Somayajulu Mallika, McClellan Sharon A, Farooq Shukkur Muhammed, Pitchaikannu Ahalya, Xu Shunbin, Hazlett Linda
Department of Ophthalmology, Visual and Anatomical Sciences, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, USA.
Pathogens. 2022 Nov 11;11(11):1327. doi: 10.3390/pathogens11111327.
This study tests the mechanism(s) of glycyrrhizin (GLY) protection against keratitis. Female C57BL/6 (B6), TLR4 knockout (TLR4KO), myeloid specific TLR4KO (mTLR4KO), their wildtype (WT) littermates, and TLR9 knockout (TLR9KO) mice were infected with KEI 1025 and treated with GLY or PBS onto the cornea after infection. Clinical scores, photography with a slit lamp, RT-PCR and ELISA were used. GLY effects on macrophages (Mϕ) and polymorphonuclear neutrophils (PMN) isolated from WT and mTLR4KO and challenged with KEI 1025 were also tested. Comparing B6 and TLR4KO, GLY treatment reduced clinical scores and improved disease outcome after infection and decreased mRNA expression levels in cornea for TLR4, HMGB1, and RAGE in B6 mice. TLR9 mRNA expression was significantly reduced by GLY in both mouse strains after infection. GLY also significantly reduced HMGB1 (B6 only) and TLR9 protein (both B6 and TLR4KO). In TLR9KO mice, GLY did not significantly reduce clinical scores and only slightly improved disease outcome after infection. In these mice, GLY significantly reduced TLR4, but not HMGB1 or RAGE mRNA expression levels after infection. In contrast, in the mTLR4KO and their WT littermates, GLY significantly reduced corneal disease, TLR4, TLR9, HMGB1, and RAGE corneal mRNA expression after infection. GLY also significantly reduced TLR9 and HMGB1 corneal protein levels in both WT and mTLR4KO mice. In vitro, GLY significantly lowered mRNA expression levels for TLR9 in both Mϕ and PMN isolated from mTLR4KO or WT mice after incubation with KEI 1025. In conclusion, we provide evidence to show that GLY mediates its effects by blocking TLR4 and TLR9 signaling pathways and both are required to protect against disease.
本研究测试了甘草酸（GLY）对角膜炎的保护机制。将雌性C57BL/6（B6）小鼠、Toll样受体4基因敲除（TLR4KO）小鼠、髓系特异性Toll样受体4基因敲除（mTLR4KO）小鼠、它们的野生型（WT）同窝小鼠以及Toll样受体9基因敲除（TLR9KO）小鼠感染KEI 1025，并在感染后将GLY或磷酸盐缓冲液（PBS）滴于角膜上。采用临床评分、裂隙灯摄影、逆转录聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）等方法。还测试了GLY对从WT和mTLR4KO小鼠分离出的巨噬细胞（Mϕ）和多形核中性粒细胞（PMN）的影响，这些细胞在受到KEI 1025攻击后进行了相关检测。比较B6和TLR4KO小鼠，GLY治疗可降低感染后的临床评分，改善疾病结局，并降低B6小鼠角膜中TLR4、高迁移率族蛋白B1（HMGB1）和晚期糖基化终末产物受体（RAGE）的mRNA表达水平。感染后，GLY可显著降低两种小鼠品系中TLR9的mRNA表达。GLY还可显著降低HMGB1（仅B6小鼠）和TLR9蛋白（B6和TLR4KO小鼠）的表达。在TLR9KO小鼠中，GLY未显著降低临床评分，仅轻微改善感染后的疾病结局。在这些小鼠中，GLY可显著降低感染后角膜中TLR4的表达，但对HMGB1或RAGE的mRNA表达水平无显著影响。相反，在mTLR4KO小鼠及其WT同窝小鼠中，GLY可显著减轻感染后的角膜疾病，降低角膜中TLR4、TLR9、HMGB1和RAGE的mRNA表达。GLY还可显著降低WT和mTLR4KO小鼠角膜中TLR9和HMGB1的蛋白水平。在体外，与KEI 1025孵育后，GLY可显著降低从mTLR4KO或WT小鼠分离出的Mϕ和PMN中TLR9的mRNA表达水平。总之，我们提供的证据表明，GLY通过阻断TLR4和TLR9信号通路发挥作用，二者对于预防疾病均不可或缺。
