Use of naringin in combination with rapamycin in the preparation of antihyperlipidemic drugs

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 pharmaceuticals, and in particular to the use of naringin in combination with rapamycin in the preparation of a drug for treating hyperlipidemia. [Background technology]

[0002] Hyperlipidemia is a major public health issue and is closely related to the development of various diseases, including cardiovascular disease, nonalcoholic fatty liver disease, osteoporosis, and tumors. Lipid accumulation and chronic inflammatory responses always coexist and interact with each other, accelerating the progression of hyperlipidemia-related diseases. Therefore, 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 considered to be the main factor causing lipid accumulation and inflammatory responses. As an intracellular pattern recognition receptor, NOD-like receptor pyrin domain-containing protein 3 (NLRP3) senses intracellular oxLp accumulation and binds downstream apoptosis-associated speck-like card protein (ASC), subsequently recruiting pro-caspase-1 to aggregate into the NLRP3 inflammasome. The activated NLRP3 inflammasome then cleaves pro-caspase-1 to form active caspase-1, which promotes the maturation and release of the inflammatory factor IL-1β, inducing an inflammatory response. Our preliminary studies have shown that upon oxLp stimulation, NLRP3 aggregates and becomes activated on oxLp, leading to the formation of an 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 an inflammatory response. This discovery provides new insights into 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 inhibiting the formation of the oxLp-NLRP3 complex. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention addresses the above-mentioned problems in the prior art and aims to provide a method for the use of naringin in combination with rapamycin in the preparation of a drug for treating hyperlipidemia. [Means for solving the problem]

[0005] Naringin also achieves anti-inflammatory and lipid-lowering effects by inhibiting the formation of the oxLp-NLRP3 complex, which is 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. It is a macrolide antibiotic immunosuppressant and is clinically used to prevent organ transplant rejection and treat autoimmune diseases. Compared to cyclosporine, which is currently widely used clinically, rapamycin has several dozen times stronger immunosuppressive effects, lower toxicity, and requires a lower dosage (2 mg / day / person). It also has a synergistic immunosuppressive effect with cyclosporine, and is used in combination with cyclosporine. Compared to cyclosporine and FK506 (tacrolimus), sirolimus is an 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 in hyperlipidemic mice does not significantly affect the regulation of lipid accumulation and inflammatory responses, 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 containing naringin and rapamycin as active ingredients.

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

[0011] The formulation further comprises a pharmaceutically acceptable carrier.

[0012] The pharmaceutically 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 adjuvants include at least one of polyethylene glycol, lecithin, silicon dioxide, and micronized silica, humectants (e.g., at least one of propylene glycol, glycerin, and ethanol), stabilizers (e.g., at least one of ethylenediaminetetraacetic acid disodium, sodium thiosulfate, sodium metabisulfite, sodium sulfite, sodium bisulfite, ethanolamine, sodium bicarbonate, sodium acetate, nicotinamide, and vitamin C), osmotic adjusters (e.g., at least one of sodium chloride and glucose), pH adjusters (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 adjuvants can be mixed with naringin and rapamycin in commonly used dosages and at commonly used ratios. After the dosages of naringin and rapamycin are determined, the ratio of each pharmaceutical adjuvant can be appropriately adjusted as needed.

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

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

[0015] Furthermore, said parenteral dosage form is specifically an injectable dosage form.

[0016] In a specific embodiment of the present invention, naringin is administered in the form of an injection formulation and rapamycin is administered in the form of a capsule, and the active capsule rapamycin is added to the diet at the same time as naringin is injected. [Effects of the Invention]

[0017] The present invention has the following advantageous effects: The present study demonstrates that naringin neutralizes oxLp, inhibiting the formation of the oxLp-NLRP3 complex and effectively intervening in the early stages of hyperlipidemia. Naringin's inhibition of the oxLp-NLRP3 complex helps unlock the therapeutic potential of rapamycin in hyperlipidemia and provides a combination strategy for the prevention and treatment of hyperlipidemia-related diseases in clinical practice. At the same time, this oxLp-directed therapeutic strategy avoids interference with the protective immune function of NLRP3, providing a rationale for the prevention and treatment of other types of oxLp-related diseases. [Brief explanation 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. [Figure 2] FIG. 1 shows the ratio of oxLDL-NLRP3 complexes to oxHDL-NLRP3 complexes in THP-1 cells. [Figure 3] FIG. 1 shows the level of IL-1β release (A) and the level of intracellular cholesterol accumulation (B) in 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. [Figure 5] FIG. 1 is a schematic diagram showing the mouse hyperlipidemia model construction and drug intervention process. [Figure 6] FIG. 1 shows the results of detecting IL-1β in mouse serum after naringin-rapamycin combination treatment. [Figure 7] FIG. 1 shows the results of arterial plaques, aortic root and liver lesions in mice 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 ORO-stained aortic root lesion area relative to the total sampled area of ​​the aortic root. [Figure 10] FIG. 1 shows the percentage of lipid accumulation in the liver stained with ORO relative to the total sampled area of ​​the liver. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention will be described in detail below with reference to the drawings and specific examples, but these should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means generally 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 our preliminary study, we found that abnormally distributed oxLp acts as an endogenous ligand, triggering the recruitment and activation of NLRP3 and forming a stable complex with activated NLRP3, promoting the continuous accumulation of oxidized lipids and excessive activation of the NLRP3 inflammasome. This unregulated process of lipid accumulation and inflammatory responses triggered 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. Naringin was selected for further study, as it showed high affinity for oxidized low-density lipoprotein (oxLDL) and oxidized high-density lipoprotein (oxHDL) and could neutralize the negative charges on the surface of oxLDL and oxHDL.

[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 the American Type Culture Collection, Manassas, VA) were cultured in RPMI 1640 and pretreated with naringin (Sigma, 91842; 100 μM) for 1 hour, then cultured in serum-free medium containing oxLDL and oxHDL (50 μg / mL) for 3 hours. Culture conditions were maintained at 37°C and 5% CO2.

[0022] Measurement of intracellular cholesterol: Measurement was performed using 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, naringin pretreatment inhibited the formation of complexes between oxLDL or oxHDL and NLRP3 (Fig. 1, Fig. 2), and naringin pretreatment 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 hyperlipidemia model was established using C57BL / 6J mice. ― / ― Mice were fed a high-fat diet (HFD) starting at 4 weeks of age for 16 weeks. Naringin (20 mg / kg) was injected intraperitoneally daily, and active capsule rapamycin (RAPA, 40 mg / kg) was also added to the diet. The intervention process is shown in Figure 5. Afterwards, mouse serum, carotid artery, liver, and aortic root tissues were collected for assays and analysis.

[0026] Evaluation of carotid artery, aortic root, and liver tissue lesions: Mouse carotid arteries, hearts, and livers were dissected and fixed overnight in 4% PFA. The carotid arteries were gently removed of adhering (adventitial) fat, laid flat, and then stained with Oil Red O. The stained aorta was placed on a non-dehiscence slide, fully unfolded, and images were captured with a high-resolution camera. The heart and liver were embedded in OCT tissue, and serial sections 7–10 μm thick were collected and stained with Oil Red O and 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 relative to the total sampled area.

[0027] Results: As shown in Figure 6, serological assays demonstrated that in hyperlipidemic mice, rapamycin alone did not significantly affect IL-1β regulation, but when used in combination with naringin, it significantly reduced IL-1β release. Naringin treatment also reduced lipid accumulation in the carotid artery, aortic root, and liver of hyperlipidemic mice, and the therapeutic effect was significantly enhanced 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 inhibiting the formation of the oxLp-NLRP3 complex, and intervenes in the progression of hyperlipidemia in synergy with rapamycin.

[0029] While preferred embodiments of the present invention have been described, those skilled in the art will be able to make further changes and modifications to these embodiments once the basic concept of inventive step is clear to them. It is therefore intended that the appended claims be interpreted to include 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 their equivalents, the present invention is intended to include these variations and variations.

Claims

1. A combination drug for treating hyperlipidemia, It contains naringin and rapamycin as active ingredients. The naringin is administered in the form of an injection preparation, and the rapamycin is administered in the form of a capsule, and hyperlipidemia is treated by injecting the naringin and simultaneously adding the active capsule rapamycin to meals; The naringin achieves anti-inflammatory and lipid-lowering effects by inhibiting the formation of the oxLp-NLRP3 complex; A compounding agent for treating hyperlipidemia, characterized in that the anti-inflammatory effect includes suppression of activation of NLRP3 inflammasome and release of downstream inflammatory cytokine IL-1β.

2. 2. The combination drug for treating hyperlipidemia according to claim 1, wherein the rapamycin is used to enhance the therapeutic effect of naringin.

3. The combination drug for treating hyperlipidemia according to claim 1, wherein the naringin and rapamycin are administered simultaneously or sequentially.

4. 2. The combination drug for treating hyperlipidemia according to claim 1, further comprising a pharmaceutically acceptable carrier.

5. 2. The compound preparation for treating hyperlipidemia according to claim 1, wherein the dosage form of the compound preparation for treating hyperlipidemia includes an oral dosage form and a parenteral dosage form.

6. The compound preparation for treating hyperlipidemia according to claim 5, wherein the oral administration dosage form is specifically granules, tablets, capsules, pills, drops or oral liquid.

7. The compound preparation for treating hyperlipidemia according to claim 5, wherein the parenteral dosage form is specifically an injection dosage form.

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