Department of Physiology, Michigan State University, East Lansing, Michigan 48824, USA.
Invest Ophthalmol Vis Sci. 2010 Jun;51(6):3253-63. doi: 10.1167/iovs.09-4731. Epub 2010 Jan 13.
The authors have previously demonstrated that DHA inhibits cytokine-induced inflammation in human retinal endothelial cells (HRECs), the resident vasculature affected by diabetic retinopathy. However, the anti-inflammatory mechanism of docosahexaenoic acid (DHA) is still not well understood. Sphingolipids represent a major component of membrane microdomains, and ceramide-enriched microdomains appear to be a prerequisite for inflammatory cytokine signaling. Acid sphingomyelinase (ASMase) and neutral sphingomyelinase (NSMase) are key regulatory enzymes of sphingolipid metabolism, promoting sphingomyelin hydrolysis to proinflammatory ceramide. The authors address the hypothesis that DHA inhibits cytokine-induced inflammatory signaling in HRECs by downregulating sphingomyelinases.
ASMase and NSMase activity was determined by sphingomyelinase assay in primary cultures of HRECs. The expression of ASMase, NSMase, ICAM-1, and VCAM-1 was assessed by quantitative PCR and Western blot analysis. Gene silencing of ASMase and NSMase was obtained by siRNA treatment.
Inflammatory cytokines TNFalpha and IL-1beta induced cellular adhesion molecule (CAM) expression and rapid increase in ASMase and NSMase activity in HRECs. DHA decreased basal and cytokine-induced ASMase and NSMase expression and activity and the upregulation of CAM expression. Anti-inflammatory effects of DHA on cytokine-induced CAM expression were mimicked by inhibition/gene silencing of ASMase and NSMase. The sphingomyelinase pathway rather than ceramide de novo synthesis pathway was important for inflammatory signaling in HRECs.
This study provides a novel potential mechanism for the anti-inflammatory effect of DHA in HRECs. DHA downregulates the basal and cytokine-induced ASMase and NSMase expression and activity level in HRECs, and inhibition of sphingomyelinases in endothelial cells prevents cytokine-induced inflammatory response.
作者先前已经证明 DHA 可抑制人视网膜内皮细胞（HRECs）中的细胞因子诱导的炎症，而 HRECs 是受糖尿病视网膜病变影响的固有血管。然而，DHA 的抗炎机制仍不清楚。神经鞘脂代表膜微区的主要成分，富含神经酰胺的微区似乎是炎症细胞因子信号传导的前提。酸性鞘氨醇酶（ASMase）和中性鞘氨醇酶（NSMase）是鞘脂代谢的关键调节酶，可促进神经鞘磷脂水解为促炎神经酰胺。作者提出假设，即 DHA 通过下调鞘氨醇酶来抑制 HRECs 中细胞因子诱导的炎症信号。
通过鞘氨醇酶测定法测定原代培养的 HRECs 中的 ASMase 和 NSMase 活性。通过定量 PCR 和 Western blot 分析评估 ASMase、NSMase、ICAM-1 和 VCAM-1 的表达。通过 siRNA 处理获得 ASMase 和 NSMase 的基因沉默。
炎症细胞因子 TNFalpha 和 IL-1beta 诱导 HRECs 中细胞黏附分子（CAM）的表达和 ASMase 和 NSMase 活性的快速增加。DHA 降低了基础和细胞因子诱导的 ASMase 和 NSMase 的表达和活性，以及 CAM 表达的上调。ASMase 和 NSMase 的抑制/基因沉默模拟了 DHA 对细胞因子诱导的 CAM 表达的抗炎作用。在 HRECs 中，鞘氨醇酶途径而不是神经酰胺从头合成途径对于炎症信号很重要。
本研究为 DHA 在 HRECs 中的抗炎作用提供了一种新的潜在机制。DHA 下调 HRECs 中基础和细胞因子诱导的 ASMase 和 NSMase 的表达和活性水平，内皮细胞中鞘氨醇酶的抑制可防止细胞因子诱导的炎症反应。
