飼糧中添加靈芝及北蟲草對日本鵪鶉肝臟脂質堆積的影響

The effect of dietary Ganoderma lucidum and Cordyceps militaris supplementation on hepatic lipid accumulation in Japanese quail

熊禹涵(Yu-Han Hsiung)

指導教授 ： 丁詩同

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201903775

摘要

肝臟為人體中重要的脂質代謝場所，當其蓄積的脂肪量大於5%的肝臟重量時，即可定義為脂肪肝(fatty liver)，而非酒精性脂肪肝(NAFLD)在現代生活習慣的影響下，已成為不可忽視的代謝疾病。另一方面，為了符合產蛋所需，經產家禽肝臟內的脂質生成(lipogenesis)作用將受到動情素調控而增加，使其容易出現天然性脂肪肝。當家禽肝臟內所蓄積的過量脂肪造成微血管破裂時，即可被診斷為出血性脂肪肝(fatty liver hemorrhagic syndrome, FLHS)，其發生除降低家禽的產蛋性能外，也可能引發突發死亡，造成蛋雞產業的損失。因產蛋家禽與人類的內源性脂質生成(de novo lipogenesis)場所皆位於肝臟的緣故，使FLHS與NAFLD 間存在相似性，並讓產蛋家禽成為良好的脂肪肝研究平台。雖然目前已有許多研究團隊在飼糧誘導的小鼠模式上證實北蟲草(Cordyceps militaris)及靈芝(Ganoderma lucidum)具有降血脂、抗發炎及降低肝臟脂質含量的成效，但卻少以產蛋家禽作為模式動物進行研究。因此，本試驗希望透過日本鵪鶉在進入產蛋周期後形成的天然脂肪肝模式，探討不同濃度的北蟲草及靈芝處理，對日本鵪鶉在肝臟脂質堆積程度上的影響。此外，也期望以日本鵪鶉的初代培養肝細胞模式探討靈芝中不同機能性成分對肝臟脂質生成作用背後分子機制的影響。
本次研究共進行兩次動物實驗：第一次使用40 週齡的日本鵪鶉進行12 週試驗，第二次則使用14 週齡的日本鵪鶉進行20 週試驗。前者共分為7 組(n=5)，分別為控制組及3 種濃度(0.5%、1%、2%)的北蟲草組與靈芝組。因在此次實驗中發現：北蟲草1%組及三組靈芝組可能具有降低肝臟脂質堆積的潛力，所以，在第二次動物實驗中，就以此4 種處理加上1 組控制組共分為5 組(n=12)，並在12週處理後(26週齡)及20週處理後(34週齡)進行兩次犧牲採樣。此次結果顯示：於12週處理下，北蟲草處理組尚未對肝臟的脂質堆積情形產生影響；而在低濃度(0.5%、1%)的靈芝處理組中則出現降低肝臟三酸甘油酯含量、肝臟組織的油紅染色區域及血漿DPP4 含量(脂肪肝生物指標)的結果，表示低濃度的靈芝處理有降低肝臟脂質堆積的潛力。相反地，高濃度(2%)的靈芝處理則讓肝臟脂質堆積情況更加嚴重，此現象在發現動情素相關基因的表現量(APOV1)會因高濃度靈芝處理而上升後得到解釋。另一方面，在20 週處理下，北蟲草1%組及靈芝1%組在肝臟組織的油紅染色區域以及肝臟中脂肪肝生物指標(DPP4、GST)的基因表現量上明顯低於控制組，表示其脂肪肝程度較控制組輕微。兩次的動物實驗呈現出北蟲草與靈芝具有改善肝臟脂質堆積的潛力，但背後機制尚不明確，為釐清其有效物質透過的影響途徑，後續實驗中也採用細胞模式進行探討。
於細胞實驗中，透過不同靈芝萃取物的處理，發現靈芝水萃物具有降低脂質合成相關基因FAS的能力，但在靈芝酒萃物的處理下，卻發現動情素相關基因如APOV1及ELOVL5的表現量會上升，表示靈芝的有效成分中存在與動情素相似的活性。因此，細胞實驗的結果說明：靈芝內含的不同有效成分對產蛋家禽的肝臟脂質生成作用造成的影響也不同。而根據動物實驗的結果，可以推測日本鵪鶉在最適濃度的靈芝處理下(1%)，有效成分間將有最佳的交互作用，出現降低肝臟脂質堆積的效果。
綜上所述，在以日本鵪鶉天然性脂肪肝模式進行評估後，發現北蟲草及靈芝具有降低肝臟內脂質堆積的潛力，因此，若能進一步的對有效成分進行探討，將有益於受到NAFLD及FLHS困擾的人類健康與家禽產業。

關鍵字

脂肪肝 ； 動情素 ； 北蟲草 ； 靈芝 ； 日本鵪鶉

並列摘要

In human, liver is not only the important place for lipid metabolism. When thehepatic lipid content exceeds 5% of the liver weight, it can be diagnosed with fatty liver. As the impact of modern way of life, non-alcoholic fatty liver syndrome (NAFLD) has become a major metabolic disorder that cannot be ignored. On the other hand, in active-laying birds, the hepatic lipogenesis increases due to secretion of estrogen to meet the need of egg production. When excess lipids cause hepatic capillaries damaged, it can be diagnosed as fatty liver hemorrhagic syndrome (FLHS). The incidence of FLHS will lead to the dramatic drop on egg production and even sudden death on laying birds, which is the great loss in poultry industry. Because the major place of de novo lipogenesis are same in laying birds and human, the similarity between FLHS and NAFLD makes the laying birds become a good research model for fatty liver. However, instead of studying on laying birds, it has been well-known that Cordyceps militaris and Ganoderma lucidum has the effect on ameliorating hyperlipidemia, anti-inflammation, and decreasing hepatic lipid accumulation on diet induced mice model so far. Thus, the aim of this study is to understand the influence of either Cordyceps militaris or Ganoderma lucidum that added in the daily diet on hepatic lipid accumulation, through the spontaneous fatty liver disease model on egg-laying Japanese quail, and furthermore, to study the role that functional ingredients from different extract plays on lipid accumulation in the liver through the primary hepatocytes of Japanese quail.
The in vivo study was repeated in two time by using age-different quails, including the 40-week-old Japanese quails in the first time and the 14-week-old Japanese quails in second time. In the first time experiment, the quails were grouped in 7 (n=5), including control group, three different concentration (0.5%, 1%, 2%) of Cordyceps militaris, and three different concentration (0.5%, 1%, 2%) of Ganoderma lucidum. It is showed that 1% C. militaris treatment and all concentration of G.lucidum treatment have a potential on reducing hepatic lipid accumulation after 12 week treated. Therefore, in the second time experiment, the quails were grouped in 5 (n=12), including control group and four treatment group which mentioned above. After 12-week-treated (26-week-old) and 20-week-treated (34-week-old), the quails were sacrificed and sampled. The result showed that under 12-week treatment, there is no significant effect on ameliorating hepatic lipid accumulation in C. militaris treatment groups. The hepatic triacylglycerol content, oil red O staining area on hepatic tissue, and plasma DPP4 (fatty liver biomarker) is reduced in G. lucidum 0.5% and 1% groups, which is implicated that the low concentration of G.lucidum treatment had potential on decrease lipid accumulation in liver. The estrogen-related gene expression increased under high concentration of G. lucidum treatment (2%), which lead to severe hepatic lipid accumulation. On the other hand, after 20-week treatment, the oil red O staining area and the gene expression such as DPP4 and GST decline in C. militaris 1% treatment and G. lucidum 1% treatment, which stands for slight level of fatty liver disease. Similarly, it is found that the hepatic lipid accumulation got severe in 2% G.lucidum treatment group. The results from in vivo study demonstrated the phenotype of reduced hepatic accumulation under C.militaris and G. lucidum treatment, but the pathway is not clear. Thus, the study continued on cell model in order to realize the effect of functional ingredients in those two hepatoprotective materials.
In the cell study, it is found that the expression of lipogenic gene like FAS is reduced under the treatment of G.lucidum water extract, while the expression of estrogen-related gene such as APOV1 and ELOVL5 is increased under the treatment of G.lucidum ethanol extract. It is indicated that the ingredients in G.lucidum has the estrogen-like activity. Therefore, the in vitro study showed that G.lucidum is composed of different ingredients that play the different role on hepatic lipid accumulation in laying birds, and according to the in vivo study, it is believed that under optimal treatment (1% G. lucidum), the hepatic lipid accumulation in Japanese quail would be ameliorated because of the best interaction between functional ingredients.
In conclusion, it is found that not only C. militaris but also G.lucidum has a potential on reducing hepatic lipid accumulation through the natural fatty liver disease in Japanese quail. Therefore, it is benefit on finding the ameliorative way to dealing with NAFLD or FLHS, if the functional ingredients in those two hepatoprotective materials could be further studied.

並列關鍵字

fatty liver disease ； estrogen ； C. militaris ； G. lucidum ； Japanese quail

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