Application of isoorientin in preparation of medicine for preventing and treating gouty arthritis

Abstract

The invention relates to the technical field of medicines, in particular to application of isoorientin in preparation of a medicine for preventing and treating gouty arthritis. According to the invention, a gouty arthritis mouse animal model is constructed, and in-vivo experiments show that the isoorientin can obviously relieve red, swollen, hot and pain caused by acute gouty arthritis. Therefore, the isoorientin can be regarded as a potential medicine for treating the acute gouty arthritis; the effects are mild; the side effects are small, and the like.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the application of isoorientin in the preparation of drugs for preventing and treating gouty arthritis. Background Art

[0002] Gouty arthritis (GA) is a metabolic and aseptic arthritis and a common cause of inflammatory arthritis. Epidemiology shows that the prevalence of GA in China is 1.1%. The characteristic of gouty arthritis is that purine catabolism disorder causes hyperuricemia, leading to the deposition of monosodium urate (MSU) crystals.

[0003] The Chinese Guidelines for the Diagnosis and Treatment of Gout (2019) recommend febuxostat, benzbromarone or allopurinol as the first-line drugs for reducing uric acid in gout patients; for the anti-inflammatory and analgesic treatment of acute gout attacks, it is recommended to use low-dose colchicine or non-steroidal anti-inflammatory drugs as early as possible. Colchicine has anti-inflammatory and analgesic effects and can effectively relieve the clinical symptoms of patients with acute gout attacks, and the curative effect is definite. It is commonly used clinically to treat patients with acute gout attacks, but the therapeutic dose of colchicine is relatively close to the toxic dose, and the incidence of adverse reactions is high. Non-steroidal anti-inflammatory drugs have definite anti-inflammatory, antipyretic and analgesic effects. These drugs act faster in the treatment of gouty arthritis, but there are more side effects, mainly including liver and kidney function damage, large gastrointestinal reactions, rashes, and even bone marrow suppression, etc. Some patients show drug resistance and other situations.

[0004] With the development of modern medicine, more and more natural small-molecule compounds in traditional Chinese medicine have been discovered. They have few adverse reactions, avoid the toxic and side effects of western medicine, are suitable for long-term application, and are expected to be used in the preparation of drugs for preventing and treating acute gouty arthritis. The molecular formula of isoorientin is C 21 H 20 O 11 , the molecular weight is 448, and the structural formula is shown in formula (I):

[0005]

[0006] Isoorientin is a flavonoid substance and has various physiological activities such as antioxidant and anti-inflammatory. There is currently no relevant report on the treatment of gouty arthritis with isoorientin. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide the application of isoorientin in the preparation of drugs for preventing and treating gouty arthritis.

[0008] The present invention provides the application of isoorientin in the preparation of drugs for preventing and treating gouty arthritis.

[0009] In the present invention, the prevention and treatment include alleviating the symptoms of gouty arthritis, enhancing the anti-inflammatory ability, and / or inhibiting the expression of inflammatory pathways.

[0010] In the present invention, the alleviation includes alleviating redness, swelling, heat, and / or pain caused by gouty arthritis.

[0011] In the present invention, the alleviation includes inhibiting synovial hyperplasia of joints and / or inhibiting inflammatory cell infiltration in the soft tissues around joints.

[0012] In the present invention, the enhancement of anti-inflammatory ability includes inhibiting the levels of inflammatory factors.

[0013] Furthermore, the inflammatory factors include IL-6, IL-18, and / or TNF-α.

[0014] In the present invention, the inflammatory pathway is the inflammatory pathway of bone marrow-derived macrophages.

[0015] Furthermore, the inhibition of the expression of the inflammatory pathway includes downregulating the NOD-like receptor signaling pathway, leukocyte migration, and / or Inflammatory Response IL-6_JAK_STAT3.

[0016] In the present invention, the gouty arthritis is gouty arthritis caused by the deposition of monosodium urate.

[0017] In the present invention, there is provided the use of isoorientin in the preparation of a drug for preventing and treating gouty arthritis with high safety. The high safety includes maintaining the body weight level of the subject and not causing damage to the liver and / or kidneys. The non-causing of damage to the liver and / or kidneys includes maintaining the tissue morphology of the liver and / or kidneys and not affecting the levels of liver function markers and / or kidney function markers. The liver function markers include alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and the kidney function markers include blood urea nitrogen (BUN) or creatinine (Cr).

[0018] In the present invention, the effective dose for the prevention and treatment is 1 mg / kg to 100 mg / kg, that is, 1 to 100 mg per kilogram of animal body weight. Preferably, it is 10 to 30 mg / kg, specifically 10, 20, or 30 mg / kg.

[0019] The present invention also provides a drug for preventing and treating gouty arthritis, which comprises isoorientin and a pharmaceutically acceptable excipient.

[0020] The present invention also provides a pharmaceutical composition, which comprises isoorientin and other therapeutic agents, and the other therapeutic agents are selected from other drugs for treating gouty arthritis.

[0021] The pharmaceutically acceptable excipients in the present invention are one or a mixture of two or more of fruit powder, edible essence, sweetening agent, souring agent, filler, lubricant, preservative, suspending agent, edible pigment, diluent, emulsifier, disintegrant or plasticizer.

[0022] The dosage forms of the drug or pharmaceutical composition include injection, granule, pill, powder, tablet, capsule, oral liquid or syrup. The injection includes injection solution or powder for injection.

[0023] The present invention also provides a method for preventing and treating gouty arthritis, which is to administer the drug described in the present invention.

[0024] In the treatment method of the present invention, the drug can be administered by a route known in the art or disclosed herein. The administration routes include but are not limited to the following: topical (including eyes, nose); inhalation (including intratracheal, intraoral, transdermal or percutaneous emulsifier or spray); oral; injection or drip (including intravenous, arterial, subcutaneous, peritoneal or intramuscular); intracranial administration (including intrathecal, intraventricular).

[0025] In some embodiments, isoorientin can also be combined with one or more other therapeutic agents or therapies to prevent and treat gouty arthritis. The administration modes of isoorientin and other therapeutic agents in the combination therapy can be separate, simultaneous or sequential, and the present invention does not make any limitation thereto.

[0026] Isoorientin is a natural flavonoid compound widely present in various plants. The present invention constructs an animal model of gouty arthritis in mice and conducts in vivo experiments, showing that isoorientin can significantly relieve the redness, swelling, heat and pain caused by acute gouty arthritis. Therefore, isoorientin can be regarded as a potential drug for treating acute gouty arthritis; and it has the characteristics of mild action and small side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Showing the body weight changes of mice in each group 7 days after modeling and treatment;

[0028] Figure 2 Showing the HE staining of liver and kidney tissues of mice in each group 7 days after modeling and treatment;

[0029] Figure 3 Showing the detection results of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (Cr) concentrations in mice in each group;

[0030] Figure 4 Showing the photographs of hind paw swelling in mice. Among them, the a row shows the photographs of hind paws of mice in each group under bright field, the b row shows the thermographic temperature measurement of hind paws of mice in each group, and the c row shows the ultrasonic imaging of hind paws of mice in each group;

[0031] Figure 5 Show the statistical results of the hind paw thickness, thermographic measurement temperature, and paw withdrawal threshold of each group of mice;

[0032] Figure 6 Show the HE staining results of the hind paws of each group of mice;

[0033] Figure 7 Show that ISO has no obvious cytotoxicity to cells;

[0034] Figure 8 Show that ISO inhibits the transcription of inflammatory factors in LPS- and MSU-treated BMDMs;

[0035] Figure 9 Show the regulation of ISO on BMDM genes in the tibia;

[0036] Figure 10 Show the KEGG analysis results;

[0037] Figure 11 Show the GO analysis results;

[0038] Figure 12 Show the GSEA analysis results. Specific embodiments

[0039] The present invention provides the application of isoorientin in the preparation of drugs for preventing and treating gouty arthritis. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications in this article without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0040] In this application, the terms "comprising", "including", and "having" can be used interchangeably, aiming to indicate the inclusiveness of the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that using "comprising", "including", and "having" to describe in this article also provides a "consisting of" solution.

[0041] In this application, the term "and / or" describes the association relationship of associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Where A and B can be singular or plural.

[0042] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single item(s) or plural item(s).

[0043] It should be understood that in various embodiments of this application, the magnitudes of the serial numbers of the above processes do not imply the order of execution. Some or all of the steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of this application.

[0044] In this application, prevention and treatment refer to prevention and / or treatment. "Treatment" refers to surgical operation or drug treatment, the purpose of which is to prevent, slow down (reduce) the undesired physiological changes or lesions in the treatment subject, such as gouty arthritis. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, attenuation of disease severity, stabilization of disease state (i.e., not worsening), delay or slowdown of disease progression, improvement or remission of disease state, and remission (whether partial remission or complete remission), whether detectable or undetectable. Subjects in need of treatment include those who already have a disorder or disease, those who are prone to developing a disorder or disease, or those who intend to prevent a disorder or disease. When referring to terms such as slow down, alleviate, attenuate, remit, etc., their meanings also include elimination, disappearance, non-occurrence, etc.

[0045] This invention uses a mouse model of acute gouty arthritis as the in vivo research object, takes bone marrow-derived macrophages (BMDMs) as the main cells for in vitro research of gouty arthritis, and uses histological, molecular biological and cytological related techniques to prove the anti-inflammatory effect of isoorientin (ISO) on the mouse model of acute gouty arthritis, and explores its mechanism of action. The test materials used in this invention are all ordinary commercially available products and can all be purchased in the market. The following further elaborates this invention in combination with embodiments:

[0046] Embodiment

[0047] 1. Experimental steps

[0048] (1) Animal experiment

[0049] Forty 6-week-old male C6BL / 7 mice were randomly divided into 5 groups, including a normal control group (control group, n = 8), a GA group (CTX, n = 0), ISO (10 mg / kg and 30 mg / kg) treatment groups (ISO, n = 8 / group), and colchicine (COL) (1 mg / kg) treatment group (COL, n = 8 / group). All experimental procedures were approved and performed in accordance with the guiding opinions of the Institutional Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine. A 3% MSU suspension (0.6 mg / 20 μL) was injected into the hind paw of male C57BL / 6 mice to establish a GA model. The mice were randomly assigned to receive a phosphate-buffered saline group (10 μL PBS injected into the hind paw, followed by an intraperitoneal injection of 20 μL of normal saline), an MSU group (20 μL of MSU injected into the hind paw, followed by an intraperitoneal injection of 200 μL of normal saline), an MSU+ISO or MSU+COL (20 μL) MSU injected into the hind paw, followed by an intraperitoneal injection of 200 μL of ISO or COL) for 7 days.

[0050] (2) Von Frey pain threshold test

[0051] Von Frey filaments were used to measure the paw withdrawal threshold (PWT). The mice were allowed to acclimatize to the test environment for 20 minutes. Starting from 0.4 g, filaments of different forces were applied to the hind paw. The response was recorded as "O" for negative and "X" for positive. The 50% PWT was calculated using the following formula: 10 [Xf+kδ] / 10 4 , where Xf is the value of the last filament used (in log units), k is determined by referring to the response pattern (according to the Dixon correction), and δ is a constant (logarithmic) of the serial force standard deviation (SD).

[0052] (3) Measurement of hind paw thickness

[0053] The thickness of the hind paw was measured daily using a vernier caliper placed vertically on the paw, with the unit of millimeters (mm).

[0054] (4) B-mode ultrasound detection of the hind paw

[0055] After 7 days of treatment, the hind paws of all mice were analyzed using B-mode ultrasound imaging. The mice were placed on the ultrasound table and adjusted to the best position where their hind paws were fully exposed and parallel to the ultrasound probe.

[0056] (5) Thermal imaging temperature measurement

[0057] After 7 days of treatment, the temperature of the hind paws of the mice was detected using a thermal imager. The mice were placed on an experimental table without heat source interference and adjusted to the best position where their hind paws were fully exposed and perpendicular to the lens for shooting.

[0058] (6) HE staining

[0059] The mice were sacrificed by spinal cord transection. The liver, kidneys, and hind paws were excised and placed in 4% tissue fixative for 24 hours. Then, the liver and kidneys were dehydrated and embedded in paraffin. The hind paws were decalcified with 13% disodium ethylenediaminetetraacetate, changed every three days for 30 days. Then, the paws were dehydrated and embedded in wax blocks. The paraffin-embedded liver, kidneys, and hind paws were heated at 60 °C for 1 h, dewaxed three times with xylene, and hydrated in a series of ethanol with decreasing concentrations. The tissue sections were stained with hematoxylin for 1 minute, placed in 1% hydrochloric acid ethanol solution for 1 minute, washed, then soaked in 2% ammonia water for 5 seconds, and then stained with eosin for 1 minute. These sections were dehydrated in a series of ethanol with increasing concentrations and made transparent with xylene. The tissue sections were mounted with neutral resin and heated at 60 °C for 2 hours. The inflammatory cell infiltration was observed under an optical microscope.

[0060] (7) Cell extraction and culture

[0061] Bone marrow-derived macrophages (BMDMs) were obtained from the femurs and tibias of 6-week-old male C57BL / 6 mice and cultured in RPMI 1640 complete medium (containing 10% fetal bovine serum and 1% double antibody (penicillin and streptomycin)), passed through a 40-μm filter, and lysed with erythrocyte lysate. The lysate was centrifuged, resuspended, and inoculated into the medium containing MCSF solution. Half of the medium was changed after 2, 4, and 6 days, and the cells were cultured for 7 days.

[0062] (8) RNA extraction and real-time reverse transcription quantitative PCR (qPCR)

[0063] BMDMs were incubated with lipopolysaccharide (LPS, 500 ng / mL), monosodium urate (MSU, 300 μg / mL), and isoorientin (ISO). Total RNA was extracted from the treated BMDMs using Trizol, mixed thoroughly with chloroform, centrifuged, and the supernatant was collected. The precipitate was resuspended in isopropanol and centrifuged. The supernatant was discarded, and then the precipitate was resuspended in 75% anhydrous ethanol and centrifuged; the ethanol supernatant was discarded. The resulting RNA was reverse transcribed into cDNA and amplified by qRT-PCR under the following conditions: 95 °C for 5 minutes, then 95 cycles of 10 seconds at 40 °C and 30 seconds at 60 °C. β-Actin was used as the internal reference for mRNA. The relative expression level was calculated by the 2- ΔΔ Ct method.

[0064] (9) CCK-8 viability assay

[0065] In a 96-well plate at 3×10 3BMDM was cultured at a density and the cells were allowed to adhere overnight. On the second day, the cells were treated with ISO at the indicated concentrations for 24, 48, and 72 hours. Then, a CCK-8 assay was performed to detect cell viability. 10 μl of CCK-8 buffer was added to each well and incubated for 1 hour. The absorbance (OD) was measured at 450 nm using an Infinite F200PRO absorbance microplate reader (Tecan Group Ltd., Männedorf, Switzerland).

[0066] (10) RNA sequencing and bioinformatics analysis

[0067] BMDM was exposed to LPS, MSU, and 20 μM ISO or an equal amount of DMSO for 48 hours. Total RNA was extracted and a NanoDrop 2000 was used to evaluate the RNA concentration and purity. RNA integrity was evaluated using an Agilent 2100 Bioanalyzer system and an RNA Nano 6000 assay kit. An mRNA library was prepared using the Hieff NGS Ultima dual-mode mRNA library preparation kit, and the transcriptome was sequenced using an Illumina NovaSeq platform. Bioinformatics analysis was performed using R software (version 4.2.2). The limma software package was used to identify differentially expressed genes (DEGs) between the control group and the drug group. Genes with a p-value < 0.05 were selected as DEGs, and volcano plots and heatmaps were drawn. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis of DEGs was performed using the clusterprofiler package, and p < 0.05 was used as the threshold to screen for significantly different terms and pathways. All results were visualized using the ggplot2 R software package. Gene set enrichment analysis (GSEA) based on GO terms was used to evaluate the enrichment of gene sets.

[0068] (11) Statistical analysis

[0069] All data are presented as the mean ± SD, with 3 replicates. Unpaired t-tests and one-way ANOVA were used to compare differences, and statistical significance was indicated by *p < 0.05, **p < 0.01, or ***p < 0.001.

[0070] 2. Experimental results

[0071] (1) ISO treatment has no toxic effect on mice

[0072] There were no significant differences in the body weight changes of mice in each group 7 days after modeling and treatment ( Figure 1) HE staining of liver and kidney tissues showed that the liver tissue structure of mice in each group was intact, the blood vessel walls were obvious, the cell nuclei were clear, and there was no degeneration or necrosis; the kidney tissue structure was intact, the boundary was clear, the cell nuclei were obvious, and there was no degeneration or necrosis, and the glomeruli on the renal cortex were clearly visible. Figure 2 ) The concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) in the sera of mice in each group were detected, and the results showed no differences among the groups. Figure 3 )

[0073] (2) ISO alleviated the symptoms of redness, swelling, heat, and pain in GA mice

[0074] Photographs of the swollen hind paws of mice showed that compared with the control group, the hind paws of mice in the MSU group were significantly red and swollen, while after drug intervention with ISO or COL, the swelling of the hind paws of mice was reduced. Figure 4 Thermal imaging temperature measurement showed that compared with the control group, the surface temperature of the hind paws of mice in the MSU group increased; compared with the MSU group, the surface temperature of mice in the drug group was significantly lower, with statistical significance. Figure 4 The results of ultrasonic imaging detection of the hind paws of mice seven days after modeling are shown in the figure: compared with the control group, the swelling degree of the hind paws of mice in the drug group and the MSU group increased; but compared with the MSU group, the swelling degree of the hind paws of mice in the drug group was significantly lower, with statistical significance. Figure 4 Statistical analysis of the thickness of the hind paws, the temperature measured by thermal imaging, and the paw withdrawal threshold of mice showed significant statistical differences. Figure 5 ) The HE staining results of the hind paws of mice showed that compared with the MSU group, the synovial hyperplasia area of the metatarsophalangeal joint of the hind paws of mice in the drug group decreased, and the inflammatory infiltration area of the soft tissues around the joint also decreased significantly. Figure 6 )

[0075] (3) ISO was non-toxic to mouse macrophages (BMMDs) in vitro

[0076] The survival rates of mouse BMDMs cells treated with ISO at concentrations of 5 - 200 μg / ml for 24 h, 48 h, and 72 h were analyzed by CCK-8. The data showed that within the concentration range of 5 - 200 μg / ml, ISO had no obvious cytotoxicity to the cells. Figure 7 )

[0077] (4) ISO inhibited the transcription of inflammatory factors in BMDMs treated with LPS and MSU

[0078] To investigate the in vitro anti-inflammatory ability of ISO, the levels of IL-6, IL-18, and TNF-α in the cell culture medium of each group were detected. The results showed that in the model group, the gene expression levels of IL-6, IL-18, and TNF-α in the cells were significantly higher than those in the normal group. After ISO intervention, the gene expression levels decreased and showed a dose-dependent manner( Figure 8 ).

[0079] (5) Pathway of ISO inhibiting inflammation in BMDMs

[0080] A total of 558 DEGs were obtained through high-throughput sequencing analysis (RNA-seq), including 152 up-regulated genes and 406 down-regulated genes( Figure 9 ). The KEGG results showed that the DEGs were mainly enriched in inflammatory pathways such as the NOD-like receptor signaling pathway( Figure 10 ). In addition, the GO analysis results showed that in the biological process, the DEGs were mainly enriched in pathways such as leukocyte migration( Figure 11 ). It is suggested that the intervention of ISO greatly affects the expression of inflammatory genes in BMDMs cells. GSEA analysis predicted that pathways such as InflammatoryResponse IL-6_JAK_STAT3 were down-regulated, and it was found that the cellular oxidative stress response was inhibited and the cell apoptosis pathway was down-regulated( Figure 12 ).

[0081] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of isoorientin in the preparation of a medicament for preventing and treating gouty arthritis.

2. The application according to claim 1, characterized in that, The prevention and treatment include relieving the symptoms of gouty arthritis, enhancing the anti-inflammatory ability and / or inhibiting the expression of inflammatory pathways.

3. The application according to claim 2, characterized in that, The relief includes relieving redness, swelling, heat and / or pain caused by gouty arthritis.

4. The application according to claim 2, characterized in that, The relief includes inhibiting synovial hyperplasia of joints and / or inhibiting inflammatory infiltration of soft tissues around joints.

5. The application according to claim 2, characterized in that The enhancement of anti-inflammatory ability includes inhibiting the levels of inflammatory factors.

6. The application according to claim 5, wherein The inflammatory factors include IL-6, IL18 and / or TNF-α.

7. The application according to claim 2, characterized in that The inflammatory pathway is the inflammatory pathway of bone marrow-derived macrophages.

8. The application according to claim 7, wherein The inhibition of the expression of the inflammatory pathway includes down-regulating the NOD-like receptor signaling pathway, leukocyte migration and / or InflammatoryResponseIL-6_JAK_STAT3.

9. The application according to claim 2, characterized in that, The gouty arthritis is gouty arthritis caused by monosodium urate deposition.

10. A drug for preventing and treating gouty arthritis, characterized in that, It includes isoorientin and pharmaceutically acceptable excipients.

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