B28號藥改善倉鼠因高脂高膽固醇飼料所引起的非酒精性脂肪肝炎涉及降低小腸與肝臟中的NPC1L1表現

Abstract

非酒精性脂肪肝 (Nonalcoholic fatty liver；NAFLD) 為最常見的非病毒性慢性肝病，全球的盛行率隨著肥胖的增加持續的在提高。其病程有不同嚴重程度的發展，由單純的脂肪堆積，到較嚴重的非酒精性脂肪肝炎 (Nonalcoholic stetohepatitis)、肝纖維化，甚至到肝硬化。到目前並沒有明確的方法可以預防及治療非酒精性脂肪肝炎，在中草藥中以許多具有保肝作用的藥材，可能會有一些藥材具有預防或治療非酒精性脂肪肝炎的作用。實驗室的學長們經過多年的篩選及測試，發現B28號藥具有預防在倉鼠以高脂高膽固醇飼料誘發的非酒精性脂肪肝與肝炎的作用。B28號藥是B65號藥經加工處理所得到的，我的實驗目的是要探討未加工的B65號藥是否就具有和B28號藥相同的效果，並建立細胞實驗模式做為B28號藥有效成分的篩選平台。 我們先以動物實驗探討未加工的B65號藥是否就具有和B28號藥相同的效果。將九週齡的倉鼠分成五組，控制組餵食一般chow diet，另外四組實驗組則餵食添加23%脂肪及0.5%膽固醇的飼料 (HFC)，為HFC、HFC+B28L、HFC+B28H及HFC+B65H組。四實驗組分別每日以胃管灌食同體積之滅菌水，或含118 mg (B28L) 或335 mg B28 (B28H) 號藥萃取物/day/kg BW或222 mg B65 (B65H) 號藥萃取物/day/kg BW，為期6週。結果顯示B28號藥會降低血液與肝臟中的膽固醇濃度，並具有改善非酒精性脂肪肝炎的效果，且這樣的效果有劑量依存性。此外，我們也發現了B28號藥不僅能夠降低小腸中與肝臟中的NPC1L1表現量，同時也會提高肝臟中CYP7A1與LDL receptor的表現量。但是，B65號藥則都沒有上述作用。 在細胞實驗上，我們建立了Caco-2腸細胞與HuS-E/2肝細胞模式來篩選B28號藥有效成分。Caco-2腸細胞在給予含不同濃度的膽固醇的脂肪微粒 (micelle) 後會提高NPC1L1 mRNA及蛋白質表現，細胞內的三酸甘油酯與膽固醇濃度以及組裝乳糜微粒相關基因的表現。在培養液中加入0.6 mg/mL B28號藥萃取物8小時後，可以觀察到細胞內的三酸甘油酯與膽固醇濃度及NPC1L1及apoB、ACAT-2、MTP mRNA表現量降低。HuS-E/2肝細胞培養液中直接加入0.6 mg/mL B28號藥萃取物8小時後，也可以觀察到NPC1L1 mRNA及蛋白質表現降低。顯示這兩種細胞模式可作為B28號藥有效成分的篩選平台。另外，我們以不同極性的四種溶劑 (90%methanol、hexane、butanol及水四種) 分離B28號藥，經過細胞實驗測試發現只有水層的萃取物具有降低NPC1L1 mRNA表現量的效果。 我們推測可能在B65號藥加工過程中產生了我們目前還不知道的化學變化，導致B28號藥在動物實驗與細胞實驗中都具有抑制NPC1L1表現的作用。後續的研究希望將水層的萃取物做更精細的分離，再透過兩種細胞模式進行篩選與找出分子作用機制，之後再以動物實驗確認。

Nonalcoholic fatty liver disease (NAFLD) is one of the most common non-viral chronic liver diseases. The global prevalence of NAFLD rises continuously with the increasing population of obesity. NAFLD has a histologic spectrum ranging from simple steatosis, nonalcoholic steatohepatitis (NASH), to fibrosis and cirrhosis. There are no medical treatments yet for NAFLD. Various Chinese herbal medicines are believed to have liver-protective functions and are widely used in clinical practice. In our previous studies, we have discovered an Herb-B28 which showed strong preventive effects in high fat/high cholesterol (HFC) diet-induced NAFLD and NASH in hamster. Herb-B28 is processed product from Herb-B65. The aims of our study are to test whether the Herb-B65 exerts the same effects as Herb-B28 in HFC diet fed hamster, and to establish cell models for screening the active component(s) in Herb-B28. First, we conducted an animal experiment to compare the effects of Herbs B28 and B65. Male gold Syrian hamsters were divided into five groups. The control group was fed with chow diet, and the four experimental groups were fed with a HFC diet for 6 weeks. The experimental groups are HFC, HFC+B28L, HFC+B28H, and HFC+B65H group, hamsters in these groups were gavaged with distilled water, 118 mg B28 extract/day/kg BW, 355 mg B28 extract/day/kg BW, and 222 mg B65 extract/day/kg BW, respectively. The results showed that Herb-B28 lowered plasma and liver cholesterol, and prevented NASH and liver fibrosis in a dose-dependent manner. We observed intestinal and hepatic NPC1L1 mRNA and protein, and up-regulate expression of hepatic CYP7A1 and LDL-receptor mRNA in Herb-B28 treated hamsters. However, Herb-B65 did not exhibit all these effects. We established cell-based models for screening active components in Herb-B28. When the CaCo-2 enterocytes were cultured with micelles containing various amount of cholesterol, the cellular cholesterol and triacylglyceride (TG), and levels of NPC1L1 mRNA and protein, and levels of mRNA of genes involved in chylomicron production were increased along with increasing amount of cholesterol in the micelle. Addition of 0.6 mg extract/ml of Herb-B28 to the cells resulted in a decrease of cellular TG and cholesterol, and mRNA levels of NPC1L1, apoB, ACAT2 and MTP. Addition of 0.6 mg/ml Herb-B28 extract also resulted in decrease of NPC1L1 mRNA and protein expression level in HuS-E/2 hepatocytes. The results suggest that these cell models can be used for screening of active compound in Herb-B28. The Herb-B28 was partitioned using solvents with different polarity (90% methanol, hexane, butanol, and water) and extracts were tested in both CaCo-2 and HuS-E/2 cells. The results show that only water-soluble fractional extract has effects on the down-regulation of NPC1L1 mRNA level. We speculated that chemical reactions may have occurred during the processing of Herb-B65 that leads to Herb-B28 having the effects of inhibiting NPC1L1 expression. The two cell models will be used for the identification of active component in the water extract of Herb-B28.