雞隻脂肪肝下膽酸濃度變化及次級膽酸對脂肪肝細胞的影響

Abstract

在蛋雞產業中，雞隻產蛋期間常於肝臟堆積脂肪造成非酒精性脂肪肝 （non-alcoholic fatty liver disease, NAFLD）的發生，而使雞隻產蛋率下降，嚴重者則使產蛋雞隻猝死。膽酸 (bile acid)由肝臟中的膽固醇合成，初級膽酸為初始合成的膽酸形式，主要產物為cholic acid、chenodeoxycholic acid，初級膽酸可藉由腸道微生物轉換成其他次級膽酸型式，例如：lithocholic、deoxycholic acid等。膽酸除涉及脂質及葡萄糖代謝的功能外，也具調節自身合成的功能來抑制過多膽酸的產生，因此，以初級膽酸或次級膽酸代謝的角度作為 NAFLD改善途徑的方法探討逐漸受到重視。 本研究利用細胞試驗及兩個動物試驗來討論蛋雞在 NAFLD 模式中膽酸的代謝情形，並評估 NAFLD 下的初級或次級膽酸在雞肝細胞中如何影響脂質代謝。動物試驗一使用低膽鹼高膽固醇飼糧 (high cholesterol and low choline diet, CLC) 將雞隻誘導成 NAFLD ，經六週飼糧誘導後，犧牲並採集肝臟、血清及盲腸內容物，進行肝臟膽酸合成及代謝相關的基因表現分析，血清與腸道內容物使用LC/MS分析各別膽酸含量，腸道內容物樣本經DNA萃取後進行NGS次世代定序。動物試驗二使用8隻羅曼蛋雞，將蛋雞依照產蛋期分為產蛋前期、初期、高峰期及產蛋末期，並收集此四階段之血清進行總膽酸含量測定，並使用LC/MS測定各別膽酸含量，建立各產蛋期的膽酸變化。在細胞試驗使用 LMH 雞肝上皮細胞為平台，以添加500μM Oleic acid方式誘導成脂肪肝模式，並以不同濃度之Taurine conjugated lithocholic acid (TLCA) 進行處理，觀察TLCA對雞隻脂肪肝的影響。 動物試驗一結果顯示合成膽酸相關基因CYP7A1、CYP8B1在CLC組顯著上升 (p<0.05)，血清中TLCA、Taurochenodeoxycholic acid (TCDCA)兩種膽酸在CLC組的濃度亦顯著上升 (p<0.001)，盲腸內容物也顯示CLC組別排出更多的次級膽酸，如：TLCA、Tauro-ursodeoxycholic acid、Taurohyocholic acid 皆顯著上升，腸道微生物組成分析顯示Alpha多樣性無顯著差異，但CLC組之膽酸耐受菌Alistipes屬之平均豐度較對照組多1.69 %。動物試驗二結果顯示，隨著週齡增長，血清中總膽酸濃度逐漸提升，產蛋末期之總膽酸濃度較產蛋前期高約兩倍，個別膽酸如：TCDCA在產蛋末期顯著高於產蛋前期，但TLCA的濃度則隨週齡增加而逐漸減少。細胞試驗結果顯示20 μM TLCA可減少脂肪油滴堆積，惟脂質代謝相關基因表現量無明顯變化，而膽酸合成基因CYP8B1在5μM TLCA處理組顯著高於對照組，且膽酸受體FXR基因表現在5μM TLCA組亦顯著上升，Western blotting結果顯示與脂質生合成有關之SREBP1蛋白質表現隨TLCA添加的濃度增加而逐漸降低。 本研究為首次闡述蛋雞在非酒精性脂肪肝下膽酸之變化，並發現CLC飼糧所誘導的脂肪肝會造成次級膽酸的增加，而自然產蛋週期下所導致的脂肪肝並不會有此現象，此外，膽酸分析資料顯示雞隻在膽酸共軛時偏向與牛磺酸結合而非甘胺酸。上述結果可作為以膽酸代謝角度改善 NAFLD之參考，然而尚有許多機制需進一步地釐清，例如：各產蛋期之腸道菌是否媒介次級膽酸的濃度改變、或是TLCA造成脂肪肝細胞中油滴減少的原因等，可透過其他試驗進一步闡明相關機制。

In the eggs industry, the accumulation of fat in the liver of chickens often causes the occurrence of non-alcoholic fatty liver disease (NAFLD) during laying periods, which reduces the egg production rate of chickens, causing egg industry in a bad situation. Bile acid is synthesized from cholesterol in the liver. Primary bile acid is the initially synthesized form of bile acid, the main products are cholic acid and chenodeoxycholic acid. Primary bile acid can be converted into other secondary bile acid forms by gut microbiome, for example: lithocholic, deoxycholic acid, etc. In addition to the functions involved in lipid and glucose metabolism, bile acid also has the function of regulating its own synthesis in order to inhibit bile acid accumulate in liver. Therefore, the method to improve NAFLD from the perspective of primary bile acid or secondary bile acid metabolism is gradually being paid attention. This study using in vivo and in vitro experiment to discuss bile acid metabolism in laying hens under NAFLD and to assess how primary or secondary bile acids under NAFLD affect lipid metabolism in chicken hepatocytes. In vivo experiment 1, the chickens were induced to NAFLD by using high cholesterol and low choline diet (CLC), after six weeks of dietary feeding, the liver、serum and cecal contents were sacrificed and collected for liver bile acid metabolism-related gene expression analysis, serum and cecal contents were analyzed by LC/MS for bile acid content, and cecal contents samples were extracted by DNA for 16s rRNA sequencing. In vivo experiment 2, using 8 Lohmann laying hens for study. The laying hens were divided into pre-laying, early, peak and later-laying period according to the eggs production rate, and the serum of these four stages was collected for the determination of total bile acid content. The content of bile acid was determined by LC/MS, and the change of bile acids concentration in each laying period was established. In the in vitro experiment, LMH cell were used as a platform to induce fatty liver model by adding 500 μM Oleic acid, and treated with different concentrations of Taurine conjugated lithocholic acid (TLCA) to observe the effect of TLCA on chicken fatty liver. The results of in vivo experiment 1 showed that the synthetic bile acid-related genes CYP7A1 and CYP8B1 were significantly increased in the CLC group (p<0.05), and the serum concentrations of TLCA and Taurochenodeoxycholic acid (TCDCA) were also significantly increased in the CLC group (p<0.001), cecal contents also showed that the CLC group excreted more secondary bile acids, such as: TLCA, Tauro-ursodeoxycholic acid, Taurohyocholic acid were all significantly increased, and the gut microbiome composition analysis showed no significant difference in Alpha diversity. The average abundance of bile acid-tolerant bacteria Alistipes was 1.69 % higher than that of the control group. The results of in vivo experiment 2 showed that with the increase of age, the concentration of total bile acids in serum gradually increased, and the concentration of total bile acid in the later laying period was twice higher than that in the pre-laying period. Individual bile acid such as TCDCA were significantly higher in the later laying period. In the pre-laying period, the concentration of TLCA decreased gradually with the increase of the age. In vitro experiment results showed that 20 μM TLCA could reduce the accumulation of fat oil droplets in Oil Red O staining, but the expression of lipid metabolic genes did not change significantly, while the bile acid synthesis gene CYP8B1 in the 5 μM TLCA group was significantly higher than that in the control group, and the expression of bile acid receptor FXR was significantly higher than control group and it also increased significantly in 5μM TLCA group. Western blotting results showed that the expression of SREBP1 protein which was related to lipid biosynthesis decreased gradually with the increase of TLCA concentration. This study is the first to describe the changes of bile acids in laying hens under non-alcoholic fatty liver disease, and found that fatty liver induced by CLC diet can increase secondary bile acids, while fatty liver induced by natural egg laying period does not happen. In addition, bile acid analysis data show that chickens prefer to bind taurine rather than glycine when bile acid is conjugated. The above results can be used as a reference for improving NAFLD from the perspective of bile acid metabolism. However, there are still many mechanisms have needed to be further clarified, such as whether the gut microbiome in each laying period mediate the concentration of secondary bile acids, or what is the mechanism of TLCA effect lipid metabolism that causing reduction of oil droplets in cells. All these doubts can be further elucidated by more experiments.