阅读说明：本技术 鼠李素-3-O-α-鼠李糖苷的药物用途及抗炎药物 (Pharmaceutical application of rhamnosine-3-O-alpha-rhamnoside and anti-inflammatory drug ) 是由 杨官娥 任凯达 周江韬 侯静 陈婕 于 2021-06-19 设计创作，主要内容包括：本发明通过探讨ARR对脂多糖(LPS)诱导的RAW264.7巨噬细胞的影响,并研究其潜在机制。通过CCK-8测定法检测了细胞毒性。进行了Griess、ELISA,RT-qPCR、蛋白质印迹、免疫荧光和免疫组化实验,以阐明炎症反应的分子机制。细胞毒性测试结果表明,最高200μg/mL的ARR对RAW264.7细胞的存活率没有明显影响。Griess、ELISA和RT-qPCR结果表明,ARR显著减轻了LPS诱导的RAW264.7细胞中炎症反应的产生。进一步的免疫印迹实验表明,ARR抑制了NF-κB通路并激活了Nrf2通路,这与免疫荧光和免疫组化实验的结果相吻合。ARR通过下调NF-κB和激活Nrf2介导的炎症反应共同发挥抗炎作用。因此,ARR可作为一种有潜力的广谱抗炎候选药物。(The invention researches the influence of ARR on RAW264.7 macrophage induced by Lipopolysaccharide (LPS) and researches the potential mechanism of the ARR. Cytotoxicity was detected by the CCK-8 assay. Griess, ELISA, RT-qPCR, western blot, immunofluorescence, and immunohistochemical experiments were performed to elucidate the molecular mechanisms of inflammatory reactions. The results of the cytotoxicity test show that ARR of up to 200. mu.g/mL has no obvious influence on the survival rate of RAW264.7 cells. Results of Griess, ELISA and RT-qPCR showed that ARR significantly reduced the production of inflammatory responses in LPS-induced RAW264.7 cells. Further immunoblotting experiments showed that ARR inhibited the NF-. kappa.B pathway and activated the Nrf2 pathway, which is consistent with the results of immunofluorescence and immunohistochemistry experiments. ARR exerts an anti-inflammatory effect through down-regulation of NF- κ B and activation of Nrf 2-mediated inflammatory responses. Therefore, ARR can be used as a potential broad-spectrum anti-inflammatory candidate drug.)

1. Application of rhamnosine-3-O-alpha-rhamnoside in preparing broad-spectrum antiinflammatory agent is provided.

2. Use according to claim 1, characterized in that: the application of the rhamnosine-3-O-alpha-rhamnoside in medicaments which play an anti-inflammatory role together by down-regulating NF-kappa B and activating Nrf2 mediated inflammatory reaction.

3. An anti-inflammatory agent characterized by: comprises the active component of rhamnosine-3-O-alpha-rhamnoside.

4. An anti-inflammatory agent as in claim 3, wherein: also comprises medically acceptable auxiliary materials.

5. An anti-inflammatory agent as in claim 3 or 4, wherein: the anti-inflammatory drug is an injection.

6. The process for preparing rhamnosine-3-O- α -rhamnoside according to any one of claims 1 to 5, which comprises:

step one, weighing the crushed north mulberry parasitic drug, and carrying out ultrasonic extraction for 3 times with 75% ethanol, wherein each time lasts for 20 min; mixing the extractive solutions, and concentrating under reduced pressure to obtain ethanol extract;

step two, kneading 1g of alcohol extract with warm water to form suspension, fixing the volume to 2ml, adding into a D101 type macroporous adsorption resin column, eluting with distilled water with the volume 3 times that of the column, and removing a large amount of water-soluble impurities; continuously eluting the flavonoid compounds with 60% ethanol in an amount which is 3 times that of the flavonoid compounds, collecting the eluent, and concentrating to obtain enriched total flavonoids;

step three, taking 1g of enriched total flavonoids extracted by 60% ethanol and 1g of 200-mesh 300-mesh silica gel, adding absolute ethanol, and mixing in a rotary evaporator;

step four, packing the sample into a column by a solvent silica gel column wet method with dichloromethane and methanol =17:2, pressing the column by using a vacuum pump or naturally settling for 4 hours, adding the sample into the silica gel column with the ratio of crude product to silica gel being 1:25, then adding 2g of silica gel, eluting by using a mobile phase with dichloromethane and methanol and formic acid =17:2:1 in an amount which is 3 times of the column volume, and collecting the eluent by dividing into 10mL test tubes; eluting with 3 times of column volume of mobile phase containing dichloromethane and methanol =4:1, and collecting eluate in 10mL test tubes; and (3) spotting a thin-layer chromatography silica gel plate, wherein a developing agent is dichloromethane, methanol and formic acid =17:2:1, mixing test tubes with consistent compounds, and performing reduced pressure concentration at 65 ℃ by using a rotary evaporator to obtain the rhamnosine-3-O-alpha-rhamnoside monomer.

Technical Field

The invention relates to the technical field of anti-inflammatory drugs, and in particular relates to a pharmaceutical application of rhamnosine-3-O-alpha-rhamnoside.

Background

Inflammation is a defense response of the human body against various damaging factors, and generally modulation of the inflammatory response plays a crucial role in dealing with pathogens and alleviating damaged tissues. The abnormal inflammatory response contributes to the development of a variety of serious diseases, including rheumatoid arthritis, chronic hepatitis, Alzheimer's disease, inflammatory bowel disease, and cancer, among others. Therefore, effective control of the inflammatory response is critical for the prevention and treatment of many diseases, including cancer. Inflammatory diseases are various complex and difficult-to-cure diseases, and establishing an inflammatory model has profound significance for screening inflammatory drugs and curing diseases. It is well known that LPS-induced inflammatory response models have been widely used in inflammatory studies.

The nuclear transcription factor κ B (NF- κ B) acts as a multi-directional functional modulator, being central to anti-inflammatory drugs. In addition, the primary approach to preventing chronic inflammation-mediated diseases is to modulate pro-inflammatory cytokines, the production or secretion of which results in the activation of NF-. kappa.B, which in turn activates the expression of transcription factors that control the gene expression of pro-inflammatory cytokines, including interleukins, Inducible Nitric Oxide Synthase (iNOS), and cyclooxygenase 2 (COX-2). NF-. kappa.B activation plays an essential role in the development of many serious diseases.

Tumor necrosis factor-related factor 6 (TRAF 6) is a key regulator of NF- κ B, a key step in the regulation of inflammation, and NLRC3 has inhibitory effects on proinflammatory signal transduction, ubiquitination of TRAF6 and nuclear translocation of NF- κ B p 65. Furthermore, NLRC3 can inhibit the major inflammatory pathway controlled by NF- κ B, which directly interacts with the molecule TRAF6 and forms a new protein complex, called "trafagome".

Nuclear factor-E2-related factor 2 (Nrf 2) is a key and important transcription factor that controls many antioxidant enzymes, including heme oxygenase-1 (HO-1), nad (p) H quinone dehydrogenase 1 (NQO 1). HO-1 plays an important role in resisting oxidation and inhibiting immune response. The literature reports that Nrf2 and a target gene thereof as an inflammation regulation system can inhibit the expression of a plurality of proinflammatory cytokines, so that the activation of NF-kB can be antagonized.

Loranthus parasiticus is a hemiparasitic plant, which uses branches of Quercus and Betula as hosts and has many biological properties including antimicrobial, antitumor, antioxidant, etc. The ethyl acetate soluble fraction of the methanol extract of Taxus chinensis shows anti-tumor activity in vitro. Rhamnin-3-O-alpha-rhamnoside (ARR, figure 1A) is a phenolic flavonoid compound, is a main active ingredient of Taxillus chinensis (Burk.) Britt.F.Chen, and has few reports about the pharmacological activity of ARR. The research on the anti-inflammatory molecular mechanism of ARR is not reported in the literature.

Disclosure of Invention

The invention aims to provide a pharmaceutical application of rhamnosine-3-O-alpha-rhamnoside and an anti-inflammatory drug obtained by the application.

Based on the above purposes, one aspect of the invention provides the application of rhamnosine-3-O-alpha-rhamnoside in preparing broad-spectrum anti-inflammatory drugs.

Preferably, the rhamnosine-3-O-alpha-rhamnoside is used in a medicament for exerting an anti-inflammatory effect by down-regulating NF-kappa B and activating Nrf2 mediated inflammatory reactions.

According to another aspect of the present invention, there is provided an anti-inflammatory agent comprising rhamnosine-3-O- α -rhamnoside as an active ingredient.

Further, in order to obtain a dosage form suitable for medical treatment, the anti-inflammatory drug further comprises medically acceptable auxiliary materials, and the dosage form can be injection, tablets, granules and the like. As the rhamnosine-3-O-alpha-rhamnoside is a water-soluble compound, the rhamnosine-3-O-alpha-rhamnoside can be directly used as an injection or dissolved in water or saline to prepare the injection.

According to another aspect of the present invention, a method for preparing rhamnosine-3-O- α -rhamnoside, comprising:

step one, weighing the crushed north mulberry parasitic drug, and carrying out ultrasonic extraction for 3 times with 75% ethanol, 20min each time. Mixing the extractive solutions, and concentrating under reduced pressure to obtain ethanol extract;

step two, kneading 1g of alcohol extract with warm water to form suspension, fixing the volume to 2ml, adding into a D101 type macroporous adsorption resin column, eluting with distilled water with the volume 3 times that of the column, and removing a large amount of water-soluble impurities; continuously eluting the flavonoid compounds with 60% ethanol in an amount which is 3 times that of the flavonoid compounds, collecting the eluent, and concentrating to obtain enriched total flavonoids;

step three, taking 1g of enriched total flavonoids extracted by 60% ethanol and 1g of 200-mesh 300-mesh silica gel, adding absolute ethanol, and mixing in a rotary evaporator;

step four, packing the sample into a column by a solvent silica gel column wet method with dichloromethane and methanol =17:2, pressing the column by using a vacuum pump or naturally settling for 4 hours, adding the sample into the silica gel column with the ratio of crude product to silica gel being 1:25, then adding 2g of silica gel, eluting by using a mobile phase with dichloromethane and methanol and formic acid =17:2:1 in an amount which is 3 times of the column volume, and collecting the eluent by dividing into 10mL test tubes; the eluate was further eluted at a column volume 3 times that of a mobile phase of dichloromethane to methanol =4 to 1, and collected in 10mL test tubes. And (3) spotting a thin-layer chromatography silica gel plate, wherein a developing agent is dichloromethane, methanol and formic acid =17:2:1, mixing test tubes with consistent compounds, and performing reduced pressure concentration at 65 ℃ by using a rotary evaporator to obtain the rhamnosine-3-O-alpha-rhamnoside monomer.

The invention researches the influence of ARR on RAW264.7 macrophage induced by Lipopolysaccharide (LPS) and researches the potential mechanism of the ARR. Cytotoxicity was detected by the CCK-8 assay. Griess, ELISA, RT-qPCR, western blot, immunofluorescence, and immunohistochemical experiments were performed to elucidate the molecular mechanisms of inflammatory reactions. The results of the cytotoxicity test show that ARR of up to 200. mu.g/mL has no obvious influence on the survival rate of RAW264.7 cells. Results of Griess, ELISA and RT-qPCR showed that ARR significantly reduced the production of inflammatory responses in LPS-induced RAW264.7 cells. Further immunoblotting experiments showed that ARR inhibited the NF-. kappa.B pathway and activated the Nrf2 pathway, which is consistent with the results of immunofluorescence and immunohistochemistry experiments. ARR exerts an anti-inflammatory effect through down-regulation of NF- κ B and activation of Nrf 2-mediated inflammatory responses. Therefore, ARR can be used as a potential broad-spectrum anti-inflammatory candidate drug.

Drawings

FIG. 1 ARR inhibits inflammatory responses. RAW264.7 cells were treated with various concentrations of ARR (0, 5, 10, 20, 40, 80, 100, 200. mu.g/mL) for 24 hours. (B) Cell viability was examined using the WST-8 assay. Data represent mean ± standard deviation (s.d.). RAW264.7 cells were pretreated with ARR (0, 25, 50, 100. mu.g/mL) at various concentrations for 2 hours and then incubated with or without LPS (100 ng/mL) for 24 hours. (C) The level of NO in the medium was determined using Griess reagent. The detection of ARR on IL-6 (D), IL-1 beta (E) and PGE by ELISA kit₂(F) The effect of cytokine production. Data represent mean. + -. standard deviation # # P<0.01, relative to LPS treated and ARR untreated controls. Relative to the LPS-treated group and the ARR-treated group,^*P <0.05 and^**P <0.01。

FIG. 2 ARR reduces proinflammatory factor levels. RAW264.7 cells were pretreated with ARR (0, 25, 50, 100. mu.g/mL) at various concentrations for 2 hours and then incubated with or without LPS (100 ng/mL) for 24 hours. RT-qPCR was performed to analyze mRNA expression of iNOS (A), IL-6 (B), IL-1. beta. (C), and COX-2 (D). The values are expressed as mean ± standard deviation. Three independent experiments of (n = 3).^##P <0.01, relative to LPS treated and ARR untreated controls.^*P <0.05 and^**P <0.01 relative to the LPS-treated group and the ARR-treated group.

FIG. 3 ARR inhibits the NF-. kappa.B pathway and activates the Nrf2 pathway. RAW264.7 cells were treated with various concentrations of ARR for 24 hours with or without LPS. Protein expression of total NF-. kappa. B p65 and phosphorylation of NF-. kappa. B p65 (A), nuclear (B) and cytosolic NF-. kappa. B p65 (C), protein expression in Nrf2 (D), HO-1 (E), NQO1 (F), were examined using ECL system and quantified using Image Lab software. CON represents the protein from the control group, LPS represents the protein from the 100 ng/mL LPS-treated group, Indo represents the protein from the 8. mu.g/mL LDO-treated group, ARR represents the protein from the 100. mu.g/mL LRR-treated group100 ng/mL LPS treated group. Data represent three independent experiments with mean ± standard deviation (n = 3). Relative to the LPS treated group and the ARR untreated control group,^#P <0.05 and^##P <0.01. relative to the LPS-treated group and the ARR-treated group,^*P <0.05 and^**P <0.01。

FIG. 4. ARR blocks nuclear translocation of NF-. kappa. B p 65. Nuclear translocation of NF- κ Bp65 was detected by immunofluorescence microscopy.

FIG. 5 ARR inhibits protein production of TRAF6 and NF- κ Bp65 by increasing the content of NLRC3 protein molecules. Expression of NLRC3, TRAF6 and NF-. kappa.Bp 65 in LPS-stimulated RAW264.7 cells was examined by immunohistochemical staining (A). The mean optical density values of NLRC3 (B), TRAF6 (C) and NF-. kappa.Bp 65 (D) were quantified using Image J software. Data represent three independent experiments with mean ± standard deviation (n = 3). Relative to the LPS-treated and ARR untreated controls,^#P <0.05 and^##P <0.01. relative to the LPS-treated group and the ARR-treated group,^*P <0.05 and^**P <0.01。

Detailed Description

The technical solution and the technical effects thereof claimed by the present invention will be explained below through experiments. Unless otherwise specifically indicated, the materials and reagents used in the present invention are available from commercial products in the art.

1. Materials and methods

1.1 reagents and chemicals

Rhamnin-3-O-alpha-rhamnoside (ARR) is separated from herba Taxilli by the following method, and its structural formula is shown in figure 1A.

Step one, weighing the crushed north mulberry parasitic drug, and carrying out ultrasonic extraction for 3 times with 75% ethanol, 20min each time. Mixing the extractive solutions, and concentrating under reduced pressure to obtain ethanol extract;

step two, kneading 1g of alcohol extract with warm water to form suspension, fixing the volume to 2ml, adding into a D101 type macroporous adsorption resin column, eluting with distilled water with the volume 3 times that of the column, and removing a large amount of water-soluble impurities; continuously eluting the flavonoid compounds with 60% ethanol in an amount which is 3 times that of the flavonoid compounds, collecting the eluent, and concentrating to obtain enriched total flavonoids;

step three, taking 1g of enriched total flavonoids extracted by 60% ethanol and 1g of 200-mesh 300-mesh silica gel, adding absolute ethanol, and mixing in a rotary evaporator;

step four, packing the sample into a column by a solvent silica gel column wet method with dichloromethane and methanol =17:2, pressing the column by using a vacuum pump or naturally settling for 4 hours, adding the sample into the silica gel column with the ratio of crude product to silica gel being 1:25, then adding 2g of silica gel, eluting by using a mobile phase with dichloromethane and methanol and formic acid =17:2:1 in an amount which is 3 times of the column volume, and collecting the eluent by dividing into 10mL test tubes; the eluate was further eluted at a column volume 3 times that of a mobile phase of dichloromethane to methanol =4 to 1, and collected in 10mL test tubes. And (3) spotting a thin-layer chromatography silica gel plate, wherein a developing agent is dichloromethane, methanol and formic acid =17:2:1, mixing test tubes with consistent compounds, and performing reduced pressure concentration at 65 ℃ by using a rotary evaporator to obtain the rhamnosine-3-O-alpha-rhamnoside monomer.

Lipopolysaccharide (LPS) and indomethacin (Indo) were purchased from Sigma-aldrich (St. Louis, Mo.). DMEM high-glucose medium, Fetal Bovine Serum (FBS) and penicillin-streptomycin were purchased from Gibco (Gathersburg, Md.). The main rabbit monoclonal antibodies, including NF-. kappa. B p65, phospho-NF-. kappa.B-p 65, Nrf2, NQO1, HO-1, NLRC3 and TRAF6, were obtained from Abcam (Cambridge, UK). Cytokine mouse enzyme-linked immunosorbent assay kit was purchased from Elabsciences (China, Wuhan).

1.2 cell culture and cell viability assays

Leukemia cells of mouse mononuclear macrophages (RAW 264.7, catalog # CL-0190) were obtained from Procell Life Science & Technology Co.Ltd. (Wuhan, China) and incubated in DMEM medium supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin in an incubator at 37 ℃ and 5% CO 2. 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-dithio-benzyl) -2H-tetrazoline sodium salt (WST-8) assay was performed to measure the effect of drug on cell viability. RAW264.7 macrophages (4X 104 cells/mL) were seeded in 96-well plates (100. mu.L/well) and treated with various amounts of ARR (5, 10, 20, 40, 80, 100, 200. mu.g/mL) for 24 hours. Then 10 μ L WST-8 reagent (Sigma-aldrich, st louis, missouri) was added to each well and incubated with the cells for an additional 2 hours. In the presence of an electron coupling agent 1-methoxy PMS, WST-8 is converted into orange yellow water-soluble formazan. The optical density was measured at 450nm using a Thermo Scientific microplate reader (Thermo Fisher Scientific, Shanghai, China).

1.3 Nitric Oxide (NO) determination

For NO analysis, 1X 10 wells were grown in 6-well plates⁶RAW264.7 macrophages. After 24 hours of incubation, LPS was added and incubated with the cells for 2 hours, followed by various amounts of ARR (25, 50, 100. mu.g/mL). After an additional 24 hours, 200 μ L Griess reagent (Sigma-aldrich, usa) and the media mixture were added to the 96-well plate and incubated for 30 minutes at 37 ℃ in the dark. Absorbance was measured at 550 nm using a Thermo Scientific Microplate Reader.

1.4 cytokine assay

Determination of cytokines IL-6, IL-1. beta., PGE in supernatants of macrophages of each group using ELISA kits according to the kit instructions₂. The absorbance was then immediately quantified in each well at 450nm by a Thermo Scientific Microplate Reader.

1.5 real-time quantitative PCR (RT-qPCR)

Total RNA was obtained from RAW264.7 cells using Trizol (TransGen Biotech, beijing, china) and the mass and concentration of the isolated RNA was determined using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, shanghai, china). First strand cDNA was synthesized using RT-qPCR synthesis kit according to the specifications of RT-qPCR synthesis kit. mRNA for IL-6, IL-1. beta., COX-2, iNOS, and GAPDH was analyzed using RT-qPCR kit (PerfectStartTM Green qPCR SuperMix, TransGen Biotech). mRNA expression for IL-6, IL-1. beta., COX-2, iNOS was normalized to GAPDH (as an internal control), and 2 was used^-ΔΔCtThe method performs the calculation. The primers are shown in Table 1.

1.6 immunoblotting

RAW264.7 cells were lysed by using a whole cell lysis assay (KeyGEN Bio TECH, jiangsu, china). Total nuclear and cytoplasmic proteins were extracted using a nuclear and cytoplasmic protein extraction kit (KeyGEN Bio TECH). Protein concentration was measured with BCA protein assay kit (KeyGEN Bio TECH). Then, 20. mu.g of protein in each sample was electrophoretically separated on 10% SDS-PAGE, and then transferred onto a polyvinylidene fluoride (PVDF) membrane. Primary antibodies specifically recognizing NF-. kappa.Bp 65, phospho-NF-. kappa.Bp 65, Nrf2, HO-1, NQO1, beta-actin and histone H3 were diluted with blocking buffer at a ratio of 1:1000, 1: 2000. The membrane was then probed with a horseradish peroxidase-conjugated anti-rabbit probe for 1 hour. After multiple washes with TBST buffer, the protein bands were visualized using Enhanced Chemiluminescence (ECL) solution and a chemiluminescence imaging system (Bio-Rad Laboratories, Inc.) and analyzed using Image Lab software (version 6.0, Bio-Rad Laboratories, Inc.).

1.7 immunofluorescence microscope

RAW264.7 cells were seeded onto glass coverslips placed on the bottom of a 6-well plate and fixed with 4% paraformaldehyde, permeabilized with 0.1% Trinton X-100, and blocked with 10% goat serum. The cells were then incubated with rabbit specific NF-. kappa. B p65 antibody (1: 1000 dilution) and goat anti-rabbit secondary IgG (H + L) (CY 3 conjugated) antibody (1: 5000 dilution). After staining the nuclei with diamino-2-phenylindole (DAPI, Pierce), a drop of an anti-fluorescence quenching fixative was added and imaged by immunofluorescence microscopy. The Image analysis and synthesis were performed using the software Image J (national institute of health, usa).

1.8 immunohistochemical staining

NLRC, TRAF6 and NF-. kappa.Bp 65 antibodies were used as primary antibodies and biotinylated antibodies were used as secondary antibodies, all samples were stained with DAB. A visualized Image was obtained using a fluorescence microscope, and the obtained Image was quantified using Image J (national institute of health).

1.9 statistical analysis

All data are expressed as mean ± standard deviation (s.d.). Statistical analysis was performed using SPSS software (version 26.0, IBM, new york, usa) and GraphPad Prism version 6.0 (GraphPad software, california, san diego). One-way anova was performed to compare various means. P <0.05 and P <0.01 are considered statistically significant and statistically very significant, respectively.

2.Results

2.1 Effect of ARR on cell viability in LPS-stimulated RAW264.7 cells

To investigate the effect of ARR on cell viability, RAW264.7 cells were incubated with 0-200. mu.g/mL ARR for 24 hours. As shown in FIG. 1B, ARR concentrations as high as 200. mu.g/mL were found to have no significant effect on the viability of RAW264.7 cells. According to previous experiments on the anti-inflammatory effect of ARR, concentrations of 25 to 100. mu.g/mL were used in the following experiments.

2.2 Effect of ARR on LPS-stimulated RAW264.7 cellular inflammatory mediators

Since LPS-induced inflammatory response models are widely used in the study of inflammation in anti-inflammatory drugs, we established LPS-induced inflammatory response models in RAW264.7 cells to evaluate the anti-inflammatory effects of ARR. After incubation with LPS, the content of NO increased significantly compared to the control group, however, addition of ARR significantly inhibited LPS-induced NO secretion (fig. 1C). High doses of ARR (100. mu.g/mL) inhibited LPS-induced NO concentrations by more than 90% compared to the LPS group. These results indicate that ARR significantly inhibited NO production in RAW264.7 cells. IL-6, IL-1 beta and PGE₂Is a key inflammatory cytokine for mediating inflammatory reaction, and uses an ELISA kit to detect IL-6, IL-1 beta and PGE in the supernatant of RAW264.7 cells₂The content of (a). As shown in FIGS. 1D-F, LPS significantly upregulated IL-6, IL-1. beta. and PGE compared to the group treated with LPS alone₂And ARR significantly reduced the expression levels of three inflammatory cytokines. Taken together, these data suggest that ARR inhibits IL-6, IL-1 β and PGE₂The release of ARR exerts an inflammatory effect, and in addition, the anti-inflammatory capacity of ARR is dose-dependent, with the best effect at a concentration of 100. mu.g/mL (FIGS. 1D-F).

2.3 Effect of ARR on LPS-stimulated RAW264.7 cellular proinflammatory factor Gene levels

To demonstrate whether the modulation of inflammatory factors by ARR is based on mRNA levels, the expression of various inflammatory factors was further examined using RT-qPCR. As shown in FIGS. 2A-D, mRNA levels of the inflammatory factors iNOS, IL-6, IL-1 β and COX-2 were significantly reduced after ARR addition compared to LPS treatment alone. The most significant ARR relief effect was achieved at 100. mu.g/mL. These results are consistent with ELISA results, indicating that ARR can exert anti-inflammatory effects by inhibiting the expression of many inflammatory factors at the mRNA and protein levels.

2.4 Effect of ARR on NF-. kappa. B p65 translocation in LPS-stimulated RAW264.7 cells

NF-. kappa.B is a well-known transcription factor involved in inflammatory responses, and therefore we examined phosphorylation of p65 and translocation of NF-. kappa. B p65 to the nucleus using Western blotting. LPS significantly increased phosphorylation of NF- κ B p65, while addition of ARR significantly inhibited phosphorylation of NF- κ B p65 in RAW264.7 cells (FIG. 3A). LPS stimulation induces translocation of NF-. kappa. B p65 to the nucleus. However, addition of ARR significantly inhibited NF- κ B p65 nuclear translocation in RAW264.7 macrophages induced by LPS (FIG. 3B). Similar results were observed with immunofluorescence microscopy for nuclear translocation of NF-. kappa. B p 65. As expected, LPS was found to significantly increase NF- κ B p65 nuclear translocation compared to the control group (fig. 4). However, the addition of ARR significantly inhibited nuclear translocation of NF- κ B p65 compared to the LPS alone group (FIG. 4). In total, it is believed that ARR exerts an anti-inflammatory effect by inhibiting NF-. kappa. B p65 translocation.

2.5 Effect of ARR on expression of NLRC3, TRAF6 and NF-. kappa. B p65 proteins in LPS-stimulated RAW264.7 cells

To further investigate the effect of ARR on the NF-. kappa.B pathway, IHC staining was performed to determine the expression of NLRC3, TRAF6 and NF-. kappa.Bp 65 in RAW264.7 cells. LPS decreased NLRC3 while increased TRAF6 and NF- κ Bp65 expression (FIG. 5). However, ARR significantly improved the expression of NLRC3, while the expression of TRAF6 and NF-. kappa.Bp 65 was significantly blocked by ARR, as compared with the LPS-alone treated group. These data indicate that ARR significantly enhances activation of NLRC3 in the inflammatory response, reducing the levels of TRAF6 and NF- κ Bp 65.

2.6 Effect of ARR on the Nrf2 pathway in LPS-stimulated RAW264.7 cells

We also determined the effect of ARR on the Nrf2 pathway in RAW264.7 cells stimulated by LPS, and the results showed that ARR significantly induced the expression levels of Nrf2 protein and its target molecules HO-1 and NQO1 compared to the control and treatment groups with LPS alone. (FIGS. 3D-3F). The results indicate that ARR may also exert anti-inflammatory effects via the Nrf2 pathway.

3. Discussion of the related Art

Inflammation is a natural reaction process of host defense and can be classified into acute and chronic depending on the length of the disease process. Acute inflammation is mainly manifested by redness, swelling, pain, etc. Chronic inflammation is caused by the persistent presence of inflammatory factors and damage to tissues, which manifest as local tissue degeneration, exudation, and proliferation. ARR is a flavonoid compound extracted from Taxus brevifolia, and the influence of ARR on inflammation and its underlying mechanism is not studied at present.

When macrophages are activated, they produce a number of inflammatory cytokines that cause inflammation. It is well known that LPS is a macrophage stimulator which causes macrophages to secrete proinflammatory factors including NO, PGE₂IL-6 and IL-1 beta. Therefore, we have established a model of LPS-induced inflammatory responses in the study to evaluate the anti-inflammatory effect of ARR on RAW264.7 cells. Exposure to high levels of NO can cause an innate immune response and result in tissue disruption or cell damage. The cytokines IL-6 and IL-1 β cause tissue damage and play an important role in mediating various inflammatory diseases. In this study, we found that ARR significantly inhibited the secretion of the proinflammatory factors IL-6 and IL-1 β.

The inflammatory response is accompanied by systemic activation of many signaling pathways. NF-. kappa.B plays a key role in the inflammatory response because it promotes the release of pro-inflammatory cytokines, and p65 translocation plays a key role in the activation of NF-. kappa.B, which has also been shown to be the major signaling pathway for LPS to induce macrophage inflammation. Inhibition of NF-. kappa.B activation has been recognized as an anti-inflammatory strategy in the treatment of inflammation. NF-. kappa.B regulates the expression of iNOS, COX-2 and other related inflammatory genes by activating transcription. NO is iNOS combined free gasThe body signaling molecule, and the iNOS-mediated excess of NO, causes an inflammatory response. COX-2 is known to produce prostaglandins (e.g., PGE), a pro-inflammatory substance₂) And cause inflammation. A variety of natural compounds including flavonoids, quercetin, genistein and kaempferol have been identified as natural COX-2 inhibitors. Min-Seon Kim demonstrated that formononetin-7-O-phosphate inhibits COX-2 by inhibiting NF-. kappa.B nuclear translocation.

We also explored whether ARR exerts anti-inflammatory effects through NF-. kappa.B signaling pathway. As expected, our results indicate that ARR not only inhibits the expression of iNOS and COX-2 mRNA, but also reduces NO and PGE in a dose-dependent manner₂The content of (a). In addition, ARR significantly inhibited the expression of NF- κ B p 65. To our knowledge, this is the first evidence that ARR can inhibit NF-. kappa. B p65 expression via the NF-. kappa.B pathway by LPS-induced RAW264.7 cells.

NLRC3 acts as a checkpoint to prevent inflammatory disorders. Upon stimulation of RAW264.7 cells with LPS, the NLRC3 molecule acts as a deubiquitinase to remove TRAF6 ubiquitination and inhibit nuclear translocation of the NF- κ B p65 subunit, thereby reducing release of IL-1 β. According to our findings, ARR increased the expression of NLRC3, thereby inhibiting the activation of NF-. kappa.B pathway.

The Nrf2 signaling pathway is another important regulator of inflammation. Activation of Nrf2 and its target molecules such as HO-1 and NQO1 are considered to be intracellular protective modulators of oxidative stress and inflammatory responses. Activation of Nrf2 can disrupt cross-talk between NF- κ B and the target molecule, thereby controlling the inflammatory response. In addition, HO-1 and NQO1 inhibit the transcription of inflammatory adhesion molecules mediated by NF-. kappa.B. In this study, our results indicate that ARR induces Nrf2 and inhibits nuclear translocation of NF- κ B. Therefore, it is believed that ARR-induced Nrf2 activation would prevent NF- κ B-mediated increase in inflammatory cytokines.

In conclusion, our studies indicate that rhamnosin-3-O- α -rhamnoside (ARR) exerts an anti-inflammatory effect in LPS-stimulated RAW264.7 cells at least in part by modulating NF- κ B and Nrf2 mediated inflammatory responses, and can be used as an active ingredient in the preparation of broad-spectrum anti-inflammatory drugs. The rhamnosine-3-O-alpha-rhamnoside ARR is water soluble, and the preparation can be injection. Of course, other medically acceptable auxiliary materials can be added to prepare the preparation formulations such as tablets, granules and the like.