Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/50938
標題: Regulation of pro-Inflammatory Cytokines Expression in Human Monocytes by Ligation of the Receptor for Advanced Glycation End Products : Inhibition by Flavonoids and Molecular Mechanisms
S100B蛋白誘發人類單核球細胞促發炎細胞激素表現之效應:介入類黃酮化合物之抑制作用及分子機制
作者: 黃祥閔
Huang, Shang-ming
關鍵字: diabete mellitus;天然抗氧化物;S100B;AGEs;pro-inflammatory cytokine;atherosclerosis;monocytes;antioxidants;flavonoid;高度糖化終產物;氧化壓力;促發炎反應;巨噬細胞;抗氧化物;發炎性;腎衰竭;心血管
出版社: 食品科學系
摘要: 
糖尿病是相當錯綜複雜的全身代謝疾病,最終是影響全身大小血管,近年來關於糖尿病併發症之發展機制,許多證據皆指向氧化壓力所造成的內皮功能缺損等發炎性疾病,如心血管疾病、腎衰竭等。此主要是因循環血液中單核球或巨噬細胞受高糖或高度糖化終產物(AGEs)等具糖毒性 (glycotoxin) 物質誘導,活化一系列促發炎因子如TNF-α、IL-1β、β2-integrin等,而產生及釋放大量發炎性物質所致。有鑑於此,本論文乃以S100B protein (ligand of the receptor for advanced glycation end products; AGEs) 誘導人類單核球細胞為模式,誘導人類單核球細胞促發炎反應,同時並介入天然抗氧化物,探討其抑制作用及調控機制,以作為防治糖尿病併發症之輔助策略。結果顯示THP-1 cells 與 S100B protein 培養 4 h 後,S100B protein 組可顯著誘發人類單核球細胞 pro-inflammatory :IL-1β、TNF-α; chemokine:MCP-1、IP-10; 黏附因子(adhension) PECAM-1;胞外整合介子(integrin):β2-Integrin 及促發炎環氧酵素 COX-2 之 mRNA 表現;而介入 20 及 50 μM 作用劑量之 quercetin 及 catechin 後,各具不同之抑制效應(p<0.05),顯示quercetin 及 catechin 之天然抗氧化物可經由抑制 S100B 所誘發之 THP-1 細胞促發炎及發炎細胞激素之分子轉錄層級而具有抗發炎特性。更進一步探討quercetin 及 catechin 對下游促發炎蛋白質表現 TNF-α、IL-1β 及 COX-2 之抑制作用,結果顯示,quercetin 及 catechin 之天然抗氧化物可有效抑制基因轉錄層級外,同時亦可抑制下游促發炎蛋白質產物(p<0.05),顯示 quercetin 及 catechin 兩種類黃酮抗氧化物確實可透過基因及下游蛋白質之抑制作用而有效逆轉促發炎現象。
本論文更進一步探討 quercetin 及 catechin 對促發炎現象之調控分子機制,包括 oxidavive stress dependent pathways 及 oxidavive stress dependent pathways(signal),結果顯示 S100B protein 組可顯著誘發人類單核球細胞 P47phox、PKC 蛋白質表現及抑制 Nrf-2 表現 。而介入 20 及 50 μM 作用劑量之 quercetin 及 catechin 後,quercetin 及 catechin 可以透過抑制 P47phox 及促進 Nrf-2 之蛋白質表現以調控 oxidavive stress dependent 路徑。而以 S100B 誘導人類單核球細胞 1 h 及 2 h 後發現,以 S100B 誘導單核球細胞 1 小時後,ROS 生成量比 NG cell(normal glucose condition) 多出 20% 左右,顯示 ROS 已逐漸開始生成,介入樣品後,以 quercetin 組對由 S100B 所誘導的單核球細胞 ROS 生成具有較佳之清除能力。以 S100B 誘導單核球細胞 2 小時,ROS 生成量比 NG cell(normal glucose)多出 50% 左右。兩種類黃酮化合物對 ROS 之清除能力為:quercetin > catechin,顯示 quercetin 在對由 S100B 所誘導的單核球細胞 ROS 生成,具最佳之清除能力。於 oxidavive stress dependent 路徑(signal)方面,介入 20 及 50 μM 作用劑量之 quercetin、catechin 後,於 PI3K 蛋白質方面,以 50 μM quercetin 及 catechin 最具顯著抑制效應 (p<0.05)。於 p38 MAPK 蛋白質方面,以50 μM catechin 最具顯著抑制效應 (p<0.05)。於 ERK 1/2 MAPK蛋白質方面,以 20 及 50 μM quercetin 最具顯著抑制效應 (p<0.05)。於 JNK MAPK 蛋白質方面,樣品組均具顯著抑制效應 (p<0.05)。顯示此 2 種天然類黃酮抗氧化物對於因 ligand of the receptor for advanced glycation end products(S100B protein)引起人類單核球細胞胞內 oxidative stress independent (signal) 訊息傳遞路徑亢進造成單核球趨化及發炎基因活化深具阻斷及調控之潛力。總括上述之結果,S100B protein 可有效促進人類單核球細胞氧化壓力上升及活性氧生成,並活化 oxidavive stress dependent pathways 及 oxidavive stress dependent pathways(signal)兩種調控路徑導致人類單核球細胞促發炎現象;而介入天然類黃酮抗氧化物 quercetin 及 catechin 後,可抑制活性氧生成並透過 oxidavive stress dependent pathways 及 oxidavive stress dependent pathways(signal)路徑之調控,有效抑制甚至逆轉促發炎基因及下游蛋白質表現。本論文之研究成果顯示此兩種類黃酮化合物具緩和糖尿病居高不下之氧化壓力及發炎現象,可作為飲食保健之參考。
關鍵字:天然抗氧化物、高度糖化終產物、氧化壓力、促發炎反應、S100B (ligand of the receptor for advanced glycation end products)

It is well known that diabetes is associated with atherosclerotic and inflammatory diseases. The adhension of monocytes to endothelium followed by transmigration into the subendothelial space is one of the key early events in the pathogenesis of atherosclerosis. These processes are further aggravated by hyperglycemia and AGEs, leading to cardiovascular complications in diabete mellitus. However, the data concerning the effects of antioxidants on AGEs-mediated oxidative stress is limited. In this study, the flavonoids were used to monitor protective effects of naturally occurring antioxidants against AGEs mediated oxidative damage and inflammatory in human monocytes.
In this study, S100B-induced pro-inflammatory cytokines and COX-2 expression were performed in THP-1 human monocytes. RT-PCR analysis comfirmed that S100B (6.5μg/ml) treatment with THP-1 significantly increased the gene expression of pro-inflammatory cytokine TNF-α, IL-1β; chemokine MCP-1, IP-10; adhension factor PECAM-1;β2-integrin and pro-inflammatory cyclooxygenase COX-2. Treatment of quercetin and catechin (20-50 μM) with S100B in THP-1 had inhibitory effects on the expression of pro-inflammatory genes and protein levels. The results showed for the first time that multiple inflammatory cytokines and chemokines relevant to the pathogenesis of diabetes complication were induced by ligation of the advanced glycation end products and then inhibited by flavonoids.
To further investigate the effects of flavonoids on the expression of S100B stimulated molecule mechanisms either oxidative stress dependent or independent (signal) pathways in THP-1, we found that S100B increased the activities of proteins of oxidative stress dependent pathway including P47phox and PKC, and decreased the expression of antioxidant protein Nrf-2. S100B also increased the activities of proteins of oxidative stress independent pathway including PI3K, P38 MAPK, ERK1/2 MAPK and JNK MAPK as indicated by increased levels of the phosphorylated forms of MAPK (pP38, pERK1/2 and pJNK). Treatment of quercetin and catechin with S100B in THP-1 could block the expression of proteins of oxidative stress dependent and independent (signal) pathways, and increased the expression of antioxidant protein Nrf-2. We also found that S100B treatment with THP-1 for 1 h and 2 h led to a significant increase in ROS levels. Furthermore, treatment of quercetin and catechin could eliminate ROS to reduce oxidative stress stimulated by S100B in THP-1.
Thus, the overall results showed that S100B could stimulate inflammatory responses by regulating oxidative stress dependent and independent pathways, leading to monocyte and inflammatory genes activation relevant to the pathogenesis of diabetes complications. Therefore, flavonoids might be beneficial for diabetes inflammatory conditions and its complications.
Keywords: diabete mellitus; S100B; AGEs; pro-inflammatory cytokine; atherosclerosis; monocytes; antioxidants; flavonoid
URI: http://hdl.handle.net/11455/50938
Appears in Collections:食品暨應用生物科技學系

Show full item record
 

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.