Application of aurantiamide alcohol ester in preparation of immunosuppressant

Abstract

The invention provides application of aurantiamide alcohol ester in preparation of an immunosuppressant, and relates to the field of biological medicine. The structural formula of the golden amide alcohol ester is as shown in formula I: (I). Researches show that the golden amide alcohol ester compound has the advantages of remarkably inhibiting ConA-induced T cell proliferation and being low in cytotoxicity, can remarkably reduce release of T cell activation related cytokines IFN-gamma and IL-6, shows concentration dependence, and can be well used for preparing immunosuppressive drugs for organ transplantation and autoimmune diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of golden amide alcohol ester in the preparation of immunosuppressants. Background Art

[0002] Immunosuppressive drugs are drugs that inhibit the body's immune response and are used to treat autoimmune diseases. They can inhibit the proliferation and function of cells related to the immune response and reduce the body's immune response.

[0003] The main immunosuppressive drugs used clinically to treat autoimmune diseases include cyclosporine A and tacrolimus. Although these drugs have some clinical efficacy, they can also cause varying degrees of neurotoxicity and hepatotoxicity. Long-term use can easily lead to hyperlipidemia and metabolic bone disease. Furthermore, these immunosuppressive drugs are often expensive. Therefore, the development of low-toxic, effective, and affordable immunosuppressive drugs is of great significance. Summary of the Invention

[0004] In view of this, the present invention proposes the use of golden amide alcohol ester in the preparation of immunosuppressants, especially the use of golden amide alcohol ester in the preparation of immunosuppressants.

[0005] Golden amide alcohol ester was first isolated from plants. Studies have found that golden amide alcohol ester has anti-inflammatory activity, antioxidant activity, and anti-influenza virus effects, but there have been no research results on its use as an immunosuppressive drug.

[0006] The technical solution of the present invention is achieved as follows: Golden amide alcohol ester is used in the preparation of immunosuppressants.

[0007] The structural formula of the golden amide alcohol ester is shown in Formula I: (I).

[0008] Furthermore, the golden amide alcohol ester is used in the preparation of an immunosuppressant for inhibiting Con A-induced splenocyte proliferation.

[0009] Furthermore, the golden amide alcohol ester is used in the preparation of an immunosuppressant for inhibiting the content of IFN-γ or IL-6.

[0010] Furthermore, the golden amide alcohol ester is used in the preparation of an immunosuppressant for inhibiting the content of IFN-γ and IL-6.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The research of the present invention proves that golden amide alcohol ester has the advantages of significantly inhibiting ConA-induced T cell proliferation and low cytotoxicity. It can significantly reduce the release of T cell activation-related cytokines IFN-γ and IL-6, showing concentration dependence. It can be well used to prepare immunosuppressive drugs for organ transplantation and autoimmune diseases. It is also low in price and can be better used in the preparation of immunosuppressive drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The cytotoxic effect of LJ-43 on mouse spleen cells; Figure 2 LJ-43 inhibits Con A-induced splenocyte proliferation; Figure 3 Effects of LJ-43 on IFN-γ and IL-6 in cell supernatants; In the figure, Splenocyte viability means in Chinese: spleen cell activity; Concentration Chinese meaning: concentration; Cell viability Chinese meaning: cell viability; Con A Chinese meaning: Concanavalin A; production Chinese meaning: output. DETAILED DESCRIPTION

[0013] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0014] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0015] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0016] The golden amide alcohol ester used in the embodiment of the present invention is a commercially available product, and the structural formula of the golden amide alcohol ester is as follows:

[0017] Example 1. Study on the immunosuppressive activity of LJ-43 on mouse spleen cells Animal materials: Female BLAB / c mice aged 6-8 weeks were housed under SPF conditions.

[0018] Experimental reagents: RPMI1640 culture medium (Punosai), fetal bovine serum (Punosai), CCK-8 reagent (Biosharp), LPS (Biosharp), DMSO (Biosharp), concanavalin A (Sigma), erythrocyte lysate (Biosharp), IL-6 ELISA kit (Shanghai Fusheng), IFN-γ ELISA kit (Shanghai Fusheng), golden amide alcohol ester (code name LJ-43), etc.

[0019] 1.1 Obtaining mouse spleen cells Prepare a dissection box, 5 mL syringe, and other equipment in a clean bench. After killing the mouse by cervical dislocation, immerse it in alcohol for disinfection, spray it with alcohol, and move it to the workbench. A small incision was made in the middle of the left ventral side of the mouse to expose the abdominal wall. The peritoneum was lifted, and the spleen was removed and placed in a 15 mL centrifuge tube containing 5 mL of culture medium. The spleen was placed on a 200-mesh stainless steel mesh and cut into several sections. The spleen tissue was gently ground with a syringe needle, while the mesh was rinsed with culture medium to obtain a spleen cell suspension. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded, and red blood cell lysis buffer was added for 5 minutes, followed by another centrifugation for 5 minutes. Then, 4 mL of RPMI1640 complete culture medium was added to prepare a cell suspension. The cells were counted and the cell density was adjusted to 1 × 10^3. 7 pieces / mL.

[0020] 1.2 LJ-43 toxicity test on mouse spleen cells 96-well plates were used for cell culture, and 100 μL of 1×10^ 7 A mouse spleen cell suspension with a density of cells / mL was cultured overnight at 37°C and 5% CO2 to allow the cells to reach a stable state. Subsequently, LJ-43 or CsA solution diluted in a complete culture medium was added to each well to a final concentration of 1, 5, 10, 15, 20, 40, 60 and 80 μM. The control group only added complete culture medium, and 3 parallel wells were set up for each treatment. After 72 hours of culture, 20 μL of CCK-8 solution was injected into each well and incubated for another 4 hours. Finally, the absorbance value at 450 nm was measured using a microplate reader to calculate the cell survival rate. The calculation formula is: Cell viability = OD 样品- OD 样品空白 / OD 空白对照组 ×100%.

[0021] Effect of LJ-43 on Con A-induced immunosuppressive activity of mouse splenocytes The experiment was performed using a 96-well plate. The cell density was adjusted to 1×10^7 cells / ml of suspension, 100 μl was added to each well, and the cells were cultured at 37°C and 5% CO2. The experiment was divided into several groups: the blank control group was added with 200 μl of RPMI-1640 complete medium; the negative control group consisted of 100 μl of cell suspension and 100 μl of RPMI-1640 complete medium; the Con A positive control group contained 100 μl of cell suspension and 100 μl of RPMI-1640 complete medium containing Con A (final concentration of 5 μg / ml); the drug control group was configured with 100 μl of RPMI-1640 complete medium, 50 μl of Con A (final concentration of 5 μg / ml) and 50 μl of different concentrations of LJ-43; the drug experimental group consisted of 100 μl of cell suspension, 50 μl of RPMI-1640 complete medium containing Con A (final concentration of 5 μg / ml) and 50 μl of LJ-43 (final concentrations of 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, and 40 μM, respectively). Three replicate wells were set up for each group. After incubation at 37°C, 5% CO₂ for 72 hours, 20 μl of CCK-8 reagent was added to each well and incubated in the dark for 4 hours. Finally, the absorbance of each well was measured at 450 nm using a microplate reader.

[0022] ConA inhibition rate % = 1-(OD 样品- OD 样品空白 / OD ConA对照组 -OD 空白对照组 )×100%.

[0023] 1.4. ELISA detection of cytokines in cell supernatants Prepare the spleen cell suspension using the same procedure as in step 1.1, adjusting the cell density to 1 × 10^7 cells / mL. Inoculate 1 ml of the cell suspension into a 6-well plate and incubate overnight at 37°C in a 5% CO2 incubator.

[0024] The experimental groups and treatments were as follows: negative control wells were supplemented with 1 mL of RPMI-1640 complete medium; Con A positive control wells were supplemented with 1 mL of RPMI-1640 complete medium containing 5 μg / mL Con A; the CsA experimental group received 1 mL of RPMI-1640 complete medium containing 5 μg / mL Con A and 5 μM CsA; and the drug experimental groups received 1 mL of RPMI-1640 complete medium containing 5 μg / mL Con A and different concentrations of LJ-43 (7.5 μM, 12.5 μM, and 17.5 μM). All treatments were incubated at 37°C, 5% CO₂ for 48 hours.

[0025] Collect the supernatant using EP tubes and centrifuge at 2000 rpm for 5 minutes. The supernatant was aspirated and diluted according to the instructions for each cytokine standard. Sample processing was as follows: A blank control well was set up, with 50 μL of sample diluent added alone, without sample or enzyme-labeled reagent. 50 μL of standard solution was added to the standard wells. 40 μL of sample diluent and 10 μL of the test sample were added to each well. After incubation at 37°C in the dark for 30 minutes, the wells were discarded, the plates were shaken dry, and the wells were washed five times with diluted wash buffer. 50 μL of enzyme-labeled reagent was added to each well except the blank well. The wells were incubated at 37°C in the dark for 30 minutes, shaken dry, and washed again five times. Subsequently, 50 μL of chromogen A and 50 μL of chromogen B were added to each well, incubated at 37°C in the dark for 10 minutes, and the reaction was terminated by adding 50 μL of stop solution. The blank well was used as the zero setting, and the absorbance of each well was measured at 450 nm.

[0026] 2. Results and Analysis like Figure 1 As shown, the IC value of compound LJ-43 on mouse spleen lymphocyte cytotoxicity is 50 =31.01±1.49 μM, indicating that LJ-43 has low toxicity to spleen cells within a certain concentration range.

[0027] like Figure 2 As shown in the Con A-induced spleen cell proliferation experiment, all experimental groups used Con A at a final concentration of 5 μg / ml to induce mouse spleen cell proliferation. LJ-43 had a certain inhibitory effect on Con A-induced spleen lymphocytes. The higher the LJ-43 concentration, the lower the cell survival rate, showing a concentration-dependent effect. The half-inhibitory concentration of LJ-43 on Con A-induced spleen lymphocytes is IC 50 =11.87±1.07 μM.

[0028] like Figure 3 As shown in the figure, after mouse spleen cells were induced by Con A, the cells were rapidly activated, and the levels of IFN-γ and IL-6 increased significantly. Different concentrations of LJ-43 and positive control (CsA) were added to treat the cells. The results showed that LJ-43 could significantly reduce the levels of IFN-γ and IL-6. The higher the concentration, the greater the inhibitory effect, showing a concentration-dependent effect.

[0029] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the scope of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of golden amide alcohol ester in the preparation of immunosuppressants.

2. The use according to claim 1, characterized in that The structural formula of the golden amide alcohol ester is shown in Formula I: （I）。 3. The use according to claim 1, characterized in that The golden amide alcohol ester is used in the preparation of an immunosuppressant for inhibiting Con A-induced splenocyte proliferation.

4. The use according to claim 1, characterized in that The golden amide alcohol ester is used in the preparation of an immunosuppressant for inhibiting the content of IFN-γ or IL-6.

5. The use according to claim 1, characterized in that The golden amide alcohol ester is used in the preparation of immunosuppressants for inhibiting the content of IFN-γ and IL-6.

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