This study aimed to explore the functional regulatory pathways of Qihuang Jianpi Zishen Granule (QJZG) in the treatment of primary Sjögren's disease (pSjD) by applying single-cell RNA sequencing (scRNA-seq) technology, and to verify its therapeutic efficacy and underlying molecular mechanism through in vivo animal experiments. The research intended to clarify the core targets and signaling pathways of QJZG in intervening pSjD, thereby providing a solid theoretical and experimental foundation for the clinical application of QJZG in pSjD treatment and offering new ideas for traditional Chinese medicine (TCM) intervention in autoimmune diseases with abnormal T cell activation. 

Peripheral blood mononuclear cells (PBMCs) were collected from healthy volunteers, untreated pSjD patients, and pSjD patients with 3 months of QJZG treatment for scRNA-seq detection. Bioinformatics analyses including cell subpopulation identification, pseudotime trajectory analysis, high-dimensional weighted gene co-expression network analysis (hdWGCNA), as well as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to explore the changes in immune cell composition and the key signaling pathways regulated by QJZG. NOD/LtJ mice were used as the pSjD animal model and randomly divided into the normal control group, pSjD model group, and QJZG treatment group (3.9 g/kg/d for consecutive 8 weeks). Hematoxylin-eosin (HE) staining was used to observe lymphocyte infiltration in the submandibular gland tissue of mice; the stimulated salivary flow rate was measured to evaluate the secretory function of salivary glands; enzyme-linked immunosorbent assay (ELISA) was adopted to detect the serum concentrations of autoantibodies (SSA, SSB) and pro-inflammatory cytokines (TNF-α, IL-6, IFN-γ); flow cytometry was used to analyze the proportion changes of T cell subsets (Th1, Th17, Tregs) in mouse PBMCs; Western blot was employed to detect the protein expression levels of key molecules in the T cell receptor (TCR) signaling pathway in the submandibular gland. All experimental data were statistically analyzed using GraphPad Prism 10 software, with ANOVA combined with Tukey’s multiple comparisons test for inter-group differences, and P< 0.05 considered statistically significant.

ScRNA-seq results revealed that QJZG treatment significantly altered the immune cell subpopulation composition in pSjD patients: the proportions of CD16_NK cells and CCR7_CD8T cells were markedly reduced, while the proportion of LEF1_CD4T cells was significantly increased. Pseudotime trajectory analysis showed that QJZG could effectively reverse the disorder of T cell differentiation trajectory induced by pSjD, and hdWGCNA identified eight gene co-expression modules in core cell subsets, with QJZG regulating the co-expression level of multiple modules. GO and KEGG enrichment analyses indicated that the differentially expressed genes after QJZG treatment were mainly enriched in immune regulation, cell activation and differentiation, and the TCR signaling pathway was identified as the key pathway targeted by QJZG. Animal experiment results confirmed that QJZG significantly reduced the number of lymphocyte infiltration foci in the submandibular gland of pSjD mice, effectively restored the secretory function of salivary glands and increased the stimulated salivary flow rate. ELISA results showed that QJZG notably decreased the serum concentrations of SSA, SSB autoantibodies and pro-inflammatory cytokines (TNF-α, IL-6, IFN-γ) in pSjD mice. Flow cytometry demonstrated that QJZG upregulated the proportion of immunosuppressive Tregs in PBMCs, while downregulating the proportions of pro-inflammatory Th1 and Th17 cells, restoring the balance of T cell subsets. Western blot further verified that QJZG significantly downregulated the protein expression levels of key molecules (FYN, GRB2, ITK, NFATC2, NFATC3) in the TCR signaling pathway in the submandibular gland of pSjD mice, thus inhibiting the abnormal activation of this pathway. 

The therapeutic effect of QJZG on pSjD is mainly mediated by targeting and inhibiting the TCR signaling pathway. QJZG can regulate the differentiation of T cells and restore the balance of Th1/Th17 and Treg subsets, inhibit the excessive activation of the TCR signaling pathway in the target tissue (submandibular gland), reduce the production of pSjD-related autoantibodies and the secretion of pro-inflammatory cytokines, thereby alleviating the immune-inflammatory infiltration and damage of exocrine glands, and ultimately exerting a therapeutic effect on pSjD. This study clarifies the specific molecular mechanism of QJZG in the treatment of pSjD, provides direct experimental evidence for its clinical application, and enriches the research on the immunomodulatory effects of TCM herbal formulas in autoimmune diseases, also offering a potential therapeutic target for the clinical intervention of pSjD.