評估山竹酮素減緩腎臟纖維化之機制

腎臟纖維化是慢性腎臟病（CKD）導致不可避免的最終結果，慢性腎臟病的全球患病率在未來十年仍會大幅增加。台灣是全世界終末期腎病（ESRD）盛行率最高的地區，因此設法延緩或逆轉慢性腎臟病的發生是台灣醫學發展上刻不容緩的目標。先前有許多研究發現來自山竹（Garcinia mangostana）果皮的山酮類化合物α-山竹酮素（Alpha-mangostin, α-MG）與β-山竹酮素（Beta-mangostin, β-MG）具有抗氧化、抗發炎與抗細菌感染的功效，文獻指出α-山竹酮素可以保護腎臟免受藥物毒性傷害。然而α-山竹酮素影響腎臟纖維化的作用機制與β-山竹酮素是否會改善腎纖維化至今仍尚未被詮釋。本次研究將針對α-山竹酮素與β-山竹酮素是否抑制腎臟纖維化的發展與透過動物和細胞實驗來探討這兩種藥物影響了哪些纖維化機制中的傳遞路徑。給予C57BL/6JNarl小鼠α-山竹酮素與β-山竹酮素（10和20 mg/kg/day）共十天，於第四天操作單側輸尿管阻滯手術（UUO），犧牲後取出腎臟做組織切片染色、西方墨點法與qPCR分析，UUO組表現顯著腎功能障礙、膠原纖維沉積、腎間質纖維化、EMT蛋白與TGF-β/Smad路徑的活化，處理兩種藥物後皆能有效改善這些病理變化，我們認為這與上皮間質轉化EMT被阻斷有關。細胞實驗使用TGF-β1預處理腎小管上皮細胞HK-2兩小時後，加入兩種藥物共同處理，利用MTT assay測試細胞毒性、傷口癒合試驗觀察細胞移動能力、西方墨點法和免疫螢光染色分析蛋白表達與qPCR觀察基因表現量。結果顯示，TGF-β1顯著誘導HK-2細胞爬行、纖維化與EMT蛋白表達，而兩種藥物皆能減少TGF-β1所誘導纖維化相關的表徵。為了探討藥物影響的MAPK路徑，從西方墨點法結果得知α-山竹酮素抑制p-ERK表現；β-山竹酮素抑制p-JNK表現。於是加入MEK1/2抑制劑U0126與α-山竹酮素共同處理HK-2；JNK抑制劑SP600125與β-山竹酮素共同處理HK-2，西方墨點法與傷口癒合試驗結果顯示，上述藥物合併處理更顯著抑制HK-2的細胞移動能力、纖維化、EMT與TGF-β/Smad路徑的活化。在UUO動物實驗也證實α-山竹酮素影響p-ERK；β-山竹酮素影響了p-JNK。進一步合併AST-120與α-山竹酮素處理UUO小鼠證實AST-120與α-山竹酮素下調ERK的活化，並顯著改善腎臟纖維化指標。綜合以上，α-山竹酮素透過抑制ERK/MAPK；β-山竹酮素透過抑制JNK/MAPK來阻斷TGF-β/Smad路徑、抑制EMT的發生藉此改善腎纖維化。因此，α-山竹酮素與β-山竹酮素可能作為慢性腎臟病的潛在抗纖維化治療方法。

Renal fibrosis is a fundamental pathological process of chronic kidney disease (CKD) leading to end-stage renal failure (ESRD), a devastating disorder that requires dialysis or kidney transplantation. According to the annual report from USRDS, Taiwan reported the highest-ranking of incidence and prevalence of dialysis worldwide. Therefore, inhibiting the process of renal fibrosis is an urgent problem to be solved in Taiwan’s medical development. Alpha-mangostin (α-MG) and Beta-mangostin (β-MG) are natural xanthones isolated from the nutritious Asian tropical fruit Mangosteen (Garcinia mangostana Linn), which has been investigated for biological properties including antioxidant, anti-inflammatory, and antibacterial activities. Previous studies have shown that α-MG attenuates lung fibrosis and protects renal function from drug toxicity. However, the precise effects of α-MG and β-MG to ameliorate renal fibrosis remain unclear. Our study investigated whether α-MG and β-MG inhibit renal fibrosis and explore the possible mechanism through animal and cell models. C57BL/6JNarl mice were administered α-MG and β-MG (10 mg/kg/day；20mg/kg/day) for 10 days then unilateral ureteral obstruction (UUO) surgery was performed to stimulate fibrosis. Histopathological changes and collagen deposition were analyzed via hematoxylin-eosin staining and Masson staining. Immunochemistry staining, western blotting, and qPCR were used to determine protein or mRNA expression, respectively. The UUO group showed significant renal dysfunction, collagen fiber deposition, tubulointerstitial fibrosis, activation of EMT and TGF-β/Smad pathway. These pathological changes were effectively attenuated by α-MG and β-MG. Human proximal tubule epithelial cell HK-2 was used as in vitro model. The cell proliferative rate was determined by MTT, cell migration by wound healing assay, and protein or gene expression by western blotting, qPCR, and immunofluorescence. In cell models, TGF-β1 was used to stimulate EMT process and enhance the cell migration ability of HK-2 cells. α-MG and β-MG reduced TGF-β1-induced expression of fibrotic proteins. Moreover, α-MG alleviated renal fibrosis by inhibiting the phosphorylation of ERK, and β-MG by inhibiting the phosphorylation of JNK. Combined AST-120 with α-MG ameliorated renal fibrosis by inhibiting the ERK-MAPK pathway. Therefore, α-MG and β-MG may serve as potential anti-fibrotic treatments for renal fibrosis.