The Ocular Surface Institute, University of Houston, College of Optometry, Houston, Texas, United States.
Invest Ophthalmol Vis Sci. 2018 Apr 1;59(5):1741-1750. doi: 10.1167/iovs.17-23363.
To determine high-mobility group box 1 (HMGB1) expression during experimental dry eye (EDE) and dry eye-like culture conditions and elucidate its role in corneal dry eye-related inflammation.
EDE was induced in 8- to 12-week-old C57BL/6 mice. Corneal tissue sections and lysates from EDE and untreated mice were evaluated for HMGB1 expression by immunostaining and quantitative real-time PCR (qPCR). For in vitro studies, human corneal epithelial cells (HCEC) were treated with hyperosmolar media, toll-like receptor (TLR) agonists, or proinflammatory cytokines to determine HMGB1 expression. HCEC were also treated with human recombinant HMGB1 (hrHMGB1) alone or in combination with inflammatory stimuli, and TNFα, IL-6, and IL-8 expression evaluated by qPCR and ELISA. Nuclear factor-κB (NF-κB) p65 nuclear translocation was determined by immunostaining.
EDE mice had higher corneal HMGB1 RNA and protein expression compared to untreated animals. In HCEC, hyperosmolar stress and TNFα treatment stimulated HMGB1 production and secretion into culture supernatants. However, in vitro stimulation with hrHMGB1 did not induce secretion of TNFα, IL-6, or IL-8 or NF-κB p65 nuclear translocation. In addition, the inflammatory response elicited by TLR agonists fibroblast-stimulating lipopeptide-1 and lipopolysaccharide was not enhanced by hrHMGB1 treatment.
HMGB1 expression was enhanced by dry eye conditions in vivo as well as in vitro, during hyperosmolar stress and cytokine exposure, suggesting an important role for HMGB1 in dry eye disease. However, no direct inflammatory effect was observed with HMGB1 treatment. Therefore, under these conditions, HMGB1 does not contribute directly to dry eye-induced inflammation and its function at the ocular surface needs to be explored further.
确定高迁移率族蛋白 B1（HMGB1）在实验性干眼（EDE）和类似干眼的培养条件下的表达，并阐明其在角膜干眼相关炎症中的作用。
在 8-12 周龄 C57BL/6 小鼠中诱导 EDE。通过免疫染色和实时定量 PCR（qPCR）评估 EDE 和未处理小鼠的角膜组织切片和裂解物中 HMGB1 的表达。对于体外研究，用高渗介质、 Toll 样受体（TLR）激动剂或促炎细胞因子处理人角膜上皮细胞（HCEC），以确定 HMGB1 的表达。还单独或联合炎症刺激物处理 HCEC 用人重组 HMGB1（hrHMGB1），通过 qPCR 和 ELISA 评估 TNFα、IL-6 和 IL-8 的表达。通过免疫染色测定核因子-κB（NF-κB）p65 核易位。
与未处理动物相比，EDE 小鼠的角膜 HMGB1 RNA 和蛋白表达更高。在 HCEC 中，高渗应激和 TNFα 处理刺激 HMGB1 的产生和分泌到培养上清液中。然而，体外用 hrHMGB1 刺激不会诱导 TNFα、IL-6 或 IL-8 的分泌或 NF-κB p65 的核易位。此外，TLR 激动剂纤维母细胞刺激脂肽-1 和脂多糖引起的炎症反应不会因 hrHMGB1 处理而增强。
HMGB1 的表达在体内和体外、高渗应激和细胞因子暴露时均增强，表明 HMGB1 在干眼病中起重要作用。然而，HMGB1 治疗没有观察到直接的炎症作用。因此，在这些条件下，HMGB1 不会直接导致干眼引起的炎症，其在眼表面的功能需要进一步探索。
