Eucommia lipidosome, preparation method thereof and medicine for treating sepsis lung injury

Abstract

The invention relates to eucommia ulmoides liposome and a preparation method thereof and a medicine for treating sepsis lung injury, the preparation method comprises the following steps: soaking eucommia ulmoides in water for reflux extraction, concentrating the obtained extract, and performing vacuum freeze drying to obtain eucommia ulmoides extract; adding the eucommia ulmoides extract into water, carrying out ultrasonic dissolution, centrifuging, and collecting supernatant, namely a water phase; adding soybean lecithin and cholesterol into an organic solvent, and performing ultrasonic dissolution to obtain an oil phase; mixing the water phase and the oil phase, performing ultrasonic treatment to form primary emulsion, and dropwise adding the primary emulsion into water in a stirring state to form W / O / W type multiple emulsion; (4) stirring the multiple emulsion in a water bath at 30-80 DEG C for 5-60 minutes to obtain a crude liposome suspension, performing probe ultrasonic treatment in an ice-water bath, and centrifuging to obtain supernate; and mixing the supernate with trehalose, pre-freezing, and carrying out vacuum freeze drying to obtain the eucommia ulmoides liposome. The eucommia ulmoides liposome and the medicine for treating the sepsis lung injury can reduce and improve the lung tissue pathology of animals infected with sepsis, improve lung inflammation and improve the survival rate of the animals infected with sepsis lung injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an eucommia ulmoides liposome, a preparation method thereof, and a drug for treating sepsis-induced lung injury. Background Art

[0002] Sepsis is one of the most common causes of death in critically ill patients globally. Its essence is the imbalance between the pro-inflammatory and anti-inflammatory processes in the body, leading to a systemic inflammatory cascade reaction and specific immune dysfunction, and ultimately evolving into multiple organ dysfunction syndrome.

[0003] In addition to conventional anti-infection and organ function support therapies, the clinical treatment measures for sepsis-related acute lung injury (ALI) are very limited. It is urgent to find more effective treatment measures to save the lives of more sepsis patients. Currently, antibiotics, fluid resuscitation, blood purification, mechanical ventilation, and multi-organ support therapies are widely used in the clinical treatment of sepsis. However, these treatment methods are prone to increase bacterial drug resistance, cause adverse reactions in the body, and have poor treatment effects. Compared with the existing treatment measures, traditional Chinese medicine has the advantages of small side effects and multi-molecule multi-target effects, and has gradually become an effective complementary alternative therapy.

[0004] PANoptosis is a form of cell death involving the simultaneous activation of multiple cell death pathways (including apoptosis, necrosis, pyroptosis, etc.). In recent years, studies have found that the PANoptosis pathway plays an important role in the process of sepsis-induced lung injury.

[0005] Eucommia ulmoides Oliv. (scientific name) is the dried bark of plants in the genus Eucommia of the family Eucommiaceae. It is a commonly used tonic medicinal material in traditional Chinese medicine. Its main components include pinoresinol diglucoside, eucommia gum, iridoid compounds, flavonoids, polysaccharides, amino acids, etc. It is included in the Chinese Pharmacopoeia 2020 edition, and its functions and indications are to tonify the liver and kidney, strengthen tendons and bones, and prevent miscarriage, and it is used for liver and kidney deficiency, lumbar and knee soreness, weakness of tendons and bones, dizziness, vaginal bleeding during pregnancy, and threatened abortion. Although eucommia ulmoides has been widely used in the treatment of various diseases in traditional Chinese medicine, there is relatively little research on its treatment of sepsis. Basically, it is used as a traditional Chinese medicine ingredient in a drug formula and needs to be compounded with other traditional Chinese medicines to exert its medicinal effects. Currently, there are no clinical research reports on the use of eucommia ulmoides alone in the treatment of sepsis.

[0006] Liposomes are spherical vesicles composed of cholesterol and phospholipids, with good biocompatibility. The hydrophilic end inside and the hydrophobic end in the lipid bilayer enable them to encapsulate both hydrophilic and lipophilic drugs simultaneously, making them suitable for various drug types; liposomes have good biocompatibility, based on natural phospholipids and cholesterol, significantly enhancing the cellular uptake rate and therapeutic effect of drugs, while having low toxicity. Through appropriate surface modification, the circulation time of liposomes in the blood can be extended. Liposomes can achieve sustained and controlled release of drugs, reducing the accumulation of drugs in normal tissues. As an efficient drug delivery carrier, liposomes have great potential in the biomedical field. Summary of the Invention

[0007] Based on the above-mentioned drawbacks and deficiencies in the prior art, one of the objectives of the present invention is to at least solve one or more of the above problems existing in the prior art. In other words, one of the objectives of the present invention is to provide a Eucommia ulmoides liposome, its preparation method, and a drug for treating sepsis-induced lung injury that meet one or more of the foregoing requirements.

[0008] To achieve the above-mentioned invention objectives, the present invention adopts the following technical solutions:

[0009] A preparation method of Eucommia ulmoides liposomes, comprising the following steps:

[0010] (1) Eucommia ulmoides is soaked in water for reflux extraction, and the obtained extract is concentrated and freeze-dried under vacuum to obtain Eucommia ulmoides extract;

[0011] (2) The Eucommia ulmoides extract is added to water and dissolved by ultrasonic treatment. After centrifugation, the supernatant is collected, which is the aqueous phase; soybean lecithin and cholesterol are added to an organic solvent and dissolved by ultrasonic treatment to obtain an oil phase;

[0012] (3) The aqueous phase and the oil phase are mixed and ultrasonicated to form a primary emulsion. The primary emulsion is added dropwise to water under stirring to form a W / O / W type multiple emulsion;

[0013] (4) The W / O / W type multiple emulsion is stirred in a water bath at 30 - 80 °C for 5 - 60 min to obtain a crude liposome suspension; the crude liposome suspension is treated by probe sonication in an ice-water bath, and the supernatant is obtained after centrifugation;

[0014] (5) The supernatant is mixed with trehalose, pre-frozen, and then freeze-dried under vacuum to obtain Eucommia ulmoides liposomes.

[0015] As a preferred embodiment, in the step (1), the solid-liquid ratio of Eucommia ulmoides to water is 1:(1 - 30) mg / mL;

[0016] The technological process of reflux extraction includes: boiling with strong fire first, then turning to gentle fire for reflux, and the reflux time is 0.5 - 3 h; the residue is refluxed 1 - 3 times under the same reflux conditions.

[0017] As a preferred embodiment, in the step (2), the solid-liquid ratio of eucommia ulmoides extract to water is (8-12) mg / mL, the centrifugation speed is 1000-16000 rpm, and the time is 1-30 min.

[0018] As a preferred embodiment, in the step (2), the mass ratio of soy lecithin to cholesterol is (1-20):1;

[0019] The organic solvent is at least one of chloroform, ether, dichloromethane, and ethyl acetate.

[0020] As a preferred embodiment, in the step (3), the volume ratio of the aqueous phase to the oil phase is (0.4-2.5):1;

[0021] The ultrasonic frequency is 10-60 kHz, and the time is 0.5-10 min.

[0022] As a preferred embodiment, in the step (3), the volume ratio of the primary emulsion to water is (0.4-2.5):1.

[0023] As a preferred embodiment, in the step (4), the frequency of probe ultrasonic treatment is 25-500 W, and the time is 1-30 min.

[0024] As a preferred embodiment, in the step (5), the solid-liquid ratio of trehalose to the supernatant is 0.5-5% mg / mL.

[0025] The present invention also provides eucommia ulmoides liposomes prepared by the preparation method described in any one of the above embodiments.

[0026] The present invention also provides a drug for treating sepsis-induced lung injury, comprising the eucommia ulmoides liposomes described in the above embodiments.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] The eucommia ulmoides liposomes of the present invention and the drug for treating sepsis-induced lung injury can reduce and improve the lung tissue pathology of animals infected with sepsis, improve pulmonary inflammation, and increase the survival rate of animals with sepsis-induced lung injury; they can reduce the expression and secretion levels of serum inflammatory factors IL-1β, IL-6, and TNF-α in cell models stimulated by LPS and animals infected with sepsis, inhibit the expression of proteins related to the pan-apoptosis signaling pathway such as apoptosis, necroptosis, and pyroptosis in cells and lung tissues, and have potential therapeutic effects on sepsis, and the effects are significantly higher than those of eucommia ulmoides extract. Description of the Drawings

[0029] Figure 1HPLC chromatograms (A) of Eucommia ulmoides extract (DZ), Eucommia ulmoides liposomes (Nano-LP-DZ), and free Eucommia ulmoides (Free DZ) in the liquid after coating Eucommia ulmoides extract with liposomes, and TEM image (B) of Eucommia ulmoides liposomes for Example 1 of the present invention;

[0030] Figure 2 Bio-distribution imaging in vitro (A) and in vivo (B) of coumarin-6 fluorescently labeled Eucommia ulmoides liposomes for Example 1 of the present invention;

[0031] Figure 3 Cytotoxicity test results graphs of Eucommia ulmoides extract DZ (A) and Eucommia ulmoides liposomes Nano-LP-DZ (B) against different macrophages for Example 1 of the present invention;

[0032] Figure 4 Comparison graph of in vitro anti-inflammatory effects between Eucommia ulmoides extract DZ and Eucommia ulmoides liposomes Nano-LP-DZ for Example 1 of the present invention;

[0033] Figure 5 Comparison graph of in vivo anti-inflammatory effects between Eucommia ulmoides extract DZ and Eucommia ulmoides liposomes Nano-LP-DZ for Example 1 of the present invention; A: Comparison graph of gross morphology of lung tissue, B: Comparison graph of HE-stained tissue sections of lung tissue, C: Statistical graph of wet / dry weight ratio of lung and tissue injury score, D: Comparison graph of levels of inflammatory factors IL-1β, IL-6, and TNF-α in lung tissue;

[0034] Figure 6 Schematic diagram of Eucommia ulmoides liposomes Nano-LP-DZ regulating the pan-apoptosis process in a sepsis model to relieve cell death for Example 1 of the present invention; among them, A is the fluorescence microscope image of cell death, B: Western blot detection of the expression of key proteins of PANoptosis (in vitro experiment), C: Western blot detection of PANoptosis-related proteins (in vivo experiment, mouse lung tissue);

[0035] Figure 7 Schematic diagram of Eucommia ulmoides liposomes Nano-LP-DZ regulating and inhibiting the pan-apoptosis (PANoptosis) process in a sepsis model through TGF-β1 for Example 1 of the present invention; among them, A: Correlation analysis graph between TGF-β1 and key factors related to PANoptosis, B: Regulatory effect of Nano-LP-DZ on the expression level of TGF-β1 (Western blot), C: Western blot analysis of Nano-LP-DZ regulating the expression of key proteins of PANoptosis through the TGF-β1 signaling pathway. Detailed implementation mode

[0036] The Eucommia ulmoides liposomes of the present invention, their preparation methods, and drugs for treating septic lung injury will be further described below.

[0037] The present invention uses a liposome drug carrier to prepare Eucommia ulmoides liposomes from traditional Chinese medicine Eucommia ulmoides, which have small particle size, uniform distribution, high bioavailability, and good cell uptake effect. Compared with traditional Eucommia ulmoides extracts, the Eucommia ulmoides liposomes of the present invention have better application effects in treating sepsis. By reducing the expression and secretion levels of cellular inflammatory factors, inhibiting the pan-apoptosis signaling pathway of cells, and improving the lung injury tissues of septic mice, the survival rate of septic mice is increased, showing good application prospects for the clinical treatment of sepsis. As an old drug used in a new way, Eucommia ulmoides greatly shortens the R & D cost and risk during the drug development process for sepsis. Further combined with liposome technology, it provides a new way to solve sepsis-related acute lung injury.

[0038] For the Eucommia ulmoides liposomes of the present invention, as an old drug used in a new way, Eucommia ulmoides shows good therapeutic effects in sepsis models beyond the records in the Chinese Pharmacopoeia and has development value in the field of clinical sepsis. Moreover, the Eucommia ulmoides liposomes have better effects in sepsis models than Eucommia ulmoides extracts, verifying that Eucommia ulmoides can act in a new field and its efficacy is better after liposome formation. In addition, Eucommia ulmoides liposomes can effectively improve the pathological damage caused by septic lung injury by regulating the pan-apoptosis pathway and increase the survival rate of septic mice, clarifying the action mechanisms of Eucommia ulmoides and Eucommia ulmoides liposomes in treating septic lung injury.

[0039] In one embodiment, the above-mentioned sepsis also includes septic lung injury-related diseases, such as endotoxemia, severe sepsis, and septic shock, which further cause symptoms such as multiple organ dysfunction syndrome throughout the body. Their pathogenic causes and treatment methods are similar.

[0040] In one embodiment, the above-mentioned drug for treating septic lung injury further includes at least one pharmaceutically acceptable excipient, and the excipients include but are not limited to fillers, disintegrants, lubricants, stabilizers, or combinations thereof.

[0041] In one embodiment, the above-mentioned drug for treating septic lung injury is a drug that can improve the lung tissue pathology of animals with septic lung injury.

[0042] In one embodiment, the above-mentioned drug for treating septic lung injury is a drug that can increase the survival rate of animals with septic lung injury.

[0043] In one embodiment, the above-mentioned drug for treating septic lung injury is a drug that can improve the pulmonary edema of septic animals.

[0044] In one embodiment, the drug for treating septic lung injury is a drug capable of inhibiting the expression levels of inflammatory factors in the lung tissues of animals infected with sepsis. Among them, the inflammatory factors include IL-1β, IL-6, and TNF-α.

[0045] In one embodiment, the drug for treating septic lung injury is a drug capable of inhibiting the pan-apoptosis signaling pathway of cells. The involved pan-apoptosis signaling pathway includes apoptosis, necrosis, and pyroptosis.

[0046] Specifically, the preparation method of the eucommia ulmoides liposome of the present invention includes the following steps:

[0047] (1) Soak eucommia ulmoides in water for reflux extraction, concentrate the obtained extract, and vacuum freeze-dry it to obtain eucommia ulmoides extract.

[0048] (2) Add the eucommia ulmoides extract to water and dissolve it by ultrasonic treatment. After centrifugation, collect the supernatant, which is the aqueous phase.

[0049] Add soybean lecithin and cholesterol to an organic solvent and dissolve it by ultrasonic treatment to obtain an oil phase.

[0050] (3) Mix the aqueous phase and the oil phase, and form a primary emulsion by ultrasonic treatment. Drop the primary emulsion into the stirred water drop by drop to form a W / O / W type multiple emulsion.

[0051] (4) Stir the W / O / W type multiple emulsion in a water bath at 30-80°C for 5-60 minutes to obtain a crude liposome suspension; subject the crude liposome suspension to probe ultrasonic treatment in an ice-water bath, and centrifuge to obtain a supernatant.

[0052] (5) Mix the supernatant with trehalose, pre-freeze it, and then vacuum freeze-dry it to obtain eucommia ulmoides liposome.

[0053] In one embodiment, in the above step (1), the solid-liquid ratio of eucommia ulmoides to water can be 1:(1-30) mg / mL, and it can be specifically determined according to actual application requirements.

[0054] The technological process of the reflux extraction includes: after boiling with strong fire, switch to slow fire for reflux, and the reflux time is 0.5-3 hours; the residue is refluxed 1-3 times under the same reflux conditions.

[0055] In one embodiment, in the above step (2), the solid-liquid ratio of the eucommia ulmoides extract to water can be (8-12) mg / mL, the centrifugation speed can be 1000-16000 rpm, and the time can be 1-30 minutes, and it can be specifically determined according to actual application requirements.

[0056] In one embodiment, in the above step (2), the mass ratio of soybean lecithin to cholesterol can be (1-20):1, which can be specifically determined according to actual application requirements;

[0057] The above organic solvent is at least one of chloroform, ether, dichloromethane, and ethyl acetate, which can be specifically determined according to actual application requirements.

[0058] In one embodiment, in the above step (3), the volume ratio of the aqueous phase to the oil phase can be (0.4-2.5):1, which can be specifically determined according to actual application requirements;

[0059] The frequency of ultrasonic wave can be 10-60 kHz and the time can be 0.5-10 min, which can be specifically determined according to actual application requirements.

[0060] In one embodiment, in the above step (3), the volume ratio of the primary emulsion to water can be (0.4-2.5):1, which can be specifically determined according to actual application requirements.

[0061] In one embodiment, in the above step (4), the frequency of probe ultrasonic treatment can be 25-500 W and the time can be 1-30 min, which can be specifically determined according to actual application requirements.

[0062] In one embodiment, in the above step (5), the solid-liquid ratio of trehalose to Nano-LP-DZ can be 0.5-5% mg / mL, which can be specifically determined according to actual application requirements.

[0063] The present invention also provides Eucommia ulmoides liposomes prepared by the above preparation method.

[0064] The present invention also provides a drug for treating sepsis-induced lung injury, including the above Eucommia ulmoides liposomes.

[0065] The following further explains the Eucommia ulmoides liposomes of the present invention, its preparation method, and the drug for treating sepsis-induced lung injury through specific examples.

[0066] Example 1:

[0067] The preparation method of the Eucommia ulmoides liposomes in this example includes the following steps:

[0068] (1) Preparation of Eucommia ulmoides extract;

[0069] The dried Eucommia ulmoides was soaked in water at a solid-liquid ratio of 1:10 mg / mL, boiled with strong fire, and then refluxed with gentle fire for 60 min to obtain the extract. The residue was refluxed twice under the same conditions, and the extracts were combined, concentrated, and freeze-dried under vacuum to obtain the Eucommia ulmoides extract DZ; stored at room temperature in a sealed manner for later use;

[0070] (2) Preparation of aqueous phase;

[0071] Accurately weigh 50.00 mg of Eucommia ulmoides extract DZ and dissolve it in 5 mL of pure water. Sonicate to dissolve, centrifuge at 10000 rpm for 10 min to remove large particles, and collect the supernatant, which is the aqueous phase and reserved for later use;

[0072] (3) Preparation of the oil phase;

[0073] Accurately weigh 50.00 mg of soybean lecithin and 15.00 mg of cholesterol and dissolve them in 10 mL of the organic solvent dichloromethane. Sonicate to dissolve to obtain the oil phase;

[0074] (4) Preparation of the W / O / W multiple emulsion;

[0075] Mix the aqueous phase from step (2) and the oil phase from step (3) at a volume ratio of 4:5, and sonicate at 53 kHz for 10 min to form the primary emulsion; gradually add 15 mL of the primary emulsion dropwise to 7.5 mL of pure water under stirring (2500 rpm) to form the W / O / W multiple emulsion;

[0076] (5) Preparation of the Eucommia ulmoides liposome supernatant;

[0077] Stir the W / O / W multiple emulsion in a water bath at 55 °C for 30 min to remove the organic solvent to obtain a crude liposome suspension; subject the crude liposome suspension to probe sonication treatment in an ice-water bath (100 W, 5 min, on: 5 s, off: 4 s), and take the supernatant after centrifugation to obtain the Eucommia ulmoides liposome supernatant;

[0078] (6) Freeze-drying of the Eucommia ulmoides liposome supernatant;

[0079] Mix trehalose with the Eucommia ulmoides liposome supernatant at a solid-liquid ratio of 2% mg / mL, pre-freeze at -80 °C and then vacuum freeze-dry, collect the freeze-dried sample for later use to obtain Eucommia ulmoides liposome Nano-LP-DZ.

[0080] The Eucommia ulmoides liposomes and intermediate products prepared in the above examples were tested, analyzed, and characterized as follows:

[0081] 1. In this example, a high-performance liquid chromatography (HPLC) system (LC-20A, Shimadzu) was used to analyze the free DZ content in Eucommia ulmoides extract DZ, Nano-LP-DZ, and the liquid after coating with Eucommia ulmoides liposomes. The chromatographic column was a WondaSil-C18-WR column (150 mm × 4.6 mm, 5 μm), the mobile phase was acetonitrile (A) and 0.1% phosphoric acid aqueous solution (B), and gradient elution was carried out: 0 - 35 minutes, 6 - 28% A; 35 - 60 minutes, 28 - 35% A; 60 - 70 minutes, 35% A; 70 - 75 minutes, 35 - 6% A; the detection wavelength was 230 nm, the flow rate was 0.8 mL / min, the column temperature was 25 °C, and the injection volume was 10 μL. The HPLC system detection results are shown inFigure 1 As shown in Figure A, the x-axis represents the retention time and the y-axis represents the absorbance. By comparing the liquid chromatograms of DZ and Nano-LP-DZ, the retention times of the peaks are almost the same, indicating that the liposome carrier material has no significant effect on the detection of Eucommia ulmoides components. By comparing the liquid chromatograms of Nano-LP-DZ and the liquid after coating Eucommia ulmoides liposomes, it can be seen that the liposomes can encapsulate no less than 80% of the Eucommia ulmoides extract DZ.

[0082] 2. Calculate the encapsulation efficiency of Nano-LP-DZ by ultrafiltration centrifugation method;

[0083] The operation steps are as follows: Take 400 μL of liposome suspension and place it in the inner tube of a 100 kDa ultrafiltration tube. Centrifuge at 6000 revolutions per minute for 20 minutes for ultrafiltration separation. After collecting the filtrate, dilute it with purified water and make the volume up to 10 mL, and then perform quantitative analysis using a high performance liquid chromatography (HPLC) system;

[0084] The encapsulation efficiency is calculated according to the following formula: Encapsulation efficiency (%) = (1 - amount of unencapsulated DZ / total amount of DZ) × 100;

[0085] The encapsulation efficiency of the Eucommia ulmoides liposomes in this example is 80%.

[0086] 3. Particle size and Zeta potential analysis;

[0087] Use a laser particle size analyzer to measure the particle size distribution and Zeta potential of Eucommia ulmoides liposomes Nano-LP-DZ to evaluate its physical stability and particle size characteristics. At the same time, observe the microstructure of the liposomes through a transmission electron microscope (TEM). The specific operation is as follows: After diluting the Nano-LP-DZ sample with purified water to an appropriate concentration, take an appropriate amount of the suspension and drop it on the surface of a carbon-supported copper mesh (C-Cu). Gently touch the filter paper to remove the excess liquid, and let it dry naturally at room temperature. After drying, observe its morphological structure under the TEM. As Figure 1 shown in Figure B, Nano-LP-DZ presents uniformly distributed spherical-like particles in the TEM image, with clear boundaries and good dispersibility; the Eucommia ulmoides liposome particles are indicated by the black arrows in the figure, and their size is less than 200 nm, indicating that the Eucommia ulmoides liposomes have ideal particle size characteristics and are suitable for use in in vivo drug delivery systems.

[0088] The particle size, polydispersity index (PDI), and Zeta potential of liposomes were detected using a laser particle size analyzer; the particle size, PDI, and Zeta potential distributions of Nano-LP-DZ and blank liposomes in this example are shown in Table 1. The particle size of Nano-LP-DZ (125.53 ± 1.81 nm) was slightly smaller than that of the blank liposomes (131.17 ± 0.91 nm), because the amphiphilic compounds in Eucommia ulmoides extract reduced the surface tension of the liposome membrane. The smaller particle size was beneficial for cell uptake and improved absorption efficiency. The PDI values of Nano-LP-DZ and blank liposomes (Blank Liposome) in this example were both lower than 0.3, indicating uniform particle dispersion. In addition, the Zeta potential of the blank liposomes was -45.33 ± 2.87 mV, and that of Nano-LP-DZ was -40.57 ± 2.25 mV. A Zeta potential value exceeding ±30 mV indicates strong electrostatic repulsion between particles, which helps maintain storage stability.

[0089] Table 1 Comparison of parameters between Eucommia ulmoides liposomes Nano-LP-DZ and blank liposomes Nano-LP in this example

[0090] Sample Particle Size / nm PDI Zeta Potential / mV Nano-LP 131.17±0.91 0.186±0.016 -45.33±2.87 Nano-LP-DZ 125.53±1.81 0.156±0.022 -40.57±2.25

[0091] Among them, the difference between the preparation method of the above blank liposomes Nano-LP and that of Eucommia ulmoides liposomes Nano-LP-DZ is only that the aqueous phase does not contain Eucommia ulmoides extract, and other steps remain the same.

[0092] 4. Preparation of Eucommia ulmoides liposomes loaded with fluorescent probes and in vitro and in vivo biodistribution imaging;

[0093] (a) In vitro biodistribution imaging of Eucommia ulmoides liposomes loaded with fluorescent probes;

[0094] The cell uptake effect was analyzed by fluorescently labeling Eucommia ulmoides liposomes with Coumarin-6. MH-S cells were seeded in 6-well plates and incubated with Coumarin-6-labeled Eucommia ulmoides liposomes Nano-LP-DZ (100 μg / mL) at 37°C for 1, 2, 4, and 6 hours, respectively. At each time point, the cells were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, and imaged using a fluorescence microscope in the green fluorescence channel (FITC). The results are shown in Figure 2 Figure A. The uptake of Coumarin-6-labeled Nano-LP-DZ in MH-S macrophages was time-dependent; the fluorescence intensity gradually increased after 1 hour, reached a peak at 2 hours, and then gradually decreased, and the fluorescence signal almost disappeared at 6 hours. This result indicates that Nano-LP-DZ can be rapidly and efficiently taken up by cells and may be degraded or released intracellularly, resulting in a decrease in fluorescence intensity.

[0095] (b) In vivo biodistribution imaging;

[0096] DiR-labeled Nano-LP-DZ (2.5 mg or 5 mg) was instilled into anesthetized mice via intratracheal injection. After 1 hour, whole-body fluorescence images were collected and major organs were isolated, and fluorescence intensity was measured to evaluate biodistribution. The in vivo biodistribution results are shown as Figure 2 shown in B. After 1 hour of intratracheal administration, Nano-LP-DZ was mainly enriched in the lung tissue, while the distribution amounts in other organs such as the heart, liver, kidney, and spleen were extremely low.

[0097] 5. Cytotoxicity experiments of eucommia bark extract DZ and eucommia bark liposome Nano-LP-DZ;

[0098] The CCK-8 method was used to evaluate the cell viability after treatment with DZ (concentrations of 10–1000 μg / mL) and Nano-LP-DZ (concentrations of 100 and 200 μg / mL).

[0099] MH-S, RAW 264.7, and NR8383 macrophages were seeded at 5×10 3 cells / well in 96-well plates and allowed to adhere overnight. The cells were treated with different concentrations of eucommia bark extract DZ (10, 100, 200, 500, 1000 μg / mL), blank liposome Nano-LP (100 μg / mL), and eucommia bark liposome Nano-LP-DZ (100 and 200 μg / mL) for 24 hours. 10 μL of CCK-8 solution was added to each well and incubated at 37 °C for 2 hours. The absorbance was measured at 450 nm using a microplate reader, and the cell viability was expressed as a percentage of the control group. As shown in Figure 3 A and Figure 3 B, the cytotoxicity experiments showed that eucommia bark liposome Nano-LP-DZ had no obvious toxicity to macrophages at concentrations of 100 and 200 μg / mL.

[0100] 6. Comparison of in vitro anti-inflammatory effects of eucommia bark extract DZ and eucommia bark liposome Nano-LP-DZ;

[0101] Mouse MH-S macrophage cell line was cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37 °C and 5% CO2. The cells were treated as follows: control group, lipopolysaccharide (LPS, 1 μg / ml), LPS + DZ (100 μg / ml), and LPS + Nano-LP-DZ (100 μg / ml);

[0102] The cytokine levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the cell culture supernatant of LPS-stimulated MH-S macrophages treated with DZ (100 μg / mL) or Nano-LP-DZ (100 μg / mL) were detected using an ELISA kit. As Figure 4 shown, in LPS-stimulated MH-S macrophages, Nano-LP-DZ significantly reduced the levels of IL-1β, IL-6 and TNF-α.

[0103] 7. Comparison of the in vivo anti-inflammatory effects of eucommia ulmoides extract DZ and eucommia ulmoides liposome Nano-LP-DZ;

[0104] Male C57BL / 6 mice at 8 weeks of age (weighing 22 - 26 g) were acclimated to the environment for one week before the experiment and received different experimental treatments, including the sham operation group Sham, the cecal ligation and puncture group CLP, the group given eucommia ulmoides extract DZ (10 mg) by oral gavage 2 hours after the operation, and the group given eucommia ulmoides liposome Nano-LP-DZ (2.5 mg or 5 mg) by intratracheal instillation 2 hours after the operation;

[0105] Lung tissue samples were collected, weighed and the ratio of wet weight to dry weight of the lung tissue was calculated for subsequent analysis. The lung tissue samples were fixed in 10% neutral buffered formalin for 24 hours and then embedded in paraffin; 5-μm-thick sections were prepared and fixed on glass slides; after dewaxing and hydration of the sections, the cell nuclei were stained with hematoxylin and the cytoplasm was stained with eosin; after staining, the sections were observed under an optical microscope and images were taken for morphological analysis;

[0106] The quantitative analysis of the inflammatory injury score was blindly evaluated based on the following criteria: alveolar congestion, hemorrhage, neutrophil infiltration into the alveoli or blood vessel walls, and alveolar wall thickening. Each parameter was scored from 0 (no injury) to 4 (severe injury), and the sum of the scores of each sample reflected the degree of inflammation; the results were as Figure 5 A, Figure 5 B and Figure 5 C shown, indicating that in the CLP-induced acute lung injury model, eucommia ulmoides liposome Nano-LP-DZ more effectively alleviated pulmonary edema and hemorrhage, and reduced alveolar wall thickening, neutrophil infiltration and hemorrhage.

[0107] The animals were sacrificed 24 hours after the operation, and blood was collected from the orbital cavity to separate serum for ELISA detection. The levels of inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the serum of each group were detected. The results were as Figure 5As shown in D, in the CLP-induced septic mouse model, Nano-LP-DZ more significantly reduced the levels of serum inflammatory factors IL-1β, IL-6, and TNF-α than DZ.

[0108] 8. Eucommia ulmoides Oliv. liposomes Nano-LP-DZ regulate the pan-apoptosis process in the sepsis model and alleviate cell death;

[0109] (a) Cell viability was detected by propidium iodide (PI) staining. Mouse macrophages MHS were seeded in 6-well plates (1×10 5 cells / well), treated with 200 μM oxalate (Oxo), 1 μg / mL lipopolysaccharide (LPS), and Nano-LP-DZ (100 μg / mL) for 24 h, and then the cells were collected. After washing with cold PBS, PI dye (1 μg / mL) was added and the cells were stained for 15 min in the dark. Subsequently, fluorescence microscopy images were taken in the red fluorescence channel, and bright-field images were collected simultaneously. The results are shown in Figure 6 the fluorescence microscopy images of cell death shown in A, which show the morphological changes and cell death of RAW264.7 macrophages under different treatment conditions (Ctrl, Oxo+LPS, Oxo+LPS+Nano-LP-DZ). The FITC channel was used to detect live cell staining, and the PI channel was used to detect dead cell nucleus staining. The results showed that Nano-LP-DZ could significantly reduce the number of PI-positive cells, revealing its alleviation of Oxo+LPS-induced cell death; the combined stimulation of Oxo and LPS could significantly increase the mortality rate of MHS cells, manifested as an increase in the proportion of PI-positive cells; after intervention with Nano-LP-DZ, the PI fluorescence intensity was significantly reduced, indicating that Eucommia ulmoides Oliv. liposomes Nano-LP-DZ had a protective effect on cell death and damage to the plasma membrane integrity.

[0110] (b) Western blot was used to analyze the protein expression changes of pyroptosis, apoptosis, and necroptosis markers in MH-S macrophages in different treatment groups (control group, Oxo+LPS group, and Oxo+LPS+Nano-LP-DZ group), with three biological replicates in each group. β-actin was used as an internal reference; the results are shown in Figure 6As shown in Figure B, Western blot was used to detect the expression of key PANoptosis proteins (in vitro experiment), which demonstrated the representative molecular expression of three programmed cell death pathways at the cellular level, including: pyroptosis (CASP1, GSDMD, GSDME), apoptosis (CASP8, CASP3, CASP7), and necroptosis (pMLKL / tMLKL); the expression of these molecules was significantly downregulated after Nano-LP-DZ treatment, suggesting its ability to inhibit PANoptosis through multiple pathways; compared with the Oxo+LPS stimulation group, the protein cleavage activation levels of pyroptosis markers [(Caspase-1, CASP1), (Gasdermin D, GSDMD), (Gasdermin E, GSDME)], apoptosis markers [(Caspase-8, CASP8), (Caspase-3, CASP3), (Caspase-7, CASP7)] and necroptosis markers [(phosphorylated Mixed Lineage Kinase Domain-Like, pMLKL) and total MLKL (tMLKL)] in the Nano-LP-DZ treatment group were significantly downregulated.

[0111] (c) Western blot analysis was used to examine the changes in the protein expression of pyroptosis, apoptosis, and necroptosis markers in the lung tissues of the sham operation group, CLP group, and CLP groups treated with Nano-LP-DZ (2.5 mg and 5 mg). Each group included three biological replicates, and β-actin was used as an internal reference; the results are shown as Figure 6 As shown in Figure C, Western blot was used to detect PANoptosis-related proteins (in vivo experiment, mouse lung tissue). By detecting the lung tissue samples of CLP model mice, it was further verified that Nano-LP-DZ could also significantly inhibit the expression of proteins such as CASP1, GSDMD, GSDME, CASP8, CASP3, CASP7, and pMLKL in vivo, confirming its effective alleviation of the PANoptosis process in lung tissues; in the lung tissues of CLP-induced sepsis mice, the expression of PANoptosis-related markers was significantly upregulated, manifested as increased protein levels of CASP1, GSDMD, GSDME, CASP8, CASP3, CASP7, and pMLKL / tMLKL. Nano-LP-DZ intervention could inhibit the expression of these markers in a dose-dependent manner, and the inhibitory effect of the 5 mg / kg dose group was significantly better than that of the 2.5 mg / kg group.

[0112] 9. Eucommia ulmoides Oliv. liposome Nano-LP-DZ inhibits PANoptosis in a sepsis model by regulating TGF-β1;

[0113] (a) Correlation analysis between core genes and PANscore. The color of the connecting lines indicates the degree of correlation (red indicates positive correlation, and green indicates negative correlation), while the size of the gene nodes reflects their connectivity in the network. As Figure 7 shown in A, the correlation analysis diagram between TGF-β1 and key factors related to PANoptosis, which is drawn based on multi-component transcriptome data, shows the Pearson correlation between TGF-β1 and multiple core molecules of the PANoptosis pathway (including CASP3, CASP8, etc.). The colors from green to red represent negative to positive correlation, and the molecules with stronger correlations are presented by obvious connecting lines; it can be seen in the figure that TGF-β1 has a significant correlation with multiple cell death regulatory genes, suggesting its key regulatory role in PANoptosis regulation; 7 key regulatory factors related to pan-apoptosis were identified through analysis, including interleukin-1β (IL-1β / IL1B), transforming growth factor-β1 (TGF-β1 / TGFB1), nuclear factor κB p65 subunit (RELA), phosphatase and tensin homolog (PTEN), caspase-8 (CASP8), signal transducer and activator of transcription 3 (STAT3), and STAT1. Among them, TGF-β1 was identified as a key regulatory node of the pan-apoptosis pathway.

[0114] (b) Analysis of the difference in TGF-β1 protein expression in MH-S macrophages by Western blot: The experimental groups were the control group, the Oxo+LPS group, and the Oxo+LPS+Nano-LP-DZ group, with GAPDH as the internal reference. The results are as Figure 7 shown in B, the regulatory effect of Nano-LP-DZ on the expression level of TGF-β1 (Western blot). This figure is the Western blot result of TGF-β1 protein expression in in vitro cell experiments. Compared with the Oxo+LPS induction group, the expression of TGF-β1 was significantly upregulated after Nano-LP-DZ treatment, indicating that Nano-LP-DZ can restore the TGF-β1 level reduced by inflammatory stimulation. Under the condition of Oxo+LPS stimulation, the expression level of TGF-β1 was significantly inhibited, while Nano-LP-DZ intervention could restore it to the baseline level, suggesting that Nano-LP-DZ exerts its biological effects by regulating TGF-β1.

[0115] (c) Western blot analysis was used to analyze the differences in the protein expression of pyroptosis, apoptosis, and necroptosis markers in MH-S macrophages under the following conditions (control group, Oxo+LPS group, Oxo+LPS+Nano-LP-DZ group, and Oxo+LPS+Nano-LP-DZ+si-TGF-β1 treatment group), with β-actin and GAPDH as internal references. The siRNA-TGF-β1 silencing experiment found that knockdown of the TGF-β1 gene could reverse the inhibitory effect of Nano-LP-DZ on pan-apoptosis, specifically manifested as a significant increase in the protein cleavage activation levels of pyroptosis-related markers (CASP1, GSDMD, GSDME), apoptosis-related markers (CASP8, CASP3, CASP7), and necroptosis markers (pMLKL, tMLKL). The results are as Figure 7 shown in C. Western blot analysis of the regulation of key PANoptosis protein expression by Nano-LP-DZ through the TGF-β1 signaling pathway further demonstrated whether Nano-LP-DZ could inhibit the expression of key proteins in three programmed cell death pathways by regulating TGF-β1 under Oxo+LPS induction conditions, including:

[0116] Pyroptosis: CASP1, GSDMD, GSDME;

[0117] Apoptosis: CASP8, CASP3, CASP7;

[0118] Necroptosis: pMLKL, tMLKL;

[0119] The above results confirmed that TGF-β1 is in a core regulatory position in the pan-apoptosis cascade reaction, and Nano-LP-DZ exerts its cytoprotective effect by targeting the TGF-β1 signaling axis.

[0120] Given that there are numerous embodiments in the solution of the present invention, the raw materials and dosages involved can be selected according to actual needs within the limited range. The experimental data of each embodiment are huge and numerous, and it is not suitable to list them one by one here. However, the contents to be verified and the final conclusions obtained in each embodiment are similar. Therefore, the verification contents of each embodiment will not be described one by one here.

[0121] The above is only a detailed description of the preferred embodiments and principles of the present invention. For those of ordinary skill in the art, according to the idea provided by the present invention, there will be changes in the specific implementation manners, and these changes should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of eucommia ulmoides liposomes, characterized in that It includes the following steps: (1) Eucommia ulmoides is soaked in water for reflux extraction, and the obtained extract is concentrated and vacuum freeze-dried to obtain Eucommia ulmoides extract; (2) The Eucommia ulmoides extract is added to water and dissolved by ultrasonic treatment. After centrifugation, the supernatant is collected, which is the aqueous phase; Soybean lecithin and cholesterol are added to an organic solvent and dissolved by ultrasonic treatment to obtain an oil phase; (3) The aqueous phase and the oil phase are mixed and ultrasonicated to form primary emulsion, and the primary emulsion is added dropwise to the water under stirring to form a W / O / W type multiple emulsion; (4) The W / O / W type multiple emulsion is stirred in a water bath at 30-80 °C for 5-60 min to obtain a crude liposome suspension; the crude liposome suspension is treated by probe sonication in an ice-water bath, and the supernatant is obtained after centrifugation; (5) The supernatant is mixed with trehalose, pre-frozen and then vacuum freeze-dried to obtain Eucommia ulmoides liposomes.

2. The preparation method according to claim 1, wherein In the step (1), the solid-liquid ratio of Eucommia ulmoides to water is 1:(1-30) mg / mL; The technological process of reflux extraction includes: after boiling with strong fire, turning to gentle fire for reflux, and the reflux time is 0.5-3 h; The residue is refluxed 1-3 times under the same reflux conditions.

3. The preparation method according to claim 1, characterized in that, In the step (2), the solid-liquid ratio of Eucommia ulmoides extract to water is (8-12) mg / mL, the centrifugation speed is 1000-16000 rpm, and the time is 1-30 min.

4. The preparation method according to claim 1, characterized in that, In the step (2), the mass ratio of soybean lecithin to cholesterol is (1-20):1; The organic solvent is at least one of chloroform, ether, dichloromethane, and ethyl acetate.

5. The preparation method according to claim 1, characterized in that, In the step (3), the volume ratio of the aqueous phase to the oil phase is (0.4-2.5):1; The ultrasonic frequency is 10-60 kHz, and the time is 0.5-10 min.

6. The preparation method according to claim 1, characterized in that, In the step (3), the volume ratio of the primary emulsion to water is (0.4-2.5):

1.

7. The preparation method according to claim 1, characterized in that In the step (4), the frequency of probe sonication treatment is 25-500 W, and the time is 1-30 min.

8. The preparation method according to claim 1, wherein, In the step (5), the solid-liquid ratio of trehalose to the supernatant is 0.5-5% mg / mL.

9. Eucommia ulmoides liposomes prepared by the preparation method according to any one of claims 1-8.

10. A drug for treating septic lung injury, characterized in that, It includes Eucommia ulmoides liposomes as described in claim 9.

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