香芹酚在氧化壓力下透過調節自噬作用以保護IPEC-J2豬小腸上皮細胞

Abstract

早期離乳為現代養豬產業中降低生產成本之常見手段，然而，在離乳的過程中經常會造成仔豬嚴重的緊迫，進而造成腸道的氧化壓力及功能受損，導致仔豬之生長表現降低，影響經濟利益。香芹酚（carvacrol）為一種具有酚類、單萜結構之天然精油，過去許多研究已經證實了其對於仔豬腸道之抗氧化及抗發炎功效。然而，過去的研究多採用成分複雜之粗萃或混合精油，導致香芹酚對於豬隻腸道的具體保護機制仍不明確。因此，本研究之目的在於透過體外模型，探討香芹酚在離乳誘導之氧化壓力下對於豬隻腸道潛在的調控路徑。 在本研究中，我們以 H2O2 （hydrogen peroxide）處理豬隻腸道上皮細胞（IPEC-J2, porcine intestinal epithelial cells），誘導嚴重之腸道氧化壓力，並在此體外模型中探討香芹酚在氧化壓力下可能的保護機制。結果指出，香芹酚可以在 H2O2 誘導的氧化壓力下顯著提升細胞存活率、減少細胞凋亡（apoptosis），並且降低超氧化物（ROS, reactive oxygen species）及脂質過氧化物（MDA, malondialdehyde）之含量，顯示香芹酚在 H2O2 處理的情況下可以幫助 IPEC-J2 存活並且減緩過度的氧化壓力。並且，香芹酚在 H2O2 處理的情況下也能夠顯著降低粒線體 ROS 生成及提升檸檬酸合成酶（citrate synthase）之活性，顯示香芹酚可以在氧化壓力下有助於改善 IPEC-J2 細胞之粒線體功能。此外，我們進一步發現香芹酚在 H2O2 處理的情況下會明顯減少溶酶體（lysosomes）及自噬溶酶體（autolysosomes）之堆積，並顯著降低自噬（autophagy）相關指標 LC3II/I （Light chain 3）之比值。同時也發現，香芹酚可以在 H2O2 處理的情況下顯著降低粒線體自噬（mitophagy）相關指標 PINK1 （PTEN-induced kinase 1）之蛋白質表現量，顯示香芹酚可能會在氧化壓力下調控 IPEC-J2 細胞中的自噬作用。最後，我們以二甲雙胍（metformin）作為自噬活化劑，以探討香芹酚在氧化壓力下是否參與調控自噬作用。結果指出，在香芹酚及 H2O2 處理下，活化自噬作用會顯著造成 IPEC-J2 細胞存活率降低及 MDA 含量上升，證實香芹酚對於 IPEC-J2 細胞之保護功效，需仰賴其對於自噬作用的調控機制。 綜上所述，本研究成功證實了香芹酚對於豬隻腸道上皮細胞 IPEC-J2 之保護效果，可能是透過緩解腸道過度的自噬作用以達到其抗氧化之功效，此結果也間接指出了香芹酚改善離乳仔豬腸道健康的可能機制。

Early weaning is a common practice to reduce the cost in the intensive pig industry. But piglets usually experience severe stress during this process and result in intestinal oxidative stress which further cause poor growth performance due to impaired intestinal function. Carvacrol is a natural essential oil with a phenolic monoterpene structure and its anti-oxidative and anti-inflammatory abilities on the intestine of piglets have been proposed in many studies. However, the underlying mechanisms of its protection on piglet intestines under oxidative stress are still unclear. The aim of this study is to investigate the possible pathway of carvacrol involved in the porcine intestine under oxidative stress via an in vitro model. In this study, porcine intestinal epithelium cells (IPEC-J2) were treated with carvacrol and hydrogen peroxide (H2O2), an oxidative stress inducer, to investigate the protective mechanisms of carvacrol under oxidative stress. We found that carvacrol ameliorated H2O2-induced cell viability loss, apoptosis, and oxidative stress markers, intracellular ROS (reactive oxygen species) and MDA (malondialdehyde) levels, indicating the protective ability of carvacrol under oxidative stress in IPEC-J2 cells. Consistently, carvacrol significantly reduced mitochondrial ROS generation and increased citrate synthase activity during H2O2 treatment, indicating carvacrol protects mitochondrial function in IPEC-J2 cells during oxidative stress. Furthermore, carvacrol significantly attenuated the increase of autophagy marker, LC3-II to I (Light chain 3) ratio, and reduced the accumulation of lysosomes and autolysosomes induced by H2O2. Also, the increased protein expression of mitophagy marker, PINK1 (PTEN-induced kinase 1), induced by H2O2 was also significantly reduced by carvacrol treatment. These results indicated carvacrol effectively regulated oxidative stress-induced excessive autophagy. Finally, metformin was used as an autophagy activator to clarify the involvement of carvacrol in the autophagic pathway. Activation of autophagy by metformin significantly diminished carvacrol’s protection in cell viability and MDA level under H2O2 treatment, indicating modulation of autophagy is necessary for carvacrol’s protective function in IPEC-J2 cells during oxidative stress. In conclusion, this study revealed the underlying mechanism that carvacrol exerted its anti-oxidative ability in porcine intestinal epithelium cells via relieving excessive autophagy during weaning stress.