和厚朴酚於細胞與動物模式中減緩腎纖維化之探討

Abstract

近年來台灣地區末期腎臟病之盛行率、發生率及死亡率，均不斷攀升，已逐漸與高血壓、糖尿病等慢性病同列為台灣國人之重要疾病。許多不同的腎臟疾病都有可能演變為末期腎臟病，台灣地區則是目前全世界末期腎臟病新生率最高以及盛行率第二的地方。末期腎臟病的最重要特徵為腎臟間質纖維化，而腎臟間質纖維化的特徵為正常的腎臟結構被不正常堆積的細胞外基質蛋白所取代、腎小管萎縮、腎小管擴張和腎絲球硬化。由於大多數末期腎病變的治療方法包括了血液透析、腹膜透析和腎臟移植，這些都所費不貲，且唯有腎臟移植可使患者腎功能恢復到近似正常人。 和厚朴酚(honokiol)是從中國草本植物厚朴所萃取出來的一種物質，具有抗血栓形成、抗焦慮、抗氧化、抗發炎、血管放鬆等等藥理特性。近期的研究也發現其具有抗癌的作用。是個非常具有發展潛力的藥物或保健食品。因此本篇研究想要探討和厚朴酚是否能成為有效預防及治療腎纖維化的藥物。 首先我們先檢測在生物體外和厚朴酚的藥效。利用細胞存活率測定（MTT assay），我們採用10 μM的和厚朴酚及5 ng/mL的轉形生長乙形因子一(TGF-β1)來處理細胞。我們使用的細胞有大鼠近曲小管上皮細胞細胞株NRK-52E、大鼠纖維母細胞細胞株NRK-49F以及由病人檢體得到正常的腎臟部分培養出的纖維母細胞。實驗結果可以發現，TGF-β1可以誘導在細胞的另一個纖維發生因子：結締組織生長因子的表現量上升，同時和厚朴酚具有抑制的效果。另外和厚朴酚也可抑制在NRK-52E中被誘發的上皮間質轉分化以及NRK-49F被誘發的活化反應。而因腎小管上皮間質轉分化以及纖維母細胞活化產生的肌纖維母細胞，其造成的細胞外基質蛋白累積也會被和厚朴酚所抑制住。因此在生物體外的實驗，和厚朴酚是具有減緩腎纖維化特徵的效果。 而後我們採用單側輸尿管阻塞誘發的大鼠腎臟纖維化模式來評估和厚朴酚的療效。有許多抑制腎臟纖維化的藥物都是用這種方法來進行研究。實驗分成四組，分別為：假性單側輸尿管阻塞手術、假性單側輸尿管阻塞手術合併餵食和厚朴酚、單側輸尿管阻塞、單側輸尿管阻塞合併餵食和厚朴酚。和厚朴酚給予劑量為每天個體每公斤給予5毫克，一天餵食兩次。經過手術後兩週犧牲動物。實驗顯示，餵食和厚朴酚並不會影響實驗動物的體重、心跳、血壓、血中尿素氮、肌酸酐、肝指數GPT等生化指標。單側輸尿管阻塞後阻塞側腎臟的腎小管擴大、腎間質體積、腎間質膠原蛋白沉積和α平滑肌肌動蛋白的嚴重程度積分都被和厚朴酚有意義的減少。核糖核酸的分析也顯示和厚朴酚可以有效的降低在單側輸尿管阻塞後阻塞側腎臟細胞外基質蛋白累積，以及降低細胞間黏附因子(ICAM-1)與單核細胞趨化蛋白一(MCP-1)表現。 整體而言，和厚朴酚在生物體外及體內的實驗，都有減緩腎纖維化特徵的表現，因此我們認為，和厚朴酚可能具有能延緩人類腎臟纖維化的療效。

In Taiwan, from 1991 to 2000, the prevalence and incidence of the end-stage renal diseases (ESRD) showed an increase in multiple. Many kinds of renal disease possibly develop ESRD. ESRD, hypertension, diabetes, and other chronic diseases are progressively became important diseases that Taiwanese may suffer from. Taiwan holds the highest incidence of ESRD in the world; however, be next to the Japan, the prevalence existed in the second. Almost all forms of kidney diseases that progress to ESRD are characterized by diffuse fibrosis, in which tubulointerstitial fibrosis is considered the most important determinant of progressive renal injury. Renal tubulointerstitial fibrosis is characterized by the accumulation of extracellular matrix (ECM) proteins, tubular dilatation, tubular atrophy and glomerulosclerosis. The most therapies for ESRD are hemodialysis, peritoneal dialysis, and kidney transplantation, and all of them may be a heavily financial load. Moreover, transplant is the only way to make patients recover similar to ordinary people. Honokiol (HK) is a major bioactive component isolated from the bark of ‘Houpo’ (Magnolia officinalis). HK has several important medicinal uses, such as antithrombotic, anxiolytic, antioxidative, anti-inflammatory, and vasodilative effects. Recent studies showed that HK also has anti-cancer ability. Therefore, HK may be valuably to develop into potential drug or healthy food. So in this study we want to investigate the possible therapeutic effect of HK on ESRD. We first examined the effects of HK in vitro. The dosage we administrated to cells was according to the results of MTT assay: 10 μM HK and 5 ng/mL TGF-β1. We examined these agents in renal cell lines NRK-52E (normal rat kidney proximal tubular epithelial cells) and NRK-49F (normal rat kidney fibroblasts), and primary human renal fibroblasts form normal kidney of patients. Our findings revealed that TGF-β1 induced overexpression of a fibrogenic factor, CTGF, which could be inhibited by HK. HK also blocked TGF-β1-induced epithelial-mesenchymal transdifferentiation (EMT) in NRK-52E and activation in NRK-49F. ECM synthesis and accumulation resulted from myofibroblasts, which came from EMT in tubular epithelial cells and activation in fibroblasts, and cell proliferation could be also blocked by HK. Hence, HK has ability on attenuating renal interstitial fibrosis in vitro. On the other hand, we examined the effects of HK on unilateral ureteral obstruction (UUO) animal model, a widely used model for progressive renal fibrosis that is independent of hypertension or systemic immune disease. In experimental sets, the rats were divided into four groups: (1) SHM, the rats underwent sham operations and were treated with vehicle; (2) SHM/HK, the rats underwent sham operations and were treated with HK; (3) UUO, the rats underwent UUO and were treated with vehicle; (4) UUO/HK, the rats underwent UUO and were treated with HK. The dosage we treated to animals was 5 mg/kg/day and administrated twice per day. Rats were sacrificed in 14 days after operation. We found that HK did not affect body weight, mean blood pressure, heartbeat rate, and the level of BUN, creatinine, and GPT in each group. Moreover, HK significantly attenuated tubular atrophy, interstitium, interstitial collagen deposition and α-smooth muscle actin (α-SMA) expression. Northern blot analyses for mRNA level also showed that HK decreased ECM, ICAM-1, and MCP-1 mRNA expression in the kidneys of UUO rats. In conclusion, HK is capable of attenuating renal fibrosis both in vitro and in vivo. HK may possess curative effects on postponing renal fibrosis in human.