芪黄健脾滋肾颗粒可改善小鼠系统性红斑狼疮血小板减少：基于Ca2+/CaMKK2/AMPK/mTOR信号通路介导的自噬

doi:10.12122/j.issn.1673-4254.2024.12.08

摘要/Abstract

摘要：

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。

关键词: 芪黄健脾滋肾颗粒, 系统性红斑狼疮, 血小板自噬, Ca²⁺/CaMKK2/AMPK/mTOR信号通路

Abstract:

Objective 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. Conclusion 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.

Key words: Qihuang Jianpi Zishhen Granules, systemic lupus erythematosus, platelet autophagy, Ca²⁺/CaMKK2/AMPK/mTOR signaling pathway

李云飞, 庞利君, 束龙武, 李明, 黄传兵. 芪黄健脾滋肾颗粒可改善小鼠系统性红斑狼疮血小板减少：基于Ca²⁺/CaMKK2/AMPK/mTOR信号通路介导的自噬[J]. 南方医科大学学报, 2024, 44(12): 2327-2334.

Yunfei LI, Lijun PANG, Longwu SHU, Ming LI, Chuanbing HUANG. Qihuang Jianpi Zishen Granules improves thrombocytopenia in mice with systemic lupus erythematosus by suppressing platelet autophagy via the Ca²⁺/CaMKK2/AMPK/mTOR signaling pathway[J]. Journal of Southern Medical University, 2024, 44(12): 2327-2334.

图/表 7

参考文献 41

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Gene	Amplicon size (bp)	Forward primer (5'→3')	Reverse primer (5'→3')
β-actin	120	AGTGTGACGTTGACATCCGT	TGCTAGGAGCCAGAGCAGTA
CaMKK2	103	GAAATTGGAAAGGGCTCCTA	GTCGGATCAGCTTCTTTTTG
AMPK2α	92	AGTGAAGACTACCAGGTGAT	TTGCAGATGTAGTCGAACAA
mTOR	156	ATTGACTTTGGGGACTGCTT	GAGCACTTCCATCACGGT
Beclin1	119	GCTGGAAGATGTGGAAAAGA	CTCACTATACTCCCGCTGGTAC
p62	88	AGGGCCTGCACAGGGAACAC	CGAAGCCAGCGGCTATGAGA

Gene	Amplicon size (bp)	Forward primer (5'→3')	Reverse primer (5'→3')
β-actin	120	AGTGTGACGTTGACATCCGT	TGCTAGGAGCCAGAGCAGTA
CaMKK2	103	GAAATTGGAAAGGGCTCCTA	GTCGGATCAGCTTCTTTTTG
AMPK2α	92	AGTGAAGACTACCAGGTGAT	TTGCAGATGTAGTCGAACAA
mTOR	156	ATTGACTTTGGGGACTGCTT	GAGCACTTCCATCACGGT
Beclin1	119	GCTGGAAGATGTGGAAAAGA	CTCACTATACTCCCGCTGGTAC
p62	88	AGGGCCTGCACAGGGAACAC	CGAAGCCAGCGGCTATGAGA

Group	PLT (×10⁹/L)	PCT (%)	PDW (%)	MPV (fl)
Control	924.50±82.64	0.80±0.08	10.56±0.72	32.44±2.36
Model	348.00±72.63**	0.32±0.08**	14.47±1.11**	58.72±3.81**
QJZG	559.50±40.62^##	0.38±0.10	12.42±0.90^##	44.43±2.20^##
Pred	536.00±35.05^##	0.33±0.03	14.28±0.68	53.42±1.65^#
CaMKK2	154.70±12.82^##	0.07±0.02^##	17.77±0.97^##	77.72±3.39^##

Group	PLT (×10⁹/L)	PCT (%)	PDW (%)	MPV (fl)
Control	924.50±82.64	0.80±0.08	10.56±0.72	32.44±2.36
Model	348.00±72.63**	0.32±0.08**	14.47±1.11**	58.72±3.81**
QJZG	559.50±40.62^##	0.38±0.10	12.42±0.90^##	44.43±2.20^##
Pred	536.00±35.05^##	0.33±0.03	14.28±0.68	53.42±1.65^#
CaMKK2	154.70±12.82^##	0.07±0.02^##	17.77±0.97^##	77.72±3.39^##

Group	TPO (ng/mL)	IL-6 (pg/mL)	IL-10 (pg/mL)	TNF-α (pg/mL)	IFN-γ (pg/mL)
Control	103.22±8.29	131.08±3.08	210.13±3.26	517.24±22.86	70.88±6.56
Model	290.31±28.49**	292.31±5.93**	89.01±10.03**	953.83±28.68**	217.11±13.05**
QJZG	164.72±6.79^##	166.81±3.82^##	122.02±3.89^##	715.61±10.02^##	121.76±10.39^##
Pred	177.27±3.16^##	183.88±2.44^##	101.61±2.42^##	799.03±25.77^##	134.02±8.75^##
CaMKK2	310.50±17.73	312.01±18.01^##	44.12±4.39^##	1643.13±17.71^##	301.13±9.09^##

芪黄健脾滋肾颗粒可改善小鼠系统性红斑狼疮血小板减少：基于Ca²⁺/CaMKK2/AMPK/mTOR信号通路介导的自噬

Qihuang Jianpi Zishen Granules improves thrombocytopenia in mice with systemic lupus erythematosus by suppressing platelet autophagy via the Ca²⁺/CaMKK2/AMPK/mTOR signaling pathway

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