Application of cycloastragenol-flower lactose in preparation of medicine for treating acute viral myocarditis

Abstract

The invention discloses an application of cycloastragenol-flower type lactose in preparation of a medicine for treating acute viral myocarditis. Cycloastragenol serving as an active ingredient of astragalus membranaceus is used for treating viral myocarditis, cycloastragenol carried by a nano-carrier is used for treating viral myocarditis, the immune storm of a body can be inhibited by regulating an immune system, and the antiviral effect can be achieved by stimulating the activity of an internal defense mechanism of target organ cells; the traditional Chinese medicine composition has a remarkable improvement effect on an experimental coxsackie B3 virus (CVB3) mouse myocarditis model.

Description

Technical Field

[0001] The invention relates to the use of cycloastragenol-floral lactose, and in particular to the use of cycloastragenol-floral lactose in preparing a medicine for treating acute viral myocarditis. Background Art

[0002] In recent years, the incidence of viral myocarditis has been gradually increasing. Enterovirus infection, especially Coxsackievirus (CVB) infection, accounts for the majority of viral myocarditis cases. People are generally susceptible to this type of virus, especially children and adults under 40 years old. Some patients may develop end-stage heart diseases such as dilated cardiomyopathy and heart failure. The pathogenesis of viral myocarditis has not yet been thoroughly elucidated. It is currently recognized that cell-mediated and autoimmune-mediated myocardial damage after viral infection is an important pathogenic mechanism of myocardial damage after viral infection. There is no specific treatment for this disease, and most treatments are symptomatic. In recent years, with the in-depth study of the disease, the combination of Chinese and Western medicine treatment measures, including Astragalus and its active ingredients, has achieved significant therapeutic effects, showing a good prospect for the treatment of the disease. Cycloastragenol is the aglycone of astragaloside, the active ingredient of Astragalus, and belongs to the cyclobromethane-type tetracyclic triterpenoid compound ( Figure 1 ), studies have shown that cycloastragenol has anti-aging, antiviral, anti-inflammatory and immunomodulatory effects, but its water insolubility limits its clinical use.

[0003] Recently, flower-shaped lactose microparticles containing nanostructured pores have been prepared for medicinal use ( Figure 2 , Figure 3 ), and its adsorption and desorption capacity for cycloastragenol was preliminarily explored ( Figure 4 ), further prepared into cycloastragenol-floral lactose preparations, which greatly improve the water solubility and bioavailability of the drug and reduce the toxicity of the drug. The nano preparation has been authorized for invention patent (patent number ZL202111026322.X). Astragalus is one of the effective ingredients of various Chinese medicine preparations for the treatment of viral myocarditis by combining traditional Chinese and Western medicine. However, there are currently no reports on the use of cycloastragenol, an effective ingredient of Astragalus, in the treatment of viral myocarditis. There is even a blank in the clinical and basic research on the use of nanocarriers to transport cycloastragenol to treat viral myocarditis. The present invention mainly found that cycloastragenol-floral lactose particles have a significant improvement effect on the experimental Coxsackie B3 virus (CVB3) mouse myocarditis model. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide the use of cycloastragenol-floral lactose in the preparation of drugs for treating acute viral myocarditis.

[0005] The invention discloses that cycloastragenol, an effective ingredient of astragalus, is used to treat viral myocarditis, and a nanocarrier is used to carry cycloastragenol to treat viral myocarditis. The invention mainly finds that cycloastragenol-floral lactose particles have a significant improvement effect on an experimental Coxsackie B3 virus (CVB3) mouse myocarditis model.

[0006] Technical solution: The present invention provides the use of the cycloastragenol-floral lactose in the preparation of a drug for treating acute viral myocarditis.

[0007] Furthermore, the therapeutic effect was dose-dependent.

[0008] Furthermore, the dosage is 5 mg / kg, 10 mg / kg, and 20 mg / kg.

[0009] Furthermore, the dosage is 1 mg / kg or 20 mg / kg.

[0010] Furthermore, the dosage is 20 mg / kg.

[0011] Furthermore, the drug reduces the content of inflammatory factors IL-6, IL-17, TNF-α and neutrophil extracellular traps (NETs).

[0012] Furthermore, the purity of the cycloastragenol raw material drug is greater than 98%.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention studies the treatment of viral myocarditis by cycloastragenol, an effective ingredient of Astragalus membranaceus. The treatment of viral myocarditis by using nanocarriers to carry cycloastragenol can not only suppress the body's immune storm by regulating the immune system, but also exert antiviral effects by stimulating the activity of the intrinsic defense mechanism of target organ cells, and has a significant improvement effect on the experimental Coxsackie B3 virus (CVB3) mouse myocarditis model. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the structural formula of cycloastragenol (CAG);

[0016] Figure 2 It is a medicinal flower-shaped carrier lactose microparticle structure containing nanostructured pores;

[0017] Figure 3 It is a medicinal flower-shaped carrier lactose microparticle structure II containing nanostructured pores;

[0018] Figure 4 It is the adsorption and desorption capacity curve of flower-shaped lactose carrying cycloastragenol;

[0019] Figure 5Figure A shows the general appearance of the hearts of mice in each group. The hearts of mice in the CVB3 infection model control group had white flaky inflammatory plaques on the surface of the hearts, and the texture was stiff and inelastic. There were a small number of scattered punctate inflammatory plaques on the surface of the hearts of mice in the high, medium and low dose groups of CAG-FL, and the texture was slightly inelastic. Figure B shows the HE staining and scoring results of mice in each group. The mice were killed 7 days after modeling, and the myocardial tissues were fixed and embedded in paraffin for pathological sections. HE staining was performed to observe pathological changes. The scores were 0 to 4 points according to the degree of inflammatory cell infiltration and alveolar tissue lesions. There were a large number of inflammatory cells infiltrating in the myocardial tissue gaps of mice in the CVB3 infection model control group, and focal lesions were visible. CAG-FL can significantly improve the degree of myocardial tissue damage and inflammatory cell infiltration in the model mice, and this improvement showed obvious dose-dependency. Compared with the normal control group, the pathological score of the model group (CVB3 group) was significantly increased. CAG-FL 5mg / kg, 10mg / kg, and 20mg / kg groups can dose-dependently reduce the HE staining pathological scores, among which, #: Group A, Group B vs Group C, p<0.0001 (Student's-t test). *: Group D, Group E, Group F vs Group C, p<0.01 (Student's-t test);

[0020] Figure 6 The levels of IL-6, IL-17, TNF-α, and neutrophil extracellular traps (NETs) in the peripheral blood of mice in each group were significantly increased in the model group (CVB3 group) compared with the normal control group; compared with the CVB3 model group, the CAG-FL treatment group was able to significantly reduce the expression levels of inflammatory factors and neutrophil extracellular traps (NETs) after CAG-FL administration; while there was no difference between the normal control group and the normal control + high-dose drug group;

[0021] Figure 7 The results of CVB3 titer detection in myocardial tissue lysate indicate that CAG-FL administration can significantly reduce the toxicity of CVB3;

[0022] Figure 8 In the in vitro cell model, compared with the normal control group, the cell activity of the model group (CVB3 infection group) was significantly decreased. After administration of CAG-FL, the cell survival rate was significantly improved and the cell activity was increased. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the technical solution of the present invention and implement it, the present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the scope of protection of the present invention.

[0024] The method for preparing cycloastragenol-floral lactose particles in the following experiments adopts the method of patent number ZL202111026322.X.

[0025] 1. Pharmacological experiments of cycloastragenol-floral lactose nanoparticles

[0026] The experimental Coxsackie B3 virus (CVB3) mouse myocarditis model was established with reference to the method of Professor Yang Yingzhen's team from Zhongshan Hospital Affiliated to Fudan University (reference for the method of establishing the mouse model of acute viral myocarditis: Yang Yingzhen, ed., "Viral Heart Disease". Shanghai Science and Technology Press, 2001.10:352).

[0027] Male purebred Balb / C mice aged 4 to 6 weeks were housed in an SPF animal room at a room temperature of 20°C ± 2°C, with free access to water and food, a 12-h day and night cycle, and a relative humidity of 50% ± 10%. All experimental procedures involving animals were approved by the Ethics Review Committee of the Laboratory Animal Science Department of Fudan University.

[0028] Coxsackie virus B3 (CVB3) Nancy strain (stored in our laboratory) was replicated in human embryonic kidney cells and frozen and thawed three times. TCID was measured on Wish cells. 50 =10 6.7 , 50μl. The above virus was diluted and inoculated into mice intraperitoneally aseptically and fed conventionally. The target organ lesions were most severe 7 to 10 days after mice were infected with CVB3, and gradually recovered after 14 days. The mice were randomly divided into 6 groups, normal group, normal mouse cycloastragenol-floral lactose granules high-dose group, model group (CVB3 infection group), cycloastragenol-floral lactose granules (CAG-FL) low, medium and high dose groups (5mg / kg, 10mg / kg, 20mg / kg, calculated based on the effective ingredient of cycloastragenol). The normal group and the model group were given an equal amount of normal saline, and the rest of the groups were gavaged according to the dose, once a day, 0.1ml each time. After 3 consecutive days of administration, except for the normal control group and the normal mouse + cycloastragenol-floral lactose granules high-dose group, the rest of the groups of mice were intraperitoneally inoculated with CVB3 virus for modeling. After modeling, continue to administer the above dose for 7 days.

[0029] On the 7th day after modeling, blood was collected from the mouse eyeballs and centrifuged at 3000 rpm for 5 minutes. The serum was separated and stored at -80°C for later use. The detection indicators were TNF-α, IL-6, and IL-17. After the mice were killed, a part of the heart tissue was fixed with 10% formaldehyde, embedded in paraffin, and sectioned, and HE staining was used to observe pathological changes. The inflammatory cell infiltration and tissue lesions were scored from 0 to 4 points, 0 point: no lesions and inflammatory cell infiltration; 1 point, mild lesions or inflammatory cell infiltration; 2 points, moderate lesions or inflammatory cell infiltration; 3 points, severe lesions or inflammatory cell infiltration; 4 points, very severe lesions or inflammatory cell infiltration; another part of the myocardial tissue was stored at -80°C for later use, and the virus titer in the tissue lysate was detected.

[0030] Intervention effect of CAG-FL on in vitro pneumonia model

[0031] Experimental groups: Primary isolated SD rat neonatal cardiomyocytes were cultured at 5×10 5 / ml density inoculated 96-well plates, 100μl per well, 37℃, 5% CO2, saturated humidity conditions for 48 hours. The cells were divided into normal control group, virus infection group, CAG-FL low-dose group (1μg / ml), medium-dose group (2μg / ml), and high-dose group (4μg / ml), all calculated based on the effective ingredient of cycloastragenol. There were 3 samples in each group, and 2 replicates were set for each sample.

[0032] The cells were treated with high, medium and low doses of CAG-FL, and 100 μl was added to each well. The normal control group and the virus-infected group were added with the same volume of saline. After 12 hours, the cells in each group were added with TCID 50 =10 -6.7 CVB3 virus solution, 100μl / well, and discard the virus solution after infecting cells for 1 hour. The normal control group and virus infection group added 100μl / well of culture medium without bovine serum, and the intervention group added the same volume of high, medium, and low doses of CAG-FL and continued to culture for 48 hours. CCK8 reagent 10μl was added to each well, reacted at 37℃ for 1 hour, and the OD value was measured at 450nm to calculate the cell activity of each group.

[0033] like Figure 5 As shown in A, the hearts of normal control mice and normal mice + high-dose CAG-FL group mice were ruddy in appearance, plump and elastic in texture; the hearts of mice in the CVB3 infection model control group had white flaky inflammatory plaques on the surface, and the texture was stiff and inelastic; the hearts of mice in the high-, medium-, and low-dose CAG-FL groups had a small number of scattered punctate inflammatory plaques on the surface, and the texture was slightly less elastic.

[0034] like Figure 5B. HE staining results of pathological sections showed that there were a large number of inflammatory cells infiltrating in the myocardial tissue gap of mice in the CVB3 infection model control group, and focal lesions were visible; CAG-FL could significantly improve the degree of myocardial tissue damage and inflammatory cell infiltration in the model mice, and this improvement showed obvious dose-dependency. Compared with the normal control group, the pathological score of the model group (CVB3 group) was significantly increased; CAG-FL 5mg / kg, 10mg / kg, and 20mg / kg groups could dose-dependently reduce the HE staining pathological score. There was no difference in the HE staining pathological score between the normal control group and the normal control + high-dose drug group.

[0035] like Figure 6 As shown in the figure, compared with the normal control group, the levels of inflammatory factors IL-6, IL-17, TNF-α and neutrophil extracellular traps (NETs) in the serum of the model group (CVB3 group) were significantly increased; compared with the CVB3 model group, the CAG-FL treatment group could significantly reduce the levels of inflammatory factors and neutrophil extracellular traps (NETs) after CAG-FL administration. s ) expression level and CVB3 titer in myocardial tissue lysate; while there was no difference between the normal control group and the normal control + high-dose drug group.

[0036] like Figure 7 As shown, the myocardial tissue was homogenized and centrifuged to measure the virus titer of the supernatant. Compared with the model group, the high, medium and low doses of CAG-FL treatment groups could significantly reduce the virus titer in the myocardial tissue, indicating that CAG-FL has the effect of inhibiting the virulence of the virus.

[0037] like Figure 8 As shown in the in vitro cell model, compared with the normal control group, the cell activity of the model group (CVB3 group) was significantly decreased, and CAG-FL administration was able to significantly increase the cell activity. This suggests that cycloastragenol-floral lactose nanoparticles can not only suppress the body's immune storm by regulating the immune system, but also may play an antiviral role by stimulating the vitality of the intrinsic defense mechanism of target organ cells.

[0038] According to the present invention, the cycloastragenol-floret lactose nanoparticles can be used for treating diseases related to acute viral myocarditis and preparing corresponding medicines.

[0039] Note: Cycloastragenol API was purchased from Nanjing Dosfu Biotechnology Co., Ltd. with a purity greater than 98%; CVB3 virus (Nancy strain) was preserved by this laboratory for scientific research.

[0040] result

[0041] In the CVB3-induced acute viral myocarditis mouse model, intragastric administration of 5 mg / kg, 10 mg / kg, and 20 mg / kg of cycloastragenol-floral lactose particles can significantly reduce the HE staining pathological score, reduce target organ tissue damage and inflammatory cell infiltration, reduce the content of IL-6, IL-17, TNF-α, and neutrophil extracellular traps (NETs) in peripheral blood, and downregulate the CVB3 titer in myocardial tissue lysate, and the effect is dose-dependent. In vitro cell experiments showed that compared with the CVB3 infection group, CAG-FL administration can significantly improve cell survival and increase cell activity.

[0042] The above results suggest that cycloastragenol-floral lactose particles can improve the symptoms of CVB3-induced acute viral myocarditis in mice, so they can be used as a therapeutic drug for acute viral myocarditis-related diseases.

[0043] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any technician in the relevant technical field, without departing from the scope of the technical solution of the present invention, makes any form of equivalent replacement or modification to the technical solution and technical content disclosed in the present invention, which does not depart from the content of the technical solution of the present invention and still falls within the protection scope of the present invention.

Claims

1. Use of cycloastragenol-floral lactose in preparing a drug for treating acute viral myocarditis.

2. The use according to claim 1, characterized in that The drug effect is dose-dependent.

3. The use according to claim 2, characterized in that: The dosage is 5 mg / kg, 10 mg / kg, and 20 mg / kg.

4. The use according to claim 3, characterized in that The dosage is 10 mg / kg and 20 mg / kg.

5. The use according to claim 4, characterized in that The dose was 20 mg / kg.

6. The use according to claim 1, characterized in that: The drug reduces the content of inflammatory factors IL-6, IL-17, TNF-α and neutrophil extracellular traps (NETs).

7. The use according to claim 1, characterized in that The purity of the cycloastragenol raw material drug is greater than 98%.

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