Application of silibinin to enhancement of stem cell immunomodulatory function

Abstract

The invention discloses an application of silibinin in enhancing the immunomodulatory function of stem cells, and relates to the technical field of biological medicines. The silybin is applied to the composition for enhancing the immunoregulation function of the stem cells, the composition comprises the silybin and umbilical cord-derived mesenchymal stem cells, and the immunoregulation function of UC-MSCs is enhanced by adjusting the interaction of ICOS / ICOSL axes based on the combined action of the silybin and the umbilical cord-derived mesenchymal stem cells. The silybin disclosed by the invention is competitively combined with the ICOS, so that the interaction of the ICOS / ICOSL is blocked, and the immune regulation function of the umbilical cord-derived mesenchymal stem cells is remarkably enhanced; therefore, the UC-MSCs show a stronger immunosuppression effect in both in-vitro and in-vivo experiments; silybin not only acts on an ICOS / ICOSL axis, but also inhibits secretion of inflammatory factors through a PI3K / AKT signal channel, so that a multi-mechanism synergistic treatment effect is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of silymarin in enhancing the immunomodulatory function of stem cells. Background Technology

[0002] Over the past decade, human umbilical cord-derived mesenchymal stem cells (UC-MSCs), as pluripotent stem cells, have demonstrated good safety and efficacy in treating autoimmune and inflammatory diseases, emerging as a potential new therapeutic approach. Although UC-MSCs possess the ability to modulate various immune cells, some studies have also reported cases where their treatment was ineffective.

[0003] For example, in a 12-month multicenter clinical follow-up study of SLE, approximately 40% of patients did not show clinical improvement after MSC infusion. In a study of UC-MSCs for RA, 28 patients experienced significant symptom relief, decreased disease activity, and reduced medication dosage within 12 weeks post-transplantation, but 24 patients still showed no significant improvement. Among the factors influencing the efficacy of MSC therapy, the heterogeneity of UC-MSC cell subsets is one of the key determinants, but its full understanding remains lacking.

[0004] Besides the inherent heterogeneity of stem cells, patient-specific immune backgrounds also significantly influence treatment response. For example, we found a close correlation between IFN-γ levels in patients and the efficacy of UC-MSCs. Furthermore, the complex immune dysregulation characteristics of RA patients increase disease heterogeneity and the risk of treatment failure. Multiple molecular epidemiological studies have shown that gene polymorphisms of CD28, CTLA-4, and inducible co-stimulatory molecules (ICOS) are closely related to susceptibility to RA. Mechanistically, ICOS signaling plays a crucial role in the pathogenesis and persistence of collagen-induced arthritis (CIA) in mouse models. Therefore, identifying the immune characteristics of patients with poor responses to stem cell therapy is of great significance. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing an application of silymarin in enhancing the immune regulatory function of stem cells.

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

[0007] An application of silybin in enhancing the immunomodulatory function of stem cells involves incorporating silybin into a composition that enhances the immunomodulatory function of stem cells. The composition comprises silybin and umbilical cord-derived mesenchymal stem cells. Based on the combined effect of silybin and umbilical cord-derived mesenchymal stem cells, the immunomodulatory function of UC-MSCs is enhanced by regulating the interaction of the ICOS / ICOSL axis.

[0008] Preferably, the concentration of silymarin is 100 μmol / L to bind ICOS and block the ICOS / ICOSL interaction.

[0009] Preferably, the combined action of silymarin and umbilical cord-derived mesenchymal stem cells includes their use in an in vitro co-culture system to evaluate their immunomodulatory function.

[0010] Preferably, the umbilical cord-derived mesenchymal stem cells are co-cultured in vitro with peripheral blood mononuclear cells from RA patients to evaluate their immunomodulatory function.

[0011] Preferably, the silybin competitively binds to ICOS to inhibit the ICOS / ICOSL interaction, thereby enhancing the immunomodulatory function of umbilical cord-derived mesenchymal stem cells; the silybin is extracted from the traditional Chinese medicine milk thistle, with a purity controlled at 98%.

[0012] Preferred method: The combined effect of silymarin and umbilical cord-derived mesenchymal stem cells was verified experimentally. The experimental steps included:

[0013] S1: Collection of data from RA patients, healthy controls, and PBMCs;

[0014] S2: Construction and treatment of CIA mouse model;

[0015] S3: In vitro co-culture system of UC-MSCs, silymarin and PBMCs;

[0016] S4: Construction of ICOS knockdown, overexpression and ICOSL knockdown cell lines;

[0017] S5: Real-time quantitative PCR;

[0018] S6: Assess the activation status of signaling pathways;

[0019] S7: Assess the effectiveness of immune regulation.

[0020] Preferably, in S2, the animal experiment is divided into two parts:

[0021] 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.

[0022] On day 0, bovine type II collagen was emulsified with complete Freund's adjuvant and subcutaneously injected at the base of the tail to induce the CIA model; on day 21, immunization was boosted again with bCII / incomplete Freund's adjuvant.

[0023] On day 1, cells from passages 5-7 were used; on days 18 and 25, 5 × 10⁵ cells were injected via tail vein. 5 UC-MSCs or sh-ICOSLUC-MSCs; mice in the CIA+UC-MSC+PI3K group were simultaneously administered 1 mg / kg PI3K inhibitor orally via gavage.

[0024] 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.

[0025] The immunization procedure was the same as in Part 1, and 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.

[0026] Preferably, the umbilical cord-derived mesenchymal stem cells are subjected to ICOSL knockdown treatment before combined action; during the combined action of silymarin and umbilical cord-derived mesenchymal stem cells, the immunomodulatory effect is monitored in real time by detecting cytokine levels.

[0027] Preferably, in the combined action of silymarin and umbilical cord-derived mesenchymal stem cells, Western blotting is used to detect p-AKT and S6K expression to assess the activation of the signaling pathway.

[0028] Preferably, in the combined action of silymarin and umbilical cord-derived mesenchymal stem cells, ELISA is used to detect the level of inflammatory factors to accurately assess the immunomodulatory effect; flow cytometry is used to detect changes in the proportion of immune cell subsets to evaluate the immunomodulatory effect in detail.

[0029] The beneficial effects of this invention are as follows:

[0030] 1. The silymarin of the present invention significantly enhances the immunomodulatory function of umbilical cord-derived mesenchymal stem cells by competitively binding to ICOS and blocking the ICOS / ICOSL interaction; this makes UC-MSCs exhibit stronger immunosuppressive effects in both in vitro and in vivo experiments.

[0031] 2. The silymarin of the present invention not only acts on the ICOS / ICOSL axis, but also inhibits the secretion of inflammatory factors through the PI3K / AKT signaling pathway, thereby achieving a synergistic therapeutic effect through multiple mechanisms.

[0032] 3. The addition of silymarin in this invention makes the treatment effect of UC-MSCs more stable in different patients and reduces the fluctuation of treatment effect due to individual differences; due to the wide availability and relatively low cost of silymarin, the combination therapy not only improves the efficacy but also reduces the overall treatment cost. Attached Figure Description

[0033] Figure 1 This is a schematic diagram illustrating how Silybin significantly inhibited the expression of p-AKT and S6K proteins in the co-culture system of RA patient PBMCs and UC-MSCs of the present invention;

[0034] Figure 2 This is a schematic diagram illustrating how Silybin of the present invention significantly reduces the secretion levels of inflammatory cytokines such as IL-2, IL-21, and IFN-γ in PBMCs of RA patients;

[0035] Figure 3 This diagram illustrates how the inhibitory effect of Silybin on the PI3K / AKT / mTOR pathway depends on ICOS expression, thus proving that the inhibitory effect of Silybin on the PI3K / AKT / mTOR pathway is dependent on ICOS expression. Detailed Implementation

[0036] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0037] Example 1:

[0038] An application of silymarin in enhancing the immunomodulatory function of stem cells, including...

[0039] S1: Collection of data from RA patients, healthy controls, and PBMCs

[0040] 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). TM Peripheral 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.

[0041] S2: Construction and treatment of CIA mouse model

[0042] 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:

[0043] 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.

[0044] 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.

[0045] On day 1, UC-MSCs were obtained from the Jiangsu Provincial Stem Cell Center, using passage 5-7 cells. 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.

[0046] 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.

[0047] 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.

[0048] Silybin, C 25 H 22 O 10 Silybin (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.

[0049] 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).

[0050] S3: In vitro co-culture system of UC-MSCs, silymarin, and PBMCs

[0051] After counting, UC-MSCs were seeded at the bottom of wells and allowed to adhere to the plate. Then, 100 μmol / L Silybin and 10 times the amount of PBMCs were added. After 24 hours, the suspended PBMCs and culture medium were collected for subsequent experiments.

[0052] S4: Construction of ICOS knockdown, overexpression, and ICOS knockdown cell lines

[0053] Plasmid construction:

[0054] Human peripheral blood cDNA was extracted, and the ICOS and ICOSL genes were amplified by PCR. They were then cloned into a third-generation lentiviral vector via homologous recombination and identified by Sanger sequencing.

[0055] The shRNA target sequences of ICOS and ICOSL were designed, primers were synthesized and annealed to form double-stranded DNA, and cloned into the PLKO.1-TRC vector.

[0056] Lentiviral Packaging and Infection:

[0057] HEK293T cells were seeded in T25 culture flasks and transfected with PSPAX, PMD2g, and plasmids using Lipofectamine 3000. Viral supernatant was collected, filtered, and used to infect Jurkat cells or UC-MSCs. Forty-eight hours after infection, puromycin was added for selection to establish stable knockdown or overexpression cell lines.

[0058] S5: Real-time quantitative PCR (qPCR)

[0059] Cells were lysed with 1 mL TRIzol, incubated on ice for 5 min, then mixed with 200 μL chloroform by vortexing, centrifuged at 12000 g for 10 min at 4 °C, and the supernatant was mixed with an equal volume of isopropanol and centrifuged again. The obtained cDNA was diluted 10-fold with DEPC water and analyzed using the 2-(ΔΔCt) method.

[0060] S6: Assess signaling pathway activation using Western blotting.

[0061] Cells were collected, lysed with RIPA containing protease and phosphatase inhibitors, proteins were extracted, separated by SDS-PAGE, and transferred to a PVDF membrane. Cells were blocked for 2 hours and incubated overnight with primary antibody.

[0062] The secondary antibody was HRP-labeled anti-mouse or rabbit IgG. ECL imaging was used, and quantification was performed using ImageJ software.

[0063] S7: Assess the effectiveness of immune regulation.

[0064] The evaluation of immunomodulatory effects includes:

[0065] S71: ELISA Detection

[0066] 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.

[0067] S72: micro-CT imaging

[0068] Mice were sacrificed on day 32, and their hind legs were fixed in paraformaldehyde for micro-CT scans.

[0069] S73: Flow Cytometry and Antibodies

[0070] 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.

[0071] S74: Surface Plasmon Resonance (SPR) Experiment

[0072] 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.

[0073] The ligand protein was diluted to 20 μg / mL with acetate buffer at different pH values ​​(pH 4.5–5.5), and pH 5.5 was determined to be the optimal coupling condition after testing. Silybin samples were serially diluted (200 to 6.25 μM, including 0 concentration), with an injection contact time of 120 seconds, an elution time of 120 seconds, and a flow rate of 10 μL / min. Data were analyzed using BiacoreInsightEvaluation software.

[0074] S75: Molecular Simulation

[0075] 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.

[0076] S76: Histopathological analysis

[0077] 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.

[0078] Statistical analysis was performed using GraphPad or R software. See figure captions for details of the statistical methods used and sample sizes (n). Box plots show the median, quartiles, and extreme values; bar charts are presented as mean ± SEM. In vitro experiments had 3–4 technical replicates per group; animal experiments used a design based on in-arm variability. Data points represent technical replicates (in vitro) or biological replicates (mice). A p-value and FDR value less than 0.05 were considered statistically significant.

[0079] result:

[0080] Silybin enhances the immunomodulatory effect of UC-MSCs in RA treatment by binding to ICOS and inhibiting the PI3K / AKT / mTOR signaling pathway.

[0081] Molecular docking simulations showed that Silybin can bind to two sites on the ICOS protein, suggesting its potential targeting ability.

[0082] Surface plasmon resonance (SPR) experiments verified the binding ability of Silybin and ICOS.

[0083] ( Figure 1 In a co-culture system of PBMCs and UC-MSCs from RA patients, Silybin significantly inhibited the expression of p-AKT and S6K proteins, with effects similar to those of PI3K inhibitors.

[0084] ( Figure 2 Silybin significantly reduced the secretion levels of inflammatory cytokines such as IL-2, IL-21, and IFN-γ in PBMCs of RA patients.

[0085] ( Figure 3In co-culture of UC-MSCs with wild-type Jurkat T cells and ICOS-deficient cells, the inhibitory effect of Silybin on the PI3K / AKT / mTOR pathway was verified to be dependent on ICOS expression.

[0086] The results showed that Silybin can competitively bind to ICOS on the surface of T cells, blocking its binding to ICOSL on UC-MSCs, thereby inhibiting the activation of downstream PI3K / AKT / mTOR signaling pathways, reducing the secretion of inflammatory factors, and improving the therapeutic effect of UC-MSCs.

[0087] 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 in enhancing the immunomodulatory function of stem cells, characterized in that, Silybin is applied to a composition that enhances the immunomodulatory function of stem cells. The composition comprises silybin and umbilical cord-derived mesenchymal stem cells. Based on the combined effect of silybin and umbilical cord-derived mesenchymal stem cells, the immunomodulatory function of UC-MSCs is enhanced by regulating the interaction of the ICOS / ICOSL axis.

2. The application of silymarin in enhancing the immunomodulatory function of stem cells according to claim 1, characterized in that, The concentration of silymarin was 100 μmol / L to bind ICOS and block the ICOS / ICOSL interaction.

3. The application of silymarin in enhancing the immunomodulatory function of stem cells according to claim 1, characterized in that, The combined use of silymarin and umbilical cord-derived mesenchymal stem cells, including in an in vitro co-culture system, was evaluated for its immunomodulatory function.

4. The application of silymarin in enhancing the immunomodulatory function of stem cells 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 to assess their immunomodulatory function.

5. The application of silymarin in enhancing the immunomodulatory function of stem cells according to claim 1, characterized in that, The silybin competitively binds to ICOS to inhibit the ICOS / ICOSL interaction, thereby enhancing the immunomodulatory function of umbilical cord-derived mesenchymal stem cells; the silybin is extracted from the traditional Chinese medicine milk thistle, with a purity controlled at 98%.

6. The application of silymarin in enhancing the immunomodulatory function of stem cells according to claim 1, characterized in that, The combined effect of silymarin and umbilical cord-derived mesenchymal 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: Assess the activation status of signaling pathways; S7: Assess the effectiveness of immune regulation.

7. The application of silymarin in enhancing the immunomodulatory function of stem cells according to claim 6, characterized in that, In S2, the animal 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. On day 0, bovine type II collagen was emulsified with complete Freund's adjuvant and subcutaneously injected at the base of the tail to induce the CIA model; on day 21, immunization was boosted again with bCII / incomplete Freund's adjuvant. On day 1, cells from passages 5-7 were used; on days 18 and 25, 5 × 10⁵ cells were injected via tail vein. 5 UC-MSCs or sh-ICOSLUC-MSCs; mice in the CIA+UC-MSC+PI3K group were simultaneously orally administered 1 mg / kg of PI3K inhibitor; 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. The immunization procedure was the same as in Part 1, and 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.

8. The application of silymarin in enhancing the immunomodulatory function of stem cells according to claim 6, characterized in that, The umbilical cord-derived mesenchymal stem cells were subjected to ICOSL knockdown treatment before the combined action; during the combined action of silymarin and umbilical cord-derived mesenchymal stem cells, the immunomodulatory effect was monitored in real time by detecting cytokine levels.

9. The application of silymarin in enhancing the immunomodulatory function of stem cells according to claim 6, characterized in that, In the combined action of silymarin and umbilical cord-derived mesenchymal stem cells, Western blotting was used to detect p-AKT and S6K expression to assess the activation of signaling pathways.

10. The application of silymarin in enhancing the immunomodulatory function of stem cells according to claim 6, characterized in that, In the combined action of silymarin and umbilical cord-derived mesenchymal stem cells, ELISA was used to detect the level of inflammatory factors to accurately assess the immunomodulatory effect; flow cytometry was used to detect changes in the proportion of immune cell subsets to evaluate the immunomodulatory effect in detail.

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