Application of drunkanin A in preparation of macrophage macropinocytosis inhibitor

Abstract

The invention discloses an application of drunkanin A in preparation of a macropinocytosis inhibitor of macrophages. It is found for the first time that drunkanin A plays an inhibiting role in inhibiting RPL37 in macrophage macropinocytosis. Compared with the existing macrophage macropinocytosis inhibitor mipalmine, the withanidin A as a natural product has good biological safety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of withaferin A in preparation of a macrophage macropinocytosis inhibitor. BACKGROUND

[0002] In the pathophysiological mechanism of atherosclerotic plaque formation, due to early endothelial damage, low-density lipoprotein cholesterol (LDL-C) in the blood will penetrate into the inner layer of the blood vessel wall (intima) through the damaged endothelium and deposit. These cholesterol trapped in the blood vessel wall will be oxidatively modified, becoming more inflammatory and toxic. The blood vessel initiates an inflammatory response upon sensing these oxidized lipids, recruiting monocytes into the blood vessel wall and transforming into macrophages, and phagocytizing these lipids. When macrophages phagocytize too much lipid, they become lipid droplet-filled foam cells. This is a hallmark lesion of atherosclerosis. Therefore, inhibiting macrophage phagocytosis of a large amount of LDL-C and foam formation is an important means of early atherosclerosis treatment.

[0003] However, the diameter of a single LDL-C is mostly between 10-30 nm, while the endocytosis process mostly phagocytizes substances within 60 nm in diameter. In the environment of cholesterol deposition in the blood vessel wall, LDL-C can cross-link and fuse with each other, and part of the larger diameter LDL-C cannot be phagocytized by the endocytosis pathway. Macropinocytosis is an important pathway for macrophages to phagocytize 100-200 nm diameter substances, and it is naturally important for macrophages to phagocytize LDL-C. However, so far, only Hui-Ping Lin et al. (Receptor-independent fluid-phase macropinocytosis promotes arterial foam cell formation and atherosclerosis, Sci. Transl. Med. 14, eadd2376 (2022)) have reported that Imipramine can improve atherosclerosis by inhibiting macrophage macropinocytosis. Imipramine is a tertiary amine tricyclic antidepressant, and its main effect is to improve depression in the nervous system. As a macropinocytosis inhibitor, it has certain side effects and the mechanism is not clear. SUMMARY

[0004] The primary purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide application of withaferin A in preparation of a macrophage macropinocytosis inhibitor.

[0005] The purpose of the present application is achieved by the following technical solutions: Application of solanine A in the preparation of macrophage macropinocytosis inhibitors. Macropinocytosis is an important biological phagocytic pathway of macrophages. This physiological process is not limited to atherosclerosis; macrophage macropinocytosis plays an important role in various aspects such as immunity, metabolism, and cancer.

[0006] The solanine A mentioned above inhibits macrophage macropinocytosis by inhibiting RPL37.

[0007] The application of solanine A in the preparation of drugs for the treatment of atherosclerosis demonstrates that solanine A can be the sole active ingredient with therapeutic effects on atherosclerosis.

[0008] The structural formula of Withaferin A (CAS 5119-48-2) is shown below:

[0009] I.

[0010] The drug is preferably in the form of powder, granules, tablets, powder, soft capsules, pills, ointment, or injection.

[0011] The present invention has the following advantages and effects compared with the prior art: (1) This invention is the first to discover that solanine A inhibits macrophage macropinocytosis by inhibiting RPL37. The inhibitory effect of solanine A on macropinocytosis is not clear in the prior art, and RPL37 has not been found to be its target.

[0012] (2) Previous reports have indicated that solanine A has the effect of suppressing appetite and improving obesity. Compared with the existing macrophage macropinocytosis inhibitor imipramine, solanine A, as a natural product, has good biocompatibility. Attached Figure Description

[0013] Figure 1 The images show fluorescence micrographs and fluorescence intensity statistics of THP-1 macrophages induced to differentiate in vitro after treatment with 1 μM, 2 μM solanine A or control solvent and 1 mg / mL 70kDa FITC-DEXTRAN for 72 hours, respectively; where *** indicates P<0.001.

[0014] Figure 2The images show fluorescence micrographs and fluorescence intensity statistics of THP-1 macrophages induced to differentiate and overexpress RPL-37 after 48 hours of in vitro treatment with 1 μM, 2 μM solanine A or control solvent and 1 mg / mL 70kDa FITC-DEXTRAN. In the figures, ns indicates no statistical significance, ** indicates P < 0.01, and *** indicates P < 0.001.

[0015] Figure 3 These are photographs and fluorescence intensity statistics of macropinocytosis in the aorta of RPL37-overexpressing ApoE- / - mice treated with solanine A; where * indicates P<0.05, ** indicates P<0.01; Veh+OEcon indicates overexpression of the empty vector with solvent control, WFA+OEcon indicates overexpression of the empty vector with solanine A, and WFA+OErpl37 indicates overexpression of RPL37 with solanine A, with 8 replicates for each group.

[0016] Figure 4 This is a staining result of aortic atherosclerosis in RPL37-overexpressing ApoE- / - mice treated with solanine A; where * indicates P<0.05, ** indicates P<0.01; Veh+OEcon indicates overexpression of empty vector with solvent control, WFA+OEcon indicates overexpression of empty vector with solanine A, and WFA+OErpl37 indicates overexpression of RPL37 with solanine A, with 8 replicates for each group. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0018] Example 1 (1) Solanine A effectively inhibits macrophage macropinocytosis: Given that macropinocytosis can form vesicles with a diameter of 200 nm, we used a large volume of 70 kDa FITC-DEXTRAN (70 kDa FITC-DEXTRAN, purchased from MCE, catalog number HY-128868E) as an indicator of macropinocytosis formation. It was dissolved to 1 mg / mL in Pronosei RPMI 1640 medium (catalog number PM150110) as the working medium.

[0019] Solanine A was dissolved in 1 mM and 2 mM 1000× concentrates using DMSO (catalog number 0219605580) from MP Company. In actual drug administration, the concentrates were added at a ratio of 1000:1 according to the volume of cell culture medium to ensure that the final solution contained 1 μM and 2 μM of solanine A, but the DMSO content did not exceed 1 / 1000 of the total volume. The same volume of DMSO was added as a control solvent group.

[0020] Human monocytic leukemia cell line THP-1 suspension cells were cultured using standard methods (culture medium: RPMI 1640 medium + 20% v / v fetal bovine serum + 1% v / v mercaptoethanol). The fetal bovine serum was from Pronossa (catalog number 164210-50), and the mercaptoethanol was from Thermo Fisher Scientific (catalog number 21985023).

[0021] THP-1 cells were induced into adherent macrophages using the classic phorbol 12-myristate 13-acetate (MCE HY-18739) induction method. The specific steps are as follows: THP-1 suspension cells were treated with 200 ng / mL phorbol 12-myristate for 24-72 hours, and the THP-1 suspension cells adhered and differentiated into macrophages.

[0022] Based on the detailed implementation plan above, we seeded 500,000 THP-1 suspension cells per well in 6-well plates and induced differentiation for 24 hours in 1640 medium containing 70 kDa FITC-DEXTRAN with 200 ng / mL phorbol ester. Subsequently, 1 μM, 2 μM solanine A, or the control solvent (Veh group) were added, and the cells were incubated together for 72 hours. We found that solanine A effectively inhibited macrophage pinocytosis in a concentration-dependent manner. Figure 1 ).

[0023] (2) Solanine A inhibits macropinocytosis by binding to and inhibiting RPL37: The RPL37 overexpression plasmid was purchased from Wuhan Miaoling Biotechnology Co., Ltd., and its full name is pLV3-CMV-RPL37(human)-3×FLAG-mCherry-Puro. The control empty vector plasmid was also purchased from Wuhan Miaoling Biotechnology Co., Ltd., and its full name is pLV3-CMV-3×FLAG-mCherry-Puro.

[0024] As described above, differentiation was induced for 24 hours in 1640 medium (2 ml / well) containing 200 ng / mL phorbol ester in 500,000 THP-1 suspension cells per well. Then, 125 μL of opti-MEM medium (Pronosai, catalog number 31985-070) was added to a 1.5 mL sterile centrifuge tube, along with 7.5 μL of Lipomaster 3000 Reagent (Novizan, catalog number TL301-01), to obtain a Lipomaster 3000Reagent / opti-MEM mixture. In another 1.5 mL sterile centrifuge tube, 125 μL of opti-MEM was added, along with 2 μg of the aforementioned RPL37 overexpression plasmid or 2 μg of the control empty vector plasmid, and gently mixed to obtain a plasmid / opti-MEM mixture. This plasmid / opti-MEM mixture was then added dropwise to the Lipomaster 3000 Reagent / opti-MEM mixture, gently mixed with a pipette, and incubated at room temperature for 10 hours. After 24 hours, THP-1 adherent cells induced to differentiate were added, followed by transfection for 24 hours to induce overexpression of either the empty vector (NC group) or RPL37 (OE-RPL37 group). 24 hours after overexpression, the medium was replaced with RPMI 1640 medium containing 1 mg / mL 70 kDa FITC-DEXTRAN, and 1 μM, 2 μM solanine A, or control solvent were added, followed by incubation for another 48 hours.

[0025] Four-week-old male C57BL / 6J ApoE- / - mice were purchased from Jiangsu Jicui Pharmaceutical Co., Ltd. RPL37-overexpressing AAV9 virus (serotype 9) (abbreviated as RPL37-AAV9, obtained by packaging the F4_80.mRpl37-P2A-mCherry.WPRE3.SV40LpA plasmid with adenovirus-associated virus AAV9 virus, expressing RPL and AAV9 [ssAAV.F4_80.mRpl37-P2A-mCherry.WPRE3.SV40LpA]) and empty vector control AAV9 virus (obtained by packaging the F4_80.mCherry.WPRE3.SV40LpA plasmid with AAV9 virus, AAV9 [ssAAV.F4_80.mCherry.WPRE3.SV40LpA]) were constructed by Paizhen Biotechnology Co., Ltd. All titers were 10. 9 GC / mL. For overexpression, ApoE- / - mice fed for 16 weeks were injected via tail vein with 100 μL of virus per mouse, achieving a 10- GC / mL ratio. 8GC viral concentration infection. Four weeks after tail vein injection of RPL37-AAV9 virus or empty AAV9 virus, mice were treated with solvent control (DMSO) or 1250 μg / kg solanine A (WFA, dissolved in DMSO) via intraperitoneal injection daily for four weeks. Three experimental groups were set up, with eight mice in each group: Veh+OEcon group (overexpressing empty AAV9 and given solvent control), WFA+OEcon group (overexpressing empty AAV9 and given solanine A), and WFA+OErpl37 group (overexpressing RPL37 and given solanine A to ApoE- / - mice). On the day of euthanasia, a large volume of 70 kDa FITC dextran was prepared into a tracer solution with a concentration of 20 mg / mL using physiological saline. 50 μL of the tracer solution was injected into the inner canthus of the left eye of each mouse. Three hours later, 50 μL of the tracer solution was injected into the inner canthus of the right eye of each mouse. After another three hours, the mice were euthanized. The internal organs of mice were dissected, and fluorescence imaging was performed in an IVIS in vivo optical imaging system with excitation / emission parameters of 480 / 520 nm and an exposure time of 1 s.

[0026] from Figure 2 It is evident that plasmid overexpression of RPL37 can block the inhibitory effect of solanine A on macropinocytosis; from Figure 3 It is evident that overexpression of RPL37 with adeno-associated virus in ApoE- / - mice effectively blocks the inhibitory effect of solanine A on macropinocytosis of mouse aortic macrophages.

[0027] (3) Solanine A effectively improves atherosclerosis by binding to and inhibiting RPL37: The administration of ApoE- / - mice and AAV9 infection were the same as described above.

[0028] After euthanizing the mice, aortic valve sections were obtained by dissection and the atherosclerosis of the mouse aorta was observed by Oil Red O staining and Masson staining.

[0029] Further experiments on mice overexpressing RPL37 with adeno-associated virus revealed that RPL37 overexpression effectively blocked the effect of solanine A on improving aortic atherosclerosis in mice. Figure 4 ).

[0030] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of withaferin A in the preparation of an inhibitor of macrophage phagocytosis.

2. Use according to claim 1, characterized in that: The withaferin A inhibits macrophage phagocytosis by inhibiting RPL37.

3. Use of withaferin A in the preparation of a drug for treating atherosclerosis.

4. Use according to claim 1, characterized in that: The drug is a powder, granules, tablets, granules, soft capsules, drop pills, ointments or injections.

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