This study aimed to systematically investigate the therapeutic efficacy of Qihuang Jianpi Zishen Granules (QJZG), a traditional Chinese medicine formula, in the treatment of Sjögren’s syndrome (SS) and to elucidate its underlying molecular mechanisms, with a specific focus on the regulatory effects of QJZG on the TLR4/MyD88/NF-κB signaling pathway, a key axis mediating immune-inflammatory responses in autoimmune diseases. Additionally, the study sought to verify the multi-target and multi-pathway characteristics of QJZG, and to provide a scientific and experimental basis for its clinical application in SS treatment, as well as to explore a novel integrated research paradigm combining computational biology and experimental validation for traditional Chinese medicine (TCM) mechanism studies. 

An integrated research strategy combining network pharmacology, molecular docking, clinical research, and animal experiments was adopted to conduct a multi-level and multi-dimensional exploration of QJZG’s anti-SS mechanism. First, network pharmacology was used to screen the active components of the eight medicinal herbs in QJZG and their predicted target proteins, intersect with SS-related disease targets to identify potential therapeutic targets, and perform GO functional enrichment and KEGG pathway enrichment analyses to predict the core signaling pathways involved. Molecular docking was then employed to verify the binding affinity and stability between the core active components of QJZG and key target proteins of the TLR4/MyD88/NF-κB pathway. For clinical validation, 8 primary SS patients meeting the diagnostic criteria and 8 age- and gender-matched healthy volunteers were enrolled; SS patients received 4 weeks of QJZG treatment, and ELISA, RT-qPCR, and flow cytometry were used to detect peripheral blood inflammatory cytokine levels, key gene expression, and immune cell subset proportions before and after treatment. Furthermore, NOD/LtJ mice were used to construct the SS animal model, which was randomly divided into normal control, model, QJZG low/medium/high-dose, and hydroxychloroquine positive drug groups, with continuous gavage administration for 8 weeks. HE staining, ELISA, RT-qPCR, and Western blot were applied to evaluate salivary gland pathological changes, serum inflammatory cytokine levels, and the expression of TLR4/MyD88/NF-κB pathway-related genes and proteins in mouse salivary gland tissues and peripheral blood mononuclear cells (PBMCs), and to analyze the dose-effect relationship of QJZG’s therapeutic effects. All experimental data were statistically analyzed using GraphPad Prism 8.0, with appropriate statistical methods selected according to experimental design, and P < 0.05 considered statistically significant. 

Network pharmacology and molecular docking results showed that QJZG contained 75 active components corresponding to 912 predicted targets, with 187 common targets overlapping with SS-related targets; KEGG enrichment analysis identified the TLR4/MyD88/NF-κB pathway as the core regulatory pathway, and core active components such as dihydroquercetin, eburicol, and stigmasterol exhibited strong binding activity (binding energy ≤ -7 kcal/mol) with key targets including TLR4, MyD88, IL-6, TNF-α, and IFN-γ, forming stable hydrogen bond interactions. Clinical study results indicated that QJZG treatment significantly reduced the serum levels of IL-6, TNF-α, and IFN-γ in SS patients and downregulated their mRNA expression in PBMCs; flow cytometry showed that QJZG effectively restored immune homeostasis, increasing the proportions of CD3⁺ and CD4⁺ T cells and decreasing the proportions of CD8⁺ T cells and CD19⁺ B cells in peripheral blood of SS patients. Western blot and RT-qPCR confirmed that QJZG markedly inhibited the overexpression of TLR4, MyD88, and p-NF-κB at both the mRNA and protein levels in PBMCs of SS patients, with no significant change in total NF-κB expression. Animal experiment results further verified the therapeutic effects and mechanism of QJZG: QJZG improved the general condition of SS model mice in a dose-dependent manner, alleviating weight loss, increasing stimulated salivary flow rate, and reducing lymphocyte infiltration and acinar structure damage in submandibular gland tissues. Meanwhile, QJZG significantly downregulated serum inflammatory cytokine levels and the expression of TLR4/MyD88/NF-κB pathway-related proteins in salivary gland tissues of model mice, with the high-dose QJZG group showing therapeutic effects comparable to the hydroxychloroquine positive control group. 

 Qihuang Jianpi Zishen Granules exerts a significant therapeutic effect on Sjögren’s syndrome by inhibiting the abnormal activation of the TLR4/MyD88/NF-κB signaling pathway: it downregulates the expression of downstream pro-inflammatory cytokines , corrects the imbalance of T/B lymphocyte subsets in peripheral blood, restores the body’s immune homeostasis, and alleviates inflammatory infiltration and structural damage of exocrine glands such as salivary glands. This study confirms the multi-target and multi-pathway regulatory characteristics of QJZG, clarifies its specific molecular mechanism in the treatment of SS, and provides direct experimental and clinical evidence for its clinical application. In addition, the integrated research method combining network pharmacology, molecular docking, clinical research, and animal experiments adopted in this study establishes an effective research paradigm for exploring the mechanism of TCM compound formulas, and provides a new idea and experimental basis for the development of novel safe and effective therapeutic drugs for SS.