Macrophage membrane vesicle-modified salvia miltiorrhiza exosomes and preparation method and application thereof

Abstract

The present invention relates to the field of biomedical technology, in particular, to macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes and a preparation method and application thereof; the preparation method of the macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes comprises: mixing Salvia miltiorrhiza exosomes, macrophage membrane vesicles and PBS buffer to obtain a mixed solution; extruding the mixed solution through a filter membrane with a first preset pore size for a first preset number of times to obtain macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes. The macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes provided by the present invention have the effect of improving acute skeletal muscle injury, can effectively improve the problem of acute skeletal muscle injury induced by drugs and physical factors (such as acute skeletal muscle injury such as exercise, car accidents, falls and crush injuries, etc.), thereby promoting the recovery process of acute skeletal muscle injury, and can be used to prepare a drug for treating acute skeletal muscle injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes, and a preparation method and application thereof. Background Art

[0002] Acute skeletal muscle injury is the most common disease in fracture trauma and daily sports injuries, so in-depth research on this injury is of great significance in many aspects. First of all, acute skeletal muscle injury not only affects the competitive ability of athletes, but also affects the quality of life of the general population. Acute skeletal muscle injury can cause pain, dysfunction, and inflammatory response in the muscle injury area, causing short-term and long-term discomfort and distress to individuals.

[0003] At present, the treatment of acute skeletal muscle injury mainly uses nonsteroidal anti-inflammatory drugs, muscle relaxants, analgesics, and physical therapy methods such as ice compresses and electrical stimulation. However, the commonly used therapeutic drugs have low utilization rates and obvious toxic side effects, and most of them cannot be used for a long time. Therefore, it is necessary to explore a new substance for the treatment of acute skeletal muscle injury. Summary of the invention

[0004] The technical problem solved by the invention is to provide a new substance for treating acute skeletal muscle injury.

[0005] In order to solve the above technical problems, the present invention provides a method for preparing macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes, comprising:

[0006] Step S1, mixing Salvia miltiorrhiza exosomes, macrophage membrane vesicles and PBS buffer to obtain a mixed solution;

[0007] Step S2, extruding the mixed solution through a filter membrane with a first preset pore size for a first preset number of times to obtain Salvia miltiorrhiza exosomes modified with macrophage membrane vesicles.

[0008] Optionally, in step S1, in the mixed solution, the ratio of the protein concentration of the Salvia miltiorrhiza exosomes to the protein concentration of the macrophage membrane vesicles is (3-4):2; in step S2, the first preset pore size is 190-210 nm, the filter membrane is a polycarbonate membrane, and the first preset number of times is more than 20 times.

[0009] Optionally, the method for preparing the Salvia miltiorrhiza exosomes comprises:

[0010] Step M1, soaking the Danshen decoction pieces in water for incubation, and then filtering to obtain a first solution;

[0011] Step M2, subjecting the first solution to a first centrifugation treatment, extracting a supernatant, and obtaining a first supernatant;

[0012] Step M3, subjecting the first supernatant to a second centrifugation treatment, extracting the supernatant, and obtaining a second supernatant;

[0013] Step M4, filtering the second supernatant with a filter, performing a third centrifugation treatment, extracting the supernatant, and obtaining a third supernatant;

[0014] Step M5, subjecting the third supernatant to a fourth centrifugation treatment, extracting the supernatant, and obtaining a fourth supernatant;

[0015] Step M6, centrifuging the fourth supernatant solution for the fifth time, and the resulting precipitate is the Salvia miltiorrhiza exosomes.

[0016] Optionally, in step M1, the mass of the Danshen decoction piece is m grams and the volume of the water is v milliliters, and the ratio of m to v is 1: (8-10).

[0017] Optionally, the first centrifugal treatment is carried out at a rotation speed of 1000-4000g and a time of 10-20min, the second centrifugal treatment is carried out at a rotation speed of 4000-6000g and a time of 10-20min, the third centrifugal treatment is carried out at a rotation speed of 9000-11000g and a time of 10-20min, the fourth centrifugal treatment is carried out at a rotation speed of 90000-110000g and a time of 70-100min, and the fifth centrifugal treatment is carried out at a rotation speed of 90000-110000g and a time of 70-100min.

[0018] Optionally, the method for preparing the macrophage membrane vesicles comprises:

[0019] Step N1, after resuspending the macrophages with water, adding a membrane protein extraction reagent to obtain a cell suspension;

[0020] Step N2, subjecting the cell suspension to an ice bath treatment, ultrasonic treatment, and centrifugation treatment, and resuspending the obtained precipitate with Tris buffer to obtain a macrophage membrane fragment solution;

[0021] Step N3, extruding the macrophage membrane fragment solution through a filter membrane with a second preset pore size for a second preset number of times to obtain macrophage membrane vesicles.

[0022] Optionally, in the step N2, the ultrasonic frequency used in the ultrasonic treatment is 20-30kHz, the amplitude is 5-10%, and the time of the ultrasonic treatment is 5-10min; the rotation speed of the centrifugal treatment is 13000-14000g, and the time is 20-30min; in the step N3, the second preset pore size is 390-410nm, the filter membrane is a polycarbonate membrane, and the second preset number of times is more than 20 times.

[0023] The present invention also provides macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes, which are prepared by the method for preparing macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes as described above.

[0024] The present invention also provides the use of the above-mentioned macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes in the preparation of a drug, wherein the drug is used for treating acute skeletal muscle injury.

[0025] Optionally, the drug is used for targeted treatment of acute skeletal muscle injury.

[0026] Compared with the prior art, the present invention provides a macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes, and experiments have shown that the macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes have the effect of improving acute skeletal muscle injury, can effectively improve the problem of acute skeletal muscle injury induced by drugs (such as TNF-a) and physical factors, thereby promoting the recovery process of acute skeletal muscle injury, and can be used to prepare drugs for the treatment of acute skeletal muscle injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a transmission electron microscope image of the Salvia miltiorrhiza exosomes prepared in Example 1;

[0028] Figure 2 This is a diagram showing the NTA analysis results of the Salvia miltiorrhiza exosomes prepared in Example 1;

[0029] Figure 3 This is a transmission electron microscope image of the macrophage membrane vesicles prepared in Example 2;

[0030] Figure 4 This is a diagram showing the NTA analysis results of the macrophage membrane vesicles prepared in Example 2;

[0031] Figure 5 This is a transmission electron microscopy image of the Salvia miltiorrhiza exosomes modified with macrophage membrane vesicles prepared in Example 3;

[0032] Figure 6 This is a diagram showing the NTA analysis results of the macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes prepared in Example 3;

[0033] Figure 7 This is a comparison chart of the absorbance (OD value) test results of the experimental group EMT, the control group N, the control group T and the control group ET in Example 4;

[0034] Figure 8 This is a comparison chart of the test results of apoptosis rates of cells in the experimental group EMT, the control group N, the control group T and the control group ET in Example 5;

[0035] Fig. 9This is a physical picture of the WB results of the expression levels of ErK1,2, a cell inflammation injury-related factor, in C2C12 cells in the experimental group EMT, the control group N, the control group T, and the control group ET of Example 6;

[0036] Fig.10 Comparison of the relative protein expression of ErK1,2, a factor related to cell inflammation and injury, relative to the reference factor a-tubulin in C2C12 cells in the experimental group EMT, the control group N, the control group T, and the control group ET of Example 6;

[0037] Fig.11 The WB results of the expression level of the cell inflammation-related factor P65 in C2C12 cells in the experimental group EMT, the control group N, the control group T and the control group ET of Example 6 are shown in FIG.

[0038] Fig.12 This is a comparison chart of the relative protein expression of the cellular inflammation-related factor P65 relative to the reference factor a-actin in C2C12 cells in the experimental group EMT, the control group N, the control group T, and the control group ET of Example 6;

[0039] Fig.13 This is a physical picture of the WB results of the expression level of the cell inflammation-related factor P38 in C2C12 cells in the experimental group EMT, the control group N, the control group T and the control group ET of Example 6;

[0040] Fig.14 It is a comparison chart of the relative protein expression of the cellular inflammation-related factor P38 relative to the reference factor a-tubulin in C2C12 cells in the experimental group EMT, the control group N, the control group T and the control group ET of Example 6;

[0041] Fig.15 This is a physical picture of the WB results of the expression level of the muscle cell damage and repair-related factor MyoD1 in C2C12 cells in the experimental group EMT, the control group N, the control group T and the control group ET of Example 6;

[0042] Fig.16 This is a comparison chart of the relative protein expression of the muscle cell damage repair-related factor MyoD1 relative to the reference factor a-tubulin in C2C12 cells in the experimental group EMT, the control group N, the control group T, and the control group ET of Example 6;

[0043] Fig.17 This is a physical picture of the WB results of the expression level of Myogenin, a factor related to myocyte damage and repair, in C2C12 cells in the experimental group EMT, the control group N, the control group T, and the control group ET of Example 6;

[0044] Fig.18This is a comparison chart of the relative protein expression of the muscle cell damage repair-related factor Myogeni relative to the reference factor a-tubulin in C2C12 cells in the experimental group EMT, the control group N, the control group T, and the control group ET of Example 6;

[0045] Fig.19 This is a fluorescence comparison diagram of the isolated tissues and organs of mice in the experimental group and the control group of Example 7. DETAILED DESCRIPTION

[0046] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0047] It should be noted that, in the absence of conflict, the features in the embodiments of the present invention can be combined with each other. The meanings of the terms "comprising", "including", "containing", and "having" are non-restrictive, that is, other steps and other ingredients that do not affect the results can be added. The above terms cover the terms "consisting of..." and "essentially consisting of...". Unless otherwise specified, materials, equipment, and reagents are all commercially available. It should be noted that when the centrifugal speed in the present invention is expressed as "g", it refers to the relative centrifugal force, that is, the multiple of the centrifugal force relative to the earth's gravitational acceleration (g).

[0048] The present invention provides a method for preparing macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes, comprising:

[0049] Step S1, mixing Salvia miltiorrhiza exosomes, macrophage membrane vesicles and PBS buffer to obtain a mixed solution;

[0050] Step S2, extruding the mixed solution through a filter membrane with a first preset pore size for a first preset number of times to obtain Salvia miltiorrhiza exosomes modified with macrophage membrane vesicles.

[0051] The embodiment of the present invention provides a macrophage membrane vesicle-modified Salvia miltiorrhiza exosome, and experiments have proved that the macrophage membrane vesicle-modified Salvia miltiorrhiza exosome has the effect of improving acute skeletal muscle injury, can effectively improve the problem of acute skeletal muscle injury induced by drugs (such as TNF-a) and physical factors, thereby promoting the recovery process of acute skeletal muscle injury, and can be used to prepare a drug for the treatment of acute skeletal muscle injury.

[0052] In some embodiments of the present invention, in step S1, in the mixed solution, the ratio of the protein concentration of the Salvia miltiorrhiza exosomes to the protein concentration of the macrophage membrane vesicles is (3-4):2.

[0053] In some embodiments of the present invention, in step S2, the first preset pore size is 190-210 nm, the filter membrane is a polycarbonate membrane, and the first preset number of times is more than 20 times.

[0054] In some embodiments of the present invention, the method for preparing the Salvia miltiorrhiza exosomes comprises:

[0055] Step M1, soaking the Danshen decoction pieces in water for incubation, and then filtering to obtain a first solution;

[0056] Step M2, subjecting the first solution to a first centrifugation treatment, extracting a supernatant, and obtaining a first supernatant;

[0057] Step M3, subjecting the first supernatant to a second centrifugation treatment, extracting the supernatant, and obtaining a second supernatant;

[0058] Step M4, filtering the second supernatant with a filter, performing a third centrifugation treatment, extracting the supernatant, and obtaining a third supernatant;

[0059] Step M5, subjecting the third supernatant to a fourth centrifugation treatment, extracting the supernatant, and obtaining a fourth supernatant;

[0060] Step M6, centrifuging the fourth supernatant solution for the fifth time, and the resulting precipitate is the Salvia miltiorrhiza exosomes.

[0061] In some embodiments of the present invention, in step M1, the mass of the Danshen decoction piece is m grams and the volume of the water is v milliliters, and the ratio of m to v is 1: (8-10).

[0062] In some embodiments of the present invention, in the step M1, the incubation temperature is 30-60° C. and the time is 12-24 hours.

[0063] In some embodiments of the present invention, in step M4, the pore size of the filter is 40 μm.

[0064] In some embodiments of the present invention, the first centrifugal treatment is carried out at a speed of 1000-4000g and a time of 10-20min, the second centrifugal treatment is carried out at a speed of 4000-6000g and a time of 10-20min, the third centrifugal treatment is carried out at a speed of 9000-11000g and a time of 10-20min, the fourth centrifugal treatment is carried out at a speed of 90000-110000g and a time of 70-100min, and the fifth centrifugal treatment is carried out at a speed of 90000-110000g and a time of 70-100min.

[0065] In some embodiments of the present invention, the method for preparing the macrophage membrane vesicles comprises:

[0066] Step N1, after resuspending the macrophages with water, adding a membrane protein extraction reagent to obtain a cell suspension;

[0067] Step N2, subjecting the cell suspension to an ice bath treatment, ultrasonic treatment, and centrifugation treatment, and resuspending the obtained precipitate with Tris buffer to obtain a macrophage membrane fragment solution;

[0068] Step N3, extruding the macrophage membrane fragment solution through a filter membrane with a second preset pore size for a second preset number of times to obtain macrophage membrane vesicles.

[0069] In some embodiments of the present invention, in step N2, the ice bath treatment time is 1-2h, the ultrasonic frequency used in the ultrasonic treatment is 20-30kHz, the amplitude is 5-10%, and the ultrasonic treatment time is 5-10min; the centrifugal treatment speed is 13000-14000g, and the time is 20-30min.

[0070] In some embodiments of the present invention, in step N3, the second preset pore size is 390-410 nm, the filter membrane is a polycarbonate membrane, and the second preset number of times is more than 20 times.

[0071] The embodiment of the present invention also provides a macrophage membrane vesicle-modified Salvia miltiorrhiza exosome, which is prepared by the method for preparing the macrophage membrane vesicle-modified Salvia miltiorrhiza exosome as described above.

[0072] The embodiment of the present invention also provides the use of the above-mentioned macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes in the preparation of a drug, wherein the drug is used to treat acute skeletal muscle injury.

[0073] In some embodiments of the present invention, the drug is used for targeted treatment of acute skeletal muscle injury.

[0074] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. It should be noted that the PBS buffer used in the embodiments of the present invention is purchased from Wuhan Pu Nuo Sai Life Science Technology Co., Ltd., model PB180327, the manufacturer of the DMEM high-glucose basal medium used is Gibco, USA, model 41401, and the manufacturer of TNF-a used is Sigma Aldrich (Shanghai) Trading Co., Ltd., model NM013693, and in the accompanying drawings of the present invention, "*", "**", "***" and "****" represent the difference between each group and the dexamethasone group, "*" represents p<0.05, "**" represents p<0.01, "***" represents p<0.001; "****" represents p<0.0001; "ns" represents no significant difference. It should be noted that. Figure 7-18 In the figure, the data above “N” correspond to the control group N, the data above “T” correspond to the control group T, the data above “ET” correspond to the control group ET, and the data above “EMT” correspond to the experimental group EMT.

[0075] Example 1: Extraction of Salvia miltiorrhiza exosomes

[0076] 1.1. Soak 10 g of Danshen slices in 80 ml of double distilled water, incubate at 37° C. for 20 h, filter with medical gauze, and filter the filtrate again through a 70 μm sieve to obtain a first solution;

[0077] 1.2. The first solution is subjected to a first centrifugal treatment to extract the supernatant to obtain a first supernatant; the first centrifugal treatment is performed at a speed of 1000 g and a time of 20 min;

[0078] 1.3. The first supernatant is subjected to a second centrifugal treatment, and the supernatant is extracted to obtain a second supernatant; the second centrifugal treatment is performed at a speed of 4000 g and a time of 20 min;

[0079] 1.4. After filtering the second supernatant with a filter having a pore size of 40 μm, centrifugation is performed for a third time to extract the supernatant to obtain a third supernatant; the third centrifugation is performed at a speed of 10,000 g for 20 min;

[0080] 1.5. The third supernatant is subjected to a fourth centrifugation treatment, and the supernatant is extracted to obtain a fourth supernatant; the fourth centrifugation treatment is performed at a speed of 100,000 g and a time of 70 min;

[0081] 1.6. The fourth supernatant solution was centrifuged for the fifth time, and the resulting precipitate was Salvia miltiorrhiza exosomes (SELNs). The precipitate was resuspended in PBS buffer and filtered using a 0.22 μm bacterial sieve to obtain a Salvia miltiorrhiza exosome suspension; the fifth centrifugation was performed at 100,000 g for 70 min.

[0082] Transmission electron microscopy was used to characterize the exosomes of Salvia miltiorrhiza. Figure 1 ,from Figure 1 It can be seen that the morphology of Danshen exosomes is a typical membrane "cup-and-disc" structure, proving that the structure of the extracted Danshen exosomes is intact. The particle size of Danshen exosomes in Danshen exosome suspension was measured by nanoparticle tracking analyzer (NTA). Figure 2 ,from Figure 2 It can be seen that the average diameter of Salvia miltiorrhiza exosomes is 138.7nm.

[0083] Example 2: Preparation of macrophage membrane vesicles (MM)

[0084] 2.1. After resuspending the macrophages with pure water, add membrane protein extraction reagent A containing PMSF to obtain a cell suspension; wherein, the manufacturer of the macrophages is Wuhan Pronocell Life Science Technology Co., Ltd., model RAW264.7, and the manufacturer of the membrane protein extraction reagent A containing PMSF is Beijing Solebow Technology Co., Ltd., model EX1500.

[0085] 2.2. After the cell suspension was treated in an ice bath for 1 hour, ultrasonic treatment and centrifugation were performed, and the obtained precipitate was resuspended in Tris buffer to obtain a macrophage membrane fragment solution; the ultrasonic frequency used in the ultrasonic treatment was 25 kHz, the amplitude was 5%, and the ultrasonic treatment time was min; the speed of the centrifugation was 14000g, and the time was 30 min;

[0086] 2.3. The macrophage membrane fragment solution was extruded through a polycarbonate membrane with a pore size of 400 nm 20 times using a liposome extruder to obtain macrophage membrane vesicles (MM).

[0087] Macrophage membrane vesicles were characterized using transmission electron microscopy. Figure 3 The particle size of macrophage membrane vesicles was determined using nanoparticle tracking analyzer (NTA). Figure 4 ,from Figure 4 It can be seen that the average diameter of macrophage membrane vesicles is 127.9nm.

[0088] Example 3: Preparation of Salvia miltiorrhiza exosomes modified with macrophage membrane vesicles (MM-SELNs)

[0089] 3.1. The Salvia miltiorrhiza exosomes prepared in Example 1, the macrophage membrane vesicles prepared in Example 2 and PBS buffer were mixed to obtain a mixed solution; wherein, in the mixed solution, the ratio of the protein concentration of the Salvia miltiorrhiza exosomes to the protein concentration of the macrophage membrane vesicles was 3:2.

[0090] 3.2. The mixed solution was extruded through a polycarbonate membrane with a pore size of 200 nm 20 times using a liposome extruder to obtain macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes (MM-SELNs).

[0091] The macrophage membrane vesicles modified Salvia miltiorrhiza exosomes were characterized by transmission electron microscopy. Figure 5 ,from Figure 5 It can be seen that the exosomes of Salvia miltiorrhiza modified by macrophage membrane vesicles present a typical membrane structure. The particle size of the exosomes of Salvia miltiorrhiza modified by macrophage membrane vesicles was measured by nanoparticle tracking analyzer (NTA). Figure 6 ,from Figure 6 It can be seen that the average diameter of Salvia miltiorrhiza exosomes is 140.8nm.

[0092] Example 4: Experiment on the effect of MM-SELNs on the proliferation of C2C12 cells.

[0093] The CCK-8 (Cell Counting Kit-8) method is a commonly used method for detecting cell viability. The CCK-8 reagent is based on the reduction reaction of tetrazolium salt (WST-8) and indirectly evaluates cell proliferation by detecting the metabolic activity of cells.

[0094] EMT in experimental group

[0095] After culturing C2C12 cells to the logarithmic growth phase, they were seeded into a 96-well plate at a density of 5000 cells / well, and 100μL of DMEM high-glucose basal medium was added to each well for culture. After the cells adhered to the wall, the medium in each well was aspirated, and 100μL of the first culture medium was added. The cells were incubated for 48 hours at a temperature of 37°C and a carbon dioxide concentration of 5%. 10μL of CCK-8 reagent was added to each well, and the 96-well plate was gently shaken to evenly distribute the reagent. The cells were incubated for 2 hours at a temperature of 37°C and a carbon dioxide concentration of 5%. One of the wells was randomly selected, and the absorbance (OD value) of the well at 450nm was measured using an enzyme marker; wherein the first culture medium consists of DMEM high-glucose basal medium, TNF-a and MM-SELNs, and the concentration of TNF-a in the first culture medium is 1μg / ml, and the concentration of MM-SELNs is 25μg / ml.

[0096] It should be noted that TNF-a is a cytokine that can cause acute damage to C2C12 cells.

[0097] Control group N

[0098] The difference from the EMT in the experimental group is that the first culture medium used during the experiment was replaced with DMEM high-glucose basal medium.

[0099] Control group T

[0100] The difference from the EMT of the experimental group is that the first culture medium used in the experiment is replaced by a second culture medium; the second culture medium consists of DMEM high-glucose basal medium and TNF-a, and the concentration of TNF-a in the second culture medium is 1 μg / ml.

[0101] Control group ET

[0102] The difference from the EMT of the experimental group is that the first culture medium used in the experiment was replaced by a third culture medium; the third culture medium consisted of DMEM high-glucose basal medium, SELNs and TNF-a, and the concentration of TNF-a in the second culture medium was 1 μg / ml, and the concentration of SELNs was 25 μg / ml.

[0103] The absorbance (OD value) test results of the experimental group EMT, control group N, control group T and control group ET are shown in Figure 7 ,from Figure 7 It can be seen that compared with the control group N, the proliferation of C2C12 cells in the control group T was significantly inhibited. Compared with the control group T, the proliferation of C2C12 cells in the control group ET and the experimental group EMT was significantly promoted, and the proliferation of C2C12 cells in the experimental group EMT was more strongly promoted. From the above analysis, it can be seen that MM-SELNs have a significant pro-proliferation effect on C2C12 cells whose proliferation is inhibited by TNF-a intervention, and the effect of promoting proliferation is better than that of SELNs.

[0104] Example 5: Experiment on the effect of MM-SELNs on C2C12 cell apoptosis

[0105] Flow cytometry is a common and efficient method for measuring cell apoptosis. Flow cytometry can distinguish early apoptotic cells, late apoptotic cells, and necrotic cells by labeling phosphatidylserine (Annexin V) on the cell membrane and DNA (PI) in the cell.

[0106] EMT in experimental group

[0107] (1) After culturing C2C12 cells to the logarithmic growth phase, they are seeded into a culture dish at a density of 20,000 cells / mL, and 1 ml of DMEM high-glucose basal medium is added for culturing. After the cells adhere to the wall, the medium in the culture dish is aspirated, and 1 ml of a first culture medium is added. The cells are incubated at a temperature of 37° C. and a carbon dioxide concentration of 5% for 24 hours to obtain a cell suspension; wherein the first culture medium consists of DMEM high-glucose basal medium, TNF-a and MM-SELNs, and the concentration of TNF-a in the first culture medium is 1 μg / ml, and the concentration of MM-SELNs is 25 μg / ml.

[0108] (2) Transfer the cell suspension to a centrifuge tube and centrifuge at 300 g for 5 min. Discard the supernatant and collect the cell pellet. After washing, resuspend the cell pellet in 100 µL of 1X Annexin V binding buffer, add 5 µL of Annexin V-FITC and 5 µL of PI, mix gently, incubate at room temperature in the dark for 15 min to complete Annexin V / PI staining, then add 400 µL of 1X Annexin V binding buffer to obtain the sample to be tested; use flow cytometry to detect the apoptosis rate of cells in the sample to be tested.

[0109] Control group N

[0110] The difference from the EMT in the experimental group is that the first culture medium used during the experiment was replaced with DMEM high-glucose basal medium.

[0111] Control group T

[0112] The difference from the EMT of the experimental group is that the first culture medium used in the experiment is replaced by a second culture medium; the second culture medium consists of DMEM high-glucose basal medium and TNF-a, and the concentration of TNF-a in the second culture medium is 1 μg / ml.

[0113] Control group ET

[0114] The difference from the EMT of the experimental group is that the first culture medium used in the experiment was replaced by a third culture medium; the third culture medium consisted of DMEM high-glucose basal medium, SELNs and TNF-a, and the concentration of TNF-a in the second culture medium was 1 μg / ml, and the concentration of SELNs was 25 μg / ml.

[0115] The results of the apoptosis rate test of cells in the experimental group EMT, control group N, control group T and control group ET are shown in Figure 8 ,from Figure 8It can be seen that compared with the control group N, the apoptosis of C2C12 cells in the control group T was significantly promoted. Compared with the control group T, the apoptosis of C2C12 cells in the control group ET and the experimental group EMT was significantly inhibited, and the apoptosis of C2C12 cells in the experimental group EMT was more strongly inhibited. From the above analysis, it can be seen that TNF-a can significantly increase the apoptosis of C2C12 cells, and MM-SELNs has a significant inhibitory effect on the apoptosis of C2C12 cells intervened by TNF-a, and the effect of inhibiting apoptosis is better than that of SELNs.

[0116] Example 6: WB analysis of the effect of MM-SELNs on C2C12 cell function

[0117] Western Blot (WB) is a commonly used protein detection method. By separating, transferring and detecting target proteins, information such as protein expression, molecular weight and modification status can be analyzed.

[0118] EMT in experimental group

[0119] (1) After culturing C2C12 cells to the logarithmic growth phase, they are seeded into a culture dish at a density of 20,000 cells / mL, and DMEM high-glucose basal medium is added for culturing. After the cell density reaches 80%, the original culture medium is removed, and an equal volume of cell induction medium containing 2% horse serum is added. The cells are cultured for 6 days to differentiate into mature myotube cells. The original culture medium is removed, and an equal volume of a first cell induction medium is added. The cells are incubated at a temperature of 37° C. and a carbon dioxide concentration of 5% for 24 hours to obtain a cell fluid; wherein the first cell induction medium consists of a cell induction medium containing 2% horse serum, TNF-a and MM-SELNs, and the concentration of TNF-a in the first cell induction medium is 1 μg / ml, and the concentration of MM-SELNs is 25 μg / ml.

[0120] (2) RIPA lysis buffer was added to the cell solution to lyse the cells for 30 minutes, and the solution was centrifuged at 12000 g for 10 minutes. The total cell protein concentration of the obtained supernatant was determined using a BCA protein assay kit, and the supernatant was balanced with protein loading buffer, and then boiled in 100° C. boiling water for 10 minutes to obtain a test liquid.

[0121] (3) Test of ErK1,2 protein expression: 15 μg of protein was extracted from the test liquid and added to 10% SDS-PAGE gel, and separated by electrophoresis at a constant voltage of 80 V for 2 h; after separation, the protein was transferred to a PVDF membrane, and then the membrane was blocked in 5% skim milk for 2 h. After blocking, the membrane was divided into two test membranes according to the location of the target protein and the internal reference protein, and the two test membranes were placed in ErK1,2 (1:1000) and internal reference a-tubulin (1:1000) specific primary antibodies respectively and incubated at 4°C overnight; after recovering the primary antibody, the two test membranes were washed with TBST for 30 min respectively, and the two test membranes were incubated with goat anti-rabbit secondary antibodies (1:5000) at room temperature for 1-2 h, and then rinsed with TBST again for 30 min. Use rapid chemiluminescent liquid to evenly cover the two test membranes and leave them for 2 minutes. Then take out the two test membranes and place them in a chemiluminescent imager for detection to obtain the expression of ErK1, 2 proteins.

[0122] (4) The same method as step (3) was used to test the expression of proteins such as P65, P38, MyoD1, and Myogenin.

[0123] Control group N

[0124] The difference from the EMT experimental group was that the first cell induction medium used in the experiment was replaced with a cell induction medium containing 2% horse serum.

[0125] Control group T

[0126] The difference from the EMT of the experimental group is that the first cell induction medium used in the experiment is replaced with a second cell induction medium; the second cell induction medium consists of a cell induction medium containing 2% horse serum and TNF-a, and the concentration of TNF-a in the second medium is 1 μg / ml.

[0127] Control group ET

[0128] The difference from the EMT of the experimental group is that the first cell induction medium used in the experiment is replaced with a third cell induction medium; the third cell induction medium consists of a cell induction medium containing 2% horse serum, SELNs and TNF-a, and the concentration of TNF-a in the third cell induction medium is 1 μg / ml, and the concentration of SELNs is 25 μg / ml.

[0129] The test results in the experimental group EMT, control group N, control group T and control group ET are shown in Figure 9-18 ,from Fig. 9 and Fig.10It can be seen that the content of ErK1,2, a factor related to cell inflammation damage, has obvious statistical significance. Compared with the control group N, the expression of ErK1,2 protein in the control group T was significantly increased, and the expression of ErK1,2 protein in the control group ET and the experimental group EMT was significantly reduced, among which the reduction in the experimental group EMT was more significant. Fig.11 and Fig.12 It can be seen that the content of cellular inflammation-related factor P65 has obvious statistical significance. Compared with the control group N, the expression of P65 protein in the control group T was significantly increased, and the expression of P65 protein in the control group ET and the experimental group EMT was significantly reduced, among which the reduction of EMT in the experimental group was more significant. Fig.13 and Fig.14 It can be seen that the content of cellular inflammation-related factor P38 has obvious statistical significance. Compared with the control group N, the expression of P38 protein in the control group T was significantly increased, and the expression of P38 protein in the control group ET and the experimental group EMT was significantly reduced, among which the reduction of EMT in the experimental group was more significant. Fig.15 and Fig.16 It can be seen that the content of MyoD1, a factor related to myocyte damage and repair, has significant statistical significance. Compared with the control group N, the expression of MyoD1 protein in the control group T was significantly reduced, and the expression of MyoD1 protein in the control group ET and the experimental group EMT was significantly increased, among which the increase in the experimental group EMT was more significant. Fig.17 and Fig.18 It can be seen that the content of Myogenin, a factor related to muscle cell damage and repair, has obvious statistical significance. Compared with the control group N, the expression of Myogenin protein in the control group T was significantly reduced, and the expression of Myogenin protein in the control group ET and the experimental group EMT was significantly increased, among which the increase in the experimental group EMT was more significant.

[0130] From the above analysis, it can be seen that after TNF-a was used to induce acute inflammatory damage in myogenically differentiated C2C12 cells, MM-SELNs could reduce the expression of inflammation-related proteins ErK1,2, P65, and P38, indicating that MM-SELNs had a significant anti-inflammatory and repair effect; and MM-SELNs could promote the expression of muscle damage repair-related proteins MyoD1 and Myogenin. In addition, MM-SELNs had a significant anti-inflammatory and repair effect on acute inflammatory damage in C2C12 myoblasts, and the effect was better than that of SELNs. This indicates that MM-SELNs have a good therapeutic effect on acute muscle damage, and the effect is better than that of SELNs.

[0131] Example 7: Verification of the targeting of MM-SELNs to the acute skeletal muscle injury mouse model.

[0132] Experimental Group

[0133] Healthy ICR mice aged 8 weeks and weighing about 25 g were selected. A specific method was used to induce acute skeletal muscle injury symptoms in the right lower limbs of the mice. 24 hours later, 300 μg of DIR-labeled MM-SELNs were injected intraperitoneally into the mice. Two hours after the drug injection, the following tissues and organs of the mice were removed: heart, lung, liver, kidney, spleen, left lower limb (normal control) and right lower limb (acute skeletal muscle injury modeling site). Fluorescence photography was performed using the mouse in vivo imager SIL to record the fluorescence distribution of each organ and tissue.

[0134] Control group

[0135] Healthy ICR mice aged 8 weeks and weighing about 25 g were selected. A specific method was used to induce acute skeletal muscle injury symptoms in the right lower limbs of the mice. 24 hours later, 200 μg of DIR-labeled SELNs were injected intraperitoneally into the mice. Two hours after the drug injection, the following tissues and organs of the mice were removed: heart, lung, liver, kidney, spleen, left lower limb (normal control) and right lower limb (acute skeletal muscle injury site). Fluorescence photography was performed using the mouse in vivo imager SIL to record the fluorescence distribution of each organ and tissue.

[0136] The test results of the experimental group and the control group are shown in Fig.19 Middle A and Fig.19 Middle B, Fig.19 A is a fluorescent image of isolated tissues and organs of mice in the control group. Fig.19 B is the fluorescence image of the isolated tissues and organs of mice in the experimental group. Fig.19 Middle A and Fig.19 From the fluorescence signal in B, we can see that MM-SELNs are obviously aggregated at the acute skeletal muscle injury site of the right lower limb, while SELNs are not obviously aggregated at the acute skeletal muscle injury site of the right lower limb. This indicates that MM-SELNs have the characteristics of aggregating at the acute skeletal muscle injury site and can be used for targeted treatment of acute skeletal muscle injury.

[0137] It should be noted that Fig.19 The part indicated by the arrow X is the right lower limb of the mouse, the part indicated by the arrow Y is the left lower limb of the mouse, and the part indicated by the arrow Z is the spleen of the mouse.

[0138] In addition, it should be noted that, although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A method for preparing Salvia miltiorrhiza exosomes modified with macrophage membrane vesicles, characterized in that: include: Step S1, mixing Salvia miltiorrhiza exosomes, macrophage membrane vesicles and PBS buffer to obtain a mixed solution; in the mixed solution, the ratio of the protein concentration of the Salvia miltiorrhiza exosomes to the protein concentration of the macrophage membrane vesicles is (3-4):2; Step S2, extruding the mixed solution through a filter membrane with a first preset pore size for a first preset number of times to obtain Salvia miltiorrhiza exosomes modified with macrophage membrane vesicles; the first preset pore size is 190-210 nm, the filter membrane is a polycarbonate membrane, and the first preset number of times is more than 20 times; The preparation method of the Salvia miltiorrhiza exosomes comprises: Step M1, soaking the Danshen decoction pieces in water for incubation, and then filtering to obtain a first solution; Step M2, subjecting the first solution to a first centrifugation treatment, extracting a supernatant, and obtaining a first supernatant; Step M3, subjecting the first supernatant to a second centrifugation treatment, extracting the supernatant, and obtaining a second supernatant; Step M4, filtering the second supernatant with a filter, performing a third centrifugation treatment, extracting the supernatant, and obtaining a third supernatant; Step M5, subjecting the third supernatant to a fourth centrifugation treatment, extracting the supernatant, and obtaining a fourth supernatant; Step M6, centrifuging the fourth supernatant solution for the fifth time, and the resulting precipitate is the Salvia miltiorrhiza exosomes.

2. The method for preparing macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes according to claim 1, characterized in that: In the step M1, the mass of the Danshen decoction piece is m grams and the volume of the water is v milliliters, and the ratio of m to v is 1: (8-10).

3. The method for preparing macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes according to claim 1, characterized in that: The first centrifugal treatment is carried out at a rotation speed of 1000-4000g and a time of 10-20min, the second centrifugal treatment is carried out at a rotation speed of 4000-6000g and a time of 10-20min, the third centrifugal treatment is carried out at a rotation speed of 9000-11000g and a time of 10-20min, the fourth centrifugal treatment is carried out at a rotation speed of 90000-110000g and a time of 70-100min, and the fifth centrifugal treatment is carried out at a rotation speed of 90000-110000g and a time of 70-100min.

4. The method for preparing macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes according to claim 1, characterized in that: The method for preparing the macrophage membrane vesicles comprises: Step N1, after resuspending the macrophages with water, adding a membrane protein extraction reagent to obtain a cell suspension; Step N2, subjecting the cell suspension to an ice bath treatment, ultrasonic treatment, and centrifugation treatment, and resuspending the obtained precipitate with Tris buffer to obtain a macrophage membrane fragment solution; Step N3, extruding the macrophage membrane fragment solution through a filter membrane with a second preset pore size for a second preset number of times to obtain macrophage membrane vesicles.

5. The method for preparing macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes according to claim 4, characterized in that: In the step N2, the ultrasonic frequency used in the ultrasonic treatment is 20-30kHz, the amplitude is 5-10%, and the time of the ultrasonic treatment is 5-10min; the rotation speed of the centrifugal treatment is 13000-14000g, and the time is 20-30min; in the step N3, the second preset pore size is 390-410nm, the filter membrane is a polycarbonate membrane, and the second preset number of times is more than 20 times.

6. A macrophage membrane vesicle-modified Salvia miltiorrhiza exosome, characterized in that: The exosomes are prepared by the method for preparing macrophage membrane vesicles modified with Salvia miltiorrhiza as described in any one of claims 1 to 5.

7. Use of the macrophage membrane vesicle-modified Salvia miltiorrhiza exosomes according to claim 6 in the preparation of a drug for treating acute skeletal muscle injury.

8. The use according to claim 7, wherein the drug is used for targeted treatment of acute skeletal muscle injury.

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