Electroacupuncture Alleviates Functional Constipation by Activating HSF1-Dependent Mitochondrial Unfolded Protein Response in Colonic Smooth Muscle Cells.

Background

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Aims

Functional constipation (FC) is closely associated with impaired gastrointestinal motility and mitochondrial dysfunction. Electroacupuncture (EA) has shown therapeutic potential in FC; however, the underlying cellular and molecular mechanisms remain incompletely understood. We investigated whether EA alleviates FC through regulation of heat shock factor 1 (HSF1)-dependent mitochondrial unfolded protein response (UPRmt) signaling in colonic smooth muscle cells (SMCs).

Methods

A loperamide-induced FC mouse model was established in male mice and treated with EA at Tianshu (ST25) and Shangjuxu (ST37). Gastrointestinal motility and neurohumoral mediators were evaluated. Single-cell RNA sequencing (scRNA-seq) was performed to identify EA-responsive colonic cell populations and molecular pathways. Mitochondrial ultrastructure and function, as well as HSF1-dependent UPRmt signaling, were assessed using transmission electron microscopy, immunofluorescence staining, flow cytometry, colorimetric assays, qRT-PCR, and western blotting. AAV-based HSF1 knockdown and overexpression under a smooth muscle-enriched promoter were further used for mechanistic validation.

Results

EA markedly improved gastrointestinal motility and partially restored neurohumoral homeostasis in FC mice. scRNA-seq identified colonic SMCs as a principal EA-responsive cell population and revealed transcriptional remodeling associated with mitochondrial homeostasis and contractile function. EA attenuated mitochondrial ultrastructural damage, restored mitochondrial membrane potential and ATP production, reduced mtROS accumulation, and enhanced mitochondrial respiratory chain complexes activities in colonic SMCs. These effects were accompanied by increased nuclear accumulation of HSF1 and upregulation of UPRmt-associated proteins, including HSP60, mtHSP70, and LONP1. HSF1 knockdown aggravated mitochondrial dysfunction and constipation-associated phenotypes, whereas HSF1 overexpression largely mimicked the therapeutic effects of EA.

Conclusions

EA alleviated FC, at least in part, through activation of HSF1-dependent UPRmt signaling and restoration of mitochondrial homeostasis in colonic SMCs.