Application of isochlorogenic acid C in medicine for inhibiting synovial fibroblast-like cell invasion and inflammatory response

Abstract

The invention belongs to the technical field of medicine technology, particularly relates to application of isochlorogenic acid C in medicine for treating inflammation, and provides novel application of isochlorogenic acid C in preparation of anti-inflammation medicine, and particularly has a good inhibition effect on abnormal behaviors such as invasion of synovial fibroblasts and secretion of inflammatory factors. Isochlorogenic acid C is provided as an effective medicine component for treating arthritis, and the main action mechanism is to inhibit invasion of RA-FLS and secretion of IL-6 so as to relieve clinical symptoms; the innovation of the invention lies in that: the invention provides treatment of inflammation based on isochlorogenic acid C, and through application of a specific dosage, joint inflammation of patients can be effectively relieved and joint functions can be improved.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to a pharmaceutical composition with isochlorogenic acid C (3,5-dicaffeoylquinic acid) as an active ingredient and its application in inhibiting the pathological behavior of synovial fibroblast-like cells (RA-FLS) in patients with rheumatoid arthritis (RA), especially providing a new treatment method for the invasive ability and pro-inflammatory cytokine secretion of RA-FLS. Background Art

[0002] The core pathological driver of rheumatoid arthritis (RA) is the invasive phenotypic transformation of synovial fibroblasts (RA-FLS), which is characterized by: (1) abnormal migration and matrix invasion: penetrating the cartilage-bone junction to form erosive pannus; (2) inflammatory amplifier function: continuous secretion of chemokines such as IL-6 and IL-8, recruiting immune cells to form a vicious cycle. The core of RA pathology lies in the abnormal activation of synovial fibroblasts (RA-FLS), which manifests as pathological invasion of cartilage and bone tissue and large secretion of proinflammatory cytokines such as IL-6 and TNF-α, causing joint destruction and loss of function. Existing NSAIDs, glucocorticoids, and immunosuppressants mostly relieve symptoms, have limited targeted inhibitory effects on RA-FLS, and long-term use has significant toxic and side effects. Biological agents (such as TNF-α inhibitors) can inhibit systemic inflammation but have no direct blocking effect on the invasive ability of resident RA-FLS.

[0003] Isochlorogenic acid C (3,5-dicaffeoylquinic acid) is a phenolic acid compound with multi-target bioactivity. It is widely found in plants of the Asteraceae and Caprifoliaceae families, including natural Chinese herbs such as dandelion, Echinacea, honeysuckle, Houttuynia cordata, and Artemisia argyi. Its polyphenolic structure imparts potent antioxidant activity. Modern research has demonstrated that the core mechanism of action of isochlorogenic acid C stems from the potent antioxidant and molecular regulatory capabilities conferred by the caffeoyl group. Its multi-target effects demonstrate significant potential in anti-inflammatory, antioxidant, organ protection, and anti-infective fields, with promising therapeutic effects in various chronic inflammatory diseases (such as hepatitis and arthritis) and metabolic syndrome. However, research on this compound has focused on overall inflammatory scores in animal models (such as joint swelling index) and immune cell regulation (such as macrophage polarization and T cell differentiation). To date, no research has examined its mechanism of action, directly inhibiting RA-FLS invasion and destructive cytokine secretion, and drug development is underway. Summary of the Invention

[0004] In response to the problem that the existing technology has not yet effectively inhibited RA synovial invasion and inflammatory response, the present invention provides a new use of isochlorogenic acid C in the targeted inhibition of RA-FLS invasive phenotype and destructive inflammatory circuit, solving the problem that existing drugs cannot block the local pathological process of the synovium.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides the use of isochlorogenic acid C in a drug for inhibiting the invasion of synovial fibroblasts and the secretion of IL-6 (or IL-8 and MMP-9).

[0007] Furthermore, the effective concentration of isochlorogenic acid C on synovial fibroblasts is 50-100 μM, with an effective inhibitory effect lasting 24-48 hours. It can significantly reduce the invasion rate of RA-FLS and IL-6 secretion, while having no significant toxicity to normal fibroblasts. After 24-48 hours of treatment, the RA-FLS invasion inhibition rate is ≥40%, and the IL-6 inhibition rate is ≥25%.

[0008] Furthermore, the dosage forms of the drug include tablets, capsules, gels, ointments, and microneedle patches to achieve systemic or local administration.

[0009] Furthermore, the isochlorogenic acid C is derived from an extract of a plant of the Asteraceae or Caprifoliaceae family.

[0010] The present invention also provides a pharmaceutical composition for inhibiting the invasion and inflammatory secretion of synovial fibroblasts. The pharmaceutical composition contains isochlorogenic acid C as the active ingredient (or as the sole active ingredient) at an effective concentration of 50 to 100 μM. It inhibits the Transwell invasion ability of RA-FLS (inhibition rate ≥40%, 100 μM, P < 0.05) and downregulates the secretion of pro-inflammatory factors such as IL-6 by RA-FLS (secretion decreased by >25%, 100 μM, P < 0.01).

[0011] Furthermore, the active ingredient may also include a pharmaceutically acceptable salt of isochlorogenic acid C.

[0012] Furthermore, the pharmaceutical composition is a thermosensitive hydrogel or liposome microneedle patch suitable for local delivery to joints.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention provides isochlorogenic acid C, which has a direct effect on synovial fibroblasts, specifically inhibits the invasion and migration behavior of RA-FLS and the secretion of inflammatory factors, and controls the progression of synovial joint diseases.

[0015] (1) Cell safety: confirmed by CCK-8 assay, no cytotoxicity within the concentration range of 1.56-100 μM;

[0016] (2) Blocking RA-FLS invasion: xCELLigence RTCA system detection showed that the cell invasion ability decreased by >40% after 24 hours of treatment with 100 μM (P < 0.05), inhibiting the cartilage infiltration and erosion ability of synovial cells; it was confirmed that isochlorogenic acid C could inhibit RA-FLS invasion (IC 50 =85 μM);

[0017] (3) Inhibition of inflammatory factor secretion: ELISA test showed that after 24 hours of treatment at a concentration of 100 μM, the IL-6 secretion of RA-FLS cells decreased by >25% (P<0.01), effectively alleviating synovial inflammation.

[0018] (4) Inhibit joint inflammation: In the CFA arthritis model experiment in rats, after 3 mg / μl of the drug was injected into the rat joint, the swelling of the foot joint decreased by >50% (P<0.01), effectively relieving joint inflammation.

[0019] The present invention discloses for the first time the dual-targeted regulatory effect of isochlorogenic acid C on RA-FLS, which not only significantly inhibits cell invasion but also reduces the secretion of key pro-inflammatory factors, providing a new idea for precise intervention in RA; the multi-dosage administration method expands clinical applicability and enhances efficacy and stability; the natural source, high safety and low cost enable it to be used in the long-term treatment of chronic RA. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure 3 is a cell safety experiment diagram of isochlorogenic acid C on human dermal fibroblasts and RA-FLS, where A is the cell proliferation experimental curve of different concentrations of isochlorogenic acid C on human dermal fibroblasts HFF; B is the normalized cell index of HFF cells under different concentrations of isochlorogenic acid C after 24 hours and 48 hours of drug intervention; C is the change in the relative proliferation rate of cells before and after intervention with different concentrations of isochlorogenic acid C; D is the effect of three different concentrations of isochlorogenic acid C on the cell activity of RA-FLS; E is the proliferation curve of RA-FLS cells with isochlorogenic acid C; F is the normalized cell index after 24 hours and 48 hours of isochlorogenic acid C intervention, as well as the change in the relative proliferation rate of cells after intervention;

[0021] Figure 2 Figure 2 is the inhibitory effect of isochlorogenic acid C on RA-FLS invasion, where A is the schematic diagram of the invasion experiment and the cell nucleus staining counting chamber; B is the cell invasion curve of RA-FLS cells treated with isochlorogenic acid C; C is the relative invasion rate of RA-FLS treated with isochlorogenic acid after normalization of cell nucleus counts;

[0022] Figure 3Figure 2 is a diagram showing the regulation of RA-FLS inflammatory factor IL-6 secretion by isochlorogenic acid C, where A is the schematic diagram of ELISA detection of interleukin IL-6 and the standard curve fitting; B is the change in the relative IL-6 inflammatory factor secretion content of RA-FLS after normalization of cell nuclear staining counts;

[0023] Figure 4 The diagram shows the inhibition of inflammatory response of the foot joints of rats with CFA model by isochlorogenic acid C, where A is a schematic diagram of inflammatory response of the foot joints of rats under the intervention of intraplantar injection of isochlorogenic acid; B is a schematic diagram of the change of relative foot circumference over time; C is a schematic diagram of HE staining of changes in the foot joints. DETAILED DESCRIPTION

[0024] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] Example 1

[0026] The cell safety test of isochlorogenic acid C on dermal fibroblasts and synovial fibroblast-like cells is shown in the figure below. Figure 1 shown.

[0027] 1. Experimental material conditions

[0028] (1) Cell sources: human dermal fibroblasts (HFF, ATCC CRL-4026), primary isolated human rheumatoid arthritis synovial fibroblast-like cells (RA-FLS);

[0029] (2) Culture conditions: HFF cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS), and RA-FLS cells were cultured in DMEM medium (containing 15% FBS). The cells were placed in a 37°C, 10% CO2 constant temperature incubator.

[0030] (3) Detection platform and methods: xCELLigence RTCA E-plate system; CCK-8 cell viability assay kit (CCK8, Elabscience);

[0031] (4) Cell seeding density: HFF seeding density 5,000 cells / well ( Figure 1 , A), RA-FLS cells 2500 cells / well ( Figure 1 , E).

[0032] 2. Treatment method and drug concentration setting

[0033] Cells were treated with isochlorogenic acid C at seven concentrations, 1.56, 3.15, 6.25, 12.5, 25, 50, and 100 μM, along with a blank control group. Drugs were added after cells entered the logarithmic growth phase, and the cells were monitored continuously in real time for 48 hours.

[0034] 3. Real-time data collection and indicator evaluation

[0035] (1) Time-dependent curve of RTCA cell proliferation ( Figure 1 A, E): To evaluate the effects of test compounds on cell proliferation, real-time cell analysis (RTCA) was used. The procedure was as follows: cells were seeded onto a dedicated RTCA electrode plate. After the cells adhered, the RTCA detection system was activated and set to automatically record the Cell Index (CI) every 15 minutes. This CI value reflects the changes in the number of cells on the electrode surface in real time. Through continuous monitoring, a time-dependent curve of cell proliferation under drug intervention can be plotted.

[0036] (2) Normalized Cell Index ( Figure 1 B): To facilitate the comparison of proliferation effects under different experimental conditions, the CI value at a specific time point before drug intervention (denoted as t1 or t0) was selected for normalization. That is, the ratio of the CI value at any time point t to the CI value at time point t0 (CI(t) / CI(t0)) was calculated. This ratio was defined as the "normalized cell index (Normalized CI). (e.g. Figure 1 C, F). This ratio can intuitively reflect the changing trend of the relative cell proliferation rate under the action of drugs;

[0037] (3) Cell proliferation rate ratio ( Figure 1 C, F): The cell proliferation rates (ΔCI_pre and ΔCI_post) were selected 24 hours before drug intervention and 24 hours and 48 hours after intervention, and their ratios (ΔCI_pre / ΔCI_post) were calculated to represent the changes in the relative cell proliferation rates under drug intervention.

[0038] (4) CCK-8 cell viability assay ( Figure 1D): Cell viability reflects the metabolic activity of cells through the CCK-8 absorbance value (OD). Cells in the logarithmic growth phase were seeded into 96-well plates at 5,000 cells / well, with 100 μl per well. After 48 hours of incubation, the test compound was added to the test wells and incubated for another 48 hours. Subsequently, 10 μl of CCK8 reagent was added to each well and incubated for 3 hours at 37°C and 10% CO2. The optical density (OD) was measured at 450 nm using a microplate reader (CLARIOstar Plus, BMG LABTECH).

[0039] 5. Experimental Results

[0040] When treated with ≤100 μM isochlorogenic acid C, cell viability remained above 90% for both HFF and RA-FLS cells, with no significant toxicity observed. CCK-8 and RTCA results were consistent, demonstrating good biosafety for RA-FLS cells within this concentration range.

[0041] Example 2

[0042] Inhibitory effect of isochlorogenic acid C on invasion of synovial fibroblast-like cells (RA-FLS)

[0043] 1. Materials and Methods

[0044] (1) Cell source: Primary isolated human synovial fibroblast-like cells (RA-FLS) from patients with rheumatoid arthritis;

[0045] (2) Culture conditions: DMEM medium (containing 15% FBS), 37°C, 10% CO2 constant temperature incubator;

[0046] (3) Detection platform: xCELLigence RTCADPlus system + CIM-Plate 16 invasion analysis system.

[0047] 2. Drug treatment

[0048] (1) Isochlorogenic acid C concentration: 100 μM and blank control;

[0049] (2) Isochlorogenic acid C intervention / detection time: After the cells enter the logarithmic growth phase, the drug is treated for 24 hours, followed by invasion experiments.

[0050] 3. Monitoring of invasive ability

[0051] (1) Coating of CIM invasion chambers: Dilute Corning Matrigel concentrate (HC) 1:100 with serum-free medium. Take 20 μl of the mixture and evenly spread it in the upper chamber of the CIM plate. Incubate the plate at 37°C for 12 hours to allow the Matrigel to solidify.

[0052] (2) Isochlorogenic acid C drug intervention: Cells in the logarithmic growth phase were pretreated with the test compound for 24 hours, and the cells were collected and the density was adjusted to 2×10 ^5 / ml, and then in serum-free medium containing the corresponding test compound, at a concentration of 2 × 10 ^4 The cells were seeded into the upper chamber at a density of 100 cells / mL. The lower chamber was filled with serum-containing medium containing the test compound to establish a serum gradient. Real-time detection continued incubation for 48 hours. Subsequently, 10 μL of CCK8 reagent was added to each well and incubated at 37°C, 10% CO2 for 3 hours. The optical density (OD) was measured at 450 nm using a microplate reader (CLARIOstar Plus, BMG LABTECH).

[0053] (3) Real-time detection of invasive ability ( Figure 2 A): The CIM-plate was mounted on the RTCA system for real-time monitoring of the invasion process, with measurements taken every 15 minutes for 24 hours. To eliminate possible differences in cell seeding density, cells were simultaneously seeded into 96-well plates for cell counting to standardize the invasion cell index. The cells were then fixed and their nuclei were stained with H33342. Cell number was quantified by counting nuclei using a Zeiss (Germany) Cell Discoverer 7 imaging system.

[0054] 4. Data Processing Methods

[0055] (1) RTCA time dependence curve analysis ( Figure 2 ,B): Delta CI = control group CI / treatment group CI × 100%, CI_norm(t) = [CI(t)-CI(0)] / [CI(24h)-CI(0)]×100%, the 24h and 48h time points were selected for inter-group comparison;

[0056] (2) Calculation of normalized cell index ( Figure 2 , C): To ensure accurate data on cytokine concentrations, we determined the number of cells in each well using H33342 nuclear staining to normalize the results.

[0057] 5. Experimental Results

[0058] The experimental results showed that the transmembrane invasion ability of RA-FLS in the 100 μM isochlorogenic acid C group was reduced by more than 40% compared with the control group (P<0.05), indicating that it significantly inhibited the abnormal migration and invasion ability of synovial fibroblasts.

[0059] Example 3

[0060] Study on the regulation of inflammatory factor IL-6 secretion by isochlorogenic acid C in synovial fibroblast-like cells (RA-FLS)

[0061] 1. Materials and Methods

[0062] (1) Cell source: Primary isolated human synovial fibroblast-like cells (RA-FLS) from patients with rheumatoid arthritis;

[0063] (2) Culture conditions: DMEM medium (containing 15% FBS), 37°C, 10% CO2 constant temperature incubator;

[0064] (3) Detection method: Enzyme-linked immunosorbent assay-interleukin IL-6 ELISA detection kit (Abclonal)

[0065] 2. Drug treatment

[0066] (1) Isochlorogenic acid C concentration: 100 μM and blank control;

[0067] (2) Isochlorogenic acid C intervention / detection time: After the cells enter the logarithmic growth phase, the drug is treated for 48 hours.

[0068] 3. Monitoring of inflammatory factor secretion capacity

[0069] Interleukin-6 (IL-6, Abclonal) protein levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit. Cells in the logarithmic growth phase were collected and plated at 5×10 ^4 The cells were seeded at a density of 100 μl / well in a 96-well cell culture plate. Forty-eight hours after seeding, the test compound was added to the wells and cultured for another 48 hours. The supernatant of the cultured cells was collected, diluted 50-fold, and then assayed by ELISA according to the kit instructions.

[0070] 4. Secretion Assessment and Data Analysis

[0071] (1) ELISA standard curve fitting Figure 3 ,A): CI(t)=(Z(t)-Z(blank)) / (Z(max)-Z(blank))×1000; where Z is the transwell impedance value and Z(blank) is the blank well baseline value;

[0072] (2) Calculation of normalized cell index ( Figure 3 ,B): To ensure accurate data of cytokine concentrations, we determined the number of cells in each well using H33342 nuclear staining to normalize the results;

[0073] 5. Experimental Results

[0074] The experimental results showed that compared with the control group, after treatment with 100 μM isochlorogenic acid C, the level of IL-6 secreted by RA-FLS decreased by more than 25% compared with the control (P<0.01), indicating that isochlorogenic acid C can effectively inhibit the release of proinflammatory factors mediated by RA-FLS and has the potential to regulate the synovial inflammatory microenvironment.

[0075] Example 4

[0076] Interventional study of isochlorogenic acid C by joint injection in rats with rheumatoid arthritis CFA model

[0077] 1. Experimental Animals and Grouping Labeling

[0078] The experimental animals were Wistar rats, weighing 180-220 g, of either sex. The animals were sourced in accordance with the Standards for Laboratory Animal Care and were acclimated for 7 days prior to the experiment. The animals were divided into two groups (n = 8 each) (normal control group and isochlorogenic acid C treatment group):

[0079] 2. CFA modeling operation

[0080] (1) Weigh the animal, calculate the injection dose of complete Freund's adjuvant (CFA) (0.75 mg / kg), and dispense the drug using a 1 mL disposable syringe;

[0081] (2) Start the isoflurane anesthesia machine, set the anesthetic concentration to 1.5%, and the anesthetic pressure to 1, and operate two rats at a time;

[0082] (3) After the rat's limbs relax and lose consciousness, remove it and place it on the operating table. Disinfect the plantar part of the left hind paw with a 75% alcohol cotton ball.

[0083] (4) 0.1 mL of CFA solution was slowly injected subcutaneously through the sole of the left hind foot. The needle was advanced from the heel along the sole to the midfoot. The needle tip was faintly visible through the skin during injection. After confirming that there was no local blood backflow, the injection was slowly injected to avoid tissue rupture.

[0084] (5) After the injection, the rats were returned to a clean cage and the inflammatory response was continuously observed.

[0085] 3. Local intervention with isochlorogenic acid C

[0086] 48 hours after the model was established, the ankles were confirmed to be significantly red and swollen, and the rats in the isochlorogenic acid C group were given drug injection intervention:

[0087] (1) Prepare isochlorogenic acid C injection at a concentration of 3 mg / μL by dissolving it in a small amount of DMSO and then diluting it in sterile PBS to a final concentration of DMSO <1%;

[0088] (2) Under aseptic operation, the skin surface of the left ankle joint was disinfected again with 75% alcohol, and a 1 mL syringe was used to puncture the posterior edge of the medial malleolus. Each rat was injected with 100 μL (300 μg) of isochlorogenic acid C solution once;

[0089] (3) The control group was injected with an equal amount of PBS solution.

[0090] 4. Efficacy evaluation indicators

[0091] (1) Ankle joint redness and swelling score: The degree of ankle redness and swelling was recorded daily according to the swelling grade (0-4 points) to evaluate the progression and relief trend of inflammation;

[0092] (2) Ankle thickness measurement: Use a vernier caliper to measure the vertical diameter of the thickest part of the ankle joint daily, record the data for 7 consecutive days, and assess the degree of inflammation and swelling;

[0093] (3) Synovial tissue sampling and HE staining: After the treatment, the animals were killed, and the synovial tissue of the left ankle joint was fixed, paraffin-sectioned, and then stained with HE to evaluate the degree of synovial layer proliferation, pannus formation, and inflammatory cell infiltration;

[0094] 5. Experimental Results

[0095] result( Figure 4 ) showed that ankle swelling was significantly reduced in the group treated with isochlorogenic acid C, ankle thickness decreased by over 50% (P<0.01), and inflammatory cell infiltration in the synovial tissue was significantly reduced, suggesting that isochlorogenic acid C significantly inhibited local inflammatory responses and intervened in synovial pathology. HE staining showed that it effectively blocked synovial hyperplasia and neovascularization, protecting the structural integrity of the joint.

[0096] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0097] It should be noted that the above content merely illustrates the technical idea of ​​the present invention and cannot be used to limit the scope of protection of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications all fall within the scope of protection of the claims of the present invention.

Claims

1. Application of isochlorogenic acid C in inhibiting the invasion and IL-6 secretion of synovial fibroblasts.

2. The use of isochlorogenic acid C in a drug for inhibiting synovial fibroblast invasion and IL-6 secretion according to claim 1, characterized in that: The effective concentration of the isochlorogenic acid C acting on synovial fibroblasts is 50-100 μM, and the effective inhibitory effect time is 24-48 hours.

3. The use of isochlorogenic acid C in a drug for inhibiting synovial fibroblast invasion and IL-6 secretion according to claim 1, characterized in that: The dosage forms of the drug include tablets, capsules, gels, ointments, and microneedle patches.

4. The use of isochlorogenic acid C in a drug for inhibiting synovial fibroblast invasion and IL-6 secretion according to claim 1, characterized in that: The isochlorogenic acid C is derived from an extract of a plant of the Asteraceae family or the Honeysuckle family.

5. A pharmaceutical composition for inhibiting the invasion and inflammatory secretion of synovial fibroblasts, characterized in that: The pharmaceutical composition is a pharmaceutical composition with isochlorogenic acid C as an active ingredient, and the effective concentration is 50 to 100 μM.

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