Total Phenylethanoid Glycosides from Cistanche tubulosa Alleviate 5-Fluorouracil-Induced Fatigue-Like Behavior and Skeletal Muscle Injury in Mice

Objectives: This study investigated the effects of total phenylethanoid glycosides from Cistanche tubulosa (CPhGs) on 5-fluorouracil (5-FU)-induced fatigue-like behavior and tissue injury in mice and examined associated molecular changes. Methods: Public transcriptomic datasets (GSE80473, GSE121972, and GSE77745) were analyzed to provide exploratory pathway-level context for hypothesis-driven markers associated with chemotherapy- or cachexia-related skeletal muscle injury. Forty tumor-free BALB/c mice were randomly assigned to a normal control group or treated with 5-FU (20 mg/kg, intraperitoneally) for five consecutive days. The 5-FU-treated mice were subsequently divided into model, low-dose-CPhG (250 mg/kg), and high-dose-CPhG (500 mg/kg) groups and treated orally for 14 days. Exercise endurance, forelimb grip strength, serum markers of tissue injury and inflammation, and histopathological changes in skeletal muscle, epididymal adipose tissue, and liver were evaluated. Proteins involved in Akt/FoxO3a signaling, muscle protein degradation, and mitochondrial metabolism were measured in skeletal muscle. Molecular docking was used to assess the predicted interactions of echinacoside and acteoside with selected proteins. Results: Exploratory transcriptomic analysis suggested alterations in muscle contraction, fatty acid oxidation and energy metabolism, with external cachexia data providing context for FoxO/ubiquitin–proteasome signaling and inflammation in skeletal muscle injury. Compared with control mice, 5-FU-treated mice showed body weight loss, reduced swimming endurance, decreased forelimb grip strength, elevated serum CK, CK-MB, LDH, LDH1, TNF-α, and IL-6 levels, and pathological changes in skeletal muscle, adipose tissue, and liver. CPhGs attenuated body weight loss, prolonged swimming time, improved forelimb grip strength, reduced serum markers of tissue injury and inflammation, and improved tissue morphology. CPhGs were also associated with lower skeletal muscle MuRF1 and Atrogin-1 expression and higher p-Akt/Akt and p-FoxO3a/FoxO3a ratios and PGC-1α expression. These effects were generally more pronounced at the higher dose. Docking analysis generated exploratory poses of echinacoside and acteoside with AKT1, MuRF1, and IL-6; the docking scores do not establish binding affinity or target engagement. Conclusions: CPhGs alleviated 5-FU-induced fatigue-like behavior and associated skeletal muscle, adipose tissue, and hepatic abnormalities in mice. These effects were associated with regulation of Akt/FoxO3a signaling, lower expression of proteolysis-associated proteins, higher PGC-1α expression, and reduced systemic inflammation. The in vivo findings concern the complete CPhG preparation; the contributions and direct molecular targets of echinacoside and acteoside require experimental confirmation.