富含三萜類山苦瓜葉萃取物對高果糖飲食誘導腎臟損傷之保護作用

Abstract

近年來加工食品和飲料中添加大量果糖，加劇代謝症候群的患病率，進而導致慢性腎病等器官疾病。然而，部分治療藥物存在副作用，尋找新的治療方法是必須且必要的。本研究主要探討具有抗發炎特性且傳統上被用作中草藥的山苦瓜株相關製品是否對於高果糖飲食誘導的腎臟疾病具有保護作用，利用山苦瓜葉中富含三萜類化合物區分物triterpenoid-enrichment extracts (TEE)的萃取物，探索其潛在的治療機制。 動物實驗方面，每天給予小鼠30%果糖水與TEE (150 mg/kg)，持續14週，與單純給予30%果糖水的組別比較，腎功能的多項指標，包括血壓、血清肌酸酐和尿白蛋白均得到改善。藉由組織學觀察，TEE能夠改善高果糖飲食誘導之小鼠腎絲球、近曲小管和腎小管間質區域的損傷。此外，果糖透過增加NADPH oxidase 1 (NOX1)造成腎臟的氧化壓力上升，TEE則促進NAD(P)H quinone dehydrogenase 1 (NQO1)與穀胱甘肽(Glutathione, GSH)的表現以抗衡增加的氧化壓力。除了抵抗氧化壓力之外，給予TEE能緩解由果糖引起之細胞激素前驅分子nuclear factor-kappa B (NF-κB)的活化，進而抑制細胞激素interlukin-1beta (IL-1β)、interlukin-18 (IL-18)、tumor necrosis factor-alpha (TNF-α)的表達。當氧化壓力被TEE緩解時，腎臟的發炎反應也隨之下降。隨後透過觀察肌纖維母細胞(Myofibroblast)標記:波形蛋白(Vimentin)、α-smooth muscle actin (α-SMA)與組織纖維染色(Trichrome stain)，可以發現TEE改善了由果糖所引起之小鼠腎臟纖維化。 藉由體外試驗觀察，發現近端腎小管上皮細胞HK-2受果糖和TEE的影響與小鼠實驗相似，TEE透過降低NOX1、提升NQO1以及促進穀胱甘肽(GSH)消耗來改善氧化壓力，當受果糖引起之氧化壓力被抑制後，下游發炎反應也隨之停擺。除此之外，透過HK-2進一步探討發炎反應相關之程序性細胞死亡機制:細胞焦亡(Pyroptosis)，其特性包含caspase-1活化、細胞膜產生孔洞、發炎細胞激素大量製造與釋放，果糖能夠誘發細胞焦亡與發炎反應，而給予TEE則能夠改善。此外，過去研究已證實三萜類化合物擁有促進粒線體活性的功效，為探討TEE是否擁有相同效果，首先觀察粒線體生合成與脂肪酸代謝相關分子: AMP activated protein kinase (AMPK)訊息路徑的調控，發現TEE能夠活化AMPK並開啟下游路徑，後續使用MitoView 633與MitoView Green觀察細胞粒線體功能與數量變化，果糖刺激會降低HK-2粒線體的活性與數量，而TEE的加入則能夠阻止果糖引起之傷害，大幅增加粒線體數量與功能而達到保護功效。當果糖引起之壓力被TEE消除後，HK-2轉換為肌纖維母細胞的比例減少亦與體內試驗相呼應，暗示著管道間質區域纖維化有所緩解。另一方面，系膜細胞SV40 MES13作為腎絲球血管內皮間質區域的纖維生產者，在病理形態下會增生與促進纖維製造，當腎絲球內堆積大量纖維會造成腎絲球硬化(Glomerulosclerosis)並減少過濾功能，果糖刺激後，MES13有明顯增生、纖維化以及發炎的狀況，不過這些負面的影響都在TEE加入後有所緩解。總結，在生物體方面，TEE可以保護果糖誘導之腎臟形態與功能損傷，細胞層面則能緩解果糖在近端腎小管上皮細胞以及腎絲球系膜細胞造成之傷害，故TEE在果糖引起之腎臟疾病擁有作為治療藥物的潛力。

In recent years, fructose enriched manufactured foods and beverages had exacerbated the incidence of metabolic syndrome, leading to chronic kidney disease and other organ diseases. However, current treatments either are not efficient or have side effects, finding new treatments is necessary. This study aims to investigate the effects of a special leaf extract [triterpenoid-enrichment extracts (TEE)] of Momordica charantia L. var. abbreviate Seringe, which is known for the anti-inflammatory properties and traditionally used as a herbal medicine, on high fructose-induced kidney disease and explore the potential therapeutic mechanisms. In the in vivo experiments, 30% fructose water and TEE 150 mg/kg were given to mice daily for 14 weeks. Compared to the 30% fructose group, several kidney function indicators, including blood pressure, serum creatinine, and urine albumin, were improved in Fructose+TEE group. Histological observation showed that TEE ameliorated the fructose-induced damages on mouse renal glomeruli, proximal convoluted tubules, and renal tubulointerstitial area. Fructose increased kidney oxidative stress by upregulating NADPH oxidase 1 (NOX1), and TEE promoted the expression of NAD(P)H quinone dehydrogenase 1 (NQO1) and glutathione (GSH) to counteract excessive oxidative stress. TEE also alleviated the activation of the pro-inflammatory molecule nuclear factor-kappa B (NF-κB) induced by fructose, thereby inhibiting the expression of cytokines such as interlukin-1beta (IL-1β), interlukin-18 (IL-18), and tumor necrosis factor-alpha (TNF-α). In addition, TEE inhibited the fructose-induced the myofibroblast markers, vimentin and α-smooth muscle actin (α-SMA), and renal fibrosis. In the in vitro experiments, the effects of TEE on fructose treated proximal tubular epithelial cells HK-2 were similar to those in the in vivo experiments. TEE decreased fructose-induced oxidative stress by reducing NOX1, enhancing NQO1, and promoting glutathione (GSH) consumption. When oxidative stress induced by fructose is suppressed, the downstream inflammatory response also decreased by TEE. Additionally, fructose can induce pyroptosis, which is characterized by the activation of caspase-1, hole formation in the cell membrane, and massive manufacture and release of inflammatory cytokines in HK-2, and TEE can significantly inhibit both phenomena. Fructose also decreased the activity and quantity of mitochondria in HK-2, while the addition of TEE can prevent fructose's negative regulation on mitochondria to achieve the protective effect by activacting AMP activated protein kinase (AMPK). The transformation of HK-2 to myofibroblasts was increased by fructose and decreased by TEE administration, which suggesting an alleviation in interstitial fibrosis. On the other hand, Fructose stimulation causes notable proliferation, fibrosis, and inflammation in mesangial cells SV40 MES13, are fiber producers in the glomerular vascular endothelium interstitial region, but these negative effects were alleviated with the addition of TEE. In conclusion, on the mouse level, TEE can protect the fructose-induced morphological and functional renal damages . On the cellular level, TEE can alleviate the damaged caused by fructose in proximal tubular epithelial cells and glomerular mesangial cells. Therefore, TEE had the potential serving as a therapeutic agent for fructose-induced kidney diseases.