Mechanism of Shenzhu components in alleviating chemotherapy-induced diarrhea via mitochondria-associated endoplasmic reticulum membranes (MAMs) regulation.

ETHNOPHARMACOLOGICAL RELEVANCE

Chemotherapy-induced diarrhea (CID) is a common side effect of 5-fluorouracil (5-FU). Traditional Chinese medicine (TCM) is responsible for the core pathogenesis of spleen qi deficiencies. Therefore, the herbal pair of the rhizomes of Atractylodes macrocephala Koidz. (AM) and the roots of Panax ginseng C. A. Mey. (PG) known for its effects on tonifying qi and strengthening the spleen, has been traditionally used to improve gastrointestinal function.

AIM OF THE STUDY

This study aimed to elucidate how the combination of Atractylodis Macrocephalae volatile oil (AMO) and Panax ginseng total saponins (PGS) (AP) regulated mitochondria-associated endoplasmic reticulum membranes (MAMs) to restore intestinal barrier function in CID.

METHODS

A 5-FU-induced CID mouse model was used to evaluate the effects of AP on diarrhea scores and body weight. Hematoxylin-eosin (HE) staining was used to examine colon pathological changes. Transmission electron microscopy (TEM) was used to observe MAMs, endoplasmic reticulum (ER), and mitochondrial ultrastructure. Immunohistochemistry was performed to detect ER stress markers (CHOP and GRP78) and biochemical assays were used to assess mitochondrial function (membrane potential, ROS, and ATP). The TUNEL assay was conducted to measure cell apoptosis. RNA sequencing (RNA-seq) and bioinformatics analysis were combined to explore the underlying mechanisms, followed by validation of related proteins (cGMP, PKG, IP3R, GRP75, VDAC1, MCU, Mfn2, ZO-1, Occludin, Bcl-2 and Bax) using ELISA, Western blotting(WB), and immunofluorescence.

RESULTS

AP significantly alleviated 5-FU-induced body weight loss, diarrhea, and colonic pathological damage in mice, while restoring intestinal barrier permeability markers. TEM revealed that AP reversed 5-FU-induced mitochondrial swelling, cristae loss, and ER dilation and fragmentation. Further studies demonstrated that 5-FU disrupted MAMs homeostasis, whereas AP restored the normal MAMs structure by downregulating Mfn2 expression. At the mechanistic level, RNA-seq analysis demonstrated that AP alleviated CID by coordinating the regulation of multiple factors, including mitochondrial function and endoplasmic reticulum homeostasis, with activation of the cGMP-PKG signaling pathway identified as a central therapeutic mechanism, which was verified by WB. AP activated the cGMP-PKG pathway to suppress IP3R-mediated ER calcium release and downregulated the MAMs calcium channel complex (IP3R-GRP75-VDAC1-MCU), thereby ameliorating mitochondrial dysfunction and ER stress while reducing mitochondrial calcium overload. Ultimately, AP inhibited apoptosis by modulating the Bcl-2/Bax balance and restored intestinal barrier integrity through the upregulation of tight junction proteins.

CONCLUSION

This study revealed for the first time that 5-FU induced intestinal barrier damage by disrupting the structure and function of MAMs. In contrast, AP corrected calcium imbalance and contacted abnormality through the cGMP-PKG-MAMs axis, resulting in multidimensional relief of CID. This finding provided a novel therapeutic target (MAMs) for CID and highlighted the unique advantage of Chinese herbal complexes in modulating organelle interactions.