Application of calycosin-7-glucoside in preparation of medicine for inhibiting Treg cell infiltration in liver cancer

Abstract

The invention discloses application of calycosin-7-glucoside in preparation of a medicine for inhibiting Treg cell infiltration in liver cancer, relates to the field of biological medicine, and discloses application of calycosin-7-glucoside in preparation of a medicine for inhibiting regulatory T cell (Treg) infiltration in hepatocellular carcinoma. The calycosin-7-glucoside realizes the application by inhibiting the activity and / or expression of matrix metalloproteinase 9 (MMP9); according to the application of calycosin-7-glucoside in preparation of the medicine for inhibiting Treg cell infiltration in liver cancer, the new functions of targeting tumor immune microenvironment and specifically inhibiting Treg cell infiltration of the traditional Chinese medicine active ingredient CG are found and confirmed for the first time, the medicinal value of CG is expanded, and a brand new direction is provided for clinical transformation of CG.

Description

Technical Field

[0001] This invention relates to biomedical technology, specifically to the application of verbascoside in the preparation of drugs for inhibiting Treg cell infiltration in liver cancer. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the main type of primary liver cancer, with persistently high global incidence and mortality rates. Despite advancements in treatment methods such as surgical resection, local ablation, transarterial chemoembolization (TACE), and liver transplantation, the high postoperative recurrence and metastasis rate remains a significant clinical challenge. In recent years, immune checkpoint inhibitors, represented by programmed death receptor-1 (PD-1) and its ligand (PD-L1) inhibitors, have brought breakthroughs in the treatment of advanced HCC. However, the overall objective response rate (ORR) remains limited, and most patients face primary or secondary drug resistance.

[0003] The suppressed state of the tumor immune microenvironment (TIME) is a key factor leading to immunotherapy failure. Regulatory T cells (Tregs) that overexpress the transcription factor Foxp3 are abnormally enriched in HCC tumor tissues. Through multiple mechanisms (such as expression of inhibitory molecules CTLA-4 and LAG-3, and secretion of cytokines like IL-10 and IL-35), they strongly suppress the function of effector T cells, driving immune escape. Studies have shown that the degree of Treg cell infiltration is significantly correlated with poor prognosis and immunotherapy resistance in HCC patients. Therefore, developing novel drugs that can specifically target and weaken Treg-mediated immunosuppression is an important direction for improving the efficacy of HCC immunotherapy.

[0004] Verbena isoflavone glycoside (CG) is one of the main active components of Astragalus membranaceus, a traditional Chinese medicine. Existing studies have reported the pharmacological effects of CG in anti-inflammatory, antioxidant, and metabolic regulation, suggesting its potential to improve non-alcoholic fatty liver disease and liver fibrosis. A few studies have indicated that CG has an inhibitory effect on the proliferation of certain tumor cells in vitro. However, to date, no published literature or patents have demonstrated that CG has the function of regulating the immune microenvironment of HCC, particularly targeting Treg cell infiltration. Whether it regulates tumor immunity by affecting specific molecular targets, and its potential as an immunomodulator in combination with existing immunotherapies, remain unreported.

[0005] Matrix metalloproteinase 9 (MMP9) is highly expressed in hepatocellular carcinoma (HCC), and not only promotes tumor invasion and metastasis by degrading the extracellular matrix, recent studies have also revealed its important immunomodulatory functions. MMP9 can activate latent transforming growth factor-β (TGF-β), a core factor inducing the differentiation of naive CD4+ T cells into Treg cells and promoting Treg chemotaxis towards tumor sites. Therefore, MMP9 may be a key node connecting extracellular matrix remodeling and the formation of an immunosuppressive microenvironment.

[0006] Based on this, this invention proposes and verifies for the first time a novel application of CG in reversing the immunosuppressive microenvironment of HCC by directly targeting and inhibiting MMP9, thereby disrupting the MMP9 / TGF-β / Treg axis. This provides a novel drug candidate and strategy for the immunotherapy of HCC. Summary of the Invention

[0007] This invention aims to provide novel pharmaceutical uses for verbascoside (CG), specifically its application in the preparation of medicaments for inhibiting regulatory T cell (Treg) infiltration in hepatocellular carcinoma (HCC) and improving the immunosuppressive tumor microenvironment. Another objective of this invention is to provide an immunomodulatory pharmaceutical composition for HCC.

[0008] To achieve the above objectives, the present invention provides the following technical solution: Through systematic in vitro and in vivo experiments, it has been demonstrated that verbascoside (CG) can inhibit the progression of HCC through the following mechanism:

[0009] Directly targets and inhibits MMP9: CG can specifically bind to the FNII-3 domain of the MMP9 protein, effectively inhibiting its proteolytic enzyme activity.

[0010] Blocking TGF-β activation: By inhibiting MMP9, CG reduces its cleavage and activation of latent TGF-β, thereby downregulating the level of active TGF-β in the tumor microenvironment.

[0011] Inhibition of Treg cell enrichment: Reduced active TGF-β levels lead to weakened differentiation induction of Treg cells and affect the expression of related chemokines, ultimately reducing Treg infiltration in HCC tumor tissues.

[0012] Reversing the immunosuppressive microenvironment: The reduction in Treg cell infiltration relieves its inhibition of effector T cells (such as CD8+ T cells), causing the tumor microenvironment to shift from an immunosuppressive state to an immunoactivated state, thereby enhancing the body's anti-tumor immune response.

[0013] Synergistic immunotherapy: Based on the above mechanism, the combined use of CG with immune checkpoint blockers such as PD-1 / PD-L1 inhibitors can produce a significant synergistic anti-tumor effect.

[0014] Based on the above-mentioned applications, the present invention provides the following specific technical solutions:

[0015] The use of a verbascoside in the preparation of a drug for inhibiting regulatory T cell (Treg) infiltration in hepatocellular carcinoma.

[0016] Preferably, the verbascoside isoflavone glycoside achieves the application by inhibiting the activity and / or expression of matrix metalloproteinase 9 (MMP9).

[0017] More preferably, the verbascoside achieves its application by inhibiting the activation of transforming growth factor-β (TGF-β) by MMP9, thereby reducing the differentiation and intratumoral recruitment of Treg cells.

[0018] The present invention also provides a pharmaceutical composition for treating hepatocellular carcinoma, comprising a therapeutically effective amount of vernix isoflavone glycoside and a pharmaceutically acceptable carrier.

[0019] Preferably, the pharmaceutical composition further comprises an immune checkpoint inhibitor.

[0020] More preferably, the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0021] The pharmaceutical composition may be formulated into any pharmaceutically acceptable dosage form, including but not limited to: oral formulations (such as tablets, capsules, granules), injections (such as injection solutions, lyophilized powder for injection), or targeted delivery systems (such as nanoparticles, liposomes).

[0022] Compared with the prior art, the application of the verbascoside provided by the present invention in the preparation of drugs for inhibiting Treg cell infiltration in liver cancer has the following beneficial effects:

[0023] This invention is the first to discover and confirm that the active ingredient CG in traditional Chinese medicine has a novel function of targeting the tumor immune microenvironment and specifically inhibiting Treg cell infiltration, which expands the medicinal value of CG and provides a new direction for its clinical translation.

[0024] This invention elucidates for the first time the mechanism by which CG regulates the MMP9 / TGF-β / Treg signaling axis by directly binding to and inhibiting MMP9. This mechanism links extracellular matrix metabolism with immune regulation, providing a new perspective for understanding HCC immune escape.

[0025] This invention confirms that the combination of CG and PD-1 inhibitors can produce a synergistic anti-tumor effect, which provides an effective integrated traditional Chinese and Western medicine treatment strategy to overcome the bottleneck of low single-drug response rate in current HCC immunotherapy.

[0026] As a natural small molecule compound, CG has the potential advantages of lower cost and relatively lower toxicity. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0028] Figure 1 A schematic diagram of the predicted interaction and binding sites between CG and MMP9 provided in an embodiment of the present invention;

[0029] Figure 2 A diagram showing the intersection of the MMP9 interacting protein network and its genes related to HCC metastasis and Treg chemotaxis, provided for embodiments of the present invention.

[0030] Figure 3 The figure shows the experimental results of CG inhibiting the growth of HCC orthotopic tumors and Treg infiltration in mice, as provided in the embodiments of the present invention.

[0031] Figure 4 The diagram illustrates the synergistic anti-tumor effect of CG combined with anti-PD-1 antibody, as provided in this embodiment of the invention. Detailed Implementation

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0033] As attached Figure 1 To be continued Figure 4 As shown:

[0034] Figure 1 : Schematic diagram of the predicted interaction between CG and MMP9 and their binding sites;

[0035] The molecular docking results of verbascoside (CG) and matrix metalloproteinase 9 (MMP9) are presented, including:

[0036] The binding sites of CG and MMP9 (Ser342, Ala343, Gly365, Arg366, Gly367, Asp368, Arg370, Trp385, Phe387).

[0037] The binding site is located within the FNII-3 domain (342-390) of MMP9;

[0038] The diagram includes a structural model of the CG-MMP9 complex and a binding energy score (Vina Score < -7 kcal / mol).

[0039] Figure 2 : MMP9 interacting protein network and its intersection analysis with HCC metastasis and Treg chemotaxis-related genes;

[0040] This presentation showcases the MMP9 interacting protein network obtained through bioinformatics analysis, highlighting the following key features:

[0041] The interaction between MMP9 and transforming growth factor-β (TGF-β) and nuclear factor κB (NF-κB).

[0042] Figure 3 Figure: Experimental results of CG inhibiting the growth of HCC in situ tumors and Treg infiltration in mice;

[0043] Figure 3 Figure A shows a comparison of the average tumor weight of mice in each group at the end of the experiment. Figure 3 The diagram in B shows the proportion of Treg cells infiltrating within the tumor.

[0044] Figure 4 : Synergistic antitumor effect diagram of CG combined with anti-PD-1 antibody;

[0045] The tumor growth curve of the combined treatment group (CG+αPD-1) in the mouse HCC model was significantly lower than that of the single-drug treatment group (CG or αPD-1) and the model control group.

[0046] Example 1:

[0047] CG inhibits the growth of hepatocellular carcinoma in situ and Treg invasion in mice.

[0048] Animal model establishment: Male C57BL / 6 mice aged 6-8 weeks were selected and injected with mouse hepatocellular carcinoma cells Hepa1-6 (5×10⁶ cells) via in situ liver injection. 5 (1 mouse / mouse) to establish an orthotopic HCC xenograft model.

[0049] Grouping and administration: On day 3 after modeling, mice were randomly divided into 4 groups (n=8): (1) Model control group (Vehicle, saline); (2) Low-dose CG group (10 mg / kg); (3) High-dose CG group (30 mg / kg); (4) Positive control group (Sorafenib, 30 mg / kg).

[0050] Each group was given the medication by gavage once daily for 21 consecutive days.

[0051] Indicator Testing:

[0052] Tumor growth: Mouse weight was measured every 3 days, and mice were sacrificed 24 hours after the last administration. The tumor was completely removed and weighed.

[0053] Treg cell infiltration analysis: Fresh tumor tissue was taken, a single-cell suspension was prepared, and flow cytometry staining was performed using anti-mouse CD4-FITC and Foxp3-PE antibodies to detect the proportion of CD4+Foxp3+Treg cells in tumor-infiltrating lymphocytes.

[0054] Result: As Figure 3 As shown, compared with the model group, the high-dose CG group (30 mg / kg) significantly inhibited tumor growth (tumor weight decreased by approximately 52%, p < 0.001), and its effect was comparable to that of the positive control drug sorafenib. Simultaneously, the proportion of Treg cells in the tumor of the high-dose CG group decreased significantly by approximately 40% compared with the model group (p < 0.01), indicating that CG has the ability to inhibit Treg infiltration.

[0055] Example 2:

[0056] CG inhibits TGF-β-induced Treg cell differentiation in vitro

[0057] Cell isolation and culture: Human primary CD4+ naïve T cells were isolated from peripheral blood of healthy volunteers (using the Naïve CD4+ T cell isolation kit).

[0058] Treg differentiation induction: Cells were seeded in culture plates coated with anti-CD3 / anti-CD28 antibodies and induced to differentiate in medium containing IL-2 (100 U / mL) and TGF-β1 (5 ng / mL). Different concentrations of CG treatment groups (1 μM, 5 μM, 10 μM) were also set up.

[0059] Detection: After 5 days of culture, cells were collected, and the expression rate of Foxp3 was detected by flow cytometry. The concentration of IL-10 in the culture supernatant was detected by ELISA kit.

[0060] Results: CG treatment dose-dependently inhibited TGF-β1-induced Foxp3 expression (the proportion of Foxp3+ cells in the 10 μM CG group was 65% lower than that in the control group, p < 0.001), and significantly reduced the secretion of the inhibitory cytokine IL-10 (p < 0.01). This indicates that CG can directly interfere with TGF-β signaling-mediated Treg cell differentiation.

[0061] Example 3:

[0062] CG directly binds to and inhibits MMP9 enzyme activity.

[0063] Molecular docking: Molecular docking simulations were performed using AutoDock Vina software. The results showed that CG could stably bind to the FNII-3 domain (residues 342-390) of MMP9, with a binding energy (Vina Score) of -8.2 kcal / mol, indicating strong and stable binding ability.

[0064] like Figure 1 and Figure 2 As shown, key binding sites include Ser342, Ala343, Gly365, Arg366, Asp368, Trp385, etc.

[0065] Enzyme activity inhibition assay: The effect of CG on the hydrolytic activity of recombinant human MMP9 protein and fluorescent substrate (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) was detected in vitro. Different concentrations of CG (0 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) and the broad-spectrum MMP inhibitor GM6001 (10 μM) were set up as positive controls.

[0066] Results: CG inhibited the fluorescent substrate hydrolysis activity of MMP9 in a concentration-dependent manner, with an IC50 of 8.5 ± 0.7 μM. The positive control GM6001 showed an inhibition rate of over 95% at 10 μM. These results confirm that CG is an effective inhibitor of MMP9.

[0067] The molecular docking results are only predictions, and their binding ability has been verified by SPR experiments (determining the binding constant KD).

[0068] Example 4:

[0069] CG blocks TGF-β activation by inhibiting MMP9.

[0070] Cell model: Human HCC cell line Huh-7 was used. Control group, MMP9 overexpression group, and MMP9 overexpression + CG (10 μM) treatment group were set up.

[0071] Detection of active TGF-β: Cell culture supernatant was collected and detected using an ELISA kit that specifically recognizes the active form of TGF-β1.

[0072] Results: Compared with the control group, MMP9 overexpression significantly increased the level of active TGF-β1 in the cell supernatant (approximately 2.8-fold increase, p < 0.001).

[0073] CG treatment almost completely reversed this effect, restoring the level of active TGF-β1 to near that of the control group. This indicates that CG effectively blocked the activation of TGF-β by inhibiting MMP9.

[0074] Example 5:

[0075] Synergistic therapeutic effect of CG combined with anti-PD-1 antibody

[0076] Animal models and grouping: The mouse HCC orthotopic tumor model was established as in Example 1. The mice were divided into 4 groups (n=8): (1) Model group (IgG control); (2) CG monotherapy group (30 mg / kg, gavage); (3) Anti-PD-1 monotherapy group (αPD-1, 10 mg / kg, intraperitoneal injection, once every 3 days); (4) CG and αPD-1 combination group.

[0077] Treatment efficacy assessment: Tumor volume was measured periodically, and growth curves were plotted. After treatment, the tumor inhibition rate was calculated, and spleen and tumor tissue were collected for flow cytometry analysis of changes in immune cell subsets.

[0078] Result: As Figure 4 As shown, the combination therapy exhibited the strongest tumor growth inhibition effect, with a tumor inhibition rate (approximately 78%) significantly higher than that of the CG monotherapy group (approximately 52%) and the αPD-1 monotherapy group (approximately 45%) (p < 0.01). Flow cytometry analysis revealed that the combination group had the highest ratio of CD8+ T cells to Treg cells within the tumor, indicating that the immune microenvironment was improved to the greatest extent. These results demonstrate the synergistic value of combining CG with immune checkpoint inhibitors.

[0079] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. The use of verbascoside or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the infiltration of regulatory T cells (Tregs) in the tumor microenvironment of hepatocellular carcinoma.

2. The application according to claim 1, characterized in that, The verbascoside inhibits Treg cell infiltration by suppressing the function of matrix metalloproteinase 9 (MMP9).

3. The application according to claim 2, characterized in that, The function of inhibiting MMP9 refers to inhibiting the protease activity of MMP9.

4. The application according to claim 3, characterized in that, The verrucoside reduces Treg cell differentiation or intratumoral recruitment by inhibiting the activation of transforming growth factor-β (TGF-β) by MMP9.

5. The application according to any one of claims 1-4, characterized in that, The drug is used in combination with immune checkpoint inhibitors for the treatment of hepatocellular carcinoma.

6. The application according to claim 5, characterized in that, The immune checkpoint inhibitor is a programmed death receptor-1 (PD-1) inhibitor, a programmed death receptor ligand-1 (PD-L1) inhibitor, or a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) inhibitor.

7. A pharmaceutical composition for treating hepatocellular carcinoma, characterized in that, It contains a therapeutically effective amount of vernix isoflavone glycoside or a pharmaceutically acceptable salt thereof, and an immune checkpoint inhibitor.

8. The pharmaceutical composition according to claim 7, characterized in that, The immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition is prepared as a combination drug formulation for simultaneous, separate, or sequential administration.

10. Use of verbascoside or a pharmaceutically acceptable salt thereof in the preparation of a medicament for enhancing the sensitivity of hepatocellular carcinoma to immune checkpoint inhibitor therapy.

Images