Objectives: To explore the mechanism of Qihuang Jianpi Zishen Granules (QJZG) for improving thrombocytopenia in a mouse model of systemic lupus erythematosus (SLE).
Methods: Twenty-four MRL/lpr lupus mice were randomized equally into 4 groups for treatment with daily gavage of saline, QJZG or prednisone (Pred) or intraperitoneal injection (twice a week) of CaMKK2 activator, with 6 C57BL/6 mice with saline gavage as the control group. After 8 weeks of treatment, the mice were examined for PLT, PCT, PDW, MPV, serum levels of TPO, IL-6, IL-10, TNF-α and IFN-γ, and calcium ion fluorescence intensity using ELISA or flow-through assay. RT-qPCR was used to detect platelet CaMKK2, AMPK2α, mTOR, Beclin1 and p62 mRNA expression levels, and the protein expressions of CaMKK2, p-CaMKK2, AMPK, p-AMPK, mTOR, p-mTOR, LC3, Beclin1 and p62 were detected using Western blotting.
Results: The saline-treated MRL/lpr lupus mice showed significantly lowered levels of PLT, PCT, IL-10, mTOR, p62 mRNA, p-mTOR and P62 with increased PDW, MPV, serum TPO, IL-6, TNF-α and IFN-γ levels, and platelet expressions of CaMKK2, AMPK, Bcl-1 mRNA, p-CaMKK2, p-AMPK, LC3II and Beclin1. These abnormalities were significantly improved in QJZG group and Pred group but worsened after treatment with the CaMKK2 activator.
Conclusions: QJZG can ameliorate thrombocytopenia in mouse models of SLE by reducing inflammation and inhibiting platelet autophagy via regulating the Ca2+/CaMKK2/AMPK/mTOR signaling pathways.
目的: 基于Ca2+/CaMMK2/AMPK/mTOR信号通路介导的自噬探讨芪黄健脾滋肾颗粒（QJZG）对小鼠系统性红斑狼疮血小板减少的影响。方法: 将24只MRL/lpr狼疮小鼠随机分为模型组、QJZG组、醋酸泼尼松（Pred）组、CaMKK2激活剂组，6只/组；另将6只C57BL/6小鼠设为正常对照（Control）组。Control组、模型组：予10 mL/（kg·d）生理盐水灌胃；QJZG组：芪黄健脾滋肾颗粒+生理盐水配成0.39 g/mL溶液灌胃，剂量3.9 g/（kg·d）；Pred组：予小鼠醋酸泼尼松片加生理盐水配成0.273 mg/mL的溶液灌胃，剂量2.73 mg/（kg·d）；激活剂组：予10 mL/（kg·d）生理盐水灌胃，另将小鼠腹腔注射CaMKK2激活剂，5 mg/kg，2次/周。检测血小板计数（PLT）、血小板压积（PCT）、血小板分布宽度（PDW）、平均血小板体积（MPV）；ELISA法检测血清血小板生成素（TPO）、白介素-6（IL-6）、白介素-10（IL-10）、肿瘤坏死因子-α（TNF-α）、γ干扰素（IFN-γ）水平；流式细胞术检测钙离子荧光强度；Real-time PCR法检测血小板CaMKK2、AMPK2α、mTOR、Beclin1、p62 mRNA表达水平；Western blotting检测血小板CaMKK2、p-CaMKK2、AMPK、p-AMPK、mTOR、p-mTOR、LC3、Beclin1、P62蛋白表达水平。结果: 与Control组相比，模型组PLT、PCT、IL-10、mTOR、p62 mRNA、p-mTOR、P62水平降低（P<0.01），PDW、MPV、TPO、IL-6、TNF-α、IFN-γ、CaMKK2、AMPK 、Bcl-1 mRNA、p-CaMKK2、p-AMPK及血小板自噬蛋白（LC3Ⅱ蛋白、Beclin1蛋白）在血小板内表达量升高（P<0.01）。与模型组比较，QJZG组及Pred组PLT、IL-10、mTOR、p62 mRNA、p-mTOR、P62水平升高（P<0.01），MPV、TPO、IL-6、TNF-α、IFN-γ、CaMKK2、AMPK、Bcl-1 mRNA、p-CaMKK2、p-AMPK及血小板自噬蛋白（LC3Ⅱ蛋白、Beclin1蛋白）在血小板内表达量降低（P<0.05）；CaMKK2激活剂组PLT、PCT、IL-10、mTOR、p62 mRNA、p-mTOR、P62水平降低（P<0.01），PDW、MPV、IL-6、TNF-α、IFN-γ、CaMKK2、AMPK、Bcl-1 mRNA、p-CaMKK2、p-AMPK及血小板自噬蛋白（LC3Ⅱ蛋白、Beclin1蛋白）在血小板内表达量升高（P<0.01）。结论: QJZG可通过减轻炎症及影响Ca2+/CaMKK2/AMPK/mTOR信号通路抑制血小板自噬改善系统性红斑狼疮血小板减少。.
Keywords: Ca 2+/CaMKK2/AMPK/mTOR signaling pathway; Qihuang Jianpi Zishhen Granules; platelet autophagy; systemic lupus erythematosus.