何首烏之活性成分在氧化壓力相關疾病的保護作用

氧化壓力反應(Oxidative stress, OS)為生物體活性氧成分與抗氧化系統之間平衡失調所引起的一系列適應性的反應。廣泛認為是各種疾病(神經疾病、肝臟疾病、腎臟疾病、心血管疾病、肺臟疾病、免疫疾病、發炎等)的發生和進展之病生理機制。何首烏（Polygonum multiflorum Thunb, PME）是普遍使用已久的中藥材，在中國、東亞及歐美被用於治療與衰老、高脂血症和氧化壓力反應相關的疾病。至今已有上百種的成分被萃取出來，PME其中的活性成分(包括THSG) 對氧化壓力相關疾病的療效及機制目前尚不清楚。在此研究中有以下目的: 首先，研究PME在OS相關器官損傷中的有效性和副作用。其次，確定PME中潛在的活性成分於OS相關器官損傷的治療機轉。由於肝臟和腎臟是OS相關器官損傷的主要器官，我們專注於此來研究PME和相關活性化合物的作用。在肝臟研究中，我們證實PME對氧化壓力導致的肝損傷有護肝作用。細胞實驗中，PME誘導HepG2細胞中ARE相關基因的表達呈現劑量依賴性。用PME前處理HepG2細胞抑制了H2O2-和對APAP誘導的ROS產生和細胞毒性。而動物實驗裡，在APAP誘導的小鼠肝損傷中，PME前處理顯示出提高存活率和降低肝損傷嚴重程度的能力。PME治療透過增加了MRP4和減少OATP的表達來降低膽汁淤積，進而減弱了膽管結紮誘導的肝外膽汁淤積性肝損傷。上述動物實驗的療效機轉與用PME處理後觀察到Nrf2的核轉位增加有關。THSG是從何首烏中提取的活性化合物。在腎臟實驗中探究了THSG在AD誘導的FSGS小鼠模型中的保護作用以及體外系統的潛在機制。結果顯示出THSG有著改善AD誘導的FSGS進展的濃度依賴性保護作用。THSG還可抑制白蛋白尿和高膽固醇血症，並降低尿液、血液和腎組織中脂質過氧化的狀態。此外，THSG可防止足細胞損傷，減少腎纖維化基因表達，並減輕腎小球硬化的嚴重程度。用THSG處理小鼠繫膜細胞則使Nrf2核轉位增加，也增加HO-1和NQO-1的基因表達，並減少細胞硫醇氧化和AD誘導的細胞毒性。抑制Nrf2或是Keap1則消除了THSG的保護作用，證實了THSG的作用機制與Nrf2/Keap1相關聯。總結來說，PME的器官保護作用是通過Nrf2激活，THSG是PME中降低OS相關器官損傷的活性化合物。在肝臟中，PME通過Nrf2激活調節肝損傷中的氧化還原狀態，並調控阻塞性膽汁淤積中的膽汁恆定。在腎臟中，THSG可通過激活Nrf2-Keap1抗氧化途徑在改善FSGS的過程中發揮保護作用。 PME和THSG的器官保護作用可以進一步擴展到其他器官或系統，開展OS相關器官損傷的治療新局。

關鍵字

氧化壓力反應；何首烏；肝損傷； FSGS ； PME ； THSG ； Nrf2

並列摘要

Polygonum multiflorum Thunb (PM), officially listed in the Chinese Pharmacopoeia, has long been used in traditional Chinese medicine. It is known as He Shou Wu in China and East Asia. It has been used to treat diseases associated with aging, hyperlipidemia, and oxidative stress related disease. Oxidative stress (OS) is widely recognized as a detrimental pathological mechanism for the initiation and progression of various diseases. Reactive oxygen species (ROS) are mainly generated by cytochrome P450 enzymes in the mitochondria of hepatocytes. When excess ROS are generated, they interact with proteins, DNA, and lipids, resulting in cell injury. Disruption of the balance between oxidant and antioxidant mechanisms is common in the progressing course of organopathy, either from inflammatory or metabolic insults. There are two main purposes in my project. First, to study the effectiveness and possible side effect of PM extracts in OS related organ damage. Second, identify potential active ingredient in PM extracts for OS related organ damage and potential therapeutic mechanism. Since liver and kidney are the two major organs of OS related injury, we focused on these two organs to study the effect of PM extracts and relevant active compounds. In the first part of my study, we examined the hepatoprotective effects of the ethanolic extract of PM (PME) in vitro and in vivo models. The PME induced expression of antioxidant response element (ARE)-related genes in HepG2 cells dose-dependently. Pretreatment of HepG2 cell with PME suppressed H2O2- and acetaminophen (APAP)-induced cellular ROS generation and cytotoxicity. In APAP-induced mouse liver injury, pretreatment with PME also showed ability to increase the survival rate and reduce the severity of liver injury. Treatment with PME attenuated bile duct ligation-induced extrahepatic cholestatic liver injury, and further increased the expression of multidrug resistance protein 4 (MRP4) and reduced organic anion-transporting polypeptide (OATP). Furthermore, increased nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2) was observed after treatment with PME in both in vivo models. 2,3,5,4′-Tetrahydroxystilbene-2-O-β-D-glucoside (THSG) is an active compound extracted from Polygonum multiflorum Thunb. In the second part of my study, we investigated the protective effects of THSG in an Adriamycin (AD)-induced focal segmental glomerulosclerosis (FSGS) mouse model and the underlying mechanisms in an in vitro system. Mice were treated with THSG (2.5 and 10 mg/kg, oral gavage) for 24 consecutive days. On the third day, mice were intravenously given a single dose of AD (10 mg/kg). At the end of the experiment, plasma and kidney samples were harvested to evaluate the therapeutic effects of THSG. The potential mechanisms of THSG in protecting against AD-induced cytotoxicity were examined using a real-time polymerase chain reaction (PCR), immunoblots, lactate dehydrogenase (LDH) assay, and a cellular oxidized-thiol detection system in a mouse mesangial cell line. THSG showed concentration-dependent protective effects in ameliorating the progression of AD-induced FSGS in our study. THSG also suppressed albuminuria and hypercholesterolemia and reduced the status of lipid peroxidation in urine, plasma, and kidney tissue. Furthermore, THSG protected against podocyte damage, reduced renal fibrotic gene expressions, and alleviated the severity of glomerulosclerosis. Treatment of mouse mesangial cells with THSG induced nuclear translocation of nuclear factor erythroid-derived 2-like 2 (Nrf2), increased gene expressions of heme oxygenase (HO)-1 and NAD(P)H:quinone oxidoreductase (NQO)-1, and reduced cellular thiol oxidation and AD-induced cytotoxicity. Silencing Nrf2 abolished these protective effects of THSG, also silencing Kelch-like ECH-associated protein 1 (Keap1), the repressor protein of Nrf2, confirmed the working mechanism of THSG. In conclusion, organ protective effect of PME is through Nrf2 activation, and THSG is the active compound in reducing OS related damage in PME. In liver, PME regulates the redox state in liver injury through Nrf2 activation, and controlling hepatic bile acid homeostasis in obstructive cholestasis. In kidney, THSG can play a protective role in ameliorating the progression of FSGS through activation of the Nrf2-Keap1 antioxidant pathway. The organ protective effect of PME and THSG may be further extended to other organs or systems, provide new therapeutic strategy in treating oxidative related disease.

並列關鍵字

oxidative stress ； Polygonum multiflorum Thunb ； liver injury ； FSGS ； PME ； THSG ； Nrf2 ； Keap1

參考文獻

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何首烏之活性成分在氧化壓力相關疾病的保護作用

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