Astragaloside lipidosome for treating viral pneumonia and preparation method of astragaloside lipidosome

Abstract

The invention relates to the technical field of pharmaceutical preparations, in particular to astragaloside lipidosome for treating viral pneumonia and a preparation method of the astragaloside lipidosome. The astragaloside lipidosome for treating viral pneumonia comprises astragaloside and lipidosome loaded with the astragaloside, wherein the lipidosome comprises lecithin, cholesterol and distearoyl phosphatidyl ethanolamine-polyethylene glycol. The lipidosome provided by the invention can well wrap astragaloside so as to improve the stability of the astragaloside, and animal model experiments prove that the astragaloside lipidosome provided by the invention can improve the viral pneumonia lung injury, significantly improve the survival rate of influenza A viral pneumonia mice and improve the curative effect of the influenza A viral pneumonia mice. Therefore, the traditional Chinese medicine composition can be well used for treating viral pneumonia caused by the influenza A virus.

Description

Technical Field

[0001] The present application belongs to the technical field of pharmaceutical preparations, and in particular relates to astragaloside liposomes for treating viral pneumonia and a preparation method thereof. Background Art

[0002] Currently, vaccination and anti-influenza medications are the primary means of preventing and treating influenza. Vaccine effectiveness depends on its match with the currently circulating influenza virus strain. Because influenza viruses are highly susceptible to antigenic drift and antigenic shift, the role of vaccines in influenza prevention and treatment remains relatively limited. Anti-influenza drugs fall into four main categories: M2 ion channel protein blockers, neuraminidase inhibitors, hemagglutinin inhibitors, and RNA polymerase inhibitors. Oseltamivir, the first orally effective, specific influenza virus neuraminidase inhibitor, has become the current antiviral drug of choice. However, there have been reports of severe side effects in some patients after taking oseltamivir.

[0003] While existing anti-influenza drugs are widely used clinically, their single target, high frequency of use, and frequent drug resistance, coupled with certain toxic side effects, severely limit their effectiveness in clinical treatment. Viral pneumonia, a lung inflammatory disease caused by an upper respiratory viral infection that spreads downward, is particularly in need of a safe and effective antiviral drug. Summary of the Invention

[0004] The purpose of this application is to provide an astragaloside liposome for treating viral pneumonia and a preparation method thereof, aiming to solve the technical problem of how to safely and effectively treat viral pneumonia.

[0005] To achieve the above application objectives, the technical solutions adopted in this application are as follows:

[0006] In a first aspect, the present application provides an astragaloside liposome for treating viral pneumonia, comprising: astragaloside IV and liposomes loaded with the astragaloside IV, wherein the liposomes comprise lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol.

[0007] In some embodiments, the mass ratio of the lecithin, the cholesterol and the distearoylphosphatidylethanolamine-polyethylene glycol is (32-48): (4-6): (8-12); or,

[0008] The mass ratio of the astragaloside IV, the lecithin, the cholesterol and the distearoylphosphatidylethanolamine-polyethylene glycol is 6:(32-48):(4-6):(8-12).

[0009] In some embodiments, the average particle size of the astragaloside liposomes is 60 nm to 120 nm.

[0010] In a second aspect, the present application provides a method for preparing the above-mentioned astragaloside liposomes, comprising:

[0011] The astragaloside IV liposomes are prepared by using astragaloside IV, lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol as raw materials through a thin film hydration method.

[0012] In some embodiments, the steps of preparing the astragaloside IV liposomes using astragaloside IV, lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol as raw materials by thin film hydration method include:

[0013] The astragaloside IV, the lecithin, the cholesterol and the distearoylphosphatidylethanolamine-polyethylene glycol are dissolved in an alcohol solvent, and then the solvent is removed by rotary evaporation to form a film; the film is mixed with a glucose aqueous solution for hydration treatment, and then ultrasonically dispersed and extruded to obtain the astragaloside IV liposomes.

[0014] In some embodiments, the mass ratio of the astragaloside IV to the alcohol solvent is 1 mg: 4-6 mL; and / or

[0015] The mass fraction of the glucose aqueous solution is 4-6%, and the volume ratio of the mass of astragaloside IV to the glucose aqueous solution is 1 mg:0.1-0.2 mL.

[0016] In some embodiments, the ultrasonic dispersion conditions include: 180-220W ice bath ultrasound for 5-10 minutes.

[0017] In a third aspect, the present application provides an application, namely, the application of astragaloside IV in the preparation of a drug for treating viral pneumonia.

[0018] In some embodiments, the viral pneumonia comprises viral pneumonia caused by influenza A virus.

[0019] In some embodiments, the drug for treating viral pneumonia includes the astragaloside liposomes provided in the first aspect of this application and / or the astragaloside liposomes prepared by the preparation method provided in the second aspect of this application.

[0020] The first aspect of the present application provides astragaloside liposomes, wherein the liposome components used to load astragaloside IV include lecithin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol. The liposomes formed by the above components can well encapsulate astragaloside IV to improve its stability. At the same time, animal model experiments have shown that the astragaloside liposomes of the present application can improve lung damage caused by viral pneumonia and significantly increase the survival rate of mice with influenza A viral pneumonia. Therefore, they can be well used to treat viral pneumonia caused by influenza A virus.

[0021] The second aspect of this application provides a preparation method for preparing astragaloside IV liposomes using a thin film hydration method using astragaloside IV, lecithin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol as raw materials. This preparation method is not only simple, but also provides excellent stability for the resulting astragaloside IV liposomes, which can be used to treat viral pneumonia caused by influenza A virus.

[0022] The application provided in the third aspect of this application is based on the fact that animal model experiments have shown that astragaloside IV can improve lung damage caused by viral pneumonia and significantly increase the survival rate of mice with influenza A viral pneumonia. Therefore, astragaloside IV can be used to prepare drugs for the treatment of viral pneumonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0024] Figure 1 This is a transmission electron micrograph of the astragaloside liposome provided in the examples of the present application;

[0025] Figure 2 This is a stability test chart of the astragaloside liposomes provided in the examples of this application;

[0026] Figure 3 This is a comparison chart of the changes in mouse body weight and lung index after treating mice with influenza virus pneumonia with astragaloside liposomes provided in the examples of the present application;

[0027] Figure 4 This is a comparison chart of the weight changes and survival rates of mice after influenza virus pneumonia mice were treated with astragaloside liposomes provided in the examples of the present application;

[0028] Figure 5 This is a comparison chart of the viral load results of lung tissues of mice with influenza virus pneumonia treated with astragaloside liposomes provided in the examples of this application;

[0029] Figure 6 This is a comparison of the staining results of lung tissue pathology sections of mice with influenza virus pneumonia treated with astragaloside liposomes provided in the examples of the present application (HE, ×100);

[0030] Figure 7 This is a comparison chart (mRNA level) of inflammatory and chemokine expressions in various groups of mice with influenza virus pneumonia treated with astragaloside liposomes provided in the examples of this application;

[0031] Figure 8This is a comparison chart (protein level) of the inflammatory and chemokine expressions in each group of mice with influenza virus pneumonia treated with astragaloside liposomes provided in the examples of the present application. DETAILED DESCRIPTION

[0032] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0033] In this application, the term "and / or" describes the relationship between associated objects, indicating that three possible relationships exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0034] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0035] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0036] The terms used in the embodiments of the present application are for the purpose of describing specific embodiments only and are not intended to limit the present application. The singular forms "a", "an", "the" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0037] The weights of the relevant components mentioned in the examples of this application may not only refer to the specific content of each component, but also represent the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the examples of this application, it is within the scope disclosed in the examples of this application. Specifically, the mass described in the examples of this application may be a mass unit known in the chemical industry, such as μg, mg, g, kg, etc.

[0038] The terms "first" and "second" are used solely for descriptive purposes to distinguish objects, such as substances, from one another and should not be understood to indicate or imply relative importance or to implicitly specify the quantity of the technical features being referred to. For example, without departing from the scope of the embodiments of this application, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX. Thus, features defined as "first" or "second" may explicitly or implicitly include one or more of such features.

[0039] At present, there is a clinical need to develop safe and effective drugs for the treatment of viral pneumonia caused by influenza A virus. Traditional Chinese medicine has a long history of experience in treating influenza. Traditional Chinese medicine treatment reduces fever faster than simple antiviral treatment, and the time for patients receiving integrated Chinese and Western medicine treatment to turn negative for the virus is shorter. Astragaloside IV (AS-IV) is a lanolin alcohol-type tetracyclic triterpenoid saponin and is the main material basis for the efficacy of the traditional Chinese medicine Astragalus. AS-IV has pharmacological effects such as immunomodulation, organ protection, anti-inflammatory, antiviral, hypoglycemic, anti-aging, and improvement of blood rheology. However, there are currently no research reports on the use of AS-IV and its preparations against influenza A virus.

[0040] Based on this, the present invention develops an astragaloside liposome that can safely and effectively treat viral pneumonia and a preparation method thereof. The specific scheme is as follows.

[0041] The first aspect of the present invention provides an astragaloside liposome for treating viral pneumonia, comprising: (1) astragaloside IV, i.e., the active pharmaceutical ingredient; (2) liposomes for loading astragaloside IV and forming a stable nanoparticle with astragaloside IV; the materials of the liposomes include lecithin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG). Lecithin has two relatively long hydrophobic hydrocarbon chains and one hydrophilic group. When lecithin is added to water or a buffer solution, the lecithin molecules can be oriented, with the hydrophilic groups facing the aqueous phase on both sides, and the hydrophobic hydrocarbon chains are relatively associated with each other to form a bilayer, forming a liposome. Therefore, lecithin can be used as the main material of the liposome loaded with astragaloside IV. Cholesterol is also an amphiphilic substance. When mixed with lecithin, it can regulate the fluidity of the bilayer and reduce the permeability of the liposome membrane, thereby improving the stability of the liposome. DSPE-PEG is an amphiphilic macromolecular compound composed of a hydrophobic phosphate head and a hydrophilic PEG tail. It has good water solubility, good biocompatibility and biodegradability. Together with lecithin and cholesterol, it constitutes a liposome structure, which can well assist the release of astragaloside IV in the liposome, thereby improving the biocompatibility and stability of the astragaloside IV liposome preparation. On the one hand, astragaloside IV can exert its own pharmacological effect, and on the other hand, astragaloside IV itself can also be used as a membrane material for liposomes to improve liposome stability. Therefore, the astragaloside IV liposome preparation of the present application can further maintain and improve the stability of the liposome structure through the combined action of the astragaloside IV active ingredient and the liposome material, so that the drug can better exert its pharmacological effect.

[0042] The astragaloside liposomes provided in the examples of the present application include phosphatidylcholine, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol, which can well encapsulate astragaloside IV to improve its stability. Moreover, the astragaloside liposomes of the examples of the present application can improve lung damage caused by viral pneumonia and significantly increase the survival rate of mice with influenza A viral pneumonia, and thus can be well used to treat viral pneumonia caused by influenza A virus.

[0043] In some embodiments, the mass ratio of lecithin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol can be (32-48): (4-6): (8-12). For example, the mass ratio of lecithin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol can be 32:4:8, 40:5:10, or 48:6:12. The liposomes formed by the above mass ratios have good stability and can well load astragaloside IV. Specifically, the above lecithin can be egg yolk lecithin.

[0044] Furthermore, the mass ratio of astragaloside IV, lecithin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol is 6:(32-48):(4-6):(8-12). The mass ratio of astragaloside IV combined with the above liposome materials can not only well encapsulate astragaloside IV, but also form stable nanoliposome particles with good dispersibility.

[0045] In some embodiments, the average particle size of the astragaloside liposomes formed by the composite of astragaloside IV and liposomes is 60 nm to 120 nm, specifically approximately 70 nm. Furthermore, the polydispersity index (PDI) of the astragaloside liposomes is less than 0.3, approximately 0.28, and the zeta potential is approximately -22 mV. The astragaloside liposomes formed with the above parameters can form stable nanoliposome particles with good dispersion.

[0046] In some embodiments, the astragaloside liposomes are dispersed in a glucose aqueous solution. Specifically, the glucose aqueous solution has a mass fraction of 4-6%, and the volume ratio of astragaloside IV to the glucose aqueous solution is 1 mg:0.1-0.2 mL. Once the astragaloside liposomes are dispersed in the glucose aqueous solution to a certain concentration, they can be used as an injection.

[0047] A second aspect of the present invention provides a method for preparing astragaloside liposomes, comprising the following steps:

[0048] S01: provides astragaloside IV, lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol;

[0049] S02: Astragaloside IV liposomes were prepared by thin film hydration method using astragaloside IV, lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol as raw materials.

[0050] In the examples of the present application, astragaloside IV, lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol are used as raw materials, and astragaloside IV liposomes are prepared by a thin film hydration method. Not only is the process simple, but the obtained astragaloside IV liposomes are also stable and can be used to treat viral pneumonia caused by influenza A virus.

[0051] In some embodiments, the specific mass ratio of astragaloside IV, lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol can be 6: (32-48): (4-6): (8-12).

[0052] In some embodiments, the steps of preparing astragaloside IV liposomes using astragaloside IV, lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol as raw materials by a thin film hydration method include: dissolving astragaloside IV, lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol in an alcohol solvent, and then rotary evaporating the solvent to form a thin film; mixing the thin film with a glucose aqueous solution for hydration treatment, and then ultrasonically dispersing and extruding to obtain astragaloside IV liposomes.

[0053] In some embodiments, the mass ratio of astragaloside IV to the volume ratio of the alcohol solvent is 1 mg: 4-6 mL. Astragaloside IV, lecithin, cholesterol, and DSPE-PEG are weighed in corresponding proportions and dissolved in the alcohol solvent in the aforementioned proportions. The resulting mixed solution can be readily rotary evaporated into a thin film. Furthermore, the alcohol solvent can be ethanol, for example.

[0054] In some embodiments, the mass fraction of the glucose aqueous solution is 4-6%, and the volume ratio of astragaloside IV to the glucose aqueous solution is 1 mg: 0.1-0.2 mL. Astragaloside IV, lecithin, cholesterol, and DSPE-PEG in corresponding proportions are weighed, dissolved in an alcohol solvent, and then rotary evaporated into a thin film. The film is then mixed with the glucose aqueous solution in the above proportions for hydration and ultrasonic dispersion, so that the liposomes can well encapsulate astragaloside IV. The hydrated mixed system can be extruded using an extruder to obtain the final astragaloside IV liposomes, which are then stored at 4°C for use.

[0055] In some embodiments, the film is mixed with the glucose aqueous solution for hydration and then ultrasonically dispersed under conditions including: 180-220W ice bath ultrasonic treatment for 5-10 minutes. This condition can make the mixed system more uniformly dispersed.

[0056] The astragaloside liposomes prepared in the examples of the present application have an average particle size of 60 nm to 120 nm, a polydispersity index (PDI) of less than 0.3, and a zeta potential of approximately -22 mV, and can form a uniformly dispersed nanoparticle preparation.

[0057] A third aspect of the present application provides an application, namely, the use of astragaloside IV in the preparation of a drug for treating viral pneumonia. Based on animal model experiments demonstrating that astragaloside IV can improve lung injury in viral pneumonia and significantly increase the survival rate of mice with influenza A viral pneumonia, the present application provides an application for the preparation of a drug for treating viral pneumonia.

[0058] In some embodiments, the viral pneumonia comprises viral pneumonia caused by influenza A virus.

[0059] In some embodiments, the drug for treating viral pneumonia prepared by astragaloside IV includes the astragaloside liposomes provided by the first aspect of the above-mentioned embodiment of the present application and / or the astragaloside liposomes prepared by the preparation method provided by the second aspect of the embodiment of the present application.

[0060] The present application example constructed a mouse viral pneumonia model, and verified through experiments that the astragaloside liposomes of the present application example can effectively alleviate the weight loss of mice infected with influenza A virus, effectively reduce the lung index of mice infected with influenza A virus, and reduce the lung virus titer accordingly. Further lung pathology examination found that the lung tissue pathological inflammation of different doses of astragaloside liposomes was improved compared with the virus control group, with only a small amount of congestion and less inflammatory cell infiltration in the lung interstitium. Moreover, the astragaloside liposomes of the present application example inhibited the large-scale production of inflammatory mediators in the lungs of mice with influenza A virus pneumonia, thereby improving the viral pneumonia lung damage caused by influenza A virus and exerting antiviral efficacy.

[0061] The following describes the details in conjunction with specific embodiments.

[0062] Example

[0063] 1 Materials and Methods

[0064] 1.1 Main materials and instruments

[0065] 1.1.1 Cell lines

[0066] MDCK cells were purchased from ATCC and subcultured using DMEM / F-12 medium containing 10% FBS.

[0067] 1.1.2 Virus strain

[0068] Influenza A virus A / PR / 8 / 34 strain (H1N1) was screened and preserved in our laboratory as a mouse lung-adapted strain.

[0069] 1.1.3 Animals

[0070] SPF female BALB / c mice, 6-8 weeks old (16-18 g), were purchased from Guangdong Weitonglihua Laboratory Animal Technology Co., Ltd. (License No. SCXK2022-0063). All animal experiments were conducted and completed at the Institute of Testing and Analysis of the Guangdong Academy of Sciences (Guangzhou Analysis and Testing Center, China) (License No. SYXK2019-0201) in strict accordance with the guidelines of the International Committee for Assessment and Accreditation of Laboratory Animal Care. Animals were acclimated for one week before the experiments began. The animal room was maintained at a temperature of 20-28°C, a relative humidity of 40%-70%, and a light intensity of 12 h / d. IVC cages, bedding, and feed were sterilized.

[0071] 1.1.4 Preparation of drugs

[0072] Preparation of astragaloside IV liposomes: 6 mg of astragaloside IV (AS-IV), 40 mg of egg yolk phosphatidylcholine, 5 mg of cholesterol, and 10 mg of DSPE-PEG were weighed and dissolved in 5 mL of ethanol. The organic solvent was then rotary evaporated to form a thin film. 1 mL of 5% glucose solution was added for hydration. The mixture was ultrasonically dispersed in an ice bath at 200 W for 5 min, and then extruded using an extruder to obtain astragaloside IV liposomes (denoted as Lip-AS). The mixture was then stored at 4°C.

[0073] Oseltamivir phosphate was purchased from Sigma, batch number LRAC6504. Preparation of the experimental agent: Accurately weigh oseltamivir phosphate and add PBS to prepare a 7.5 mg / mL solution (the dosage for mice, based on oseltamivir, corresponds to 75 mg / kg / day, oral administration). Mix thoroughly to obtain a clear liquid.

[0074] 1.1.5 Main reagents

[0075] The reagents used in the experiment are shown in Table 1 below.

[0076] Table 1

[0077]

[0078]

[0079] The room temperature in the examples of the present application refers to 18 to 27°C.

[0080] 1.2 Experimental methods

[0081] 1.2.1 Establishment of a viral pneumonia model in BALB / c mice and drug intervention

[0082] (1) Establishment of mouse viral pneumonia model

[0083] 1) After one week of adaptive feeding, the mice were randomly divided into a normal control group (CON or Con, i.e., not treated with the virus), a virus group (PR8, i.e., after virus treatment and modeling, no drug treatment), an oseltamivir group (OSE, i.e., after successful virus treatment and modeling, the mice were treated with oseltamivir at a daily dosage of 75 mg / kg / d, calculated as the weight of the active ingredient of oseltamivir), a high-dose astragaloside IV group (Lip-AS-H, i.e., after successful virus treatment and modeling, the mice were treated with liposome astragaloside IV at a daily dosage of 60 mg / kg / d, calculated as the weight of the active ingredient of astragaloside IV), and a low-dose astragaloside IV group (Lip-AS-L, i.e., after successful virus treatment and modeling, the mice were treated with liposome astragaloside IV at a daily dosage of 30 mg / kg / d, calculated as the weight of the active ingredient of astragaloside IV).

[0084] 2) Experimental mice were anesthetized with isoflurane in a biosafety cabinet. Mice in the virus group, oseltamivir group, high-dose astragaloside IV group, and low-dose astragaloside IV group were infected with 25 μL of influenza A virus A / PR / 8 / 34 strain virus solution at a median lethal dose (LD50) via intranasal instillation. The normal control group was given an equal volume of PBS via nasal instillation (this was defined as day 0 of viral infection). The influenza viral pneumonia model was successfully established when mice showed no weight gain and anorexia on days 1-2 after viral infection, and weight loss on days 3-4. Virus culture of mouse lung tissue homogenate was positive.

[0085] (2) Drug intervention:

[0086] 1) After successful viral infection, the timing began, and every two hours, the experimental mice were administered medication: the high-dose and low-dose astragaloside IV groups received intramuscular injections of the same concentration and varying volumes of the drug daily. The oseltamivir group received oral gavage of 0.2 mL of oseltamivir daily. The normal control and virus-treated mice were gavage-treated with an equal volume of PBS. After successful viral infection, medication was continued for 5 consecutive days. The mice in each group were observed, photographed, and weighed daily.

[0087] 1.2.2 Effects of drugs on lung injury and survival rate of mice with influenza virus pneumonia

[0088] (1) Detection of mouse lung index and lung viral load

[0089] Lung index and lung viral load detection experiments were conducted using the above-mentioned modeling and drug intervention methods.

[0090] 1) Calculation of Mouse Lung Index: After sampling, lung tissue was rinsed with PBS and dried with sterile gauze. The lung index was calculated as follows: lung index = (lung weight / body weight) × 100%. The entire procedure was performed aseptically. The weighed lung tissue was placed in a sterile EP tube and temporarily stored on ice before being transferred to -80°C for storage.

[0091] 2) Lung viral load determination: 1 mL of pre-chilled PBS and three sterile magnetic beads were added to each EP tube. The lung tissue was thoroughly homogenized in a Jingxin homogenizer for 3 minutes. The homogenate was centrifuged at 4°C and 5000 rpm for 5 minutes. The precipitate was discarded, and the upper lung homogenate was retained. The tube was aliquoted into 200 μL / tubes and stored in an ultra-low temperature refrigerator for later use. Penicillin-streptomycin ("double antibiotic") was added to the DMEM / F-12 basal medium to a final antibiotic concentration of 1%. TPCK trypsin was added to the DMEM / F-12 basal medium to a final TPCK concentration of 1.5. g / mL; dilute the lung homogenate in a 10-fold serial dilution using a "double-antibody" culture medium containing TPCK trypsin, add 100 μL / well of the lung homogenate dilution to a previously prepared 96-well monolayer MDCK cell plate, and set up 3 replicates; incubate at 37°C, 5% CO2 for 48 hours; observe the cytopathic effect under a microscope, observe the entire cell plate under a microscope, and record the dilution corresponding to the wells with cytopathic effect; calculate the virus TCID50 according to the Spearman-Karber method, and finally convert it into a 1g value, which is the lung virus titer.

[0092] (2) Detection of mouse survival percentage

[0093] The death protection experiment was observed until the 15th day after the successful viral infection model was established. Furthermore, the death protection experiment used 25uL of influenza A virus A / PR / 8 / 34 strain virus solution at two half-lethal doses (LD50) via intranasal instillation. Other drug administration methods were the same as above. Body weights were recorded daily until the 15th day after the successful viral infection model was established. The death limit for this study was set at 30% weight loss, and the mice were euthanized. Each mouse was weighed daily, and the time of death and number of mice in each group were recorded.

[0094] (3) Lung pathology detection in mouse lung tissue

[0095] Pathological HE staining: Place the lung tissue in a 15mL EP tube containing 4% paraformaldehyde and fix it for 24 hours; Place the tissue in an automatic dehydrator and dehydrate it according to the set dehydration program; After embedding the lung tissue, correct the wax block; Place the wax block with the embedded lung tissue on a microtome, cut out slices with a thickness of about 3μm, float and unfold the slices; Remove the slices and place them on a slide, and dry-bake them at 60℃ for 10 hours; Dewax and rehydrate: Place the slices in an automatic stainer, stain them according to the set program, and seal them with neutral gum; Carefully observe and photograph the inflammatory cell infiltration and other pathological manifestations of lung injury in the lung tissue of each mouse under an optical microscope: The cytoplasm is stained red, the cell nucleus is stained blue, the elastic fibers and collagen fibers are light pink, the muscle tissue and cellulose are dark pink, and the cartilage tissue is dark blue.

[0096] (4) Detection of inflammatory factors and chemokines in lung tissue by qPCR and ELISA

[0097] Add 1ml Trizol to 150μL of lung tissue homogenate supernatant, let it stand at room temperature for 5 minutes to fully lyse, then add 200μL chloroform, shake and mix, let it stand at room temperature for 10 minutes, centrifuge at 4℃ and 13000rpm for 15 minutes, and aspirate the upper aqueous phase to a new EP tube. Add 500μL isopropanol, mix, let it stand at room temperature for 15 minutes, centrifuge at 4℃ and 12000rpm for 10 minutes, discard the supernatant, and retain the precipitate. Add 1ml of anhydrous ethanol, shake, centrifuge at 4℃ and 8000rpm for 5 minutes, discard the supernatant, retain the precipitate, and repeat this step twice. Dry at room temperature for 10 minutes, and dissolve RNA with 40μL DEPC water. Measure its concentration on thermo scientific Nanodrop one. Total RNA (1000ng) was purified by HiScriptⅢRT SuperMix for qPCR The cDNA was reverse transcribed on a PCR System 9700 and then amplified by PCR using an ABI 7500 system. -ΔΔCt The relative gene expression was calculated by the method, and the corresponding PCR amplification system could be prepared by referring to the PCR kit.

[0098] Move all reagents to room temperature (18-25°C) and equilibrate for at least 30 minutes; set up standard wells and test sample wells respectively, add 100 μL of standard and lung homogenate supernatant to each well, shake gently to mix, cover with plate sticker, and incubate at 37°C for 2 hours; discard the liquid, spin dry, and do not wash. Add 100 μL of biotin-labeled antibody working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour. Discard the liquid in the well, spin dry, wash the plate 5 times, soaking for 2 minutes each time, 200 μL / well, and spin dry. Add 90 μL of substrate solution to each well in sequence, and develop color at 37°C in the dark for 15-30 minutes. Add 50 μL of stop solution to each well in sequence to terminate the reaction. Measure the optical density (OD value) at a wavelength of 450 nm using a microplate reader within 5 minutes after the reaction is terminated.

[0099] 1.2.3 Data processing:

[0100] Statistical analysis was performed using IBM SPSS Statistics 21 software. All data were expressed as mean ± standard deviation. One-way analysis of variance was used for data between multiple groups, and t-test was used for data between two groups. A P value < 0.05 was considered statistically significant.

[0101] 2 Results

[0102] 2.1 Characterization of Astragaloside Liposomes

[0103] The astragaloside liposomes were diluted with ultrapure water, and the particle size and potential were measured using a Malvern particle size analyzer. The results are shown in Table 2. The average particle size of the astragaloside liposomes was about 70 nm, the PDI was less than 0.3, and the Zeta potential was about -22 mV.

[0104] Table 2

[0105]

[0106] The morphology of astragaloside IV liposomes was observed using a transmission electron microscope. The method was as follows: Astragaloside IV liposome solution was mixed with phosphomolybdic acid staining solution in a volume ratio of 1:1, then added dropwise to the front of the copper mesh, kept in the dark for more than 2 hours, and then excess water was absorbed with filter paper and observed using a transmission electron microscope. The results are shown in Figure 2. Figure 1 As shown, the nanoparticles of astragaloside liposomes are uniformly dispersed spheres.

[0107] 2.2 Stability of Astragaloside Liposomes

[0108] The particle size of the prepared astragaloside liposomes was measured every other day, and then a curve of the relationship between the particle size and the number of days was drawn. The results are as follows: Figure 2 The data show that after the preparation of astragaloside liposomes, the particle size of astragaloside liposomes did not change much within 7 days. Although there was a trend of increase, it was always below 100 nm, indicating that astragaloside liposomes have good stability.

[0109] 2.3 Effects of astragaloside liposomes on lung injury, survival rate, and viral load in mice with viral pneumonia

[0110] To evaluate the anti-influenza virus efficacy of astragaloside liposomes in vivo, a viral pneumonia model was established in BALB / c mice using 1 LD50 of influenza A virus A / PR / 8 / 34 (H1N1) via intranasal inoculation. Following successful A / PR / 8 / 34 (H1N1) infection, mice in the virus-treated group exhibited typical influenza infection symptoms, including ruffled hair, laziness, hunched back, unsteady gait, and difficulty breathing. Both the high- and low-dose groups of astragaloside liposomes showed improvements.

[0111] like Figure 3 and Figure 4As shown, astragaloside liposome treatment slowed the weight loss of mice induced by influenza virus infection. The weight of mice in the virus group began to decline continuously on day 3 after infection, while the weight of mice in the Lip-AS-H group decreased more slowly after virus infection and began to recover on day 11. In addition, high- and low-dose Lip-AS treatment significantly reduced the lung index and alleviated pulmonary edema in mice on day 5 after virus infection. In addition, the Lip-AS-H group significantly improved the survival rate of mice with influenza virus pneumonia due to the increased dose of astragaloside IV (0% survival rate in the virus group vs. 20% survival rate in the Lip-AS-H group).

[0112] like Figure 5 As shown, the cytopathic effect assay was further used to detect the viral load in the mouse lung tissue homogenate. The results showed that high and low doses of Lip-AS intervention reduced the viral load in the mouse lungs.

[0113] In summary, on the 5th day after influenza virus infection, the lung index of mice increased due to pulmonary edema, consolidation, etc. After intervention with astragaloside liposomes, the lung index of mice was significantly reduced, the pulmonary edema of mice was alleviated, and the viral load in the lung tissue was reduced on the 5th day after viral infection.

[0114] 2.4 Pulmonary pathology results

[0115] like Figure 6 HE staining of lung tissue sections showed that mice in the virus-treated group developed interstitial pneumonia, characterized by extensive alveolar atrophy, thickened alveolar septa, extensive inflammatory cell infiltration of the alveolar walls and bronchioles, and dilated and congested alveolar and interstitial blood vessels. Compared with the virus-treated group, high-dose astragaloside IV in the Lip-AS-H group significantly inhibited the development of interstitial pneumonia, with no significant alveolar atrophy and marked improvement in alveolar and interstitial blood vessel congestion. Low-dose Lip-AS-L also demonstrated some improvement.

[0116] 2.5 Expression of inflammatory and chemokines in each group

[0117] The examples of the present application further explore the potential mechanism by which Lip-AS improves lung damage and improves the survival rate of mice with influenza virus pneumonia, and studies the effect of Lip-AS on inflammatory factors.

[0118] The results of mRNA level detection were as follows Figure 7 As shown in the results, after influenza virus infection, the mRNA levels of IL-6, IL-1β, CXCL-1, MCP-1, MIP-1β, and IFN-γ in the lungs of mice in the Lip-AS-H and Lip-AS-L groups were significantly reduced, and the effect of the Lip-AS-H group was better, which could significantly inhibit the expression of these lung inflammatory factors.

[0119] Granulocyte-macrophage colony-stimulating factor (GM-CSF) can promote the differentiation of M1 macrophages and lead to the production of higher levels of inflammatory cytokines (such as IL-1, IL-6 and TNF-α). These factors are involved in the inflammatory factor storm, causing serious damage to the body. Chemokine that regulates the expression and secretion of activated normal T cells (RANTES / Rantes) is a member of the CC subfamily of chemokines, also known as CCL5. It has a wide range of sources. Most tissues can release CCL5 under the stimulation of inflammatory factors IL-1β or TNF-α. CCL5 can activate inflammatory cells, prolong and aggravate the inflammatory response. Therefore, the protein levels of GM-CSF and RANTES can well reflect the level of inflammatory factors in the body. The test results of the two proteins are shown in the figure below. Figure 8 As shown in the figure, both the Lip-AS-H group and the Lip-AS-L group could reduce the levels of the two proteins, and the Lip-AS-H group had a better overall effect.

[0120] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. An astragaloside liposome for treating viral pneumonia, characterized in that: include: Astragaloside IV and liposome loaded with the astragaloside IV, wherein the liposome comprises phosphatidylcholine, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol.

2. The astragaloside liposome according to claim 1, wherein The mass ratio of the lecithin, the cholesterol and the distearoylphosphatidylethanolamine-polyethylene glycol is (32-48): (4-6): (8-12); or, The mass ratio of the astragaloside IV, the lecithin, the cholesterol and the distearoylphosphatidylethanolamine-polyethylene glycol is 6:(32-48):(4-6):(8-12).

3. The astragaloside liposome according to claim 1 or 2, characterized in that The average particle size of the astragaloside IV liposome is 60 nm to 120 nm.

4. A method for preparing astragaloside liposomes according to any one of claims 1 to 3, characterized in that: include: The astragaloside IV liposomes are prepared by using astragaloside IV, lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol as raw materials through a thin film hydration method.

5. The method for preparing astragaloside liposomes according to claim 4, wherein: The steps of preparing the astragaloside IV liposomes by using astragaloside IV, lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol as raw materials through a thin film hydration method include: The astragaloside IV, the lecithin, the cholesterol and the distearoylphosphatidylethanolamine-polyethylene glycol are dissolved in an alcohol solvent, and then the solvent is removed by rotary evaporation to form a film; the film is mixed with a glucose aqueous solution for hydration treatment, and then ultrasonically dispersed and extruded to obtain the astragaloside IV liposomes.

6. The method for preparing astragaloside liposomes according to claim 5, wherein: The mass ratio of the astragaloside IV to the alcohol solvent is 1 mg: 4-6 mL; and / or The mass fraction of the glucose aqueous solution is 4-6%, and the volume ratio of the mass of astragaloside IV to the glucose aqueous solution is 1 mg:0.1-0.2 mL.

7. The method for preparing astragaloside liposomes according to claim 5 or 6, characterized in that: The ultrasonic dispersion conditions include: 180-220W ice bath ultrasonication for 5-10 minutes.

8. Application of astragaloside IV in the preparation of drugs for treating viral pneumonia.

9. The use according to claim 8, characterized in that The viral pneumonia includes viral pneumonia caused by influenza A virus.

10. The use according to claim 8, characterized in that The drug for treating viral pneumonia comprises the astragaloside liposomes according to any one of claims 1 to 4 and / or the astragaloside liposomes prepared by the preparation method according to any one of claims 5 to 8.

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