蓮蓬萃取物降低順鉑導致腎損傷之體內與體外研究

The study of nephroprotective effect of lotus seedpod extract against cisplatin-induced renal injury in vivo and in vitro

張妤宣(Yu-Hsuan Chang)

指導教授 ： 翁玉青

中山醫學大學/健康管理學院/營養學系/碩士(2019年)

https://doi.org/10.6834/CSMU201900195

摘要

順鉑(cisplatin)常用於卵巢癌、頭頸部癌等許多癌症的化療用藥，但因cisplatin具有腎毒性而影響癌症病患健康。為了降低cisplatin的腎毒性，本研究欲尋找有效天然物來達到保護腎臟的作用。蓮蓬(lotus seedpod)為傳統中藥材，富含多酚(polyphenol)成分，在過去研究指出，蓮蓬具抗氧化、抗老化及抗癌之活性，但是否能改善cisplatin所造成的腎毒性仍未知。因此本篇研究利用體外與體內實驗來探討蓮蓬萃取物(lotus seedpod extracts, LSE)是否能改善cisplatin所造成的腎損傷。在細胞實驗中，預先給予LSE，再加入cisplatin誘導大鼠腎小管上皮細胞(NRK-52E)損傷，結果發現LSE能減少細胞死亡，達到保護功效。經流式細胞儀和DAPI螢光染色分析發現，LSE能降低cisplatin所導致的細胞凋亡(apoptosis)。以西方墨點法分析，在cisplatin誘導NRK-52E細胞損傷之下，LSE能降低凋亡蛋白caspase-3、caspase-9、CAD、Bax、t-Bid的表現量，以及促進抗凋亡蛋白Bcl2和p-Bad的表現。在動物實驗中，以裸鼠(BALB/c nu/nu mice)進行實驗，先給予1% LSE飲食持續三週，再以腹腔注射cisplatin誘導腎損傷，並且持續給予四週1% LSE飲食觀察。結果發現cisplatin損傷下會增加血清中尿素氮(blood urea nitrogen, BUN)和血清肌酸酐(creatinine)含量，若合併LSE飲食下則有明顯降低。再以H&E及TUNEL切片染色觀察腎臟組織，發現LSE飲食能改善cisplatin所導致腎臟組織結構異常和細胞凋亡程度。綜合以上研究結果顯示，LSE可能有潛力應用於改善cisplatin化療所導致的腎損傷。

關鍵字

順鉑 ； 蓮蓬 ； 腎損傷 ； 細胞凋亡

並列摘要

Cisplatin is commonly used in the chemotherapy of many cancers such as ovarian cancer and head and neck cancer, but it affects the health of cancer patients because of its nephrotoxicity. In order to reduce the nephrotoxicity of cisplatin, this study sought to find effective natural substances to protect the kidneys. Lotus seedpod is a traditional Chinese herbal medicine, which is rich in polyphenols. In the past, it was pointed out that lotus seed has anti-oxidation, anti-aging and anti-cancer activities, but whether it can improve the nephrotoxicity caused by cisplatin is still unknown. Therefore, this study uses in vitro and in vivo experiments to investigate whether lotus seedpod extracts (LSE) can improve kidney damage caused by cisplatin. In vitro assay, experiments were performed using rat renal tubular epithelial cells (NRK-52E). NRK-52E cells were pre-administered to LSE, and then cisplatin was added to induce damage. It was found that LSE can reduce cell death and achieve protective effects. Flow cytometry and DAPI fluorescence staining analysis showed that LSE can reduce apoptosis caused by cisplatin. Further analysis by Western blotting revealed that when cisplatin was used to induce NRK-52E cell damage, LSE can reduce the expression of caspase-3, caspase-9, CAD, Bax, t-Bid, and promote the expression of anti-apoptotic proteins Bcl2 and p-Bad. In animal experiments, experiments were performed in nude mice (BALB/c nu/nu mice). Nude mice were pre-treated with LSE (1%, op. once daily) for 3 weeks, and then treated with cisplatin (5 mg/kg BW, ip. once weekly) with or without LSE (1%, op. once daily) for 4 weeks. It was found that cisplatin increased the levels of serum urea nitrogen (BUN) and serum creatinine (Cre), but it was significantly reduced when given LSE diet. In histological studies, using H&E and TUNEL staining to observe kidney tissue, it was found that LSE diet can improve the abnormality of renal tissue and the degree of apoptosis caused by cisplatin. Based on these findings, LSE may have potential to improve renal damage caused by cisplatin chemotherapy.

並列關鍵字

cisplatin ； lotus seedpod extracts ； renal injury ； apoptosis

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