制可能与提高肝脏LDLR基因表达,降低ApoB基因表达;增强抗氧化酶的活力,提高机体的抗氧化能力;降低主动脉LOX-1、VCAM-1基因表达等有关。此成果不仅为荜茇宁的进一步研发提供了科学实验依据,而且对阐明中蒙药的药理作用及其机制,以及对调脂、抗AS创新药的研发具有重要意义。
Long Pepper is the grown ear-fruits of which is frequently used in Chinese medicine and Mongolian medicine.That can warm the middle energizer to disperse cold and regulating qi to alleviate pain.It may be not only used in clinic to therapy gastric abscess、disgorging、diarrhea、abdominal pain、toothache and headache.In the traditional Mongolian medicine,Piper Longum L.was used as for treating hyperlipemia and coronary heart disease.Recently,piperlonguminine and it's effective component were studied increasingly,and it's pharmacologic action became a focus for many scholars.After a few year's investigation,our team obtained a kind of chemical compound named as piperlonguminine from Long Pepper,and we found out that the piperlonguminine was the most important compound having the hypolipidemic activity for the first time.In order to study piperlonguminine's effect on accommodating lipid metabolism and anti-artherosclerosis,and to investigate its mechanism of action in molecular level,the chemical,medical science,biological and pharmacological methods were adopted."The following studies were carried out in this thesis.Firstly,the piperlonguminine was prepared using piperineas a starting material,and the structure was determined with high performance liquid chromatogram(HPLC),nuclear magnetic resonance hydrogen spectra(H-NMR) and carbon-spectra(C-NMR).Secondly,we observed piperlonguminine's influence on rat's lipid metabolic disorder using hyperlipemia model,and carried out acute toxicity test study in mouse to confirm piperlongguminine's availability about accommodating lipid metabolism and security.We systematically observed piperlonguminine's effect on HLP and AS rabbits,lipid,NO and apoprotein's contents in serum,antioxidation enzymes activities in serum and tissue.The arteriae aorta,heart and liver's pathomorphology was observed and liver's lipid was determined.Thirdly,we detected piperlonguminine's effect on LDLR,ApoB and HMG-CoAR mRNA expression in HLP rats using semi quantitative RT-PCR technique;and on LOX-1 and VCAM-1 gene expression in HLP and AS rabbits using Real-time PCR.Follow study outcomes were obtained by above-mentioned investigation:
1.Piperlonguminine was synthesized successfully by semisynthetic method,and the purity was more than 98%,which not only satisfied pharmacological and toxicological experiment,but also provide good way for producing it in the future.
2.Animal studies confirmed that Piperlonguminine may lower significantly TC, TG and LDL-C,and heighten HDL-C in HLP rat dose dependently,and can prevent availably serum lipid to heighten and degrade AI.
3.Toxic reaction was not found in mouse acute toxicity when 5g/kg piperlonguminine was administered one time,which manifest piperlonguminine was little toxic,safe and reliable chemical compound,deserving to further lucubrate.
4.Piperlongumnine's effect on serum lipid contents in HLP and AS rabbits was observed dynamically,and found that piperlongumnine can lower TC,TG,LDL-C,and AI and more than positive control Simvastatin in TG;Piperlongumnine can lessen aorta athermatous plaque's areas and intima thickening in rabbits that was induced by high-fat feeds,and relieve the degree of aorta pathology injury and coronary artery stenosis,and had better effect on anti-atherosclerosis.
5.Piperlongumnine had heighten AI in HLP rats and AS rabbits,and lower ApoB and ApoB/ApoAl,and had effect on accommodating lipoprotein metabolism.
6.Piperlongumnine lessened liver lipid contents in HLP and AS rabbits,and relieved liver adipose degeneration extent in HLP rabbits.Piperlongumnine not only caused lipid's accretion accumulation in liver,but also lowered TC and TG;which indicated initially had preventive and therapeutic effected on adiposis hepatica.
7.Piperlongumnine heightened serum NO in AS rabbits,and protected vascular endothelial cell.
8.Piperlonguminine enhanced SOD,CAT,GSH-Px activity,reduced MDA contents in HLP and AS animals,elevate antioxidation capability,and decreased free radical and lipid peroxidate damage to blood vessel endothelium.
9.LDLR,ApoB,HMG-CoAR gene expression in HLP rat's liver was detected by RT-PCR technique,which indicated that piperlonguminine could accommodate serum lipid through elevating LDLR'gene transcriptional level and lowering ApoB m RNA expression,and had nothing to do with HMG-CoAR m RNA expression.
10.Aorta LOX-1,VCAM-1 gene expression in HLP and AS rabbits could be inhibited by piperlonguminine and decrease 42%and 51%respectively comparing with model group,which is possibly a molecule mechanism for piperlonguminine to resist AS.
All in all,we found firstly that piperlonguminine can accommodate lipid metabolic disorder and resist AS and its toxicity is very little and it is a kind of safe, reliable chemical compound that can accommodate lipid metabolic disorder and resist AS,which mechanism of action is possibly concerned with elevating liver LDLR and lowering ApoB gene expression,and enhancing antioxidase activity and anti-oxidation ability,and degrading aorta LOX-1 and VCAM-1 gene expression and so on.The outcome provided scientific experiment base for piperlonguminine's further investigation and development.Furthermore,it has important significance for elucidating Chinese and Mongolian drug's molecule mechanism and breakthrough drug's study and exploitation on accommodating lipid metabolism and anti-artherosclerosis.
引文
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