- 學位論文
以倉鼠為動物模式探討中草藥複方B及單方B28之降低密度脂蛋白膽固醇之作用
LDL–C lowering effects of mixture B and B28 herbs in Syrian Golden Hamster
摘要
近年來，國人由於飲食習慣的改變，常在未察覺情況下吃下過量的膽固醇，使得高膽固醇血脂症的病人 (hypercholesterolemia) 有增加的趨勢，而高血脂症是造成心血管疾病的危險因子。臨床上常用來治療這類的人的藥物大致可分兩大類，主要是抑制膽固醇合成關鍵酵素-HMG-CoA reductase的抑制劑(statins)，以及抑制腸道膽固醇的吸收-Ezetimibe；前者降低血脂效果很好，但是在某些病人會有如肌炎、橫紋肌溶解症、肝功能異常等的副作用；後者是阻礙膽固醇經由小腸 Niemann-Pick C1 Like 1 (NPC1L1) 的吸收，目前市面上唯有 Ezetimibe 是抑制膽固醇吸收的藥物，因此開發能抑制膽固醇吸收的藥物是非常有發展空間的。 我們先前的研究發現，利用細胞實驗篩選出具有抑制脂肪酸合成和促進脂肪酸氧化分解的中草藥，經與中醫師討論由三個藥材組成一個中草藥配方 (複方BM)。在C57BL/6小鼠的高油脂高膽固醇動物模式中，發現複方BM可以有效降低肝臟和血漿中的膽固醇以及三酸甘油酯濃度。這個動物模式在大量膽固醇餵食下HMG-CoA reductase 基因表現已受到抑制，作用可能不是經由抑制HMG-CoA reductase。因此我們推測複方BM可能含有抑制腸道吸收膽固醇的成分，因而降低肝臟及血液中膽固醇。為了探討其可能性及找出複方BM中那種藥材有這樣的作用，因此我將三個藥材B14、B28和B50，以及複方BM以胃管餵食六週大倉鼠 (Syrian golden Hamster)。實驗分成七組，控制組餵食正常飼料 (chow diet) 以及灌食滅菌水，另外六組實驗組則都餵食含 23% 油脂 0.2% 膽固醇飼料，其中五組分別灌食B14、B28、B50、複方BM和三倍藥量複方BM (3XBM)，對照組則灌食滅菌水 (HFC)。飼養六週後，結果顯示複方BM和單方B28可以顯著降低血漿的總膽固醇，以及血漿中ALT活性，而B14和B50組則沒有顯著作用。餵食複方BM和B28藥明顯降低血漿LDL-cholesterol，但B14和B50組則沒有顯著影響。另外，複方BM和單方B28都能有效降低肝臟總膽固醇量，肝臟組織切片亦顯示複方B和單方B28具有減輕脂肪油滴堆積、發炎現象及纖維化之情形。我們由這些結果看到複方BM和單方B28有顯著降低LDL- cholesterol及肝臟膽固醇的效果，所以我們接著分析這它們在肝臟和腸道運送或吸收膽固醇相關基因的表現，RT-Q-PCR 結果顯示複方BM和B28藥可以顯著降低腸道 Niemann-Pick C1 Like 1 (NPC1L1) mRNA 並提升肝臟 LDL-Receptor mRNA 的表現，表示複方BM和B28藥可能是抑制膽固醇的吸收，減少肝臟膽固醇，使肝臟LDL-Receptor表現增加，導致血漿LDL-cholesterol降低。糞便膽固醇與中性固醇分析發現灌食倉鼠複方BM和B28組的糞便中，膽固醇顯著高於HFC組，且B28組糞便中性固醇量也顯著高於HFC組。 我們的結果顯示，複方BM中的單方B28具抑制腸道NCP1L1表現的作用，透過抑制吸收能達到降血脂、預防肝臟發炎與纖維化形成的功效，後續研究可望從B28中鑑定並純化出具抑制膽固醇吸收作用的成分，並探討其作用機制。
並列摘要
In recent years, the prevalence of hypercholesterolemia in Taiwan is increased due to change of diet and lifestyle. Statins and ezetimibe are two major cholesterol-lowing drugs of hypercholesterolemia. Statins are HMG-CoA reductase inhibitors, they inhibit cholesterol synthesis and have good cholesterol-lowing effects. But statins cause side effects, including myalgias, rhabdomyolysis and liver damage in some patients. Ezetimibe, block cholesterol absorption by targeting NPC1L1, reduces cholesterol by inhibiting cholesterol absorption in small intestine. Currently, ezetimibe is the only one absorption inhibitor; therefore, developing of medicines for hypercholesterolemia by absorption inhibition has great potential. In our previous study, we found mixture B (BM) that contained herbs able to inhibit fatty acid (FA) synthesis, and induced FA oxidation in HepG2. BM had cholesterol-lowing effect in liver and plasma in high fat/cholesterol fed C57BLL/6 mice. Since dietary cholesterol inhibits HMG-CoA reductase, we speculated that BM may reduce plasma and liver cholesterol by inhibiting cholesterol absorption. In this study, six weeks old Syrian golden Hamsters were divided into seven groups. The control group was fed with chow diet and given distilled water by gavage. The experimental groups were fed with high fat/high cholesterol diet, and HFC was gavaged with distilled water and the other five groups were gavaged with either B14, B28, B50, BM or 3XBM daily for 6 weeks. After 6 weeks of experiment, BM and B28 not only lowered plasma total cholesterol, LDL-cholesterol and liver cholesterol but also reduced plasma ALT activities significantly. However, no significant changes were found in B14 and B50 groups. Histological analyses of hepatic tissues show that BM and B28 improved liver steatosis, inflammation and fibrosis. Moreover, RT-Q-PCR analysis revealed that mixture B and B28 decreased mRNA levels of NPC1L1 in the small intestine and increased mRNA the levels of LDL-Receptor in the liver. Analysis of fecal cholesterol and neutral sterols showed that cholesterol contents were increased in the feces of BM and B28 groups, and neutral sterols were increased in the feces of B28 group. The results suggested that BM and B28 inhibited cholesterol absorption in small intestine and thus reduce liver and plasma cholesterol. Our results showed that B28 of the BM was effective in prevention of high fat/high cholesterol diet-induced hypercholesterolemia, NASH and fibrosis through the inhibition of cholesterol absorption. Further studies will focus on the identification and isolation of the functional component(s) in B28, and investigation of the underlined molecular mechanisms.