Oleanolic acid amide derivative for treating inflammatory immune diseases, preparation method thereof and use thereof

Abstract

The present invention discloses an oleanolic acid amide derivative for treating inflammatory immune diseases and a preparation method thereof. The method comprises the following steps: reacting natural structure oleanolic acid with methylamine under catalyst conditions to produce an amide reaction, thereby obtaining an oleanolic acid amide derivative. Acute toxicity and pharmacokinetic experiments have demonstrated that the modified oleanolic acid derivative is a safe and effective drug, and the modified oleanolic acid derivative has better water solubility and pharmacokinetic characteristics; pathological experiments have shown that the oleanolic acid amide derivative can significantly improve the skin phenotype and severity index of psoriasis model mice, thereby effectively alleviating the clinical symptoms of psoriasis; the oleanolic acid amide derivative can significantly improve the arthritis symptoms of adjuvant arthritis rats. The oleanolic acid amide derivative of the present invention can be used to prepare drugs for treating inflammatory immune diseases such as psoriasis and rheumatoid arthritis, and has good social and economic benefits.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to a method for preparing oleanolic acid amides and an application thereof in preparing drugs for treating inflammatory diseases (taking psoriasis and rheumatoid arthritis as examples). Background Art

[0002] Oleanolic acid (OA) has the chemical formula 3β-hydroxyoleanolic acid-12-ene-17-carboxylic acid, a molecular weight of 456.700, a CAS number of 508-02-1, and the structural formula is as follows.

[0003]

[0004] Inflammation and immune response are basic pathological processes that occur when various immune cells of the body's immune system are stimulated by various damaging factors and are mainly defensive reactions. Inflammation and immune response play an important role in the host defense and the progression of immune-mediated diseases. Under normal circumstances, it is a normal physiological response of the immune system to injury, infection, and stress. This process acts as a signal to prompt the immune system to heal and repair damaged tissues to protect the body from infectious factors such as viruses and bacteria. However, excessive or persistent inflammatory immune responses can lead to and aggravate the occurrence of diseases. [1] Autoimmune diseases are a type of immune disease in which the immune system induces tissue damage and inflammatory response, ultimately leading to target organ damage. [2] Autoimmune diseases accompanied by chronic inflammation, such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis, bring pain and distress to thousands of patients.

[0005] Natural medicines are considered to be an important resource for the treatment of inflammatory and immune diseases. Oleanolic acid is a pentacyclic triterpenoid substance widely found in plants of the Oleaceae family. It exists in daily food in the form of free acid or triterpenoid saponin aglycone. To date, a variety of pharmacological activities of oleanolic acid have been studied and reported, including its antioxidant, anti-inflammatory, anti-asthma, anti-diabetic, anti-tumor, liver protection and immunomodulatory effects. [3] At present, most of the research on the pharmacological activity of oleanolic acid focuses on its anti-tumor and liver-protective effects. Studies have found that oleanolic acid can induce apoptosis and autophagy of tumor cells, inhibit their migration and invasion, and thus achieve the effect of inhibiting tumor development. [4] Recent studies have found that oleanolic acid has a certain therapeutic effect on inflammatory immune diseases. [5][6] .

[0006] However, oleanolic acid has extremely poor water solubility, is difficult to absorb in the intestine, and has low solubility, which leads to low bioavailability and also makes it difficult to conduct mechanism studies in vitro. Based on this, researchers have modified natural oleanolic acid, trying to improve its water solubility and drugability by modification to improve its bioavailability. Studies have found that the structural modification of OA has a great influence on its biological activity, among which the carboxyl group at the C-17 position can undergo esterification, amide and other reactions, connecting various groups to achieve the effect of modification without losing its pharmacological activity. The comprehensive drug database shows that more than 25% of known drugs have amide as a structural feature. The presence of amide bonds in drugs may enable them to better form hydrogen bonds with the amino acid fragments of their target proteins, thereby firmly binding. [7] The -COOH group at the C-17 position creates a high steric hindrance for OA, preventing it from effectively binding to its target protein. Amidation of the -COOH group may eliminate or reduce the high steric hindrance caused by the original -COOH group, making it easier to form hydrogen bonds with target proteins and exhibiting better pharmacological activity. Therefore, the synthesis of oleanolic acid amide derivatives has broad application prospects.

[0007] Psoriasis is an inflammatory, autoimmune, papular, scaly skin disease that affects more than 60 million adults and children worldwide. [8] Psoriasis not only brings physical health problems to people, but also seriously affects their mental health. Up to 30% of psoriasis patients suffer from anxiety and depression. [9] It can be seen that the physical and psychological risks brought by psoriasis are huge. The pathological characteristics of psoriasis show that epidermal keratinocytes, T cells, dendritic cells, etc. play an important role.

[10] . In particular, the IL-23 / Th17 pathway plays an important role in psoriasis. Helper T cells 17 (Th17 cells) differentiate from precursors under the regulation of interleukin-1β (IL-1β), IL-6, transforming growth factor-β (TGF-β) and transcription factor RORγt. The expansion and survival of Th17 cells depend on IL-23 produced by bone marrow cells. Once activated, Th17 cells produce cytokines such as IL-17A, IL-17F and IL-22, which induce keratinocyte proliferation and participate in the course of psoriasis.

[11] Currently, psoriasis treatments include steroids, retinoids, methotrexate, cyclosporine, biologics, and targeted small molecule drugs (TNF-α inhibitors, IL-17, and IL-23 inhibitors). However, these drugs have significant side effects or are less effective than expected. Therefore, exploring and developing targeted drugs with proven efficacy and minimal adverse reactions is an urgent research goal for clinicians and pharmaceutical experts.

[0008] Recent studies have found that SHP2 is a non-receptor protein tyrosine kinase containing two SH2 domains and a catalytic protein tyrosinephosphatase (PTP) domain. It is encoded by PTPN11 and plays a key role in cell proliferation, differentiation, and survival. SHP2 is closely associated with the pathogenesis of systemic lupus erythematosus (SLE). Studies have shown that in cultured human SLE T cells, inhibition of SHP2 can reduce the levels of IFN-γ and IL-17A, suggesting that SHP2 inhibition can be used to improve SLE.

[12] Similarly, SHP2 plays a key role in the proliferation and activation of T cells in the course of psoriasis. SHP2 enhances the activation of TLR7 / NF-κB, leading to further increase in the expression of psoriasis-related inflammatory cytokines IL-23A, TNF-α, IL-6, and IL-1β, thereby accelerating psoriasis-like skin inflammation.

[13] At the same time, the SHP2 allosteric inhibitor SHP099 suppressed skin inflammation caused by myeloid cells, especially macrophages. In addition, the optimized SHP2 inhibitor TK-453 significantly improved imiquimod-induced skin-like inflammation in mice by inhibiting the IL-23 / Th17 pathway.

[14] Therefore, exploring inhibitors targeting SHP2 from traditional Chinese medicine natural medicines to treat psoriasis will have potential clinical value.

[0009] Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory, autoimmune disease with joints as the main target organs. It has a high disability rate and seriously affects the normal life and work of patients. In addition to joints, patients may also suffer damage to multiple organs such as lungs and blood vessels. According to statistics, about 1 in every 200 adults in the world is diagnosed with RA, and the incidence rate in women is 2 to 3 times that of men.

[15] .

[0010] Although RA manifests as a systemic autoimmune reaction, its clinical features are mainly synovial inflammation and joint damage. Fibroblast-like synovial cells (FLS) are a highly specific mesenchymal cell located in the synovial lining. As the main effector cell for proliferation and inflammation of RA synovial tissue, FLS has the biological characteristics of abnormal proliferation, migration, invasion and secretion. In RA, FLS proliferates rapidly under the stimulation of platelet-derived growth factor, TGF-β, IL-1β, and TNF-α produced by various immune cells in the inflamed joints, transforming the synovial lining into invasive hyperplastic tissue. FLS also produces many inflammatory cytokines such as IL-1β, IL-6, TNF-α, etc., leading to synovial inflammation. More importantly, activated FLS will produce active substances such as matrix metalloproteinases (MMPs) to destroy collagen-rich joint tissue.

[16] Therefore, inhibiting the abnormal function of FLS cells in RA can effectively control the proliferation of synovial inflammation, prevent the erosion of cartilage and ultimately alleviate the progression of RA. Targeting FLS therapy may become a promising treatment method.

[0011] Currently, clinically used RA medications primarily include nonsteroidal anti-inflammatory drugs (NSAIDs), immunosuppressants, biologics, and glucocorticoids. These drugs are primarily used to control and alleviate symptoms, but they are associated with numerous adverse reactions and high treatment costs. Therefore, the search for highly effective, low-toxic, and cost-effective medications is crucial. Studies have shown that oleanolic acid, a plant extract, not only significantly inhibits paw swelling and the release of inflammatory factors in RA rats but also inhibits TNF-α-induced expression of inflammatory factors such as IL-6 and IL-1β in FLS in a concentration-dependent manner. Furthermore, it inhibits TNF-α-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and nuclear translocation of nuclear factor kappa-B (NF-κB). Therefore, further investigation of the mechanisms of action of oleanolic acid and its modified molecules has broad clinical application value.

[0012] The pro-inflammatory cytokine TNF-α is a key driver of various inflammatory diseases such as RA. It can drive two independent receptors, tumor necrosis factor receptor 1 (TNFR1) and TNFR2, to control different signal transduction pathways. Activation of TNFR1 leads to activation of NF-κB and MAPK signal transduction, driving inflammatory responses and is closely related to the occurrence and development of RA.

[17] Recent studies have found that TNF-α affects mitochondrial function and drives mtDNA release. Under inflammatory conditions, when the cytoplasmic DNA sensor cyclic GMP-AMP (cGAS) senses DNA that should not be present in the cytoplasm, it catalyzes ATP and GTP to synthesize a small molecule, cyclic GMP-AMP (cGAMP). cGAMP binds to the dimerized interferon stimulating gene (STING), causing its conformation to change and translocate from the endoplasmic reticulum to the Golgi apparatus, where it recruits and phosphorylates TANK-binding kinase 1 (TBK1) and inhibitor of NF-κB (IKK), subsequently activating interferon regulatory transcription factor 3 (IRF3) and NF-κB, respectively, thereby promoting the production of type I interferon (IFN-I). Studies have shown that damaged mitochondria increase in FLS of RA patients and high expression of cGAS. At the same time, in TNF-α-stimulated FLS, overexpression of cGAS enhances the production of proinflammatory cytokines and matrix metalloproteinases (MMPs).

[18] Therefore, it is of great clinical value to find natural drugs that target TNFR1 and inhibit the cGAS-STING axis in FLS to treat RA.

[0013] References

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[0031]

[18] WANG Y, SU GH, ZHANG F, et al. Cyclic GMP-AMP Synthase Is Required for Cell Proliferation and Inflammatory Responses in Rheumatoid ArthritisSynoviocytes[J]. Mediators Inflamm, 2015, 2015: 192329. Summary of the Invention

[0032] This invention modifies the C-17 position of OA through an amide reaction to produce a novel oleanolic acid derivative, code-named OA-17. Its toxicology, pharmacokinetics, and pharmacodynamics were studied and compared with the parent OA. Results showed that OA-17 exhibits improved water solubility and greater drug development potential compared to OA. Most importantly, OA-17 demonstrated superior efficacy compared to OA in psoriasis pharmacodynamic studies and effectively alleviated the progression of adjuvant arthritis in rats by inhibiting FLS. The invention also investigated the mechanism of action of OA-17. In psoriasis, OA-17 significantly inhibited the expression of SHP2 and PI3K / mTOR on T cells, thereby suppressing imiquimod-induced skin inflammation in psoriatic mice in pathological experiments. In RA, OA-17 inhibited the TNF-α receptor TNFR1 in FLS, significantly suppressing the expression of its downstream proteins cGAS and STING, thereby alleviating inflammatory changes in FLS and ultimately alleviating the progression of RA. Based on this, the present invention provides an oleanolic acid derivative that can be used to prepare a method for treating psoriasis and RA, which has good social and economic benefits.

[0033] Therefore, the present invention aims to provide an oleanolic acid amide derivative effective in treating psoriasis and a method for its preparation. This method uses natural oleanolic acid as a lead compound to design an oleanolic acid amide derivative, OA-17 (modified at the C-17 position). OA-17 can significantly inhibit the expression of SHP2 on T cells, thereby inhibiting imiquimod-induced skin inflammation in psoriatic mice in pathological experiments. This oleanolic acid derivative has the following molecular formula:

[0034]

[0035] The present invention thus provides an oleanolic acid amide derivative having the effect of treating psoriasis, and its molecular structure is shown in formula (II).

[0036] The present invention provides stereoisomers, solvent compounds, prodrugs, metabolites, pharmaceutically acceptable salts or cocrystals of the oleanolic acid amide derivatives with the effect of treating psoriasis.

[0037] The present invention further provides a pharmaceutical composition containing the oleanolic acid amide derivatives, or stereoisomers, solvent compounds, prodrugs, metabolites, pharmaceutically acceptable salts or cocrystals thereof.

[0038] Specifically, it also includes a pharmaceutically acceptable carrier.

[0039] The present invention also provides the use of the oleanolic acid amide derivatives, or their stereoisomers, solvent compounds, prodrugs, metabolites, pharmaceutically acceptable salts or cocrystals in the preparation of drugs for treating or preventing psoriasis.

[0040] The present invention also provides a method for preparing the oleanolic acid amide derivatives, which comprises the following steps:

[0041] (1) Take the raw material oleanolic acid, the reactant methylamine hydrochloride, add the catalyst 2-(7-azobenzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and the catalyst N,N-diisopropylethylamine, and stir at a constant speed at 20-25°C for 6-15 hours;

[0042] (2) dissolving the mixture from step 1 in an organic solvent, adding saturated aqueous NaCl solution and saturated aqueous potassium hydrogen sulfate solution, and extracting with ethyl acetate 2-3 times;

[0043] (3) Collect the organic phase to obtain the target product.

[0044] Preferably, in step (1), the equivalent ratios of oleanolic acid to methylamine hydrochloride, 2-(7-azobenzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and N,N-diisopropylethylamine are 1:1, 1:1.3, and 1:2, respectively.

[0045] Specifically, in step (2), the organic solvent is ethyl acetate.

[0046] Specifically, in step (3), the organic phase is collected, concentrated using a rotary evaporator, and silica gel powder is added and rotary evaporated to obtain a solid powder.

[0047] More specifically, the obtained solid powder is purified by silica gel column chromatography; preferably, gradient elution is performed using a mixture of petroleum ether and ethyl acetate as the eluent to obtain a purified solution of the target product, which is concentrated and vacuum dried to obtain the target product; more preferably, the volume ratios of the eluents petroleum ether and ethyl acetate in the silica gel purification are 3:1 and 1:1.

[0048] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0049] 1. Toxicology and pharmacokinetic studies have shown that OA-17 is safe and effective, has no toxic effects on any organ, and has better water solubility and pharmacokinetic characteristics than its precursor OA.

[0050] 2. Pharmacodynamic experiments have shown that OA-17 is superior to OA in the treatment of psoriasis. It can significantly improve the skin inflammatory response of mice with psoriasis and significantly reduce the levels of psoriasis-related inflammatory factors such as IL 17, IFN-γ, TNF-α, and IL-23a.

[0051] 3. Molecular docking revealed that OA-17 binds to SHP2, a key drug target in the psoriasis disease process, with a higher binding affinity than its precursor OA. Molecular biology experiments demonstrated that OA-17 significantly inhibits SHP2 expression on T cells, thereby suppressing imiquimod-induced skin inflammation in psoriatic mice in pathological experiments.

[0052] 4. The present invention provides a new medication option for the treatment of psoriasis patients. The parent drug has a wide range of sources, simple synthesis steps, low cost and few by-products, clear ingredients and dosage, safe medication without obvious side effects, and has good social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 HE staining of the heart, liver, spleen and kidney of mice in each group.

[0054] Figure 2 .Standard curve of OA-17 in rat plasma.

[0055] Figure 3 .Average blood concentration-time curve of rats after oral administration of 100 mg / kg OA-17.

[0056] Figure 4 .Skin phenotype changes of mice in each group.

[0057] Figure 5 Effects of OA-17 and OA on the PASI scores of mice in each group, including erythema, scales, thickness, and cumulative scores (n=4, Mean±SD) (*P<0.05 and **P<0.01 OA-17 (25 mg / kg) vs. Model, #P<0.05 and ##P<0.01 OA-17 (25 mg / kg) vs. OA (25 mg / kg)).

[0058] Figure 6Changes in spleen and spleen index of mice in each group (n=4, Mean±SD). **P<0.01 vs. Model, #P<0.05 OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg) vs. OA (25 mg / kg, 50 mg / kg, 100 mg / kg), &&P<0.01 vs. Normal).

[0059] Figure 7 Pathological changes of skin in mice in each group

[0060] Figure 8 .Expression of PCNA in the skin tissue of mice in each group.

[0061] Figure 9 Effects of OA-17 and OA on IMQ-induced serum IL-17A levels in mice (n=4, Mean±SD). **P<0.01 and ***P<0.0001 vs. Model, ##P<0.01 and ###P<0.001 OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg) vs. OA (25 mg / kg, 50 mg / kg, 100 mg / kg), &&&P<0.001 Model vs. Normal.

[0062] Figure 10 Effects of OA and OA-17 on the expression of IL-17a and IL-23 mRNA in IMQ-induced mouse skin tissue (n=4, Mean±SD). ***P<0.001 vs. Model, ###P<0.001 vs. OA (25 mg / kg, 50 mg / kg, 100 mg / kg), &&P<0.01 and &&&P<0.001 vs. Normal.

[0063] Figure 11 .Molecular docking results of OA, OA-17 and SHP2.

[0064] Figure 12 .Effects of OA-17 and SHP2 inhibitors on the activity of mouse spleen T cells (n=4, Mean±SD) (**P<0.01 vs.ConA).

[0065] Figure 13 Effects of OA-17 on the expression of SHP2 and PI3K / mTOR pathway-related proteins in skin tissue

[0066] Figure 14Effects of OA-17 on clinical manifestations in AA rats (n=6, Mean±SD). (A) Body weight; (B) Paw swelling; (C) Arthritis index; (D) Number of swollen joints; (E) Whole-body score. (#P<0.05, ##P<0.01 vs Normal; *P<0.05, **P<0.01 vs AA model)

[0067] Figure 15 Effects of OA-17 on spleen and thymus indexes in AA rats (n=6, Mean±SD). #P<0.05, ##P<0.01 vs Normal; **P<0.01 vs AA model.

[0068] Figure 16 Effects of OA-17 on the expression of TNF-α and IL-1β in the serum of AA rats (n=6, Mean±SD). **P<0.01 vs Normal; ##P<0.01 vs AA model.

[0069] Figure 17 Effects of OA-17 on the expression of CRP, CCP, and RF in AA rats (n=6, Mean±SD). **P<0.01 vs Normal; ##P<0.01 vs AA model.

[0070] Figure 18 Th17 / Treg cell ratio in rat spleen (n=6, Mean±SD). **P<0.01 vs Normal; ##P<0.01 vs AA model).

[0071] Figure 19 .X-ray changes of the ankle joint and paw on the secondary side of AA rats.

[0072] Figure 20 OA-17 inhibits MH7A cell migration and invasion. (A) Cell migration; (B) Cell invasion.

[0073] Figure 21 OA-17 regulates the expression of mitochondrial damage-related genes and related inflammatory factors mRNA in MH7A cells (n=6, Mean±SD). **p<0.01, ***p<0.001 vs TNF-α group.

[0074] Figure 22 OA-17 regulates the expression of mitochondrial damage-related proteins cGAS and STING in MH7A cells.

[0075] Figure 23.Molecular docking results of OA-17 and TNFR1. DETAILED DESCRIPTION

[0076] The technical solution of the present invention will be clearly described below in conjunction with the accompanying drawings. Obviously, the described embodiments are not all embodiments of the present invention. All other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0077] Example 1: Preparation of OA-17

[0078] (1) Take 200 mg of oleanolic acid, add 216.46 mg of the catalyst 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and 152.56 μL of N,N-diisopropylethylamine, respectively, and react at 20-25°C for 1 hour. Then, add 29.56 mg of methylamine hydrochloride at a 1:1 ratio and stir at a constant speed for 6-15 hours.

[0079] (2) The mixture of step 1 was dissolved in an organic solvent, saturated aqueous NaCl solution and saturated aqueous potassium hydrogen sulfate solution were added, and the mixture was extracted with ethyl acetate 2-3 times. The organic phase was collected and concentrated using a rotary evaporator, silica gel powder was added and rotary evaporated, and the resulting solid powder was subjected to silica gel column chromatography. Gradient elution was performed using a mixture of petroleum ether and ethyl acetate in a volume ratio of 3:1 and 1:1 as the eluent to obtain a purified solution of the target product, which was concentrated and vacuum dried to obtain the target product. The yield was 82%.

[0080] Example 2: Acute toxicity study of OA-17

[0081] 1. Experimental Methods

[0082] 7-8-week-old C57BL / 6 mice, weighing 20±2 g, were randomly divided into three groups: CMC-Na, OA-17 (1 g / kg), and OA-17 (10 g / kg), with 10 mice in each group. OA-17 / CMC-Na was administered orally twice within 24 hours, 0.2 mL each time, for 7 days. The mice's behavior, fur, and mental state were observed twice daily. Water and food intake were recorded daily, and body weight was measured. On day 4, the heart, liver, kidney, and spleen were washed in PBS buffer, dehydrated on filter paper, and the spleen index was calculated. The mice were then immersed in 4% formalin for 24 hours, sectioned, and stained with hematoxylin and eosin for microscopic examination. Peripheral blood (50 μL) was collected for biochemical analysis, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN), to assess the effects of OA-17 on the liver and kidneys.

[0083] 2. Experimental Results

[0084] During the observation period, the oral administration group had no erect hair, changes in body color, normal movements, no spasms, movement disorders, convulsions, normal body reflexes, smooth fur, no abnormal secretions in the eyes, nose, and mouth, and normal feces and urine compared with the control group. There was no obvious constriction or dilation of the pupils, and eye examination signs such as proptosis were all normal. No mice died in all groups during the 7-day observation period. The weight of mice in each group changed to varying degrees with the increase in feeding time, but no obvious abnormalities were found. After the observation period, the mice were killed, and the results of macroscopic observation and dissection showed that there were no obvious abnormalities in the main tissues and organs of the mice in the oral administration group, such as the heart, liver, spleen, lungs, and kidneys. Figure 1 The results of main tissue pathology staining showed that the cell structure and morphology of liver lobules in the drug-treated group were normal, without lesions or abnormalities, and the germinal center, marginal zone and red and white pulp junction in the spleen were normal. At the same time, the spleen tissue structure of the drug-treated group was basically the same as that of the control group, the lung tissue structure was clear, without edema and effusion, the alveolar structure was normal, the alveolar septum was slender and not widened, and the glomerular and renal unit cell structure was normal.

[0085] Biochemical index detection showed that the ALT, AST, and BUN indicators of the mice in the drug-treated group were normal, and OA-17 did not affect the liver and kidney functions (Table 1).

[0086] Table 1. Effects of OA-17 on blood biochemical parameters in mice

[0087]

[0088] Example 3: Pharmacokinetic study of OA and OA-17 after single oral administration

[0089] 1. Experimental Methods

[0090] (1) Construction of standard curve

[0091] Blood was collected from the ophthalmic venous plexus of a healthy male SD rat and placed in a 1.5 mL centrifuge tube prefilled with 20 μL of sodium heparin solution. The tube was centrifuged at 3500 rpm and 4°C for 10 min. Accurately aspirate 50 μL of the upper plasma layer and add OA-17 standard control solution at concentrations of 160, 320, 640, 1280, and 2560 ng / mL, respectively, and mix for 1 min. Then, 5 μL of 100 ng / mL internal standard control solution was added and mixed for 1 min. Then, 200 μL of acetonitrile was added and mixed for 2 min. The tube was centrifuged at 14000 rpm for 10 min. 200 μL of the supernatant was transferred to a 1.5 mL centrifuge tube, diluted with 800 μL of acetonitrile, filtered, and 2 μL was injected for analysis.

[0092] (2) Detection of blood drug concentration and calculation of pharmacokinetic parameters after single oral administration of OA-17 in rats

[0093] Three healthy male SD rats were fasted for 12 hours and treated with OA-17 by gavage at a dose of 100 mg / kg. Orbital blood was collected at 10, 20, 40, 60, 120, 180, 240, 360, 480, 600, 720, and 1440 minutes after administration. Approximately 200 μL of blood was collected from each rat at each time point and placed in a 1.5 mL centrifuge tube pre-filled with 20 μL of heparin sodium solution. The blood was centrifuged at 3500 rpm and 4°C for 10 minutes. 50 μL of the upper plasma was accurately aspirated and placed in a 1.5 mL centrifuge tube and mixed for 1 minute. 5 μL of 100 ng / mL internal standard control solution was added and mixed for 1 minute. 200 μL of acetonitrile was added and mixed for 2 minutes. The supernatant was centrifuged at 14000 rpm for 10 minutes. 200 μL of the supernatant was placed in a 1.5 mL centrifuge tube and diluted with 800 μL of acetonitrile. The tube was filtered and 2 μL was injected for analysis.

[0094] 2. Experimental Results

[0095] (1) Construction of standard curve

[0096] The concentration of OA-17 in the sample (ng / mL) was used as the horizontal axis, and the peak area ratio of OA-17 to the internal standard was used as the vertical axis. The weighted least square method (W = 1 / C 2 ) to perform regression calculation to obtain a standard curve, and the relative standard deviation (RSD) should be less than 20%. The results show that this method has good linearity in the range of 1-1000ng / mL. Figure 2 is the standard curve.

[0097] (2) Detection of blood drug concentration and calculation of pharmacokinetic parameters after single oral administration of OA-17 in rats

[0098] The average blood concentration-time curve of rats given 100 mg / kg by gavage is shown in Figure 3 .

[0099] The measured data were processed using DAS 3.0 pharmacokinetic software and the two-compartment model analysis method to obtain the main pharmacokinetic parameters of OA-17 in rats under extravascular administration. The results are shown in Table 2.

[0100] Table 2 Pharmacokinetic parameters of OA-17 in rats after administration

[0101]

[0102] Example 4: Effects of OA and OA-17 on Imiquimod-induced Psoriasis Model in Mice

[0103] 1. Experimental Methods

[0104] BALB / c mice weighing 20±2g were acclimated for one week and then anesthetized with 1.5% isoflurane inhalation. The hair on the back of the mice was removed with a depilatory cream, leaving a 2cm×3cm denuded area. After depilation, the mice were randomly divided into nine groups: a normal group, a model group, low-, medium-, and high-dose OA groups (25mg / kg, 50mg / kg, and 100mg / kg), a low-, medium-, and high-dose OA-17 group (25mg / kg, 50mg / kg, and 100mg / kg), and a positive drug MTX (2mg / kg) group, with three mice in each group. Mice in each treatment group were premedicated. The low-, medium-, and high-dose OA groups, the low-, medium-, and high-dose OA-17 groups, and the MTX group were each given a pre-prepared drug suspension. The OA and OA-17 groups were gavaged once daily, while the MTX group was gavaged every two days, with 0.2mL administered each time. The normal and model groups were gavaged with an equal volume of CMC-Na. On day 6 of oral administration, mice in the model group, OA low-, medium-, and high-dose groups (25 mg / kg, 50 mg / kg, and 100 mg / kg), OA-17 low-, medium-, and high-dose groups (25 mg / kg, 50 mg / kg, and 100 mg / kg), and the positive drug MTX (2 mg / kg) group were treated with 62.5 mg of 5% imiquimod (IMQ) cream on the exposed skin. The same amount of petroleum jelly cream was applied to the backs of mice in the normal group at a fixed time daily. Mice in each group were weighed and sacrificed by dislocation until day 12.

[0105] 2. Evaluation indicators

[0106] (1) Psoriasis Area and Severity Index (PASI): Lesions were scored and photographed daily to record changes in the lesions. The mice were given scores of 0-4 for erythema, scaling, and thickening of the lesions. The total score was obtained by adding the three scores. The PASI scoring system is as follows: none = 0; mild = 1; moderate = 2; severe = 3; very severe = 4; total score = scaling + thickening + erythema (0-12 points).

[0107] (2) Spleen index = mouse spleen weight (mg) / mouse body weight (g)

[0108] (3) Mouse skin histopathological observation and epidermal thickness detection: The skin on the back of the mouse was cut with clean scissors and divided into four pieces. One piece of skin was placed in 4% paraformaldehyde, dehydrated, paraffin-embedded, sectioned, and stained with hematoxylin and eosin. The pathological changes in the mouse skin tissue were observed under a microscope. The hematoxylin and eosin stained sections were selected for observation and photography under a microscope to analyze the thickening of the epidermis.

[0109] (4) Immunohistochemical observation of epidermal cell proliferation: A piece of skin tissue from the back of each mouse was fixed in pre-prepared 4% formaldehyde, embedded in paraffin, and sliced. After dewaxing and antigen retrieval, it was labeled with immunohistochemical marker antibody PCNA. After sealing, the slices were observed under a microscope to analyze the proliferation of epidermal cells in the skin of each group.

[0110] (5) ELISA assay for IL-17A levels in mouse serum: Mice were sacrificed by cervical dislocation, and blood was collected from the eyeballs and placed in EP tubes. The tubes were placed in an ice box for 2 h and centrifuged at 4°C (2000 rpm, 10 min). The supernatant was collected as the mouse serum. ELISA assay was performed according to the kit instructions.

[0111] (6) Detection of IL-17a and IL-23 mRNA expression in mouse skin tissue by qPCR: RNA was extracted from the skin tissue of each group of mice using the Trizol method. -ΔΔCT The relative quantitative analysis of il-17a and il-23 mRNA in the skin tissue of each group of mice was performed by the method.

[0112] 3. Experimental Results

[0113] (1) Psoriasis skin phenotype and severity index (PASI) in mice

[0114] After modeling, mice were photographed and scored daily. Figure 4 The results showed that the skin of normal mice was smooth and delicate from day 6 to 12; the IMQ-induced mice showed slight scaling and slight redness on day 6, and the skin lesions gradually worsened from day 7 to 12, with the scales eventually becoming dense and thickened, and the erythema becoming darker. Compared with the model group mice, the OA (25 mg / kg, 50 mg / kg) group had a slight but no significant improvement in the distribution of dandruff on the back. OA (100 mg / kg), OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg) and MTX (2 mg / kg) were able to significantly improve the erythema, scaling and thickening of the mouse skin.

[0115] Depend on Figure 5The results showed that the scales, erythema, thickening and total scores of the normal group mice were all 0 on days 6-12; the scales, thickening, erythema and total scores of the skin of the mice in the IMQ-induced model group continued to increase on days 6-12; there was no significant difference between the OA (25 mg / kg, 50 mg / kg) groups and the model group, and the scales and thickening of the back skin of the mice in the OA (100 mg / kg) group were slightly improved compared with the model group, and the scales, erythema, thickening and total scores were reduced on day 12. The skin scaling, thickening, erythema and total scores of mice in the OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg) and MTX (2 mg / kg) groups continued to increase from d6 to 11, but the values ​​were lower than those in the model group. On d12, OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg) and MTX (2 mg / kg) could significantly reduce the IMQ-induced skin scaling, thickening, erythema and total scores of mice.

[0116] (2) Spleen index measurement results

[0117] Depend on Figure 6 Compared with the normal group, the spleens of the model group mice were significantly enlarged, and the spleen index was significantly increased, indicating successful modeling. Compared with the model group, the spleen enlargement of the mice in the OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg) and MTX (2 mg / kg) groups was significantly alleviated, and the spleen index was significantly reduced. The higher the dose of OA-17, the lower the spleen index. However, the spleen size of the OA (25 mg / kg and 50 mg / kg) groups did not decrease significantly compared with the model group. However, the spleen size of the OA (100 mg / kg) group was significantly smaller than that of the model group, and the spleen index was also significantly reduced. This indicates that OA (100 mg / kg), OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg), and MTX (2 mg / kg) all have therapeutic effects on IMQ-induced psoriasis mice, but OA-17 (100 mg / kg) has the best therapeutic effect, second only to the positive control drug MTX.

[0118] (3) Pathological observation of mouse skin tissue and detection of epidermal thickness The skin of each group of mice was removed and stained with HE, and the pathological changes of skin tissue were observed under a microscope. Figure 7The results showed that the skin tissue structure of mice in the normal group was normal, with only two to three layers of epidermal cells. Compared with the normal group, the epidermis of mice in the IMQ-induced model group was significantly thickened, with capillary hyperplasia and inflammatory cell infiltration in the superficial layer of the epidermis. The stratum corneum showed parakeratosis, thickening of the stratum spinosum, and epidermal protrusions extending downward. Compared with the model group, the epidermal thickness, parakeratosis, capillary hyperplasia, and inflammatory cell infiltration in the OA (100 mg / kg), OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg), and MTX (2 mg / kg) groups were significantly reduced, while there was no significant difference between the OA (25 mg / kg) and OA (50 mg / kg) groups and the model group.

[0119] (4) Immunohistochemical observation of epidermal cell proliferation

[0120] Proliferating Cell Nuclear Antigen (PCNA) can reflect the proliferation of keratinocytes. Immunohistochemically positive expression is located in the cell nucleus and appears brownish yellow. Figure 8 The results showed that PCNA in the normal group of mice was only arranged linearly in the basal layer of the epidermis; compared with the normal group, the number of PCNA-positive cells in the model group of mice was significantly increased; compared with the model group, the number of PCNA-positive cells in the OA (100 mg / kg), OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg) and MTX (2 mg / kg) groups of mice was significantly reduced, while there was no significant difference between the OA (25 mg / kg) and OA (50 mg / kg) groups and the model group.

[0121] (5) Detection of mouse serum IL-17A levels by ELISA

[0122] IL-17A is an important inflammatory cytokine in psoriasis patients and is involved in the development of psoriasis. The ELISA kit was used to detect the level of IL-17A in mouse serum. Figure 9 The results showed that compared with the normal group of mice, the expression of serum IL-17A in the model group mice was significantly increased; compared with the model group, the serum IL-17A levels of mice in the OA (100 mg / kg), OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg) and MTX (2 mg / kg) groups were significantly downregulated, and among the OA-17-treated groups, the therapeutic effect of the OA-17 (100 mg / kg) group was the most obvious, second only to the positive control drug MTX group.

[0123] (6) Detection of IL-17a and IL-23 mRNA expression in mouse skin tissue by qPCR

[0124] The expression of il-17a and il-23 mRNA in mouse skin tissue was detected by qPCR. Figure 10 The results showed that compared with normal mice, the expression of IL-17a and IL-23 mRNA in the skin tissue of the model mice was significantly increased; compared with the model mice, the expression of IL-17a and IL-23 mRNA in the OA (25mg / kg, 50mg / kg) groups did not change significantly; compared with the model mice, OA (100mg / kg), OA-17 (25mg / kg, 50mg / kg, 100mg / kg), and MTX (2mg / kg) significantly reduced the expression of IL-17a and IL-23 mRNA, and the effect was most significant in the OA-17 (100mg / kg) group. This suggests that OA-17 can alleviate psoriasis symptoms by inhibiting the expression of inflammatory factors and cytokine mRNA.

[0125] Example 5: Study on the Mechanism of OA-17 in Treating Psoriasis

[0126] 1. Experimental Methods

[0127] (1) Discovery Studio molecular docking technology was used to investigate the binding ability of OA and OA-17 with SHP2: 4OHL human non-receptor type 11 protein tyrosine phosphatase protein subunit was retrieved from the RCSB PDB website and imported into Discovery Studio for processing, including removal of water molecules, side chain repair, main chain end treatment, and identification of the active pocket. The chemical structure of OA-17 was drawn using ChemDraw software and imported into the Discovery Studio small molecule window to prepare the ligand molecule. After successful preparation, CDOCKER in Discovery Studio was used to perform simulated molecular docking experiments between small molecules and proteins, and the docking results of OA and OA-17 with proteins were analyzed respectively.

[0128] (2) Effects of OA-17 and SHP2 inhibitors on mouse spleen T cell activity: Normal C57BL / 6 mouse spleens were isolated in a clean bench, cleaned with 75% ethanol, and half was placed in a culture dish. 5 mL of mouse lymphocyte separation solution was added, and the spleen was wrapped with sterile gauze and fully ground with a homogenizer to prepare a spleen lymphocyte suspension. 3 × 10 6 The spleen lymphocyte suspension collected in vitro was divided into control group, ConA (5 ng / mL) stimulation group, OA-17 (25 mg / kg, 50 mg / kg, 100 mg / kg groups), and SHP2 inhibitor group. After 24 h of culture, the CCK8 method was used to detect the lymphocyte viability of each group.

[0129] (3) Effect of OA-17 on the expression of SHP2 and PI3K / mTOR pathway-related proteins in skin tissue: 1% protease inhibitor (PMSF) and 1% phosphatase inhibitor were added to RIPA lysis buffer and mixed well. 50 mg of mouse skin from the normal group, MTX group, and OA-17 (100 mg / kg, 50 mg / kg, 25 mg / kg) group was weighed and added to 500 μL of the prepared lysis buffer for thorough grinding. After centrifugation at 12,000 g for 15 min at 4°C, the supernatant was aspirated. After BCA quantification and denaturation with loading buffer, the expression of SHP2, PI3K, mTOR, and p-mTOR proteins in the skin of each group of mice was detected by Western blot.

[0130] 2. Experimental Results

[0131] (1) Molecular docking technology showed that OA-17 has a stronger affinity with SHP2 than OA

[0132] Figure 11 The following are the molecular docking results of OA and OA-17 with SHP2. The green bonds are hydrogen bonds. Hydrogen bonds are a type of intermolecular force, a force between permanent dipoles. Hydrogen bonds are particularly important for biopolymers. They are part of the reason why the secondary, tertiary, and quaternary structures of proteins and nucleic acids are stabilized. Therefore, the more hydrogen bonds in the molecular docking results, the stronger the affinity between the two molecules. Figure 11 As shown, there are three hydrogen bonds between OA-17 and SHP2, while there is only one hydrogen bond between OA and SHP2. This may preliminarily indicate that the modified OA, namely OA-17, has a stronger affinity for the target site SHP2.

[0133] (2) OA-17 and SHP2 inhibitors inhibit abnormal proliferation of mouse spleen T cells

[0134] Depend on Figure 12 The results showed that compared with the control group, the T cell activity in the ConA-stimulated group was significantly enhanced, while the OA-17 (10 μM) and SHP2 inhibitor groups were significantly reduced in the spleen T cell activity compared with the ConA-stimulated group. This indicates that OA-17 and SHP2 inhibitors can inhibit the abnormal proliferation of T cells.

[0135] (3) OA-17 inhibits the expression of SHP2 and PI3K / mTOR pathway-related proteins in skin tissue

[0136] Depend on Figure 13The results showed that compared with the normal group, the expression of PI3K / AKT-related proteins and SHP2 proteins in the skin of the model group mice was significantly increased. Compared with the model group mice, the expression of PI3K / mTOR-related proteins and SHP2 proteins in the skin of the OA-17 (25mg / kg, 50mg / kg, 100mg / kg) and MTX (2mg / kg) groups mice was significantly reduced. This suggests that OA-17 can alleviate psoriasis by inhibiting the expression of SHP2 and PI3K / mTOR.

[0137] Example 6: Effects of OA and OA-17 on AA rat model

[0138] 1. Experimental Methods

[0139] Dissolve chicken type II collagen in 0.1 mol / L glacial acetic acid to a final concentration of 2 mg / mL. Pipette thoroughly to dissolve, and refrigerate at 4°C overnight. The next day, remove the prepared collagen solution and mix it with an equal volume of complete Freund's adjuvant. Continue grinding. Alternatively, repeatedly pipette with a 1 mL syringe without a needle to emulsify the mixture to a milky white color.

[0140] The rat's right hind paw was disinfected with a 75% ethanol cotton ball. 0.1 mL of the mixture was drawn up into a 1 mL syringe and injected intradermally into the rat's paw from the bottom up. A bulge formed after injection, and pressure was applied to prevent outflow. A control group of rats in the normal paw was injected with the same volume of saline. The model was established on day 0, and inflammatory symptoms began to appear between days 14 and 16.

[0141] Based on overall evaluation after model establishment, rats were randomly divided into four groups: model group, OA-17 (25 mg / kg) group, OA-17 (50 mg / kg) group, and OA-17 (100 mg / kg) group. A normal control group was established, with eight rats in each group. Before dosing, rats were weighed, and the daily dose was calculated based on the dose. Dosing began on day 17 and continued until day 30. The normal control group received the appropriate saline solution as a control. After the dosing cycle, the rats were anesthetized and sacrificed, and various relevant indicators were observed and recorded.

[0142] 2. Evaluation indicators

[0143] (1) Rat weight

[0144] After model establishment (from day 0), the same experimenter weighed and recorded the weight of rats in each group every 3 days.

[0145] (2) Joint swelling number and arthritis index score

[0146] After modeling (starting from day 0), the same experimenter scored the paw lesions of each group of rats every 3 days according to the scoring criteria, and counted the number of swollen joints. Each rat's toes were counted for 5 finger joints and 1 ankle joint, with a maximum of 24 swollen joints and a maximum score of 24 points. At the same time, the same experimenter evaluated the arthritis index of the ankle joints of each group of rats every 3 days according to the scoring criteria, with a maximum score of 16 points. The scoring details are as follows:

[0147] Table 2. Arthritis index scoring criteria (maximum 16 points per rat)

[0148]

[0149] Table 3. Joint swelling scoring criteria (maximum 24 points per rat)

[0150]

[0151]

[0152] (3) Measurement of the secondary paw volume

[0153] Before modeling, the volume of the left hind paw (non-inflammation side) of the rats was measured and recorded as the initial value. Starting from the 16th day, the same experimenter measured the volume of the left hind paw of each group of rats every 3 days. Each rat was measured 3 times in a row using a toe volume meter. The average value was the joint volume, and the corresponding initial value was subtracted when calculating the swelling degree.

[0154] (4) Calculation of rat spleen and thymus index

[0155] The night before sacrifice, rats were deprived of food. Each rat's weight was weighed and recorded before the experiment. Rats were anesthetized and sacrificed. Thymus and spleen tissue were removed in a cleanroom, cleanly dissected, and placed in pre-labeled sterile centrifuge tubes (centrifuge tubes must be weighed in advance). The total weight was then measured using an electronic balance. The thymus or spleen index for each rat was calculated as the ratio of the net thymus or spleen weight to the corresponding body weight.

[0156] (5) Detection of inflammatory factors and related reaction proteins in rat serum by enzyme-linked immunosorbent assay

[0157] Femoral arterial blood was collected from each rat, allowed to rest for 2 hours, and centrifuged at 2500 rpm for 10 minutes. Serum samples were collected and frozen at -80°C until further use. Supernatants from FLS cell cultures were collected from each group of rats and centrifuged at 2500 rpm for 10 minutes. The supernatants were collected and frozen at -80°C until further use. Serum levels of inflammatory factors and related reactive proteins were assayed using ELISA kits.

[0158] (6) Flow cytometry analysis of the effect on Th17 / Treg immune balance in rats

[0159] Place the spleen tissue on a gauze containing lymphocyte separation fluid, grind it evenly until the spleen cells are fully detached, rinse the spleen cells adhering to the filter with PBS, and collect the washed spleen cells. Centrifuge the prepared single-cell suspension at 500g for 5 minutes, remove the supernatant, and take the precipitate. Resuspend the cell pellet with 2mL of hemolysin and react at room temperature for 10 minutes to lyse the red blood cells. Centrifuge at 500g for 5 minutes, remove the supernatant, and take the precipitate. Add 2mL of PBS to wash the cell pellet, resuspend it and centrifuge at 500g for 5 minutes, remove the supernatant, and take the precipitate. Use 1mL of RPMI culture medium to mix and resuspend the cells, dilute the cells at 1:10 or 1:20 times, and count the cells. Take 5×10 6 Each tube was stimulated with T cell co-stimulator and incubated at 37°C for 4 hours. After stimulation, the cells were centrifuged at 500g for 5 minutes, the supernatant discarded, and the cells were resuspended in 5mL of PBS and centrifuged at 500g for 5 minutes. The supernatant was discarded. The cells were resuspended in 100μL of PBS and 1μL of FITC-CD4 antibody was added, vortexed to mix, and incubated at room temperature in the dark for 20 minutes. 2mL of cell fixative solution was added to each tube, incubated at room temperature in the dark for 20 minutes, centrifuged at 600g for 5 minutes, and the supernatant discarded. 2mL of cell permeabilization solution was added to each tube, incubated at room temperature in the dark for 10 minutes, centrifuged at 600g for 5 minutes, and the supernatant discarded. 2mL of cell permeabilization solution was added to each tube, centrifuged at 600g for 5 minutes, and the supernatant discarded. 1μL of PE-IL17A antibody was added, vortexed to mix, and incubated at room temperature in the dark for 30 minutes. 2mL of cell permeabilization solution was added to each tube, centrifuged at 600g for 5 minutes, and the supernatant discarded. Finally, add 300 μL PBS to each tube, vortex to mix, and then test on the instrument.

[0160] (7) X-ray scanning of rat ankle joints to observe bone destruction

[0161] X-ray imaging of the secondary ankle joints of AA rats was performed using an X-ray imaging system (Leica, Germany). Rats in the experimental group were anesthetized with isoflurane inhalation and placed on an imaging board. The secondary inflamed ankle joints were photographed using a GTI-2000 imaging system.

[0162] 3. Experimental Results

[0163] (1) Effects of OA-17 on clinical manifestations in rats

[0164] On the 16th day after modeling, the body weight, arthritis index, number of paw swellings and degree of paw swelling of the rats were measured and recorded every 3 days. The results are shown in the figure below ( Figure 14). From the 14th to 16th day after modeling, the paws of the rats began to swell and reached a peak around the 27th day. The body weight of AA model rats was significantly lower than that of normal rats (P<0.01). Daily gavage was started from the 18th day after modeling. Compared with the model group, the arthritis index, paw swelling number and paw swelling degree of the OA-17 (50mg / kg / day) group and the OA-17 (100mg / kg / day) group were significantly lower than those of the AA rat group on the 10th day after administration (the 26th day of modeling) (P<0.05). In summary, OA-17 can significantly improve the arthritis index, paw swelling number and paw swelling degree of AA rats. The results are shown below. Figure 14 , the body weight changes of AA rats ( Figure 14 Middle A), paw swelling ( Figure 14 Middle B), arthritis index ( Figure 14 Middle C), number of joint swelling ( Figure 14 D) and whole body score ( Figure 14 Middle E).

[0165] (2) Effects of OA-17 on the spleen and thymus indexes of rats

[0166] The results are as follows Figure 15 As shown in the figure, compared with normal rats, the thymus and spleen of AA model rats were significantly enlarged and the index was increased; compared with the model group, the OA-17 administration group significantly reduced the spleen and thymus index of AA rats (P<0.01).

[0167] (3) Detection of TNF-α and IL-6 levels in rat serum by enzyme-linked immunosorbent assay

[0168] ELISA was used to detect the changes in the levels of inflammatory factors TNF-α and IL-6 in the serum of rats in each group. Figure 16 ), compared with normal rats, the serum IL-6 level of AA model rats was significantly increased (P<0.01); compared with AA group rats, the serum IL-6 level of OA-17 treatment group was significantly decreased (P<0.01).

[0169] (4) Detection of CRP, CCP and RF levels in rats by enzyme-linked immunosorbent assay

[0170] ELISA was used to detect the changes in the levels of C-reactive protein (CRP), CCP (anti-cyclic citrullinated polypeptide antibody) and rheumatoid factor (RF) in rats of each group. Figure 17 ), compared with normal rats, the levels of CRP, CCP and RF in AA model rats were significantly increased (P<0.01); compared with AA group rats, the high-dose OA-17 group significantly reduced the levels of CRP, CCP and RF in rats (P<0.01).

[0171] (5) Flow cytometry analysis of the effect of Th17 / Treg immune balance in rat spleen

[0172] like Figure 18 The Th17 / Treg ratio in the OA-17 medium and high dose groups was significantly lower than that in the AA model group (P < 0.05), while there was no significant change in the low dose group compared with the AA group.

[0173] (6) X-ray scanning of rat ankle joints to observe bone destruction

[0174] AA model rats have swelling of the secondary side joint soft tissue, osteoporosis, decreased bone density, obvious bone erosion lesions, and narrowing or even disappearance of the joint space. Figure 19 As shown in the figure, compared with the model group, the OA-17 treatment group showed relief of symptoms such as soft tissue swelling, bone erosion, and joint space narrowing, especially the medium and high dose treatment groups showed significant differences compared with the model group.

[0175] Experimental Example 7: Study on the mechanism of OA-17 in treating arthritis

[0176] 1. Experimental Methods

[0177] (1) Transwell assay to evaluate the effect of OA-17 on the invasion ability of MH7A cells

[0178] Matrigel glue (0.1%) was evenly applied to the bottom membrane of the upper chamber of the Transwell plate, dried at 37°C for 30 min, and washed three times with PBS. Cells were collected by trypsin digestion and washed 1-2 times with PBS, resuspended in serum-free medium, and diluted with serum-free medium according to the required density. 600 μL of DMEM medium containing 20% ​​FBS was added to the lower chamber of the 24-well plate; 100 μL of cell suspension (1×10 5 The cells were incubated with TNF-α (20 ng / mL) and OA-17 (1 μM, 5 μM) for 24 hours. The upper chamber was carefully removed with tweezers and washed with PBS. The cells were fixed with 4% paraformaldehyde for 10 minutes, washed three times with PBS, stained with crystal violet dye for 5 minutes, and rinsed several times with clean water. Excess cells on the bottom membrane surface of the upper chamber were carefully wiped off with a wet cotton swab. Nine randomly selected fields of view were photographed under an optical microscope, and the invading cells were statistically analyzed.

[0179] (2) Scratch assay to determine the effect of OA-17 on the migration ability of MH7A cells

[0180] MH7A cells (5×10 5Cells were seeded in 6-well plates (100 cells / well). When the cells grew to cover the entire bottom surface, they were cultured in serum-free medium for another 12 hours to eliminate the influence of normal cell growth on the scratch test results. A 200 μL sterile pipette tip was used to scratch the bottom of the well plate, and the scratched cells were gently washed three times with PBS to remove the scratched cells. The cells were stimulated with TNF-α (20 ng / mL) and treated with different concentrations of OA-17 (1 μM, 5 μM) for 24 hours. The culture medium was aspirated, the cells were washed three times with PBS, and then fixed with 4% paraformaldehyde for 20 minutes. The fixative was aspirated, the cells were washed three times with PBS, and the cells were stained with crystal violet, washed with water, dried, and photographed. The distance changes in the scratch area were then measured and analyzed.

[0181] (3) OA-17 regulates the expression of cgas, sting and various inflammatory factors mRNA in MH7A cells

[0182] MH7A cells (5×10 5 Each well was inoculated with Trizol and then the cells were lysed and RNA was extracted using 2 -ΔΔCT The method was used to quantitatively analyze the mRNA of cgas, sting and various inflammatory factors in MH7A.

[0183] (4) OA-17 regulates the expression of mitochondrial damage-related proteins cGAS and STING in MH7A cells

[0184] MH7A cells (5×10 5 / well) were added to a six-well plate and cultured for 12 hours. TNF-α (20 ng / mL) and OA-17 (1 μM, 5 μM) were added and co-cultured for 24 hours. The supernatant was removed and washed three times with pre-chilled PBS. RIPA lysis buffer and protease inhibitors were mixed in proportion, added to the well plate, and lysed on crushed ice for 30 minutes. The lysate was collected on ice, centrifuged at 4°C and 12,000 rpm for 10 minutes, and the supernatant was retained as the total protein sample. The total protein concentration of each group was detected using the microplate method in the BCA protein quantification kit. A BSA standard curve was established, and the total protein concentration was calculated based on the absorbance of the sample to be tested. The total protein content was adjusted to the same concentration with PBS. The sample was mixed with 5× Loding buffer at a ratio of 4:1, boiled in boiling water for 5 minutes to denature the protein, and stored at -80°C.

[0185] 2. Experimental Results

[0186] (1) Effects of OA-17 on the migration and invasion of MH7A cells

[0187] The migration and invasion of MH7A cells were detected by scratch assay and Transwell chamber. The results showed that OA-17 (1μM, 5μM) could inhibit the migration and invasion of MH7A cells in a concentration-dependent manner. Figure 20 ), the scratches made by the pipette tip in the normal group and TNF-α group were almost filled with MH7A cells, while the migration ability of the OA-17 treatment group decreased. The scratches made by the pipette tip in the normal group and TNF-α group were almost filled with MH7A cells, while the migration ability of cells in the OA-17 (1μM, 5μM) treatment group decreased. At the same time, the Transwell results showed that compared with the TNF-α group, OA-17 (1μM, 5μM) had a significant inhibitory effect on the invasiveness of MH7A cells (p<0.001) ( Figure 20 B).

[0188] (2) Real-time fluorescence quantitative PCR (qPCR) was used to detect the levels of cGAS, STING and related inflammatory factors. Figure 21 As shown, compared with normal MH7A cells, all tested inflammatory factors such as IL-1β and IL-6 were significantly increased under TNF-α stimulation (p<0.001), while OA-17 (0.1μM, 1μM, 10μM) significantly reduced the increased expression of cgas, sting, and related inflammatory factors (p<0.01). This indicates that OA-17 can significantly inhibit the expression of cgas, sting, and related inflammatory factor mRNA in MH7A.

[0189] (3) OA-17 regulates the expression of mitochondrial damage-related proteins cGAS and STING in MH7A cells

[0190] like Figure 22 As shown in the results, compared with the TNF-α group, the OA-17 treatment groups (0.1 μM, 1 μM, and 10 μM) led to downregulation of cGAS and STING expression. The mRNA levels of related factors in qPCR were consistent, confirming the regulatory effect of OA-17 on MH7A mitochondrial damage-related proteins.

[0191] (4) Discovery Studio molecular docking analysis showed that OA-17 has a strong affinity with TNFR1

[0192] Figure 23 The figure shows the molecular docking results of OA-17 and TNFR1. The green bonds are hydrogen bonds. Hydrogen bonds are a type of intermolecular force, a force between permanent dipoles. Hydrogen bonds are particularly important for biopolymers, as they are part of the reason why the secondary, tertiary, and quaternary structures of proteins and nucleic acids are stabilized. Figure 23As shown, there is a strong intermolecular hydrogen bond between OA-17 and TNFR1. This also proves that OA-17 can directly interact with TNFR1, thereby inhibiting the expression of downstream mitochondrial damage-related cGAS / STING by inhibiting TNFR1, ultimately inhibiting the inflammatory changes of MH7A.

[0193] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Use of an oleanolic acid amide derivative in the preparation of a medicament for treating psoriasis, characterized in that: The molecular structure of the oleanolic acid amide derivatives is shown in formula (II):

2. The use according to claim 1, characterized in that The drug further includes a pharmaceutically acceptable carrier.

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