Application of sinomenine in medicine preparation

Abstract

The invention relates to the technical field of new application of sinomenine, and discloses application of sinomenine in preparation of drugs. The invention relates to application of sinomenine in preparation of drugs for inhibiting RA synovial fibroblast proliferation and the like. The sinomenine can significantly inhibit the proliferation of RA-FLS, and plays a role in inducing cell apoptosis at the G0 / G1 phase by retarding the cell cycle. The sinomenine can also be used for remarkably inhibiting the phosphorylation level of downstream effector molecules p70S6K and 4E-BP1 of the mTOR. The sinomenine can also be combined with RNF7 in a targeted manner to inhibit an mTOR signal channel.

Description

Technical Field

[0001] The present invention relates to the technical field of new uses of sinomenine, and particularly relates to the application of sinomenine in the preparation of drugs. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease mainly involving joints, and its main pathological feature is progressive symmetrical inflammation, cartilage destruction and bone erosion of the affected joints. Sinomenine (SIN), as a natural compound, shows significant anti-RA potential.

[0003] The imbalance between the excessive proliferation and apoptosis of fibroblast-like synoviocytes (FLS) is the pathological basis of RA. They promote the development of the disease by promoting the formation of invasive granulation tissue, secreting pro-inflammatory cytokines and matrix degrading enzymes. Therefore, in-depth exploration of the internal mechanism of RA-FLS dysfunction is of great significance for the development of RA treatment strategies.

[0004] The Cullin-RING ligase (CRL) complex consists of a scaffold protein cullin, an adaptor protein, a substrate recognition protein and a RING component. The various compositions of these components constitute the largest family of E3 ubiquitin ligases, which are responsible for the ubiquitination and degradation of various key regulatory proteins, and thus regulate various important biological processes including the cell cycle, signal transduction, angiogenesis, DNA damage response and tumorigenesis. There are only two family members of RING proteins in humans or mice, namely RBX1 / ROC1 and SAG / RBX2 / ROC2 / RNF7, which perform catalytic functions in CRL. By binding to the E2 ubiquitin-conjugating enzyme, they promote the transfer of ubiquitin to the substrate protein, thereby marking these proteins for degradation. Ring finger protein 7 (RNF7) was first found to be related to apoptosis sensitivity. It has been found to be overexpressed in cancer and is related to tumor growth and anti-apoptotic ability. Therefore, RNF7 has become a potential anti-cancer target.

[0005] The mammalian target of rapamycin (mTOR) signaling pathway plays a central role in cell proliferation, metabolism, and growth regulation. The mTOR signaling pathway consists of two complexes, mTORC1 and mTORC2. Among them, mTORC1 regulates cell growth and proliferation, while mTORC2 is involved in the regulation of cell survival and metabolism. mTORC1 affects protein synthesis and the cell cycle process by regulating downstream effector molecules such as p70 ribosomal protein S6 kinase (p70S6K) and Eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). Summary of the Invention

[0006] The object of the present invention is to provide a new use of sinomenine.

[0007] The first aspect of the present invention provides the use of sinomenine in the preparation of a drug for inhibiting the proliferation of RA-FLS. Sinomenine significantly inhibits the proliferation of RA-FLS and acts by arresting the cell cycle at the G0 / G1 phase and inducing apoptosis.

[0008] The second aspect of the present invention provides the use of sinomenine in the preparation of a drug for regulating the mTOR / p70S6K / 4E-BP1 pathway. The transcriptomic analysis of the present invention reveals that multiple signaling pathways are regulated after treatment with sinomenine, especially the mTOR / p70S6K / 4E-BP1 pathway, which has a particularly prominent effect on cell proliferation and apoptosis.

[0009] The third aspect of the present invention provides the use of sinomenine in the preparation of a drug for inhibiting the phosphorylation level of mTOR.

[0010] The fourth aspect of the present invention provides the use of sinomenine in the preparation of a drug for inhibiting the phosphorylation levels of the downstream effector molecules p70S6K / 4E-BP1 of mTOR. Sinomenine can significantly inhibit the phosphorylation levels of mTOR and its downstream effector molecules p70S6K and 4E-BP1. The present invention confirmed the key role of mTOR in the inhibition of RA-FLS mediated by sinomenine through a rescue experiment using the mTOR agonist MHY1485. Activating mTOR can reverse the effects of sinomenine on proliferation, cell cycle arrest, and apoptosis.

[0011] The fifth aspect of the present invention provides the use of sinomenine in the preparation of a drug for targetedly binding to RNF7. RNF7 is a component of the CRL complex, which promotes the ubiquitination of substrates by interacting with the E2 ubiquitin ligase and transferring ubiquitin to the substrates. Sinomenine has a strong binding affinity for RNF7. Knockdown of RNF7 can inhibit mTOR, inhibit cell proliferation, and promote apoptosis. Protein chip and surface plasmon resonance (SPR) analysis show that RNF7 is a direct binding target of sinomenine. The RNF7 knockdown experiment shows that it can significantly inhibit the proliferation of RA-FLS, promote apoptosis, and block the cell cycle by inhibiting the mTOR signaling pathway. Sinomenine inhibits the mTOR signaling pathway by targeting RNF7, thereby inhibiting the proliferation of RA-FLS cells and inducing apoptosis. The present invention provides a new strategy for the treatment of RA.

[0012] The sixth aspect of the present invention provides the use of sinomenine in the preparation of a drug for inhibiting the ubiquitination of p27 Kip1 . p27 Kip1 is one of the substrates of the CRL complex. By binding to RNF7, sinomenine can inhibit the ubiquitination modification of p27 Kip1 , thereby inhibiting the activity of mTOR.

[0013] The inventors of the present invention found through HuProt TM Human Protein Microarray chip technology that sinomenine can directly bind to RNF7. This finding provides a clue for determining that RNF7 is a potential target in synovial hyperplasia.

[0014] The inventors of the present invention found that sinomenine can effectively inhibit the proliferation of RA-FLS, induce cell cycle arrest, and promote apoptosis. Transcriptomics analysis shows that this inhibitory effect is related to the mTOR signaling pathway.

[0015] The present invention further found that the mTOR activator MHY1485 can partially reverse the inhibitory effect of sinomenine in RA-FLS.

[0016] To explore the specific mechanism of sinomenine regulating the mTOR pathway, the present invention used HuProt TM Human protein microarray technology to screen for proteins that directly bind to sinomenine, and verified these interactions through SPR technology and pull-down experiments. The results show that sinomenine has a strong binding affinity for RNF7. Knockdown of RNF7 inhibited mTOR, inhibited cell proliferation, and promoted apoptosis.

[0017] In addition, further research of the present invention shows that by binding to RNF7, sinomenine inhibits p27Kip1 Ubiquitination modification, thus inhibiting the activity of mTOR.

[0018] Therefore, RNF7 was identified as the direct action target of sinomenine, and sinomenine inhibited the mTOR signaling pathway by regulating its kinase activity, effectively inhibiting the tumor-like growth of RA-FLS. Brief Description of the Drawings

[0019] Figure 1 It is a graph showing the effect of SIN on the proliferation of RA-FLS cells.

[0020] Figure 2 It is a graph showing the effect of SIN on the cell cycle and apoptosis of RA-FLS cells.

[0021] Figure 3 It is an analysis graph revealing the potential mechanism of SIN inhibiting RA-FLS by transcriptome sequencing after RA-FLS cells are co-incubated with SIN for 24 hours.

[0022] Figure 4 It is a graph showing the effect of SIN on the mTOR pathway in RA-FLS after RA-FLS cells are co-incubated with SIN for 24 hours.

[0023] Figure 5 It is a graph showing the effect of MHY1485 on the proliferation, cell cycle and apoptosis of SIN-treated RA-FLS cells after RA-FLS cells are co-incubated with SIN and MHY1485 for 24 hours.

[0024] Figure 6 It is a graph showing the verification result of the binding of SIN and RNF7 proteins. Figure 6 A in [reference] shows the schematic diagram of biotin labeling of SIN.

[0025] Figure 7 It is a graph showing the function of RNF7 in RA-FLS cells and its effect on cell biological characteristics.

[0026] Figure 8 It is about the effect of SIN and RNF7 on p27 Kip1 ubiquitination and the effect of p27 Kip1 knockdown on mTOR activation. Detailed Implementation Manner

[0027] The endpoints and any values disclosed in this text for a range are not limited to the exact range or value. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the values between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this text.

[0028] The present invention will be described in detail below by way of examples. In the following examples, unless otherwise specified, the raw materials used are ordinary commercially available products.

[0029] Sinomenine (SIN, purity 99.89%, T2726) was purchased from TargetMol (Shanghai, China).

[0030] EdU-488 Cell Proliferation Detection Kit (C0071S) was purchased from Betta Pharmaceuticals (Shanghai, China).

[0031] Cell Cycle Detection Kit (CA1510) was purchased from Solarbio (Beijing, China).

[0032] Annexin V-FITC / PI Apoptosis Detection Kit (E-CK-A258) was purchased from Elabscience (Wuhan, China).

[0033] Cyclin D1 antibody (#55506), CDK4 antibody (#12790), p27 Kip1 antibody (#3886), mTOR antibody (#2972), p-mTOR antibody (#2971), p70S6K antibody (#2708), p-p70S6K antibody (#9234), p-Akt antibody (#9271), Akt antibody (#9272), p-4E-BP1 antibody (#2855) and 4E-BP1 antibody (#9644) were purchased from Cell Signaling Technology (Danvers, MA, USA).

[0034] RNF7 antibody (ab181986) was purchased from Abcam (UK).

[0035] Rheumatoid arthritis synovial fibroblasts (RA-FLS) and healthy human fibroblast synovial cells (HFLS) cell lines were purchased from BeNa Culture Collection (China). All cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) and placed in a humidified incubator at 37°C and 5% CO2.

[0036] Example 1

[0037] 1. Cell proliferation detection

[0038] Cell viability was evaluated by the CCK-8 method (Elabscience, China). Cells were seeded in 96-well plates at a density of 4×10 3 cells per well and allowed to adhere overnight. After incubation with SIN at concentration gradients (150 μM, 300 μM, 600 μM, 1200 μM) for 24 hours and 48 hours, 10 μL of CCK-8 solution was added to each well, and incubation continued at 37 °C for 2.5 hours. Absorbance was measured at 450 nm using a microplate reader. The absorbance value is proportional to the number of live cells. Cell viability was calculated as a percentage with the untreated group (0 μM SIN) as the control. The EdU incorporation assay was used to evaluate cell proliferation. Cells were seeded in 24-well plates at a density of 4×104 cells / mL and co-cultured with 10 μM EdU for 48 hours to incorporate it into newly synthesized DNA. After culturing, the cells were processed according to the manufacturer's instructions. Two methods were used for detection: one was to stain with DAPI and observe using a fluorescence microscope; the other was to digest with trypsin, collect the cells, further process according to the manufacturer's guidelines, and analyze EdU-positive cells using flow cytometry.

[0039] 2. Cell cycle detection

[0040] Cells were seeded at a density of 4×104 cells / mL, incubated overnight, and then treated with different concentrations of SIN (0 μM, 150 μM, 300 μM, 600 μM). After 24 hours of treatment, the cells were collected, washed twice with 1 mL of cold PBS phosphate buffer (purchased from Sevier Bio, product number G2156-1L), and fixed overnight at 4 °C with 1 mL of 70% (v / v) ethanol aqueous solution. After fixation, the cells were washed again with 1 mL of PBS and treated with 100 μL of 100 μg / mL RNase A in a 37 °C water bath for 30 minutes. Subsequently, propidium iodide (PI) with a final concentration of 50 μg / mL was added, and incubation was carried out in the dark at 4 °C for 30 minutes. The cell cycle distribution was analyzed by flow cytometry (BD LSR Fortessa, USA), and the proportions of cells in G0 / G1, S, and G2 / M phases were calculated using ModFit software.

[0041] 3. Cell apoptosis detection

[0042] Cell apoptosis was evaluated using an Annexin V-APC / DAPI apoptosis detection kit according to the manufacturer's instructions. Briefly, the treated cells were collected and washed twice with cold PBS. Then, the cells were resuspended in 1× Annexin V binding buffer. 5 μL of Annexin V-APC and 5 μL of DAPI were added to 500 μL of the cell suspension, gently vortexed, and incubated in the dark at room temperature for 15 minutes. After incubation, 400 μL of 1× binding buffer was added, and apoptosis analysis was performed using a flow cytometer (BD LSR Fortessa, USA). The data were analyzed using FlowJo software to quantify the proportions of early apoptotic cells (Annexin V positive, DAPI negative) and late apoptotic cells (Annexin V positive, DAPI positive).

[0043] 4. RNA Sequencing and Bioinformatics Analysis

[0044] Total RNA was extracted from RA-FLS and RA-FLS treated with SIN for 24 hours using TRIzol reagent. After extraction, the purity, concentration, and integrity of the RNA were evaluated by NanoDrop and Agilent 2100 Bioanalyzer. A cDNA library was constructed by reverse transcription of enriched mRNA into cDNA, ligation of adapters, and PCR amplification. The effective concentration of the library (library effective concentration > 2 nM) was accurately quantified using Q-PCR to ensure library quality. Sequencing was performed using the Illumina platform (BioMarker Technologies, Beijing, China). The reads for each gene were quantified using the HTSeq-count tool. Differentially expressed genes (DEGs) were analyzed using edgeR software, and the screening thresholds were false discovery rate (FDR) < 0.05 and |fold change (FC)| > 2. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses were performed using the BMKCloud platform (www.biocloud.net). Finally, a heatmap of DEGs was plotted using the pheatmap package, and a volcano plot was generated using the ggplot2 package in R studio.

[0045] 5. Western Blot Analysis

[0046] Cells were lysed using RIPA lysis buffer (Solarbio, China) containing protease and phosphatase inhibitors. Protein samples were separated by SDS-PAGE electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with Tris-buffered saline containing 0.1% Tween-20 (TBST) and 5% non-fat dry milk (2 g non-fat dry milk added to 40 ml TBST) for 1 hour at room temperature, and then incubated with primary antibodies overnight. After washing with 10 mL of TBST, the membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies for 1 hour at room temperature. After washing 3 times with 10 mL of TBST, protein bands were visualized using enhanced chemiluminescence (ECL) reagent (Bio-Rad, USA), and images were captured using a ChemiDoc imaging system (Bio-Rad, USA). Band intensities were quantified using ImageJ software.

[0047] 6. Quantitative real-time polymerase chain reaction (qRT-PCR)

[0048] Total RNA was extracted using TRIzol reagent (Thermo), and cDNA was synthesized by reverse transcription using NovoScript Plus All-in-One 1stStrand cDNA Synthesis SuperMix (Novoprotein Scientific Inc., catalog number E047-01B). qRT-PCR was performed using NovoStat SYBR qPCR SuperMix Plus (Novoprotein Scientific Inc., catalog number E96-01A) on a CFX96 TM real-time system. The loading system and amplification program for qRT-PCR were specifically referred to the reagent instructions provided by the above-mentioned kit. Human GAPDH gene was used as an endogenous control, and semi-quantitative analysis was performed using the 2 -ΔΔCT method. The primers used are shown in Table 1.

[0049] Table 1

[0050]

[0051] 7. Biotin-labeled SIN

[0052] Biotin, diethanolamine, and 4-dimethylaminopyridine (DMAP) were dissolved in dichloromethane (DCM), and the reaction was stirred at room temperature for 12 hours. After stirring, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (Xinweier, catalog number C184173) to obtain a white powder product, Biotin-PEG. Biotin-PEG, carbon tetrabromide (CBr4), and triphenylphosphine (PPh3) were dissolved in dichloromethane, and the reaction was stirred at room temperature for 8 hours. After stirring, the organic layer was extracted 3 times with DCM (dichloromethane). The combined organic phases were concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain a white powder product, Biotin-PEG-Br. SIN, potassium carbonate (K2CO3), and Biotin-PEG-Br were dissolved in anhydrous N,N-dimethylformamide (DMF), and the reaction was stirred at 50 °C for 12 hours. After the reaction was completed, 20 mL of ice water was added to the system. The organic phase was extracted with DCM, the solvent was evaporated under reduced pressure, and the product was purified by silica gel column chromatography to obtain Biotin-SIN.

[0053] 8. HuProt TM Screening of the direct targets of SIN by HuProt

[0054] The biotinylated SIN was incubated with the HuProt 20K human proteome microarray (purchased from CDI Laboratories, catalog number CDI9034370). Cy5-labeled streptavidin (Cy5-SA-Biotin) was used as a tracer to identify the proteins that directly bind to SIN. The microarray was scanned using a GenePix 4000B scanner.

[0055] For data analysis, the median fluorescence signal of each point was divided by the background signal for further analysis, denoted as F635Median / B635 Median, where F635 Median represents the median fluorescence signal of each point and B635 Median represents the median background signal. After removing 5,144 non-detectable (ND) and 1,280 control points, 46,312 points were retained. The original signal intensity of each point was calculated as I = F635 Median / B635 Median. For each chip, 24 regions were normalized by assuming a uniform distribution of the signal intensity (I) within each region, and the median signal intensity of each region was set to 1. The normalization process was performed using the following formula to obtain the normalized signal intensity, denoted as

[0056]

[0057] 9. Computer-aided molecular docking technology

[0058] Molecular docking simulations were performed using AutoDock Vina to predict the binding interaction between SIN and the target protein RNF7. The 3D structure of RNF7 was obtained from the Protein Data Bank (PDB), and the structure of SIN was obtained from the PubChem database. Before docking, the protein and ligand structures were prepared by removing water molecules, adding polar hydrogen atoms, and calculating the Gasteiger charges using AutoDockTools. The docking grid was defined to cover the active site of RNF7. The docking results were sorted according to the binding affinity, and the conformation with the lowest binding energy was selected for further analysis. The docking complex was visualized using PyMOL software to identify the key interacting residues.

[0059] 10. SPR technology

[0060] The protein was immobilized on the sensor chip using the amine coupling method. First, recombinant human RNF7 was immobilized on the chip, and then SIN was diluted to different concentrations (0.15625 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 10 μM) and added to a 96-well plate. The diluted SIN solution was injected into the chip in order from low to high concentration, with a flow rate set at 30 μL / min and an interaction time of 150 seconds for each concentration. After each concentration point, data were collected and the chip was regenerated with 10 mM glycine chloride (pH 2.0) solution for 5 minutes. This regeneration process was repeated until all concentration points of the target sample were processed. Data were collected using the BIAcore T200 control software (v.2.0, GE Healthcare), and the background signal of the reference channel (the reference channel was the uncoupled RNF7 protein) was removed. The obtained data were globally fitted using the BIAcore T200 Evaluation software (v.2.0, GE Healthcare) through a 1:1 Langmuir binding model to determine the binding constant and dissociation constant.

[0061] 11. Cell transfection

[0062] Cell transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Specifically, cells were seeded into 6-well plates and allowed to attach overnight. On the day of transfection, small interfering RNA (siRNA) of the target gene was mixed with Lipofectamine 3000 reagent in Opti-MEM medium. Then the siRNA-Lipofectamine 3000 complex was added to the cells, with a final siRNA concentration of 50 nM per well, and incubated for 48 hours. After transfection, fresh complete growth medium (purchased from Thermo Fisher Scientific, catalog number C11995500BT) was replaced, and the cells were continued to be cultured for subsequent analysis.

[0063] 12. Pull-down assay

[0064] Streptavidin magnetic beads (Beyotime, P2151) were incubated with 20 μM biotin or SIN-biotin overnight at 4°C. After incubation, the magnetic beads were washed three times with TBS buffer (purchased from Beyotime Biotechnology, catalog number ST663), and then added to the RA-FLS cell lysate and incubated overnight at 4°C. After incubation, the magnetic beads were washed three times again with TBS buffer (the same as above) to remove non-specific binders. Finally, the bound proteins were analyzed by SDS-PAGE and detected and quantified by Western blot.

[0065] 13. p27 Kip1 Detection of endogenous ubiquitination

[0066] RA-FLS cells were seeded into 10-mm culture dishes at a density of 6×104 cells / mL. 4 mL of culture medium (containing 90% DEME medium and 10% fetal bovine serum) was added to each dish, and 0 μM, 150 μM, 300 μM, or 600 μM of SIN was added respectively. After incubation for 24 hours, 10 μM of MG-132 (purchased from MCE, a potent cell-permeable 26S proteasome inhibitor that prevents the degradation of ubiquitinated proteins) was added and incubated for another 4 hours. Each dish was lysed with lysis buffer (300 μL) containing 1 mM protease inhibitor (purchased from Solarbio, catalog number P0100). The lysate was gently shaken to ensure complete cell lysis, and then the cells were scraped with a cell scraper and incubated on a rotary shaker at 4 °C for 10 minutes. After lysis, the supernatant was collected by centrifugation at 12,000×g for 5 minutes, and approximately 50 μL of the supernatant was reserved as the input sample. The remaining supernatant was incubated overnight with specific antibodies, and then protein A magnetic beads were added for immunoprecipitation. The immune complexes were incubated at 4 °C, followed by magnetic separation and washing three times. After washing, the proteins were denatured using SDS-PAGE sample loading buffer, subjected to SDS-PAGE electrophoresis, and finally the ubiquitination level of p27 was detected by Western blot analysis Kip1 of p27

[0067] 14. Statistical analysis

[0068] Experimental data were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using paired t-tests or one-way analysis of variance (ANOVA) to compare differences between experimental groups. Statistical significance was set at P < 0.05. All data were analyzed using GraphPad Prism 8.0.1 (GraphPad Software, Inc.)

[0069] 15. Results

[0070] 15.1. SIN inhibits the proliferation of RA-FLS cells

[0071] To evaluate the effect of SIN on the proliferation of RA-FLS, the EdU incorporation assay was used for detection, and the results are shown in Figure 1 ; the results were demonstrated by both fluorescence microscopy imaging and flow cytometry and are shown in Figure 1 .

[0072] Figure 1Part A shows the chemical structure of SIN and a schematic diagram of its source; it can be seen from Part A that SIN is derived from the traditional Chinese medicine Sinomenium acutum, a plant of the Menispermaceae family.

[0073] Figure 1 Part B shows the effects of SIN at different concentrations on the viability of RA-FLS cells after 48 hours of treatment detected by the CCK-8 method (n = 4); it can be seen from Part B that as the concentration increases, the cell viability gradually decreases. Among them, the cell viability in the 150 μM and 300 μM groups is relatively high, the cell viability in the 600 μM group is greater than 70%, while the cell viability in the 1200 μM group is significantly reduced to about 50%. Generally, IC30 (i.e., the cell survival rate of about 70%) is used as the upper limit concentration without obvious toxic effects to avoid interference of excessive cytotoxicity on the experimental results. Based on these results, three concentrations of 150 μM, 300 μM, and 600 μM are selected as the subsequent experimental concentrations.

[0074] Figure 1 Part C shows the proliferation of RA-FLS cells detected by the EdU incorporation method combined with fluorescence microscopy imaging after co-incubating with 10 μM EdU and different concentrations of SIN for 48 hours. Green fluorescence labels EdU-positive cells (n = 3); it can be seen from Part C that SIN inhibits the proliferation of RA-FLS cells.

[0075] Figure 1 Parts D and E show the quantitative analysis of the proportion of EdU-positive cells by flow cytometry. The proportion of EdU-positive cells in the SIN treatment group is significantly reduced, indicating that SIN effectively inhibits the abnormal proliferation of RA-FLS. It can be seen from Parts D and E that SIN inhibits the proliferation of RA-FLS cells.

[0076] Figure 1 The data are expressed as Mean ± SEM. Compared with the control group, *** P < 0.001, **** P < 0.0001, ### P < 0.001, #### P < 0.0001.

[0077] 15.2. SIN blocks the cell cycle of RA-FLS cells and induces apoptosis

[0078] Figure 2 This is the result graph of the effects of SIN on the cell cycle and apoptosis of RA-FLS cells.

[0079] Figure 2Parts A and B show the cell cycle distribution of RA-FLS cells after SIN treatment for 24 hours by flow cytometry (n = 3); it can be seen from Parts A and B that SIN blocks the cell cycle progression of RA-FLS. The analysis results show that with the increase in the concentration of SIN during incubation, the effect of cell cycle arrest of RA-FLS cells in the G0 / G1 phase is enhanced.

[0080] Figure 2 Parts C and D show the Western blot analysis of the expression levels of cyclin Cyclin D1, CDK4, and p27 Kip1 (n = 5); it can be seen from Parts C and D that SIN treatment leads to a significant decrease in the expression of cyclin Cyclin D1, while the expression level of the cell cycle inhibitor p27 Kip1 increases significantly, and this effect becomes more obvious with the increase in the concentration of SIN. This indicates that SIN effectively blocks the cell cycle progression of RA-FLS cells by regulating the expression of key cell cycle proteins in a concentration-dependent manner.

[0081] Figure 2 Parts E and F show the apoptosis flow cytometry detection after SIN treatment for 48 hours (n = 3); it can be seen from Parts E and F that the proportion of apoptotic cells in the SIN treatment group increases significantly with the increase in the concentration of SIN, indicating that SIN can effectively induce apoptosis of RA-FLS cells.

[0082] Figure 2 The data in the figure are expressed as Mean ± SEM. Compared with the control group without SIN treatment, * P < 0.05, ** P < 0.01, *** P < 0.001.

[0083] 15.3. The inhibition of SIN on RA-FLS may be related to the mTOR signaling pathway

[0084] To explore the specific mechanism of SIN in inhibiting the proliferation and inducing apoptosis of RA-FLS, the high-throughput technical means of transcriptomics was used to analyze the differentially expressed genes (DEGs) after SIN treatment and perform bioinformatics analysis.

[0085] Figure 3 This is the analysis result diagram revealing the potential mechanism of SIN in inhibiting RA-FLS through transcriptome sequencing after co-incubation of RA-FLS cells with SIN for 24 hours.

[0086] Figure 3 Part A in the figure shows the expression heat map of DEGs; Figure 3Part B in it shows a volcano plot of gene expression in the control group and the experimental group with SIN (600 μM), and the genes marked in the figure meet the conditions of |log2FC| > 3 and FDR < 0.05; from Figure 3 Parts A and B in it, it can be seen that through transcriptomics screening, the expression of multiple genes has changed significantly after SIN treatment, among which 578 genes are up-regulated and 565 genes are down-regulated.

[0087] Figure 3 Part C in it shows a Venn diagram display of DEGs in the experimental groups with different concentrations of SIN; among them, compared with the control group, a total of 1,143 differentially expressed genes were detected in the SIN (600 μM) group.

[0088] Figure 3 Part D in it shows a GO functional enrichment analysis, showing the enrichment of differential genes in biological processes; the results show that there are significant differences between the SIN (600 μM) group and the control group in multiple GO classifications, especially in key biological processes such as apoptosis and angiogenesis.

[0089] Figure 3 Part E in it shows a KEGG signaling pathway enrichment analysis, highlighting the main enriched signaling pathways of DEGs. It is found that these differential genes are mainly enriched in signaling pathways such as MAPK and PI3K / Akt. Particularly noteworthy is that the downstream mTOR signal of the PI3K / Akt signaling pathway plays an important role in regulating cell proliferation and apoptosis.

[0090] 15.4. SIN inhibits the mTOR / p70S6K signaling pathway

[0091] Figure 4 is a result diagram showing the effect of SIN on the mTOR pathway in RA-FLS cells after co-incubation with SIN for 24 hours.

[0092] Figure 4 Part A in it shows that transcriptome analysis shows the change in the expression level of genes related to the mTOR pathway after SIN treatment; it can be seen from Part A that after SIN treatment, the genes enriched on the mTOR signal show expression differences.

[0093] Figure 4 Part B in it shows that qRT-PCR verifies the expression trend of genes related to the mTOR pathway; it can be seen from Part B that after SIN treatment, the genes enriched on the mTOR signal show expression differences. And its expression trend is consistent with the transcriptome data.

[0094] Figure 4Parts C and D show the results of Western blot analysis indicating the effect of SIN on the phosphorylation levels of mTOR and its downstream effectors p70S6K and 4E-BP1. As can be seen from Parts C and D, SIN significantly inhibits the phosphorylation of mTOR and its downstream effectors p70S6K and 4E-BP1, suggesting that SIN may affect the proliferation of RA-FLS by inhibiting the mTOR signaling pathway.

[0095] Figure 4 The data in are presented as Mean±SEM. Compared with the control group treated with SIN, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

[0096] 15.5. Activation of mTOR can restore the inhibitory effect of SIN on RA-FLS

[0097] Figure 5 is a graph showing the effects of MHY1485 (a synthetic small molecule compound purchased from Selleck) on the proliferation, cell cycle, and apoptosis of RA-FLS cells after co-incubation with SIN for 24 hours.

[0098] Figure 5 Parts A and B show the results of EdU incorporation assay for detecting cell proliferation; as can be seen from Parts A and B, activation of the mTOR signal using MHY1485 can partially reverse the inhibitory effect of SIN on RA-FLS proliferation.

[0099] Figure 5 Parts C and D show the cell cycle results; as can be seen from Parts C and D, activation of the mTOR signal using MHY1485 can partially reverse the cell cycle arrest effect of SIN on RA-FLS.

[0100] Figure 5 Parts E and F show the apoptosis results. As can be seen from Parts E and F, activation of the mTOR signal using MHY1485 can partially reverse the pro-apoptotic effect of SIN on RA-FLS.

[0101] Figure 5 The data in are presented as Mean±SEM. Compared with the control group, * P<0.05, *** P<0.001. Figure 5Flow cytometry detection results showed that MHY1485 could significantly rescue the inhibitory effects of SIN on the proliferation and cell cycle of RA-FLS cells, and at the same time reverse the promoting effect of SIN on apoptosis. This indicates the key role of the mTOR pathway in SIN regulation of RA-FLS proliferation and apoptosis.

[0102] 15.6, SIN targets RNF7

[0103] Figure 6 It is a result graph of the binding characteristics analysis of SIN and RNF7 protein. SIN was linked to biotin through a polyethylene glycol (PEG, molecular weight 105.08) chain, and the addition of this chain increased the distance between biotin and the active molecule, thereby improving the accuracy of binding.

[0104] Figure 6 Part A in it shows the schematic diagram of the structure of biotin-labeled SIN.

[0105] Figure 6 Part B in it shows HuProt TM Schematic diagram of the experimental principle of the HuProt Human Protein Microarray chip.

[0106] Figure 6 Part C in it shows HuProt TM chip (HuProt TM Human Protein Microarray chip) directly visualizes the binding of SIN to various target proteins. The results showed that RNF7 is the protein target with the strongest binding to SIN.

[0107] Figure 6 Parts D and E in it further show the results of RNF7 as the main binding protein of SIN; it can be seen from Parts D and E that RNF7 is the protein target with the strongest binding to SIN.

[0108] Figure 6 Part F in it shows that computer-aided molecular docking analysis confirmed that the specific binding site of SIN and RNF7 is amino acid VAL-76.

[0109] Figure 6Part G in it shows that the SPR technology verified the direct binding of SIN and RNF7. Part G shows the measurement results of the dissociation constant (KD), the association rate constant (Ka), and the dissociation rate constant (Kd). Among them, the smaller the KD value, the stronger the affinity between SIN and RNF7; the larger the Ka value, the faster the binding rate; the larger the Kd value, the faster the dissociation rate. In addition, the surface plasmon resonance (SPR) technology was used to verify the direct binding between SIN and RNF7, and the measured dissociation constant (KD) was 3.84×10 -6 M, indicating a high binding affinity between SIN and RNF7.

[0110] Figure 6 Part H in it shows that the pull-down experiment verified the direct binding of SIN and RNF7; it can be seen from Part H that SIN can directly bind to RNF7.

[0111] 15.7. Knockdown of RNF7 inhibits the mTOR signaling pathway, inhibits the proliferation and cell cycle progression of RA-FLS, and promotes apoptosis

[0112] To explore whether SIN mediates the inhibition of the mTOR signaling pathway through RNF7, RNF7 was knocked down. Figure 7 It is the result diagram of the function of RNF7 in RA-FLS cells and its influence on cell biological characteristics.

[0113] Figure 7 Part A in it shows the expression results of RNF7 mRNA detected by qRT-PCR; it can be seen from the qRT-PCR results in Part A that, compared with normal FLS, the expression of RNF7 in RA-FLS is significantly increased.

[0114] Figure 7 Parts B and C in it show the expression results of RNF7 protein detected by Western Blot; it can be seen from Parts B and C that, compared with HFLS, RNF7 is highly expressed in RA-FLS.

[0115] Figure 7 Part D in it shows the fluorescence image results of siRNA transfection; it can be seen from Part D that siRNA can be successfully transfected into cells.

[0116] Figure 7 Part E in it shows the results of screening the siRNA with the highest knockdown efficiency; after knocking down RNF7, cell proliferation was detected ( Figure 7 Parts F and G in it), cell cycle (the results are shown in Figure 7 Parts J and K in it), apoptosis (the results are shown in Figure 7 Parts H and I in it); from Figure 7It can be seen from parts E, F, G, J, K, H, and I that after knocking down RNF7, the proliferation of RA-FLS cells is inhibited, the apoptosis rate is significantly increased, and the cell cycle is arrested in the G0 / G1 phase.

[0117] Figure 7 Parts L and M show that Western blot analysis reveals the effect of SIN on the phosphorylation levels of mTOR and its downstream effector molecules p70S6K and 4E-BP1; it can be seen from parts L and M that the activity of the mTOR signaling pathway is significantly decreased.

[0118] Figure 7 Parts N and O show the expression levels of cyclin Cyclin D1, CDK4, and p27 analyzed by Western blot Kip1 ; it can be seen from parts N and O that the expression levels of cyclin Cyclin D1 and CDK4 decrease, while the expression level of p27 Kip1 increases.

[0119] Figure 7 The data in are presented as Mean±SEM. Compared with the si-NC group, * P<0.05, ** P<0.01, **** P<0.0001.

[0120] Figure 7 These results in indicate that RNF7 may be a key target protein for SIN to exert its inhibitory effect on RA-FLS. These results suggest that SIN affects the mTOR signaling pathway by regulating RNF7, thereby inhibiting the proliferation of RA-FLS cells and promoting apoptosis.

[0121] 15.8, SIN inhibits the ubiquitination of p27 Kip1 and knockdown of p27 activates the mTOR signaling pathway

[0122] Figure 8 is the result graph of the effect of SIN on the ubiquitination of p27 Kip1 and the effect of p27 Kip1 knockdown on mTOR activation.

[0123] Figure 8 Part A in shows a pull-down experiment to explore the interaction between RNF7 and p27 Kip1 and the result shows that there is no direct binding between the two.

[0124] Figure 8 Parts B and C in show the Western blot analysis of p27 after RNF7 knockdown and SIN (600 μM, 24 hours) treatmentKip1 Expression results; It can be seen from parts B and C that whether RNF7 is knocked down or SIN is treated, the expression level of p27 can be increased, and the levels of both are comparable; SIN affects the stability of p27 in cells through its interaction with RNF7. Kip1 Kip1

[0125] Figure 8 Parts D and E in show the expression of p27 after treatment with MG-132 (10 μM, 4 hours) and SIN (600 μM, 24 hours); It can be seen from parts D and E that after MG-132 treatment, the level of p27 increases, suggesting that there is a connection between the level of p27 and ubiquitination in RA-FLS cells. Kip1 Kip1 Kip1

[0126] Figure 8 Part F in shows the detection of the endogenous ubiquitination level of p27 by Co-Immunoprecipitation (Co-IP); It can be seen from part F that SIN can inhibit the ubiquitination modification of p27. Kip1 Kip1

[0127] Figure 8 Part G in shows the transfection efficiency of the siRNA transfection system; It can be seen from part G that the transfection system successfully transfected siRNA into cells.

[0128] Figure 8 Part H in shows the results of knocking down p27 by three siRNAs respectively; It can be seen from part H that #1 has the highest knockdown efficiency, so #1 was selected for verification in subsequent experiments. Kip1

[0129] Figure 8 Parts I and J in show the activation of mTOR after knocking down p27 by Western blot analysis; It can be seen from parts I and J that the phosphorylation level of mTOR increases after knocking down p27. Kip1 Kip1

[0130] Figure 8 The data in are expressed as Mean ± Standard Error of the Mean (Mean ± SEM). Compared with the siRNA negative control group (si-NC), * P < 0.05, ** P < 0.01.

[0131] In summary, the present invention studied the inhibitory effects of SIN on the proliferation, cell cycle progression, and apoptosis of RA-FLS, and revealed the mechanism by which it achieves this function by regulating the mTOR signaling pathway. The results showed that SIN can inhibit the proliferation of RA-FLS, arrest the cell cycle, and promote cell apoptosis, demonstrating its potential application value in the treatment of RA.

[0132] The study of the present invention clarified the inhibitory effect of SIN on the proliferation of RA-FLS through EdU incorporation experiments. SIN can significantly reduce the proportion of EdU-positive cells, indicating its effective inhibition of the abnormal proliferation of RA-FLS. Combining with cell cycle arrest experiments, the inventors found that SIN downregulates the expression of Cyclin D1 and CDK4, while upregulating the cell cycle inhibitor p27 Kip1 , arresting the cells in the G0 / G1 phase. These results indicate that SIN can effectively inhibit the abnormal proliferation of RA-FLS by regulating the expression of cell cycle-related proteins.

[0133] In addition, the promotion of cell apoptosis by SIN is also one of the core contents of the present invention. Flow cytometry analysis showed that with the increase in the concentration of SIN, the apoptosis rate of RA-FLS increased significantly. Further transcriptomic analysis revealed that the mTOR / p70S6K signaling pathway played a key role in SIN-mediated proliferation inhibition and apoptosis promotion. This finding is consistent with previous studies showing the important role of mTOR in regulating cell proliferation and apoptosis. The present invention further verified this mechanism through Western blot experiments, and the results showed that SIN treatment significantly inhibited the phosphorylation levels of mTOR and its downstream effector molecules p70S6K and 4E-BP1. To confirm the role of the mTOR pathway in SIN regulation of RA-FLS, the present invention used the mTOR agonist MHY1485 for a rescue experiment. The results showed that MHY1485 could partially reverse the inhibitory effect of SIN on the proliferation of RA-FLS and rescue SIN-induced cell cycle arrest and apoptosis. This result further supports the key role of the mTOR signaling pathway in the anti-RA effect of SIN.

[0134] The direct interaction between SIN and RNF7 was further verified by SPR and pull-down experiments. As an E3 ubiquitin ligase, RNF7 promotes the ubiquitination and subsequent degradation of the cell cycle inhibitor p27 Kip1 . Knockdown of RNF7 led to a significant accumulation of p27 in RA-FLS Kip1 (shown in the N part of Figure 7 ).

[0135] The research results of the present invention showed that there was no significant difference in p27 between the SIN treatment group and the RNF7 knockdown group Kip1There was no significant difference in the expression level. In addition, the effect of the combined treatment of SIN and RNF7 knockdown did not show an additive effect compared with the individual treatment ( Figure 8 as shown in part B of

[0136] These results suggest that SIN increases intracellular p27 by binding to RNF7 Kip1 level. In addition, SIN treatment reduced the ubiquitination of p27 Kip1 .

[0137] In the present invention, knockdown of p27 Kip1 led to a significant increase in the phosphorylation level of mTOR. In addition, the data of the present invention also showed that the expression of RNF7 in RA-FLS was significantly higher than that in HFLS ( Figure 7 as shown in parts A to C of Figure 7 as shown in parts F to M of

[0138] Collectively, the studies of the present invention showed that SIN binds to RNF7 and inhibits RNF7-mediated p27 Kip1 ubiquitination, thereby counteracting RNF7-driven abnormal proliferation and apoptosis of RA-FLS.

[0139] In summary, the studies of the present invention showed that SIN exerts its therapeutic effect by regulating the mTOR signaling pathway, thereby inhibiting the proliferation of RA-FLS, inducing cell cycle arrest, and promoting apoptosis. In addition, the present invention confirmed the direct binding of SIN to RNF7 and inhibited the ubiquitination of p27 Kip1 , ultimately inhibiting mTOR activity, providing new insights into its mechanism of action in RA. This is the first to elucidate the unique molecular mechanism by which SIN regulates the biological behavior of RA-FLS by targeting RNF7. The research results not only provide strong evidence for the therapeutic potential of SIN in RA, but also highlight that RNF7 can serve as a therapeutic target.

[0140] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. Use of sinomenine in the preparation of a drug for inhibiting the proliferation of RA synovial fibroblasts.

2. Use of sinomenine in the preparation of a drug for regulating the mTOR / p70S6K / 4E-BP1 pathway.

3. Use of sinomenine in the preparation of a drug for inhibiting the phosphorylation level of mTOR.

4. Use of sinomenine in the preparation of a drug for inhibiting the phosphorylation levels of the downstream effector molecules p70S6K / 4E-BP1 of mTOR.

5. Use of sinomenine in the preparation of a drug for targeting and binding to RNF7.

6. Application of sinomenine in the preparation of a drug for inhibiting the ubiquitination of p27 Kip1 ​

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