HMGB1-TLR4和Müller细胞在视网膜血管生成中的作用

Mechanism effect of HMGB1-TLR4 and Müller cells in retinal angiogenesis

目的探讨高迁移率族蛋白1-Toll样受体4(HMGB1-TLR4)信号通路及Müller细胞在视网膜新生血管中的作用。方法将小鼠分为3组,分别是常氧组、正常视网膜缺血模型组(OIR组)和TLR4基因敲除OIR(TLR4^(-/-)OIR)组。通过视网膜FITC-Dextran荧光灌注铺片染色和GS-isolectin B4染色评估新生血管情况。通过免疫荧光染色,观察HMGB1在小鼠视网膜中的表达情况,观察HMGB1和TLR4、TLR4和GFAP的共表达情况。通过PCR检测两组OIR小鼠出生后12天(P12)和出生后17天(P17)时视网膜组织HMGB1 mRNA表达水平。结果通过两种视网膜血管平铺片染色方法观察到P17正常OIR小鼠新生血管面积最大,TLR4^(-/-)OIR小鼠的新生血管面积相对较少,这提示TLR4基因缺失可以抑制血管的生成。通过免疫荧光染色结果发现在TLR4^(-/-)OIR小鼠中,HMGB1、TLR4和GFAP的表达均较OIR组减少。PCR检查结果发现P12和P17时,TLR4^(-/-)OIR小鼠中的HMGB1的表达水平均较正常OIR组低。结论HMGB1-TLR4可以通过活化Müller细胞促进视网膜新生血管的生成,因此靶向抑制HMGB1-TLR4信号通路,可以降低Müller细胞的活性,抑制视网膜新生血管的生成。

Objective:To explore the role of HMGB1-TLR4 signal pathway and Müller cells in retinal neovascularization.Methods:The mice were divided into three groups:normoxic group,normal OIR model group and TLR4 gene knockout(TLR4^(-/-))OIR model group.Retinal FITC-Dextran fluorescence perfusion and GS-isolectin B4 staining were used to evaluate the neovascularization.Immunofluorescence staining was used to observe the expression of HMGB1 in mouse retina,the co-expressions of HMGB1 and TLR4,and the co-expressions of TLR4 and GFAP.The mRNA expressions of HMGB1 in the retina of OIR mice in P12 and P17 groups were detected by PCR.Results:It was observed that the neovascularization area of P17 normal OIR mice was the largest,while that of TLR4^(-/-)OIR mice was relatively small,which suggested that TLR4 gene deletion could inhibit angiogenesis.The results of immunofluorescence staining showed that the expressions of HMGB1,TLR4 and GFAP in TLR4^(-/-)OIR mice were lower than those in normal OIR group.The results of PCR examination showed that the expression levels of HMGB1 in TLR4^(-/-)OIR mice at P12 and P17 were lower than those in normal OIR group.These results suggested that inhibition of HMGB1-TLR4 signal pathway could weaken the activation of Müller cells and reduce neovascularization.Conclusion:This study explores the mechanism of HMGB1-TLR4 activating Müller cells participating in neovascularization,and reveals the effect of HMGB1-TLR4 on retinal neovascularization by activating Müller cells.It is suggested that blocking HMGB1-TLR4 pathway can weaken the activity of Müller cells and provide a new method for the treatment of retinal neovascularization.