Application of BV2 exosomes in the preparation of drugs for the treatment of postoperative delirium

Abstract

The present invention provides the use of BV2 exosomes in the preparation of a drug for treating postoperative delirium. A rat postoperative delirium model was successfully constructed, and the role of BV2 exosomes in treating postoperative delirium was studied by injecting BV2 exosomes into the rat postoperative delirium model. The results showed that BV2 exosomes can improve the damage of hippocampal nerve cells and reduce cell apoptosis, thereby partially reversing the postoperative delirium in rats.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to the application of BV2 exosomes in preparing a drug for treating postoperative delirium. Background Art

[0002] Postoperative delirium (POD) refers to a reversible, fluctuating acute brain disorder syndrome that occurs within a few days after anesthesia and surgery, with functional disorders in consciousness, cognitive function, orientation, logical thinking ability, memory, and sleep as the main manifestations. It is an important risk factor for postoperative falls, depression, and self-harm in elderly patients. It can also increase the incidence and mortality of postoperative lung infections, bedsores, and venous thrombosis, prolong hospitalization, increase the workload of medical staff, and bring huge economic burdens to families and society.

[0003] Currently, POD in elderly patients has received widespread attention both at home and abroad. However, there is still no definite conclusion on whether the depth of anesthesia is a related factor for the occurrence of POD in elderly patients, whether different anesthesia depths will affect the incidence of postoperative POD in elderly patients, and what its occurrence mechanism is. Summary of the invention

[0004] Based on the above, the purpose of the present invention is to provide the use of BV2 exosomes in the preparation of drugs for the treatment of postoperative delirium, and to study the occurrence and mechanism of cognitive impairment in the rat brain under long-term deep anesthesia by preparing mouse microglial BV2 exosomes and constructing a rat postoperative delirium model.

[0005] In order to achieve the purpose of the present invention, the technical solution adopted by the present invention is:

[0006] The present invention provides use of BV2 exosomes in preparing a medicament for treating postoperative delirium.

[0007] Preferably, the drug treats postoperative delirium by improving damage to nerve cells in hippocampal tissue and reducing cell apoptosis.

[0008] Preferably, the application includes constructing a postoperative delirium model in rats, and the construction method includes: anesthetizing SD rats with isoflurane anesthesia machine and then performing surgical modeling, making a longitudinal midline incision from the sternum to 0.5CM proximal to the pubic symphysis through the skin, abdominal muscles and peritoneum, partially exposing the abdominal organs for 2 minutes, and then gradually suturing with 5-0 sutures, returning to the anesthesia room for anesthesia for 2 hours after surgery, and applying EMLA cream to each rat before, after and one day after surgery.

[0009] Preferably, the method comprises injecting BV2 exosomes into a rat postoperative delirium model.

[0010] Preferably, the BV2 exosomes are prepared by ultracentrifugation.

[0011] More preferably, the method for preparing BV2 exosomes comprises:

[0012] 1) Rapidly thaw the cell supernatant at 37°C.

[0013] 2) Move the cell supernatant to a new centrifuge tube and centrifuge at 2000×g, 4°C, for 30 min.

[0014] 3) Carefully transfer the supernatant to a new centrifuge tube and centrifuge again at 12,000×g, 4°C, 45 min to remove larger vesicles.

[0015] 4) Take the supernatant, filter it through a 0.45 μm filter membrane, and collect the filtrate.

[0016] 5) Transfer the filtrate to a new centrifuge tube, select an ultraspeed rotor, and centrifuge at 110,000×g for 70 min at 4°C.

[0017] 6) Remove the supernatant, resuspend with 10 mL of pre-cooled 1× PBS, select an ultracentrifuge rotor, and centrifuge again at 4°C, 110,000×g for 70 min.

[0018] 7) Remove the supernatant and resuspend in 90 μL pre-cooled 1× PBS.

[0019] The beneficial effects of the present invention are:

[0020] A rat model of postoperative delirium was successfully constructed, and the role of BV2 exosomes in the treatment of postoperative delirium was studied by injecting BV2 exosomes into the rat model of postoperative delirium. The results showed that BV2 exosomes could improve the damage of hippocampal neurons and reduce cell apoptosis, thereby partially reversing postoperative delirium in rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the transmission electron microscopy imaging result of the exosome sample in the embodiment of the present invention.

[0022] Figure 2 This is a schematic diagram of long-term preoperative pure oxygen anesthesia in an embodiment of the present invention.

[0023] Figure 3 Schematic diagram of surgical injury in an embodiment of the present invention.

[0024] Figure 4 This is the open field test result after the model is constructed in the embodiment of the present invention.

[0025] Figure 5 This is the experimental result of burying food after the model is constructed in the embodiment of the present invention.

[0026] Figure 6 This is the result of the open field test after drug administration in the model of the present invention.

[0027] Figure 7 These are the results of the food burial experiment after drug administration in the model of the present invention.

[0028] Figure 8 The results of HE staining and apoptosis detection of hippocampal tissue in the embodiment of the present invention are shown. DETAILED DESCRIPTION

[0029] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0030] Example

[0031] 1. Extraction and identification of mouse microglial cells BV2

[0032] 1. Extraction of exosomes by ultracentrifugation

[0033] 1) Rapidly thaw the cell supernatant of mouse microglial cells BV2 at 37° C. (The source and preparation method of the cell supernatant are not limited in this application. The cell supernatant can be directly aspirated with a pipette according to conventional techniques in the art.)

[0034] 2) Move the cell supernatant to a new centrifuge tube and centrifuge at 2000×g, 4°C, for 30 min.

[0035] 3) Carefully transfer the supernatant to a new centrifuge tube and centrifuge again at 12,000×g, 4°C, 45 min to remove larger vesicles.

[0036] 4) Take the supernatant, filter it through a 0.45 μm filter membrane, and collect the filtrate.

[0037] 5) Transfer the filtrate to a new centrifuge tube, select an ultraspeed rotor, and centrifuge at 110,000×g for 70 min at 4°C.

[0038] 6) Remove the supernatant, resuspend with 10 mL of pre-cooled 1× PBS, select an ultracentrifuge rotor, and centrifuge again at 4°C, 110,000×g for 70 min.

[0039] 7) Remove the supernatant and resuspend in 90 μL pre-cooled 1× PBS.

[0040] 2. Transmission electron microscopy observation of exosome samples

[0041] 1) Take out 10 μL of exosomes.

[0042] 2) Pipette 10 μL of the sample and drop it onto a copper grid to precipitate for 1 min, then remove the floating liquid with filter paper.

[0043] 3) Add 10 μL of uranyl acetate onto the copper grid and let it precipitate for 1 min. Then remove the floating liquid with filter paper.

[0044] 4) Dry at room temperature for several minutes.

[0045] 5) Use electron microscope to detect and image at 100kV.

[0046] 6) Obtain transmission electron microscopy imaging results, such as Figure 1 As shown in Figure 2, electron microscopy results showed that exosomes were vesicle-like.

[0047] 1. Experimental Materials

[0048] Experimental animals: 12 SD rats, female, 12 weeks old.

[0049] Experimental materials: 2-0 suture, angle needle and round needle, homemade pull line hook, normal saline, medium and small surgical scissors, curved forceps, needle holder, cotton swab, gauze, complex iodine disinfectant, oxygen (100%), isoflurane anesthesia machine, spontaneous activity box.

[0050] 2. Operating procedures

[0051] 2.1 Experimental animal grouping: The experimental SD rats were randomly divided into 2 groups: a normal control group and a model group.

[0052] 2.2 Preoperative preparation: All animals were given maintenance feed and fasted but not water for 12 hours before surgery.

[0053] 2.3 Model establishment: All animals were given maintenance feed. Rats in the normal group were placed in a cage with 99.9% oxygen for 2 hours without any other treatment, such as Figure 2 As shown, except for the normal group, the mice were anesthetized with isoflurane anesthesia machine and then surgically modeled. A longitudinal midline incision was made from the sternum to 0.5 cm proximal to the pubic symphysis through the skin, abdominal muscles and peritoneum, as shown in Figure 3 As shown, the abdominal organs were partially exposed for 2 minutes and then gradually sutured with 5-0 sutures. After surgery, the mice were returned to the anesthesia chamber for anesthesia for 2 hours. Each mouse was given EMLA cream before, after and one day after surgery.

[0054] 2.4 Model Validation

[0055] 1) Open field test: Figure 4 As shown in the figure, compared with the control group, the total distance and average speed of the model group at 6h and 24h were significantly decreased (P<0.05), indicating that after a certain depth of anesthesia, the rats in the model group had reduced activity and tended to develop delirium.

[0056] 2) Food burial experiment: Figure 5 As shown in the figure, compared with the control group, the model group spent significantly more time searching for food at 6h and 24h (P<0.05), indicating that after a certain depth of anesthesia, the rats in the model group had reduced activity and tended to develop delirium.

[0057] 3. Exosome therapy and validation

[0058] 1. Treatment methods:

[0059] Mouse microglial BV2 exosomes were injected into the model group rats at 100 μg / rat (100 μg of exosomes were dissolved in 100 μl PBS).

[0060] 2. Experimental verification:

[0061] 1) Open field test: Figure 6 As shown, compared with the model group, the total distance and average speed after 6h and 24h of exosome treatment increased (P>0.05), indicating that exosome treatment can partially reverse postoperative delirium in rats.

[0062] 2) Food burial experiment: Figure 7 As shown in the data, compared with the model group, the time of searching for food was significantly reduced after 6h and 24h of exosome treatment (P<0.05), indicating that exosome treatment can partially reverse postoperative delirium in rats.

[0063] 3) HE staining and apoptosis detection:

[0064] In order to further observe the effect of mouse microglial BV2 exosomes treatment on the hippocampus of rats with postoperative delirium, HE staining and apoptosis detection were performed on the hippocampus tissue. Figure 8 A concluded that the structure of the neurons in the hippocampus of the rats in the control group was complete and clear, with a tight and orderly arrangement, a large number of neurons, and complete development; the neurons in the hippocampus of the rats in the model group were significantly atrophied, with a reduced number of neurons, and the structure was damaged, with shrunken nuclei; compared with the model group, the damage to the neurons in the hippocampus of the mice in the microglia BV2 exosome group was improved, and it can be seen that the structure of the neurons was clearer and more complete. Figure 8 B results show that the blue fluorescence is the cell nucleus (corresponding to Figure 8 The second row in B), red fluorescence indicates apoptotic cells (corresponding to Figure 8 Compared with the control group, the apoptotic cells in the model group increased significantly, and compared with the model group, the apoptotic cells in the mouse microglia BV2 exosome group decreased significantly, indicating that the mouse microglia BV2 exosome group can significantly reduce cell apoptosis.

[0065] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. Application of BV2 exosomes in the preparation of drugs for the treatment of postoperative delirium.

2. The use according to claim 1, characterized in that: The BV2 exosomes were prepared by ultracentrifugation.

3. The use according to claim 2, characterized in that: The preparation method of the BV2 exosomes comprises: 1) Rapidly thaw the cell supernatant at 37°C; 2) Move the cell supernatant to a new centrifuge tube and centrifuge at 2000 × g, 4 °C, 30 min; 3) Carefully transfer the supernatant to a new centrifuge tube and centrifuge again at 12,000 × g, 4 °C, 45 min to remove larger vesicles. 4) Take the supernatant, filter it through a 0.45 μm filter membrane, and collect the filtrate; 5) Transfer the filtrate to a new centrifuge tube, select an ultraspeed rotor, and centrifuge at 110,000 × g for 70 min at 4 °C; 6) Remove the supernatant, resuspend with 10 mL of pre-cooled 1× PBS, select an ultracentrifuge rotor, and centrifuge again at 4°C, 110,000 × g, for 70 min; 7) Remove the supernatant and resuspend in 90 μL of pre-cooled 1× PBS.

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