Mitigation of Harmful Factors in Porcine Intestinal Epithelium Through Natural products : 천연물을 통한 돼지 장 상피의 유해 인자 완화

다국어 초록 (Multilingual Abstract)

The intestinal health of livestock and humans is threatened by various harmful factors such as mycotoxins and heat stress. Mycotoxins disrupt intestinal barrier integrity and impair nutrient absorption, leading to a decline in overall physiological performance. Similarly, heat stress induces oxidative damage and inflammatory responses that compromise epithelial function. These harmful stimuli act through complex molecular networks involving key regulatory genes and proteins, ultimately disturbing cellular homeostasis.

Natural products have attracted significant attention as promising resources capable of protecting biological systems from these detrimental effects or alleviating the resulting damage. Beyond their traditional role as therapeutic agents, natural compounds have gained recognition as valuable functional materials in fields such as nutritional genomics and immunology, particularly as the utilization of natural-product-derived antibiotics expands.

Therefore, elucidating the molecular mechanisms underlying the effects of harmful factors such as mycotoxins and heat stress and identifying natural compounds capable of mitigating these effects represent crucial research goals. Such studies are essential not only for improving livestock productivity but also for ensuring food safety and animal welfare.

다국어 초록 (Multilingual Abstract)

Citrinin (CTN) is a mycotoxin that adversely affects livestock by contaminating
stored grains, leading to significant health and economic impacts. This study
investigates the toxicological effects of CTN on porcine small intestinal epithelial
cells (IPEC-J2) and explores potential mitigation strategies using natural products
and chemical inhibitors. Our study demonstrates that CTN induces cytotoxicity
through the transforming growth factor beta (TGF-β) signaling pathway, triggering
apoptosis and G2/M phase cell cycle arrest. We examined cell viability, cell cycle
progression, and gene expression changes in IPEC-J2 cells treated with CTN, 4′
Hydroxydehydrokawain (4-HDK), and LY-364947, a TGF-β receptor inhibitor. LY
364947 treatment confirmed that CTN-induced toxicity is mediated through TGF-β
signaling. Although 4-HDK alleviated CTN-induced cytotoxicity by improving cell
viability and reducing apoptosis, its direct involvement in TGF-β inhibition remains
unclear. These results suggest that CTN disrupts intestinal epithelial cell homeostasis
via TGF-β activation, whereas 4-HDK may exert protective effects through an
alternative mechanism. Our study provides novel insights into CTN-induced toxicity
mechanisms and highlights the therapeutic potential of 4-HDK as a mitigator of
mycotoxin-induced cellular damage.

다국어 초록 (Multilingual Abstract)

Citrinin (CTN), a mycotoxin commonly found in contaminated food and animal
feed, impairs intestinal barrier integrity through oxidative stress and cytotoxicity.
However, its link to ferroptosis, an iron-dependent form of regulated cell death,
remains unclear. This study investigated whether CTN induces ferroptosis in
intestinal epithelial cells and evaluated the protective role of Euphorbia hypericifolia
(EH) against CTN-induced oxidative damage and tight junction (TJ) disruption.
Using IPEC-J2 cells exposed to CTN, intracellular ferrous ion (Fe2+) levels, reactive
oxygen species (ROS) accumulation, and TJ integrity were assessed using
FerroOrange and DCFH-DA staining, RT-qPCR, immunofluorescence, and WST-1
assays. Additionally, a high-throughput screen of 459 natural products identified EH
extract as a top candidate in mitigating CTN toxicity. The CTN treatment
significantly elevated intracellular Fe²⁺ and ROS levels, downregulated antioxidant
genes (notably catalase (CAT)) and disrupted Zonula Occludens-1 (ZO-1) expression
and TJ morphology in IPEC-J2 cells, all hallmarks of ferroptosis-like cell death. Co
treatment with EH extract substantially counteracted these alterations, restoring
antioxidant gene expression, reducing Fe²⁺ and ROS accumulation, and preserving
TJ structure. Phytochemical profiling of EH extract revealed several bioactive
compounds potentially responsible for its protective effects. These findings suggest
that CTN induces ferroptosis-related cytotoxicity in IPEC-J2 cells, but EH alleviates
this toxicity by modulating oxidative stress and iron homeostasis, supporting its
potential use as a natural feed additive for intestinal protection

다국어 초록 (Multilingual Abstract)

Heat stress (HS) severely compromises intestinal epithelial integrity by
promoting oxidative stress, apoptosis, and the loss of tight junction (TJ) structure.
This study aimed to determine whether activation of the aryl hydrocarbon receptor
(AHR) and treatment with Pinus koraiensis (PK) extract could alleviate HS-induced
cellular damage in IPEC-J2 cells. Cells were exposed to HS (43 °C) with or without
flavone (FLA, an AHR agonist) and PK treatment, and the effects on oxidative stress,
apoptosis, TJ integrity, and antioxidant gene expression were evaluated. Both AHR
activation and PK treatment markedly decreased intracellular reactive oxygen
species (ROS) accumulation, restored NRF2 expression, and reduced apoptotic cell
populations. PK also produced a stronger induction of antioxidant genes (SOD1,
CAT, and GCLM) than FLA, indicating additional antioxidant capacity beyond AHR
activation. Moreover, PK and FLA treatments alleviated HS-induced loss of the TJ
protein ZO-1, preserving epithelial barrier integrity. In contrast, AHR inhibition
increased apoptosis and aggravated TJ disruption. These findings demonstrate that
PK and AHR activation cooperatively protect intestinal epithelial cells against HS
induced oxidative and structural damage, suggesting that PK-mediated AHR
activation may serve as a potential strategy to improve intestinal resilience under
thermal stress conditions.

국문 초록 (Abstract)

다양한 천연물질이 외부 유해 인자에 의한 세포 손상을 완화하거나 해독하는 전략으로 주목받고 있다. 특히 천연물에서 유래한 생리활성물질은 항산화, 항염, 세포 보호 등의 기능을 통해 영양유전체학과 면역학 등 여러 분야에서 잠재적 기능성 소재로서 높은 가치를 지닌다.
곰팡이독소는 사람과 가축의 장 상피 기능을 저하시켜 영양 흡수율과 장벽 기능을 약화시키며, 이는 전반적인 건강과 생산성 감소로 이어진다. 이러한 독소는 세포 내 산화적 스트레스, 세포사멸, 그리고 타이트 결합(tight junction)의 붕괴를 유도하는 복합적인 분자생물학적 경로를 통해 작용한다. 따라서, 곰팡이독소의 독성 기전을 규명하고 이를 완화할 수 있는 천연물질을 발굴하는 것은 가축의 장 건강 유지를 위해 매우 중요하다.
본 연구에서는 돼지 소장 상피세포(IPEC-J2)를 이용하여 곰팡이독소 및 환경적 스트레스에 의한 세포 손상 메커니즘과 이를 완화하는 천연물질의 작용 기전을 규명하였다. 시트리닌(Citrinin)은 대표적인 곰팡이독소로, 세포 내 산화적 손상과 세포사멸을 유도하여 세포 생존율을 저하시켰다. 이에 대해 카바락톤 유도체인 4'-Hydroxydehydrokawain (4-HDK)은 시트리닌에 의해 유발된 세포사멸(apoptosis)과 세포주기(cell cycle) 정지를 완화시켜 세포 생존율을 회복시켰다. 이러한 결과는 4-HDK가 시트리닌으로 인한 세포 독성에 대해 세포사멸 경로를 조절함으로써 보호 효과를 나타냄을 시사한다. 또한, 천연 식물인 Euphorbia hypericifolia 추출물은 시트리닌에 의해 붕괴된 타이트 결합 단백질(ZO-1)을 회복시키고, 페롭토시스(ferroptosis) 관련 인자의 발현을 조절함으로써 장 상피세포의 장벽 기능을 유지하는 효과를 보였다.
한편, 환경적 요인인 열스트레스(Heat stress)는 가축에서 생산성과 면역력을 저하시켜 산업적 손실을 초래한다. 본 연구에서는 Pinus koraiensis (PK)로부터 유래한 플라본 (flavone)을 이용하여 Aryl hydrocarbon receptor (AHR)의 활성화가 열스트레스에 의한 세포 손상을 완화할 수 있음을 확인하였다. PK 유래 플라본은 AHR을 활성화시켜 NRF2 신호경로를 통한 항산화 유전자 발현을 유도함으로써 세포의 방어 능력을 강화시켰으며, AHR 억제제(CH-223191)의 병용 처리 시 이러한 보호 효과가 소실되는 것을 통해 AHR 경로의 기여를 확인하였다.
이상의 결과들은 서로 다른 스트레스 요인(곰팡이독소, 열스트레스)에 대해 천연물질이 공통적으로 세포사멸 억제, 페롭토시스 조절, 그리고 타이트 결합 유지 등의 경로를 통해 장 상피세포를 보호함을 보여준다. 본 연구는 4-HDK, Euphorbia hypericifolia, 및 Pinus koraiensis 유래 플라본과 같은 천연물질 기반의 장 보호 전략을 분자 수준에서 제시함으로써, 향후 기능성 사료 첨가제나 장 건강 개선 소재 개발에 기초자료로 활용될 수 있을 것이다.

목차 (Table of Contents)

• GENERAL INTRODUCTION 1
• CHAPTER Ⅰ LITERATURE REVIEW 5
• CHAPTER II 4'-Hydroxydehydrokawain Mitigate the Cytotoxicity of Citrinin in Porcine Intestinal Epithelial Cells 16
• 1. Introduction 17
• 2. Materials and methods 20
• 3. Results 27
• 4. Discussion 43
• 5. Conclusions 48
• CHAPTER III Euphorbia hypericifolia Attenuates Citrinin-Induced Oxidative Stress and Maintains Tight Junction Integrity in Porcine Intestinal Epithelial Cells 49
• 1. Introduction 50
• 2. Materials and methods 54
• 3. Results 60
• 4. Discussion 77
• 5. Conclusions 83
• CHAPTER IV Pinus koraiensis containing flavonoids alleviate heat stress by activating the AHR in IPEC-J2 cells 84
• 1. Introduction 85
• 2. Materials and methods 88
• 3. Results 94
• 4. Discussion 110
• 5. Conclusions 113
• GENERAL DISCUSSION 114
• REFERENCES 118
• SUMMARY IN KOREAN 170