洛神葉對腎纖維化的抑制作用之體內外研究

高尿酸血症(hyperuricemia)是引發起慢性腎病(chronic kidney disease, CKD)的危險因子，進而導致腎纖維化(renal fibrosis)。在高尿酸(uric acid, UA)環境中造成的氧化壓力(oxidative stress)會直接誘發腎間質細胞(renal mesangial cells)纖維化可能是促進CKD的主要機轉。有研究指出天然抗氧化(antioxidant)物質具有開發為護腎之輔助試劑的潛力，已知洛神植株(Hibiscus sabdariffa Linn)葉子具有降血糖、抗氧化、抗動脈硬化及抗癌之功效，本研究擬探討洛神葉萃取物(H. sabdariffa leaf extracts, HLE)及其主要多酚成份-鞣花酸(ellagic acid, EA)是否具有抑制腎纖維化之作用。首先，以UA誘發CKD之細胞模式中，HLE和EA能有效保護小鼠腎間質細胞SV40MES13存活，以及抑制基質金屬水解蛋白酶(matrix metalloproteinases, MMPs)的活性。HLE和EA也具有抗氧化和抗發炎作用，並減少纖維化指標蛋白--轉化生長因子(transforming growth factor beta, TGF)表現，同時抑制促纖維化相關因子(fibrotic factor)生成、上皮間質轉化(epithelial-mesenchymal-transition, EMT)和細胞外基質(extracellular matrix, ECM)過度沉積，以及調控TGFβ/Smad訊息路徑，進而降低UA所誘導的細胞損傷和纖維化作用；進一步，以Smad7 siRNA基因轉染確認HLE的護腎作用是藉由Smad7的表現來執行。在體內試驗為利用腺嘌呤(adenine)誘發CKD小鼠之動物模式，研究證實HLE可顯著改善腎功能指標，對於蛋白尿也有降低的效果。以腎臟組織病理切片染色分析顯示HLE能改善腎絲球萎縮、基底膜增厚，以及慢性損傷病徵之纖維化形成。分子機制顯示HLE也能抑制adenine誘發之TGF和發炎因子表現增加，並提升體內抗氧化防禦系統。總結，我們提出HLE具有調控TGFβ/Smad纖維化途徑和抗發炎作用，以保護腎間質細胞免於損傷，並透過降低氧化壓力達到改善CKD之功效。

Hyperuricemia is an independent risk factor for causing chronic kidney disease (CKD) and contributes to renal fibrosis. Uric acid (UA) has been proven to directly cause renal mesangial cell oxidative stress and fibrosis in pathogenesis of hyperuricemia nephropathy. Some antioxidants can be used as chemopreventive agents of CKD. Hibiscus sabdariffa leaf extracts (HLE), rich in polyphenols, has been shown to possess hypoglycemic, antioxidant, hypolipidemic, antiatherosclerotic and anticancer effects. The aim of the study is to examine the inhibitory effect of HLE and its main component ellagic acid (EA) on renal fibrosis. In vitro, mouse renal glomerular mesangial SV40MES13 cells pre-treated with UA were demonstrated to trigger obvious morphological changes and viability loss, as well as affect the activities of matrix metalloproteinases (MMPs). Non-cytotoxic doses of HLE and EA abolished the UA-induced cell injury, and the secretion of MMP-2/9. In addition, HLE and EA exhibited the antioxidant and anti-inflammatory effects on the UA-treated cells with a reduction in transforming growth factor beta (TGF) production. Next, the UA-activated the fibrotic factors, epithelial-mesenchymal-transition (EMT) and extracellular matrix (ECM) deposition was inhibited by HLE or EA. Molecular data indicated that anti-fibrotic effects of HLE might be mediated via TGF/Smad signaling, as confirmed by the transfection of Smad7 siRNA. In vivo, HLE and EA supplementations significantly alleviated CKD development, which may result from inhibiting adenine-induced TGF production following oxidative stress and inflammation, as well as fibrogenesis. Our data imply that EA-enriched HLE regulates the TGF/Smad signals, which in turn led to reduce renal mesangial cell injury and fibrosis in CKD, and provide a new mechanism for its nephroprotective activity.