Single-nucleus RNA sequencing and spatial transcriptomics reveal the mechanism by which Xiaozhiling injection treats internal hemorrhoids.

Background: Hemorrhoids, a prevalent chronic condition globally, significantly impact patients' quality of life. While various surgical interventions, such as external stripping and internal ligation, procedure for prolapse and hemorrhoids, and tissue selecting technique, are employed for treatment, they are often associated with postoperative complications, including unsatisfactory defecation, bleeding, and anal stenosis. In contrast, Xiaozhiling injection, a traditional Chinese medicine-based therapy, has emerged as a minimally invasive and effective alternative for internal hemorrhoids. This treatment offers distinct advantages, such as reduced dietary restrictions, broad applicability, and minimal induction of systemic inflammatory responses. Additionally, Xiaozhiling injection effectively eliminates hemorrhoid nuclei, prevents local tissue necrosis, preserves anal cushion integrity, and mitigates postoperative complications, including bleeding and prolapse. Despite its clinical efficacy, the molecular mechanisms underlying its therapeutic effects remain poorly understood, warranting further investigation.

Aim: To investigate the molecular mechanism underlying the therapeutic effect of Xiaozhiling injection in the treatment of internal hemorrhoids.

Methods: An internal hemorrhoid model was established in rats, and the rats were randomly divided into a modeling group [control group (CK group)] and a treatment group. One week after injection, Stereo-seq and electron microscopy were used to study the changes in gene expression and subcellular structures in fibroblasts.

Results: Single-cell sequencing revealed differences in the expression and transcript levels of the genes collagen 3 alpha 1, decorin, and actin alpha 2 in fibroblasts between the CK group and the treatment group. Spatial transcriptome analysis revealed that genes of the sphingosine kinase 1 (Sphk1)/sphingosine-1-phosphate (S1P) pathway spatially overlapped with key genes of the transforming growth factor beta 1 pathway, namely, Sphk1, S1P receptor, and transforming growth factor beta 1, in the treatment group. The proportion of fibroblasts was lower in the treatment group than in the CK group, and Xiaozhiling treatment had a significant effect on the proportion of fibroblasts in hemorrhoidal tissue. Immunohistochemistry revealed a significant increase in the expression of a fibroblast marker. Electron microscopy showed that the endoplasmic reticulum of fibroblasts contained a large amount of glycogen, indicating cell activation. Fibroblast activation and the expression of key genes of the Sphk1-S1P pathway could be observed at the injection site, suggesting that after Xiaozhiling intervention, the Sphk1-S1P pathway could be activated to promote fibrosis.

Conclusion: Xiaozhiling injection exerts its therapeutic effects on internal hemorrhoids by promoting collagen synthesis and secretion in fibroblasts. After Xiaozhiling intervention, the Sphk1-S1P pathway can be activated to promote fibrosis.