Laboratory Animal Center, Nantong University, Nantong, 226001, China.
Department of Thoracic Surgery, Affiliated Hospital of Nantong University, Nantong, 226001, China.
Inflammation. 2021 Apr;44(2):633-644. doi: 10.1007/s10753-020-01363-1. Epub 2020 Nov 10.
The regulatory role of toll-like receptor 4 (TLR4) in the inactivate staphylococcus epidermidis (ISE)-induced cornea inflammation is not well investigated. Here, TLR4 silence could decrease inflammatory cytokines in corneal epithelial cells treated with ISE. The mouse corneal epithelial cells were exposed to ISE for 24 h, either alone or with the NF-κB inhibitor, TLR4 lentivirus to bilaterally (knock-down or and overexpression). The expression of TLR4 in mouse corneal epithelial cells was investigated using western blot and qRT-PCR assay. The inflammatory cytokine levels were evaluated by qRT-PCR and ELISA, respectively. The relative impact factors of TLR4-mediated NF-κB signaling detected using western blot assay. Results show the expression levels of TLR4 and some inflammatory cytokines were significantly increased in corneal epithelial cells treated with ISE. TLR4 Silence markedly decreased ISE-induced production of IL12, TNF-α, CCL5, and CCL9 in corneal epithelial cells. Furthermore, the nuclear translocation of NF-κB p65 and myeloid differentiation protein 88 (MyD88) in the cells treated with ISE were further reduced by silencing TLR4. Inhibition of TLR4-mediated NF-κB signaling by using BAY11-7082 also alleviated ISE-induced inflammation. In the rescue experiment, transfected the stable TLR4 silenced corneal epithelial cells with TLR4 overexpression lentivirus, we found that TLR4 overexpression can restore the down-regulation of TLR4 and inflammatory cytokines (IL12, TNF-α, CCL9) caused by TLR4 knocked down. Therefore, ISE-induced cornea inflammation was due to the activation of the TLR4/MyD88/NF-κB signaling pathway, and dramatically stimulated IL12, TNF-α, CCL9 secretion. TLR4 silence presented mitigates damage in corneal epithelial cells treated with ISE.
TLR4 在灭活表皮葡萄球菌（ISE）诱导的角膜炎症中的调节作用尚未得到充分研究。在这里，沉默 TLR4 可以减少 ISE 处理的角膜上皮细胞中炎症细胞因子的表达。将小鼠角膜上皮细胞暴露于 ISE 中 24 小时，单独或与 NF-κB 抑制剂、TLR4 慢病毒一起（敲低或过表达）。使用 Western blot 和 qRT-PCR 检测法检测 TLR4 在小鼠角膜上皮细胞中的表达。通过 qRT-PCR 和 ELISA 分别评估炎症细胞因子水平。Western blot 检测法检测 TLR4 介导的 NF-κB 信号的相对影响因素。结果表明，ISE 处理后的角膜上皮细胞中 TLR4 和一些炎症细胞因子的表达水平显著升高。TLR4 沉默显著降低了 ISE 诱导的角膜上皮细胞中 IL12、TNF-α、CCL5 和 CCL9 的产生。此外，TLR4 沉默进一步降低了 ISE 处理的细胞中 NF-κB p65 和髓样分化蛋白 88（MyD88）的核转位。用 BAY11-7082 抑制 TLR4 介导的 NF-κB 信号也缓解了 ISE 诱导的炎症。在挽救实验中，用 TLR4 过表达慢病毒转染稳定沉默 TLR4 的角膜上皮细胞，我们发现 TLR4 过表达可以恢复 TLR4 敲低引起的 TLR4 和炎症细胞因子（IL12、TNF-α、CCL9）的下调。因此，ISE 诱导的角膜炎症是由于 TLR4/MyD88/NF-κB 信号通路的激活，并显著刺激了 IL12、TNF-α、CCL9 的分泌。TLR4 沉默减轻了 ISE 处理的角膜上皮细胞的损伤。
