利用金奈米粒子胜肽探針檢測糜蛋白酶活性並探討糜蛋白酶於馬兜鈴酸誘導之腎臟病變的角色

Abstract

金奈米粒子(gold nanoparticles, AuNPs)因擁有獨特的光學特性與表面易修飾生物分子的優點，為目前最常被使用的生醫檢測奈米材料，其擁有表面電漿共振(surface plasmon resonance, SPR)和螢光共振能量轉移(fluorescence resonance energy transfer, FRET)特性，使其可根據與螢光物質之距離造成螢光淬熄。據此，在本研究中我們藉由在AuNPs表面修飾胜肽螢光探針開發一靈敏且有效率之酵素檢測方法，此方法設計目標為檢測糜蛋白酶(chymase)，以及將之應用於腎臟病變之診斷。馬兜鈴酸(aristolochic acid, AA)為一中草藥組成份，其會造成腎臟間質永久性之損傷。在腎臟組織之腎素-血管擴張素系統(renin-angiotensin system, RAS)中，血管收縮素轉換酶(angiotensin conversing enzyme, ACE)為將血管收縮素I (angiotensin I, Ang I)轉換成血管收縮素II (angiotensin II, Ang II)的主要途徑，然而在許多腎臟疾病中，已有證據指出chymase會取代ACE將Ang I轉換成Ang II。本研究目的即在於探討chymase在AA誘導之腎臟病變過程中是否扮演重要角色。

本研究首先利用13-nm AuNPs建構一可測得chymase活性的胜肽螢光探針(即AuNPs-peptide probe)，此螢光探針序列為FITC-Acp-DRVYIHPFHLDDDDDC，其中包含了在C端的螢光基團(fluoresceine isothiocyanate, FITC)、酵素受質胜肽段(DRVYIHPFHL)、帶負電之胜肽間隔子(DDDDD)及一含有硫醇基之半胱胺酸(cysteine, C)來與AuNPs形成金硫鍵。製備完成之AuNPs-peptide probe，其可見光吸收波峰從520 nm紅移至525 nm，當酵素水解探針上胜肽受質後，探針上的螢光基團將遠離AuNPs表面，使得螢光訊號得以於485 nm被激發且於515 nm被偵測到。實驗動物方面，C57BL /6野生型(Wild-type, WT)與第二型血管收縮素轉換酶(angiotensin conversing enzyme II, ACE2)基因剔除(ACE2 KO)小鼠被利用於進行腎臟病變模型實驗。藉由腹腔注射AAI (累積劑量為30或100 mg/kg小鼠體重)，經過2週和4週病情發展期後，犧牲小鼠收集其血液和腎臟樣本，進行血液生化、組織病理及分子檢測。

實驗結果顯示，AuNPs-peptide probe用於chymase活性檢測時間只需15分鐘，檢測反應在10-120 ng/mL chymase間有線性關係，檢測低限值為5 ng/mL，在檢測反應中加入chymase拮抗劑(chymostatin)後，酵素反應會被明顯抑制。在專一性實驗中，本實驗所建構的AuNPs-peptide probe用於高濃度的胰蛋白酶(trypsin)和胰凝乳蛋白酶(chymotrypsin)檢測時，並無代表蛋白質酶活性的螢光值被檢測到。根據以上結果，證明我們已成功利用AuNPs-peptide probe建立一靈敏且專一性高的chymase活性檢測方法。

在AAI誘發腎臟受損後，可發現小鼠的體重嚴重下降，血清肌酐酸(creatinine)和尿素氮(blood urine nitrogen, BUN)上升，在腎臟組織的H&E染色切片中，可發現大量白血球細胞浸潤的炎症反應，而在腎臟組織Masson’s trichrome染色切片中，則發現明顯的腎臟間質纖維化病變。上述的血液生化值和病理病變，在AAI處理的ACE2 KO小鼠中顯著嚴重於AAI處理的WT小鼠。在WT小鼠中，AAI處理使小鼠的腎臟組織MMP-2活性顯著上升，MMP-9活性則下降，chymase活性顯著上升，但是腎臟ACE及ACE2活性卻未因AAI誘發腎臟病變而有顯著變化。在ACE2 KO小鼠中，AAI處理對小鼠腎臟組織的MMP-2、MMP-9及ACE活性變化趨勢與WT小鼠相似，但上升的chymase活性和表現較WT小鼠顯著為大。

依據上述研究結果顯示，我們可利用AuNPs-peptide probe來靈敏且有效率地測得腎臟中chymase活性。而在AAI誘發小鼠腎臟病變實驗中，我們發現ACE及ACE2活性並無明顯變化，但腎臟chymase表現與活性卻顯著上升，此顯現chymase在腎臟病變機轉中的重要性，再則ACE2的缺失會加速AAI誘發腎臟病程，這些結果皆有助於醫師與研究者們更了解RAS在腎臟疾病中之角色，並據此研發新的腎臟疾病標的藥物。

Gold nanoparticles (AuNPs) are the most popular nanomaterials due to its unique optical properties and feasibility of surface modifying with biomolecules. AuNPs with the characteristics of surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) have the ability of quenching fluorescence with distant dependece. Based on this, we have established a sensitive and efficient biosensing method by modifying peptide-probe onto the AuNPs to detect enzyme activity in this study. The biosensing method is designed for chymase activity detection and applied in renal disease diagnosis. Aristolochic acid (AA) is an extract from Chinese herbs which causes progressive interstitial nephritis. In kidney, angiotensin converting enzyme (ACE) is the primary enzyme which converts angiotensin I (Ang I) to angiotensin II (Ang II) in renin-angiotensin system (RAS). However, in some renal diseases, several evidences have showed that chymase may play a role to convert Ang I into Ang II independent to ACE. The aim of this research is to investigate whether chymase play the crucial role in AA-induced nephropathy.

In this study, 13-nm AuNPs were used to establish the AuNPs-based fluorescence peptide probe (named AuNPs-peptide probe) for chymase detection. The peptide sequence is FITC-Acp-DRVYIHPFHLDDDDDC which contains a fluorophore at the C-terminal end, enzyme (chymase) substrate (DRVYIHPFHL), a spacer (DDDDD) and a cysteine (C) used for conjugating to AuNPs surface. A red shift of O.D. was observed from 520 nm to 525 nm after the 13-nm AuNPs modified with the peptide into be AuNPs-peptide probe. When the enzyme catalyzed the substrate sequence, the fluorophore drifted away from AuNPs and the fluorescence emitting signal can be excited at 485 nm and detected at 515 nm.

In the animal experiments, animal model of nephropathy was performed in wild type (C57BL/6, WT) and ACE2 knockout (ACE2 KO) mice. The mice were treated with aristolochic acid I (AAI), which is the major and toxic constituent of AA, via intraperitoneal (ip) injection and the accumulated AAI dosages are 30 and 100 mg/kg of body weight. The animals were sacrificed after another 2 or 4 weeks for nephropathy development, and blood and kidney were sampled for the further biochemical, pathologic and molecular assays.

The results show, detection time of the AuNPs-peptide probe used for the activity detection of chymase only needed 15 min and a linear correlation from 10 to 120 ng/mL of chymase is acquired. Moreover, detection limit of the AuNPs-peptide probe is 5 ng/mL. The chymase reaction would be obviously inhibited by adding the chymase antagonist (chymostatin). The AuNPs-peptide probe was also to detect high concentration of trypsin and chymotrypsin, but no emitting fluorescence intensity was detected. According to the results, sensitivity and specificity of the AuNPs-peptide probe using for chymase detection have been approved.

The mice after the treatment of AAI, decreased bodyweight and enhanced serum creatinine as well as increased blood urea nitrogen (BUN) were observed. In the renal tissue sections, high amount of inflammatory cells were found by hematoxylin and eosin (H&E) stain, and increased blue areas of fibrosis in renal interstitial tissue were observed by Masson’s trichrome stain. The biochemical and pathologic characterizations in ACE2 KO mice were more severe than those in WT mice. In WT mice, increased MMP-2 and decreased MMP-9 activity were found in the AAI-treated mice compared with those untreated controls. More, an significantly increase in chymase activity was also found treated with AAI. However, in the AAI-treated kidney tissue, both ACE and ACE2 activity did not show significant changes, indicating the chymase may play the important role in the nephropathy progress. In ACE2 KO mice, the changes of MMP-2, MMP-9 and ACE activity were similar with those in WT mice. However, the increased chymase activity in ACE2 KO mice was more significant compared with that in WT mice.

According to the results, sensitivity and specificity of the AuNPs-peptide probe using for chymase detection have been approved and it is effective to detect chymase activity in kidney tissue. In the AAI-induced nephropathy, ACE and ACE2 activity in kidney tissue did not show significant change, but chymase did, showing that the chymase may play a crucial role in the AAI-induced kidney damage. Moreover, it was also observed that the deficiency of ACE2 would accelerate the disease development of AAI-induced nephropathy. These results may help physicians and researchers to know more information about the role of RAS in kidney disease and can be applied in developing new drug targets for nephropathy.