线粒体 DNA 泄漏通过 cGAS-STING 通路诱导成牙本质细胞炎症,Cell Communication and Signaling

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线粒体 DNA 泄漏通过 cGAS-STING 通路诱导成牙本质细胞炎症
Cell Communication and Signaling ( IF 8.2 ) Pub Date : 2021-05-20 , DOI: 10.1186/s12964-021-00738-7
Lu Zhou ₁ , Yi-Fei Zhang ₁ , Fu-Hua Yang ₁ , Han-Qing Mao ₁ , Zhi Chen _{1,

2} , Lu Zhang _{1,

2}

Affiliation

当线粒体 DNA (mtDNA) 从受损的线粒体泄漏到细胞质中时，线粒体 DNA (mtDNA) 是炎症的重要驱动因素。mtDNA 应激可能有助于传染病中干扰素基因 (STING) 通路激活的环 GMP-AMP 合酶 (cGAS) 刺激物。成牙本质细胞是第一个受到致龋细菌攻击的细胞，参与维持牙髓对侵入牙本质的病原体的免疫和炎症反应。在这项研究中，我们研究了 mtDNA 作为一种重要的炎症驱动因子，通过 cGAS-STING 通路在成牙本质细胞中参与防御细菌入侵。通过蛋白质印迹和免疫组织化学染色测量正常组织、龋齿组织和牙髓组织。在体外建立牙髓炎模型，以评估 cGAS-STING 通路在成牙本质细胞样细胞系 (mDPC6T) 在炎症下的作用。进行蛋白质印迹和实时荧光定量PCR检测cGAS-STING通路和促炎细胞因子的表达。线粒体功能使用 MitoSOX Red 染料染色评估线粒体产生的活性氧 (ROS)。在 LPS 刺激后，通过免疫荧光染色和实时 PCR 在 mDPC6T 细胞中评估细胞溶质 DNA。此外，mDPC6T 细胞用溴化乙锭 (EtBr) 处理以消耗 mtDNA 或用分离的 mtDNA 转染。测量cGAS-STING通路和促炎细胞因子的表达。cGAS和STING在患者龋齿和牙髓炎组织中的高表达，与炎症进展有关。cGAS-STING 通路在发炎的 mDPC6T 中被激活。STING 敲低抑制了 p65 和 IRF3 的核输入，并限制了 LPS 诱导的炎性细胞因子 CXCL10 和 IL-6 的分泌。LPS 在 mDPC6T 中引起线粒体损伤，从而促进 mtDNA 泄漏到细胞质中。mtDNA 的消耗抑制了 cGAS-STING 通路和 p65 和 IRF3 的核转位。此外，mtDNA 的补充挽救了被 STING 敲低抑制的炎症反应。我们的研究系统地确定了 LPS 诱导的成牙本质细胞炎症的新机制，其涉及 mtDNA 从受损的线粒体泄漏到细胞质中，刺激 cGAS-STING 通路和炎性细胞因子 IL-6 和 CXCL10 的分泌。

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Mitochondrial DNA leakage induces odontoblast inflammation via the cGAS-STING pathway

Mitochondrial DNA (mtDNA) is a vital driver of inflammation when it leaks from damaged mitochondria into the cytosol. mtDNA stress may contribute to cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) pathway activation in infectious diseases. Odontoblasts are the first cells challenged by cariogenic bacteria and involved in maintenance of the pulp immune and inflammatory responses to dentine-invading pathogens. In this study, we investigated that mtDNA as an important inflammatory driver participated in defending against bacterial invasion via cGAS-STING pathway in odontoblasts. The normal tissues, caries tissues and pulpitis tissues were measured by western blotting and immunohistochemical staining. Pulpitis model was built in vitro to evaluated the effect of the cGAS-STING pathway in odontoblast-like cell line (mDPC6T) under inflammation. Western blot and real-time PCR were performed to detect the expression of cGAS-STING pathway and pro-inflammatory cytokines. The mitochondrial function was evaluated reactive oxygen species (ROS) generated by mitochondria using MitoSOX Red dye staining. Cytosolic DNA was assessed by immunofluorescent staining and real-time PCR in mDPC6T cells after LPS stimulation. Furthermore, mDPC6T cells were treated with ethidium bromide (EtBr) to deplete mtDNA or transfected with isolated mtDNA. The expression of cGAS-STING pathway and pro-inflammatory cytokines were measured. The high expression of cGAS and STING in caries and pulpitis tissues in patients, which was associated with inflammatory progression. The cGAS-STING pathway was activated in inflamed mDPC6T. STING knockdown inhibited the nuclear import of p65 and IRF3 and restricted the secretion of the inflammatory cytokines CXCL10 and IL-6 induced by LPS. LPS caused mitochondrial damage in mDPC6T, which promoted mtDNA leakage into the cytosol. Depletion of mtDNA inhibited the cGAS-STING pathway and nuclear translocation of p65 and IRF3. Moreover, repletion of mtDNA rescued the inflammatory response, which was inhibited by STING knockdown. Our study systematically identified a novel mechanism of LPS-induced odontoblast inflammation, which involved mtDNA leakage from damaged mitochondria into the cytosol stimulating the cGAS-STING pathway and the inflammatory cytokines IL-6 and CXCL10 secretion. The mtDNA-cGAS-STING axis could be a potent therapeutic target to prevent severe bacterial inflammation in pulpitis.

更新日期：2021-05-20

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