Application of isomagnoflorine in pharmacy

Abstract

The invention discloses application of isomagnoflorine in pharmacy, and particularly relates to application of isomagnoflorine in preparation of a medicine for treating rheumatoid arthritis. Through experiments, the applicant finds that the isomagnoflorine can effectively relieve swelling of foot joints of CIA mice, reduce arthritis indexes and the content of IL-1beta and IL-6 in serum and improve the disease symptoms of RA, the isomagnoflorine has a remarkable treatment effect, the possible action mechanism of the isomagnoflorine is to inhibit activation of an inflammatory signal channel NF-kB, and the isomagnoflorine can be used for preventing and treating RA. The compound is expected to be used for preparing medicines for treating rheumatoid arthritis.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and in particular relates to the use of isomaltine in medicine making. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovitis of the joints, which often causes damage to the synovium, cartilage, bone and other tissues, accompanied by complications such as pericarditis, pleurisy, arteritis and peripheral neuropathy. According to statistics, 1 in every 200 adults in the world suffers from rheumatoid arthritis, and the incidence rate in women is 2 to 3 times that in men. Studies have shown that patients with rheumatoid arthritis live 6 to 11 years less than normal people. It can be seen that rheumatoid arthritis seriously endangers people's physical health and brings a serious burden to families and society.

[0003] The etiology and pathogenesis of RA are not completely clear at present, and may be the result of multiple factors such as environment, heredity, and immunity. In modern medicine, the western medicine treatment of RA is mainly non-steroidal anti-inflammatory drugs, anti-rheumatic drugs modified by glucocorticoids, immunosuppressants and immunomodulators, etc. These drugs can relieve patient symptoms in the short term, but long-term use can cause some adverse reactions, such as infection, bone marrow suppression, liver and kidney function damage, etc. Compared with western medicine, the side effect of Chinese medicine prescription is relatively small. Celastrus orbiculatus has a long history of clinical anti-rheumatic treatment in my country, and the invention patent applications of publication numbers CN1823870A and CN103006747A all point out that the extract of the root of Celastrus orbiculatus or its stem has a certain therapeutic effect on rheumatoid arthritis. Therefore, in the Chinese medicine prescriptions for the current clinical treatment of RA, many contain Celastrus orbiculatus ingredients.

[0004] Isomagnolone has a molecular formula of C 18 H 18 O3, with a molecular weight of 282.33, is a lignan compound, which is polymerized from two molecules of phenylpropanoid derivatives (i.e., C6-C3 monomers). The compound was originally isolated from the bark of star anise by Kouno I et al. The structural formula is shown below:

[0005]

[0006] It is reported that isomalin has a wide range of biological activities, can remove free radicals in the body, play an antioxidant role, and has certain antifungal and immunosuppressive activities. Invention patent applications with publication numbers CN112552258A and CN116768825A point out that isomalin derivatives with higher antibacterial activity can be obtained by structurally modifying isomalin. However, there are no reports on the use of isomalin to treat rheumatoid arthritis. Summary of the invention

[0007] The technical problem to be solved by the invention is to provide the use of isomaltine in medicine.

[0008] The technical scheme of the present invention is: application of isomaltine in preparing medicine for treating rheumatoid arthritis.

[0009] Furthermore, the rheumatoid arthritis is collagen-induced arthritis (CIA).

[0010] The invention also provides a medicine for treating rheumatoid arthritis, which contains a therapeutically effective dose of isomaltine.

[0011] Furthermore, the above-mentioned medicine also contains pharmaceutically acceptable carriers and / or excipients.

[0012] Furthermore, the dosage form of the drug can be a pharmaceutically acceptable dosage form, such as tablets, capsules, oral liquids, lozenges, granules, suspensions, suppositories, injections, powder injections, pills, sustained-release agents, controlled-release agents or targeted preparations and other conventional dosage forms.

[0013] The applicant found through experiments that isomaltine can effectively reduce the swelling of the foot joints of CIA mice, reduce the arthritis index and the content of IL-1β and IL-6 in the serum, improve the symptoms of RA disease, and has a significant therapeutic effect. It is expected to be used to prepare drugs for the treatment of rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the induction of CIA mouse model and intervention with isomaltine.

[0015] Figure 2 Figure 2 shows the improvement of arthritis symptoms in CIA mice by pretreatment in each treatment group; (A) Representative photo of the left hind paw of CIA mice on day 28 of administration; (B) Arthritis index score; (C) Paw thickness (mm). Data are expressed as mean ± SEM (n = 5), *p < 0.05, **p < 0.01, compared with the CIA group.

[0016] Figure 3The secretion of inflammatory factors (A) IL-1β and (B) IL-6 in the serum of CIA mice was inhibited in each treatment group. Data are expressed as mean ± SEM (n = 5). #p < 0.05, ##p < 0.01, compared with the NC group; *p < 0.05, **p < 0.01, compared with the CIA group.

[0017] Figure 4 The effect of each treatment group on the histopathological changes of the left hind paw of CIA mice; ← Indicates cartilage hyperplasia; ▲ Indicates inflammatory cell infiltration; ★ Indicates the presence of neutrophils in the exudate; magnification 100×.

[0018] Figure 5 Effects of each treatment group on the expression of TLR4, p-p65, p65 and MyD88 proteins in the foot muscle tissue of mice (mean ± SEM, n = 3); (A) The expression level of proteins in each group was analyzed by western blotting; (B-D) Quantitative analysis of immunoblot signals, relative to the expression content of β-actin protein in three independent experiments. ###p<0.001, compared with the NC group; **p<0.01, ***p<0.001, compared with the CIA group. DETAILED DESCRIPTION

[0019] The following is a specific experiment to illustrate the application of isomaltine in the treatment of rheumatoid arthritis.

[0020] Example 1

[0021] 1. Construction and drug administration of DBA / 1 mouse CIA model

[0022] Eight-week-old male DBA / 1 mice were provided by Beijing HFK Biotechnology Co., Ltd. (Beijing, China, certification number: SCXK(Jing)2019-0008). 25 Mice were allowed to acclimate to the environment one week before the start of the study and were allowed free access to food and water. All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee Guidelines and approved by the Laboratory Animal Management and Ethics Committee of the Guangxi Institute of Botany, Chinese Academy of Sciences.

[0023] After acclimating to the laboratory conditions for 7 days, 25 mice were randomly divided into 5 groups, 5 mice in each group: CIA group; normal (NC) group; methotrexate group (MTX); isoglossine low-dose group (IMGL-L); isoglossine high-dose group (IMGL-H). Except for the normal group, bovine type II collagen (BIIC, 20022, Redmond) dissolved in complete Freund's adjuvant (CFA, F5881, Sigma) was injected into the base of the tail of the mice at a ratio of 1:1 (total 100 μL) on day 0. On day 21, BIIC booster immunization with incomplete Freund's adjuvant (IFA, F5506, Sigma) was performed proximal to the first injection site. IMGL-L and IMGL-H were treated with isomaltine (PCS2277, Chengdu Zhibiao Huachun Biotechnology Co., Ltd.) 10 and 20 mg / kg (gavage, once a day, 4 weeks), respectively; the MTX group was treated with methotrexate (036201005, Shanghai SPH Pharmaceuticals Co., Ltd.) 1 mg / kg (gavage, once a day, 4 weeks); the normal group and CIA group were given an equal volume of normal saline at the same time. The whole experimental process was as follows Figure 1 shown.

[0024] 2. Clinical evaluation of arthritis

[0025] Joint swelling and arthritis index are two important indicators for judging the anti-RA effect of drugs, reflecting the inflammatory response in the joints. From the 21st to the 49th day of the experiment, the redness, swelling and deformity of the joints were observed every 3 days. The size of the toes of each group was measured using a toe volume meter, and the severity of arthritis was measured using an arthritis scoring method. The results are as follows: Figure 2 shown.

[0026] Depend on Figure 2 It can be seen that the swelling degree and arthritis index of the IMGL-H group, IMGL-L group and MTX group were significantly lower than those of the CIA group, and the RA-like clinical symptoms of mice were improved. Among them, the efficacy of the IMGL-L group was equivalent to that of the MTX treatment group. These results show that IMGL has a strong inhibitory effect on the inflammation of the foot joints of CIA mice.

[0027] 3. Determination of serum inflammatory factors IL-6 and IL-1β

[0028] IL-6 (E-EL-M0044c, Eliorite) and IL-1β (E-EL-M0037c, Eliorite) were detected using ELISA kits, and the operation was strictly carried out according to the instructions. The specific operation process is as follows:

[0029] 1) Before use, mix all reagents thoroughly and prepare the washing solution according to the instructions;

[0030] 2) Add 5 μL of the diluted standard and 5 μL of the sample to be tested into each reaction well, and make duplicates for each standard and blank well;

[0031] 3) Immediately add 5 μL of biotin-labeled antibody, cover the membrane plate, gently shake to mix, and incubate in a 37°C water bath for 90 min;

[0032] 4) Shake off the liquid in the wells, add 350ul of washing solution to each well, let it stand for 1 minute, shake off the washing solution, pat dry with absorbent paper, and repeat this operation 3 times;

[0033] 5) Add 100 μL of HRP enzyme conjugate working solution to each well, gently shake to mix, and incubate at 37°C for 30 min;

[0034] 6) Shake off the liquid in the wells, add 350ul of washing solution to each well, let it stand for 1 minute, shake off the washing solution, pat dry with absorbent paper, and repeat this operation 5 times;

[0035] 7) Add 5 μL of substrate A and B to each well; shake gently to mix; incubate in a 37°C oven for 15 min. Avoid light.

[0036] 8) Take out the ELISA plate, quickly add 50 μL of stop solution, and immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using an ELISA reader.

[0037] The results are as follows Figure 3 As shown in the figure, the levels of inflammatory factors IL-1β and IL-6 in the serum of mice in the CIA group increased significantly, aggravating the inflammatory response, while the levels of IL-1β and IL-6 in the IMGL group were significantly reduced, which was equivalent to the positive drug methotrexate. This shows that IMGL can inhibit the release of inflammatory factors in CIA mice and reduce the occurrence of inflammatory response.

[0038] 4. Histological Evaluation

[0039] All mice were subjected to tissue sampling 28 days after secondary immunization and administration for histological analysis. The left hind limb of the mouse was fixed with 4% (v / v) neutral formalin for 24 hours, and the paraffin-embedded hind limb samples were cut into 5 μm slices using a microtome. The paraffin sections were stained with hematoxylin-eosin (HE), and the inflammation of the sliced ​​tissue was observed under a microscope.

[0040] Synovitis and bone lesions are the main pathological changes of RA. Figure 4As shown in the results, compared with the NC group, the ankle joint cavity of rats in the CIA group had more inflammatory cell infiltration, severe synovial hyperplasia, and severe bone damage; while after treatment with IMGL-L, IMGL-H, and MTX, the infiltration of inflammatory cells and the accumulation of neutrophil exudates in the joint cavity were reduced. In addition, the cartilage and bone surface damage in the IMGL-L, IMGL-H, and MTX groups was milder than that in the CIA group. The results showed that IMGL had a strong inhibitory effect on joint inflammation and bone lesions in CIA mice.

[0041] 5. Inhibitory effect of IMGL on TLR4 / NF-κB pathway in CIA mice

[0042] In the pathological mechanism of RA, abnormal activation of NF-κB (dimer of p65) is closely related to inflammatory proliferation of synovial tissue and erosion of local joint tissue. TLR4 is an important intermediate of inflammatory response, and MyD88 is a key signal transduction adaptor of TLR4. Both are involved in the activation of downstream NF-κB signaling pathway and play a clear and important role in inflammatory response. In view of this, we used Western blot to detect the expression of inflammatory-related proteins TLR4, MyD88, p65 and p-p65 in the foot muscle tissue of CIA mice to explore the molecular mechanism of IMGL's anti-RA effect. The specific steps are as follows: the right hind foot muscle tissue of each group of mice was taken and ground in a cold lysis solution containing protease and phosphatase inhibitors using a grinder for 1 min (fully automatic sample rapid grinder, JXFSTPRP-48L). After the homogenate was placed at 4°C for 30 min, it was centrifuged at 12000 rpm at 4°C for 10 min to remove tissue precipitation. The total protein concentration was quantified using the BCA (P0012S, Beyotime) protein detection kit. Equal amounts of protein samples were separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with blocking buffer for 1 h and then incubated with corresponding concentrations of specific antibodies (TLR4: 10005089, MyD88: 10006278, p65: 10004156, Vibio) and p-p65 (P0018AS, Beyotime)) in primary antibody dilution buffer at 4 °C overnight. The membranes were then incubated with HRP-labeled goat anti-mouse or anti-rabbit secondary antibodies (1:5000) at room temperature for 1 h. The membranes were washed three times with TBST, and the immune complexes were detected using chemiluminescence technology. The protein signals were recorded and analyzed using an imaging system (Bio-Rad, USA). The results are shown in Figure 2 Figure 5 shown.

[0043] The results showed that the CIA group induced a significant increase in the levels of TLR4 and MyD88 proteins in the sole muscles of mice, while the high- and low-dose IMGL groups significantly reduced the expression of TLR4 and MyD88 ( Figure 5 A, B&D). Similarly, compared with the CIA group, IMGL significantly inhibited the phosphorylation of p65 ( Figure 5 A&C). The results showed that IMGL had an inhibitory effect on both upstream and downstream proteins of the NF-κB signaling pathway, and its anti-RA effect may be directly inhibiting the activation of the inflammatory signaling pathway NF-κB.

Claims

1. Application of isomaltine in the preparation of drugs for the treatment of rheumatoid arthritis.

2. The use according to claim 1, characterized in that: The rheumatoid arthritis is collagen-induced arthritis.

3. A medicine for treating rheumatoid arthritis, comprising a therapeutically effective dose of isomaltine.

4. The drug according to claim 3, characterized in that: The medicine also contains pharmaceutically acceptable carriers and / or excipients.

5. The drug according to claim 3, characterized in that: The dosage form of the drug is a pharmaceutically acceptable dosage form.

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