Application of wogonin in preparation of medicine for treating or inhibiting spinal cystic echinococcosis

Abstract

The invention relates to the technical field of biological medicine, in particular to application of wogonin in preparation of a medicine for treating or inhibiting spinal cystic echinococcosis. The wogonin can accurately regulate and control the Nrf2-RTN4 signal axis and inhibit the generation of focus angiogenesis, so that the problem that the traditional medicine cannot block the key path Nrf2-RTN4-VEGF axis is solved, and the effect of treating or inhibiting the spinal cystic echinococcosis is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the application of wogonin in the preparation of a medicine for treating or inhibiting spinal cystic echinococcosis. Background Art

[0002] Clinical treatment for spinal cystic echinococcosis primarily relies on surgical resection and the antiparasitic drug albendazole. However, existing treatment options have significant drawbacks; intraoperative cyst rupture can easily lead to serious complications such as anaphylactic shock. Albendazole, the first-line drug, has poor bone barrier penetration, requiring high doses and causing hepatotoxicity. Hydatid cysts rely on angiogenesis for nutrition, but existing drugs are unable to block the key pathway, the Nrf2-RTN4-VEGF axis. Overall, there is a lack of targeted therapies for angiogenesis. Summary of the Invention

[0003] The purpose of the present invention is to provide the use of wogonin in preparing a drug for treating or inhibiting spinal cystic echinococcosis.

[0004] To achieve the above-mentioned purpose, the present invention provides the use of wogonin in the preparation of a drug for treating or inhibiting spinal cystic echinococcosis.

[0005] In the present invention, the CAS number of wogonin is: 632-85-9.

[0006] In the present invention, wogonin inhibits Nrf2 to activate RTN4, reduce VEGF expression, block lesion angiogenesis, and treat or inhibit spinal cystic echinococcosis.

[0007] The present invention also provides a drug for treating or inhibiting spinal cystic echinococcosis, comprising a drug-loaded liposome and a coating, wherein the mass ratio of the drug-loaded liposome to the coating is 1-5:10;

[0008] The drug-loaded liposomes comprise a drug phase and a lipid phase, and the drug phase comprises wogonin and albendazole.

[0009] In the present invention, the mass ratio of wogonin to albendazole is 0.5-1:0.5-1;

[0010] The lipid phase includes dipalmitoylphosphatidylcholine and cholesterol, and the molar ratio of dipalmitoylphosphatidylcholine to cholesterol is 6-8:2-4.

[0011] In the present invention, the coating is hydroxyapatite, and the particle size of the hydroxyapatite is 40-50 nm.

[0012] In the present invention, the dosage of wogonin in the drug for treating or inhibiting spinal cystic echinococcosis is 15-25 mg / kg.

[0013] The present invention also provides a method for preparing the above-mentioned drug for treating or inhibiting spinal cystic echinococcosis, comprising the following preparation steps:

[0014] S1, mixing dipalmitoylphosphatidylcholine, cholesterol and chloroform to obtain a lipid phase;

[0015] S2, mixing wogonin, albendazole and ethanol to obtain a drug phase;

[0016] S3, mixing the lipid phase with the drug phase, rotary evaporating under reduced pressure, and drying to obtain a lipid film, adding phosphate buffer, stirring, sonicating, and filtering to obtain a drug-loaded liposome suspension;

[0017] S4. After mixing hydroxyapatite with phosphate buffer, add drug-loaded liposome suspension, shake and incubate, and thus obtain a drug for treating or inhibiting spinal cystic echinococcosis.

[0018] In the present invention, the chloroform in S1 is only used as a solvent and can dissolve dipalmitoylphosphatidylcholine and cholesterol, and its amount is not limited.

[0019] In the present invention, the ethanol in S2 is only used as a solvent and can dissolve wogonin and albendazole, and its usage is not limited.

[0020] In the present invention, the role of the reduced pressure rotary evaporation in S3 is to remove chloroform and ethanol.

[0021] In the present invention, the temperature of the vacuum rotary evaporation in S3 is 40-50° C., the time of the vacuum rotary evaporation is 30-60 min, and the pH of the phosphate buffer is 7-8.

[0022] In the present invention, the purpose of stirring in S3 is to fully hydrate, swell and peel off the lipid film to form a coarse liposome suspension.

[0023] In the present invention, the stirring temperature in S3 is 50-60° C., the stirring time is 5-10 min, the ultrasonic power is 180-220 W, and the ultrasonic time is 3-8 min.

[0024] In the present invention, the temperature of ultrasound in S3 is 0°C, and the function of filtration is to remove unencapsulated drugs and large particle aggregates.

[0025] In the present invention, the shaking incubation in S4 is to allow hydroxyapatite to be bound to the surface of the drug-loaded liposomes through physical adsorption or electrostatic interaction.

[0026] In the present invention, the pH of the phosphate buffer in S4 is 7-8, the shaking incubation temperature is 36-38° C., the shaking incubation time is 1-3 h, and the shaking incubation frequency is 150-200 rpm.

[0027] The present invention has the following beneficial effects

[0028] The present invention provides the use of wogonin in the preparation of a drug for treating or inhibiting spinal cystic echinococcosis. Wogonin can precisely regulate the Nrf2-RTN4 signaling axis, inhibiting angiogenesis in lesions, addressing the problem of traditional drugs being unable to block the key pathway, the Nrf2-RTN4-VEGF axis.

[0029] The present invention also provides a drug for treating or inhibiting spinal cystic echinococcosis, comprising wogonin, albendazole, a lipid phase, and a coating. The lipid phase ensures drug accumulation at the spinal lesion site. When used in combination with albendazole, wogonin can cut off vascular nutrient supply, while albendazole can directly kill parasites, significantly reducing recurrence rates and toxicity risks.

[0030] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a comparison of spinal vesicle tissues after dissection of mice in the experimental group and the control group of the present invention;

[0032] Figure 2 This is a graph showing the results of protein level detection using the Western immunoblotting method of the present invention;

[0033] Figure 3 It is the result of protein level detection by western blotting method of the present invention;

[0034] Figure 4 This is a diagram of the hematoxylin-eosin (HE) staining result of the present invention;

[0035] in, Figure 4 a in the figure is the HE staining result of the vesicle tissue sections of the experimental group and the control group. Figure 4 b in the figure is the number of capillaries in the vesicle tissues of the experimental and control groups;

[0036] Figure 5 It is the immunohistochemical staining result diagram of the present invention;

[0037] in, Figure 5 a in the figure is the immunohistochemical staining of CD31 in the vesicle tissue sections of the experimental group and the control group. Figure 5 b in the figure is the quantification of hemoglobin content in the vesicle tissues of the experimental and control groups. Figure 5 c in the figure is the average optical density of immunohistochemical staining;

[0038] Figure 6 is an immunohistochemical analysis diagram of the present invention;

[0039] in, Figure 6a in the figure is the average optical density of PDGF immunohistochemical staining. Figure 6 b in the figure is the average optical density of VEGF immunohistochemical staining. Figure 6 Figure c is the immunohistochemical staining of PDGF in the vesicle tissue sections of the experimental group and the control group. Figure 6 d in the figure shows the immunohistochemical staining of VEGF in the vesicle tissue sections of the experimental and control groups;

[0040] Figure 7 This is a graph showing the results of an in vitro angiogenesis experiment in a PSCs-HUVECs co-culture system treated with wogonin according to the present invention;

[0041] in, Figure 7 Figure a is the result of HUVECs lumen formation detected by in vitro angiogenesis experiment. Figure 7 b in the figure is the quantitative analysis result of the number of branch points of HUVECs. Figure 7 Figure c shows the quantitative analysis results of HUVECs lumen length. DETAILED DESCRIPTION

[0042] The present invention is further described below with reference to the accompanying drawings and examples. Unless otherwise defined, technical or scientific terms used herein shall have the same meanings as those commonly understood by persons of ordinary skill in the art to which the present invention pertains. The above-mentioned features or features described in the specific examples of the present invention may be combined in any manner. These specific examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0043] Purchasing information for drugs and mice

[0044] The sources of raw materials for the prevention of spinal cystic echinococcosis are shown in Table 1.

[0045] Table 1 Source of raw materials

[0046] Reagent name Manufacturer (Country / Region) Dipalmitoylphosphatidylcholine Cordenpharma (Switzerland) cholesterol Henan Liwei Biopharmaceuticals Wogonin Xi'an Qiyue Biotechnology Albendazole Sino-US Tianjin SmithKline Pharmaceuticals Hydroxyapatite (nHA, 50 nm) Sichuan Guona Technology Phosphate-buffered saline (PBS) Solarbio (China) Chloroform Solarbio (China) Anhydrous ethanol Solarbio (China)

[0047] Experimental cells

[0048] Human umbilical vein endothelial cells (HUVECs) were purchased from Shanghai iCell Biotechnology Co., Ltd. (iCell, China). Protoscolex of Echinococcus granulosus (PSCs) were isolated from the livers of naturally infected sheep collected from the Changji slaughterhouse in Xinjiang. After sterile separation and repeated washing with PBS, the cells were observed under a microscope to determine if the protoscolex activity was ≥90% and then adjusted to a final concentration of 4 × 10 3 / mL for future use.

[0049] Example 1

[0050] A method for preparing a drug for treating or inhibiting spinal cystic echinococcosis comprises the following steps:

[0051] S1. Mix 10 mg of dipalmitoylphosphatidylcholine, 2.2 mg of cholesterol, and 5 mL of chloroform (the molar ratio of dipalmitoylphosphatidylcholine to cholesterol is 7:3) to obtain a lipid phase.

[0052] S2. Mix 5 mg of wogonin, 5 mg of albendazole, and 2 mL of ethanol to obtain a drug phase;

[0053] S3. The lipid phase and the drug phase were mixed, and vacuum evaporated at 40°C for 30 minutes, followed by drying in a vacuum drying oven for 2 hours to obtain a lipid film. A pH 7.4 phosphate buffer solution was added to the lipid film, and the mixture was stirred at 50°C for 5 minutes. The mixture was then ultrasonicated at 200 W for 5 minutes at 0°C. The ultrasonicated suspension was filtered through a 0.22 μm microporous filter membrane to obtain a 2.44 mg / mL drug-loaded liposome suspension.

[0054] S4. Mix 10 mg of 50 nm hydroxyapatite and 1 mL of pH 7.4 phosphate buffer for 1 min, then add 2 mL of the above-mentioned drug-loaded liposome suspension, and incubate at 37°C with shaking at a frequency of 150 rpm for 2 h to prepare a drug for treating or inhibiting spinal cystic echinococcosis.

[0055] Experimental example

[0056] In vivo drug administration and efficacy verification

[0057] Animal model establishment: Construction of a mouse spinal cystic echinococcosis model

[0058] experimental animals

[0059] SPF-grade C57BL / 6 wild-type male mice (aged 4-6 weeks, weighing 18-22 g, purchased from the Experimental Animal Center of Shihezi University) were used in this study. All mice were housed in an SPF-grade barrier environment (temperature 20-26°C, humidity 40-70%, 12h / 12h light / dark cycle) with free access to sterilized feed and drinking water. This study was approved by the Animal Ethics Committee of the First Affiliated Hospital of Shihezi University (approval number: A2023-186-01) and strictly adhered to the relevant provisions of the "Regulations on the Administration of Laboratory Animals" in China.

[0060] Preparation of protoscolex suspension: Place a T25 culture flask under an inverted microscope and select protoscolex with plump morphology, intact body wall and typical scolex telescopic movement. Use a pipette to transfer the culture medium to a 15mL centrifuge tube, centrifuge at 1000rpm for 5 minutes and discard the supernatant. Add 5mL of pre-cooled 1× PBS buffer, gently pipette to mix and let it stand for 3 minutes. After the protoscolex naturally settles, discard the supernatant again. Repeat this process 3 times until the liquid is transparent. Use a hemocytometer to adjust the protoscolex concentration to 10,000 heads / mL, draw 1mL of suspension into a sterile syringe, expel the bubbles and place in an ice box for use within 2 hours.

[0061] Mouse anesthesia and positioning: Place the mouse in an isoflurane induction chamber for anesthesia. Once the righting reflex disappears, quickly transfer it to a fixator and secure it in the prone position with its ventral side facing downward. Wear sterile gloves during the procedure. Disinfect the mouse's dorsal skin with an alcohol swab. Use your left index and middle fingers to gently palpate along the longitudinal axis of the spine to locate the intervertebral space between the 3rd and 5th lumbar vertebrae (feel as a depression between continuous bony protrusions).

[0062] Paravertebral injection procedure: Hold the syringe loaded with the protoscolex suspension in your right hand, ensuring the needle bevel faces upward. Insert the needle at a 30-degree angle, 2 mm lateral to the left side of the spine, and slowly penetrate the paravertebral myofascial layer. After confirming the absence of blood return, slowly and evenly inject 0.5 mL of the protoscolex suspension. This should result in a hemispherical bulge in the skin. Similarly, inject 0.5 mL of the suspension symmetrically on the right side of the spine. After the injection, gently press the needle hole with sterile gauze for 10 seconds to prevent leakage. This will establish a mouse model of spinal cystic echinococcosis.

[0063] Treatment options:

[0064] Experimental group: intraperitoneal injection of the drug prepared in Example 1 (the dose of wogonin was 20 mg / kg), 3 times a week for 4 weeks;

[0065] Control group: intraperitoneal injection of normal saline (20 mg / kg) 3 times a week for 4 weeks.

[0066] (1) Extraction and processing of spinal vesicle tissue

[0067] Mice were anesthetized: Mice in the experimental and control groups were placed in an anesthesia induction chamber and anesthetized with 4% isoflurane, followed by maintenance with 1.5% isoflurane. The depth of anesthesia was determined by the absence of pain reflexes and stable breathing (approximately 3-5 minutes).

[0068] Extraction of spinal vesicle tissue: Shave the hair in the spinal region, disinfect the skin three times with 75% alcohol, cut the skin along the edge of the vesicle, bluntly separate the adhesion tissue, and completely peel off the vesicle. Measure the diameter with a ruler and the weight with a scale. After dissecting the mice in the experimental and control groups, extract the hydatid lesion tissue as follows: Figure 1 As shown. Figure 1As can be seen, bone-targeted liposomes (HA coating) achieve higher drug concentrations in spinal lesions compared to traditional oral administration. Furthermore, when combined with albendazole, the vesicle volume was significantly reduced (p<0.01). This suggests that hydroxyapatite (HA coating) specifically adsorbs to bone tissue, resolving the issue of insufficient bone penetration by albendazole.

[0069] Half of the obtained spinal vesicle tissue was immersed in 4% paraformaldehyde (fixed at 4°C for 24 hours) for subsequent tissue embedding, and the other half was wrapped in tin foil and quickly frozen at -80°C for storage.

[0070] (2) Extraction of vesicle tissue proteins

[0071] Tissue pretreatment and disruption: Remove the quick-frozen spinal cystic cyst tissue from the -80°C freezer and thaw at room temperature until the tissue softens slightly. Use a sterile scalpel blade to cut approximately 50 mg of tissue chunks, place them in a pre-chilled grinding tube, and grind in a tissue grinder.

[0072] Ultrasound-assisted cell disruption: Transfer the ground tissue to a 1.5 mL centrifuge tube and add 1 mL of pre-chilled phosphate-buffered saline (PBS). To completely disrupt cellular structures, use an ice-bath ultrasonic disruptor (50 W) in pulse mode (3 seconds on, 10 seconds off) for 5 cycles. After sonication, remove a small amount of the suspension and examine under a microscope to confirm that no intact cells remain.

[0073] Lysis buffer preparation and mixing: Mix RIPA lysis buffer and protease inhibitor PMSF in a precise 100:1 volume ratio. Allow the mixture to stand on ice for 10 minutes to fully activate the inhibitor.

[0074] Low-temperature lysis and centrifugation enrichment: Add 300 μL of pre-chilled lysis buffer to the centrifuge tube containing the tissue suspension and gently pipette to mix thoroughly. To fully release intracellular proteins, lyse the mixture on ice for 30 minutes, briefly mixing on a vortex for 10 seconds every 5 minutes. After lysis is complete, transfer the centrifuge tube to a centrifuge pre-chilled to 4°C and centrifuge at 12,000 × g for 20 minutes. Carefully aspirate the supernatant, taking care not to touch the pellet. This supernatant is the total protein extract.

[0075] Protein quantification and storage: The protein concentration was determined by the BCA method. Based on the test results, the lysis buffer was used to adjust each sample to the same concentration for subsequent experiments. 10× SDS-PAGE loading buffer was added at a ratio of 1:9 (e.g., 90 μL protein solution plus 10 μL buffer). The samples were heated in a boiling water bath for 10 minutes to denature the protein. The samples were then aliquoted into EP tubes and stored in a -20°C refrigerator for later use.

[0076] Western Blot

[0077] SDS-PAGE gel preparation

[0078] Glass plate pretreatment: Clean the glass plates for making glue (a combination of tall and short plates) with detergent three times, rinse with double-distilled water and dry, align them in the direction of "tall plate outside and short plate inside", clamp them on the glue rack, add double-distilled water to the gap between the plates, let it stand for 10 minutes, and then test for leaks to verify the sealing.

[0079] Separation gel preparation (10% concentration, two pieces)

[0080] Mix 4.0 mL of double-distilled water, 3.3 mL of 30% propylene, 2.5 mL of Tris-HCl (pH 8.8), and 100 μL of 10% SDS. Then add 100 μL of 10% AP and 4 μL of TEMED and mix thoroughly. Pour the mixture into the gap between the glass plates to 2 cm from the top, slowly cover with 1 mL of double-distilled water to isolate the top from oxygen, and let it stand at room temperature for 30 minutes until complete polymerization.

[0081] Preparation of stacking gel (5% concentration, two pieces)

[0082] Discard the double-distilled water on the upper layer of the separation gel, use absorbent paper to dry the residual liquid, and mix 3.51mL of double-distilled water, 871uL of 30% propylene, 650uL of Tris-HCl (pH 6.8), and 52uL of 10% SDS. Then add 52uL of 10% AP and 5.2uL of TEMED and continue mixing. After mixing, quickly inject it into the glass plate until it is full, then insert a 10-hole comb (1.0mm thickness) and let it stand at room temperature for 15 minutes until it solidifies. Then put it into double-distilled water and place it in a 4°C refrigerator for use.

[0083] Protein loading

[0084] Fix the prepared SDS-PAGE gel vertically in the electrophoresis tank, and slowly inject 1× electrophoresis buffer through the middle groove. The liquid surface should cover the upper edge of the colloid and the outer tank electrode at the same time (add about 500 mL of buffer to each inner and outer tank).

[0085] Grasp the sides of the comb with your thumb and index finger and slowly pull it upwards, maintaining even pressure. Immediately inspect the integrity of the wells after removal. If any wells are offset or have damaged edges, gently adjust the well shape with a flat-tipped microsyringe needle.

[0086] Add 5uL of prestained protein marker to each lane on either side. Add denatured protein sample (volume per well ≤ 10uL) to the middle lane according to experimental grouping. When adding samples, the pipette tip should be inserted vertically into the bottom of the well. Slowly push the liquid until the liquid surface in the well is slightly convex, avoiding contact with the well wall, which may cause sample leakage or residual bubbles.

[0087] electrophoresis

[0088] Place the electrophoresis tank in a basin and surround it with ice. Connect the positive and negative electrodes correctly (black wire to the negative tank, red wire to the positive tank). Set the voltage to 60V and run for 30 minutes. At this point, the sample and marker should form a straight line at the top of the gel, and the blue indicator band (bromophenol blue) should move to the middle of the gel. Then, increase the voltage to 120V and continue electrophoresis for approximately 1 hour, until the blue band migrates to the bottom of the gel and the proteins are completely separated by molecular weight. The marker band should clearly indicate the standard molecular weight.

[0089] Electroporation

[0090] Cut a PVDF membrane (slightly larger than the gel area) according to the molecular weight of the target protein. Cut a small notch in the upper left corner of the membrane to mark the front and back (the side in contact with the gel is the front). Activate the membrane by immersing it in methanol for 2 minutes, then transfer it to pre-chilled 1× transfer buffer and soak until ready to use.

[0091] After electrophoresis, pry open the glass plates, remove the stacking gel, and cut the gel region containing the target protein according to the pre-stained marker. Pour pre-chilled transfer buffer into an ice tray. Place the transfer chuck with the black negative plate facing down. Stack the layers in the following order: soaked sponge pad → filter paper → PVDF membrane (front side up) → gel → filter paper → sponge pad. Gently roll each layer with a glass rod to remove any bubbles (especially checking the contact surface between the membrane and the gel).

[0092] Insert the assembled transfer cassette vertically into the electroporation chamber (black side facing the black electrode, red side facing the red electrode) and pour pre-chilled transfer buffer to completely cover the transfer cassette. Place the electroporation chamber in a foam box filled with ice to cool down. Set a constant current of 200 mA and electroporate for 1 hour (up to 1.5 hours for cells with a molecular weight greater than 100 kDa).

[0093] Closed

[0094] After transfer, quickly immerse the PVDF membrane in TBST buffer and wash three times (5 minutes each) on a shaker (100 rpm) to thoroughly remove any residual transfer buffer. Then, transfer the membrane to 5% skim milk blocking buffer (prepared in TBST) and block with gentle shaking on a shaker (50 rpm) for 2 hours at room temperature or overnight at 4°C.

[0095] Antibody incubation

[0096] Primary Antibody Incubation: After blocking, immerse the PVDF membrane in TBST buffer and wash on a shaker (120 rpm) for 15 minutes. Then, lay the membrane flat in an incubation box and add an appropriately diluted primary antibody (see Table 2 for primary antibodies and dilution ratios), ensuring that the antibody solution completely covers the membrane surface. Seal the edges of the incubation box with parafilm and incubate on a shaker at 4°C overnight (16-18 hours).

[0097] Table 2 Primary antibodies and dilution ratios

[0098] Antibody name Dilution ratio VEGFA 1:1000 Nrf2 1:1000 β-actin 1:1000 RTN4 1:500

[0099] Wash the membrane and incubate with secondary antibody: The next day, recover the primary antibody (store at -20°C until needed) and wash three times with TBST on a shaker (140 rpm, 10 minutes each). Select the corresponding secondary antibody based on the primary antibody species (see Table 3), dilute and evenly coat the membrane surface, incubate on a shaker (50 rpm) at room temperature for 1 hour, and repeat the TBST wash three times (as above).

[0100] Table 3 Secondary antibodies and dilution ratios

[0101] Antibody name Dilution ratio Goat anti-mouse IgG-HRP 1:20,000 Mouse anti-rabbit IgG-HRP 1:20,000

[0102] Development and imaging:

[0103] After washing, prepare ECL luminescent solution in a 1:1 ratio, react in the dark for 1 minute, mix well and add it dropwise onto the strip. Be sure to prepare it immediately before use. Use the Tanon-4600 imaging system to collect signals for WB strip exposure, and use β-actin as the internal reference to standardize the target protein expression level.

[0104] The molecular mechanism was verified (Western blot was used to detect the expression levels of Nrf2, RTN4 and VEGF in HUVECs). The results were as follows: Figure 2 As shown, from Figure 2 As can be seen, when SFN overexpressed Nrf2 under PSC stimulation, RTN4 protein expression levels significantly decreased, while Nrf2 and VEGF protein expression levels increased significantly. Conversely, when wogonin interfered with Nrf2, RTN4 protein expression levels significantly increased, while Nrf2 and VEGF protein expression levels decreased accordingly. This suggests that wogonin activates RTN4 by inhibiting Nrf2, reducing VEGF expression and blocking angiogenesis in lesions.

[0105] The protein levels of HUVECs treated with PSCs were detected by Western blotting. Figure 3 As shown (Western blot detection of Nrf2, RTN4 and VEGF expression levels in HUVECs). Figure 3 It can be seen that the protein expression levels of Nrf2 and VEGF in the hanhuangsu treatment group were significantly lower than those in the control group, while the protein expression level of RTN4 was increased. The protein expression levels of VEGF and Nrf2 in HUVECs were negatively correlated with RTN4 (p < 0.05).

[0106] Paraffin embedding and sectioning of spinal cystic echinococcosis tissue

[0107] The extracted vesicle tissue was immersed in 4% paraformaldehyde fixative at room temperature for at least 24 hours to maintain cellular morphology. After fixation, the tissue block was finely trimmed to the appropriate size using a sterile scalpel, ensuring a smooth, crack-free cut surface. The block was then placed in a clearly labeled embedding cassette (labeled with the sample number and date). Dehydration was performed sequentially from 70% ethanol (1 hour) to 80% ethanol (1 hour) to 95% ethanol (1 hour) to anhydrous ethanol (2 hours), with fresh solution replaced at each step. After dehydration, the tissue was transferred to xylene for 30 minutes, during which the solution gradually turned from turbid to transparent. After transparency, the tissue was then immersed in molten paraffin wax. Pre-wax immersion: a 60°C paraffin bath for 1 hour to allow the wax to fully penetrate the tissue interstitial spaces; the main wax immersion: transfer the tissue to a 65°C paraffin bath in an embedding machine for 40 minutes, ensuring that no bubbles remained. The tissue block was embedded in a pre-made wax tray and placed on a -20°C freezer for rapid solidification to form a homogeneous wax block. Secure the embedded wax block to a microtome and cut a continuous wax strip to a thickness of 4 μm. Gently transfer the strip to a 42°C warm water bath and flatten it. Use a non-shedding glass slide to evenly remove the tissue from the lower middle portion of the liquid, ensuring the tissue is centered. Bake at 65°C for 4 hours to solidify. Ensure that the sections are uniform in thickness and free of wrinkles or damage.

[0108] Hematoxylin-eosin (HE) staining

[0109] Section baking: Paraffin sections were placed horizontally in a 65°C electric forced-air drying oven and baked continuously for 30 minutes. Paraffin was dissolved by immersion in xylene I and xylene II for 5 minutes each. Rehydration was then performed using a gradient of ethanol, then 90% ethanol, then 80% ethanol, then 70% ethanol (5 minutes per step). Finally, sections were rinsed three times with running water (5 minutes each) to remove any residual organic solvent. After rehydration, sections were completely immersed in hematoxylin stain and allowed to stand at room temperature for 5 minutes to fully stain the nuclei. After staining, sections were washed using a "three-in, three-out" technique: vertically immersing sections in water for 5 minutes each time, repeating this three times to remove free dye. The sections were quickly immersed in 1% acidic ethanol for 3 seconds, followed by a gentle rinse in running water for 10 seconds to terminate the reaction. Sections were then transferred to pH 7.4 phosphate buffer and immersed for 5 minutes. Slides were tilted and slowly immersed in eosin stain with gentle agitation for 1 minute to ensure uniform staining of the cytoplasm. After staining, pre-rinse with a trickle of clean water for 30 seconds, and then thoroughly remove the dye by three 5-minute immersions. Dehydrate in reverse gradient through 70% ethanol → 80% ethanol → 90% ethanol → anhydrous ethanol (5 minutes per level), and then transfer to xylene I and II for 5 minutes each for transparent treatment. Be careful to keep the slices hanging vertically throughout the process to avoid tissue wrinkles. Place the transparent slices in a dust-free environment to dry naturally for 10 minutes, and use the "hanging drop method" to seal the slices: slowly add neutral gum at a 45° angle from the edge of the coverslip, and use capillary action to spread it naturally to avoid air bubbles. After sealing, let it stand for 24 hours until the medium is completely solidified, and use an inverted optical microscope to collect images.

[0110] Histological examination was performed by hematoxylin-eosin (HE) staining. Figure 4 As shown, it was confirmed that abundant capillary-like lumen structures were formed in the vesicle tissue of the control group, while this structure was rare in the capillary-like lumen structure of the wogonin combined with albendazole experimental group, indicating that wogonin combined with albendazole has the effect of cutting off vascular nutrition, and wogonin has a natural low toxicity (LD 50 >1g / kg), the dosage of albendazole can be reduced, thus lowering the risk of hepatotoxicity.

[0111] Immunohistochemical staining

[0112] Paraffin sections were placed horizontally in a 65°C electric forced-air drying oven and baked continuously for 30 minutes. Dewaxing and rehydration were performed by immersing the sections in xylene I and xylene II for 5 minutes each to dissolve the paraffin. The sections were then rehydrated in a gradient of ethanol, 90% ethanol, 80% ethanol, and 70% ethanol (5 minutes per step). Finally, the sections were rinsed three times with running water (5 minutes each) to remove any residual organic solvent. Sodium citrate / citrate buffer was prepared in advance and poured into a retrieval box. The solution was preheated in a microwave oven for 3 minutes. The sections were placed in an antigen retrieval box and heated in an autoclave at 1800W until the air valve was inflated, then increased to 800W and maintained for 8 minutes. After cooling to room temperature, the sections were transferred to an ice-water bath for 10 minutes to enhance epitope exposure. A closed circle was drawn along the edge of the tissue using an immunohistochemistry pen. Endogenous peroxidase inhibitor was added dropwise to completely cover the tissue. The sections were incubated at room temperature in the dark for 10 minutes. The sections were rinsed three times with PBS buffer (5 minutes each) to block endogenous peroxidase activity.

[0113] Primary antibody incubation: Prepare the primary antibody by diluting according to Table 2 above. After absorbing the liquid around the tissue, add 50uL of diluted antibody to each section (covering the tissue) and incubate in a humidified chamber at 4°C overnight.

[0114] Rewarming: The next day, take out the incubation box from the 4°C refrigerator and transfer it to a 37°C constant temperature incubator to rewarm for 30 minutes.

[0115] Secondary antibody incubation: rinse with PBS three times (5 minutes each time), and add HRP-labeled secondary antibody (incubate at 37°C for 30 minutes).

[0116] Wash the secondary antibody: immerse the slice vertically in PBS liquid, shake gently for 5 minutes each time, and repeat 3 times.

[0117] DAB color development control: Prepare fresh DAB working solution (A:B = 1:100) and monitor the reaction under a microscope after adding it. When brown-yellow particles appear in the tissue (usually within 5-20 seconds), rinse vigorously with running water to terminate the reaction.

[0118] Hematoxylin staining: Completely immerse the rehydrated sections in hematoxylin stain and let them stand at room temperature for 5 minutes to fully stain the nuclei. After staining, wash the sections using the "three in, three out" method: vertically immerse the sections in clean water for 5 minutes each time, repeating this three times to remove free dye.

[0119] Differentiation: Quickly immerse in 1% acidic ethanol for 3 seconds and immediately rinse gently with running water for 10 seconds to terminate the reaction.

[0120] Rebluing: Transfer sections to pH 7.4 phosphate buffer and soak for 5 minutes.

[0121] Dehydration and clearing: Dehydrate in reverse gradient from 70% ethanol to 80% ethanol to 90% ethanol to anhydrous ethanol (5 minutes per level), then transfer to xylene I and II for 5 minutes each for clearing. Keep the sections vertically hung throughout the process to avoid tissue wrinkles.

[0122] Sealing: Place the transparent sections in a dust-free environment to air dry for 10 minutes, and use the "hanging drop method" to seal the sections: slowly add neutral gum at a 45° angle from the edge of the coverslip, and use capillary action to spread it naturally to avoid air bubbles.

[0123] After the tissue samples were processed, the results were as follows Figure 5 As shown, from Figure 5 It can be seen that after treatment with wogonin, the hemoglobin concentration in the lesions of the spinal cystic echinococcosis mouse model was significantly reduced (p<0.05) and the microvessel density (CD31+) was significantly reduced.

[0124] Immunohistochemical analysis of cystic tissues in the spinal cystic echinococcosis model was performed, e.g. Figure 6 As shown. Figure 6 It can be seen that after treatment with wogonin, the positive expressions of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) in the spinal cystic echinococcosis mouse model were significantly reduced (p < 0.001).

[0125] Data statistical analysis

[0126] Data were statistically analyzed using SPSS 26.0, and quantitative data are presented as mean ± standard deviation. Two-group data with normal distribution and homogeneous variance were analyzed using independent sample t-tests, and multiple group comparisons were performed using one-way analysis of variance (ANOVA). Immunohistochemistry, in vitro angiogenesis, hematoxylin and eosin staining, and Western blot results were quantitatively analyzed using Image J, and statistical graphs were generated using GraphPad Prism 10.0. Statistical significance between group differences was determined at p < 0.05.

[0127] In vitro experiments

[0128] Construction of PSCs-HUVECs co-culture system:

[0129] (1) To simulate the microenvironment of Echinococcus granulosus infection, a PSCs-HUVECs co-culture system was constructed. Human umbilical vein endothelial cells (HUVECs, density 2×10 5 The cells were pre-cultured for 24 hours in endothelial cell complete medium (ECM) containing 10% fetal bovine serum (FBS), 1% endothelial cell growth supplement (ECGS) and 100 U / mL penicillin-streptomycin. After the cell attachment rate reached 80%, the protoscoleces of Echinococcus granulosus (PSCs, density 2×10 3 / mL, inoculation volume 100uL).

[0130] (2) During the co-culture period, the system was maintained at a constant temperature of 37°C, 5% CO2, and saturated humidity (>95%), and fresh culture medium (containing 10% FBS) pre-equilibrated to 37°C was added every 12 hours.

[0131] (3) After 48 h of co-culture, the culture medium was discarded and HUVECs were gently washed three times with pre-cooled PBS buffer (pH 7.4) to completely remove PSCs secretory vesicles and apoptotic debris for subsequent related experiments.

[0132] HUVECs in vitro angiogenesis assay

[0133] Pre-experimental preparation: 24 hours before the experiment, transfer Matrigel stored at -80°C on ice to a 4°C refrigerator and slowly thaw (to avoid repeated freeze-thaw cycles that may reduce the activity of the colloid). At the same time, seal the 96-well plate, 100µL pipette tips, pipette gun, and 1mL syringe and pre-chill at -20°C. For the actual experiment, all consumables were sterilized with UV light for 30 minutes. ECM basal medium and sterile PBS were pre-chilled to 4°C until use.

[0134] Cell status assessment: Select 4-6 passage human umbilical vein endothelial cells (HUVECs) with a confluency of 70%-80%. After discarding the old culture medium, slowly add PBS preheated to 37°C along the wall of the flask. Gently shake three times to remove residual serum. Confirm under a microscope that the cells have a typical "cobblestone" spreading morphology and are free of vacuoles.

[0135] Cell digestion: Add 1 mL of 0.25% trypsin-EDTA digestion solution and incubate at 37°C for 1 minute until the liquid turns from pink to orange-red. Tap the side of the culture flask three times to detach the cells. Immediately add 2 mL of pre-cold stop solution containing 10% FBS to terminate the digestion. Centrifuge the cell suspension at 1000 × g for 5 minutes, discard the supernatant, and gently resuspend in pre-cold serum-free ECM medium. Adjust the density to 4 × 10 by counting. 5 cells / mL, and store the cell suspension in an ice box for later use (no more than 15 minutes).

[0136] Perform the entire Matrigel plating process on ice. Mix the thawed Matrigel with pre-chilled basal medium in a 1:3 volume ratio. Mix thoroughly with a pre-chilled pipette tip by pipetting and aspirating 10 times, avoiding shear stress caused by vortexing. Hold the pipette tip vertically in the center of a 96-well plate and slowly inject 50 μL of the mixture, allowing the droplet to naturally spread to the edge of the well. If bubbles form, gently puncture them with a 1 mL sterile needle. After plating, transfer the plate to a 37°C incubator and incubate for 30 minutes. Observe the plate every 10 minutes. The gel is considered fully cured when the surface loses its reflectivity and does not flow when pressed.

[0137] Cell inoculation and culture: After the colloid solidifies, 100uL of cell suspension (containing 4×10 4 If drug intervention is required, dilute the solution to a 2× working concentration in serum-free medium in advance and mix with an equal volume of the cell suspension before inoculation. Gently tap the edge of the plate five times to evenly distribute the cells. Incubate at 37°C, 5% CO₂ for 4-6 hours. Do not move the plate during this time to prevent the tubular structures from breaking.

[0138] Imaging and quantitative analysis: After the culture, five non-edge fields were randomly selected using an inverted phase contrast microscope to photograph the tubular structure formation. Tube length and branch points were analyzed using ImageJ software to evaluate the tubular formation ability.

[0139] After the PSCs-HUVECs co-culture system was treated with the drug prepared in Example 1, the results were as follows: Figure 7 As shown. Figure 7 It can be seen that the baicalein treatment group can significantly reduce the endothelial cell vascular lumen length and branch point number compared with the use of Nrf2 activator sulforaphane (SFN) (p<0.05), and is consistent with the high expression of RTN4 (LV-RTN4).

[0140] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of wogonin in the preparation of drugs for treating or inhibiting spinal cystic echinococcosis.

2. The use of wogonin according to claim 1 in the preparation of a drug for treating or inhibiting spinal cystic echinococcosis, characterized in that: Wogonin activates RTN4 by inhibiting Nrf2, reducing VEGF expression, blocking lesion angiogenesis, and treating or inhibiting spinal cystic echinococcosis.

3. A drug for treating or inhibiting spinal cystic echinococcosis, characterized in that: The method comprises drug-loaded liposomes and a coating, wherein the mass ratio of the drug-loaded liposomes to the coating is 1-5:10; The drug-loaded liposomes comprise a drug phase and a lipid phase, and the drug phase comprises wogonin and albendazole.

4. A drug for treating or inhibiting spinal cystic echinococcosis according to claim 3, characterized in that: The mass ratio of wogonin and albendazole is 0.5-1:0.5-1; The lipid phase includes dipalmitoylphosphatidylcholine and cholesterol, and the molar ratio of dipalmitoylphosphatidylcholine to cholesterol is 6-8:2-4.

5. The drug for treating or inhibiting spinal cystic echinococcosis according to claim 3, characterized in that: The coating is hydroxyapatite, and the particle size of hydroxyapatite is 40-50nm.

6. The drug for treating or inhibiting spinal cystic echinococcosis according to claim 3, characterized in that: The dosage of wogonin in drugs for treating or inhibiting spinal cystic echinococcosis is 15-25 mg / kg.

7. A method for preparing a drug for treating or inhibiting spinal cystic echinococcosis according to any one of claims 3 to 6, characterized in that: The method comprises the following preparation steps: S1, mixing dipalmitoylphosphatidylcholine, cholesterol and chloroform to obtain a lipid phase; S2, mixing wogonin, albendazole and ethanol to obtain a drug phase; S3, mixing the lipid phase with the drug phase, rotary evaporating under reduced pressure, and drying to obtain a lipid film, adding phosphate buffer, stirring, sonicating, and filtering to obtain a drug-loaded liposome suspension; S4. After mixing hydroxyapatite with phosphate buffer, add drug-loaded liposome suspension, shake and incubate, and thus obtain a drug for treating or inhibiting spinal cystic echinococcosis.

8. The method for preparing the drug for treating or inhibiting spinal cystic echinococcosis according to claim 7, characterized in that: The temperature of the vacuum rotary evaporation in S3 is 40-50° C., the time of the vacuum rotary evaporation is 30-60 min, and the pH of the phosphate buffer is 7-8.

9. The method for preparing a drug for treating or inhibiting spinal cystic echinococcosis according to claim 7, characterized in that: The stirring temperature in S3 is 50-60° C., the stirring time is 5-10 min, the ultrasonic power is 180-220 W, and the ultrasonic time is 3-8 min.

10. The method for preparing the drug for treating or inhibiting spinal cystic echinococcosis according to claim 7, characterized in that: The pH of the phosphate buffer in S4 is 7-8, the shaking incubation temperature is 36-38° C., the shaking incubation time is 1-3 h, and the shaking incubation frequency is 150-200 rpm.

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