Qingjie Fuzheng Granules alleviates 5-fluorouracil-induced skeletal muscle injury in tumor-bearing mice by inhibiting mitochondria-dependent apoptosis and activating the AMPK-PGC-1α pathway

doi:10.12122/j.issn.1673-4254.2026.01.10

Abstract

Abstract:

Objective To explore the therapeutic mechanism of Qingjie Fuzheng granules (QFG) for alleviating 5-fluorouracil (5-FU)-induced skeletal muscle atrophy. Methods Male BALB/c mice bearing subcutaneous colorectal cancer CT26 cell xenografts were randomized into control group, model group, and treatment group. The mice in model and treatment groups were given intraperitoneal 5-FU injections every 3 days and treated with daily gavage of saline and QFG for 21 days, respectively; the mice in the control group and normally fed mice were given only saline gavage. Gripping test and hanging test of the mice were performed before and after the treatment, and on day 21, tumor weight and gastrocnemius muscle weight were measured, and histopathology and cell apoptosis in the gastrocnemius muscle were examined with HE staining, transmission electron microscopy and TUNEL assay. ATP content in the muscle was measured, and protein expressions of AMPK, PGC-1α, Cyt c, AIF, Apaf-1, Smac, Bcl-2, Bax, cleaved caspase-3 and cleaved caspase-9 were determined with immunohistochemistry. Results The tumor-bearing mice in the control group showed significantly decreased gastrocnemius muscle weight and grip and suspension test scores. The gastrocnemius muscle showed ultrastructure injuries with lowered ATP content, obvious cell apoptosis, decreased expressions of AMPK, PGC-1 α, and Bcl-2, and increased expressions of Bax, Cyto C, AIF, Apaf-1, Smac, cleaved caspase-3 and cleaved caspase-9. These changes were obviously worsened in 5-FU-treated mice, while QFG treatment significantly increased gastrocnemius muscle weight and strength, ameliorated its ultrastructural injuries, reduced cell apoptosis, and reversed the abnormal protein expressions. Conclusion QFG alleviates 5-FU-induced skeletal muscle fatigue in tumor-bearing mice by activating the AMPK/PGC-1α pathway and inhibiting mitochondria-dependent apoptosis in the gastrocnemius muscle.

Key words: Qingjiefuzheng granule, AMP-activated protein kinase, peroxisome proliferator-activated receptor γ coactivator-1α, mitochondrial biogenesis, skeletal muscle apoptosis

Jinyan ZHAO, Jiao PENG, Minghe LIN, Xiaoqin ZHU, Bin HUANG, Jiumao LIN. Qingjie Fuzheng Granules alleviates 5-fluorouracil-induced skeletal muscle injury in tumor-bearing mice by inhibiting mitochondria-dependent apoptosis and activating the AMPK-PGC-1α pathway[J]. Journal of Southern Medical University, 2026, 46(1): 94-103.

Figures/Tables 15

Tab.1 Comparison of tumor weight and gastrocnemius muscle weight in each group (g, Mean±SD, n=4)

Group	Tumor weight	Gastrocnemius weight
Normal	-	0.31±0.02
Control	10.93±1.92	0.25±0.01**
Model	2.83±0.40^##	0.21±0.01^##
Treatment	2.74±0.57	0.25±0.01^△
F	63.88	25.57
P	<0.001	<0.001

Tab.2 Comparison of grip and suspension test scores in each group (Mean±SD, n=4)

Group	Grip	Suspension score
Normal	76.37±1.48	3
Control	63.45±1.66**	2.33±0.47*
Model	54.75±1.93^##	1.85±0.33^#
Treatment	69.12±2.96^△△	2.67±0.27^△△
F	76.63	9.74
P	<0.001	<0.01

Fig.1 HE staining of the gastrocnemius muscle of the mice in each group. A: Normal group. B: Control group. C: Model group. D: Treatment group.

Fig.2 Transmission electron microscopy of the gastrocnemius muscle in each group (Original magnification: ×20 000). A: Normal group. B: Control group. C: Model group. D: Treatment group. Yellow arrows indicate the mitochondria.

Tab.3 Comparison of ATP content in the gastrocnemius muscle in each group (μmol/g, Mean±SD, n=4)

Group	Content of ATP
Normal	3.31±0.25
Control	2.06±0.11**
Model	0.89±0.12^##
Treatment	1.44±0.17^△△
F	113.85
P	<0.001

Fig.3 TUNEL staining of the gastrocnemius muscle in each group (×400). A: Normal group. B: Control group. C: Model group. D: Treatment group.

Tab.4 Comparison of cell apoptosis rate in the gastrocnemius muscle in each group (%, Mean±SD, n=3)

Group	Apoptosis rate
Normal	3.66±0.37
Control	14.13±0.60**
Model	36.39±0.53^##
Treatment	13.61±1.06^△△
F	1214.94
P	<0.001

Fig.4 Immunohistochemistry for detecting AMPK and PGC-1α protein expressions in the gastrocnemius muscle in each group (×400). A: Normal group. B: Control group. C: Model group. D: Treatment group.

Tab.5 Protein expression levels of AMPK and PGC-1α in the gastrocnemius muscle in each group (%, Mean±SD, n=3)

Group	AMPK	PGC-1α
Normal	56.46±0.62	57.90±1.40
Control	42.48±1.88**	25.67±1.01**
Model	16.27±1.07^##	14.29±0.65^##
Treatment	21.8±1.17^△△	20.67±2.00^△△
F	648.10	406.17
P	<0.001	<0.01

Fig.5 Immunohistochemistry for detecting Bax, Bcl-2 and Cyt c protein expressions in the gastrocnemius muscle in each group (×400). A: Normal group. B: Control group. C: Model group. D: Treatment group.

Tab.6 Protein expression levels of Bax, Bcl-2 and Cyt c in the gastrocnemius muscle in each group (%, Mean±SD, n=3)

Group	Bax	Bcl-2	Cyt c
Normal	29.42±0.79	71.08±1.53	20.41±0.53
Control	51.03±1.74**	55.40±0.92**	43.32±1.55**
Model	80.67±1.36^##	37.53±1.41^##	69.45±1.13^##
Treatment	33.38±1.0^△△	57.27±1.07^△△	38.02±0.96^△△
F	661.12	293.84	674.29
P	<0.001	<0.001	<0.001

Fig.6 Immunohistochemistry for detecting AIF, Smac and Apaf1 protein expressions in the gastrocnemius muscle in each group (×400). A: Normal group. B: Control group. C: Model group. D: Treatment group.

Tab.7 Protein expression levels of AIF, Smac and Apaf1 in the gastrocnemius muscle in each group (%, Mean±SD, n=3)

Group	AIF	Smac	Apaf1
Normal	17.09±0.58	18.21±0.66	19.54±0.51
Control	36.43±0.50**	36.41±0.69**	39.32±0.69**
Model	51.70±0.55^##	51.23±1.72^##	49.32±1.27^##
Treatment	27.24±0.32^△△	23.59±1.59^△△	29.63±1.68^△△
F	1719.99	270.47	252.82
P	<0.001	<0.001	<0.001

Fig.7 Immunohistochemistry for detecting cleaved caspase-9 and cleaved caspase-3 expressions in the gastrocnemius muscle in each group (×400). A: Normal group. B: Control group. C: Model group. D: Treatment group.

Tab.8 Protein expression levels of cleaved caspase-9 and cleaved caspase-3 in the gastrocnemius muscle in each group (%, Mean±SD, n=3)

Group	Cleaved-caspase9	Cleaved-caspase3
Normal	16.35±0.34	19.61±0.50
Control	44.50±0.84**	29.94±1.60**
Model	90.65±0.80^##	58.86±1.33^##
Treatment	20.08±0.43^△△	20.67±2.00^△△
F	2244.19	569.43
P	<0.001	<0.01

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