二十二碳六烯酸藉由GPR120抑制叉頭轉錄因子O1降低發炎反應

Abstract

肥胖所引起的代謝症候群包含胰島素阻抗、二型糖尿病、心血管疾病與慢性發炎等相關疾病。肥胖使脂肪組織累積油滴而分泌許多發炎因子，例如：腫瘤壞死因子-α（tumor necrosis factor-α, TNF-α）、介白素-1β（IL-1β）and IL-6。 二十二碳六烯酸（Docosahexaenoic acid, DHA）為人類必須的ω-3多元不飽和脂肪酸，其藉由G-protein-coupled receptor 120 （GPR120）降低nuclear factor kappa B （NF-κB）活性。叉頭轉錄因子（FoxO1）在胰島素阻抗性所引起的次臨床發炎反應扮演重要的角色，當被phosphoinositide 3-kinase （PI3K） and protein kinase B（PKB, Akt）路徑磷酸化後便從細胞核移出至細胞質，並減少目標基因的轉錄，例如：IL-1β和單核球趨化因子（ Monocyte chemotactic protein-1, MCP-1）。因此，我們推測DHA可藉由增加磷酸化AKT及GPR120降低FoxO1基因表達。 利用人類單核球細胞株（THP-1）待分化後加入不同濃度的DHA （50, 100, 150 or 200 μM），及200 μM 棕梠酸（palmitic acid, PA）共同培養6及24小時。PA會增加FoxO1, IL-1β, MCP-1 及TNF-α mRNA的表現。100 μM DHA處理6小時及24小時後可降低因PA引起的FoxO1mRNA表現，不同濃度DHA處裡6及24小時亦可降低因PA引起的IL-1βmRNA表現，而以100 or 150 μM DHA處理6及24小時則可降低因PA引起的TNF-α and MCP-1 mRNA表現。蛋白質方面，100 μM DHA可增加磷酸化AKT；PA可增加FoxO1表現但受DHA抑制，且隨著DHA濃度增加，可增加磷酸化FoxO1。為了確認DHA藉由PI3/AKT調控FoxO1路徑，利用PI3K 抑制劑；LY294002處理至細胞中。DHA降低因PA所引起的FoxO1及TNF-α的mRNA表現，但此抑制反應在添加LY294002抑制劑後則明顯被抑制。以上結果顯示，DHA可藉由PI3K/AKT 路徑調控FoxO1的表達與活性。 以DHA或GPR120促進劑；GW9508可降低因PA而增加的FoxO1、 IL-1β、 TNF-α 和 MCP-1 mRNA表現。但同時以DHA及GW9508則無法增強此抑制發炎因子的反應。利用RNA靜默及慢病毒系統約降低30%GPR120表現，感染空質體及GPR120 RNAi質體的組別中FoxO1 mRNA因PA的添加而增加。DHA則會降低空質體組的FoxO1, TNF-α及IL-1β mRNA表現；但在GPR120 RNAi組中，DHA無法降低其表現。結果顯示，DHA需藉由GPR120調控FoxO1及發炎因子的表現。DHA可增加AKT的表現及磷酸化，在GPR120 RNAi的組別中也有同樣的趨勢。PA處理降低磷酸化的FoxO1，而感染GPR120 RNAi的組別中DHA則可增加磷酸化的FoxO1。整體而言，感染GPR120 RNAi組中，AKT與FoxO1的表達與磷酸化皆較感染空質體低，結果表示DHA可藉由GPR120增加AKT及FoxO1活性來達到調節發炎因子。 綜上所述，DHA可能藉由GPR120及PI3K/AKT路徑抑制FoxO1功能，影響下游發炎因子表現，達到降低發炎的目的。本研究顯示DHA具有預防或改善因飽和脂肪酸引起的發炎反應。

Obesity nowdays considered as a chronic, low-grade subclinical inflammatory state in association with metabolic diseases. Relaease and production of proinflammatory adipokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β are greatly enhanced in the adipose tissue due to excessive lipid accumulation. Docosahexaenoic acids (DHA) belongs one of the essential ω-3 polyunsaturated fatty acids for mammals. It has been shown the activation of the NF-κB, a key proinflammatory transcription factor through G-protein-coupled receptor 120 (GPR120). The subclass of forkhead transcription factors O (FoxOs), FoxO1, plays an important role in inflammation. Phosphorylation of FoxO1 by phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB, Akt) to change the subcellular location of FoxO1 from nucleus into cytosol diminish FoxO1 binding on its target genes such as IL-1β and MCP-1. Hence, it is plausible that DHA can down-regulate FoxO1 expression by promoting AKT activation through GPR120. The human monocytic cell line THP-1 cells were subsequently treated with different dose of DHA ( 50, 100, 150 or 200 μM ) with or without palmitic acids (200 μM PA) co-culture for 6 or 24 hr. Expression of FoxO1, IL-1β, MCP-1, and TNF-α was increased by PA treatment. The increase of FoxO1 expression by PA was suppressed in presene of 100 μM DHA under short-term or long-term treatment. Expression of IL-1β was inhibited by 50, 100, 150 and 200 μM DHA under long-term treatment. Expression of TNF-α and MCP-1 induced by PA under short- or long-term treatment were decreased by 100 or 150 μM DHA treatment. DHA promoted AKT activation, particularly at 100 μM level in either presence or absence of PA. Expression of at protein level was increased by PA treatment but inhibited by DHA. Treatment of DHA also increased FoxO1 phosphorylation in a dose dependent fashion. In order to confirm the relationship between DHA and PI3K/AKT pathway, PI3K inhibitor, LY294002, was used in the study. Palmitic acid-induced expression of FoxO1 and TNF-α was aupressed in presence of DHA and such effect was diminished by LY294002, indicating that DHA may reduce FoxO1 expression through the PI3K/AKT pathway. Moreover, the PA-induced expression of FoxO1, IL-1β, TNF-α and MCP-1 was decreased by DHA or GW9508, an agonist of GPR120. Addition of DHA and GW9508 together, however, did not enhance their inhibitory effect on FoxO1 and proinflammatory gene expressions as compared with DHA or GW9508 treatment alone. By using small interfering RNAs with a lentivirus system, GPR120 expreesion was reduced to 30%. Expression FoxO1 was induced by PA in null-vector or GPR120 RNAi treated cells. Treatment DHA blunted the elevation of FoxO1, TNF-α and IL-1β expression by PA induction in cells with null-vector treatment but not GPR120 RNAi cells. These results suggested expression of FoxO1 is reduced by DHA act through PI3K/AKT signaling and GPR120 pathway. Treatment of DHA increased AKT activation similar to that of GPR120 RNAi transfected cells. Transfection with GPR120 RNAi suppressed both AKT transcript and phosphorylation level. Palmitic acid treatment suppressed but DHA treatment increased FoxO1phosphorylation transfected GPR120 RNAi. In general, GPR120 RNAi transfection suppress p-FoxO1/FoxO1 ratio. The results suggested that DHA regulates phosphorylation of AKT and FoxO1 is mediated through GPR120 signaling. In conclusion, the beneficial role of DHA may partially act through down-regulation of FoxO1 functions though GPR120 and/or PI3K pathway, which reduce in such inflammatory factor gene expressions. This study indicates that DHA is provided with reducing inflammation which induces by saturated fatty acids.