Application of salvianolic acid B in preparation of anti-inflammatory and anti-radiation drugs

Abstract

The invention relates to application of salvianolic acid B in preparation of anti-inflammatory and anti-radiation drugs. Experimental verification shows that within a safe concentration range, salvianolic acid B can effectively inhibit the release amount of NO in RAW264.7 cells and the expression level of proinflammatory factors TNF-alpha, IL-6, IL-1beta and IFN-gamma in the RAW264.7 cells under the co-stimulation of LPS and radiation, and meanwhile, the phagocytic ability of the cells is improved; the salvianolic acid B remarkably inhibits the H2O2 expression level of RLE-6TN cells and relieves the early apoptosis of the RLE-6TN cells, and the results show that the salvianolic acid B can be used for preparing the anti-inflammatory and anti-radiation drugs. Compared with a traditional clinical medicine, the salvianolic acid B has the advantages of low toxicity and low side effect as a natural traditional Chinese medicine product.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine, and in particular to application of salvianolic acid B in the preparation of anti-inflammatory and anti-radiation drugs. Background Art

[0002] Ionizing radiation (IR) is radiation or ion beam that can cause atoms or molecules to lose electrons and produce ions. It has the characteristics of high energy and strong penetration. It is widely used in medicine, health safety, environmental protection and industry. The number of occupational personnel exposed to ionizing radiation for a long time is also gradually increasing. Radiation sickness can cause systemic diseases such as hematopoietic system damage and other systemic lesions. The sensitivity of various tissues in the body to radiation varies greatly, which is positively correlated with the complexity of their structure. Among them, the lung, as one of the moderately sensitive tissues to radiation, can show early radiation damage within hours to days after radiation exposure. Radiation exposure causes DNA damage and reactive oxygen production in lung tissue, leading to damage and apoptosis of epithelial and endothelial cells, further causing changes in vascular permeability and uncontrolled inflammatory response, causing acute lung injury, and then forming radiation pneumonia. Macrophages are one of the most important immune cells in the body's innate immune system and play an important role in the body's resistance to viral infection. These cells can remove pathogens and respond to microbial antigens by producing inflammatory mediators. At the same time, the main task of phagocytes is to effectively identify foreign matter and apoptotic cell fragments, and to engulf and digest them as quickly and effectively as possible. As pro-inflammatory cytokines, IL-6, IL-1β and IFN-γ are crucial to the innate immune response in the body's cells; because IL-6, IL-1β and IFN-γ can stimulate macrophages, thus responding quickly to the second signal, activating the body's anti-tumor and microbial elimination activities related to immunity such as viruses. In the body, NO participates in platelet regulation and immune system responses. When expressed in large quantities, it will cause vasodilation, increased exudates such as plasma and leukocytes, and damage to blood vessels and tissues. Therefore, the content of NO can be used as a standard for assessing the severity of inflammation; within a certain range, the secretion level of NO can reflect the body's immune stress efficiency against exogenous stimuli and pathogens and cell fragments in the body. The H2O2 produced by the body under stress can directly oxidize proteins and lipids on the cell membrane, and can also freely penetrate the cell membrane and react with intracellular iron ions to obtain free radicals, thereby inducing cell apoptosis and tissue damage. It can induce oxidative stress in cells in the body, resulting in decreased cell viability, increased cell apoptosis, and serious damage to cells in the body.

[0003] In clinical practice, glucocorticoids are mostly used for drug treatment of inflammation combined with radiation stimulation. They have significant anti-inflammatory and immunosuppressive effects, but they are also accompanied by some side effects. For example, long-term use of dexamethasone in clinical practice can easily cause water and sodium retention in patients, damage the digestive system, induce or aggravate infection, cause adrenal cortex insufficiency, and endocrine and metabolic system disorders. Since the side effects of glucocorticoids are relatively extensive and serious, involving multiple systems, it is urgent to find specific, low-toxic and highly effective drugs for inflammation combined with radiation stimulation. Compared with traditional clinical drugs, the advantages of low toxicity and low side effects of natural products of traditional Chinese medicine are gradually being recognized and valued. Therefore, it is of great significance to explore natural products of traditional Chinese medicine that can systematically treat inflammation combined with radiation stimulation.

[0004] Salvia miltiorrhiza Bge. is a traditional Chinese medicine. The pharmacopoeia records that the dried roots and rhizomes of Salvia miltiorrhiza Bge. are used as medicine. It has the effects of promoting blood circulation and removing blood stasis, relieving pain, clearing the heart and eliminating troubles, cooling blood and eliminating carbuncle. Salvianolic acid compounds are one of the key indicators of Salvia miltiorrhiza quality control and one of the material bases of its efficacy. Among them, salvianolic acid B is the compound with the highest content and the best biological activity among salvianolic acids. Studies have shown that salvianolic acid B can reduce the infiltration of lymphocytes and neutrophils in lung tissue, and protect acute lung injury by inhibiting cell apoptosis and resisting oxidative stress. In addition, salvianolic acid B can stimulate the proliferation of vascular endothelial cells, enhance cell migration, and reduce cell apoptosis, playing an important role in promoting angiogenesis, accelerating blood circulation and hemostasis. Anti-radiation related studies have shown that salvianolic acid B can protect immune organs, enhance hematopoietic function, and protect the body from radiation damage. Common dosage forms on the market include lyophilized powder of Danshen, salvianolic acid capsules, salvianolic acid B injection, and compound preparations containing salvianolic acid B. The excipients of these compound preparations will depend on the specific drug combination and preparation process, and may include a variety of different excipients to meet the specific needs of the preparation, such as the addition of stabilizers (such as sodium EDTA) and pH regulators (such as hydrochloric acid or sodium hydroxide), which can ensure the stability and safety of the drug. However, the therapeutic effect of salvianolic acid B in inflammation combined with radiation stimulation has not yet been reported. Summary of the invention

[0005] In order to overcome at least one problem existing in the prior art, the present invention provides the use of salvianolic acid B in the preparation of anti-inflammatory and anti-radiation drugs. Experiments have confirmed that salvianolic acid B, a natural product derived from Salvia miltiorrhiza, has significant anti-inflammatory and anti-radiation effects.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The present invention provides the use of salvianolic acid B, an active component of salvia miltiorrhiza, in the preparation of anti-inflammatory and anti-radiation drugs; the drug is preferably used for the preparation of drugs for preventing and / or treating inflammation combined with radiation stimulation.

[0008] Furthermore, the salvianolic acid B is a commercially available product, with CAS No.: 115939-25-8, and its chemical structure is as follows:

[0009]

[0010] Furthermore, in the drug, for RAW264.7 cells, the concentration of the salvianolic acid B is 2-200 μM. Salvianolic acid B has no cytotoxicity to RAW264.7 cells at 2-200 μM, and its preferred concentration is 200 μM.

[0011] Furthermore, the salvianolic acid B is used to prepare a drug for alleviating the stress response of RAW264.7 cells under co-stimulation of lipopolysaccharide and radiation.

[0012] Furthermore, the relief of the stress response of RAW264.7 cells under the co-stimulation of lipopolysaccharide and radiation includes: inhibiting the release of NO, an inflammatory mediator of RAW264.7 cells, enhancing the phagocytic ability of macrophages, and reducing the expression level of pro-inflammatory factors in RAW264.7 cells.

[0013] Furthermore, the pro-inflammatory factors include IL-6, IL-1β, TNF-α and IFN-γ.

[0014] Furthermore, for RAW264.7 cells, the radiation is 10 Gy ionizing radiation.

[0015] Specifically, the salvianolic acid B can effectively inhibit the release of the inflammatory mediator NO under LPS combined with radiation stimulation in a dose-dependent manner in the range of 2 to 200 μM; the salvianolic acid B helps to improve the ability of macrophages stimulated by LPS combined with radiation to phagocytize FITC-labeled Escherichia coli, thereby activating immune stress and exerting an anti-inflammatory effect; the salvianolic acid B can significantly reduce the expression levels of pro-inflammatory factors TNF-α, IL-6, IL-1β, and IFN-γ in RAW264.7 cells after LPS combined with radiation stimulation.

[0016] Furthermore, in the drug, for RLE-6TN cells, the concentration of the salvianolic acid B is 1-25 μM. The salvianolic acid B has no cytotoxicity to RLE-6TN at 1-25 μM, and its preferred concentration is 25 μM.

[0017] Furthermore, the salvianolic acid B is used to prepare a drug for alleviating the stress response of RLE-6TN cells under co-stimulation of lipopolysaccharide and radiation.

[0018] Furthermore, the method of alleviating the stress response of RLE-6TN cells under the co-stimulation of lipopolysaccharide and radiation includes: inhibiting the expression of peroxide H2O2 in RLE-6TN cells and inhibiting the early apoptosis of RLE-6TN cells.

[0019] Furthermore, for RLE-6TN cells, the radiation is 18 Gy ionizing radiation.

[0020] Specifically, the salvianolic acid B, within the range of 1 to 25 μM, effectively inhibits the expression of peroxide H2O2 in RLE-6TN cells after LPS combined with radiation stimulation in a dose-dependent manner; the salvianolic acid B at 25 μM can significantly alleviate the apoptosis of RLE-6TN cells after LPS combined with radiation stimulation.

[0021] Furthermore, the active ingredient of the drug includes salvianolic acid B; the drug also includes a pharmaceutically acceptable carrier. The above active ingredient may also include other substances for anti-inflammatory and / or anti-radiation.

[0022] Furthermore, the dosage form of the drug is a solid preparation, a liquid preparation or a semisolid preparation, such as a tablet, a powder, a capsule, a solution, etc.

[0023] The above-mentioned "pharmaceutically acceptable carrier" refers to: one or more compatible solid, semi-solid, liquid or gel fillers, which are suitable for human or animal use and must have sufficient purity and sufficiently low toxicity. "Compatibility" refers to the components in the pharmaceutical composition and the active ingredients of the drug and their mutual blending without significantly reducing the efficacy of the drug. It is understood that the above-mentioned carrier can be a commonly used material in the art, or can be prepared by conventional methods, or can be purchased from the market.

[0024] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0025] The present invention verifies through experiments that within the safe concentration range, salvianolic acid B can effectively inhibit the release of NO in RAW264.7 cells and the expression level of pro-inflammatory factors in RAW264.7 cells under the co-stimulation of LPS and radiation, and improve the cell phagocytic ability; salvianolic acid B significantly inhibits the expression level of H2O2 in RLE-6TN cells and alleviates the early apoptosis of RLE-6TN cells. The above results show that salvianolic acid B can be used to prepare anti-inflammatory and anti-radiation drugs. Compared with traditional clinical drugs (such as dexamethasone), salvianolic acid B as a natural product of traditional Chinese medicine has the advantages of better safety, lower side effects and low toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0027] Figure 1The figure shows the effect of different concentrations of salvianolic acid B on the proliferation of RAW264.7 cells in an example of the present invention (n=3, ±SEM); wherein, A.* compared with the control group; B.* compared with the LPS combined with radiation co-stimulation group, *P<0.05, ***P<0.001, ****P<0.0001.

[0028] Figure 2 The figure shows the effect of different concentrations of salvianolic acid B on the production of NO in RAW264.7 cells in an example of the present invention (n=3, ±SEM). *Compared with the LPS combined with radiation costimulation group, *P<0.05, ***P<0.001, ****P<0.0001.

[0029] Figure 3 This is a fluorescence microscope image (×400) of RAW264.7 cells engulfing FITC-labeled Escherichia coli under the action of salvianolic acid B in one embodiment of the present invention.

[0030] Figure 4 This is the effect of salvianolic acid B on the mRNA level of IFN-γ in RAW264.7 cells stimulated by LPS in an example of the present invention (n=3, ±SEM). # Compared with the Con group, #### P<0.001; *Compared with the LPS group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0031] Figure 5 This is the effect of salvianolic acid B on the mRNA level of IL-6 in RAW264.7 cells stimulated by LPS in an example of the present invention (n=3, ±SEM), # Compared with the Con group, #### P<0.001; *Compared with the LPS group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0032] Figure 6 This is the effect of salvianolic acid B on the mRNA level of IL-1β in RAW264.7 cells stimulated by LPS in an example of the present invention (n=3, ±SEM). # Compared with the Con group, #### P<0.001; *Compared with the LPS group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0033] Figure 7The effect of salvianolic acid B on the mRNA level of TNF-α in RAW264.7 cells stimulated by LPS in an example of the present invention (n=3, ±SEM), # compared with the Con group, #### P<0.001; *Compared with the LPS group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0034] Figure 8 The figure shows the effect of different concentrations of salvianolic acid B on the proliferation of RLE-6TN cells in one embodiment of the present invention (n=3, ±SEM); wherein, A.* compared with the control group, ****P<0.0001; B.* compared with the LPS combined with radiation co-stimulation group, *P<0.05, ***P<0.001, ****P<0.0001.

[0035] Fig. 9 The figure shows the effects of different concentrations of salvianolic acid B on the production of H2O2 in RLE-6TN cells in an example of the present invention (n=3, ±SEM). *Compared with the LPS combined with radiation costimulation group, ****P<0.0001.

[0036] Fig.10 The figure shows the effects of different concentrations of salvianolic acid B on the apoptosis process of RLE-6TN cells in one example of the present invention (n=3). DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0038] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0039] In the following examples, the experimental cells: mouse mononuclear macrophage cell line RAW264.7 cells and rat alveolar type II cell line RLE-6TN cells were donated by the teaching and research department of the Navy Special Medical Center; 137 The Cs small source radiometer was provided by the Naval Medical Department of Naval Medical University.

[0040] In the following examples, the reagents used include: salvianolic acid B, Shanghai Yuanye Biotechnology Co., Ltd.; fetal bovine serum (FBS), Gibco, USA; sulfonamide, Sigma Aldrich, USA; N-(1-naphthyl)ethylenediamine hydrochloride, Sigma Aldrich, USA; F12 culture medium, Tianjin TBD; DMEM high glucose culture medium, Tianjin TBD; PBS buffer, Tianjin TBD; trypsin, Dalian Meilun; penicillin-streptomycin antibody, Dalian Meilun; CCK-8 kit, Dalian Meilun; Titan One Tube RT-PCR kit, ThermoFisher, USA; TB Premix ExTaqTM, Tli RNaseH plus, RR420A, TaKaRa, Japan; NaOH, Dalian Meilun; 5% Triton X-100, Shanghai Sangon Biotechnology; lipopolysaccharide (LPS), Sigma Aldrich, USA; cell culture dishes, Corning Incorporated, USA; 96-well cell culture plates, ThermoFisher, USA; RIPA lysis buffer, ThermoFisher, USA; FITC-coupled Annexin-V apoptosis detection kit, Guangzhou Dewei Biotechnology Co., Ltd.; 4% paraformaldehyde, Dalian Meilun; fluorescein-5-isothiocyanate (FITC)-labeled Escherichia coli, Gibco, USA.

[0041] In the following examples, the experimental instruments used include: ice machine (AF103AS), Scotsman, USA; double-person single-sided clean bench (SW-CJ-2FD), Suzhou Purification Equipment Factory; CO2 constant temperature incubator (MCO-15AC), Hereus, Germany; inverted phase contrast microscope (DMI3000), Olympus, Japan; ultraspeed refrigerated centrifuge (MIKRO 200), Hereus, Germany; microplate reader (MK3), ThermoFisher, USA; micro-spectrophotometer (Nano-300), Hangzhou Aosheng Company; desktop centrifuge (Centrifuge 5425), Eppendorf, Germany; fluorescence inverted microscope (LSM880), Leica, Germany; ultraviolet spectrophotometer (DU530), Beckman, Germany; laser confocal scanning microscope (TCS SP8CARS), Leica, Germany.

[0042] In the following embodiments, the preparation of the reagents includes:

[0043] 1) 5% Triton X-100: Pipette 600 μL of 30% Triton X-100 and add to 10 mL of distilled water.

[0044] 2) RAW264.7 cell culture medium (DMEM high-glucose medium): Fetal bovine serum (FBS) + double antibodies (penicillin and streptomycin) + DMEM high-glucose blank medium were mixed in a ratio of 1:1:89.

[0045] 3) RLE-6TN cell culture medium (F12 culture medium): Fetal bovine serum (FBS) + double antibody (penicillin and streptomycin) + F12 blank culture medium were mixed in a ratio of 1:1:89.

[0046] 4) Preparation of Salvianolic Acid B mother solution (10 mM): Weigh 4.4 mg of the extract using an electronic precision balance, pipette the corresponding 296.95 mL of sterile water, ultrasonically shake at 37°C until dissolved, filter with a Millipore 0.22 μm sterile syringe filter, dispense into sterile centrifuge tubes, store at -20°C for later use, and mix to the corresponding concentration using DMEM high-glucose complete medium.

[0047] 5) LPS stock solution: 4 ng / mL: Weigh 10 mg of LPS powder and dissolve it in 10 mL of double distilled water. Pass it through a microporous filter to prepare the stock solution. After diluting it 25 times, it can act on cells.

[0048] 6) Griss reagent: premix A and B solutions in equal volumes at a ratio of 1:1 (solution A: weigh 0.1 g of p-aminobenzenesulfonic acid and dissolve it in 10 mL of 30% acetic acid; solution B: weigh 0.04 g of α-naphthylamine naphthylethylenediamine and dissolve it in 10 mL of distilled water, add 300 μL of 80% phosphoric acid, and perform ultrasound at low temperature and in the dark).

[0049] 7) 1 mg / mL 100 μL FITC-labeled E. coli (pHrodoTM E. coli Bioparticles Conjugate): Weigh 1 mg / mL E. coli powder, dissolve it in 1 mL PBS, and wrap it in tin foil to protect it from light.

[0050] In the following examples, the inhibitory effect of salvianolic acid B on the stress response under LPS and radiation co-stimulation was evaluated according to internationally recognized methods. The following examples are merely illustrative of the present invention.

[0051] Example 1- Salvianolic acid B alleviates the stress response of RAW264.7 cells under LPS and radiation co-stimulation

[0052] This example verifies that salvianolic acid B can alleviate the stress response of RAW264.7 cells under LPS and radiation co-stimulation, and specifically includes the following steps:

[0053] (I) CCK-8 method was used to detect the effect of salvianolic acid B on the proliferation of RAW264.7 cells;

[0054] (1) Determine the effect of radiation dose on the proliferation of RAW264.7 cell line;

[0055] RAW264.7 cells were plated at 5 × 10 4 The density of cells was plated in a sterile 96-well plate, 100 μL per well. After the cells were cultured overnight, different doses (0, 2, 4, 6, 8, 10, 15, 20 Gy) of ionizing radiation were given and cultured for 24 h. The cell supernatant was discarded, and 100 μL of DMEM blank medium containing 10% volume of CCK-8 reagent was added to each well, and incubated at 37°C in the dark for 4 h. The absorbance at 450 nm was read on a microplate reader.

[0056] (2) Determine the cytotoxicity of salvianolic acid B on RAW264.7 cell line;

[0057] RAW264.7 cells were plated at 5 × 10 4The density of cells was spread into a sterile 96-well plate, 100 μL per well. After the cells were cultured overnight, the cell supernatant was discarded, and different doses of salvianolic acid B were mixed with DMEM complete medium and cultured for 24 hours according to the gradient concentration (3.125, 6.25, 12.5, 25, 50, 100, 200 μM). 10 μL of CCK-8 reagent was aspirated into each well and the cells were kept in the dark for 4 hours under cell culture. The absorbance at 450 nm was read on an ELISA reader.

[0058] (3) Determine the cytotoxicity of salvianolic acid B on RAW264.7 cell line under LPS combined with radiation stimulation;

[0059] RAW264.7 cells were plated at 5 × 10 4 The density of cells was spread into a sterile 96-well plate, 100 μL per well. After the cells were cultured overnight, 10 Gy of ionizing radiation was given, the cell supernatant was discarded, and LPS with a final concentration of 5 ng / mL was mixed with different doses of salvianolic acid B and cultured for 24 hours according to the gradient concentration (2, 20, 200 μM), and the positive drug dexamethasone 2 μM was given at the same time. 10 μL of CCK-8 reagent was aspirated into each well and the cells were kept in the dark for 4 hours under cell culture. The absorbance at 450 nm was read on an ELISA reader.

[0060] The above experimental results are as follows Figure 1 As shown by Figure 1 Part A shows that 10Gy radiation dose can significantly inhibit the proliferation of RAW264.7 cells; Figure 1 Part B shows that within the administered concentration range (3.125-200 μM), salvianolic acid B does not inhibit the growth of RAW264.7 cells, that is, within this concentration range, salvianolic acid B has no cytotoxicity to RAW264.7 cells; Figure 1 From part C, we can see that after LPS stimulation combined with radiation irradiation, salvianolic acid B at 200 μM promoted the growth of RAW264.7 cells, with no significant difference from the blank group.

[0061] (ii) The Griess reagent method was used to determine the effect of salvianolic acid B on the expression of NO content in the supernatant of RAW264.7;

[0062] NO is an indicator of immune enhancement activity, and its content is usually detected by Griess reagent. RAW264.7 cells were cultured at 5×10 5Inoculate in a 96-well plate, incubate overnight in a cell culture incubator at 37°C with 5% CO2, give 10Gy ionizing radiation, discard the cell supernatant, and give LPS stimulation (5ng / mL) at the same time, and divide into 7 groups according to gradient concentration (blank group; radiation group; radiation and LPS co-stimulation group; dexamethasone group 2μM; tansolic acid B administration group: 2, 20, 200μM) and treat for 24h. Take the supernatant 24h after administration, and use Griess reagent to measure the amount of nitrite. Mix 100μL of cell culture medium with 100μL of Griess reagent and let it stand in a 37°C cell incubator for 30min. Measure the absorbance at a wavelength of 540nm.

[0063] The above experimental results are as follows Figure 2 As shown, compared with the control group, salvianolic acid B at a concentration of 20-200 μM significantly downregulated the secretion of NO in RAW264.7 cells in a dose-dependent manner.

[0064] (III) Effect of salvianolic acid B on phagocytosis of RAW264.7 cells;

[0065] RAW264.7 cells were seeded in a 96-well plate, incubated overnight at 37°C in a cell culture incubator with 5% CO2, given 10Gy ionizing radiation, the cell supernatant was discarded, and LPS stimulation (5ng / mL) was given at the same time, and divided into 7 groups according to gradient concentrations (blank group; LPS group; radiation and LPS co-stimulation group; dexamethasone group 2μM; salvianolic acid B administration group: 8, 40, 200μM) for 24h. The supernatant was then removed, 100μL FITC-labeled Escherichia coli was added to each well and incubated for 2h, and the cells were rinsed twice with PBS preheated to 37°C to rinse off the FITC-labeled Escherichia coli that was not phagocytosed by macrophages, fixed with 4% paraformaldehyde, and photographed under a fluorescence microscope.

[0066] The above experimental results are as follows Figure 3 As shown. Using a fluorescence microscope to observe the fluorescence intensity of FITC-labeled E. coli can prove the phagocytic ability of cells. Compared with untreated RAW264.7 cells, the phagocytic ability of the drug-treated group and the cells treated with LPS for 24 hours on FITC-labeled E. coli was observed, and it was found that the RAW264.7 cells treated with 200μM salvianolic acid B had a higher fluorescence intensity than the LPS group. This comprehensive proof that salvianolic acid B has an enhancing effect on the phagocytic ability of macrophages.

[0067] (IV) Effect of salvianolic acid B on the levels of inflammation-related mRNA in RAW264.7 cells stimulated by LPS and radiation;

[0068] (1) Total RNA extraction: Logarithmic phase cells were collected and 5×10 5Cells were plated in 6-well cell culture plates and incubated at 37°C and 5% CO2 for 24h. Four wells were protected with drugs (2μM dexamethasone group; 2, 20, 200μM salvianolic acid B) for 4h in advance, and the supernatant was removed. The blank group, radiation group (10Gy) and LPS group were set up. The drug was combined with LPS at 5ng / mL. After 24h of incubator culture, the supernatant was discarded, 1mL of pre-cooled PBS was aspirated to each well, and the cells were blown and collected. Centrifuged at 4°C, 1000rpm, 5min, the supernatant was discarded, 200μL RIPA lysis buffer was added, shaken and mixed, and placed on ice for 5min, 4°C, 2000rpm, 10min, the supernatant was taken, and the total RNA was obtained. The detection value at 260nm / 280nm wavelength was measured by Nano, and the RNA concentration of the corresponding group was obtained, and the concentration of each group was adjusted to be consistent.

[0069] (2) Reverse transcription: Reverse transcribe the fresh RNA extracted in the previous step. In this experiment, the TaKaRa cDNA Rapid Kit (PrimeScriptTM RT Master Mix, RR036A) was used. Take 8 tubes and sample according to the following scheme.

[0070] Reverse transcription reaction system:

[0071]

[0072] Flick with your fingertips and then instantly remove from the tube. Set up the PCR instrument according to the following program; store the PCR product at -20℃.

[0073] Reverse transcription reaction procedure:

[0074]

[0075] (3) Real-time fluorescence quantitative PCR (qRT-PCR): The qRT-PCR kit used was TaKaRa (TB Premix Ex TaqTM, Tli RNaseH plus, RR420A), the qRT-PCR amplification instrument used was Applied Biosystems' Quant Studio 3, and the specific operation method is as follows.

[0076] Primer design:

[0077]

[0078] qRT-PCR reaction solution preparation system (reaction solution preparation should be carried out on ice):

[0079]

[0080]

[0081] qRT-PCR reaction conditions:

[0082]

[0083] On the basis of the above experiments, qRT-PCR was used to detect the mRNA levels of intracellular IL-6, IL-1β, TNF-α and IFN-γ, with β-actin as a reference.

[0084] The above experimental results are as follows Figure 4 to Figure 7 As shown, salvianolic acid B can effectively inhibit the mRNA transcription levels of IL-6, IL-1β, TNF-α and IFN-γ, and its inhibition efficiency is highest at high concentrations of salvianolic acid B.

[0085] Example 2- Salvianolic acid B alleviates the stress response of RLE-6TN cells under LPS and radiation co-stimulation

[0086] This example verifies that salvianolic acid B can alleviate the stress response of RLE-6TN cells under LPS and radiation co-stimulation, and specifically includes the steps of:

[0087] (A) CCK-8 method was used to detect the effect of salvianolic acid B on the proliferation of RLE-6TN cells;

[0088] (1) Determine the effect of radiation dose on the proliferation of RLE-6TN cell line;

[0089] RLE-6TN cells were plated at 8×10 4 The density of cells was plated in a sterile 96-well plate, 100 μL per well. After the cells were cultured overnight, different doses (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 Gy) of ionizing radiation were given and cultured for 48 h. The cell supernatant was discarded, and 100 μL of F12 blank culture medium containing 10% volume of CCK-8 reagent was added to each well and incubated at 37°C in the dark for 4 h. The absorbance at 450 nm was read on a microplate reader.

[0090] (2) Determine the cytotoxicity of salvianolic acid B on RLE-6TN cell line;

[0091] RLE-6TN cells were plated at 8×10 4 The density of cells was spread into a sterile 96-well plate, 100 μL per well. After the cells were cultured overnight, the cell supernatant was discarded, and different doses of salvianolic acid B were mixed with F12 complete medium and cultured for 48 hours according to the gradient concentration (3.125, 6.25, 12.5, 25, 50, 100, 200 μM). 10 μL of CCK-8 reagent was aspirated into each well and the cells were kept in the dark for 4 hours under cell culture. The absorbance at 450 nm was read on an ELISA reader.

[0092] (3) Determine the cytotoxicity of salvianolic acid B on RLE-6TN cell line under LPS combined with radiation stimulation;

[0093] RLE-6TN cells were plated at 8×10 4 The density of cells was spread into a sterile 96-well plate, 100 μL per well. After the cells were cultured overnight, ionizing radiation was given, the cell supernatant was discarded, and LPS with a final concentration of 5 ng / mL was mixed with different doses of salvianolic acid B and cultured for 48 hours according to the gradient concentration (1, 5, 25 μM), and the positive drug dexamethasone 2 μM was given at the same time. 10 μL CCK-8 reagent was drawn into each well and the cells were kept in dark for 4 hours under cell culture. The absorbance at 450 nm was read on an ELISA reader.

[0094] The above experimental results are as follows Figure 8 As shown by Figure 8 From the A part, it can be seen that the 18Gy radiation dose can significantly inhibit the proliferation of RLE-6TN cells; Figure 8 From the B part, it can be seen that within the concentration range of administration (1-25 μM), salvianolic acid B does not inhibit the growth of RLE-6TN cells, that is, within this concentration range, salvianolic acid B has no cytotoxicity to RLE-6TN. Figure 8 Part C shows that salvianolic acid B does not inhibit the growth of RLE-6TN cells within the range of 1 to 25 μM.

[0095] (ii) Elisa kit was used to determine the effect of salvianolic acid B on H2O2 expression in the supernatant of RLE-6TN cells stimulated by LPS and radiation;

[0096] RLE-6TN cells were plated at 4 × 10 5 The density of cells was plated in a sterile 6-well plate, 100 μL per well. After the cells were cultured overnight, 18 Gy ionizing radiation was given, the cell supernatant was discarded, and LPS with a final concentration of 5 ng / mL was mixed with different doses of salvianolic acid B and cultured for 48 h according to the gradient concentration (1, 5, 25 μM), and the positive drug dexamethasone 2 μM was given at the same time. The culture fluid was collected, and the culture supernatant was collected according to the instructions of the Elisa kit, and the H2O2 content was measured according to the instructions of the Elisa kit. The absorbance at 450 nm was recorded.

[0097] The above experimental results are as follows Fig. 9 As shown, at concentrations of 1 to 25 μM, salvianolic acid B significantly downregulated the secretion of H2O2 in RLE-6TN cells in a dose-dependent manner.

[0098] (III) Flow cytometry was used to detect the effect of salvianolic acid B on apoptosis of RLE-6TN cells after LPS combined with radiation stimulation;

[0099] RLE-6TN cells were plated at 4 × 10 5 The cells were plated in a sterile 6-well plate at a density of 100 μL per well. After the cells were cultured overnight, 18 Gy ionizing radiation was given, the cell supernatant was discarded, and LPS with a final concentration of 5 ng / mL was mixed with different doses of salvianolic acid B and cultured for 48 hours according to the gradient concentration (6.25, 12.5, 25 μM), and the positive drug dexamethasone 2 μM was given at the same time. The cells were digested with EDTA-free trypsin, and the following steps were completed according to the instructions of the FITC-coupled Annexin-V apoptosis detection kit: dilute the binding buffer according to the ratio of 12 mL of sterile water per 4 mL of 4x binding buffer. Resuspend the cells with 1x binding buffer and adjust the cell concentration to 1×10 6 Pipette 100 μL of cell suspension into a 5 mL flow tube, add 5 μL of Annexin V / FITC and mix, incubate at room temperature in the dark for 5 minutes, then add 10 μL of propidium iodide solution and 400 μL of PBS, mix and test on the flow tube.

[0100] The above experimental results are as follows Fig.10 As shown, salvianolic acid B at 25 μM can effectively inhibit early cell apoptosis (Q3 quadrant).

[0101] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. Application of salvianolic acid B in the preparation of anti-inflammatory and anti-radiation drugs.

2. The use according to claim 1, characterized in that: The inflammation is inflammation caused by lipopolysaccharide, and the radiation is 8-20 Gy ionizing radiation.

3. The use according to claim 1 or 2, characterized in that: In the drug, for RAW264.7 cells, the concentration of salvianolic acid B is 2-200 μM.

4. The use according to claim 3, characterized in that: The salvianolic acid B is used to prepare a drug for alleviating the stress response of RAW264.7 cells under the co-stimulation of lipopolysaccharide and radiation; wherein the alleviating the stress response of RAW264.7 cells under the co-stimulation of lipopolysaccharide and radiation includes: inhibiting the release of NO, an inflammatory mediator of RAW264.7 cells, enhancing the phagocytic ability of macrophages, and reducing the expression level of pro-inflammatory factors in RAW264.7 cells.

5. The use according to claim 4, characterized in that: The pro-inflammatory factors include IL-6, IL-1β, TNF-α and IFN-γ.

6. The use according to claim 3, characterized in that: The radiation is 10 Gy ionizing radiation.

7. The use according to claim 1 or 2, characterized in that: In the drug, for RLE-6TN cells, the concentration of salvianolic acid B is 1 to 25 μM.

8. The use according to claim 7, characterized in that: The salvianolic acid B is used to prepare a drug for alleviating the stress response of RLE-6TN cells under the co-stimulation of lipopolysaccharide and radiation; wherein the alleviating the stress response of RLE-6TN cells under the co-stimulation of lipopolysaccharide and radiation includes: inhibiting the expression of peroxide H2O2 in RLE-6TN cells and inhibiting the early apoptosis of RLE-6TN cells.

9. The use according to claim 7, characterized in that: The radiation is 18Gy ionizing radiation.

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