終期醣化產物對自體免疫疾病的免疫發炎反應及血管病變引發類似發炎老化反應的分子機制

Abstract

背景：發炎老化反應，是一種長期低度非感染性的發炎，此一現象的持續存在對於老化過程影響甚劇，並且在老化相關疾病的致病機轉中扮演重要角色。終期醣化產物是一群由單醣分子，經過非酵素性的梅納反應修飾而成的大分子。在糖尿病、老化相關疾病和自體免疫疾病的患者，常可見到患者體內終期醣化產物升高的狀況。我們認為終期醣化產物出現在老化相關疾病及慢性發炎疾病患者體內，而發炎老化反應又可能造成老化相關疾病，這兩者之間應有其相關，所以我們假設終期醣化產物可能是誘發發炎老化反應的重要因子之一。我們之前的研究發現終期醣化產物對 Th1/Th2 的抑制以及對單核細胞/巨噬細胞的刺激作用，乃經由MyD88和MAPK-ERK-NF-κB的訊息傳導路徑。然而，與終期醣化產物引發的發炎老化反應的分子機制仍有待更進一步的闡明。 方法：本研究將人類血清白蛋白和葡萄糖在37℃、5%CO2培養箱中培養0-180天，並動態觀察終期醣化產物的生合成。我們用此人工合成的終期醣化產物觀察對免疫細胞，如T細胞和巨噬細胞細胞株，及和內皮細胞功能的影響，並研究其分子機制。另外，為了研究發炎現象和醣化之間的交互作用，我們也檢測了和各種不同自體免疫疾病患者血清中的終期醣化產物濃度，並在製作終期醣化產物的過程中加入和免疫老化反應相關的各種不同細胞激素，以評估其影響。 結果：我們發現終期醣化產物在製作過程中其顏色會逐漸由透明最終變為棕色，並且其分子量也會逐漸增加。其酸鹼值也從7.2逐漸降低到5.4，但此變化與離子電荷或鈣離子濃度無關。這些變化乃因血清白蛋白分子內的鹼性氨基酸，包括離氨酸和精氨酸的逐漸醣化，從而喪失鹼性特性而趨向酸性溶液相關。我們發現，每毫升40微克的終期醣化產物，會經由抑制p-STAT3、p-STAT4 和p-STAT6的訊息傳導路徑，來抑制人類Jurkat T細胞株產生第二介白質，同時，也會增加衰老相關β-半乳糖苷酶(SA- βgal) 的表現。但與 Th1/Th2/Treg 亞群的變化無關。另外同樣濃度的終期醣化產物會增加趨化因子CCL-5、第八介白質、巨噬細胞遷移抑制因子(MIF) 和第一介白質受體拮抗分子(IL-1Ra)的產生，但會抑制巨噬細胞的SA-βgal表現量。除此之外，終期醣化產物也會抑制白蛋白對人類冠狀動脈內皮細胞的影響，包括釋放可溶性細胞間質粘附分子1 (sICAM-1)、可溶性內皮細胞選擇素和內皮素的分泌，並增強了老化相關分子SA-βgal的表現量。而在體外研究中，我們發現個別的發炎細胞激素，例如第二介白質、第六介白質、第十七介白質，乙型轉化生長因子，甲型腫瘤壞死因子等，會加速及增加終期醣化產物的生成。本發現佐證了在自體免疫疾病患者體內終期醣化產物增加的現象。 結論：本研究證實終期醣化產物具有免疫抑制、促發炎反應、及引發血管病變等發炎性老化現象。這些病態生理作用乃經由MAPK-ERK-及MyD88-STATs-NF-κB等訊息傳導路徑，並可能與產生衰老相關的 β-半乳糖苷酶相關。另外， AGE-ALB分子會喪失正常白蛋白對血管內皮細胞的正常生理機能，而引發血管病變。而各種不同的發炎性細胞激素因子本身會加速終期醣化產物的形成，而導致惡性循環。

Introduction: Inflamm-aging is a chronic, sterile, low-grade inflammation occurred during aging process. Inflamm-aging play a critical role in aging process and contribute to pathogenesis of age-related diseases. Advanced glycation end products (AGEs) are macro-molecules modified by different monocarbohydrates via non- enzymatic Maillard reaction. Increased serum levels of AGEs are commonly found in the patients with Diabetes mellitus (DM), aging-related diseases, and immune- mediated diseases. We thought that both AGEs and inflammaging existed in age-related disease may not be a coincidence and suggest that AGEs may contribute to inflammaging. We have already demonstrated that the AGE-BSA would exert inhibitory effects on Th1/Th2 cytokine expression and stimulatory effects on monocyte/macrophage lineage via MyD88- and MAPK-ERK- NF-κB signaling pathways. However, the detailed molecular bases of inflamm-aging and vasculopathy related to AGEs remains elucidation. In addition, the real mechanism(s) of inflammation-related cytokines in enhancing AGE-HSA production in autoimmune diseases need further investigation. Methods: We incubated human serum albumin (HSA) and glucose at 37℃ in 5% CO2-95% air incubator for 0-180 days to generate AGE-HSA. The immune-related cell, such as T cell and macrophage cell lines and endothelial cell were incubated with AGE-HSA to evaluate their effects on inflamm-aging and the respective signaling pathways. Furthermore, the effects and the possible molecular mechanism(s) of different inflammation-related cytokines including IL-2. IL-6, IL-17, TNF-α, and TGF-β on AGE-HSA formation were also evaluated. Results: We found the mixture of HSA and glucose gradually changing the color from transparency to brown and increased the molecular weight during incubation. The pH value also gradually decreased from 7.2 to 5.4 irrelevant to ionic charge or [Ca2+] concentration, but dependent on progressive glycation of the alkaline amino acids, lysine and arginine, in the HSA protein molecule. Functionally, 40 μg/mL of AGE-HSA decreased IL-2 production from human Jurkat T cell line via suppressing p-STAT3, p-STAT4, and p-STAT6 whereas increasing senescence-associated β-galactosidase (SA-βgal) expression irrelevant to shifting of Th1/Th2/Treg subpopulations. In contrast, AGE-HSA enhanced CC motif chemokine ligand 5 (CCL-5), IL-8, macrophage migration inhibitory factor (MIF), and interleukin 1 receptor antagonist (IL-1Ra) production but suppressed SA-βgal expression by human macrophage-like THP-1 cells. Interestingly, AGE-HSA abrogated the HSA-induced soluble intercellular adhesion molecules 1 (sICAM-1), sE-selectin and endothelin release from human coronary artery endothelial cells (HCAEC) as well as enhanced SA-βgal expression. The accelerated and increased HSA glycations by individual inflammation-related cytokine such as IL-2, IL-6, IL-17, TGF-β, or TNF-α in the in vitro study reflect increased serum AGE levels in patients with different immune-mediated diseases. Conclusions: AGE-HSA can exert immunosuppressive, inflammatory and vasculopathic effects in patients with immune-mediated disease mimicking inflamm- aging via both MyD88-, and MAPK-ERK-STAT-NF-κB signaling pathways and increasing senescence-associated β-galactosidase expression. The inflammatory cytokines per se may accelerate AGE-HSA formation as reflected by increased serum AGE-HSA levels in these patients.