| 摘要: | 泌尿道結石在台灣名列十大常見疾病之一，處理結石耗費龐大的醫療資源與支出，因此，本研究希望能找到一種有效的替代治療方法。
本研究探討中藥方劑—五苓散，對於乙二醇所誘發大鼠腎結石的預防治療效果，並使用蛋白質體學方法探索相關之蛋白質。
實驗目的乃是希望從分子生物學層面，探討腎結石致病機轉；並且評估五苓散在動物體內的結石預防效果。
四十一隻Sprague-Dawley品系雄性大鼠分成四組：對照組接受正常食物及飲水，另外三組接受0.75%乙二醇飲水誘發腎結石，其中安慰劑組接受管灌澱粉，而另外兩個藥物組分別接受375 mg/kg或1125 mg/kg不同劑量的五苓散餵食。
實驗四週後，處死動物，分析血液、尿液，摘取腎藏，並評估體重變化，檢視腎臟病理變化，針對結晶沉澱嚴重度給予客觀評分。結晶嚴重度之評分，係採用光學偏光顯微鏡分區拍攝腎臟切片，再輔以自行研發的圖片瀏覽軟體，隨機匿名顯示圖片；並採用「半定量計分方式」由六位評分者獨立給分，最後統計分析各組之差異。
選取各組具代表性的鼠腎樣本，經過研磨製備萃取，進行第一維等電點集焦電泳分離，再經第二維SDS-PAGE凝膠電泳。使用PDQuest程式比對膠片蛋白質點，挖取差異較大者進行胰蛋白酶水解，並送入MALDI-TOF質譜儀分析胜肽片段。蛋白質身份鑑定，則使用BioTools軟體並連線MASCOT搜尋網站，與各大序列資料庫連線比對確認身份。
結果顯示，安慰劑組大鼠的體重增加遲緩，而餵食五苓散的兩組體重增加情況與正常對照組相似。飲用乙二醇水的大鼠腎臟組織切片，可發現明顯的腎小管破壞、損傷或發炎反應。餵食五苓散後大鼠腎臟內結晶情況顯著地減輕，結晶評分之平均值，低劑量組是0.46，高劑量組是0.45，相較於安慰組的1.40，顯著地較低(p=0.034, Kruskal-Wallis test)。
實驗後血清尿液變化顯示，血清鈣濃度四組並無不同，但是血清游離鈣濃度，在接受五苓散餵食兩組大鼠都明顯地低於安慰劑組(p=0.001)，低劑量組是0.91±0.07 mmol/L，高劑量組是0.94±0.17 mmol/L，而安慰劑組則是1.10 ± 0.06 mmol/L。安慰劑組的血磷值是9.45 ± 2.26 mg/dl，明顯低於正常對照組的12.54±1.25 mg/dl (p=0.015)。餵食五苓散的兩組大鼠血液酸鹼值(pH)也明顯地低於安慰劑組，分別是低劑量組的6.96±0.07，高劑量組的6.96±0.17，而安慰劑組是7.13 ± 0.09 (p=0.006)。五苓散高劑量組大鼠的尿磷值是19.36±2.59 mg/dl，明顯地低於正常對照組的20.98 ± 1.12 mg/dl (p=0.021)。
在安慰劑組大鼠腎臟內發現AGT2 (丙氨酸乙醛酸轉氨酶; Alanine-glyoxylate aminotransferase 2; EC 2.6.1.44)蛋白質的存在，其mRNA表現量明顯地高於正常組，達2.69倍(p<0.05)。
本研究獲得結論如下：餵食五苓散可以減輕大鼠腎臟草酸鈣結晶嚴重程度，而罹患腎結石的危險性也相對較低，因此五苓散的確是一個預防腎結石的有效方劑。目前，五苓散的抑制結石機轉尚未明瞭，推測存在於尿液或腎臟內的蛋白質應該扮演著重要角色。從質譜儀蛋白質身份鑑定結果，發現蛋白質Alanine-glyoxylate aminotransferase 2的存在，推測應該和乙二醇解毒功能有關。
Urolithiasis is a common disease in Taiwan, and the medical expenditure is quite huge. The purposes of this study are to explore the mechanism of nephrocalcinosis using molecular technologies and to evaluate the antilithic abilities of a Traditional Chinese herbal formula, Wulingsan (WLS), by an ethylene glycol-induced nephrocalcinosis rat model. Forty one male Sprague-Dawley rats were divided into four groups. Eight rats were fed with normal chaw and water servered as the normal control, and the others received 0.75% ethylene glycol-added water as a stone inducer. The placebo group (11 rats) received a daily gastric gavage of starch, the low-dose group (11 rats) received WLS (375mg/kg), and the high-dose group (11 rats) received WLS (1125mg/kg) in stead. Four weeks later, animals were killed. The kidneys had harvested and the changes of body weight were measured. The biochemical data of urine and serum were obtained and analyzed. The kidney specimens were examined by a polarized light microscopy and the pictures were taken by a digital camera. The severity of crystal deposits in rat kidneys were evaluated by a semi-quantitative scoring method assisted with a self-composed picture browsing software named ImageScoring. Using this software, pictures were randomly and anonymously displayed allowing the investigators makeing scores. Six investigators had employed to accomplish their scoring works independently; finally those data were collected and analyzed.
Representative samples of rat kidneys were selected from each group. Isoelectric focusing gel electrophorisis (IEF) and Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to separate the protein extracts. The PDQuest software was applied for gel comparisons. The interest proteins were digested with trypsin and sent to mass spectrometry (MALDI-TOF) for peptide analysis. The peptid fragments were macthed by MASCOT web interface from several sequence databases.
Our results revealed that the rats of placebo group gained the least body weights; on the contrary, the rats of WLS-fed groups could normally grow just the same as control group did. The placebo group exhibited lower levels of urine-free calcium (p=0.059) and significantly lower serum phosphorus (p=0.015) than WLS-fed rats. Histological findings of kidneys revealed tubular destruction, damage and inflammatory reactions in the EG-water rats. The crystal deposit scores dropped significantly in the WLS groups, from 1.40 to 0.46 in the low-dose group and from 1.40 to 0.45 in the high-dose group. Overall, WLS effectively inhibited the deposition of calcium oxalate crystal and lowered the incidence of stones (p=0.034, Kruskal-Wallis test).
The serum levels of calclium were no difference in four groups. Bu the free calcium level in the WLS-fed groups(group 3 and 4) were significantly lower than the placedo group, the low-dose group is 0.91±0.07 mmol/L and the high-dose group is 0.94±0.17 mmol/L, compared with the placebo group is 1.10 ± 0.06 mmol/L (p=0.001). The serum levels of phosphorus of placebo group is 9.45 ± 2.26 mg/dl, this is much lower than the control group(12.54±1.25 mg/dl) (p=0.015)。The serum pH levels of WLS-fed rats were significantly lowerthan the placebo rats, the low-dose group is 6.96±0.07, the high-dose group is 6.96±0.17, and the placebo group is 7.13 ± 0.09 (p=0.006). |
