Aims

In this study, rat retinal Müller cells (RMCs) were cultured in vitro to investigate the protective mechanism of evolocumab on rat RMCs in diabetes mellitus (DM) and the expression of relevant inflammatory factors.

Methods

The expression of proprotein convertase subtilisin/kexin type 9 (PCSK9) in the retinal tissues of diabetic rats was detected by immunohistochemistry. Sprague-Dawley (SD) rats at 5–7 d of life were selected as the source of RMCs and divided equally into three groups of 12 rats/24 eyes each. The effect of CoCl₂ and evolocumab on the cellular activity of RMCs was determined by CCK-8 assay. The effect of CoCl₂ and evolocumab on the migration level of RMCs after 72 h was measured by scratch test and the expression of various proteins after 72 h was measured by Western blot.

Results

In STZ rats, the expression of PCSK9 was significantly upregulated in the retina, especially in the inner nuclear layer, which is mainly composed of RMCs. High glucose and CoCl₂ stimulation markedly elevated PCSK9 and GFAP expression at the protein level in RMCs (P < 0.05). Evolocumab treatment (100 μg/ml) reduced the expression and secretion of inflammatory factors in stimulated RMCs (P < 0.05). Furthermore, evolocumab downregulates toll-like receptor-4 (TLR-4) levels and inhibited nuclear transcription factor-κB (NF-κB) phosphorylation in RMCs (P < 0.05).

Conclusions

Evolocumab protects against inflammation in RMCs, at least in part, by negatively regulating the activation of the TLR-4/NF-κB signalling pathway. Evolocumab may be a promising anti-inflammatory therapy for ocular fundus diseases, such as DR.

中文翻译：

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高血糖和缺氧条件下evolocumab对大鼠视网膜Müller细胞的保护作用

目标

本研究通过体外培养大鼠视网膜Müller细胞（RMC），探讨evolocumab对糖尿病（DM）大鼠RMC的保护机制及相关炎症因子的表达。

方法

采用免疫组化法检测糖尿病大鼠视网膜组织中前蛋白转化酶枯草杆菌蛋白酶/kexin 9型（PCSK9）的表达。选择生命 5-7 天的 Sprague-Dawley (SD) 大鼠作为 RMC 来源，并平均分为三组，每组 12 只大鼠/24 只眼。_{通过CCK-8测定确定CoCl 2}和evolocumab对RMC细胞活性的影响。_{通过划痕试验检测CoCl 2}和evolocumab对72 h后RMC迁移水平的影响，并通过Western blot检测72 h后各种蛋白的表达量。

结果

在STZ大鼠中，PCSK9的表达在视网膜中显着上调，尤其是在主要由RMC组成的内核层中。高葡萄糖和CoCl ₂刺激显着升高RMCs中蛋白质水平的PCSK9和GFAP表达（P < 0.05）。Evolocumab治疗 (100 μg/ml) 减少了受刺激的 RMC 中炎症因子的表达和分泌 (P < 0.05)。此外，evolocumab 下调 RMC 中 Toll 样受体 4 (TLR-4) 水平并抑制核转录因子 -κB (NF-κB) 磷酸化 (P < 0.05)。

结论

Evolocumab 至少部分通过负调节 TLR-4/NF-κB 信号通路的激活来预防 RMC 炎症。Evolocumab 可能是治疗 DR等眼底疾病的一种有前途的抗炎疗法。