Electroacupuncture Alleviates Functional Constipation by Activating Heat Shock Factor 1-Dependent Mitochondrial...

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

A loperamide-induced functional constipation mouse model was established in male mice and treated with electroacupuncture at Tianshu and Shangjuxu. Gastrointestinal motility and neurohumoral mediators were evaluated. Single-cell RNA sequencing was performed to identify electroacupuncture-responsive colonic cell populations and molecular pathways. Mitochondrial ultrastructure and function, as well as heat shock factor 1-dependent mitochondrial unfolded protein response signaling, were assessed using transmission electron microscopy, immunofluorescence staining, flow cytometry, colorimetric assays, quantitative real-time polymerase chain reaction, and Western blotting. Adeno-associated virus-based heat shock factor 1 knockdown and overexpression under a smooth muscle-enriched promoter were further used for mechanistic validation.

Electroacupuncture markedly improved gastrointestinal motility and partially restored neurohumoral homeostasis in mice with functional constipation. Single-cell RNA sequencing identified colonic smooth muscle cells as a principal electroacupuncture-responsive cell population and revealed transcriptional remodeling associated with mitochondrial homeostasis and contractile function. Electroacupuncture attenuated mitochondrial ultrastructural damage, restored mitochondrial membrane potential and adenosine triphosphate production, reduced mitochondrial reactive oxygen species accumulation, and enhanced mitochondrial respiratory chain complexes activities in colonic smooth muscle cells. These effects were accompanied by increased nuclear accumulation of heat shock factor 1 and upregulation of mitochondrial unfolded protein response-associated proteins, including heat shock protein 60, mitochondrial heat shock protein 70, and Lon peptidase 1. Heat shock factor 1 knockdown aggravated mitochondrial dysfunction and constipation-associated phenotypes, whereas heat shock factor 1 overexpression largely mimicked the therapeutic effects of electroacupuncture.

Electroacupuncture alleviated functional constipation, at least in part, through activation of heat shock factor 1-dependent mitochondrial unfolded protein response signaling and restoration of mitochondrial homeostasis in colonic smooth muscle cells.