Department of Ophthalmology, Fifth Affiliated Hospital, Sun Yat-sen University , Zhuhai, PR China.
Department of Pathology, Fifth Affiliated Hospital, Sun Yat-sen University , Zhuhai, PR China.
Curr Eye Res. 2020 Sep;45(9):1043-1050. doi: 10.1080/02713683.2020.1716986. Epub 2020 Feb 26.
PURPOSE/AIM: Corneal stromal fibroblasts are connected to each other via gap junctions, which contribute to maintenance of corneal homeostasis. Viral infection of the corneal stroma can result in inflammation and scarring. The effects of polyinosinic-polycytidylic acid [poly(I:C)], an analog of viral double-stranded RNA, on gap junctional intercellular communication (GJIC) in cultured human corneal fibroblasts (HCFs) were examined.
Cultured HCFs were exposed to poly(I:C) in the absence or presence of inhibitors of mitogen-activated protein kinase (MAPK) signaling or the antioxidant -acetyl-L-cysteine (NAC). Expression of the gap junction protein connexin 43 (Cx43) was examined by immunoblot and immunofluorescence analyses. The level of Cx43 mRNA or microRNA-21 or -130a was determined by quantitative reverse transcription-polymerase chain reaction analysis. GJIC was measured with a dye coupling assay. The amount of malondialdehyde and the activity of superoxide dismutase (SOD) were measured with assay kits.
Exposure of HCFs to poly(I:C) resulted in down-regulation of Cx43 expression and GJIC activity as well as in up-regulation of microRNA-21 expression. Poly(I:C) increased the amount of malondialdehyde and reduced the activity of SOD in the cells, and these effects were prevented by NAC. The inhibitory effects of poly(I:C) on both Cx43 expression and GJIC activity were attenuated by NAC and by c-Jun NH-terminal kinase (JNK) inhibitor II.
Poly(I:C) inhibited Cx43 expression and GJIC in cultured HCFs, possibly as a result of the associated up-regulation of microRNA-21. Poly(I:C) also increased oxidative stress in these cells, and such stress together with signaling by the MAPK JNK was implicated in the effects of poly(I:C) on Cx43 expression and GJIC activity. Down-regulation of GJIC activity among corneal fibroblasts by double-stranded RNA may thus contribute to the disruption of stromal homeostasis during viral infection of the cornea.
角膜基质成纤维细胞通过缝隙连接相互连接，有助于维持角膜内稳态。角膜基质的病毒感染可导致炎症和瘢痕形成。本文研究了聚肌苷酸-聚胞苷酸（poly(I:C)），一种病毒双链 RNA 的类似物，对培养的人角膜成纤维细胞（HCFs）缝隙连接细胞间通讯（GJIC）的影响。
在不存在或存在丝裂原活化蛋白激酶（MAPK）信号转导抑制剂或抗氧化剂 -N-乙酰-L-半胱氨酸（NAC）的情况下，将培养的 HCFs 暴露于 poly(I:C)中。通过免疫印迹和免疫荧光分析检查缝隙连接蛋白连接蛋白 43（Cx43）的表达。通过定量逆转录聚合酶链反应分析测定 Cx43 mRNA 或 microRNA-21 或 -130a 的水平。通过染料偶联测定法测量 GJIC。用测定试剂盒测定丙二醛的量和超氧化物歧化酶（SOD）的活性。
poly(I:C) 处理 HCFs 导致 Cx43 表达和 GJIC 活性下调，以及 microRNA-21 表达上调。poly(I:C)增加了细胞内丙二醛的含量并降低了 SOD 的活性，而 NAC 可预防这些作用。NAC 和 c-Jun NH2 末端激酶（JNK）抑制剂 II 可减弱 poly(I:C)对 Cx43 表达和 GJIC 活性的抑制作用。
poly(I:C) 抑制培养的 HCFs 中的 Cx43 表达和 GJIC，可能是由于相关的 microRNA-21 上调。poly(I:C)还增加了这些细胞中的氧化应激，而这种应激以及 MAPK JNK 的信号转导与 poly(I:C)对 Cx43 表达和 GJIC 活性的影响有关。角膜基质成纤维细胞中 GJIC 活性的下调可能导致病毒感染角膜时基质内稳态的破坏。
