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

Abstract

The present application relates to eucommia lipid and its preparation method and medicine for treating sepsis lung injury, the preparation method is: eucommia is soaked in water and is carried out reflux extraction, the obtained extract is concentrated and vacuum freeze-dried, and the eucommia extract is obtained;The eucommia extract is added to water and is dissolved by ultrasonic, and the supernatant is collected after centrifugation, which is aqueous phase;Soy lecithin and cholesterol are added to organic solvent, dissolved by ultrasonic, and the oil phase is obtained;The aqueous phase and oil phase are mixed, and the initial milk is formed by ultrasonic;The initial milk is added drop by drop to the water under stirring, and the W / O / W type multiple emulsion is formed;The multiple emulsion is stirred in 30-80 DEG C water bath for 5-60 min, and the crude lipid suspension is obtained, then the probe is treated in ice water bath, and the supernatant is obtained by centrifugation;The supernatant is mixed with trehalose, pre-frozen and vacuum freeze-dried, and the eucommia lipid is obtained.The eucommia lipid and the medicine for treating sepsis lung injury can reduce and improve the lung tissue pathology of infected sepsis animal, improve lung inflammation and improve the survival rate of infected sepsis lung injury animal.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to eucommia ulmoides liposomes, a preparation method thereof, and a medicine for treating septic lung injury. Background Art

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

[0003] Beyond conventional anti-infective and organ function support therapies, clinical treatment options for sepsis-associated lung injury (ALI) are very limited, necessitating the urgent need to find more effective treatments to save the lives of more sepsis patients. Currently, clinical treatments for sepsis widely utilize antibiotics, fluid resuscitation, blood purification, mechanical ventilation, and multi-organ support. However, these treatments can easily lead to increased bacterial resistance, adverse reactions, and poor therapeutic efficacy. Compared to existing treatments, traditional Chinese medicine (TCM) offers advantages such as fewer side effects and multi-molecular and multi-target effects, making it an increasingly effective complementary alternative therapy.

[0004] Pan-apoptosis is a form of cell death that involves the simultaneous activation of multiple cell death pathways, including apoptosis, necrosis, and pyroptosis. In recent years, studies have found that the pan-apoptosis pathway plays an important role in the progression of septic lung injury.

[0005] Eucommia ulmoides Oliv. is the dried bark of the Eucommia genus of the Eucommia family. It is a commonly used tonic in traditional Chinese medicine. Its main components include pinoresinol diglucoside, eucommia gum, iridoid compounds, flavonoids, polysaccharides, amino acids, etc. It is included in the 2020 edition of the "Chinese Pharmacopoeia". Its functions and indications are to nourish the liver and kidneys, strengthen the muscles and bones, and stabilize the fetus. It is used for liver and kidney deficiency, low back and knee pain, muscle and bone weakness, dizziness, bleeding during pregnancy, and fetal movement disorder. Although Eucommia ulmoides is widely used in traditional Chinese medicine to treat a variety of diseases, there are relatively few studies on the treatment of sepsis. It is basically used as a single Chinese medicine in a drug formula and needs to be combined with other Chinese medicines to exert its pharmacological effects. Currently, there are no reports on clinical studies of Eucommia ulmoides alone in the treatment of sepsis.

[0006] Liposomes are spherical vesicles composed of cholesterol and phospholipids, exhibiting excellent biocompatibility. Their internal hydrophilic ends and hydrophobic ends within the lipid bilayer enable them to encapsulate both hydrophilic and lipophilic drugs, making them suitable for a wide range of drug types. Liposomes exhibit excellent biocompatibility, based on natural phospholipids and cholesterol, significantly enhancing drug cellular uptake and therapeutic efficacy while exhibiting low toxicity. Appropriate surface modification can prolong the time liposomes remain in the bloodstream. Liposomes enable sustained and controlled release of drugs, minimizing drug accumulation in normal tissues. As highly effective drug delivery vehicles, liposomes hold enormous potential in the biomedical field. Summary of the Invention

[0007] Based on the above-mentioned shortcomings and deficiencies in the prior art, one of the objects of the present invention is to at least solve one or more of the above-mentioned problems in the prior art. In other words, one of the objects of the present invention is to provide a Eucommia ulmoides liposome and a preparation method thereof and a drug for treating septic lung injury that meet one or more of the above-mentioned needs.

[0008] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0009] A method for preparing Eucommia ulmoides liposomes comprises the following steps:

[0010] (1) soaking Eucommia ulmoides in water for reflux extraction, concentrating the obtained extract and vacuum freeze-drying to obtain Eucommia ulmoides extract;

[0011] (2) adding the Eucommia ulmoides extract to water and dissolving it by ultrasonication, collecting the supernatant after centrifugation to obtain the aqueous phase; adding soybean lecithin and cholesterol to an organic solvent and dissolving them by ultrasonication to obtain the oil phase;

[0012] (3) mixing the aqueous phase and the oil phase, ultrasonically forming colostrum, and adding the colostrum dropwise into the stirred water to form a W / O / W type emulsion;

[0013] (4) stirring the W / O / W emulsion in a water bath at 30-80° C. for 5-60 min to obtain a crude liposome suspension; subjecting the crude liposome suspension to ultrasonic treatment in an ice-water bath, and centrifuging to obtain a supernatant;

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

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

[0016] The process of reflux extraction includes: boiling with high heat, then refluxing with low heat for 0.5 to 3 hours; and refluxing the residue 1 to 3 times under the same reflux conditions.

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

[0018] As a preferred embodiment, in step (2), the mass ratio of soybean 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 step (3), the volume ratio of the water phase to the oil phase is (0.4-2.5):1;

[0021] The frequency of ultrasound is 10 to 60 kHz and the time is 0.5 to 10 minutes.

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

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

[0024] As a preferred embodiment, in 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 of the above schemes.

[0026] The present invention also provides a medicine for treating septic lung injury, comprising the Eucommia ulmoides liposomes as described in the above scheme.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

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

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

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

[0031] Figure 3 Graph showing the cytotoxicity test results of Eucommia ulmoides extract DZ (A) and Eucommia ulmoides liposome Nano-LP-DZ (B) on different macrophages according to Example 1 of the present invention;

[0032] Figure 4 This is a comparison of the in vitro anti-inflammatory effects of the Eucommia ulmoides extract DZ and the Eucommia ulmoides liposome Nano-LP-DZ of Example 1 of the present invention;

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

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

[0035] Figure 7 This is a schematic diagram of Example 1 of the present invention showing that the Eucommia ulmoides liposome Nano-LP-DZ inhibits the pan-apoptosis (PANoptosis) process in the sepsis model by regulating TGF-β1; wherein, A: correlation analysis diagram between TGF-β1 and key factors related to PANoptosis, B: regulatory effect of Nano-LP-DZ on TGF-β1 expression level (Western blot), C: Western blot analysis of Nano-LP-DZ regulating the expression of key PANoptosis proteins through the TGF-β1 signaling pathway. DETAILED DESCRIPTION

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

[0037] The present invention adopts liposome drug carrier to prepare the traditional Chinese medicine Eucommia ulmoides into Eucommia ulmoides liposomes with small particle size, uniform distribution, high bioavailability and good cellular uptake effect. Compared with traditional Eucommia ulmoides extract, the Eucommia ulmoides liposomes of the present invention have better application effect in treating sepsis. By reducing the expression and secretion level of cellular inflammatory factors, inhibiting the pan-apoptotic signaling pathway of cells, improving the lung injury tissue of mice infected with sepsis, etc., the survival rate of mice infected with sepsis is improved, and it has good application prospects for the treatment of clinical sepsis. As an old drug with new uses, Eucommia ulmoides greatly reduces the R&D cost and R&D risk in the drug development process of sepsis, and further combines liposome technology to provide a new way to solve sepsis-related acute lung injury.

[0038] The Eucommia ulmoides liposomes of the present invention use Eucommia ulmoides as an old drug in a new way, and show good therapeutic effects in sepsis models not recorded in the "Chinese Pharmacopoeia", and have development value in the field of clinical sepsis; moreover, the effect of Eucommia ulmoides liposomes in the sepsis model is higher than that of Eucommia ulmoides extract, which verifies that Eucommia ulmoides can act in new fields and has better efficacy after liposomalization; in addition, Eucommia ulmoides liposomes can effectively improve pathological damage caused by septic lung injury by regulating the pan-apoptosis pathway, and improve the survival rate of mice infected with sepsis, clarifying the mechanism of action 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 in turn induce symptoms such as systemic multiple organ dysfunction, and their causes and treatment methods are similar.

[0040] In one embodiment, the drug for treating septic lung injury further comprises at least one pharmaceutically acceptable excipient, including but not limited to a filler, a disintegrant, a lubricant, a stabilizer, or a combination thereof.

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

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

[0043] In one embodiment, the drug for treating septic lung injury is a drug that can improve pulmonary edema in animals infected with sepsis.

[0044] In one embodiment, the drug for treating septic lung injury is a drug that can suppress the expression level of inflammatory factors in the lung tissue of animals infected with sepsis, wherein 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 that can inhibit pan-apoptotic signaling pathways, and the pan-apoptotic signaling pathways involved include apoptosis, necroptosis, and pyroptosis.

[0046] Specifically, the preparation method of the Eucommia ulmoides liposomes of the present invention comprises the following steps:

[0047] (1) soaking Eucommia ulmoides in water for reflux extraction, concentrating the obtained extract and vacuum freeze-drying to obtain Eucommia ulmoides extract;

[0048] (2) adding the Eucommia ulmoides extract to water and dissolving it by ultrasonication, and collecting the supernatant after centrifugation, which is the aqueous phase;

[0049] Soy lecithin and cholesterol are added to an organic solvent and dissolved by ultrasonication to obtain an oil phase;

[0050] (3) mixing the aqueous phase and the oil phase, ultrasonically forming colostrum, and adding the colostrum dropwise into the stirred water to form a W / O / W type emulsion;

[0051] (4) stirring the W / O / W emulsion in a water bath at 30-80° C. for 5-60 min to obtain a crude liposome suspension; subjecting the crude liposome suspension to ultrasonic treatment in an ice-water bath, and centrifuging to obtain a supernatant;

[0052] (5) The supernatant was mixed with trehalose, pre-frozen, and then vacuum-freezed to obtain Eucommia ulmoides liposomes.

[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, which can be determined according to actual application requirements;

[0054] The process of reflux extraction includes: boiling with high heat, then refluxing with low heat for 0.5 to 3 hours; and refluxing the residue 1 to 3 times under the same reflux conditions.

[0055] In one embodiment, in the above step (2), the solid-liquid ratio of Eucommia ulmoides extract to water can be (8-12) mg / mL, the centrifugal speed can be 1000-16000 rpm, and the time can be 1-30 min, which can be 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 determined according to actual application requirements;

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

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

[0059] The frequency of ultrasound can be 10 to 60 kHz, and the time can be 0.5 to 10 minutes, which can be determined according to actual application requirements.

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

[0061] In one embodiment, in the above step (4), the frequency of the probe ultrasonic treatment can be 25 to 500 W, and the time can be 1 to 30 minutes, which can be 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 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 medicine for treating septic lung injury, comprising the above-mentioned Eucommia ulmoides liposome.

[0065] The following is a further explanation of the Eucommia ulmoides liposomes, the preparation method thereof, and the drug for treating septic lung injury of the present invention through specific examples.

[0066] Example 1:

[0067] The preparation method of the Eucommia ulmoides liposomes of the present embodiment comprises 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 over high heat, and then refluxed over low heat for 60 minutes to obtain an extract. The residue was refluxed twice under the same conditions, and the extracts were combined, concentrated, and vacuum freeze-dried to obtain Eucommia ulmoides extract DZ; the extracts were sealed and stored at room temperature for future 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. Dissolve it by ultrasonication. Centrifuge at 10,000 rpm for 10 min to remove larger particles. Collect the supernatant, which is the aqueous phase, and set aside.

[0072] (3) Oil phase preparation;

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

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

[0075] The aqueous phase of step (2) and the oil phase of step (3) were mixed in a volume ratio of 4:5, and ultrasonicated at 53 kHz for 10 minutes to form colostrum; 15 mL of colostrum was added dropwise to 7.5 mL of pure water under stirring (2500 rpm) to form a W / O / W type emulsion;

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

[0077] The W / O / W emulsion was stirred in a 55°C water bath for 30 minutes to remove the organic solvent, thereby obtaining a crude liposome suspension. The crude liposome suspension was subjected to ultrasonic treatment (100W, 5 minutes, on: 5 seconds, off: 4 seconds) in an ice-water bath, and the supernatant was collected after centrifugation to obtain an Eucommia ulmoides liposome supernatant.

[0078] (6) Lyophilization of Eucommia ulmoides liposome supernatant;

[0079] Trehalose was mixed with Eucommia ulmoides liposome supernatant at a solid-liquid ratio of 2% mg / mL, pre-frozen at -80°C and then freeze-dried in vacuo. The freeze-dried sample was collected for later use to obtain Eucommia ulmoides liposome Nano-LP-DZ.

[0080] The following tests, analyses and characterizations were performed on the Eucommia ulmoides liposomes and intermediate products obtained in the above examples:

[0081] 1. In this example, a high performance liquid chromatography (HPLC) system (LC-20A, Shimadzu) was used to analyze the content of DZ in Eucommia ulmoides extract, Nano-LP-DZ, and free DZ in the liquid after coating 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 the gradient elution was: 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 results of the HPLC system test are shown in Figure 1 As shown in A, the x-axis represents retention time and the y-axis represents absorbance. Comparing the liquid phase spectra of DZ and Nano-LP-DZ, the peak retention times of the two are almost the same, indicating that the liposome carrier material has no significant effect on the detection of Eucommia components; comparing the liquid phase spectra of Nano-LP-DZ and the liquid after coating with Eucommia liposomes, it can be seen that the liposomes can encapsulate no less than 80% of the Eucommia extract DZ.

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

[0083] The operation steps are as follows: 400 μL of liposome suspension was placed in a 100 kDa ultrafiltration tube liner and centrifuged at 6000 rpm for 20 minutes for ultrafiltration separation. The filtrate was collected and diluted with purified water to 10 mL, and then quantitatively analyzed by high performance liquid chromatography (HPLC) system;

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

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

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

[0087] Laser particle size analyzer was used to measure the particle size distribution and Zeta potential of Eucommia ulmoides liposome Nano-LP-DZ to evaluate its physical stability and particle size characteristics. At the same time, the microstructure of the liposome was observed by transmission electron microscopy (TEM). The specific operation is as follows: After the Nano-LP-DZ sample is diluted to an appropriate concentration with purified water, an appropriate amount of suspension is added dropwise to the surface of the carbon-supported copper mesh (C-Cu), and the excess liquid is removed by gently touching with filter paper, and the sample is placed at room temperature to dry naturally. After drying, its morphology is observed under TEM. Figure 1 As shown in Figure B, Nano-LP-DZ appears as evenly distributed spherical particles in the TEM image, with clear boundaries and good dispersion. The black arrows in the figure indicate the Eucommia ulmoides liposome particles, which are less than 200 nm in size, 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 the liposomes were measured using a laser particle size analyzer. The particle size, PDI, and zeta potential distributions of the Nano-LP-DZ and blank liposomes of this example are shown in Table 1. The particle size of the Nano-LP-DZ (125.53±1.81 nm) was slightly smaller than that of the blank liposomes (131.17±0.91 nm). This is because the amphiphilic compounds in the Eucommia ulmoides extract reduce the surface tension of the liposome membrane. The smaller particle size is conducive to cellular uptake and improves absorption efficiency. The PDI of both the Nano-LP-DZ and the blank liposomes of this example was less than 0.3, indicating that the particles were uniformly dispersed. In addition, the zeta potential of the blank liposomes was -45.33±2.87 mV, and the zeta potential of the Nano-LP-DZ was -40.57±2.25 mV. Zeta potential values ​​exceeding ±30 mV indicate strong electrostatic repulsion between the particles, which helps maintain storage stability.

[0089] Table 1 Parameter comparison of Eucommia ulmoides liposome Nano-LP-DZ and blank liposome Nano-LP of 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] The only difference between the preparation method of the blank liposome Nano-LP and the preparation method of the Eucommia ulmoides liposome Nano-LP-DZ is that the aqueous phase does not contain the Eucommia ulmoides extract, and the 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 cellular uptake effect was analyzed by fluorescently labeling Eucommia ulmoides 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 under the green fluorescence channel (FITC). The results are shown in Figure 2. Figure 2 As shown in A, the uptake of coumarin-6-labeled Nano-LP-DZ in MH-S macrophages is time-dependent; the fluorescence intensity gradually increases after 1 hour, reaches a peak at 2 hours, and then gradually weakens, and the fluorescence signal almost disappears at 6 hours; this result indicates that Nano-LP-DZ can be taken up by cells quickly and efficiently, 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 intratracheally into anesthetized mice. One hour later, whole-body fluorescence images were collected and major organs were isolated. The fluorescence intensity was measured to evaluate the biodistribution. The in vivo biodistribution results are shown in Figure 2. Figure 2 As shown in B, 1 hour after intratracheal administration, Nano-LP-DZ was mainly enriched in the lung tissue, while the distribution amount in other organs such as the heart, liver, kidney and spleen was extremely low.

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

[0098] The CCK-8 assay was used to assess cell viability after treatment with DZ (concentrations 10–1000 μg / mL) and Nano-LP-DZ (concentrations 100 and 200 μg / mL);

[0099] MH-S, RAW 264.7 and NR8383 macrophages were cultured at 5×10 3 Cells were seeded in 96-well plates and allowed to adhere overnight. Cells were treated with different concentrations of Eucommia ulmoides extract DZ (10, 100, 200, 500, and 1000 μg / mL), blank liposome Nano-LP (100 μg / mL), and Eucommia ulmoides 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 cell viability was expressed as a percentage of the control group. Figure 3 A and Figure 3 As shown in B, the cytotoxicity experiment showed that Eucommia ulmoides liposome Nano-LP-DZ had no obvious toxicity to macrophages at concentrations of 100 and 200 μg / mL.

[0100] 6. Comparison of the in vitro anti-inflammatory effects of Eucommia ulmoides extract DZ and Eucommia ulmoides 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% CO 2 Cell treatments were as follows: control, 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 after treatment with DZ (100 μg / mL) or Nano-LP-DZ (100 μg / mL) were detected using ELISA kits. Figure 4 As shown, Nano-LP-DZ significantly reduced the levels of IL-1β, IL-6, and TNF-α in LPS-stimulated MH-S macrophages.

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

[0104] Eight-week-old male C57BL / 6 mice (weighing 22-26 g) were acclimatized for one week before the experiment and received different experimental treatments, including sham operation (Sham), cecal ligation and puncture (CLP), oral administration of Eucommia ulmoides extract DZ (10 mg) 2 hours after surgery, and intratracheal instillation of Eucommia ulmoides liposome Nano-LP-DZ (2.5 mg or 5 mg) 2 hours after surgery.

[0105] Lung tissue samples were collected and weighed to calculate the wet-to-dry weight ratio for subsequent analysis. Lung tissue samples were fixed in 10% neutral-buffered formalin for 24 hours and then embedded in paraffin. 5-μm thin sections were prepared and mounted on slides. After dewaxing and hydration, the sections were stained with hematoxylin for nuclei and eosin for cytoplasm. After staining, the sections were observed under a light microscope and images were taken for morphological analysis.

[0106] The quantitative analysis of the inflammatory damage score was performed in a blinded manner based on the following criteria: alveolar congestion, hemorrhage, neutrophil infiltration into the alveolar or vascular walls, and alveolar wall thickening. Each parameter was scored from 0 (no damage) to 4 (severe damage), and the sum of the scores for each sample reflected the degree of inflammation; the results were as follows: Figure 5 A. Figure 5 B and Figure 5 As shown in C, 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 killed 24 hours after surgery, and blood was collected from the orbits to separate the serum for ELISA testing. The levels of inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the serum of the above groups were detected. The results are shown in Figure 2. Figure 5As shown in D, in the CLP-induced sepsis mouse model, Nano-LP-DZ significantly reduced the levels of serum inflammatory factors IL-1β, IL-6, and TNF-α compared with DZ.

[0108] 8. Eucommia ulmoides liposome Nano-LP-DZ regulates the pan-apoptotic process in the sepsis model and alleviates 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 were collected after treatment with 200 μM oxalate (Oxo), 1 μg / mL lipopolysaccharide (LPS), and Nano-LP-DZ (100 μg / mL) for 24 hours, washed with cold PBS, and stained with PI dye (1 μg / mL) for 15 minutes in the dark. The cells were then imaged under the red fluorescence channel using a fluorescence microscope, and bright field images were collected at the same time. The results are shown in Figure 2. Figure 6 The cell death fluorescence microscopy images shown in A 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 nuclear staining of dead cells. The results showed that Nano-LP-DZ could significantly reduce the number of PI-positive cells, revealing that it alleviated Oxo+LPS-induced cell death; Oxo combined with LPS stimulation could induce a significant increase in the cell death rate of MHS cells, manifested as an increase in the proportion of PI-positive cells; after Nano-LP-DZ intervention, the PI fluorescence intensity was significantly reduced, indicating that Eucommia ulmoides liposome Nano-LP-DZ has a protective effect on cell death and plasma membrane integrity damage.

[0110] (b) Western blot analysis of protein expression of pyroptosis, apoptosis, and necroptosis markers in MH-S macrophages treated with different groups (control group, Oxo+LPS group, and Oxo+LPS+Nano-LP-DZ group). Each group included three biological replicates. β-actin was used as an internal control. Figure 6As shown in Figure B, Western blot analysis of the expression of key proteins in PANoptosis (in vitro experiment) showed the expression of representative molecules of the three pathways of programmed cell death at the cellular level, including pyroptosis (CASP1, GSDMD, GSDME), apoptosis (CASP8, CASP3, CASP7), and necroptosis (pMLKL / tMLKL). The expression of the above molecules was significantly downregulated after Nano-LP-DZ treatment, suggesting that it can inhibit PANoptosis through multiple pathways. Compared with the Oxo+LPS stimulation group, the expression 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 The protein cleavage activation levels of both Domain-Like (pMLKL) and total MLKL (tMLKL) were significantly downregulated.

[0111] (c) Western blot analysis was performed to analyze the protein expression of pyroptosis, apoptosis, and necroptosis markers in lung tissues in the sham group, CLP group, and Nano-LP-DZ-treated (2.5 mg and 5 mg) CLP group. Each group included three biological replicates, with β-actin as the internal control. The results are shown in Figure 4. Figure 6 As shown in Figure C, Western blot analysis of PANoptosis-related proteins (in vivo, mouse lung tissue) was performed. Testing of lung tissue samples from CLP model mice further confirmed that Nano-LP-DZ significantly inhibited the expression of proteins such as CASP1, GSDMD, GSDME, CASP8, CASP3, CASP7, and pMLKL in vivo, confirming its effective mitigation of PANoptosis in lung tissue. In the lung tissue of CLP-induced septic mice, the expression of pan-apoptosis (PANoptosis) markers was significantly upregulated, as evidenced by elevated protein levels of CASP1, GSDMD, GSDME, CASP8, CASP3, CASP7, and pMLKL / tMLKL. Nano-LP-DZ intervention inhibited the expression of these markers in a dose-dependent manner, with the 5 mg / kg dose group exhibiting significantly greater inhibitory effects than the 2.5 mg / kg group.

[0112] 9. Eucommia ulmoides liposome Nano-LP-DZ inhibits pan-apoptosis in a sepsis model through TGF-β1 regulation;

[0113] (a) Correlation analysis between core genes and PANscore. The color of the connecting line indicates the degree of correlation (red indicates positive correlation, green indicates negative correlation), while the size of the gene nodes reflects their connectivity in the network. Figure 7 Figure A shows a correlation analysis between TGF-β1 and key panoptosis-related factors. This figure, based on multi-component transcriptome data, demonstrates the Pearson correlation between TGF-β1 and several core molecules in the panoptosis pathway, including CASP3 and CASP8. Colors range from green to red, representing negative to positive correlations, respectively, with molecules with stronger correlations indicated by distinct lines. The figure shows significant correlations between TGF-β1 and multiple cell death-regulating genes, suggesting its key regulatory role in panoptosis. The analysis identified seven key pan-apoptosis-related regulatory factors, 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. TGF-β1 was identified as a key regulatory node in the pan-apoptosis pathway.

[0114] (b) Western blot analysis of TGF-β1 protein expression in MH-S macrophages: the experimental groups were control group, Oxo+LPS group, and Oxo+LPS+Nano-LP-DZ group, with GAPDH as the internal reference. The results are shown in Figure 2. Figure 7 Figure B shows the regulatory effect of Nano-LP-DZ on TGF-β1 expression levels (Western blot). This figure shows the Western blot results of TGF-β1 protein expression in in vitro cell experiments. Compared with the Oxo+LPS-induced group, Nano-LP-DZ treatment significantly upregulated TGF-β1 expression, indicating that Nano-LP-DZ can restore TGF-β1 levels that are reduced due to inflammatory stimulation. Under Oxo+LPS stimulation conditions, TGF-β1 expression levels were significantly inhibited, while Nano-LP-DZ intervention could restore them to baseline levels, suggesting that Nano-LP-DZ exerts its biological effects by regulating TGF-β1.

[0115] (c) Western blot analysis was performed to analyze the differences in 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 controls. siRNA-TGF-β1 silencing experiments found that TGF-β1 gene knockdown could reverse the inhibitory effect of Nano-LP-DZ on pan-apoptosis, as evidenced by 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). Figure 7 As shown in Figure C, Western blot analysis of Nano-LP-DZ regulating the expression of key proteins in PANoptosis 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 confirm that TGF-β1 plays a core regulatory role in the pan-apoptotic cascade reaction, and Nano-LP-DZ exerts its cytoprotective effect by targeting the TGF-β1 signaling axis.

[0120] Given the numerous embodiments of the present invention, the raw materials and amounts involved can be selected within a limited range according to actual needs. The experimental data for each embodiment is voluminous and it is not suitable to list and explain them one by one here. However, the content required for verification and the final conclusions obtained in each embodiment are similar. Therefore, the verification content of each embodiment will not be described one by one here.

[0121] The above description is only a detailed description of the preferred embodiments and principles of the present invention. For ordinary technicians in this field, based on the ideas provided by the present invention, there may be changes in the specific implementation methods, and these changes should also be considered as the scope of protection of the present invention.

Claims

1. A method for preparing Eucommia ulmoides liposomes, characterized in that: The following steps are involved: (1) soaking Eucommia ulmoides in water for reflux extraction, concentrating the obtained extract and vacuum freeze-drying it to obtain Eucommia ulmoides extract; (2) Add the Eucommia ulmoides extract to water and dissolve it by ultrasonication. After centrifugation, collect the supernatant, which is the aqueous phase; Soy lecithin and cholesterol are added to an organic solvent and dissolved by ultrasonication to obtain an oil phase; (3) The aqueous phase and the oil phase are mixed and ultrasonically formed into colostrum, which is then added dropwise into the stirred water to form a W / O / W type emulsion; (4) Stirring the W / O / W emulsion in a water bath at 30-80°C for 5-60 min to obtain a crude liposome suspension; ultrasonically treating the crude liposome suspension in an ice-water bath, and centrifuging to obtain a supernatant; (5) The supernatant was mixed with trehalose, pre-frozen, and then vacuum-freezed to obtain Eucommia ulmoides liposomes; In the step (1), the solid-liquid ratio of Eucommia ulmoides to water is 1: (1-30) mg / mL; The process of reflux extraction includes: boiling with high heat, then reflux with low heat, and the reflux time is 0.5 to 3 hours; The residue is refluxed 1 to 3 more times under the same reflux conditions; In the step (2), the solid-liquid ratio of the Eucommia ulmoides extract to water is (8-12) mg / mL, the centrifugal speed is 1000-16000 rpm, and the time is 1-30 min; 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; In the step (3), the volume ratio of the water phase to the oil phase is (0.4-2.5):1; The frequency of ultrasound is 10 to 60 kHz and the duration is 0.5 to 10 minutes; In the step (3), the volume ratio of colostrum to water is (0.4-2.5):1; In the step (4), the frequency of the probe ultrasonic treatment is 25 to 500 W and the time is 1 to 30 minutes.

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

3. The Eucommia ulmoides liposome prepared by the preparation method according to any one of claims 1 to 2.

4. A drug for treating septic lung injury, characterized in that: The method comprises the Eucommia ulmoides liposome as claimed in claim 3.

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