Microglia-derived TNF-α mediates Muller cell activation by activating the TNFR1-NF-κB pathway
Microglia-derived TNF-α mediates Muller cell activation by activating the TNFR1-NF-κB pathway
复制标题
DOI：
10.1016/j.exer.2021.108852
复制
发表时间：
2021-11-27
影响因子：
3.4
通讯作者：
Guan, Huaijin
中科院分区：
文献类型：
--
作者：
Ji, Min;Sun, Qing;Guan, Huaijin
在视网膜神经退行性变过程中，小胶质细胞及其与Muller细胞的相互作用负责视网膜的监视，然而，小胶质细胞衍生的肿瘤坏死因子(TNF)- α在视网膜Muller细胞活化中的作用和机制尚未完全阐明。本研究从新生SD大鼠中分离出原代小胶质细胞和Muller细胞，纯度分别为88.2 +/- 6.2%和92.2 +/- 2.2%。通过免疫荧光和Western blot分析，我们发现TNF受体(TNFR)-1和TNFR2在Muller细胞中表达。与小胶质细胞条件培养基（MCM）共培养后，Muller细胞中胶质原纤维酸性蛋白（GFAP）、促炎因子（tnf - α、IL-1 β、CXCL-1、CSF-1、NOS2、COX2） mRNA水平升高，CNTF mRNA水平降低。然而，用R-7050（一种tnf - α受体抑制剂）或抗tnfr1进行预处理可显著消除这些变化。同时，抗tnfr2预处理能轻微抑制mcm培养Muller细胞中GFAP的表达。同时，抗tnfr1治疗逆转了tnf - α诱导的CSF-1和IL-1 β的表达升高。与对照组相比，tnf α处理的Muller细胞NF-kappa B P65、MAPK P38和ERK1/2的磷酸化水平显著升高。然而，抗tnfr1预处理抑制NF-kappa B P65和MAPK p38的磷酸化，尤其是NF-kappa B P65。此外，Bay117082 （NF-kappa B抑制剂）预处理也显著抑制NF-kappa B P65磷酸化和GFAP表达。此外，抗tnfr1和Bay117082处理可降低MCM诱导的Muller细胞NF-kappa B P65磷酸化。这些结果表明，小胶质细胞衍生的tnf - α在视网膜神经退行性变过程中调节Muller细胞活化中起着至关重要的作用。
Microglia and its interaction with Muller cells are responsible to retinal surveillance during retinal neuro-degeneration, however, the role and mechanism of microglia-derived tumor necrosis factor (TNF)-alpha in the activation of retinal Muller cells have not been fully elucidated. In the present study, primary microglia and Muller cells were isolated from newborn Sprague-Dawley (SD) rats with purities of 88.2 +/- 6.2% and 92.2 +/- 2.2%, respectively. By performing immunofluorescence and Western blot analysis, we found that TNF receptor (TNFR)-1 and TNFR2 were expressed in Muller cells. After co-cultured with microglia-conditioned medium (MCM), the elevated mRNA levels of glial fibrillary acidic protein (GFAP), proinflammatory factors (TNF-alpha, IL-1 beta, CXCL-1, CSF-1, NOS2, COX2) and decreased CNTF mRNA levels were found in Muller cells. However, pretreatment with R-7050 (a TNF-alpha receptor inhibitor) or anti-TNFR1 significantly abrogated the changes. Simultaneously, pretreatment with anti-TNFR2 slightly inhibited the expression of GFAP in MCM-incubated Muller cells. Meanwhile, anti-TNFR1 treatment reversed the increased expression of CSF-1 and IL-1 beta induced by TNF-alpha. Compared to the control groups, the phosphorylation of NF-kappa B P65, MAPK P38 and ERK1/2 in TNF-alpha-treated Muller cells was significantly increased. Nevertheless, pretreatment with anti-TNFR1 inhibited the phosphorylation of NF-kappa B P65 and MAPK p38, especially NF-kappa B P65. Additionally, pretreatment with Bay117082 (an NF-kappa B inhibitor) also significantly inhibited NF-kappa B P65 phosphorylation and GFAP expression. Moreover, anti-TNFR1 and Bay117082 treatment reduced NF-kappa B P65 phosphorylation of Muller cells induced by MCM. These results suggested that microglia-derived TNF-alpha served as a vital role in regulating Muller cells activation during retinal neurodegeneration.