紫草素通过促进巨噬细胞Ⅰ型干扰素的产生抑制病毒感染

Shikonin inhibits virus infection by promoting production of type Ⅰ interferon in macrophages

目的:探讨紫草素对病毒感染的影响及其作用机制。方法:C57BL/6J小鼠用水疱性口炎病毒(VSV)感染后,不同浓度紫草素通过腹腔注射处理小鼠,观察小鼠的生存率以及肝、脾和肺组织中的VSV病毒滴度和VSV-G病毒基因的转录水平变化;H&E组化实验检测肺部组织的损伤和炎症细胞的浸润。RT-PCR检测小鼠肝脏、脾脏及肺中IFN-β和IL-6的mRNA表达。在细胞实验中,用VSV病毒感染经不同浓度紫草素预处理的原代巨噬细胞,利用RT-PCR及ELISA实验方法检测IFN-α和IFN-β的mRNA水平及蛋白水平。Western blot检测p38、ERK、JNK、p65、TBK1、IRF3蛋白磷酸化水平。结果:紫草素处理能够显著提高VSV病毒感染小鼠的生存率;H&E组化实验结果表明,VSV病毒感染小鼠在紫草素处理后其肺组织结构的损伤和炎症细胞的浸润与对照组相比明显减少;VSV病毒感染小鼠在紫草素处理后其肝、脾和肺组织中的VSV滴度和VSV-G mRNA与对照组相比明显降低,而IFN-β的mRNA水平显著升高;在细胞实验中,紫草素能够显著提高巨噬细胞抵抗病毒感染的能力,抑制病毒基因VSV-G mRNA的表达。紫草素能够促进病毒感染的巨噬细胞诱导IFN-α和IFN-β的产生,然而TNF-α没有明显差异。此外,利用Ⅰ型干扰素受体的阻断抗体(IFNAR1 Ab)来阻断Ⅰ型干扰素的信号通路,发现紫草素并不能抑制VSV病毒的扩增和复制。Western blot实验发现在病毒感染过程中,紫草素能够显著促进巨噬细胞IRF3的磷酸化。结论:紫草素通过促进IRF3激活来上调Ⅰ型干扰素的产生从而抑制病毒感染。

Objective:To investigate the effect of shikonin on virus infection and its mechanism.Methods:C57 BL/6 J mice were infected with VSV(vesicular stomatitis virus),and then treated with different concentrations of shikonin through intraperitoneal injection. The survival rate of the mice and the VSV titer in the liver,spleen and lung were detected. H&E histochemical analysis was used to detect the damage of mouse lung tissue and the infiltration of inflammatory cells. RT-PCR was used to detect the mouse IFN-βand IL-6 mRNA expression in the liver,spleen and lung. In vitro experiments,VSV was used to infect bone marrow derived macrophages pretreated with different concentrations of shikonin,and then RT-PCR and ELISA were used to detect IFN-α(interferon-α)and IFN-β mRNA and protein levels. p38,ERK,JNK,p65,TBK1,IRF3 protein phosphorylation levels was detected by Western blot.Results:Shikonin could significantly increase the survival rate of mice infected with VSV;H&E histochemistry showed that the damage of lung tissue structure and the infiltration of inflammatory cells were significantly reduced in shikonin treated mice after VSV infection.VSV titers and VSV-G mRNA were decreased in the liver,spleen and lung of mice after treatment with shikonin. Furthermore,the level of IFN-β was significantly increased. In addition,shikonin could significantly improve the ability of macrophages to resist viral infection and inhibit the expression of VSV-G mRNA. Shikonin could promote the production of IFN-α and IFN-β induced by viral infection,but there was no significant difference in TNF-α. Furthermore,the type Ⅰ interferon receptor blocking antibody(IFNAR1 Ab)was used to block the type Ⅰ interferon signal pathway,and found that shikonin could not inhibit the replication of the VSV virus. Shikonin could significantly promote the phosphorylation of IRF3 in macrophages after viral infection.Conclusion:Shikonin can promote the production of typeⅠinterferon and inhibit virus infection by up-regulating the activation of IRF3.