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

doi:10.12122/j.issn.1673-4254.2024.12.08

Abstract

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

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.

Figures/Tables 7

References 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^##

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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[1]	CHEN Junjie, HUANG Chuanbing, LI Ming. Jianpi Zishen granule inhibits podocyte autophagy in systemic lupus erythematosus: a network pharmacology and clinical study [J]. Journal of Southern Medical University, 2024, 44(3): 465-473.
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