1,25（OH）2D3與木犀草素在吲哚硫酸鹽所誘導之腎小管上皮細胞產生上皮間質細胞化和人類內皮細胞活性氧產生作用中扮演之角色

慢性腎臟病(Chronic kidney disease, CKD)是全球性的重要公衛議題，慢性腎臟病的特徵是腎臟功能逐漸下降，最終發展成末期腎臟病(end-stage renal disease, ESRD)，需要透析或腎臟移植等替代治療。腎臟纖維化(renal fibrosis)是CKD進展的主要病理特徵，而上皮間質轉化(epithelial-to-mesenchymal transition, EMT)則是促使腎間質纖維化的重要病理機制。EMT為腎小管上皮細胞失去其原有的上皮細胞特性，轉而表現出間質細胞的表現型，此轉化加速了腎臟間質纖維化，進一步惡化腎功能。尿毒素，尤其是硫酸吲哚酚(Indoxyl sulfate, IS)，隨著CKD進展導致無法有效排除，會大量累積在血液中進一步加速CKD的進程。IS對腎臟的毒性作用主要透過誘導氧化壓力、發炎反應、腎小管上皮細胞間質化，以及導致嚴重的血管內皮損傷。IS誘導的EMT伴隨著上皮細胞黏附分子(如E-cadherin)的表現下降，同時增加間質標記蛋白如α-smooth muscle actin (α-SMA)、N-cadherin和fibronectin的表現。活性維生素D，即1,25-二羥基維生素D3 [1,25(OH)2D3]，在慢性腎臟病的病患會隨著腎功能的惡化導致1-α hydroxylase活性下降使得活性維生素D的生成減少。過去的研究證實補充活性維生素D可減緩腎功能的惡化，第一部分研究顯示以不同濃度的IS處理HK-2細胞(人類腎小管上皮細胞)， IS能以劑量依賴的方式誘導EMT標記物的改變，包括E-cadherin的下降及α-SMA、fibronectin表現的上升。而其主要的機轉為IS能夠顯著促進Akt (Ser473)與β-catenin (Ser552)的磷酸化，進而促使β-catenin入核，啟動EMT機制。而以1,25(OH)2D3預處理HK-2細胞能顯著抑制這些機制。研究結果表明，1,25(OH)2D3主要透過抑制PI3K/Akt/β-catenin訊息傳導路徑，減緩IS誘發的EMT，可望改善CKD的腎臟纖維化病程。眾多研究指出，CKD之病患心血管疾病風險顯著增加，其機轉與IS累積造成氧化壓力以及發炎反應造成血管內皮細胞損傷相關，因發炎細胞黏附後聚集於內皮細胞造成血管發炎進而損傷為動脈粥狀硬化早期之組織學現象，而氧化壓力以及發炎反應造成血管內皮細胞損傷亦導致腎臟功能進一步的惡化。第二部分研究特別針對IS引起的EA.hy926人類內皮細胞發炎反應進行深入探討，研究發現，IS誘導EA.hy926細胞ROS生成，同時伴隨著ICAM-1 (細胞間附著分子-1)及MCP-1 (單核細胞趨化蛋白-1)蛋白的顯著增加，並可觀察到IS對於白血球黏附內皮細胞(Human monocytic, HL-60 cells)的黏附性增加。黃酮類植物成分木犀草素(Luteolin)因其抗氧化、抗發炎特性，可有效抑制IS造成的氧化壓力與發炎反應。木犀草素保護作用的機轉，與血紅素氧合酶-1 (HO-1)的誘導密切相關。HO-1的增加降低ROS誘發的NF-κB (核因子活化B細胞κ輕鏈增強子)和AP-1 (活化蛋白-1)核轉位，減少ICAM-1和MCP-1表現，進而抑制炎症反應與白血球黏附。研究進一步以HO-1基因靜默試驗證明，HO-1表現的抑制會完全消除木犀草素的保護效果，證實HO-1在抑制IS誘導內皮發炎反應中的核心角色。

Chronic kidney disease (CKD) represents a significant global public health concern. It is characterized by a progressive decline in renal function, ultimately leading to end-stage renal disease, which necessitates renal replacement therapies such as dialysis or kidney transplantation. Renal fibrosis is the primary pathological hallmark of CKD progression, with epithelial-to-mesenchymal transition (EMT) recognized as a crucial mechanism contributing to renal interstitial fibrosis. EMT involves renal tubular epithelial cells losing their inherent epithelial characteristics and acquiring a mesenchymal phenotype, thereby accelerating interstitial fibrosis and further exacerbating renal dysfunction. Uremic toxins, particularly indoxyl sulfate (IS), accumulate in the bloodstream as CKD progresses as the result of impaired renal clearance, thereby accelerating disease advancement. The nephrotoxic effects of IS are primarily mediated through the induction of oxidative stress, inflammation, renal tubular epithelial cell mesenchymal transition, and severe vascular endothelial damage. IS-induced EMT is often accompanied by a reduction in the expression of epithelial adhesion molecules (such as E-cadherin) and a concomitant increase in mesenchymal markers (such as α-smooth muscle actin [α-SMA], N-cadherin, and fibronectin). Patients with CKD experience reduced synthesis of active vitamin D, specifically 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], because 1-α hydroxylase activity declines as renal function deteriorates. Prior research has demonstrated that active vitamin D supplementation can mitigate the worsening of renal function. The first study on this topic revealed that treating HK-2 cells (human renal tubular epithelial cells) with varying concentrations of IS induced dose-dependent alterations in EMT markers, including a decrease in E-cadherin and an increase in α-SMA and fibronectin expression. The primary mechanism involved IS significantly promoting the phosphorylation of Akt (Ser473) and β-catenin (Ser552), subsequently leading to β-catenin nuclear translocation and the activation of EMT. Pretreating HK-2 cells with 1,25(OH)2D3 significantly inhibited these mechanisms. These findings suggest that 1,25(OH)2D3 primarily attenuates IS-induced EMT by suppressing the PI3K/Akt/β-catenin signaling pathway, offering a potential avenue for ameliorating renal fibrosis in CKD. Numerous studies indicate a substantially increased risk of cardiovascular disease in CKD patients. The underlying mechanisms are linked to IS accumulation leading to oxidative stress and inflammation, which subsequently cause vascular endothelial cell damage. The adhesion and aggregation of inflammatory cells on endothelial cells, resulting in vascular inflammation and subsequent damage, represents an early histological feature of atherosclerosis. Furthermore, oxidative stress and inflammatory responses inflicting vascular endothelial cell damage also contribute to the further deterioration of renal function. The second study specifically investigated IS-induced inflammatory responses in EA.hy926 human endothelial cells. This research demonstrated that IS induced reactive oxygen species (ROS) generation in EA.hy926 cells, accompanied by a significant increase in intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) protein levels. Additionally, adhesion of human monocytic (HL-60) cells to endothelial cells was increased after IS exposure. Luteolin, a flavonoid plant component, effectively inhibits IS-induced oxidative stress and inflammatory responses owing to its antioxidant and anti-inflammatory properties. The protective mechanism of luteolin is strongly associated with the induction of heme oxygenase-1 (HO-1). The upregulation of HO-1 reduces ROS-induced nuclear translocation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and AP-1 (activator protein 1), thereby decreasing ICAM-1 and MCP-1 expression and consequently suppressing inflammatory responses and leukocyte adhesion. Further investigation utilizing HO-1 gene silencing experiments confirmed that inhibition of HO-1 expression completely abrogated luteolin's protective effects, solidifying HO-1's pivotal role in suppressing IS-induced endothelial inflammatory responses.