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|標題:||以 Nrf2 及 NF-κB 路徑評估牛樟芝菌絲固態發酵物應用於肉雞飼糧之潛在抗氧化及免疫調節影響|
Evaluation of potential antioxidant and immunomodulatory effects of solid-state fermented product by Antrodia cinnamomea and the underlying molecular mechanisms via Nrf2- and NF-κB-dominated pathways in broiler chickens
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|摘要:||家禽產業中不可避免的有多種多樣的緊迫因子常導致動物的生長及產能表現不佳；因此，本研究期以台灣特有、珍貴且具抗氧化及免疫正向調節之特有食藥用真菌－牛樟芝 (Antrodia cinnamomea) 應用於肉雞飼糧中，使之活化抗氧化機制並抑制發炎路徑之異常擴大作為改善家禽負面影響的潛在防禦方法，並進而探討其中之分子調節機制。本研究分為兩部分進行；預試驗以商業培養製成之牛樟芝菌絲發酵粉末 (solid-state cultured Antrodia cinnamomea mycelial powder, ACP) 為原料進行；而第二部分則利用與預試驗相同之牛樟芝菌株固態發酵麩皮 (wheat bran, WB)，試驗最終以16天培養期之產物進行後續試驗 (fermented A. cinnamomea from Wang's lab, FAC)。ACP 及 FAC 中的機能性成分如粗三萜、粗多醣及總酚類化合物含量均初步評估以預先了解潛在的影響。預試驗將 240 隻一日齡白肉雞 (Ross 308) 依平均體重逢機分配至以下四組，分別為基礎飼糧 (Control)中添加 0%、0.1%、0.2% 以及 0.4% 之 ACP，每處理組均有四重複欄，每欄 15 隻雞，試驗共持續 35 日。而第二部分之動物試驗則將 400隻一日齡白肉雞 (Ross 308) 依平均體重逢機分配至以下五組，分別為基礎飼糧 (Control)、以及以 5% WB、10% WB、5% FAC 以及 10% FAC 取代基礎飼糧原料組，試驗同樣維持35日。以試驗全期 (1-35 日齡) 而言，試驗之三種添加組別均較Control有較重之體重。而5% 及 10% FAC 組別則較飼糧中含有 WB 之組別有較重之體增重。此外，迴腸及盲腸大腸桿菌群數在 0.1% 及 0.2% ACP 組較低；而飼糧中含 FAC 組別之大腸桿菌群及乳酸菌數分別受抑制及增加。血液抗氧化酵素－超氧化物岐化酶 (Superoxide dismutase, SOD) 在 21 及 35 天較高，而過氧化酶 (Catalase, CAT) 則在 21 天較對照組佳；以FAC 之兩個組別而言，SOD 活性在35天有顯著較其他組高，CAT 則在 21 及 35 天均顯著提高。FAC部分取代雞隻飼糧原料可使35日齡雞隻之周邊免疫單核球細胞(chicken peripheral blood mononuclear cell, cPBMCs) 在受到Lipopolysaccharide (LPS) 或 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) 刺激下，氧化性物質含量如過氧化氫及一氧化氮有相對較低的結果。此外，cPBMCs中的前列腺素E2 (PGE2) 同樣也在飼糧中含有 ACP抑或FAC 組別較低。mRNA表現在預試驗及第二個試驗分別以cPBMCs及肝臟組織進行分析討論。由Nuclear factor (erythroid-derived 2)-like-2 factor (Nrf2)主導之抗氧化基因如Heme Oxygenase 1 (HO-1) 及Glutamate—cysteine ligase catalytic subunit (GCLC)在三組ACP添加組別之35日齡雞隻均顯著提高，而在FAC兩組則以大部分基因均有顯著較高表現的結果。另一方面，由Nuclear factor-κB (NF-κB)主要調節之發炎相關基因如Interleukin 1 beta (IL-1β) 及 IL-6 則在 21及35日齡0.2% ACP 添加組別雞隻之細胞中受抑制；而這樣的結果在第二項試驗中則僅在 35 日齡餵飼含有 FAC 飼糧之雞隻較顯著。Nrf2及 NF-κB 在雞隻肝臟中的蛋白質表現量可支持前述mRNA表現的結果，顯示所有ACP組別均有較高之 Nrf2 表現，而NF-κB則相對受抑制。試驗二更進一步探討兩個轉錄因子之細胞核易位表現，顯示FAC 可促進 Nrf2 並抑制NF-κB易位至細胞核中。綜合此二試驗，較佳的菌相結果顯示ACP 及 FAC 具有改善雞隻免疫調節之潛能。此外，ACP 及 FAC可促進Nrf2 路徑且減緩NF-κB 調控之發炎傳訊路徑。抗氧化酵素及細胞耐受性均在 ACP 及 FAC 組別中顯著改善。同時，ACP 及 FAC潛在之正向作用似乎具有提高雞隻體增重之潛能。|
Antrodia cinnamomea, a precious and unique medical fungus existing exclusively in Taiwan, exhibits antioxidant and immunomodulatory properties. This study was divided into two parts, the preliminary study employed a commercial solid-state cultured Antrodia cinnamomea mycelial powder (ACP); and the second part applied the same strain of A. cinnamomea (AC) as the first part to wheat bran (WB) by solid-state fermentation for 16 days (FAC). Both experiments aimed to evaluate the beneficial effects of AC on chickens, and to further illuminate its underlying antioxidant and immunomodulation molecular mechanisms in broilers. The functional compounds of ACP and FAC - crude triterpenoids, crude polysaccharides and total phenolic content - were both assayed at first to evaluate the possible effects of these materials. In the first animal trial, 240 d-old broiler chickens (Ross 308) were assigned to 4 treatment groups receiving diet supplemented with ACP at 0%, 0.1%, 0.2% and 0.4% for 35 days. Each group had four replicate pens, with 15 birds per pen. For the second trial, 400 d-old broiler chickens were allotted into 5 treatment groups fed control diet, and control diet replaced with 5% WB, 10% WB, 5% FAC, and 10% FAC respectively. Regarding the entire experimental period, chickens in the ACP-supplemented groups demonstrated increased body weight gain compared to those had control diet. 5% and 10% FAC inclusion in diet had birds optimal weight gain than those in WB groups. Moreover, cecal and ileal coliform count were decreased in both the 0.1% and 0.2% ACP groups; and cecal coliform and lactic acid bacteria were diminished and increased respectively while diet replaced with FAC. Blood antioxidant potentiality - SOD activity, increased in birds fed ACP supplemented diet at both 21 and 35 day, accompanied by higher CAT activity at 21 day; yet for FAC inclusion in diet, SOD activity rather increased at 35 day only, with CAT elevated at 21 and 35 day. Oxidative species, in terms of H2O2 and NO levels, induced by LPS and AAPH in chicken peripheral blood mononuclear cells (cPBMCs) were compromised in chickens received FAC containing diet. Furthermore, in 35-d-old birds, PGE2 production in cPBMCs was also suppressed while offering ACP and FAC in chicken diet. mRNA expressions were detected in cPBMCs and liver for the first and the second trial respectively. Antioxidant genes dominated by Nrf2, such as HO-1, GCLC, were up-regulated in 35 day-old birds given ACP supplemented diets and mostly in 5% and 10% FAC groups. On the other hand, inflammatory-related genes, like IL-1β and IL-6, ruled mainly by NF-κB, were rather down-regulated by 0.2% ACP addition at 21 and 35 day; for the second trial, these patterns were pronounced at 35 d. Protein expression levels of Nrf2 and NF-κB in chicken liver supported the mRNA results, demonstrating that all the ACP-supplemented groups showed significantly higher Nrf2 expression, whereas the NF-κB was inhibited. Particularly, promoted Nrf2 and inhibited NF-κB nuclear translocation in chicken liver were found in FAC containing groups. In conclusion, preferable microbial balance may indicate the improvement of immunomodulatory capacity by ACP and FAC. Furthermore, ACP and FAC could induce the Nrf2-dependent pathway and decrease NF-κB dominated inflammatory signaling pathway. Antioxidant and immune capacity in terms of antioxidant enzymes and cell tolerance were also elevated by ACP and FAC. Concomitantly, body weight increased by ACP supplementation and shown commensurate by FAC replacement as comparing with the corresponding control group further implied the promising effects exerted by ACP and FAC.
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