Paeoniflorin in the preparation of GM-CSF inhibitors + Application of Th cell differentiation in drugs for the prevention and / or treatment of rheumatoid arthritis

Abstract

The application provides a preparation method of paeonol glycoside for inhibiting GM-CSF + The application belongs to the technical field of biological medicine, and relates to application of Th cell differentiation in a medicine for preventing and / or treating rheumatoid arthritis (RA). + Abnormal activation of Th cells is obviously earlier than appearance of clinical symptoms of RA in a time axis, is an early key initiating factor for driving occurrence of the RA disease, and is a core target point for implementing an early blocking strategy. + Further, it is found that paeonol glycoside (Alb) can block a gamma c / JAK3 / STAT5 signal path by targeting and combining with an interleukin-2 receptor gamma chain, down-regulate CSF2 gene transcription and GM-CSF protein expression, thereby inhibiting differentiation of Th cells, and finally achieving an effect of preventing occurrence of CIA arthritis and not rebounding after drug withdrawal. The application provides a new intervention medicine which is high in safety, suitable for long-term use, can effectively regulate immune abnormalities in a pre-RA stage, prevents rheumatoid arthritis, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically the application of paeoniflorin in the preparation of drugs that inhibit GM-CSF⁺Th cell differentiation to prevent and / or treat rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized primarily by symmetrical polyarticular synovitis, which leads to progressive destruction of articular cartilage and bone, severely impacting patients' quality of life. Epidemiological studies show that the global prevalence of RA is approximately 0.5%–1%, while the prevalence in my country is approximately 0.42%.

[0003] Studies have shown that rheumatoid arthritis (RA) does not occur suddenly, but rather through a gradual process from autoimmune abnormalities to the appearance of clinical symptoms. Several years before the clinical onset of RA, autoantibodies such as anti-citrullinated protein antibodies (ACPA) can be detected in patients' bodies, and a sustained increase in ACPA titers is closely related to the risk of future progression to clinical RA. This preclinical stage is called "pre-RA" or "high-risk state for RA." Currently, the academic community believes that if the evolution of immune abnormalities can be effectively blocked at this stage, it is possible to delay or even prevent the clinical onset of RA. However, existing intervention studies targeting high-risk pre-RA populations are not ideal: rituximab only delays the onset of arthritis in the short term but fails to achieve true prevention (Gerlag DM, Safy M, Maijer KI, et al. Effects of B-cell directed therapy on the preclinicalstage of rheumatoid arthritis: the PRAIRI study[J]. Ann Rheum Dis, 2019, 78(2): 179-185); abatacept can reduce the risk of arthritis progression to some extent during treatment, but the protective effect disappears after discontinuation, and the high relapse rate suggests that it has failed to achieve durable remodeling of immune homeostasis (Cope AP, Jasenecova M, Vasconcelos JC, et al. Abatacept in individuals at high risk of rheumatoid arthritis (APIPPRA): a randomised, double-blind, multicentre, parallel, placebo-controlled, phase 2b clinical trial[J]. Lancet, 2024, 403(10429):838-849); Hydroxychloroquine intervention did not significantly reduce the risk of RA conversion in high-risk populations and did not show significant preventive effects (Deane KD, Striebich CC, Feser ML, et al. A phase 2 trial of hydroxychloroquine in individuals at risk for rheumatoid arthritis[J].Arthritis Rheumatol, 2025; early intervention with methotrexate in patients with clinically suspected arthritis (CSA) only temporarily improved radiographic inflammation and failed to significantly reduce the long-term risk of eventually developing into diagnosed RA (Krijbolder DI, Verstappen M, Van Dijk BT, et al. Intervention with methotrexate in patients with arthralgia at risk of rheumatoid arthritis to reduce the development of persistent arthritis and its disease burden (TREAT EARLIER): a randomised, double-blind, placebo-controlled, proof-of-concept trial[J]. Lancet, 2022,400(10348): 283-294). The above studies collectively suggest that directly using existing RA treatment drugs for pre-RA intervention faces prominent problems such as limited efficacy, rebound after drug discontinuation, and a mismatch between the long-term safety (such as infection risk and immunosuppression) and benefits. Total Glucosides of Paeony (TGP) is an effective component extracted from the traditional Chinese medicine Paeonia lactiflora Pall. It has been approved by the National Medical Products Administration for use as an adjunct therapy for rheumatoid arthritis (RA), possessing dual anti-inflammatory and immunomodulatory activities. In recent years, some clinical experts have suggested using TGP for preventative intervention in high-risk RA populations. However, TGP has the following shortcomings in practical clinical application: First, TGP is a multi-component mixture containing numerous chemical components, and the key active ingredients and their molecular targets for exerting its core efficacy are not yet clearly defined, hindering in-depth elucidation of its mechanism of action and precise drug application. Second, TGP has significant gastrointestinal adverse reactions in clinical use; some patients experience symptoms such as diarrhea, abdominal pain, and increased bowel movements after taking the medication, leading to decreased medication adherence and even forced discontinuation. For pre-RA high-risk individuals without obvious clinical symptoms, their tolerance for adverse drug reactions is far lower than that of diagnosed patients, and the gastrointestinal irritation issues of TGP will severely restrict its clinical promotion in long-term preventative intervention.

[0004] Therefore, developing novel intervention drugs that are safe, suitable for long-term use, and can effectively regulate immune abnormalities in the pre-RA stage is an important technological need in this field. Summary of the Invention

[0005] In response to the prominent problems of limited efficacy, rebound after drug withdrawal, and mismatch between the safety (such as infection risk and immunosuppression) and benefits of long-term use of existing RA treatment drugs for pre-RA intervention, as well as the unclear core active ingredient and molecular target of TGP in RA treatment, the purpose of this invention is to develop a novel intervention drug that is highly safe, suitable for long-term use, and can effectively regulate immune abnormalities in the pre-RA stage to prevent rheumatoid arthritis.

[0006] This invention provides the use of an agent that inhibits GM-CSF⁺ Th cell differentiation in the preparation of medicaments for the prevention and / or treatment of rheumatoid arthritis.

[0007] Furthermore, the drug for preventing rheumatoid arthritis is a drug that inhibits the development of rheumatoid arthritis in high-risk individuals of pre-RA or reduces the risk of developing rheumatoid arthritis in high-risk individuals of pre-RA.

[0008] Furthermore, the reagent for inhibiting GM-CSF⁺ Th cell differentiation is a reagent that inhibits GM-CSF⁺ Th cell differentiation by targeting and binding to the interleukin-2 receptor γ chain.

[0009] Furthermore, the reagent for inhibiting GM-CSF⁺ Th cell differentiation is a reagent that inhibits GM-CSF⁺ Th cell differentiation by targeting and binding to the interleukin-2 receptor γ chain to block the γc / JAK3 / STAT5 signaling pathway, downregulating CSF2 gene transcription and GM-CSF protein expression.

[0010] Furthermore, the reagent used to inhibit GM-CSF⁺ Th cell differentiation is paeoniflorin.

[0011] Furthermore, the drug is a preparation made with paeoniflorin as the active ingredient and pharmaceutically acceptable auxiliary ingredients; preferably, the preparation is a tablet, capsule, granule, injection or oral liquid.

[0012] Furthermore, the effective concentration of paeoniflorin in the drug is 5–100 μM.

[0013] Furthermore, the dosage of the drug for mice, calculated as paeoniflorin, is 20–200 mg / kg / day, and the dosage for humans, calculated as paeoniflorin, is 1.6 mg / kg / day–16.2 ​​mg / kg / day.

[0014] This invention also provides the application of paeoniflorin in the preparation of formulations that inhibit GM-CSF⁺ Th cell differentiation.

[0015] Furthermore, the preparation for inhibiting GM-CSF⁺ Th cell differentiation is a drug for the prevention and / or treatment of autoimmune diseases, preferably, the autoimmune diseases include rheumatoid arthritis, multiple sclerosis, and type 1 diabetes.

[0016] This invention also provides the application of paeoniflorin in the preparation of interleukin-2 receptor γ chain inhibitors.

[0017] Furthermore, the interleukin-2 receptor γ chain inhibitor is a drug for the prevention and / or treatment of autoimmune diseases, preferably, the autoimmune diseases include rheumatoid arthritis, multiple sclerosis, and type 1 diabetes.

[0018] The present invention has achieved the following beneficial effects: First, total glucosides of paeony have been approved for clinical treatment of rheumatoid arthritis (RA), but their true active ingredients and molecular targets remain unclear. Literature reports that total glucosides of paeony are effective components extracted from white peony, containing paeoniflorin, oxypaeoniflorin, paeoniflorin glycosides, paeoniflorin lactone, benzoylpaeoniflorin, etc., with paeoniflorin accounting for over 90% of the total glucosides and being the main effective component of white peony (Pharmacological Research Progress of Total Glucosides of Paeony in Cardiovascular Disease Model Animals, Dai Shuping et al., China Pharmacy, Vol. 26, No. 10, 2015; Therapeutic potential and pharmacological insights of total glucosides of paeony in dermatologic diseases: a comprehensive review, Huige Wang et al., Frontiers in Pharmacology, January 2, 2025). Existing research almost entirely focuses on paeoniflorin, and the academic community generally accepts paeoniflorin as the core pharmacodynamic component of total glucosides (TGP). Research on paeoniflorin lactone is extremely limited, and its role in RA has been almost entirely unexplored. This invention discovers that GM-CSF + Abnormal activation of Th cells significantly precedes the onset of RA clinical symptoms on the timeline, GM-CSF + The expansion of Th cells is not only a biological marker reflecting the progression of RA, but also a reversible indicator sensitive to immunomodulatory therapy. GM-CSF +Abnormal activation of Th cells is a key early initiating factor driving the development of rheumatoid arthritis (RA) and a core target for early intervention strategies. However, small molecule drugs targeting GM-CSF⁺ Th cell differentiation are currently lacking. This invention is the first to discover that paeoniflorin is inactive in inhibiting GM-CSF⁺ Th cell differentiation, a key pathogenic step in RA, while paeoniflorin lactone, also a component of TGP, exhibits significant activity. This invention breaks the conventional thinking that "paeoniflorin is the main drug" in traditional research and achieves unexpected technical results.

[0019] Second, γc (common gamma chain, CD132) is a common signal transduction subunit of receptors for multiple cytokines, including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Its downstream JAK3 / STAT5 pathway is closely related to T cell proliferation and differentiation (The Common Cytokine Receptor γ Chain Family of Cytokines, Jian-XinLin et al., Cold Spring Harbor Perspectives in Biology, 2018 Sep 4;10(9):a028449; Disorders of the JAK / STAT Pathway in T Cell Lymphoma Pathogenesis:Implications for Immunotherapy, Thomas A. Waldmann et al., Annual Review of Immunology, Volume 35, 2017). However, there has never been a previous report linking paeoniflorin to γc. This invention is the first to discover that paeoniflorin can directly bind to γc protein (confirmed by SPR experiments), inhibiting its downstream JAK3 / STAT5 signaling pathway, thereby blocking GM-CSF⁺ Th cell differentiation. The target and mechanism of action are clearly defined. In contrast, the binding affinity of paeoniflorin to IL-2β protein via the BLI curve is weak. This discovery provides a clear molecular target explanation for the efficacy of paeoniflorin and lays the foundation for expanding its indications to other γc-related diseases.

[0020] Third, this invention is the first to discover that paeoniflorin can significantly prevent arthritis in CIA model mice, as evidenced by a significant reduction in clinical arthritis scores, a significant reduction in paw swelling, and a significant improvement in bone destruction.

[0021] Fourth, the joint-protective effect of paeoniflorin can be maintained without significant rebound after drug withdrawal, suggesting that it fundamentally blocks the differentiation of pathogenic GM-CSF⁺ Th cells in the early stage of the immune response, achieving long-lasting disease control and having the potential for disease-modifying.

[0022] In summary, this invention reveals a complete chain of evidence from target to signaling pathway to cellular function to in vivo efficacy: paeoniflorin binds to γc → inhibits JAK3 phosphorylation → inhibits STAT5 phosphorylation → blocks GM-CSF⁺ Th cell differentiation → prevents CIA arthritis without rebound after drug withdrawal. Paeoniflorin has broad application prospects in the preparation of drugs for the prevention and / or treatment of rheumatoid arthritis, especially in drugs that inhibit the development of RA in pre-RA high-risk individuals or reduce the risk of developing RA in pre-RA high-risk individuals.

[0023] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0025] Figure 1 GM-CSF + Th cells are involved in the entire course of RA in human and mouse models. (AB) Transcriptome activity analysis of synovial membrane in RA at different stages and comparison of MTX treatment before and after in newly diagnosed patients (n=10). (CD) Single-cell transcriptome analysis and subset identification of CD4⁺ T cells in GPI-induced arthritis model. (E) Enrichment analysis of common marker genes between humans and mice. (F) GSVA analysis at different time points in CIA model. Box plots show median and IQR; scatter plots represent independent samples. P <0.05, P <0.01, P <0.001.

[0026] Figure 2The differential effects of paeoniflorin and paeoniflorin on the in vitro differentiation of GM-CSF⁺ Th cells. (A) Representative scatter plot of the proportion of GM-CSF⁺ Th cells in each group as detected by flow cytometry. Naive CD4⁺ T cells were treated with different concentrations (10, 20, 40 μM) of Alb or Pae during in vitro differentiation. FMO was used as a fluorescence subtraction control to assist in gating. (B) Statistical graph of the proportion of GM-CSF⁺ Th cells in each group. Note: Data are expressed as Mean ± SD. n = 3). ns: No significant difference. P <0.05, P <0.01, P <0.0001 compared to the Ctrl group. Abbreviations: Alb, albiflorin; Pae, paeoniflorin.

[0027] Figure 3 The binding kinetics of paeoniflorin to its receptor subunits were detected using biomembrane interferometry (BLI). (AB) Analysis of the binding kinetics of paeoniflorin (Alb) with IL-2Rγ (A) and IL-2Rβ (B). The left side shows real-time sensorograms of the binding and dissociation of Alb (62.5–1000 μM) with the immobilized receptor at different concentration gradients; the solid and smooth lines represent the original data and the fitted model, respectively. The right side shows the steady-state affinity fit curve based on the equilibrium response value, used to calculate the dissociation equilibrium constant (K). D The vertical axis Response (nm) represents the optical interference displacement signal, and the horizontal axis Time (s) represents time. Abbreviations: Alb, albiflorin; K D , equilibriumdissociation constant. Figure 4Paeoniflorin (Alb) inhibits the downregulation of GM-CSF expression along the γc / JAK3 / STAT5 signaling axis. (A) RT-qPCR detection of the effect of Alb on the mRNA expression of key genes in the pathway. (BC) Representative Western blot bands (B) and quantitative analysis of relative expression levels and phosphorylation levels of each protein (C); the phosphorylated protein levels were normalized to their corresponding total protein levels. Right-hand bar chart: showing the fold change in the expression of JAK1, JAK3, STAT5, and total GM-CSF protein relative to β-actin. Note: Data are expressed as Mean ± SD (n=3). One-way ANOVA was used to test the significance between groups compared with the control group (Ctrl). P <0.05, P <0.01, P <0.001, P <0.0001 compared to the Ctrl group.

[0028] Figure 5 : Protective effect of paeonolactone (Alb) on clinical phenotype and joint bone destruction in CIA mice. (A) Schematic diagram of experimental procedure. Eight-week-old DBA / 1 mice were divided into Ctrl, CIA, and Alb (80 mg / kg / d, ip) groups. (B) Cumulative incidence curve. (C) Representative gross observation of the hind paws of mice in each group on day 49. (D) Dynamic change curve of arthritis score. (E) Micro-CT three-dimensional reconstruction images of the ankle joint and the region of interest (ROI) of the proximal metaphysis of the tibia. (F) Quantitative analysis of bone parameters, including bone mineral density (Tb.BMD) and trabecular bone number (Tb.N). Note: Data are expressed as Mean ± SD. Compared with the CIA group. Abbreviations: Alb, paeonolactone; CIA, collagen-induced arthritis; ip, intraperitoneal injection. Detailed Implementation

[0029] Unless otherwise specified, the raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0030] All statistical analyses and graphs in this invention were performed using GraphPad Prism (version 9.0) or SPSS (version 26.0) software. All statistical tests were two-tailed tests. P A difference of <0.05 is considered statistically significant. P <0.05, P <0.01, P <0.001, P <0.0001).

[0031] Example 1 GM-CSF + Spatiotemporal characteristics of Th cells in the evolution of RA 1. Experimental Methods (Bioinformatics Analysis Methods Based on Public Databases) (1) Construction and analysis of transcriptional feature score of the whole course of RA This study searched the NCBI Gene Expression Comprehensive Database (GEO) and included the synovial transcriptome dataset (GSE229449) covering the entire disease course, including healthy controls (HC), osteoarthritis (OA), pre-clinical RA (pre-RA) with arthralgia and undifferentiated arthritis (UA), treatment-naïve RA, and remission, to explore GM-CSF. + The evolutionary pattern of Th cells.

[0032] (1.1) Data standardization: Background correction and quantile normalization were performed on the Microarray chip data using the RMA algorithm; RNA-seq data were uniformly converted to TPM format. The ComBat algorithm in the sva package of R language was used to eliminate batch effects, and the correction effect was verified by principal component analysis (PCA) to ensure the consistency of data across platforms.

[0033] (1.2) Feature scoring model construction: Based on the RA synovial single-cell atlas previously constructed by our research group, differential expression analysis was performed using the FindMarkers function. The screening criteria were set as Log2FC>1 and Adjusted P <0.05, extract GM-CSF + The top 50 marker genes that are highly expressed in Th cells compared to other T cell subsets were used to construct a subset-specific characteristic gene set.

[0034] (1.3) ssGSEA scoring and statistics: Using the single-sample gene set enrichment analysis (ssGSEA) algorithm in the GSVA package (v1.40.1), the characteristic gene set was mapped to the synovial transcriptome expression matrix, and the standardized enrichment score was calculated to quantify the transcriptional activity of this subgroup at each pathological stage. Differences between groups were compared using the Wilcoxon rank-sum test or the Kruskal-Wallis test.

[0035] (2) Single-cell transcriptome reanalysis and cross-species conservation verification To confirm GM-CSF + To study the conservation of Th cell subsets across species, this study used single-cell transcriptome data (GSE263231) from the inguinal lymph node of a mouse model of arthritis induced by glucose-6-phosphate isomerase (GPI) for in-depth reanalysis.

[0036] (2.1) Standard Processing Flow for Single-Cell Data: The scRNA-seq data was processed using the Seurat R package (v4.0.0) according to a standard workflow. The following quality control (QC) standards were strictly followed: high-quality cells with a detection gene count (nFeature_RNA) between 200 and 2500 and a mitochondrial gene proportion (percent.mt) below 10% were retained. After LogNormalization, the top 2000 hypervariable genes were screened using the FindVariableFeatures function, and standardized using ScaleData to eliminate the heterogeneity effects of cell cycle scores and mitochondrial content. Principal component analysis (PCA) was then performed for dimensionality reduction. RunUMAP was used for nonlinear visualization based on the top 20 principal components, and unsupervised clustering was performed using the FindNeighbors and FindClusters (Resolution = 0.5) algorithms.

[0037] (2.2) Subgroup identification and characteristic analysis: based on classical immune marker genes (such as...) Cd3e , Cd4 Positioning CD4 + T cell population, and further detected Csf2 ( The expression level of GM-CSF was measured to identify Th cell subsets that specifically highly express GM-CSF.

[0038] (2.3) Cross-species gene set enrichment analysis (GSEA): To verify the conservation of this cell subpopulation, the human GM-CSF⁺ Th cell characteristic genes were converted into mouse homologous genes using the biomaRt package. Subsequently, the fgsea package was used to analyze mouse CD4. + A list of differentially expressed genes in each T cell subset (sorted by Log2FC) was analyzed using GSEA to calculate the normalized enrichment score (NES) and the corrected NES. P Value (Adjusted) P The value was used to assess the enrichment level of human characteristic gene sets in mouse subpopulations.

[0039] (2.4) Screening for common marker genes: GM-CSF was extracted from human and mouse samples, respectively. + Significantly differentially expressed genes in each Th cell type (screening criteria: Log2FC>0.5, Adjusted) P <0.05, taking the intersection of the two, finally identified 8 conserved cross-species shared marker genes ( Csf2, Cx3cr1, Ccr10, Tnf, Il2, Il2rb, Il2rg, Il7r ).

[0040] (3) Electron flow cytometry tracing of spatiotemporal evolution of cell subpopulations in the CIA model To track early dynamics of target cells in a mouse model of collagen-induced arthritis (CIA) lacking single-cell data, this study employed a Bulk RNA-seq-based signature scoring strategy.

[0041] (3.1) Data source and processing: Time-series Bulk RNA-seq data (GSE193335) of CIA mice covering the period from day 0 of immune induction, the incubation period to the peak of the disease were selected. The raw data were converted to TPM format after quality control.

[0042] (3.2) Scoring model construction: Based on the 8 cross-species shared marker genes identified in 2.1.1.2 (single-cell reanalysis) Csf2, Cx3cr1, Ccr10, Tnf, Il2, Il2rb, Il2rg, Il7r Constructing a simplified version of mouse GM-CSF + Th cell characteristic gene set.

[0043] (3.3) Time-series scoring and statistical analysis: The ssGSEA algorithm in the R language GSVA package (v1.40.1) was used to calculate the comprehensive expression score of the characteristic gene set in the sample at each time point, defined as the "CellActivity Score". The Kruskal-Wallis test was used to compare the differences in scores at different time points after immune induction. The Dunn's post-hoc test was used as the main method, and the Benjamini-Hochberg (BH) method was used for multiple comparison correction. The differences between the latency period before the appearance of clinical symptoms (e.g., Day 14) and the baseline group (Day 0) and the peak of the disease were examined to reveal the spatiotemporal evolution of this pathogenic subgroup as the disease progresses.

[0044] 2. Experimental Results (1) Cellular transcriptional characteristics persist throughout the entire course of the disease and are reversible in treatment. To investigate GM-CSF + This invention investigates the dynamic evolution of Th cell subsets throughout the entire course of rheumatoid arthritis (especially in the preclinical stage). Firstly, it constructs a transcriptional signature score based on the gene expression profile of this subset and maps it to transcriptomic data from human synovial tissue. Analysis throughout the entire disease course shows a significant positive correlation between the transcriptional activity of this subset and disease progression. P <0.05, Figure 1 A). Notably, this aberrant activation of the pathogenic transcriptional program is not limited to the diagnosed RA stage, but rather begins in the high-risk stage of pre-clinical RA. Specifically, in typical high-risk RA populations such as arthralgia (AG) and undifferentiated arthritis (UA), GM-CSF is present in the synovial tissue. + The characteristic scores of Th cells have shown a trend of being higher than those in the control group (Ctrl) and the osteoarthritis group (OA). This progressive upregulation before the onset of clinically overt synovitis suggests that GM-CSF... + Activation of the Th cell signaling pathway is a key early event driving the pathogenesis of rheumatoid arthritis (RA), providing a potential biological basis for preclinical intervention.

[0045] Furthermore, further analysis of clinical intervention data revealed that this transcriptional feature is significantly reversible. Figure 1B). Longitudinal follow-up data of treatment-naïve RA patients showed that clinical remission was closely associated with downregulation of transcriptional activity in this subset: GM-CSF in the synovial tissue of patients was significantly reduced after 3 months of methotrexate (MTX) treatment. + The enrichment score of Th cells decreased significantly (Signed-rank test). P = 1.9 × 10 -3 The level then returned to a level where there was no statistically significant difference compared to the control group (Ctrl). This result confirms that GM-CSF... + The expansion of Th cells is not only a biological marker reflecting disease progression, but also an indicator of reversibility that is sensitive to immunomodulatory therapy.

[0046] (2) GM-CSF + Cross-species conservation of Th cell subsets To confirm GM-CSF + To investigate the conservation of Th cell subsets across species and assess the applicability of mouse models in simulating the evolution from preclinical to clinical stages, this study retrieved and thoroughly analyzed single-cell transcriptome data from public databases for a mouse model of glucose-6-phosphate isomerase (GPI)-induced arthritis. This included data targeting CD4+ in mouse inguinal lymph nodes. + The reanalysis results of T cells clearly revealed a group of cells that specifically highly express... Csf2 The presence of Th cell subsets in (GM-CSF) Figure 1 CD).

[0047] Subsequently, through gene set enrichment analysis (GSEA), human GM-CSF was analyzed. + The characteristic gene set of Th cells was mapped to mouse data. Analysis showed that human characteristic genes were significantly enriched in this mouse subpopulation. P = 1.0 × 10⁻ 4 This indicates a high degree of consistency between the two at the transcriptional profile level. Based on this, through cross-species intersection analysis, this study identified eight conserved shared marker genes, namely: Csf2, Cx3cr1, Ccr10, Tnf, Il2, Il2rb, Il2rg and Il7r（ Figure 1 E). This cross-species validation result, based on multi-database mining, strongly confirms the GM-CSF. + Th cells exhibit high transcriptomic homology between humans and mice, providing a solid bioinformatics basis for subsequent preclinical intervention mechanism exploration using mouse models.

[0048] (3) CIA Model Validation GM-CSF + Th cells are an early initiating factor driving the pathogenesis of the disease. To further explore the universality of this subpopulation under different pathogenic induction environments and its dynamic evolution in the disease process, this study used the eight common marker genes identified above and employed electron flow cytometry to score and track the time-series transcriptome data of a mouse model of collagen-induced arthritis (CIA).

[0049] Analysis revealed that GM-CSF + Th cells exhibit unique spatiotemporal evolutionary characteristics. Figure 1 F): In the second week after primary immunization (CIA-2w), the incubation period before clinical symptoms of arthritis appeared (corresponding to the preclinical stage), the activity score of this subgroup was significantly higher than that of the control group (Wilcoxon ranked-sum test). P <0.05. With prolonged induction time, the amplification trajectory of this subpopulation showed a high degree of consistency with disease progression (Kruskal-Wallis test, ...). P = 4.8 × 10⁻ 5 This finding confirms GM-CSF. + Abnormal activation of Th cells precedes the appearance of clinical symptoms on a timeline, suggesting that it is a key early initiating factor driving the development of RA and a core target for implementing early intervention strategies.

[0050] The above results indicate that GM-CSF + Abnormal activation of Th cells significantly precedes the onset of RA clinical symptoms on the timeline, GM-CSF + The expansion of Th cells is not only a biological marker reflecting the progression of RA, but also a reversible indicator sensitive to immunomodulatory therapy. GM-CSF + Abnormal activation of Th cells is a key early initiating factor driving the development of RA and a core target for implementing early blocking strategies.

[0051] Example 2: Paeoniflorin (Alb) and Paeoniflorin (Pae) ​​on GM-CSF + In vitro intervention experiment on the effect of Th cell differentiation Based on the findings of the previous embodiment, this embodiment compares the effects of paeoniflorin (Alb) and paeoniflorin (Pae) ​​on GM-CSF. + The influence of Th cell differentiation.

[0052] 1. Experimental Methods To determine the safe dosage range of paeoniflorin (Pae) ​​and albiflorin (Alb) in in vitro experiments, the effects of different drug concentrations on Naïve CD4⁺ T cell viability were first detected using a CCK-8 assay kit. Based on the cell viability test results, 10, 20, and 40 μM, which had no significant effect on cell viability, were selected as low, medium, and high doses for subsequent pharmacodynamic evaluation. Next, the regulatory effect of the drugs on Th cell differentiation was evaluated, and the experimental steps are as follows: (1) T cell receptor (TCR) signal pre-activation: Take a 96-well flat-bottom cell culture plate, add 50 μL of DPBS solution (final concentration 2.5 μg / mL) containing purified anti-mouse CD3e monoclonal antibody (Clone 145-2C11, BioLegend, USA, Cat# 100340) to each well for coating, and incubate overnight at 4℃. Before the experiment, aspirate the coating solution and wash twice with sterile PBS to remove unbound antibodies.

[0053] (2) Resuspension of cells: The sorted and purified Naïve CD4 cells were resuspended. + T cells were resuspended in RPMI-1640 complete medium (containing 10% FBS), counted, and the cell density was adjusted to 8 × 10⁶ cells / year. 5 cells / mL.

[0054] (3) Addition of inducing factors and blocking antibodies to the induction suspension: Prepare an induction working suspension (final concentrations as follows): Co-stimulatory signal: mouse CD28 monoclonal antibody (Clone 37.51, BioLegend, USA, Cat# 102116), final concentration 1 μg / mL; Inducing factors: recombinant anti-mouse IL-2 (rmIL-2) and recombinant mouse IL-7 (rmIL-7) (both purchased from STEMCELL Technologies, Canada), final concentrations 5 ng / mL and 2 ng / mL, respectively; Lineage blocking: add anti-mouse IFN-γ neutralizing antibody (Clone XMG1.2, BioXCell, USA, Cat# BE0055) and anti-mouse IL-4 neutralizing antibody (Clone 11B11, BioXCell, USA, Cat# BE0045), final concentrations 10 μg / mL for both.

[0055] (4) Drug intervention: After thoroughly mixing the cell suspension containing the inducing factor, the mixture was seeded into 96-well plates pre-coated with CD3e antibody, with a volume of 100 μL per well (i.e., approximately 8 × 10⁻⁶ cells per well). 4 (Number of cells). The control group received only cell suspension containing the aforementioned inducing factors. The drug intervention group received different concentrations of paeoniflorin (Pae) ​​or paeoniflorin lactone (Alb) (10, 20, 40 μM) simultaneously with cell seeding. The control group received an equal volume of solvent. Cells were incubated at 37 °C in a 5% CO2 incubator for induction.

[0056] (5) Restimulation and Flow Cytometry: After culturing for 66 h, PMA (50 ng / mL), Ionomycin (1 μg / mL), and the protein transport inhibitor GolgiStop™ (BD Biosciences) were added to each well for restimulation. After 72 h of culture, cells were collected for FVS450 staining (whether the cells are viable or dead), CD4 surface staining, and GM-CSF intracellular staining. Finally, GM-CSF was detected using a BD flow cytometer. + The proportion of Th cells was used to evaluate the regulatory effect of drugs on Th cell differentiation.

[0057] 2. Experimental Results Flow cytometry results showed that Alb exhibited a potent inhibitory effect on GM-CSF⁺ Th cells. Under directed induction conditions, GM-CSF⁺ Th cells in the solvent control group (Ctrl) differentiated to (55.57 ± 5.01)%, indicating that the induction model was successfully established and the system was stable. Figure 2 A). After intervention with different concentrations of Alb, the proportions of GM-CSF⁺ Th cells in the 10, 20, and 40 μM treatment groups decreased sharply to (40.50 ± 4.75)%, (21.67 ± 2.56)%, and (13.70 ± 4.06)%, respectively. Statistical analysis showed that all Alb treatment groups had highly significant differences compared with the control group. P <0.05 or P <0.01). And it exhibits a clear dose-dependent inhibitory trend ( Figure 2 B).

[0058] In contrast, the isoform Pae did not show a significant inhibitory effect; at the same concentration gradient (10, 20, 40 μM), the proportions of GM-CSF⁺ Th cells in the Pae-treated groups were (53.83 ± 8.21)%, (48.60 ± 7.67)%, and (51.73 ± 5.86)%, respectively. There were no statistically significant differences between the Pae-treated groups and the control group at any concentration. P >0.05), indicating that Pae cannot effectively inhibit the differentiation process of GM-CSF⁺ Th cells within this dose range.

[0059] These differential results reveal the specificity of Alb as the core pharmacodynamic substance of TGP. Although Alb and Pae are structurally highly similar and both are monoterpene glycosides, they exhibit distinctly different biological effects in regulating the key pathological process of GM-CSF⁺ Th cell differentiation: Alb is a key effector monomer that exerts an immune cutoff effect, while Pae is not a direct regulator of this process. This discovery of "similar structure, distinct activity" breaks the conventional thinking that "paeoniflorin is the main drug" in traditional research.

[0060] Example 3: Determination of the binding of paeoniflorin to IL-2Rγ (γc) protein using biomembrane interferometry (BLI). To empirically demonstrate the theoretical predictions of molecular docking and pinpoint the direct molecular targets of paeoniflorin (Alb), this experiment employed biomembrane interference technology to quantitatively determine the physical binding affinity between Alb and various human recombinant receptor subunits.

[0061] 1. Experimental Methods (1) Instruments and reagents: The Octet R8 label-free molecular interaction analysis system (Sartorius, Germany) was used for detection. All human recombinant receptor proteins were purchased from SinoBiological, Beijing, China, and were all biotinylated proteins. The specific information is as follows: Human IL-2Rβ / CD122 protein (ECD, His&AVI Tag, Biotinylated), Cat# 10696-H27H-B. Human IL-7Rα / CD127 protein (ECD, His&AVI Tag, Biotinylated), Cat# 10975-H49H-B. Human IL-2Rγ / CD132 protein (ECD, His&AVI Tag, Biotinylated), Cat# 10555-H49H-B. The capture sensor used was the Streptavidin (SA) biosensor (Sartorius, Cat# 18-5019), which utilizes the high affinity of biotin-streptavidin to specifically immobilize the aforementioned ligand protein. The experimental run buffer was a PBS solution (pH 7.4) containing 0.1% BSA and 0.02% Tween-20.

[0062] (2) Experimental Procedure: The SA sensor was pre-wetted by immersing it in pre-run buffer for 10 min. The experimental parameters were set as follows: temperature 30℃, oscillation speed 1,000 rpm. The detection procedure was as follows: Baseline equilibration: The sensor was equilibrated in AssayBuffer for 60 s. Protein curing: Biotinylated receptor proteins (IL-2Rβ, IL-7Rα, or IL-2Rγ) were loaded onto the surface of the SA sensor, and the curing time was set to 120-300 s until the signal height reached saturation (approximately 1.0 nm). Baseline washing: The protein-cured sensor was washed in AssayBuffer for 60 s to remove unbound proteins. Binding and dissociation: The sensor was immersed in a solution containing different concentration gradients of paeoniflorin (Alb) for binding (60 s), and then quickly transferred to AssayBuffer for dissociation (60 s). For the IL-2Rγ assay, the Alb concentration gradient was set at 62.5, 125, 250, 500, and 1000 μM. For the weaker affinity IL-2Rβ assay, the optimized Alb concentration gradient was 350, 570, 860, 1300, and 2000 μM. Control settings: A buffer solution containing only DMSO solvent was set as a dual reference control to subtract background drift and non-specific binding signals.

[0063] (3) Data fitting: Data processing was performed using the system's accompanying Data Analysis 2019 software. After subtracting the reference sensor signal, the steady-state affinity model was used to perform nonlinear fitting between the equilibrium response value and the drug concentration based on the shape of the binding and dissociation curves, and the dissociation equilibrium constant (KD) value was calculated to evaluate the binding strength of paeoniflorin to each receptor subunit.

[0064] 2. Experimental Results Experimental results showed that Alb preferentially and specifically binds to the receptor common chain IL-2Rγ (i.e., γc). Kinetic analysis showed that with increasing Alb concentration gradient (62.5 μM - 1000 μM), its binding response to the IL-2Rγ subunit exhibited a typical concentration-dependent increasing trend. Figure 3 A). The dissociation equilibrium constant (K) of Alb and IL-2Rγ(γc) was calculated. D The concentration was 93.4 μM, confirming that the two have clear molecular recognition and moderate physical binding potential.

[0065] Comparative analysis revealed the high selectivity of Alb for different subunits. Although Alb also showed some binding activity with the IL-2Rβ subunit ( Figure 3 (B) However, at the same concentration gradient, its binding response value was relatively low, at 869 μM. In comparison, Alb's affinity for IL-2Rγ (γc) is approximately 9.3 times that for IL-2Rβ, indicating that the γc chain is its preferentially recognized core subunit. Crucially, the BLI experiment effectively eliminated false positive biases predicted by theory. Although molecular docking predicted that Alb had the strongest theoretical binding energy to IL-7Rα, under the same detection conditions, no specific binding signal was observed between Alb and IL-7Rα protein, confirming the target selectivity of Alb's binding behavior.

[0066] In summary, this experiment confirms at the molecular level that Alb exerts its therapeutic effect by targeting and binding to the shared chain IL-2Rγ. Given that IL-2Rγ is a core subunit essential for IL-2 and IL-7 signal transduction, this selective "occupation" by Alb can block the driving force of GM-CSF. + This discovery reveals a key signaling pathway for Th cell differentiation. It not only corrects the biases of static simulations but also provides conclusive biophysical evidence for Alb, a core component of TGP, regulating adaptive immunity.

[0067] To elucidate the regulatory logic of Alb on the γc pathway at the molecular level, the dynamic changes of key kinases and downstream effector GM-CSF in HuT102 cells were detected using RT-qPCR and Western blot techniques, respectively.

[0068] Example 4: Western Blot Detection of Inhibition of JAK3 and STAT5 Phosphorylation 1. Experimental Methods (1) Total protein extraction and quantification: HuT 102 cells were seeded in 24-well plates (1.2 × 10⁻⁶ m² / well). 6Cells were treated with Alb at final concentrations of 10, 20, and 40 μM (cells / mL), with a solvent control group included. Cells were incubated for 24 h. After treatment, cells were collected and washed twice with pre-chilled DPBS. RIPA lysis buffer containing 1% PMSF and a protease phosphatase inhibitor was added, and cells were lysed on ice for 30 min, vortexing every 10 min to ensure complete lysis. Cells were centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatant was carefully transferred to new EP tubes. Protein concentration was determined using a BCA protein quantification kit, and the concentration of each group was adjusted to the same level based on the results. 5× SDS-PAGE loading buffer was added, and the cells were boiled in a metal bath at 100 °C for 10 min to denature the proteins. After aliquoting, the cells were stored at -80 °C for later use.

[0069] (2) SDS-PAGE electrophoresis: Prepare a 10% SDS-PAGE separating gel and a 5% stacking gel according to the molecular weight of the target proteins. Add the prepared protein samples to the wells, with a sample volume of 5 μg per well. Connect the electrophoresis apparatus, set the initial voltage to 80 V, and after the bromophenol blue indicator band enters the separating gel, adjust the voltage to 120 V and continue electrophoresis until the bromophenol blue runs off the bottom of the gel or the target protein is separated to a suitable position.

[0070] (3) Transfer: After electrophoresis, peel off the gel and remove any excess. Activate the PVDF membrane by soaking it in methanol for 30 seconds, then equilibrate it together with filter paper and a sponge pad in transfer buffer. Assemble the transfer clamp in the order of "sponge pad-filter paper-gel-PVDF membrane-filter paper-sponge pad" (be careful to remove air bubbles) and place it in the transfer tank. Place it in an ice-water bath and transfer at a constant current of 250mA for 150 min.

[0071] (4) Blocking and Antibody Incubation: After transfer, remove the PVDF membrane and rinse it once quickly with TBST buffer. Place the membrane in 5% dedicated blocking buffer (Beyotime) and block on a shaker at room temperature for 30 min. After blocking, wash the membrane three times with TBST, 10 min each time. Dilute the target protein antibody and internal control antibody (β-actin) with the primary antibody dilution buffer according to the dilution ratio shown in the experimental materials above, and place the membrane in an antibody incubation box and incubate overnight at 4 °C.

[0072] (5) Secondary antibody incubation and development: The next day, the primary antibody was recovered, and the membrane was washed 3 times with TBST for 10 min each time. HRP-labeled secondary antibody of the corresponding species (goat anti-rabbit) (dilution ratio 1:20000) was added and incubated on a shaker at room temperature for 2 h. The secondary antibody was discarded, and the membrane was washed 3 times with TBST for 10 min each time.

[0073] Mix solution A and solution B of the ECL chemiluminescence detection kit at a 1:1 ratio, and drop the mixture evenly onto the PVDF membrane. After reacting for 1-2 minutes, use a gel imaging system (Bio-Rad ChemiDoc) for exposure imaging.

[0074] (6) Image analysis: ImageJ software was used to analyze the grayscale values ​​of the bands. The grayscale ratio of the target protein to the internal reference protein was normalized.

[0075] 2. Experimental Results At both the translational and activation levels, Alb significantly inhibited the phosphorylation switch of the JAK3 / STAT5 cascade. Western blot and grayscale analysis (…) Figure 4 B, C) reveal a deeper role: at the total protein level, JAK1 and JAK3 The expression of Alb decreased significantly with increasing Alb concentration. P <0.01), and showed a decreasing trend with increasing drug concentration; while the total protein level of STAT5 did not change significantly in the low and medium concentration groups, and only showed a slight downregulation in the high concentration (40 μM) group. P <0.05). More importantly, Alb significantly inhibited the activated state of key kinases. Quantitative results showed that both the p-JAK3 / JAK3 ratio and the p-STAT5 / STAT5 ratio decreased significantly with increasing Alb concentration ( P <0.01 or P <0.001), indicating that Alb significantly weakens JAK3's ability to drive phosphorylation of downstream STAT5.

[0076] Example 5: Real-time quantitative PCR (RT-qPCR) detection of inhibition of downstream gene expression 1. Experimental Methods HuT 102 cells were seeded in 24-well plates (1.2 × 10⁻⁶). 6Cells were treated with Alb at final concentrations of 10, 20, and 40 μM, respectively, with a solvent control group included. Cells were incubated for 24 h. After treatment, the cell culture supernatant was removed, and cells were washed twice with pre-chilled DPBS buffer. Total RNA was extracted from HuT102 cells in each group strictly according to the instructions of the cell / tissue total RNA extraction kit (Yisheng Biotechnology, Shanghai). RNA concentration and purity were measured using a NanoDrop spectrophotometer to ensure... A 260 / A280 The ratio was kept between 1.8 and 2.0 to ensure RNA quality. Subsequently, 1 μg of total RNA was used as a template and cDNA was synthesized by reverse transcription using a reverse transcription kit (Yugong Biotechnology, Beijing).

[0077] Amplification reactions were performed using the SYBR Green dye method (iScience) on a Bio-Rad CFX96 real-time quantitative PCR instrument. The reaction mixture (total 20 μL) contained 10 μL of 2 × SYBR Green Master Mix, 0.8 μL each of forward and reverse primers, 2 μL of cDNA template, and 6.4 μL of nuclease-free water. The amplification program was: 95 °C pre-denaturation for 30 s; followed by cycling: 95 °C denaturation for 5 s, 60 °C annealing / extension for 30 s, for a total of 40 cycles. After the reaction, the amplification was... GAPDH As an internal reference gene, 2 -ΔΔCt Method for calculating the target gene ( JAK1, JAK3, STAT5A and CSF2 The relative expression levels of ) are shown in Table 1. Primer sequences are detailed in Table 1.

[0078] Table 1 Primer sequences used in this experiment for real-time quantitative PCR 2. Experimental Results At the transcriptional level, Alb comprehensively downregulates the expression of core genes in the signaling axis. RT-qPCR results showed ( Figure 4 A), after treatment with Alb (10, 20, 40 μM) for 24 h, HuT102 cells JAK1, JAK3 as well as STAT5A The relative expression levels of mRNA in all samples decreased significantly. P <0.05), and showed a clear dose-dependent effect. Correspondingly, the downstream target genes encoding GM-CSF... CSF2mRNA levels also decreased synchronously. This result confirms that Alb can effectively block the activation of the γc / JAK3 / STAT5 signaling axis at the transcriptional source.

[0079] The results from Examples 4 and 5 show that Alb regulates GM-CSF production through a "cascade blockade" mechanism. Experiments confirm that Alb synergistically inhibits the transcription and protein synthesis of key kinases and blocks the crucial step of phosphorylation into the nucleus. Combined with the specific binding of Alb to γc (IL-2Rγ) confirmed by the aforementioned BLI experiment, this invention fully elucidates its mechanism of action: Alb targets and binds to γc → interferes with JAK3 recruitment and activation → blocks STAT5 phosphorylation into the nucleus → ultimately downregulates GM-CSF production at the transcriptional level.

[0080] The previous article confirmed that paeoniflorin can specifically inhibit the differentiation of GM-CSF⁺ Th cells. In order to further explore whether this cellular-level regulatory effect can be translated into actual therapeutic effect on autoimmune arthritis, the following article further conducts in vivo systemic pharmacodynamic verification in a CIA animal model.

[0081] Example 6: The effect of paeoniflorin on the arthritis phenotype in CIA mice 1. Experimental Methods (1) CIA model establishment and experimental grouping Eight-week-old male DBA / 1 mice were used to establish a collagen-induced arthritis (CIA) model according to the following method: (1.1) Experimental Animals: To simulate the pathophysiological characteristics of the preclinical stage of human rheumatoid arthritis (RA) in a controlled experimental system, 8-week-old male DBA / 1J mice (weighing 18-22 g) were purchased from Beijing Huafukang Laboratory Animal Technology Co., Ltd. All animal experiments were approved by the Laboratory Animal Ethics Committee of West China Hospital, Sichuan University (Approval No.: 20230427001). Mice were housed in an SPF-grade animal room with a constant temperature (22 ± 2 °C) and constant humidity (55% ± 5%), and a 12-hour light / dark cycle was implemented.

[0082] (1.2) Preparation of immunoemulsifier: All modeling reagents were purchased from Chondrex (USA). Immunograde chicken type II collagen (Chick Type II Collagen, Cat# 20011) was dissolved in 0.05 mol / L acetic acid solution to prepare a collagen solution of 2 mg / mL, and incubated overnight at 4 °C on a shaker to ensure complete dissolution.

[0083] Primary immunization emulsion: Under ice bath conditions, the collagen solution is mixed with an equal volume of complete Freund's adjuvant (CFA, Cat#7023, containing 5 mg / mL Mycobacterium tuberculosis) and fully emulsified using a high-speed homogenizer until the emulsion clumps and floats in water without spreading.

[0084] Enhanced immune emulsion: Emulsify the collagen solution with an equal volume of incomplete Freund's adjuvant (IFA, Cat# 7002) as described above.

[0085] (1.3) Immunization Modeling Procedure: Primary Immunization (Day 0): After isoflurane-induced anesthesia, the primary immunization emulsion (100 μL / mouse) was injected intradermally at two points in the buttocks (0.5 cm from the tail root). Booster Immunization (Day 21): 100 μL / mouse of booster immunization emulsion was injected intradermally in the buttocks, avoiding the original injection point, to induce typical arthritis. On day 7 after the primary immunization (Day 7), the mice were randomly divided into 3 groups: Normal Control Group (Ctrl, n = 5): No immunization modeling was performed; mice were fed routinely. Model Solvent Group (CIA, n = 9): Immunization modeling was performed, and an equal volume of physiological saline was administered. Paeoniflorin Intervention Group (Alb, n = 9): Immunization modeling was performed, and paeoniflorin intervention was administered.

[0086] (2) Dosing regimen and discontinuation observation strategy This experiment employed a strategy of preventative treatment followed by observation after drug withdrawal to investigate the drug's ability to block disease progression and maintain efficacy. Dosage and route of administration: The Alb group was administered 80 mg / (kg·d) via intraperitoneal injection (ip); the model group received an equal volume of physiological saline via intraperitoneal injection. Administration period: Administration began on Day 7 after initial immunization, once daily, and continued until Day 35. Afterward, administration was discontinued, and mice were fed routinely while the disease progression was monitored until the experiment concluded on Day 49.

[0087] (3) Arthritis Clinical Score Clinical scores were assessed on the joints of the limbs of mice every two days starting from Day 21. Arthritis scores were recorded using a 0–4 scale, and morbidity was calculated. Symptom fluctuations were closely monitored from Day 35–49 (drug withdrawal period) to evaluate the durability of Alb's efficacy.

[0088] 2. Experimental Results Collagen-induced arthritis (CIA) is a classic animal model of rheumatoid arthritis (RA). Clinical phenotypic assessment results showed that Alb significantly delayed the progression of arthritis in CIA mice. Figure 5 As shown in Figure B, compared with the model group, the cumulative incidence rate curve of the disease in the Alb intervention group mice shifted significantly to the right, and the incubation period of the disease was significantly prolonged. Observation of hind limb paw morphology revealed that Alb treatment significantly alleviated the redness and deformity of the hind limbs in CIA mice. Figure 5 C). Dynamic scoring analysis confirms ( Figure 5 D), the clinical arthritis score of the Alb group was significantly lower than that of the model group from the early stage of the disease, and remained at a lower level. P <0.0001).

[0089] Example 7: Effect of paeoniflorin on bone microstructure improvement in CIA mice 1. Experimental Methods Continuing the experiment from Example 1, mice were sacrificed at the experimental endpoint (Day 49), and a sample from the right hind limb ankle joint was isolated and fixed in 4% paraformaldehyde. Scanning and three-dimensional reconstruction were performed using a Micro-CT system. The region of interest (ROI) was selected at the distal tibial metaphysis and ankle joint. Parameters such as bone mineral density (Tb.BMD) and trabecular bone number (Tb.N) were calculated using the system software to quantitatively evaluate the protective effect of Alb against inflammatory bone destruction.

[0090] 2. Experimental Results Micro-CT imaging and quantitative analysis of three-dimensional reconstruction confirmed the osteoprotective effect of Alb. The ankle joints of the model group mice exhibited typical "worm-eaten" osteolytic destruction, accompanied by severe joint space fusion and cortical bone defects; Alb intervention effectively blocked this pathological process and maintained the relative integrity of the trabecular bone morphology. Figure 5 E). The corresponding quantitative analysis results show ( Figure 5 In the Alb group, both bone mineral density (Tb.BMD) and trabecular bone number (Tb.N) significantly increased compared to the model group. P <0.01 or P <0.001). The effectiveness of Alb in inhibiting bone destruction was confirmed at the microstructural level.

[0091] The results from Examples 6 and 7 show that paeoniflorin can significantly prevent arthritis in CIA model mice, as evidenced by a significant reduction in clinical arthritis scores, a significant reduction in paw swelling, and a significant improvement in bone destruction. Furthermore, the joint-protective effect of paeoniflorin can be maintained after drug withdrawal without significant rebound.

[0092] In summary, this invention discovers that GM-CSF+ Abnormal activation of Th cells significantly precedes the appearance of clinical symptoms of rheumatoid arthritis (RA) on the timeline, making them a key early initiating factor driving RA disease and a core target for early intervention strategies. Furthermore, it was discovered that paeoniflorin (Alb) can inhibit the differentiation of GM-CSF⁺ Th cells by targeting and binding to the interleukin-2 receptor γ chain to block the γc / JAK3 / STAT5 signaling pathway, downregulating CSF2 gene transcription and GM-CSF protein expression, thereby ultimately preventing the occurrence of CIA arthritis without rebound after drug withdrawal. This invention provides a novel intervention drug with high safety, suitable for long-term use, and effective in regulating immune abnormalities in the pre-RA stage, thus preventing rheumatoid arthritis, with broad application prospects.

Claims

1. Inhibiting GM-CSF + Use of agents that inhibit Th cell differentiation in the preparation of a medicament for the prevention and / or treatment of rheumatoid arthritis.

2. The application according to claim 1, characterized in that, The drugs mentioned above for preventing rheumatoid arthritis are those that inhibit the development of rheumatoid arthritis in high-risk individuals with pre-RA or reduce the risk of developing rheumatoid arthritis in high-risk individuals with pre-RA.

3. The application according to claim 1, characterized in that, The agent inhibits GM-CSF + The agent for Th cell differentiation is an agent for inhibiting GM-CSF by targeting the binding of interleukin-2 receptor gamma chain + The agent for Th cell differentiation.

4. The application according to claim 3, characterized in that, The agent for inhibiting GM-CSF + The agent for inhibiting Th cell differentiation is to block the γc / JAK3 / STAT5 signal pathway by targeting the binding of interleukin-2 receptor γ chain, down-regulate the transcription of CSF2 gene and the expression of GM-CSF protein, so as to inhibit GM-CSF + The agent for inhibiting Th cell differentiation.

5. The application according to claim 1, characterized in that, The inhibition of GM-CSF + The reagent for Th cell differentiation is paeoniflorin.

6. The application according to any one of claims 1-5, characterized in that, The drug is a preparation made of paeoniflorin as the active ingredient and pharmaceutically acceptable auxiliary ingredients; preferably, the preparation is a tablet, capsule, granule, injection or oral liquid.

7. Paeoniflorin in the preparation of GM-CSF inhibitors + Application in preparations for Th cell differentiation.

8. The application according to claim 7, characterized in that, The inhibition of GM-CSF + The preparations for Th cell differentiation are drugs for the prevention and / or treatment of autoimmune diseases, preferably including rheumatoid arthritis, multiple sclerosis, and type 1 diabetes.

9. Application of paeoniflorin in the preparation of interleukin-2 receptor γ chain inhibitors.

10. The application according to claim 9, characterized in that, The interleukin-2 receptor γ chain inhibitor is a drug for the prevention and / or treatment of autoimmune diseases. Preferably, the autoimmune diseases include rheumatoid arthritis, multiple sclerosis, and type 1 diabetes.

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