L-苏糖酸镁增强恩替卡韦对HBV感染小鼠的抗病毒作用及其机制

目的：观察L-苏糖酸镁（MLT）是否可增强恩替卡韦（ETV）对乙型肝炎病毒（HBV）感染小鼠的抗病毒作用，并初步探讨其机制。方法：采用高压水动力法经尾静脉注射携带1.3拷贝HBV基因组（ayw亚型）的重组8型腺相关病毒建立HBV持续感染的C57BL/6小鼠模型。成模后分为模型组、MLT组（1.2 g·kg^-1 L-苏糖酸镁胶囊）、ETV组（75 μg·kg^-1恩替卡韦片）、联合处理组（1.2 g·kg^-1 L-苏糖酸镁胶囊+75 μg·kg^-1恩替卡韦片），每组8只；另选取8只未经病毒感染的正常小鼠作为对照组，连续给药4周。采用ELISA法检测小鼠血清HBsAg和HBeAg水平；采用荧光定量PCR法检测小鼠肝脏组织及血清中HBV DNA拷贝数；分离小鼠外周血CD8+T细胞，检测小鼠血清及CD8+T细胞中Mg²⁺含量，并采用流式细胞仪检测小鼠外周血CD8+T细胞中表面分子程序性死亡受体1（PD-1）、自然杀伤细胞活化受体（NKG2D）、CD95和CD38表达水平。结果：与对照组比较，模型组小鼠血清和CD8+T细胞中Mg²⁺含量及CD8+T细胞中表面活化性分子NKG2D和CD38表达水平明显降低（P<0.05），而抑制性分子PD-1和CD95表达水平明显升高（P<0.05）；与模型组比较，各给药组小鼠血清HBsAg、HBeAg水平及肝脏组织和血清中HBV DNA拷贝数均明显降低（P<0.05），且联合处理组小鼠各指标改善效果最好；与模型组比较，MLT组和联合处理组小鼠血清及CD8+T细胞中Mg²⁺含量及CD8+T细胞表面活化性分子NKG2D和CD38表达水平明显升高（P<0.05），而抑制性分子PD-1和CD95表达水平明显降低（P<0.05），但ETV组小鼠各指标差异无统计学意义（P>0.05）。结论：MLT可增强ETV对HBV感染小鼠的抗病毒作用，其机制可能是MLT可引起CD8+T细胞内Mg²⁺含量增加，提高CD8+T细胞活性，进而更加有效地清除HBV。

Enhancement effect of magnesium-L-threonate for antiviral effect of entecavir in HBV-infected mice and its mechanism

Objective: To observe whether magnesium-L-threonate (MLT) can enhance the antiviral effect of entecavir (ETV) in the HBV-infected mice, and to explore its mechanism. Methods: The recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome was injected into the C57BL/6 mice by tail vein to establish the HBV persistent infection mouse models. After successful modeling, the mice were divided into model group, MLT group (1.2 g·kg^-1 magnesium-L-threonate capsules), ETV group (75 μg·kg^-1 entecavir tablets), and combined treatment group (1.2 g·kg^-1 magnesium-L-threonate caspules+75 μg·kg^-1 entecavir tablets) (n=8), and another 8 normal mice uninfected with HBV were selected as control group. Then all the mice were intragastrically administered for 4 weeks. The serum levels of HBsAg and HBeAg were detected by ELISA method, the copied of HBV DNA in liver tissue and serum of the mice were detected by quantitative real-time PCR. The peripheral blood CD8+ T cells were isolated, and the contents of Mg²⁺ in serum and CD8+ T cells were detected. The expression levels of PD-1, NKG2D, CD95 and CD38 in the CD8+ T cells were detected by flow cytometry. Results: Compared with control group, the contents of Mg²⁺ in serum and CD8+ T cells and the expression levels of surface activation molecules NKG2D and CD38 in the CD8+ T cells in model group were significantly decreased (P<0.05), while the expression levels of surface inhibitory molecules PD-1 and CD95 in the CD8+ T cells were significantly increased (P<0.05). Compared with model group, the expression levels of serum HBsAg and HBeAg, and the copied of HBV DNA in liver tissue and serum of the mice in various administration groups were significantly decreased (P<0.05), and the improvement degree of the indexes in combined treatment group was superior to other administration groups. Compared with model group, the contents of Mg²⁺ and the expression levels of NKG2D and CD38 in MLT group and combined treatment group were significantly increased (P<0.05), the expression levels of PD-1 and CD95 were significantly decreased (P<0.05), but there were no significant differences in ETV group (P>0.05). Conclusion: MLT can enhance the antiviral effect of ETV in the HBV-infected mice, and the mechanism may be that MLT can cause the increase of Mg²⁺ contents in the CD8+ T cells to improve the activity of CD8+ T cells, and thereby HBV can be removed more effectively.