Use of naringin combined with rapamycin in preparation of medications for treating hyperlipidemia

Abstract

To provide the use of naringin combined with rapamycin in preparation of medications for treating hyperlipidemia.SOLUTION: The present invention relates to the field of medicine, and specifically provides the use of naringin combined with rapamycin in the preparation of medications for treating hyperlipidemia. The naringin achieves anti-inflammatory and lipid-lowering effects by inhibiting the formation of oxLp-NLRP3 complexes. The rapamycin is used to enhance the therapeutic effect of naringin.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to the field of medicine, in particular to the use of naringin in combination with rapamycin in the preparation of a hyperlipidemic drug. [Background technology]

[0002] Hyperlipidemia is an important public health problem and is closely related to the occurrence of various diseases, such as cardiovascular disease, nonalcoholic fatty liver disease, osteoporosis, and tumors. Lipid accumulation and chronic inflammatory responses always coexist and influence each other, accelerating the progression of hyperlipidemia-related diseases, so targeted interventions against lipid accumulation and inflammatory responses are particularly important for the clinical treatment of hyperlipidemia-related diseases. During the progression of hyperlipidemia, oxidized lipoproteins (oxLp), especially oxidized low-density lipoproteins (oxLDL), have long been believed to be the main factor inducing lipid accumulation and inflammatory responses. NOD-like receptor pyrin domain-containing protein 3 (NLRP3), as an intracellular pattern recognition receptor, can sense the accumulation of intracellular oxLp and bind to downstream apoptosis-associated speck-like card protein (ASC), then recruit pro-caspase-1 to aggregate into NLRP3 inflammasome, and the activated NLRP3 inflammasome cleaves pro-caspase-1 to become active caspase-1, thereby promoting the maturation and release of inflammatory factor IL-1β and inducing inflammatory responses. The applicant's preliminary studies have shown that under oxLp stimulation, NLRP3 aggregates and activates on oxLp, causing the formation of oxLp-NLRP3 complex. This stable oxLp-NLRP3 complex can resist autophagy, leading to the continuous accumulation of oxLp and excessive activation of NLRP3, resulting in intracellular lipid accumulation and the continuous occurrence of inflammatory responses. This discovery provided new insights into the targeted treatment of hyperlipidemia-related diseases.

[0003] Therefore, the present invention provides an effective therapeutic strategy for hyperlipidemia-related diseases by intervening in the development of lipid accumulation and inflammatory responses by suppressing the formation of the oxLp-NLRP3 complex. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention aims to address the problems existing in the prior art and to provide a use of naringin in combination with rapamycin in the preparation of a hyperlipidemic drug. [Means for solving the problem]

[0005] Naringin also achieves anti-inflammatory and lipid-lowering effects by inhibiting the formation of the oxLp-NLRP3 complex formed by the aggregation and activation of NOD-like receptor pyrin domain-containing protein 3 on oxidized lipoproteins.

[0006] Furthermore, rapamycin, also known as sirolimus, is used to enhance the therapeutic effect of naringin, and is a macrolide antibiotic immunosuppressant that is clinically used to prevent rejection during organ transplantation and to treat autoimmune diseases. Rapamycin has several dozen times stronger immunosuppressive effect than cyclosporine, which is currently widely used in clinical practice, is less toxic, requires a smaller dosage (2 mg / day / person), and has a synergistic immunosuppressive effect with cyclosporine, so it is used in combination with cyclosporine. Compared to cyclosporine and FK506 (tacrolimus), sirolimus is the immunosuppressant with the lowest nephrotoxicity and no neurotoxicity.

[0007] Furthermore, the anti-inflammatory effects include activation of the NLRP3 inflammasome and the downstream release of the pro-inflammatory cytokine IL-1β.

[0008] In one embodiment of the present invention, treatment with rapamycin alone does not significantly affect the regulation of lipid accumulation and inflammatory responses in hyperlipidemic mice, but when used in combination with naringin, it significantly attenuates lipid accumulation and inflammatory responses.

[0009] Based on the technical concept of the above invention, the present invention also provides a combination drug for treating hyperlipidemia, which contains naringin and rapamycin as active ingredients.

[0010] Additionally, the naringin and rapamycin are administered simultaneously or sequentially.

[0011] In addition, the formulation also includes a pharma- ceutically acceptable carrier.

[0012] The pharma- ceutically acceptable carrier may include conventional diluents (e.g., at least one of water for injection, microcrystalline cellulose, etc.), fillers (e.g., at least one of mannitol, sucrose, lactose, polyethylene glycol, Tween 80, sorbitol, menthol, liquid paraffin, petrolatum, stearic acid, glyceryl monostearate, lanolin, mineral oil, DMSO, etc.), binders (e.g., at least one of carbomer, gum arabic, starch, cellulose, gelatin, polyvinylpyrrolidone, polyacrylamide, etc.), disintegrants (e.g., at least one of sodium carboxymethyl starch, croscarmellose sodium, hypromellose, low-substituted hydroxypropyl cellulose, etc.), lubricants (e.g., talc, magnesium stearate, calcium stearate, solid Examples of the adjuvants include at least one of polyethylene glycol, lecithin, silicon dioxide, and finely powdered silica, wetting agents (e.g., at least one of propylene glycol, glycerin, and ethanol), stabilizers (e.g., at least one of disodium ethylenediaminetetraacetate, sodium thiosulfate, sodium metabisulfite, sodium sulfite, sodium hydrogensulfite, ethanolamine, sodium bicarbonate, sodium acetate, nicotinamide, and vitamin C), osmotic pressure regulators (e.g., at least one of sodium chloride and glucose), pH regulators (e.g., at least one of triethanolamine, sodium hydroxide, and trisodium citrate), and preservatives (e.g., at least one of chlorobutanol, methylisothiazolinone, ethyl hydroxybenzoate, and benzalkonium bromide). The above adjuvants can be mixed with naringin and rapamycin in a commonly used dosage and in a commonly used mixing ratio, and after the dosages of naringin and rapamycin are determined, the mixing ratio between each pharmaceutical adjuvant can be appropriately adjusted as necessary.

[0013] The dosage form of the combination agent may be an oral dosage form or a parenteral dosage form.

[0014] Moreover, the oral dosage form is specifically granules, tablets, capsules, pills, drops or oral liquid.

[0015] Moreover, said parenteral dosage forms are specifically injectable dosage forms.

[0016] In a specific embodiment of the invention, naringin is administered in the form of an injectable formulation and rapamycin is administered in the form of a capsule, with naringin being injected and active rapamycin being added to the diet at the same time. Effect of the Invention

[0017] The present invention has the following advantageous effects: The research results of the present invention demonstrate that naringin can inhibit the formation of oxLp-NLRP3 complex by neutralizing oxLp, and effectively intervene in the early stage of hyperlipidemia. The inhibition of oxLp-NLRP3 complex by naringin helps to unlock the therapeutic potential of rapamycin in hyperlipidemia, providing a combination strategy for the prevention and treatment of hyperlipidemia-related diseases in clinical practice. At the same time, this oxLp-directed therapeutic strategy can avoid the interference of the protective immune function of NLRP3, and also provide a theoretical basis for the prevention and treatment of other types of diseases related to oxLp. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows the results of fluorescence imaging of the formation of oxLDL-NLRP3 complexes (A) and oxHDL-NLRP3 complexes (B) after naringin treatment. [Diagram 2] FIG. 1 shows the ratio of oxLDL-NLRP3 complexes and oxHDL-NLRP3 complexes in THP-1 cells. [Diagram 3] FIG. 1 shows the IL-1β release level (A) and intracellular cholesterol accumulation level (B) of THP-1 cells treated with naringin. [Figure 4] FIG. 1 is a schematic diagram showing the mechanism of action of the naringin-rapamycin combination in the treatment of hyperlipidemia. [Diagram 5] FIG. 1 is a schematic diagram showing mouse hyperlipidemia model construction and drug intervention process. [Figure 6] FIG. 1 shows the results of detection of IL-1β in mouse serum after naringin-rapamycin combination treatment. [Figure 7] FIG. 1 shows the results of mouse arterial plaque, aortic root and liver lesions after naringin-rapamycin combination treatment. [Figure 8] FIG. 1 shows the percentage of arterial plaque area stained with ORO relative to the total surface area of ​​the aortic arch. [Figure 9] FIG. 1 shows the percentage of aortic root lesion area stained with ORO relative to the total sampled area of ​​the aortic root. [Figure 10] FIG. 1 shows the percentage of lipid accumulation in liver stained with ORO relative to the total sampled area of ​​the liver. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention will be described in detail below with reference to the drawings and specific examples, but should not be construed as being limited to the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means commonly known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0020] In the preliminary study of the present invention, we found that abnormally distributed oxLp in cells, as an endogenous ligand, triggers the recruitment and activation of NLRP3, and forms a stable complex with activated NLRP3, promoting the continuous accumulation of oxidized lipids and the excessive activation of NLRP3 inflammasomes. This unregulated process of lipid accumulation and inflammatory response caused by the oxLp-NLRP3 complex accelerates the progression of hyperlipidemia. Considering the role of the oxLp-NLRP3 complex in the progression of hyperlipidemia, we used drug screening techniques to search for potential compounds among 200 natural products with the aim of neutralizing oxLp. As a result, we showed that Naringin has high affinity for oxidized low-density lipoprotein (oxLDL) and oxidized high-density lipoprotein (oxHDL) and can neutralize the negative charges on the surface of oxLDL and oxHDL, so we selected Naringin for further research.

[0021] (Example 1: Inhibitory effect of naringin on the formation of oxLp-NLRP3 complex in THP-1 cells) Cell culture and treatment: THP-1 cells (from American Type Culture Collection, Manassas, VA) were cultured in RPMI 1640 and pretreated with naringin (Sigma, 91842; 100 μM) for 1 h, then cultured in serum-free medium containing oxLDL and oxHDL (50 μg / mL) for 3 h, and maintained at 37°C and 5% CO2.

[0022] Measurement of intracellular cholesterol: It was measured with a commercially available intracellular cholesterol assay kit according to the manufacturer's instructions.

[0023] Measurement of IL-1β: IL-1β was measured using a commercially available IL-1β kit according to the manufacturer's instructions.

[0024] Results: In THP-1 cells, pretreatment with naringin suppressed the formation of complexes between oxLDL or oxHDL and NLRP3 (Fig. 1, Fig. 2), and naringin pretreated cells simultaneously reduced oxLDL- or oxHDL-induced IL-1β release and cholesterol accumulation (Fig. 3).

[0025] Example 2: Treatment of Hyperlipidemia with Naringin in Combination with Rapamycin (RAPA) To evaluate the synergistic therapeutic effect of naringin and rapamycin on hyperlipidemic mice (Figure 4), Ldrl ― / ― A hyperlipidemic model was established using C57BL / 6J mice. ― / ― Mice were intraperitoneally injected with naringin (20 mg / kg) daily while being fed a high-fat diet (HFD) for 16 weeks starting from the 4th week of age, and active capsule rapamycin (RAPA, 40 mg / kg) was also added to the diet, and the intervention process is shown in Figure 5. After that, mouse serum, carotid artery, liver and aortic root tissues were collected for assay and analysis.

[0026] Assessment of carotid artery, aortic root, and liver tissue lesions: Mouse carotid arteries, hearts, and livers were dissected and fixed overnight in 4% PFA. Carotid arteries were gently removed of adhering (adventitial) fat, laid flat, and then stained with Oil Red O. Stained aortas were placed on anti-staining slides, fully unfolded, and images were captured with a high-resolution camera. Hearts and livers were embedded in OCT tissue, and serial sections 7-10 μm thick were collected and stained with Oil Red O, hematoxylin, and eosin. Stained sections were analyzed under a light microscope. Images were quantified using ImageJ (version 2.10). Lesion area was assessed as the percentage of Oil Red O positive area in the total area sampled.

[0027] Results: As shown in Figure 6, serological assays showed that in hyperlipidemic mice, rapamycin alone did not significantly affect the regulation of IL-1β, but when used in combination with naringin, it significantly reduced the release of IL-1β. Furthermore, naringin treatment reduced lipid accumulation in the carotid artery, aortic root, and liver of hyperlipidemic mice, and the therapeutic effect was significantly improved when used in combination with rapamycin (Figures 7-10).

[0028] The above results indicate that naringin exerts dual anti-inflammatory and lipid-lowering effects by suppressing the formation of oxLp-NLRP3 complex, and intervenes in the progression of hyperlipidemia in synergy with rapamycin.

[0029] Although the preferred embodiments of the present invention have been described, those skilled in the art may make further changes and modifications to these embodiments once the basic concept of the inventive step is clear to them. It is therefore intended that the appended claims be interpreted as including the preferred embodiments and all such changes and modifications that fall within the scope of the present invention.

[0030] Obviously, those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and the equivalent technology thereof, the present invention is intended to include these variations and variations.

Claims

1. Use of naringin in combination with rapamycin in the preparation of a hyperlipidemic drug.

2. The use according to claim 1, characterized in that the naringin achieves anti-inflammatory and lipid-lowering effects by inhibiting the formation of oxLp-NLRP3 complex.

3. The use according to claim 2, characterized in that said rapamycin is used to enhance the therapeutic effect of naringin.

4. The use according to claim 3, characterized in that the anti-inflammatory effect comprises inhibition of activation of the NLRP3 inflammasome and release of the downstream pro-inflammatory cytokine IL-1β.

5. A combination drug for treating hyperlipidemia, characterized in that it contains naringin and rapamycin as active ingredients.

6. The combination according to claim 5, wherein the naringin and rapamycin are administered simultaneously or sequentially.

7. 7. The combination according to claim 6, further comprising a pharma- ceutically acceptable carrier.

8. The combination according to claim 7, wherein the dosage form of the combination includes an oral dosage form and a parenteral dosage form.

9. The combination according to claim 8, wherein the oral administration form is specifically granules, tablets, capsules, pills, drops or oral liquid.

10. The combination according to claim 8, characterized in that the parenteral dosage form is specifically an injection dosage form.

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