To elucidate the therapeutic mechanism of holistic regulation of body and mind (HRBM) acupuncture in chronic fatigue syndrome (CFS) by focusing on leptin receptor (LepR)–AMP-activated protein kinase (AMPK) signaling to address mitochondrial dysfunction and oxidative stress. A CFS rat model was established by a 4-week treadmill-based exhaustive exercise protocol. After modeling, 50 rats were randomly allocated into 5 groups (n=10), control, CFS (model), acupuncture (Acu), Acu + Allo-aca, and Acu + Allo-aca + 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) groups. Rats in the Acu group received acupuncture at bilateral Baihui (GV 20) and Zusanli (ST 36) for 30 min daily over 4 weeks. Behavioral assessments were used to confirm model validity and treatment effects. Skeletal muscle transcriptomics were applied to identify differentially expressed genes associated with mitochondrial metabolism. LepR, AMPK, phosphorylated AMPK (p-AMPK), peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), and nuclear factor erythroid 2-related factor 2 (Nrf2) were validated by RT-qPCR and Western blot analyses. In vitro, chronic fatigue-like cells were treated with EX-527 (1 µmol/L) and AICAR (500 µmol/L). Mitochondrial membrane potential was assessed using JC-1 assay, intracellular ATP and reactive oxygen species (ROS) levels were quantified, and expressions of apoptosis-related proteins, antioxidative enzymes, and mitophagy-associated proteins were evaluated by Western blot analysis. Mitochondrial ultrastructure was observed by transmission electron microscopy. Immunofluorescence and DCFH-DA probes were used to assess the LepR/AMPK co-localization and ROS dynamics. Acupuncture at GV 20 and ST 36 significantly improved fatigue-related behaviors and exercise endurance in CFS rats. Compared with the control group, rats with CFS exhibited markedly aggravated fatigue symptoms (P<0.01), whereas acupuncture treatment significantly ameliorated these deficits (P=0.0015). Transcriptomic analysis revealed that acupuncture activated LepR-AMPK signaling pathway, accompanied by enhanced mitochondrial function, increased ATP production, and improved antioxidative capacity. Furthermore, acupuncture promoted PINK1/Parkin-mediated mitophagy and stabilized mitochondrial ultrastructure. In vitro, treatment with EX-527 and/or AICAR improved mitochondrial and metabolic measures in the chronic fatigue-like cell model. HRBM acupuncture exerts therapeutic effects in CFS by activating LepR–AMPK signaling, thereby restoring mitochondrial bioenergetics, enhancing antioxidant defenses, and promoting mitophagy.
To explore the molecular mechanisms underlying the protective effect of acupuncture on ovarian function in mice with cyclophosphamide-induced premature ovarian insufficiency (POI) via transcriptomic analysis. Twenty female C57BL/6 mice were divided into 4 groups: control, model, acupuncture, and non-meridian/non-acupoint (NOMA). POI was induced in the model, acupuncture, and non-meridian/non-acupoint groups via cyclophosphamide injection. The acupuncture group received acupuncture at Guanyuan (CV 4), bilateral Guilai (ST 29), and Sanyinjiao (SP 6) for 3 weeks. After the intervention, ovarian tissue weight and ovarian coefficient were calculated, serum levels of key reproductive hormones including follicle-stimulating hormone (FSH), luteinizing hormone (LH) and anti-Müllerian hormone (AMH) were detected, and ovarian histopathological changes were observed to evaluate ovarian function. Transcriptome sequencing was performed to identify differentially expressed genes (DEGs), followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to explore key functional terms and signaling pathways. Western blot was finally applied to validate the expression of core proteins related to mitochondrial function, endoplasmic reticulum stress and inflammatory pathways. The model group showed reduced ovarian weight and elevated FSH levels. The acupuncture group exhibited significantly higher ovarian weight and coefficient, lower FSH levels, and increased E2 and AMH levels compared to the model group (all P<0.01). Transcriptomic analysis revealed 4,021 DEGs between groups. GO and KEGG analyses revealed that these DEGs were mainly involved in oocyte development, steroid hormone synthesis, and pathways related to mitochondrial function, endoplasmic reticulum stress, and inflammatory signaling. Western blot analysis showed that acupuncture partially restored mitochondrial function markers cytochrome c oxidase subunit IV and NADH dehydrogenase 1 beta subcomplex subunit 8 and reduced endoplasmic reticulum stress markers (glucose-regulated protein 78 and Calnexin, P<0.01). It also downregulated pro-inflammatory proteins (IL-17R, IL-17A, NF-κB p65, p-NF-κB p65, ERK1/2, and p-ERK1/2) and upregulated proteins related to metabolic homeostasis (peroxisome proliferator-activated receptor γ, receptor-interacting protein 140, nicotinamide phosphoribosyltransferase, and sirtuin 1, P<0.01). Acupuncture effectively alleviates cyclophosphamide-induced POI in mice, improves ovarian function and follicular quality by regulating cellular functions and inflammatory pathways, suggesting a novel therapeutic approach for POI.
To investigate the protective effects and underlying mechanisms of Babao Dan (BBD) against doxorubicin (Dox)-induced cardiomyopathy (DIC). A zebrafish model of DIC alongside an in vitro model of cardiomyocyte injury induced by Dox was utilized to elucidate the protective effects of BBD against DIC. The zebrafish and cell models were divided into control, Dox, and BBD (low-, medium-, and high-dose) plus Dox treatment groups. Reactive oxygen species (ROS) and malondialdehyde (MDA) content were determined by commercial kits. Nrf2/GPX4 signaling activity was assessed by Western blot and RT-qPCR. A comprehensive network pharmacology analysis was conducted to explore the potential mechanisms of action of BBD on DIC. Compared with Dox group, BBD treatment significantly reduced ROS levels and decreased MDA content in zebrafish (P<0.01), indicating ameliorated oxidative stress, which contributed to a reduction in Dox-induced cardiac injury. Notably, BBD was found to inhibit cardiomyocyte ferroptosis by restoring mitochondrial function in vitro. Network pharmacology analysis identified 4 core network genes, including Nrf2, which is pivotal in linking oxidative stress with ferroptosis, along with 6 key active components of BBD. Molecular docking and dynamics simulations demonstrated that these active components exhibit a strong binding affinity with the identified core targets. BBD effectively activates the Nrf2/GPX4 signaling pathway, thereby inhibiting cardiomyocyte ferroptosis and mitigating DIC. These insights provide a theoretical foundation for the future exploration of BBD as a preventive and therapeutic agent against Dox-related cardiotoxicity.
