Identifying potential biomarkers in the hippocampus of chronic fatigue syndrome rats treated with moxibustion at Zusanli (ST36): a proteomics study.

OBJECTIVE: To observe the effects of moxibustion at Zusanli (ST36) on rats with chronic fatigue syndrome (CFS) and to analyze the mechanisms of moxibustion through hippocampal Proteomics. METHODS: Male Sprague-Dawley (SD) rats were randomly divided into three groups: control group (CON), model group (MOD), and moxibustion group (MOX), with 12 rats in each group. The MOD and MOX groups underwent chronic multi-factor stress stimulation for 35 d to establish the CFS model. After modeling, the rats in the MOX group received mild moxibustion at Zusanli (ST36) (bilateral) for 10 minutes daily for 28 d. During the treatment period, rats in both the MOD and MOX groups continued modeling, while the CON group was kept under normal breeding conditions. The general condition of the rats was monitored, and behaviors were assessed using the Open Field Test (OFT), Exhaustion Treadmill Test, and Morris Water Maze (MWM). Hematoxylin and eosin (HE) staining and transmission electron microscopy (TEM) were employed to observe morphological changes in the hippocampus. Label-free Proteomics were utilized to identify differentially expressed proteins (DEPs) in the hippocampus, followed by bioinformatics analysis. The reliability of the Proteomics results was verified using Parallel Reaction Monitoring. RESULTS: A: Moxibustion at Zusanli (ST36) significantly reduced the general condition score of CFS rats, improved their behavioral performance in OFT, treadmill and MWM, and repaired the pathological and synaptic structural damage in the hippocampus.B: We identified DEPs by applying a fold change threshold of 1.2 and a significance level of P < 0.05. In the comparison between the CON and the MOD, we identified a total of 72 DEPs (31 up-regulated and 41 down-regulated) associated with the development of CFS. In the comparison between the MOX and the MOD group, we identified a total of 103 DEPs (40 up-regulated and 63 down-regulated) related to the therapeutic effects of moxibustion. Gene Ontology (GO) enrichment analysis showed that CFS and moxibustion treatment were related to multiple biological processes, molecular functions, and cellular components. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that CFS pathogenesis was linked to base excision repair, steroid biosynthesis, and systemic lupus erythematosus, Furthermore, the treatment of CFS with moxibustion was relevant to terpenoid skeleton biosynthesis.C: Compared with the two comparison groups, we identified 16 potential biomarkers, noting that moxibustion reversed the up-regulation of 14 DEPs and the down-regulation of 2 DEPs in CFS. These proteins are mainly associated with synaptic plasticity, ribosomal function, neurotransmitter secretion, glycine metabolism, and mitochondrial function. CONCLUSION: Moxibustion at Zusanli (ST36) is effective in treating CFS, the potential biomarkers identified by Proteomics confirm that the mechanisms of moxibustion involve multiple targets and pathways, which may be key to regulating the structural and functional damage in the hippocampus associated with CFS, highlighting their significant value for future research.