衣康酸衍生物在視網膜色素上皮細胞經由Nrf2/HO1訊息傳導路徑抑制因高糖所引發之氧化壓力及細胞凋亡

Abstract

背景 糖尿病視網膜病變是糖尿病常見的微血管病變，也是造成全世界失明的主要原因之一。視網膜色素上皮細胞可以維持視網膜構造及功能的穩定。糖尿病的高血糖情況會引起視網膜色素上皮細胞內的氧化壓力增加、產生過量的活性氧自由基，進而造成細胞凋亡。糖尿病視網膜病變的病理機轉就是因視網膜色素上皮細胞的凋亡引起血-視網膜障蔽的破壞，進而使血管通透性增加並造成血管滲漏及視網膜出血。因此，在糖尿病的病人中，如何防止視網膜色素上皮細胞內的氧化壓力增加及細胞凋亡，以避免糖尿病視網膜病變的發生或惡化是一個重要的課題。 Nrf2是一個轉錄因子，可調控抗氧化基因的轉錄活性，Nrf2活化後會從細胞質進入細胞核，啟動抗氧化基因的轉錄活性以移除氧化壓力，而HO-1、NQO1、GCLM則是由Nrf2調控的主要基因產物。衣康酸是檸檬酸循環中的一個中間產物，在發炎、代謝、免疫的過程都扮演重要的角色。近期的研究發現衣康酸衍生物經由活化Nrf2訊息傳遞途徑可抑制細胞凋亡與氧化壓力。衣康酸衍生物與Keap1結合後會使Keap1-Nrf2的結合分離、活化Nrf2並進入細胞核，進一步去活化產生抗氧化相關的蛋白質，減少細胞內的活性氧自由基及氧化產物。本研究目標即是探討衣康酸衍生物在視網膜色素上皮細胞中抑制因高糖環境所引發之氧化與細胞凋亡的能力。 研究方法 本研究使用人類視網膜色素上皮細胞株ARPE-19，建立高糖環境對視網膜色素上皮細胞傷害的實驗模式。添加不同濃度的衣康酸衍生物於ARPE-19細胞培養中，觀察衣康酸衍生物對高糖引起的細胞凋亡與氧化壓力的影響。透過測量其細胞活性、細胞凋亡情形與氧化產物的表現量，可以得知衣康酸衍生物對於ARPE-19細胞的抗凋亡與抗氧化的保護效果。此外，為了確認衣康酸衍生物作用於Nrf2訊息傳遞調控的機轉，使用免疫螢光染色證明衣康酸衍生物促使Nrf2從細胞質進到細胞核，並透過西方墨點法去偵測Nrf2調控之下游的抗氧化蛋白質產物。最後使用Nrf2 siRNA去抑制Nrf2的表現，觀察衣康酸衍生物的相關保護效果是否隨之消失，來證明衣康酸衍生物是透過調控Nrf2訊息傳遞來達到其影響的結果。 結果 ARPE-19細胞能耐受在各個濃度(5、10、25與50 μM)之衣康酸衍生物中。高糖環境刺激ARPE-19細胞產生活性氧自由基及各種氧化產物，而衣康酸衍生物能顯著抑制高糖環境所造成的活性氧自由基、去氧核醣核酸、蛋白質與脂質的氧化產物(分別為8-OHdG、nitrotyrosine及acrolein)。透過西方墨點法檢測cleaved caspase-3的量與Bcl-2/Bax比例以及分析TUNEL試驗，發現衣康酸衍生物能顯著減少高糖環境中ARPE-19細胞的凋亡。使用螢光染色法與核質分離萃取分析Nrf2蛋白質，可見衣康酸衍生物促使Nrf2蛋白質從細胞質進到細胞核內，且Nrf2蛋白質在細胞核中的表現量顯著增加。Nrf2所調控之下游抗氧化蛋白質產物(HO1、NQO1與GCLM)的產量則隨衣康酸衍生物之作用而顯著上升。此外，使用Nrf2 siRNA轉染之ARPE-19細胞，與對照組相比，可見衣康酸衍生物對於ARPE-19細胞的抗氧化、抗細胞凋亡作用顯著下降。 結論 本研究有助於了解衣康酸衍生物對因高糖引起之氧化壓力造成視網膜色素上皮細胞損傷具有保護作用，是透過活化Nrf2、增加Nrf2下游調控之抗氧化蛋白質的途徑。本研究結果或能對衣康酸衍生物未來用於治療或減緩糖尿病視網膜病變惡化提供參考。

Background Diabetic retinopathy (DR) is a significant microvascular complication associated with diabetes mellitus. The pathogenesis of DR encompasses increased vascular permeability, neovascularization, retinal ischemia, and edema, culminating in visual impairment and potential blindness. The heightened oxidative stress induced by hyperglycemia plays a pivotal role in the pathogenic mechanisms underlying DR. Accumulation of reactive oxygen species (ROS) disrupts retinal cellular homeostasis, triggers apoptotic cascades, compromises the integrity of the blood-retinal barrier, and contributes to the development and progression of DR. Therefore, attenuating oxidative stress has the potential to inhibit apoptosis in retinal cells and impede the progression of DR. Itaconate, an essential intermediate metabolite derived from the tricarboxylic acid (TCA) cycle, has gained recognition as a vital autocrine regulatory component implicated in cellular metabolism. Recent research indicates that derivatives of itaconate possess protective properties against oxidative damage by activating the Nrf2 signaling pathway. Our study aims to investigate the antioxidative and anti-apoptotic effects of 4-octyl itaconate (4-OI), a cell-permeable derivative of itaconate, in high glucose (HG)-induced oxidative retinal injury. Materials and methods We used human ARPE-19 cells as the in vitro model. A subset of ARPE-19 cells was cultivated in a medium containing low glucose concentration (5.5 mM), while the remaining cells were exposed to various concentrations (0, 5, or 50 μM) of 4-OI for a duration of 2 hours and transferred to a high-glucose medium (35 mM) subsequently. Analysis of ROS production, peroxidation products, mitochondrial depolarization assay, cytotoxicity, Nrf2 signaling-related antioxidative stress proteins, and expression of Nrf2 protein in nuclear lysates and cytoplasmic lysates were performed. ARPE-19 cells were also transfected with NRF2 specified siRNA or siRNA control. The transfected cells were cultured 48 hours after transfection and then used in the experiments. Results ARPE-19 cells tolerated well under the treatment of 4-OI in the concentration of 5, 10, 25, and 50 μM. Four-OI suppressed ROS, ROS-related mitochondrial damage, tested by JC-1, oxidative stress mediators including 8-OHdG, nitrotyrosine, and acrolein in HG-stimulated ARPE-19. Four-OI inhibited HG-induced apoptosis activation including cleaved caspase-3, Bcl-2/Bax, and TUNEL staining. Four-OI caused nuclear translocation of Nrf2 protein and expression of Nrf2 signaling-related antioxidative stress proteins including HO1, NQO1 and GCLM. Moreover, Nrf2 siRNA eliminated OI-mediated antioxidative and anti-apoptotic protection against HG in ARPE-19 cells. Conclusion Our study provided evidence supporting the activation of Nrf2/HO1 signaling by 4-OI protects ARPE-19 cells against oxidative injury induced by HG levels. This cytoprotective effect mediated by 4-OI was reversed when Nrf2 was knocked down using targeted siRNA in ARPE-19 cells exposed to HG. These findings contribute to an enhanced comprehension of the antioxidative properties exhibited by itaconate in HG-induced oxidative retinal injury. Furthermore, the implications of these results have the potential for future applications in the treatment of diabetic retinopathy.