Application of silibinin combined stem cells in treatment of rheumatoid arthritis

Abstract

The invention discloses application of silibinin combined stem cells in treatment of rheumatoid arthritis, and relates to the technical field of biological medicines. The silybin is applied to the composition for treating the rheumatoid arthritis, the composition comprises umbilical cord-derived mesenchymal stem cells and the silybin, and the concentration of the silybin is 10-200 [mu] mol / L. The silybin and the umbilical cord-derived mesenchymal stem cells are combined for use, and the release of inflammatory factors can be remarkably inhibited by regulating the interaction of ICOS / ICOSL axes, so that the symptoms of rheumatoid arthritis are effectively relieved; due to the synergistic effect, the treatment effect is enhanced, and the limitation of a single treatment means is reduced; the silybin is used as a flavonoid compound extracted from traditional Chinese medicines, and has the characteristics of good safety and low side effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of silymarin combined with stem cells in the treatment of rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease that primarily affects the synovium, cartilage, and bone tissue of the hands, feet, and knees. Its main pathogenic mechanism is a systemic and local inflammatory response of the synovium, caused by an imbalance in immune homeostasis. Current clinical treatments for RA include disease-modifying antirheumatic drugs (DMARDs), nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids (GCs), and traditional Chinese medicine, among other interventions; however, the overall efficacy remains unsatisfactory.

[0003] The treatment strategy for rheumatoid arthritis (RA) is complex due to its high heterogeneity, unclear pathogenesis, and tissue specificity. Therefore, combination therapy is considered a potential direction for improving efficacy. In a mouse model of coronary artery disease (CIA), combined administration of bone marrow-derived MSCs and IL-4 significantly improved RA symptoms. However, in clinical practice, combination therapy faces many challenges, such as dosage optimization, safety, potential drug interactions, and side effects, especially a lack of systematic understanding of its mechanisms of action.

[0004] To optimize the strategy of treating rheumatoid arthritis (RA) with UC-MSCs, this invention discovered significantly elevated ICOS expression in peripheral immune cells of non-responsive patients. Through in vitro co-culture systems and cytokine detection, this invention confirmed that ICOS binding to ICOSL on the surface of UC-MSCs activates T cells and promotes the release of inflammatory factors through the PI3K / AKT pathway. This invention further validated this mechanism using engineered cell lines and patient samples. This invention aims to explore novel strategies for managing rheumatoid arthritis by inhibiting this pathway. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing an application of silymarin combined with stem cells in the treatment of rheumatoid arthritis.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] An application of silybin combined with stem cells in the treatment of rheumatoid arthritis, wherein silybin is applied to a composition for treating rheumatoid arthritis, the composition comprising umbilical cord-derived mesenchymal stem cells and silybin, wherein the concentration of silybin is 10-200 μmol / L.

[0008] Preferably, the umbilical cord-derived mesenchymal stem cells are 5th-7th generation cells.

[0009] Preferably, the method for preparing the composition includes the following steps:

[0010] Silymarin was dissolved in a mixture containing 35% PEG400, 15% Cremophor EL, 5% ethanol and 45% physiological saline to form a solution.

[0011] Preferably, the umbilical cord-derived mesenchymal stem cells are co-cultured in vitro with peripheral blood mononuclear cells from RA patients, and silymarin is added to the co-culture system to evaluate their immunomodulatory function.

[0012] Preferably, the silymarin reduces the release of inflammatory factors IL-2, IL-21, and IFN-γ by competitively binding to ICOS.

[0013] Preferred method: The therapeutic effect of silymarin combined with stem cells is verified through experiments. The experimental steps include:

[0014] S1: Collection of data from RA patients, healthy controls, and PBMCs;

[0015] S2: Construction and treatment of CIA mouse model;

[0016] S3: In vitro co-culture system of UC-MSCs, silymarin and PBMCs;

[0017] S4: Construction of ICOS knockdown, overexpression and ICOSL knockdown cell lines;

[0018] S5: Real-time quantitative PCR;

[0019] S6: Western Blot;

[0020] S7: ELISA detection and micro-CT imaging;

[0021] S8: Flow cytometry and antibodies;

[0022] S9: Surface plasmon resonance experiment;

[0023] S10: Molecular simulation;

[0024] S11: Statistical analysis.

[0025] Preferably, in S2, the experiment is divided into two parts:

[0026] Part 1: Mice were randomly divided into 5 groups of 8 mice each: control group, CIA group, CIA+UC-MSC group, CIA+sh-ICOSLUC-MSC group, and CIA+UC-MSC+PI3K inhibitor group.

[0027] Part 2: Mice were randomly divided into 7 groups of 8 mice each: control group, CIA group, CIA+UC-MSC group, CIA+Silybin group, CIA+UC-MSC+Silybin group, CIA+anti-ICOSL antibody group and CIA+anti-ICOSL antibody+UC-MSCs group.

[0028] Preferably, in step S4, the construction of ICOS knockdown, overexpression, and ICOS knockdown cell lines specifically includes: plasmid construction and lentiviral packaging and infection, wherein the lentiviral packaging and infection are performed in the following manner:

[0029] HEK293T cells were seeded in T25 culture flasks. When the cell density reached 70-80%, PSPAX, PMD 2g, and plasmid were transfected with Lipofectamine 3000 in a molar ratio of 1:1:1, with a total amount of 5μg. After 48 hours, the viral supernatant was collected, filtered through a 0.45μm filter membrane, and used to infect Jurkat cells or UC-MSCs. After 48 hours of infection, puromycin was added for selection for 1 week to establish stable knockdown or overexpression cell lines.

[0030] Preferably, in step S6, Western blotting specifically includes:

[0031] Cells were collected, lysed with RIPA containing protease and phosphatase inhibitors, proteins were extracted, separated by SDS-PAGE, and transferred to a 0.45 μm PVDF membrane; blocked with 5% skim milk powder for 2 hours, and incubated overnight at 4°C with primary antibody; the secondary antibody was HRP-labeled anti-mouse or rabbit IgG; ECL was used for imaging, and ImageJ software was used for quantification.

[0032] Preferably, in step S7, ELISA detection and micro-CT imaging specifically include:

[0033] The levels of cytokines IL-2, IL-21, IFN-γ, IL-17, TNF-α, and MMP-9 in cell culture supernatant and mouse serum were measured according to the ELISA kit instructions. Mice were sacrificed on day 32, and their hind legs were fixed in paraformaldehyde for micro-CT scanning.

[0034] The beneficial effects of this invention are as follows:

[0035] 1. The present invention combines silymarin with umbilical cord-derived mesenchymal stem cells, which can significantly inhibit the release of inflammatory factors by regulating the interaction of the ICOS / ICOSL axis, thereby effectively alleviating the symptoms of rheumatoid arthritis; this synergistic effect not only enhances the therapeutic effect, but also reduces the limitations of single treatment methods.

[0036] 2. In a mouse CIA model, the combined strategy of this invention not only significantly alleviated arthritis symptoms but also delayed the disease progression and improved bone structure. These results indicate that the combined approach has superior performance in immune regulation and joint protection, demonstrating strong clinical translational potential.

[0037] 3. The silymarin used in this invention is a flavonoid compound extracted from traditional Chinese medicine, which has good safety and low side effects. Therefore, the combined treatment regimen improves efficacy while ensuring high safety. Attached Figure Description

[0038] Figure 1 A flowchart for establishing the CIA model of this invention;

[0039] Figure 2 This is a comparison chart of arthritis scores and foot swelling between the combined treatment group and the single treatment group of the present invention;

[0040] Figure 3 This is a comparison image of the Micro-CT scan of the present invention;

[0041] Figure 4 This is a graph showing the analysis of bone mineral density parameters in this invention;

[0042] Figure 5 This is a histopathological analysis diagram of the present invention. Detailed Implementation

[0043] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0044] Example 1:

[0045] An application of silymarin combined with stem cells in the treatment of rheumatoid arthritis was investigated. The therapeutic efficacy of silymarin combined with stem cells for rheumatoid arthritis was verified experimentally. The specific experimental methods are as follows:

[0046] S1: Collection of data from RA patients, healthy controls, and PBMCs

[0047] All RA patients and healthy controls were from Gulou Hospital affiliated with Nanjing University School of Medicine. Twenty-six RA patients were diagnosed according to the 2010 American College of Rheumatology diagnostic criteria for rheumatoid arthritis. Exclusion criteria included concurrent infection or tumors. Ten healthy controls were age-matched women with normal blood tests and no history of autoimmune diseases or infections. RA was diagnosed using Ficoll density gradient centrifugation (Lymphoprep). TMPeripheral blood mononuclear cells (PBMCs) were isolated from RA patients and healthy individuals using the 07851 STEMCELL (STEMCELL). This study was approved by the Ethics Committee of Gulou Hospital Affiliated to Nanjing University School of Medicine (Approval No.: 2021-544-01), and all participants signed informed consent forms.

[0048] S2: Construction and treatment of CIA mouse model

[0049] Eight-week-old female DBA / 1J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in a specific pathogen-free (SPF) environment. The animal experiments consisted of two parts:

[0050] Part 1: Mice were randomly divided into 5 groups of 8 mice each: control group, CIA group, CIA+UC-MSC group, CIA+sh-ICOSLUC-MSC group, and CIA+UC-MSC+PI3K inhibitor group.

[0051] On day 0, bovine type II collagen (bCII) was emulsified with complete Freund's adjuvant (CFA) and subcutaneously injected at the base of the tail to induce the CIA model; on day 21, bCII / incomplete Freund's adjuvant (IFA) was used for a booster immunization.

[0052] On day 1, UC-MSCs were obtained from the Jiangsu Provincial Stem Cell Center, using passages 5-7. On days 18 and 25, 5×10⁵ cells were injected via tail vein. 5 Mice in the CIA+UC-MSC+PI3K group were administered 1 mg / kg of PI3K inhibitor orally during the same period.

[0053] Part 2: Mice were randomly divided into 7 groups of 8 mice each: control group, CIA group, CIA+UC-MSC group, CIA+Silybin group (150 mg / kg), CIA+UC-MSC+Silybin group (150 mg / kg), CIA+anti-ICOSL antibody group (3 mg / kg) and CIA+anti-ICOSL antibody+UC-MSCs group.

[0054] The immunization procedure was the same as above. UC-MSCs were administered via tail vein injection on days 18 and 25; the anti-ICOSL antibody group received injections twice weekly at a dose of 3 mg / kg.

[0055] Silybin, C 25 H 22 O 10Silybin (98% purity) was purchased from Shanghai Maclean Chemical Technology Co., Ltd.; PEG400 was purchased from Tokyo Chemical Co., Ltd.; CremophorEL was purchased from Aladdin Reagent Co., Ltd. Silybin was dissolved in a mixture containing 35% PEG400, 15% CremophorEL, 5% ethanol, and 45% physiological saline. From day 1 to day 32, silybin was administered by gavage at a dose of 150 mg / kg daily.

[0056] Following immunization, mice were observed daily for arthritis scores (0 = normal, 1 = single toe joint swelling, 2 = multiple toe joint swelling or mild paw swelling, 3 = entire paw swelling, 4 = severe swelling). All animal experiments complied with the requirements of the Ethics Committee of Nanjing University Gulou Hospital (Approval No.: 2020AE01061).

[0057] S3: In vitro co-culture system of UC-MSCs, silymarin, and PBMCs

[0058] After counting, UC-MSCs were seeded at the bottom of a plate and allowed to adhere to the walls before PBMCs were added. After a period of time, the suspended PBMCs and culture medium were collected for subsequent experiments.

[0059] S4: Construction of ICOS knockdown, overexpression, and ICOS knockdown cell lines

[0060] Plasmid construction:

[0061] Human peripheral blood cDNA was extracted, and the ICOS and ICOSL genes were amplified by PCR. These genes were then cloned into a third-generation lentiviral vector via homologous recombination and identified by Sanger sequencing. ShRNA target sequences for ICOS and ICOSL were designed, primers were synthesized and annealed to form double-stranded DNA, which was then cloned into the PLKO.1-TRC vector.

[0062] Lentiviral Packaging and Infection:

[0063] HEK293T cells were seeded in T25 culture flasks. When the cell density reached 70-80%, PSPAX, PMD 2g, and plasmids (pLKO.1 or pLenti) were transfected with Lipofectamine 3000 at a molar ratio of 1:1:1, with a total amount of 5 μg. Viral supernatant was collected after 48 hours, filtered through a 0.45 μm filter, and used to infect Jurkat cells or UC-MSCs. Puromycin was added 48 hours after infection, and selection was performed for one week to establish stable knockdown or overexpression cell lines.

[0064] S5: Real-time quantitative PCR (qPCR)

[0065] Cells were lysed with 1 mL of TRIzol, placed on ice, and then chloroform was added, followed by vortexing and centrifugation. The supernatant was collected, mixed with an equal volume of isopropanol, and centrifuged again. The obtained cDNA was diluted with DEPC water, and the results were analyzed.

[0066] S6: Western Blot

[0067] Cells were collected, lysed with RIPA containing protease and phosphatase inhibitors, proteins were extracted, separated by SDS-PAGE, and transferred to a 0.45 μm PVDF membrane. The membrane was blocked with 5% skim milk powder for 2 hours, and then incubated overnight at 4°C with primary antibody.

[0068] Major antibodies: β-Actin, S6K, AKT, p-AKT (S473), ICOS, ICOSL (all purchased from Abcam). Secondary antibody was HRP-labeled anti-mouse or rabbit IgG. Development was performed using ECL, and quantification was performed using ImageJ software.

[0069] S7: ELISA Detection and Micro-CT Imaging

[0070] The levels of cytokines such as IL-2, IL-21, IFN-γ, IL-17, TNF-α, and MMP-9 in cell culture supernatant and mouse serum were detected according to the ELISA kit instructions.

[0071] Mice were sacrificed on day 32, and their hind legs were fixed in paraformaldehyde for micro-CT scans.

[0072] S8: Flow Cytometry and Antibodies

[0073] Cellular apoptosis dyes and surface antigens were first used for staining, followed by fixation and permeabilization. Intracellular or nuclear labeling was then performed using staining buffer provided by Thermo Fisher Scientific. Flow cytometry analysis was performed using a BDFACSAriaII instrument, and data were analyzed using FlowJov 10.0.7.

[0074] S9: Surface Plasmon Resonance (SPR) Experiment

[0075] Install the CM5 chip using the Biacore8k system and calculate Rmax (maximum binding value) according to the formula: Rmax = analyte molecular weight / ligand molecular weight × RL × Sm.

[0076] The ligand protein was diluted with acetate buffer at different pH values, and the optimal pH for coupling conditions was determined after testing. Silybin samples were serially diluted (200 to 6.25 μM, including 0). Data were analyzed using Biacore Insight Evaluation software.

[0077] S10: Molecular Simulation

[0078] Obtain the ICOS protein structure from https: / / www.rcsb.org and the silymarin molecular structure from https: / / pubchem.ncbi.nlm.nih.gov. Use Discovery Studio software to predict its binding site.

[0079] Histopathological analysis

[0080] On day 32, mouse hind legs were harvested, fixed in paraformaldehyde, and subjected to HE staining and Safranin-Fix Green double staining. In HE staining, cell nuclei appeared blue and cytoplasm red; in Safranin-Fix Green staining, cartilage appeared red or orange-red, bone tissue appeared green, and some connective tissue appeared red.

[0081] S11: Statistical Analysis

[0082] Statistical analysis was performed using GraphPad or R software; p-values ​​and FDR values ​​less than 0.05 were considered statistically significant.

[0083] result:

[0084] ICOS / ICOSL expression and cytokine dynamics determine the efficacy of UC-MSCs in RA treatment.

[0085] To explore the characteristics of RA patients who did not improve after UC-MSC transplantation, an in vitro co-culture system was established. This system categorized RA patients based on the levels of IL-21, IL-2, and IFN-γ in the supernatant after 24 hours of culture. These cytokines are closely associated with RA progression, and their levels typically decrease significantly during clinical symptom relief. Based on this, 12 of the 26 subjects were identified as UC-MSCs responders, with significantly decreased levels of IFN-γ, IL-21, and IL-2; the remaining 14 were defined as non-responders due to elevated cytokine levels.

[0086] After grouping by cytokine, the expression of co-stimulatory molecules was further examined. These key factors can enhance immune cell activity and may drive the excessive secretion of cytokines such as IFN-γ. qPCR detection of co-stimulatory factors such as CD28, CD27, ICOS, OX-40, and 4-1BB revealed significant differences in their expression between responders and non-responders, and a positive correlation with cytokine levels. Flow cytometry further validated these differences, suggesting that they may affect the therapeutic efficacy of UC-MSCs.

[0087] Considering that the activity of co-stimulatory molecules depends on ligand binding, the expression of ligands such as CD80 / CD86, ICOSL, OX-40L, CD70, and 4-1BBL on UC-MSCs was examined. The results showed that CD80 and ICOSL were highly expressed in MSCs, with ICOSL being the most significant in both expression profile and function. Subsequently, Western blotting and flow cytometry confirmed that ICOS levels were significantly elevated in non-responders of UC-MSCs, and that ICOSL expression in UC-MSCs was higher than in PBMCs and 293T cells. When ICOSL-knockdown UC-MSCs were constructed and used in an in vitro co-culture system, the secretion of IFN-γ, IL-21, and IL-2 was significantly reduced, further demonstrating that the ICOS / ICOSL axis plays a crucial role in regulating therapeutic response.

[0088] In summary, the combined application of UC-MSCs and silybin (SB) demonstrated a synergistic therapeutic effect in a CIA mouse model, as detailed below:

[0089] ( Figure 1 Flowchart for establishing the CIA model: DBA / 1 mice were induced to develop rheumatoid arthritis by intradermal injection of type II collagen on day 0 and day 21, and UC-MSCs, SB or a combination of both were administered during the induction period.

[0090] ( Figure 2 The combined treatment group showed significant improvement in arthritis scores and foot swelling compared to the single treatment group, and the onset of the disease was delayed by about one week.

[0091] ( Figure 3 Micro-CT scans showed that the combined treatment group had the least joint damage and the best preservation of bone structure.

[0092] ( Figure 4 Bone mineral density parameter analysis: The combined treatment group of mice showed increased trabecular bone thickness (Tb.Th) and decreased bone surface area / bone volume ratio (BS / BV), suggesting a reduction in bone destruction.

[0093] ( Figure 5 Histopathological analysis showed that the combined treatment group had significant relief of synovial inflammation and cartilage destruction, indicating that the treatment effect was optimal.

[0094] The results suggest that UC-MSCs combined with Silybin can significantly improve arthritis symptoms and bone destruction in CIA mice, with better efficacy than single-drug treatment.

[0095] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An application of silymarin combined with stem cells in the treatment of rheumatoid arthritis, characterized in that, Silybin is used in a composition for treating rheumatoid arthritis, the composition comprising umbilical cord-derived mesenchymal stem cells and silybin, the concentration of silybin being 10-200 μmol / L.

2. The application of silymarin combined with stem cells in the treatment of rheumatoid arthritis according to claim 1, characterized in that, The umbilical cord-derived mesenchymal stem cells are from the 5th to 7th generation.

3. The application of silymarin combined with stem cells in the treatment of rheumatoid arthritis according to claim 1, characterized in that, The method for preparing the composition includes the following steps: Silymarin was dissolved in a mixture containing 35% PEG400, 15% Cremophor EL, 5% ethanol and 45% physiological saline to form a solution.

4. The application of silymarin combined with stem cells in the treatment of rheumatoid arthritis according to claim 1, characterized in that, The umbilical cord-derived mesenchymal stem cells were co-cultured in vitro with peripheral blood mononuclear cells from RA patients, and silymarin was added to the co-culture system to evaluate its immunomodulatory function.

5. The application of silymarin combined with stem cells in the treatment of rheumatoid arthritis according to claim 1, characterized in that, Silymarin reduces the release of inflammatory factors IL-2, IL-21, and IFN-γ by competitively binding to ICOS.

6. The application of silymarin combined with stem cells in the treatment of rheumatoid arthritis according to claim 1, characterized in that, The therapeutic effect of silymarin combined with stem cells was verified through experiments. The experimental steps included: S1: Collection of data from RA patients, healthy controls, and PBMCs; S2: Construction and treatment of CIA mouse model; S3: In vitro co-culture system of UC-MSCs, silymarin and PBMCs; S4: Construction of ICOS knockdown, overexpression and ICOSL knockdown cell lines; S5: Real-time quantitative PCR; S6: Western Blot; S7: ELISA detection and micro-CT imaging; S8: Flow cytometry and antibodies; S9: Surface plasmon resonance experiment; S10: Molecular simulation; S11: Statistical analysis.

7. The application of silymarin combined with stem cells in the treatment of rheumatoid arthritis according to claim 6, characterized in that, In S2, the experiment is divided into two parts: Part 1: Mice were randomly divided into 5 groups of 8 mice each: control group, CIA group, CIA+UC-MSC group, CIA+sh-ICOSLUC-MSC group, and CIA+UC-MSC+PI3K inhibitor group. Part 2: Mice were randomly divided into 7 groups of 8 mice each: control group, CIA group, CIA+UC-MSC group, CIA+Silybin group, CIA+UC-MSC+Silybin group, CIA+anti-ICOSL antibody group and CIA+anti-ICOSL antibody+UC-MSCs group.

8. The application of silymarin combined with stem cells in the treatment of rheumatoid arthritis according to claim 6, characterized in that, In S4, the construction of ICOS knockdown, overexpression, and ICOS knockdown cell lines specifically includes: plasmid construction and lentiviral packaging and infection, wherein the lentiviral packaging and infection are performed in the following manner: HEK293T cells were seeded in T25 culture flasks. When the cell density reached 70-80%, PSPAX, PMD 2g, and plasmid were transfected with Lipofectamine 3000 in a molar ratio of 1:1:1, with a total amount of 5μg. After 48 hours, the viral supernatant was collected, filtered through a 0.45μm filter membrane, and used to infect Jurkat cells or UC-MSCs. After 48 hours of infection, puromycin was added for selection for 1 week to establish stable knockdown or overexpression cell lines.

9. The application of silymarin combined with stem cells in the treatment of rheumatoid arthritis according to claim 6, characterized in that, In S6, Western Blot specifically includes: Cells were collected, lysed with RIPA containing protease and phosphatase inhibitors, proteins were extracted, separated by SDS-PAGE, and transferred to a 0.45 μm PVDF membrane; blocked with 5% skim milk powder for 2 hours, and incubated overnight at 4°C with primary antibody; the secondary antibody was HRP-labeled anti-mouse or rabbit IgG; ECL was used for imaging, and ImageJ software was used for quantification.

10. The application of silymarin combined with stem cells in the treatment of rheumatoid arthritis according to claim 6, characterized in that, In S7, ELISA detection and micro-CT imaging specifically include: The levels of cytokines IL-2, IL-21, IFN-γ, IL-17, TNF-α, and MMP-9 in cell culture supernatant and mouse serum were measured according to the ELISA kit instructions. Mice were sacrificed on day 32, and their hind legs were fixed in paraformaldehyde for micro-CT scanning.

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