Wedelolactone-loaded exosome and application thereof in preparation of drugs for preventing and treating sepsis

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a wedelolactone-loaded exosome and application thereof in preparation of a medicine for preventing and treating sepsis. The wedelolactone loaded exosome (WEL at Exo) is obtained by loading wedelolactone in a macrophage exosome, and the mass ratio of the wedelolactone to the macrophage exosome is 1: (5-8). Animal experiments prove that the WEL-coated Exo can relieve hepatoma injury, systemic inflammation of sepsis and systemic oxidative stress of sepsis, so that the WEL-coated Exo can be further developed into the medicine for preventing sepsis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to exosomes loaded with wedelolactone and their application in the preparation of drugs for preventing and treating sepsis. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome caused by severe infections such as bacteria. So far, there is no specific drug or therapy, and the fatality rate is as high as 20% to 45%, which is regarded as a huge threat to human health. With the continuous increase of antibiotic resistance, the development of effective anti-sepsis drugs has become a major proposition in the medical field.

[0003] Wedelolactone, chemical name: 7-methoxy-5,11,12-trihydroxy, molecular formula: C 16 H 10 O 7 , molecular weight: 314.249. Wedelolactone is a natural small molecule compound derived from Eclipta prostrata and Wedelia chinensis, and is a traditional Chinese medicine additive for treating diseases such as osteoporosis, liver diseases, kidney damage, hyperlipidemia, snake bites, and hair loss. However, wedelolactone is not easily soluble in water, has low bioavailability, and has little retention in local liver tissues, which limits its application effect. Summary of the Invention

[0004] The purpose of the present invention is to provide exosomes loaded with wedelolactone and their application in the preparation of drugs for treating sepsis-induced liver injury, so as to lay a foundation for improving the biological application effect of wedelolactone and thus better reducing liver injury inflammation.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions: In the first aspect, the present invention provides exosomes loaded with wedelolactone (Exo@WEL), which are obtained by loading wedelolactone (WEL) into macrophage exosomes (Exo), and the mass ratio of the wedelolactone to the macrophage exosomes is 1:5 - 8.

[0006] In the present invention, Exo@WEL is obtained by loading WEL into Exo, and then DiR-labeled Exo and Exo@WEL are intraperitoneally injected into mice. The results show that compared with Exo, Exo@WEL has a significantly greater drug localization in the livers of mice, indicating that Exo@WEL has strong liver targeting. In terms of the therapeutic effect of sepsis, Exo@WEL > Exo > WEL, indicating that WEL@Exo can be further developed into a drug for preventing sepsis.

[0007] Furthermore, the macrophage exosomes are exosomes derived from macrophage RAW264.7.

[0008] In a second aspect, the present invention provides a method for preparing the exosomes loaded with wedelolactone, which is to load wedelolactone into the macrophage exosomes by using an ultrasonic incubation treatment method.

[0009] Further, the ultrasonic incubation treatment method includes: mixing wedelolactone with a macrophage exosome suspension having a concentration of 1-1.5 mg / mL, and performing 4-5 cycles of ultrasonic treatment on the obtained mixture, with one cycle process being 40-50 s of ultrasonic treatment, 1.5-2 min of rest, and 4-5 min of cooling, the ultrasonic power being 200-300 W, and the ultrasonic frequency being 40-50 KHz.

[0010] In a third aspect, the present invention provides the use of the exosomes loaded with wedelolactone in the preparation of a drug for preventing and treating sepsis.

[0011] In a fourth aspect, the present invention provides the use of the exosomes loaded with wedelolactone in the preparation of a drug for preventing and treating target organ damage caused by sepsis.

[0012] Further, the target organ includes the liver.

[0013] In a fifth aspect, the present invention provides a drug for preventing and treating liver injury caused by sepsis, including the exosomes loaded with wedelolactone as described above, and pharmaceutically acceptable excipients.

[0014] Further, the excipients are selected from one or more of conventional diluents, fillers, stabilizers, osmotic pressure regulators, pH regulators, and preservatives.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The exosomes loaded with wedelolactone (WEL@Exo) provided by the present invention can alleviate the inflammation of septic liver injury and play a hepatoprotective role.

[0016] The present invention proves through animal experiments that in a sepsis mouse model induced by cecal ligation puncture (CLP), WEL@Exo significantly reduces the expression levels of inflammatory factors TNFα, IL6, and IL-1β in the serum of septic mice, reduces the expression level of the biochemical index MDA, increases the expression levels of GSH and SOD, and reduces the expression levels of ALT and AST, and has an obvious effect of preventing and treating septic liver injury, indicating that WEL@Exo can be further developed into a drug for preventing sepsis. Description of the Drawings

[0017] Figure 1It is the characterization diagram of WEL@Exo provided by the present invention. Among them, A is the transmission electron microscope observation result of WEL@Exo; B is the particle size distribution result of WEL@Exo; C is the Zeta potential distribution diagram of WEL@Exo.

[0018] Figure 2 It is the result diagram of the in vitro toxicity experiment of WEL@Exo provided by the present invention.

[0019] Figure 3 It is the result diagram of the in vitro targeting experiment of WEL@Exo provided by the present invention. Among them, A is the fluorescence analysis of exosome distribution in mice in vivo and in vitro; B is the fluorescence analysis of exosome distribution in the mouse liver; C is the quantitative fluorescence result of exosomes in the mouse liver.

[0020] Figure 4 It is the anti-inflammatory and antioxidant result diagram of WEL@Exo provided by the present invention. Among them, A is the statistical chart of the content of TNF-α in serum, B is the statistical chart of the content of IL-6 in serum, C is the statistical chart of the content of IL-1β in serum, D is the statistical chart of the content of MDA in serum, E is the statistical chart of the content of GSH in serum, F is the statistical chart of the content of SOD in serum. Compared with the Sham group, ** P <0.01; compared with the CLP group, * P <0.05, ** P <0.01.

[0021] Figure 5 It is the result diagram of the liver morphology of mice in each group.

[0022] Figure 6 It is the result diagram of HE staining of liver tissues of mice in each group, and the scale bar is 50 μm.

[0023] Figure 7 It is the liver function index diagram of mice in each group, which is the influence of WEL@Exo on the expression level of liver function indexes in the serum of septic mice. Among them, A is the statistical chart of the content of AST in serum, B is the statistical chart of the content of ALT in serum. Compared with the Sham group, ** P <0.01; compared with the CLP group, * P <0.05, ** P <0.01.

[0024] Figure 8 It is the result diagram of the observation of tissue immunofluorescence of mice in each group. A is the result diagram of Nrf2 immunofluorescence staining; B is the statistical chart of Nrf2 immunofluorescence staining. Compared with the Sham group, ** P <0.01; compared with the CLP group, * P <0.05, ** P <0.01.

[0025] Protein expression in the livers of mice in each group in Figure 9. A shows the expression of Nrf2 and HO-1 proteins in the liver, B is the statistical chart of the content of Nrf2 in the liver, and C is the statistical chart of the content of HO-1 in the liver. Compared with the Sham group, ** P < 0.01; compared with the CLP group, * P < 0.05, ** P < 0.01. Specific implementation mode

[0026] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0027] Example 1: Preparation and verification of Wedelolactone-loaded exosomes (WEL@Exo).

[0028] 1. Experimental method (1) Preparation of WEL@Exo The first step: Culture macrophages RAW264.7 for 24 h and collect the cell supernatant. Specifically: The cells are cultured in a special medium containing normal serum (RAW264.7 complete medium) for a certain period of time. When the density of RAW264.7 cells reaches 70% - 80%, the original medium is removed and replaced with a new medium without exosomes for continued culture. After culturing for 24 h, use a sterile cell scraper to gently scrape close to the bottom of the cell culture flask until the foggy layer at the bottom of the cell culture flask completely disappears. Then, collect the cells and cell suspension into a 15 mL centrifuge tube with a 1 mL pipette gun, centrifuge at 1100 rpm / min, collect the cell supernatant, combine the supernatant samples of the same cell culture medium, put them into a sterile centrifuge tube, and store them at -80 °C.

[0029] The second step: Centrifuge to obtain macrophage exosomes (Exo) by ultracentrifugation method. Specifically: Take out the supernatant from -80 °C and thaw the sample at medium speed at 37 °C; transfer the sample to a new centrifuge tube and centrifuge at 300 × g, 4 °C for 10 min; take the supernatant to a new centrifuge tube and centrifuge at 3000 × g, 4 °C for 15 min; take the supernatant to a new centrifuge tube and centrifuge again at 10,000 × g, 4 °C for 30 min to remove larger vesicles; take the supernatant, transfer it to a new centrifuge tube, select an ultracentrifuge rotor, and centrifuge at 100,000 × g, 4 °C for 70 min; remove part of the supernatant, resuspend the remaining supernatant and precipitate in PBS, filter through a 0.22 μm filter membrane, and collect the filtrate; transfer the filtrate to a new centrifuge tube, select an ultracentrifuge rotor, and centrifuge again at 100,000 × g, 4 °C for 70 min; remove the supernatant, and the obtained precipitate is exosomes (Exo). Resuspend the precipitate with pre-cooled 1×PBS to obtain an Exo suspension with a concentration of 1.2 mg / mL.

[0030] Step 3: Then use the ultrasonic incubation treatment method to load WEL into Exo to obtain Exo@WEL. Specifically, add WEL to the above Exo suspension so that the mass ratio of Exo to WEL is 6:1. The obtained mixture is subjected to 4 cycles of ultrasonic treatment, with a cycle process of ultrasonic treatment for 45 s, standing for 2 min, and cooling for 5 min. The ultrasonic power is 300 W, and the ultrasonic frequency is 40 KHz. After ultrasonic treatment, to restore the stability of the exosome membrane, the obtained mixed solution is incubated in the dark at 37 °C for 1 h. Transfer to a 100 kDa ultrafiltration tube, centrifuge at 4 °C, 4000 rpm, wash with PBS, centrifuge a total of 3 times, 20 min each time. After centrifugation, carefully aspirate the supernatant and retain the precipitate in the centrifuge tube. Repeatedly pipette the precipitate in the ultracentrifuge tube with a sterile PBS solution to obtain Exo@WEL.

[0031] (2)Identification of WEL@Exo.

[0032] Reconstitute Exo@WEL with PBS, add an equal volume of chromatographic methanol, mix well, break the membrane by ultrasound (30 min), take 0.2 ml of the filtrate in a liquid phase bottle, transfer the Exo@WEL solution to an HPLC injection vial, inject 10 μL respectively, and detect according to the standard curve chromatographic conditions. As Figure 1 The transmission electron microscopy, particle size, and Zeta potential results show that Exo successfully encapsulates WEL.

[0033] Example 2: In vitro cytotoxicity experiment.

[0034] 1. Experimental method Count the AML12 cells in the logarithmic growth phase and seed them in a 96-well plate. Add WEL@Exo with final concentrations of 0.4 μg / mL, 0.8 μg / mL, 1.6 μg / mL, 3.2 μg / mL, 6.4 μg / mL, 12.8 μg / mL, 25.6 μg / mL, and 51.2 μg / mL in sequence. After incubating for 24 h, add CCK-8 reagent to measure the toxicity.

[0035] 2. Experimental results As Figure 2 shown, WEL@Exo did not show any toxic effects on AML12 cells within the concentration range of 0.4 - 51.2 μg / mL.

[0036] Example 3: In vivo distribution of WEL@Exo.

[0037] 1. Experimental method Stain the exosomes with the lipophilic dye DiR. Take 100 μL of Exo or Exo@WEL solution, add DiR dye at a volume ratio of 1:100, mix well, and incubate in a 37°C incubator for 1 h. Use an Exosome Spin Column gel column to filter out the excess dye. Prepare the DiR staining solution according to the mouse body weight and number of mice, and inject the DiR-labeled exosomes into the mice via intraperitoneal injection. Inject 50 mg of Exo or Exo@WEL per kilogram of mouse body weight. Sacrifice the mice 24 h after administration, take out the liver, and observe the distribution of exosomes in the liver.

[0038] 2. Experimental results As Figure 3 shown, in this example, the in vivo distribution of Exo and Exo@WEL drugs was measured. DiR-labeled exosomes were used to monitor the in vivo drug distribution. Compared with the Exo group, WEL@Exo was widely distributed in the mouse peritoneal cavity in the Exo@WEL group, and the drug localization in the mouse liver in the Exo@WEL group was significantly greater, indicating that WEL@Exo retained more in the local liver tissue and had good targeting to the liver.

[0039] Example 4: Animal experiments to verify the effect of WEL@Exo in treating sepsis.

[0040] 1. Experimental method (1) Experimental grouping After 3 days of adaptive feeding, female C57BL / 6 mice weighing 18 - 22 g were randomly divided into 4 groups of 12 mice each, namely the Sham group, the CLP group, the CLP + WEL group, and the CLP + Exo group (WEL@Exo group). Except for the Sham group, the remaining mice were induced with sepsis by cecal ligation and perforation (CLP). In the Sham group, the cecum was only exposed and then returned to the abdominal cavity without ligation and perforation. The CLP group was sacrificed 24 h after the operation. In the treatment groups (CLP + WEL group, CLP + Exo group, WEL@Exo group), WEL, Exo, or Exo@WEL (diluted with sterile water for injection) were administered intraperitoneally to the mice before the operation. Calculated based on the body weight of the mice, the dosage was 50 mg / kg. The operation was performed 24 h after the administration started, and the samples were taken 24 h after the operation.

[0041] (2)Pathological section detection.

[0042] Since pathological sections can visually reflect the degree of tissue lesions in the body. We further investigated the effect of WEL@Exo on the target liver injury in septic mice. The liver tissues of the mice were dissected and fixed with 10% neutral formalin solution. After 48 h, they were paraffin-embedded, sectioned at a thickness of 4 μm, dewaxed with xylene, and then stained with hematoxylin-eosin. The pathological changes of each tissue sample were observed under an inverted microscope and photographed.

[0043] (3)In vivo inflammation detection.

[0044] In addition to histological and transaminase changes, the change of verification factors is another major indicator in sepsis-induced inflammation. During the occurrence of inflammation, inflammatory cells are activated and recruited, secreting a large number of pro-inflammatory factors such as TNF-α, IL-6, and IL-1β. At 24 h after drug administration to the mice, serum was separated from the mouse blood, and the contents of TNF-α, IL-6, and IL-1β inflammatory factors in the serum were measured according to the operating steps of the ELISA kit.

[0045] (4)Transaminase level detection.

[0046] During the occurrence of sepsis, the transaminase level in the liver is one of the important indicators. At 24 h after drug administration to the mice, serum was separated from the mouse blood, and ALT and AST were detected.

[0047] (5)MDA, GSH, and SOD level detection.

[0048] During the occurrence of sepsis, oxidative stress injury of the liver is also one of the pathologies of liver injury. At 24 h after administering the drug to the mice, serum was separated from the mouse blood, and MDA, GSH, and SOD were detected.

[0049] (6)Detection of Nrf2 expression level.

[0050] In the immunofluorescence assay, according to body weight, the mice were randomly divided into 4 groups of 3 each, and were set as the Sham group, the CLP group, the WEL group, and the WEL@Exo group respectively. The primary antibody Nrf2 (rabbit, 1:200) was incubated at 4 °C for 12 h. The secondary antibody (goat anti-rabbit IgG[H+L] labeled with Nrf2) was incubated at 25 °C at 1:250 for 1 h. The DAPI staining solution was applied for 20 minutes. Finally, the images were observed with an inverted confocal laser microscope.

[0051] (7)Expression of Nrf2 and HO-1 proteins.

[0052] According to the molecular weights of Nrf2 and HO-1, a separating gel with the corresponding percentage content was selected to electrophoretically separate the protein samples. The gel was taken out and the stacking gel was removed. The PVDF membrane of the same size was activated in methanol, and the protein was transferred from the gel to the PVDF membrane. At room temperature, the prepared blocking solution was incubated with the PVDF membrane for 1.5 h. Nrf2 (rabbit anti, 1:800), HO-1 (rabbit anti, 1:800), and GAPDH (rabbit anti, 1:8000) were incubated with the PVDF membrane on a shaker at 4 °C overnight. After the incubation of the primary antibody was completed, the membrane was washed three times with TBST buffer for 5 min each time, and the rabbit secondary antibody was prepared with TBST buffer. After the membrane washing was completed, the PVDF membrane and the secondary antibody were incubated on a shaker at room temperature for 1 h. After the incubation of the secondary antibody was completed, the membrane was washed again three times with TBST buffer for 5 min each time. Finally, the A solution and the B solution in the ECL chemiluminescence kit were mixed in equal amounts to prepare the luminescence reagent. The PVDF membrane was developed and photographed using a gel imaging system, and the gray value of the protein band was calculated using ImageJ software and statistically analyzed.

[0053] 2. Experimental results As Figure 4 shown, after CLP-induced sepsis injury in mice, the levels of inflammatory factors TNF-α, IL-6, and IL-1β in the serum of the CLP group were significantly increased ( P < 0.05), indicating the inflammatory response that occurred in mice under the action of CLP. Compared with the CLP group, the levels of inflammatory factors were reduced to varying degrees after administration of WEL, Exo, and WEL@Exo ( P<0.05), the effect of Exo@WEL was the most significant. The above results indicate that Exo@WEL can improve tissue inflammation in mice induced by CLP and has a significant anti-inflammatory effect. Kits were used to detect MDA, SOD, and GSH to reflect the oxidative stress injury in mice. After CLP-induced liver injury, the activities of GSH and SOD in the serum of mice in the CLP group were significantly decreased, and the content of MDA was significantly increased ( P <0.05), indicating that oxidative stress occurred in mice under the action of CLP. Compared with the CLP group, the activities of GSH and SOD in the WEL, Exo, and Exo@WEL administration groups increased to varying degrees, and the content of MDA decreased to varying degrees ( P <0.05), especially the effect of Exo@WEL was the most significant. The results show that Exo@WEL can significantly enhance the activity of antioxidant enzymes, improve the antioxidant stress level of mice, and reduce the oxidative stress injury in mice induced by CLP.

[0054] As Figure 5 shown, the results of the liver morphology of mice showed that the liver appearance in the CLP group was rough, with nodules on the surface and the liver was pale. Administration of WEL, Exo, and Exo@WEL made the liver tissue surface gradually smooth and the color gradually approach that of the control group. Among them, the effect of Exo@WEL was the most significant, indicating that Exo@WEL can improve the morphological damage of liver tissue.

[0055] As Figure 6 shown, the histological observation of HE staining in the animal experiment. HE staining showed that the liver tissue morphology of mice in the Sham group was normal, the hepatocytes were arranged neatly, and there was no obvious damage. The arrangement of hepatocytes in the CLP group was less neat than that in the control group, and obvious inflammatory infiltration was observed. Administration of WEL, Exo, and WEL@Exo improved the arrangement of liver tissue to varying degrees, and the inflammatory cells decreased to varying degrees. The results indicate that Exo@WEL can significantly improve the pathological damage of liver tissue.

[0056] As Figure 7 shown, the levels of ALT and AST in the serum are widely used in the detection of liver function indicators. The results show that the levels of ALT and AST in the serum of mice in the CLP group were significantly increased ( P <0.05), indicating that CLP-induced abnormal liver function in mice, indicating that the experimental model was successfully established. Compared with the CLP group, the expression levels of ALT and AST gradually decreased after administration of WEL, Exo, and WEL@Exo ( P <0.05), indicating that Exo@WEL can significantly inhibit the increase of liver function indicators induced by CLP and relieve the target organ damage induced by sepsis.

[0057] As Figure 8As shown, the expression of Nrf2 in mouse liver tissue was detected by immunofluorescence staining. The results showed that the green intensity of Nrf2 immunofluorescence staining in the liver tissue of mice in the CLP group was significantly weakened. Compared with the CLP group, the green fluorescence gradually increased after the intervention of WEL, Exo, and Exo@WEL in the liver tissue of mice, and the effect of Exo@WEL was the most significant. The results indicate that Exo@WEL can improve CLP-induced septic liver injury.

[0058] As Figure 9 shown, the expressions of Nrf2 and HO-1 proteins in mouse liver tissue were detected by Western bloting. The results showed that the expression levels of Nrf2 and HO-1 proteins in the liver tissue of mice in the CLP group were significantly decreased ( P < 0.05). Compared with the CLP group, the levels of Nrf2 and HO-1 in the liver tissue of mice increased to varying degrees after the intervention of WEL, Exo, and Exo@WEL ( P < 0.05), and the effect of Exo@WEL was the most significant. The results indicate that Exo@WEL can improve CLP-induced septic liver injury.

[0059] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0060] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0061] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. Exosomes loaded with wedelilactone, characterized in that: The method is obtained by loading wedelolactone into macrophage exosomes, wherein the mass ratio of wedelolactone to macrophage exosomes is 1:5-8.

2. The exosomes loaded with wedelilactone according to claim 1, characterized in that: The macrophage exosomes are exosomes derived from macrophage RAW264.

7.

3. The method for preparing exosomes loaded with wedelilactone according to claim 2, characterized in that: The method uses an ultrasonic incubation method to load the wedelia lactone into the macrophage exosomes.

4. The method for preparing exosomes loaded with wedelilactone according to claim 3, characterized in that: The ultrasonic incubation treatment method comprises: mixing wedelia lactone with a macrophage exosome suspension having a concentration of 1-1.5 mg / mL, subjecting the resulting mixture to 4-5 cycles of ultrasonic treatment, with ultrasonic treatment for 40-50 seconds, rest for 1.5-2 minutes, and cooling for 4-5 minutes as one cycle process, with an ultrasonic power of 200-300 W and an ultrasonic frequency of 40-50 KHz.

5. Use of the exosomes loaded with wedelia lactone according to any one of claims 1 to 2 in the preparation of a medicament for preventing and treating sepsis.

6. Use of the exosomes loaded with wedelilactone according to any one of claims 1 to 2 in the preparation of a medicament for preventing and treating target organ damage caused by sepsis.

7. The use of the exosomes loaded with wedelilactone according to claim 6 in the preparation of a drug for preventing and treating target organ damage caused by sepsis, characterized in that: The target organ includes the liver.

8. A drug for preventing and treating liver damage caused by sepsis, characterized in that: It comprises the exosomes loaded with wedelilactone according to any one of claims 1 to 2, and pharmaceutically acceptable excipients.

9. The drug for preventing and treating liver damage caused by sepsis according to claim 8, characterized in that: The auxiliary material is selected from one or more of a diluent, a filler, a stabilizer, an osmotic pressure regulator, a pH regulator and a preservative.

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