Tang Wenyi, Ma Jun, Gu Ruiping, Ding Xinyi, Lei Boya, Wang Xin, Zhuang Hong, Xu Gezhi
Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, China.
Research Center, Eye and ENT Hospital of Fudan University, Shanghai, China.
Cell Physiol Biochem. 2018;46(1):375-388. doi: 10.1159/000488472. Epub 2018 Mar 23.
BACKGROUND/AIMS: Lipocalin 2 (LCN2), an important mediator of a variety of cellular processes, is involved in regulating the inflammatory response, but its roles in different inflammatory diseases are controversial. Because the role of LCN2 in ocular inflammation has been unclear until now, we explored the function of LCN2 in lipopolysaccharide (LPS)-induced ocular inflammation in vivo and in vitro.
Endotoxin-induced uveitis (EIU) was induced in male Sprague Dawley rats by the intravitreal injection of LPS. The expression and location of LCN2 in the retina were detected with western blotting and immunohistochemistry, respectively. We determined the clinical scores for anterior inflammation, quantified the infiltrated inflammatory cells, and measured the pro-inflammatory factors to determine the anti-inflammatory effects of LCN2 in EIU eyes. Cultured primary rat Müller cells were stimulated with LPS and the expression and secretion of LCN2 were measured with real-time PCR, western blotting, and an ELISA. After Müller cells were cotreated with LPS and LCN2 or PBS, the expression and secretion of TNF-α, IL-6, and MCP-1 were examined with realtime PCR, western blotting, and ELISAs. Western blotting and immunofluorescence were used to detect the phosphorylation and cellular distribution of nuclear factor kappaB (NF-κB) subunit p65.
In EIU, the expression of LCN2 was significantly upregulated in the retina, especially in the outer nuclear layer (mainly composed of Müller cells). LPS stimulation of cultured Müller cells also markedly elevated LCN2 expression. Intravitreal injection of LCN2 significantly reduced the clinical scores, inflammatory infiltration, and protein leakage in EIU, which correlated with the reduced levels of proinflammatory factors in the aqueous humor and retina. LCN2 treatment also reduced the expression and secretion of TNF-α, IL-6, and MCP-1 in LPS-stimulated Müller cells. LCN2 inhibited the inflammatory response by inhibiting the phosphorylation and translocation of NF-κB p65.
LCN2 protects against ocular inflammation, at least in part, by negatively regulating the activation of the NF-κB signaling pathway. LCN2 may be a promising anti-inflammatory therapy for ocular diseases, such as uveitis.
背景/目的：脂联素2（LCN2）是多种细胞过程的重要介质，参与调节炎症反应，但其在不同炎症性疾病中的作用存在争议。由于LCN2在眼部炎症中的作用至今尚不清楚，我们在体内和体外研究了LCN2在脂多糖（LPS）诱导的眼部炎症中的功能。
通过玻璃体内注射LPS在雄性Sprague Dawley大鼠中诱导内毒素性葡萄膜炎（EIU）。分别用蛋白质印迹法和免疫组织化学法检测视网膜中LCN2的表达和定位。我们确定前部炎症的临床评分，量化浸润的炎症细胞，并测量促炎因子，以确定LCN2在EIU眼中的抗炎作用。用LPS刺激培养的原代大鼠Müller细胞，并用实时PCR、蛋白质印迹法和酶联免疫吸附测定法测量LCN2的表达和分泌。在Müller细胞与LPS和LCN2或PBS共同处理后，用实时PCR、蛋白质印迹法和酶联免疫吸附测定法检测肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）和单核细胞趋化蛋白-（MCP-1）的表达和分泌。用蛋白质印迹法和免疫荧光法检测核因子κB（NF-κB）亚基p65的磷酸化和细胞分布。
在EIU中，视网膜中LCN2的表达显著上调，尤其是在外核层（主要由Müller细胞组成）。LPS刺激培养的Müller细胞也显著提高了LCN2的表达。玻璃体内注射LCN2显著降低了EIU的临床评分、炎症浸润和蛋白质渗漏，这与房水和视网膜中促炎因子水平的降低相关。LCN2处理还降低了LPS刺激的Müller细胞中TNF-α、IL-6和MCP-1的表达和分泌。LCN2通过抑制NF-κB p65的磷酸化和易位来抑制炎症反应。
LCN2至少部分通过负向调节NF-κB信号通路的激活来预防眼部炎症。LCN2可能是葡萄膜炎等眼部疾病有前景的抗炎治疗药物。
