Supplementary file 3_Omega 3 fatty acid docosahexaenoic acid (DHA) mitigates inflammatory responses in experimental sepsis.zip

BackgroundSepsis is a life-threatening condition characterized by organ dysfunction resulting from a dysregulated host response to infection. Sepsis induces systemic inflammation and increases adhesion molecule expression and activation, promoting leukocyte adhesion to the endothelium. In addition, sepsis leads to the disruption of vascular integrity with fluid leakage and migration of leukocytes across the compromised endothelial barrier, leading to organ damage. Bioactive food compounds such as DHA, an essential omega 3 polyunsaturated fatty acid (PUFA) in the Mediterranean Diet (MedDiet), are known for their anti-inflammatory and pro-resolving properties. Thus, the supplementation of DHA may affect sepsis development, protecting the host.
MethodsTo investigate the role of DHA in neutrophil function, we conducted flow chamber assays using isolated neutrophils from mice and humans treated with DHA. To assess whether similar effects occur in vivo, we performed intravital microscopy of the TNF-stimulated cremaster muscle. Finally, we employed the cecal ligation and puncture (CLP) model to evaluate the therapeutic potential of DHA in experimental sepsis, and we applied intravital microscopy to assess cerebral vascular perfusion and the cerebral microcirculation in septic mice.
ResultsWe found a significant reduction in neutrophil rolling and adhesion in DHA-treated neutrophils compared to controls in flow chamber assays, which can be mechanistically explained by a substantial reduction in adhesion markers, such as PSGL-1, CD11a, and CXCR4. Next, we employed intravital microscopy in the mouse cremaster muscle, stimulating it with tumor necrosis factor, and found a significant reduction in leukocyte rolling and adhesion in DHA-treated mice, confirming the in vitro flow chamber results. We also used a CLP model of sepsis. We found that DHA treatment ameliorated CLP-related sepsis parameters, including mortality, clinical score, total leukocyte and neutrophil transmigration, cytokine levels in peritoneal exudate, plasma, and brain tissue, and lactate levels. DHA treatment also improved cerebral microcirculatory perfusion and exhibited anti-inflammatory and pro-resolving effects, reflected by increased plasma and brain tissue resolving D1 and D2 levels.
ConclusionTogether, we identify DHA as a promising anti-inflammatory therapeutic agent that mitigates sepsis-related vascular dysfunction and prevents organ failure.

【背景】脓毒症（Sepsis）是一种危及生命的疾病，其特征为宿主对感染的反应失调引发的器官功能障碍。脓毒症可诱发全身性炎症，上调黏附分子的表达与活化，促进白细胞黏附于内皮细胞。此外，脓毒症会破坏血管完整性，引发液体渗漏以及白细胞穿越受损内皮屏障的迁移，最终导致器官损伤。二十二碳六烯酸（DHA）作为地中海饮食（MedDiet）中的必需ω-3多不饱和脂肪酸（PUFA）这类生物活性食品成分，其抗炎及促消退特性已广为人知。因此，补充DHA或可影响脓毒症的发生发展，对宿主起到保护作用。

【方法】为探究DHA对中性粒细胞功能的作用，我们采用经DHA处理的小鼠及人类分离中性粒细胞开展流动腔实验（flow chamber assays）。为评估体内是否存在类似效应，我们对肿瘤坏死因子（TNF）刺激的提睾肌进行了活体显微镜成像（intravital microscopy）。最后，我们运用盲肠结扎穿刺术（CLP）模型评估DHA在实验性脓毒症中的治疗潜力，并通过活体显微镜成像检测脓毒症小鼠的脑血管灌注与脑微循环情况。

【结果】在流动腔实验中，与对照组相比，DHA处理组的中性粒细胞滚动与黏附现象显著减少，这一现象可通过黏附分子标志物（如PSGL-1、CD11a及CXCR4）的大幅下调得到机制层面的解释。随后，我们在经TNF刺激的小鼠提睾肌中开展活体显微镜成像，发现DHA处理组小鼠的白细胞滚动与黏附显著降低，验证了体外流动腔实验的结果。我们还采用了脓毒症CLP模型，结果显示DHA治疗可改善CLP相关脓毒症参数，包括死亡率、临床评分、白细胞及中性粒细胞总跨迁移量、腹腔渗出液、血浆与脑组织中的细胞因子水平，以及乳酸水平。此外，DHA治疗可改善脑微循环灌注，并表现出抗炎及促消退效应，这一点可通过血浆与脑组织中消退素D1、D2水平的升高得到印证。

【结论】综上，本研究确认DHA是一种极具潜力的抗炎治疗剂，可减轻脓毒症相关血管功能障碍，预防器官衰竭。