Application of 3,4-dihydropsoralen in preparing medicine for preventing or treating rheumatoid arthritis

Abstract

The present invention discloses the use of 3,4-dihydropsoralen in the preparation of a drug for preventing or treating rheumatoid arthritis. The present invention simulates RA by constructing TNF-a-induced SW982 cells. The results show that 3,4-dihydropsoralen (DP) can downregulate the mRNA expression of inflammatory factors, chemokines, and matrix degrading enzymes through the IL-17 signaling pathway, and affect the IL-17 signaling pathway and the NF-κB signaling pathway to jointly exert an anti-inflammatory effect, thereby effectively improving rheumatoid arthritis. This provides a theoretical basis for the application of 3,4-dihydropsoralen in the clinical treatment of rheumatoid arthritis and opens up new therapeutic drugs and approaches.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicines, and in particular to application of 3,4-dihydropsoralen in preparing medicines for preventing or treating rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic joint inflammation, pain, swelling, and deformity, which can lead to joint dysfunction and severely impact quality of life. In the pathological state of RA, new microvessels, inflammatory factors, and abnormally proliferating synovial cells collectively form pannus, causing osteoarticular lesions and cartilage damage. RA patients produce large amounts of inflammatory factors, chemokines, and matrix-degrading enzymes, which contribute to the initiation and maintenance of RA. These factors, in turn, recruit and stimulate neutrophils, macrophages, and lymphocytes to secrete inflammatory mediators into the synovium, further exacerbating RA progression. Currently, nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and antirheumatic drugs are the primary treatments for RA, primarily to prevent or delay the progression of RA, reduce inflammation, and alleviate chronic pain. However, long-term use can easily lead to side effects, impacting quality of life. Therefore, the search for natural plant ingredients to treat or prevent RA has become a research priority.

[0003] Ficus erecta (Thunb.) var. beecheyana (Hook. et Arn.) King, a member of the Moraceae family, primarily grows as a shrub or tree. It is also known as milky pulp because its branches, leaves, and fruit release a milky white liquid resembling milk when broken. The entire Ficus erecta is used medicinally, with the root being the primary medicinal part. Its properties are pungent, sour, astringent, and warm, and it is primarily used to treat rheumatic joint pain, traumatic injuries, menstrual irregularities, lower back pain, leukorrhea, and swelling, as well as to detoxify and reduce swelling. Its roots and meat are often used in soups or mixed with rice wine, which can help prevent and alleviate rheumatoid arthritis. Numerous studies have reported on the chemical constituents of the root, stem, leaf, and fruit of Ficus erecta, but little research has been conducted on the underlying pharmacological mechanisms and mechanisms of action. 3,4-Dihydropsoralen is a compound isolated from the root of Ficus erecta, but its use in the treatment of rheumatoid arthritis has yet to be reported. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a new use of 3,4-dihydropsoralen in the preparation of drugs for preventing or treating rheumatoid arthritis, thereby providing a theoretical basis for its application in the clinical treatment of rheumatoid arthritis and developing new therapeutic drugs and approaches.

[0005] The compound 3,4-dihydropsoralen (DP) provided by the present invention is a reduced derivative of psoralen, a furanocoumarin compound, and has anti-tumor, antibacterial and immunomodulatory effects. Its English name is 3,4-dihydropsoralen, and its molecular formula is C 11 H8O3, its structural formula is as follows:

[0006]

[0007] The 3,4-dihydropsoralen (DP) of the present invention can be isolated and purified from the root of the angelica dahurica. The specific preparation method comprises the following steps:

[0008] (1) The roots of the angelica dahurica were crushed and extracted with 60% ethanol to obtain a total extract, which was then extracted with petroleum ether, ethyl acetate, and n-butanol in sequence according to a polarity gradient to obtain a petroleum ether fraction, an ethyl acetate fraction, and an n-butanol fraction, respectively;

[0009] (2) The ethyl acetate fraction was subjected to silica gel column chromatography and eluted with petroleum ether-ethyl acetate as a solvent to obtain 8 fractions Fr.1-8;

[0010] (3) Fr.4 was subjected to ODS column chromatography and eluted with methanol-water to separate Fr.4.1-5;

[0011] (4) Fr.4.2 was eluted by semi-preparative liquid chromatography using methanol-water with a volume ratio of 40:60 as the mobile phase to separate the compound 3,4-dihydropsoralen.

[0012] This study simulated RA in SW982 cells by inducing TNF-α. Results showed that 3,4-dihydropsoralen (DP) effectively reduced the release of inflammatory factors including NO, IL-6, IL-8, and IL-1β. RT-PCR results also revealed a significant downregulation of IL-6, IL-8, and IL-1β mRNA expression, demonstrating 3,4-dihydropsoralen's anti-inflammatory and RA-alleviating effects. High-dose DP was then administered to the cells, and RNA-seq was used to identify differentially expressed genes and pathways in response to DP treatment. Comparative analysis revealed 352 targets enriched in the transcriptome, including 33 overlapping genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched in the IL-17 signaling pathway, chemokine signaling pathway, and NOD-like receptor signaling pathway. RT-PCR results showed that DP reduced the expression of MMP1, MMP3, CCL2, CXCL5, and CXCL11 mRNA (P < 0.05, 0.01) and significantly downregulated the p-IκBα / IκBα ratio (P < 0.001). GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) results revealed a significant increase in the IL-17 signaling pathway, suggesting that it may be the primary mechanism of action of 3,4-dihydropsoralen (DP) against RA. These results suggest that 3,4-dihydropsoralen can downregulate the mRNA expression of inflammatory factors, chemokines, and matrix degrading enzymes through the IL-17 signaling pathway, and influence the IL-17 and NF-κB signaling pathways to exert anti-inflammatory effects, thereby effectively improving rheumatoid arthritis.

[0013] The inflammatory factors of the present invention are NO, IL-6, IL-8, and IL-1β; the chemokines are CCL2, CXCL5, or CXCL11; and the matrix degrading enzymes are MMP1 or MMP3.

[0014] The medicine of the present invention comprises an effective amount of 3,4-dihydropsoralen and a pharmaceutically acceptable carrier.

[0015] Preferably, the dosage form of the drug is tablet, capsule, powder, oral solution, powder, pill or granule.

[0016] The pharmaceutically acceptable carriers of the present invention include, but are not limited to, lubricants, fillers, binders, disintegrants, pH regulators, surfactants, antioxidants, etc., and their usage amounts are conventional amounts in the art.

[0017] The pharmaceutical dosage form of the present invention can be prepared according to conventional methods in the art.

[0018] Compared with the prior art, the present invention has the following excellent effects:

[0019] The present invention provides a new use of 3,4-dihydropsoralen in the preparation of a drug for preventing or treating rheumatoid arthritis. The present invention simulates RA by constructing TNF-a-induced SW982 cells. The results show that 3,4-dihydropsoralen (DP) can downregulate the expression of inflammatory factors, chemokines, and matrix degrading enzyme mRNAs through the IL-17 signaling pathway, and influence the IL-17 signaling pathway and the NF-κB signaling pathway to jointly exert an anti-inflammatory effect, thereby effectively improving rheumatoid arthritis. This provides a theoretical basis for the application of 3,4-dihydropsoralen in the clinical treatment of rheumatoid arthritis and opens up new therapeutic drugs and approaches. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of different concentrations of 3,4-dihydropsoralen on cytotoxic activity;

[0021] Figure 2 The effect of TNF-a at different concentrations on cell proliferation activity and IL-6 content;

[0022] Figure 3 The effect of 3,4-dihydropsoralen on the NO content in the supernatant of SW982 cells induced by TNF-a;

[0023] Figure 4 The effect of DP on the release of IL-6, IL-8, and IL-1β in the supernatant of SW982 cells induced by TNF-a;

[0024] Figure 5 The effect of DP on the mRNA expression of IL-6, IL-8, and IL-1β in SW982 cells induced by TNF-a;

[0025] Figure 6 It is the differential gene volcano map, GO function enrichment map and KEGG enrichment pathway map;

[0026] Figure 7 The effect of DP on the mRNA expression of MMP1, MMP3, CCL2, CXCL5, and CXCL11 in SW982 cells induced by TNF-a;

[0027] Figure 8 The results show that DP has an effect on NF-κB activation induced by TNF-a in SW982 cells. DETAILED DESCRIPTION

[0028] The present invention will be further described below by way of specific embodiments. The following examples are specific embodiments of the present invention, but the embodiments of the present invention are not limited to the following examples.

[0029] Example 1:

[0030] 1 Materials and Methods

[0031] 1.1 Raw materials

[0032] The angelica fruit (collected from Zhentou Mountain in Youxi County) was identified by Professor Huang Mingqing of Fujian University of Traditional Chinese Medicine as the root of the Ficus erecta Thunb.var.beecheyana (Hook.et Arn.) King plant of the Moraceae family.

[0033] 1.2 Cell lines

[0034] Human synovial sarcoma cells (SW982) were obtained from the ATCC cell bank in the United States and purchased from the Cell Bank of the Chinese Academy of Sciences.

[0035] 1.3 Drugs and reagents

[0036] Anhydrous ethanol, petroleum ether, and ethyl acetate (analytical grade, Guangdong Xilong Chemical Co., Ltd.); methanol (chromatographic grade, Sigma-Aldrich, USA); reversed-phase C18 silica gel for column chromatography (Qingdao Bangkai High-Tech Materials Co., Ltd.); deuterated methanol and deuterated DMSO (Shanghai Zhongwei Chemical Co., Ltd.); Dulbecco's modified Eagle's medium (C11965500BT, GIBCO, USA); fetal bovine serum (A5669701, GIBCO, USA); penicillin-streptomycin solution (ABK023C509, Abcam Biopharmaceutical Technology Co., Ltd.); recombinant human TNF-α protein (10602-HNAE, Sino Biological, China); trypsin (J121103, Shanghai Yuanpei Biotechnology Co., Ltd.); dimethyl sulfoxide (196055, MP Biopharmaceuticals, USA) Biomedicals Company); Trizol (15596026CN, Thermo Fisher Scientific); p-IκBα antibody (HY-P80826, MedChemExpress Company, USA); IκBα antibody (HY-P80718, MedChemExpress Company, USA); α-Tubulin (T9026, Sigma-Aldrich Company, USA).

[0037] 2 Methods

[0038] 2.1 Compound separation and purification

[0039] 2.1.1 Extraction and separation

[0040] 6.3 kg of washed, impurity-free, and dried angelica roots were crushed and extracted continuously for 2 hours with 60% ethanol at a solid-liquid ratio of 1:23 by heating reflux method for a total of 3 times to obtain a total extract. The total extract was then extracted with petroleum ether, ethyl acetate, and n-butanol in sequence according to a polarity gradient to obtain the petroleum ether fraction, ethyl acetate fraction, and n-butanol fraction, respectively.

[0041] The ethyl acetate fraction was eluted by silica gel column chromatography (petroleum ether-ethyl acetate) to obtain 8 fractions Fr.1 to 8; Fr.4 was separated by ODS column chromatography (methanol-water) to obtain Fr.4.1 to 5; Fr.4.2 was separated by semi-preparative liquid chromatography (methanol-water = 40:60) to obtain compound 3,4-dihydropsoralen (DP) (20 mg).

[0042] 2.2 Cell culture

[0043] Human synovial sarcoma cells (SW982) were cultured in DMEM high-glucose complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. The medium was changed every 1-2 days. When the cell density reached approximately 80%, the cells were digested with trypsin and passaged. Passages 3-7 were used for experiments.

[0044] 2.2.1 CCK-8 assay for cell viability

[0045] After conventional cell culture, the confluence reached about 80%, and the cells were digested with trypsin and centrifuged. New culture medium was added and mixed by pipetting. The cells were seeded into 96-well plates at a rate of 1*105 / well and cultured in an incubator (37°C, 5% CO2) for 24 h. 14 sample solutions were added, all at a concentration of 10 μmol·L -1 For treatment, an equal volume of complete medium was added to the blank control group. After 12 and 24 hours of incubation, the original medium was discarded and 100 μL of complete medium (containing 10% CCK-8 solution) was added to each well. The blank control group was treated with complete medium containing 10% CCK-8 solution but without cells. After incubation for 2 hours, the optical density (D(λ)) was measured at 450 nm on a microplate reader, and cell viability was calculated.

[0046] 2.2.2 TNF-a induced SW982 cell inflammation model

[0047] When the cells reached approximately 80% confluency, they were digested and centrifuged, then seeded at 1 x 105 cells / well in a 96-well plate and cultured in an incubator (37°C, 5% CO2) for 24 hours. After 24 hours of cell attachment, a blank control group without any drug treatment was established, and inflammatory models were established using different concentrations of TNF-α (40, 20, 10, 5, and 2.5 ng mL⁻¹). After 24 hours of induction, cell viability was measured according to the method in 2.2.1. The assay was performed according to the kit's instructions, with the measured OD value plotted as the vertical axis and the corresponding standard concentration plotted as the horizontal axis to construct a standard curve. The cell supernatant was collected and the OD value of IL-6 was measured. The amount of IL-6 released was calculated based on the standard curve and the OD value.

[0048] 2.2.3 Griess method for NO content detection

[0049] SW982 cells in the logarithmic growth phase were obtained, digested and centrifuged, and seeded into 96-well plates at 1*105 / well. They were cultured in an incubator (37°C, 5% CO2) for 24 hours. The next day, after the cells adhered to the wall, they were divided into a blank group without any drug treatment and a control group with 10 ng·mL -1 TNF-a was used as the model group, 10 μmol·L-1DXM was added as the positive drug group, and 10 μmol·L-1DXM was added as the pre-prepared compound. -1 SW982 cells were treated with the drug group, and 10 ng mL -1 TNF-a was induced, and an equal volume of complete culture medium was added to the blank group and the cells were placed in an incubator for further culture. After incubation for 24 h, the cell supernatant was collected and Griess reagent was added and mixed. After protection from light for 30 min, the UV absorbance value was detected at 548 nm using a microplate reader.

[0050] 2.2.4 ELISA to detect inflammatory factor IL-6 in cell supernatant

[0051] Take the cells in logarithmic growth phase, digest and centrifuge them, and inoculate them into 96-well plates at 1*105 / well. Culture them in an incubator (37°C, 5% CO2). After 24 hours of attachment, use 10ng·mL -1 The inflammatory model was established by TNF-a and divided into blank group, model group, positive drug group and drug group. After treating SW982 cells for 2 h, 10 ng·mL -1 Inflammation was induced by TNF-a. An equal volume of complete culture medium was added to the blank control group and the cells were cultured in an incubator. After 24 h, the cell supernatant was collected to detect the release of the inflammatory factor IL-6 in the cell supernatant (this step was performed according to the kit instructions).

[0052] 2.2.5 qRT-PCR detection of inflammatory cytokine expression

[0053] After grouping and drug administration according to 2.2.3, RT-PCR was used to detect the expression of related genes in cells. Total RNA was extracted using TRIzol using an RNA extraction kit and converted into cDNA using a reverse transcriptase kit. Actin was used as an internal reference control, and RT-PCR was used to detect the mRNA expression of related inflammatory genes. The primer sequences are shown in Table 1. -△△CT The expression levels of related genes were calculated.

[0054] Table 1

[0055]

[0056] 2.2.6 Transcriptome Sequencing

[0057] Total RNA was extracted from normal SW982 cells, TNF-α-induced SW982 cells, and a high-dose DP-treated group. PE100 / PE150 sequencing was performed using combined probe-anchored polymerization (cPAS) on a G400 / T7 / T10 sequencer (BGI-Shenzhen, China). The raw data was filtered using SOAPnuke, and clean data was aligned to the reference genome.

[0058] 2.2.7 Western blot

[0059] Western blot was used to detect the expression levels of corresponding proteins in SW982 cells after administration of different concentrations of DP. Cells were digested and centrifuged at a density of 80%, and plated at 20 μL / well in 6-well plates. These cells were designated as the normal group, model group, and high-, medium-, and low-dose DP groups. After 24 hours of treatment, RIPA lysis buffer with protease inhibitors and phosphoinhibitors was added to each group. The cells were fully lysed on ice for 30 minutes and then centrifuged (4°C, 12,000 rpm, 5 minutes). The supernatant was collected and the protein concentration was determined using a BCA kit (refer to the instructions). The protein concentration was adjusted to the same level with PBS, and 150 μL of SDS-PAGE protein loading buffer (1x) was added, mixed, and boiled at 100°C for 5 minutes before loading.

[0060] 2.2.8 Statistical analysis

[0061] Data were pre-processed and statistically analyzed using Graphpad Prism 9.5 software. Measurement data were expressed as x ± s, and comparisons between groups were performed using one-way analysis of variance (ANOVA). P < 0.05 indicated statistical significance.

[0062] 3 Results

[0063] 3.1 Structure identification

[0064] Compound 3: white powder. ESI-MS m / z: 187 [M+H] + ; Molecular formula: C 11 H8O3. 1 H-NMR (600 MHz, MeOD) δ H :7.51(1H,dd,J=2.2,1.0Hz,H-3'),7.28(1H,s,H-8),6.89(1H,s,H-5),6.63(1H ,dt,J=2.2,1.0Hz,H-2'),2.95(2H,t,J=7.7Hz,H-3),2.62(2H,t,J=7.7Hz,H-4); 13 C-NMR (151 MHz, MeOD) δC : 27.47 (C-3), 35.48 (C-4), 98.21 (C-8), 107.16 (C-3'), 120.96 (C-10), 122.21 (C-5), 125.00 (C-6), 144.56 (C-2'), 154.55 (C-9), 156.16 (C-7), 177.58 (C-2). This data is basically consistent with the literature report, so compound 3 was identified as 3,4-dihydropsoralen (DP).

[0065] 3.2 Effects of different concentrations of 3,4-dihydropsoralen on cytotoxic activity

[0066] TNF-a induced SW982 cells, and a blank control group, model group, positive control group, low-dose, medium-dose, and high-dose drug groups were set up. After 24 hours, the cell survival rate was detected by CCK-8 method (compared with the blank group, ### P<0.001; compared with the model group, *P<0.05; **P<0.01; ***P<0.001, n=3).

[0067] The results of CCK-8 showed that the different concentrations of 30, 10, and 3 μmol·L -1 After 24 hours of treatment, SW982 cells showed no cytotoxic reaction compared with the blank group. Figure 1 , which can be further studied later.

[0068] 3.3 Establishment of TNF-a-induced SW982 cell inflammation model

[0069] SW982 cells were given 2.5, 5, 10, 20, and 40 ng·mL -1 After 24 hours, the CCK-8 assay was used to detect the effect of different concentrations of TNF-a on cell proliferation activity; the ELISA kit was used to detect the IL-6 content in the cell supernatant. The main purpose was to ensure the stability of the inflammation model and provide a suitable TNF-a concentration for subsequent modeling. The CCK-8 assay results showed that compared with the blank control group, the administration of TNF-a 10, 20, and 40 ng·mL -1 It can significantly improve the viability of SW982 cells and promote cell proliferation, and the difference is statistically significant (P < 0.05). The results of IL-6 content detection showed that compared with the blank control group, the TNF-a-induced SW982 cell inflammation model was successfully established, and 2.5, 5, 10, 20, and 40 ng·mL -1All of them significantly promoted the release of IL-6, and the difference was statistically significant (P < 0.05). The results of CCK-8 and ELISA test data showed that the subsequent selection of 10 ng·mL -1 As the dosage concentration for inflammation model. Figure 2 .

[0070] 3.4 Effect of 3,4-dihydropsoralen on NO content in the supernatant of SW982 cells induced by TNF-α

[0071] SW982 cells were induced by TNF-a and the blank control group, model group, positive control group, high, medium and low (30, 10, 3 μmol·L -1 ) dose group were pretreated for 2 h and 10 ng mL -1 TNF-a was used to induce inflammation. After 24 hours, the NO content in the cell supernatant was detected by Griess method (compared with the blank group, ### P<0.001; compared with the model group, *P<0.05; **P<0.01; ***P<0.001, n =3).

[0072] The results showed that compared with the blank control group, the NO content in the model group increased significantly after TNF-a induction (P < 0.001), indicating that the inflammation model was successfully established. Compared with the model group, the NO expression level decreased significantly after administration of different concentrations of DP (P < 0.01). Figure 3 .

[0073] 3.5 Effect of 3,4-dihydropsoralen on the expression of inflammatory factors IL-6, IL-8, and IL-1β in SW982 cells induced by TNF-a

[0074] The blank control group, model group, positive control group, DP high, medium and low (30, 10, 3 μmol·L -1 ) dose group were pretreated for 2 h and 10 ng mL -1 TNF-a was used to induce inflammation. After 24 hours, the cell supernatant was collected and the contents of IL-6, IL-8 and IL-1β were detected by ELISA (compared with the blank group, ### P<0.001; compared with the model group, *P<0.05; **P<0.01; ***P<0.001, n =3).

[0075] The results of ELISA test showed that compared with the blank control group, -1Under the induction of TNF-a, the levels of inflammatory factors IL-6, IL-8, and IL-1β increased significantly. When different concentrations of DP (30, 10, 3 μmol·L -1 ) after the treatment, the levels of inflammatory factors decreased ( Figure 4 The results showed that DP could inhibit the production of inflammatory factors induced by TNF-a and down-regulate the levels of IL-6, IL-8, and IL-1β in a dose-dependent manner.

[0076] 3.6 Effect of 3,4-dihydropsoralen on TNF-a-induced inflammatory cytokine IL-6, IL-8, and IL-1β mRNA expression in SW982 cells

[0077] The blank control group, model group, positive control group, DP high, medium and low (30, 10, 3 μmol·L -1 ) dose group were pretreated for 2 h and 10 ng mL -1 TNF-a was used to induce inflammation. Q-PCR was used to detect the effects of different concentrations of DP on the expression of IL-6, IL-8, and IL-1β mRNA (compared with the blank group, ### P<0.001; compared with the model group, *P<0.05; **P<0.01; ***P<0.001, n =3).

[0078] qRT-PCR data showed that compared with the blank control group, -1 Under the induction of TNF-a, the mRNA expression of inflammatory factors IL-6, IL-8, and IL-1β was significantly upregulated. When different concentrations of DP (30, 10, 3 μmol·L -1 ) after the inflammatory factor mRNA expression was significantly downregulated ( Figure 5 The results showed that DP significantly downregulated the expression of IL-6, IL-8, and IL-1β inflammatory factors mRNA.

[0079] 3.7 Screening of differentially expressed genes

[0080] Figure 6Middle A: Difference Venn diagram (a: normal group vs. model group; b: DP high-dose group vs. model group); B: Difference volcano diagram of normal group vs. model group; C: Difference volcano diagram of DP high-dose group vs. model group; (X-axis represents the difference fold value after log2 transformation, and Y-axis represents the significance value after -log10 transformation. Red represents upregulated DEG, blue represents downregulated DEG, and gray represents non-DEG.); D: GO function enrichment map of differential genes, GO analysis of differential genes was performed on the normal group and model group, DP high-dose group and model group (dark blue: biological process; orange-red: cellular component; light blue: molecular function); E: KEGG pathway map of differential genes.

[0081] The differential gene volcano plot showed that there were 264 differentially expressed genes between the normal group and the model group, including 206 up-regulated genes and 58 down-regulated genes ( Figure 6 There were 121 differentially expressed genes between the DP group and the model group, including 14 upregulated genes and 107 downregulated genes. Interaction analysis of the differentially expressed genes between the two groups revealed a total of 33 genes in common.

[0082] 3.8 GO function enrichment map

[0083] GO functional enrichment is mainly composed of biological process (BP), cellular component (CC), and molecular function (MF), which is used to describe the properties of genes and gene products and provide functional distribution characteristics of different genes. This study mainly performed GO functional enrichment analysis on the 33 intersection genes obtained by screening. The results showed that ( Figure 6 ), 33 intersection genes may be involved in biological processes such as cellular processes, biological regulation, regulation of biological processes, positive regulation of biological processes, and metabolic processes; CC analysis showed significant enrichment in cells, organelles, cell membranes, and the cell periphery; MF analysis showed that binding, catalytic activity, molecular function regulation, and structural molecular activity were the main enriched functions. These results suggest that high-dose DP can regulate differential gene expression levels through targeted regulation of GO function enrichment, thereby participating in the above-mentioned series of molecular functions and biological processes, exerting a modulating effect on inflammation.

[0084] 3.9 KEGG enrichment pathway map

[0085] Based on the KEGG pathway annotation classification, enrichment analysis was performed using the phyper function in R software. The P value was calculated and then the P value was corrected for FDR to obtain the Q value. Generally, functions with a Q value ≤ 0.05 were considered significantly enriched. KEGG results showed that differentially expressed genes were primarily enriched in nine signaling pathways, primarily the IL-17 signaling pathway, the chemokine signaling pathway, the rheumatoid arthritis signaling pathway, and the NOD-like receptor signaling pathway.

[0086] 3.10 Transcriptomics analysis

[0087] GO function enrichment and KEGG pathway enrichment analysis revealed that significantly differentially expressed pathways between the normal and model groups, and between the model and high-dose DP groups, were primarily enriched in the IL-17 and chemokine signaling pathways. This suggests that DP may exert an anti-inflammatory effect on TNF-α-induced SW982 cells, possibly through its involvement in regulating the IL-17 signaling pathway. Further analysis of differentially expressed genes in inflammatory signaling pathways was conducted. Comprehensive analysis revealed significant differences in the expression of genes encoding CSF3, CCL2, CXCL5, and CXCL11 in the IL-17 and chemokine signaling pathways. Because the IL-17 signaling pathway was the primary enriched pathway, it will be investigated as a target pathway in future studies. Studies have shown that in the pathological state of RA, a large number of chemokines, inflammatory factors, and matrix-degrading enzymes are produced in the body. These factors, through recruitment and stimulation, induce the secretion of inflammatory mediators by neutrophils, macrophages, and lymphocytes into the synovium, thereby creating a microenvironment conducive to the infiltration and survival of inflammatory cells, leading to cell aggregation, activation, and release, further exacerbating RA. This process is associated with the involvement of the IL-17 signaling pathway. Therefore, based on the target pathway enrichment and literature search, further RT-PCR verification of genes MMP1, MMP3, CCL2, CXCL5, and CXCL11 will be carried out in the future. (Table 2)

[0088] Table 2 KEGG enriched signaling pathways of differentially expressed genes

[0089]

[0090] 3.11 Effect of 3,4-dihydropsoralen on TNF-α-induced mRNA expression of key targets in SW982 cells

[0091] The blank control group, model group, positive control group, high, medium and low (30, 10, 3 μmol·L -1 ) dose group were pretreated for 2 h and 10 ng mL -1 TNF-a was used to induce inflammation. Q-PCR was used to detect the effects of different concentrations of DP on the expression of MMP1, MMP3, CCL2, CXCL5, and CXCL11 mRNA (compared with the blank group, ### P<0.001; compared with the model group, *P<0.05; **P<0.01; ***P<0.001, n =3).

[0092] RT-PCR data showed that compared with the blank control group, -1Under the induction of TNF-a, the mRNA expression of inflammatory factors MMP1, MMP3, CCL2, CXCL5, and CXCL11 was significantly upregulated. When different concentrations of DP (30, 10, 3 μmol·L -1 ) after the inflammatory factor mRNA expression was significantly downregulated ( Figure 7 The results showed that DP significantly downregulated the expression of MMP1, MMP3, CCL2, CXCL5, and CXCL11 mRNA.

[0093] 3.12 Effects of 3,4-dihydropsoralen on NF-κB signaling pathway in SW982 cells induced by TNF-α

[0094] The normal control group, model group, low, medium and high (3, 10, 30 μmol·L -1 ) dose group were pretreated for 2 h and 10 ng mL -1 TNF-a was used to induce inflammation. Western Blot was used to detect the expression levels of IκBα and p-IκBα proteins after different concentrations of DP, with a-tubulin as the internal reference. (Compared with the blank group, ### P<0.001; compared with the model group, ***P<0.001, n=3).

[0095] To further verify that DP activates NF-κB through the IL-17 signaling pathway and improves the relationship between TNF-a-induced SW982 cells and the NF-κB signaling pathway, the expression levels of p-IκBα and IκBα proteins at low, medium and high concentrations of DP were detected ( Figure 8 The results showed that compared with the normal control group, at 10 ng·mL -1 Under the action of TNF-a, the expression of IκBα protein in the model group did not change significantly, but the expression of p-IκBα was significantly increased. When different concentrations of DP (3, 10, 30 μmol·L -1 ), the ratio of p-IκBα / IκBα was significantly downregulated (P<0.001), suggesting that DP may activate and significantly inhibit the NF-κB signaling pathway through the IL-17 signaling pathway.

Claims

1. Use of 3,4-dihydropsoralen as the sole active ingredient in the preparation of a drug for preventing or treating rheumatoid arthritis.

2. The use according to claim 1, characterized in that The preparation method of the 3,4-dihydropsoralen comprises the following steps: (1) The roots of the angelica dahurica were crushed and extracted with 60% ethanol to obtain a total extract. The total extract was then extracted with petroleum ether, ethyl acetate, and n-butanol in sequence according to a polarity gradient to obtain a petroleum ether fraction, an ethyl acetate fraction, and an n-butanol fraction, respectively; (2) The ethyl acetate fraction was subjected to silica gel column chromatography and eluted with petroleum ether-ethyl acetate as solvent to obtain 8 fractions Fr.1~8; (3) Fr.4 was subjected to ODS column chromatography and eluted with methanol-water to separate Fr.4.1~5; (4) Fr.4.2 was eluted by semi-preparative liquid chromatography using methanol-water with a volume ratio of 40:60 as the mobile phase to separate the compound 3,4-dihydropsoralen.

3. The use according to claim 1, characterized in that The 3,4-dihydropsoralen can downregulate the mRNA expression of inflammatory factors, chemokines, and matrix degrading enzymes through the IL-17 signaling pathway, and affect the IL-17 signaling pathway and the NF-κB signaling pathway to jointly exert an anti-inflammatory effect, thereby effectively improving rheumatoid arthritis.

4. The use according to claim 3, characterized in that The inflammatory factors are NO, IL-6, IL-8, and IL-1β; the chemokines are CCL2, CXCL5, or CXCL11; and the matrix degrading enzymes are MMP1 or MMP3.

5. The use according to claim 1, characterized in that The medicine comprises an effective amount of 3,4-dihydropsoralen and a pharmaceutically acceptable carrier.

6. The use according to claim 5, characterized in that The dosage form of the medicine is tablet, capsule, powder, oral solution, powder, pill or granule.

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