Use of salvia miltiorrhiza external vesicles in preparation of anti-pneumonia drugs

Abstract

The present application relates to the technical field of molecular biology, more particularly to the application of Danshen external vesicles in the preparation of anti-pneumonia drugs, and a preparation method of the Danshen external vesicles, comprising the following steps: collecting juice after fresh Danshen is sliced and squeezed, sequentially centrifuging at 3000g-3500g for 30-40 min, 8000g-9000g for 20-30 min, and 10000g-11000g for 20-30 min, collecting the supernatant of the third time, centrifuging at 200000g-220000g for 120-150 min, collecting the precipitate, resuspending the precipitate, and obtaining a crude extract. The Danshen external vesicles have the biological activity of resisting pneumonia and repairing lung tissue vascular endothelial barrier, and the curative effect is better than that of tanshinone II A and salvianolic acid B extracted from Danshen in the same dose.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically, to the application of tanshinone exovesicles in the preparation of anti-pneumonia drugs. Background Technology

[0002] Extracellular vesicles (EVs) are vesicles containing nucleic acids, proteins, and other substances, ranging in size from approximately 30 to 200 nm, and are widely distributed in various body fluids. They possess highly efficient "information communication" functions, including the delivery of functional substances and natural attraction to parent cells. They participate in various physiological processes such as cell communication, cell migration, angiogenesis, and anti-tumor immunity, and are closely related to the occurrence and development of many diseases. Plant vesicles are various nanoscale membrane vesicles actively released by plant cells, playing a crucial role in intercellular and interspecies information and substance transfer.

[0003] For example, prior art CN 117925501 A provides a platycodon exosome and its application in the preparation of drugs for pulmonary diseases. The platycodon exosomes obtained by the method of this invention can be efficiently taken up by A549 cells and RAW264.7 cells in the lungs, and possess biological activities against lung tumor cell proliferation and lung inflammation, demonstrating that platycodon exosomes can provide insights for the development of new drugs for targeted therapy and anti-tumor treatment in the clinical treatment of pulmonary diseases.

[0004] Danshen (Salvia miltiorrhiza) is the dried root and rhizome of the plant *Salvia miltiorrhiza*, belonging to the Lamiaceae family. It possesses properties that promote blood circulation and remove blood stasis, regulate menstruation and relieve pain, clear the mind and relieve irritability, cool the blood and reduce swelling. It is commonly used to treat chest pain, abdominal pain, abdominal masses, hot arthralgia, insomnia, irregular menstruation, dysmenorrhea, and amenorrhea. Danshen is bitter and slightly cold in nature, entering the heart, pericardium, and liver meridians. Its cold nature clears heat and cools the blood, while its bitter taste can purge and dry. Entering the heart and liver meridians, it excels at promoting blood circulation and removing blood stasis, and also clears the mind and calms the nerves. However, there is currently no technology to study the uses of the exovesicles of Danshen. Summary of the Invention

[0005] The purpose of this invention is to provide the application of Danshen exovesicles in the preparation of anti-pneumonia drugs.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The application of Danshen external vesicles in the preparation of anti-pneumonia drugs, wherein the preparation method of Danshen external vesicles includes the following steps:

[0008] Fresh Salvia miltiorrhiza slices were juiced and the juice was collected. The juice was then centrifuged sequentially at 3000g-3500g for 30-40 min, 8000g-9000g for 20-30 min, and 10000g-11000g for 20-30 min. The supernatant from the third centrifugation was collected and centrifuged at 200000g-220000g for 120-150 min. The precipitate was collected and resuspended to obtain a crude extract. The crude extract was added to a gradient concentration sucrose solution containing an 8% sucrose solution layer and centrifuged at 200000g-220000g for 120-150 min. The solution containing bands between 30% and 45% was collected and centrifuged again at 200000g-220000g for 120-150 min to obtain Salvia miltiorrhiza vesicles.

[0009] The tanshinone vesicles obtained by this invention have good biosafety, biocompatibility, biotargeting and excellent anti-pneumonia effect, and the efficacy is better than that of an equivalent dose of tanshinone extract.

[0010] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows:

[0011] In one preferred embodiment, the gradient concentration sucrose solution containing the 8% sucrose solution layer is prepared by adding 8%, 30%, 45% and 60% sucrose solutions by mass fraction sequentially from the bottom of a centrifuge tube to prepare a gradient concentration sucrose solution.

[0012] In one preferred embodiment, the operation of adding the crude extract to a gradient concentration sucrose solution containing an 8% sucrose solution layer is as follows: the crude extract is added to a sucrose solution layer containing an 8% mass fraction of the gradient concentration sucrose solution.

[0013] In one preferred embodiment, the centrifugation temperature is 3-10 °C for all steps.

[0014] In one preferred embodiment, the centrifugation temperature is 3-8 °C for all steps.

[0015] In one preferred embodiment, the diameter of the tanshinone exovesicles is 30-150 nm.

[0016] In one preferred embodiment, pneumonia includes LPS-induced inflammation of human umbilical vein endothelial cells or LPS-induced pneumonia.

[0017] This invention applies tanshinone extracellular vesicles to an LPS-induced human umbilical vein endothelial cell (HUVEC) inflammatory injury model. HUVEC cells, as microvascular endothelial cells, can serve as an important target cell closely related to inflammation, congestion, and exudation in pneumonia. The invention examines whether these extracellular vesicles can be taken up by HUVEC cells, whether their therapeutic effect is superior to that of tanshinone IIA and salvianolic acid B extracted from tanshinone at the same dose, and whether these extracellular vesicles can inhibit the expression of inflammatory cytokines, suppress cell damage, and promote vascular repair.

[0018] This invention applies tanshinone exovesicles to an LPS-induced mouse model of acute pneumonia to detect whether they have therapeutic effects against acute pneumonia and their bioactivity.

[0019] Based on the same inventive concept, this invention also claims protection for a miRNA, the sequence of which is shown in SEQ ID NO.1.

[0020] The sequence of miRNA PC-3p-8407_77 (SEQ ID NO.1) is: TCGAAACCGGGCGGAAACAC.

[0021] Based on the same inventive concept, the present invention also claims protection for the use of the miRNA in the preparation of anti-pneumonia reagents.

[0022] In one preferred embodiment, pneumonia includes LPS-induced inflammation of human umbilical vein endothelial cells or LPS-induced pneumonia.

[0023] This invention designs mimics and inhibitors of the newly discovered miRNA PC-3p-8407_77. The mimics and inhibitors demonstrate that PC-3p-8407_77 can significantly alleviate LPS-induced vascular damage in HUVEC cells and inflammatory damage caused by the expression of IL-1β, IL-6, and TNF-α, thus verifying that the miRNA PC-3p-8407_77 can treat lung injuries such as acute pneumonia caused by LPS.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] 1. The method of this invention yields standard-compliant and stable-quality tanshinone vesicles, which exhibit good biosafety, biocompatibility, and biotargeting properties. These vesicles can be efficiently taken up by HUVEC cells and possess anti-pneumonia and lung tissue vascular endothelial barrier repair bioactivity, demonstrating superior efficacy compared to tanshinone IIA and salvianolic acid B extracted from equal doses of tanshinone. Therefore, the tanshinone vesicles extracted by the method described in this invention can provide insights into new drugs for the clinical treatment of pneumonia.

[0026] 2. This invention yielded miRNA PC-3p-8407_77 in the external vesicles of *Salvia miltiorrhiza*, which can alleviate LPS-induced vascular damage and inflammatory response, and is beneficial for further follow-up research. Attached Figure Description

[0027] Figure 1 The results represent the characterization of the external vesicles of *Salvia miltiorrhiza*, among which... Figure 1 A is a transmission electron microscope image depicting the morphology of external vesicles in *Salvia miltiorrhiza*. Figure 1 B is a graph showing the determination of protein content in the outer vesicles of *Salvia miltiorrhiza*. Figure 1 C is an image of proteins in the supernatant analyzed by a gel imaging analysis system; Figure 1 D is a diagram showing the RNA composition of the external vesicles of *Salvia miltiorrhiza* analyzed by agarose gel electrophoresis.

[0028] Figure 2 The results show the pathological effects of SMEVs on major tissues in mice;

[0029] Figure 3 The effects of different concentrations of SMEVs on zebrafish eggs;

[0030] Figure 4 Results of hemolysis experiments for SMEVs;

[0031] Figure 5 The distribution of SMEVs in mice;

[0032] Figure 6 Laser confocal imaging results for HUEVC cells to take up tanshinone exovesicles;

[0033] Figure 7 The effects of tanshinone exovesicles on HUVEC cells and LPS-induced HUVEC cell activity;

[0034] Figure 8 The effects of different Danshen extracts on LPS-induced HUVEC cell activity;

[0035] Figure 9 The results of the scratch assay on LPS-induced HUVEC cells by the external vesicles of *Salvia miltiorrhiza*.

[0036] Figure 10 The results of RT-qPCR experiments on LPS-induced inflammatory factors in HUVEC cells from the external vesicles of *Salvia miltiorrhiza*.

[0037] Figure 11 The changes in body mass index and lung index of mice are shown. Figure 11 A represents weight change over 5 days; Figure 11 B represents the body mass index on day 5; Figure 11 C represents the change in the lung index;

[0038] Figure 12 The results of RT-qPCR experiments on inflammatory factors in mouse lung tissue;

[0039] Figure 13 To collect lung tissue from each group of mice, HE staining was used to observe the pathological changes in the lung tissue of each group of mice.

[0040] Figure 14 The expression of vascular endothelial marker protein (CD31) and vascular endothelial barrier marker protein (VE-cadherin) in the lung tissue of mice in each group was detected by immunofluorescence. Figure 14 A shows the fluorescence results of the expression of vascular endothelial marker protein CD31 and vascular endothelial barrier marker protein VE-cadherin in the lung tissue of mice in each group. Figure 14 B is a bar chart showing the quantitative analysis of the expression of vascular endothelial marker protein CD31 and vascular endothelial barrier marker protein VE-cadherin in the lung tissue of mice in each group.

[0041] Figure 15 The results of RT-qPCR experiments on LPS-induced inflammatory factors in HUVEC cells using five newly discovered highly expressed miRNAs.

[0042] Figure 16 The results of the scratch assay of miRNA PC-3p-8407_77 on LPS-induced HUVEC cells are shown. Figure 16 A shows the fluorescence results of the LPS-induced scratch assay on HUVEC cells using miRNA PC-3p-8407_77. Figure 16 B represents the quantitative analysis of the scratch test results.

[0043] Note: In the statistical analysis, compared with the normal group, ** P<0.01, * P<0.05; compared with the model group, ## P<0.01, # P<0.05 Detailed Implementation

[0044] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0045] Example 1

[0046] Preparation and characterization analysis of Salvia miltiorrhiza-derived extracellular vesicles (SMEV)

[0047] The method for isolating extracellular vesicles from *Salvia miltiorrhiza* according to the present invention includes the following preparation and identification steps:

[0048] 1. Isolation of external vesicles of Salvia miltiorrhiza

[0049] (1) After washing the fresh Salvia miltiorrhiza, slice it, add 5 times the amount of water and juice it. Filter the juice through gauze and collect the juice.

[0050] (2) Centrifuge the salvia miltiorrhiza filtrate obtained in step (1) at 4°C, centrifuging at 3,000 ×g for 30 min to remove large residues; centrifuging at 8,000 ×g for 30 min to remove small residues; centrifuging at 10,000 ×g for 30 min to remove small plant fragments, and retain the supernatant.

[0051] (3) At 4℃, the supernatant was centrifuged at 200,000 g for 120 min, the precipitate was collected, and the precipitate was resuspended with PBS to obtain the crude salvia miltiorrhiza vesicles.

[0052] 2. Purification of Salvia miltiorrhiza exovesicles

[0053] (1) Prepare sucrose solutions with mass fractions of 8%, 30%, 45% and 60% using ultrapure water respectively;

[0054] (2) Using a long needle, add 8%, 30%, 45% and 60% sucrose solutions sequentially from the bottom of the centrifuge tube to prepare a discontinuous sucrose density gradient solution. Transfer the resuspended crude extract of the tanshinone exovesicles to the uppermost layer of the sucrose density gradient solution, which is 8% sucrose solution.

[0055] (3) Centrifuge at 200,000 g for 120 min at 4℃, collect the bands between 30%-45% and 45%-60% respectively, transfer them to a new ultracentrifuge tube, centrifuge at 200,000 g for 120 min to remove sucrose, resuspend the precipitate with PBS to obtain purified tanshinone vesicles.

[0056] 3. Characterization analysis of external vesicles of Salvia miltiorrhiza

[0057] a. Electron microscopy analysis

[0058] The vesicles of *Salvia miltiorrhiza* were fixed and examined by transmission electron microscopy using standard procedures. The resulting transmission electron micrographs are shown below. Figure 1 A and Figure 1As shown in Figure B, electron microscopy results indicate that the purified bands between 30% and 45% exhibit round or oval vesicle structures that are uniform in size and intact, with diameters mainly between 30 and 150 nm, indicating successful extraction of tanshinone exovesicles. However, the purified bands between 45% and 60% show unclear bilayer membrane structures with uneven sizes and numerous impurities. Therefore, the purified bands between 30% and 45% were selected for subsequent experiments.

[0059] b. Protein analysis

[0060] Electrophoresis was performed using a 4×SDS polyacrylamide gel. The 10% SDS-PAGE gel was cut and stained with Coomassie blue for 2 hours, then washed 2-3 times, 1-2 hours each time (wash buffer: 2250 mL 95% ethanol + 250 mL glacial acetic acid + 2500 mL distilled water). Proteins in the supernatant were then analyzed using a gel imaging system. Results are as follows: Figure 1 As shown in C.

[0061] c. RNA component analysis

[0062] The exovesicles of *Salvia miltiorrhiza* were resuspended in PBS, and RNA was extracted from the EVs using an exosome RNA purification kit (Hangzhou Xinjing Bioreactor Development Co., Ltd., Cat. No. 5202050, batch number: 20210410). RNA agarose gel electrophoresis was then performed on a 1.7% agarose gel, and the results are shown below. Figure 1 As shown in D.

[0063] It is evident that the main components of the external vesicles of Danshen are proteins and RNA.

[0064] 4. Safety and stability of Danshen exovesicles

[0065] a. Toxicity detection of Salvia miltiorrhiza external vesicles on mouse tissues

[0066] C57 mice were administered tanshinone vesicles at a dose of 25 mg / kg via gavage, while maintaining a standard diet. After three consecutive days of administration, the mice were sacrificed, and heart, lung, liver, spleen, and kidney tissues were collected. HE staining was used to observe the pathological changes in each tissue. Results are as follows: Figure 2 As shown, SMEVs (external vesicles of Danshen) showed no toxicity to major tissues of mice after continuous gavage for 3 days.

[0067] b. Toxicity detection of Salvia miltiorrhiza exovesicles in zebrafish

[0068] Different concentrations (0 µg / mg, 50 µg / mg, 100 µg / mg, 150 µg / mg, 200 µg / mg, 500 µg / mg) of *Salvia miltiorrhiza* vesicles were added to culture dishes containing zebrafish eggs. The zebrafish eggs were then exposed to different concentrations of SMEVs and cultured for 7 consecutive days. The changes in the zebrafish eggs were then observed. The results are as follows: Figure 3 As shown, different concentrations of SMEVs (tanshinone exovesicles) intervention had no effect on zebrafish eggs.

[0069] c. Hemolysis test of external vesicles of Salvia miltiorrhiza

[0070] A 2% chicken erythrocyte suspension was prepared and incubated with different concentrations of SMEVs (0 µg / mg, 10 µg / mg, 30 µg / mg, 50 µg / mg, 100 µg / mg, 500 µg / mg, 1000 µg / mg) for 1 h. Positive and negative precipitates (PBS) were prepared. After centrifugation, the absorbance at 540 nm was measured. The hemolysis rate was calculated using the formula: Hemolysis rate = (OD experimental group - OD negative control) / (OD positive control - OD negative control) × 100%.

[0071] The results are as follows Figure 4 As shown, the hemolysis rate of different concentrations of SMEVs was not significantly different from that of the negative control group in pure water PBS (P<0.05), and was lower than 10%.

[0072] d. Distribution of SMEVs in mice

[0073] Incubate 100 μM DiR dye reagent at a concentration ratio of SMEVs:DiR (10:1) at 37 °C for 30 min, then centrifuge at 200,000 g for 120 min to remove excess unbound DiR. Collect the precipitate, resuspend in PBS, and obtain DiR-labeled *Salvia miltiorrhiza* vesicles. Administer the DIR-labeled *Salvia miltiorrhiza* vesicles to C57 mice by gavage at a dose of 25 mg / kg. Two hours after administration, perform in vivo distribution imaging of the mice using a small animal in vivo imaging system. The results are as follows: Figure 5 As shown in the figure. The results indicate that DIR-labeled Tanshinone exovesicles concentrate on targeting the lungs, spleen, liver, kidneys, and heart. This demonstrates that the present invention has successfully extracted Tanshinone exovesicles that meet the standards and are of stable quality. Furthermore, these vesicles exhibit non-toxicity, good biocompatibility, and targeted targeting to in vivo tissues.

[0074] Example 2

[0075] Comparison of the effects of tanshinone exovesicles and tanshinone-derived compounds on an LPS-induced human umbilical vein endothelial cell (HUEVC) inflammatory injury model.

[0076] Cellular experiments confirmed that the external vesicles of *Salvia miltiorrhiza* have an anti-LPS-induced effect against human umbilical vein endothelial cell (HUVEC) damage, and the therapeutic effect is superior to other *Salvia miltiorrhiza* extracts extracted at the same dosage. The verification process is as follows:

[0077] 1. Cellular uptake

[0078] a. SMEV fluorescent labeling

[0079] (1) Dilute the purified SMEVs (tanshinone exovesicles) with 200 μl Dilution C and mix well. Take another 2 μl PKH67 dye and mix it with 200 μl Dilution C.

[0080] (2) Mix the two and incubate at 25 °C for 1-5 min;

[0081] (3) Add an equal volume (400 μl) of serum (or 1% BSA) and incubate for 1 min to terminate staining;

[0082] (4) Add 800 μl of medium culture, transfer to an ultracentrifuge tube, incubate at 4 °C, 200,000 ×g, for 120 min;

[0083] (5) Wash with PBS at 4 °C, 200,000×g for 120 min to remove unbound dye;

[0084] (6) Repeat once.

[0085] b. Cell uptake experiment

[0086] (1) Mix the labeled SMEVs (green) with HUVEC cells, add them to a 12-well plate with a spreader, and incubate for 24 hours;

[0087] (3) Fixation: The cytoskeleton (red) was stained with rhodamine-labeled phalloidin dye, and the nuclei (blue) were stained with ready-to-use DAPI dye. The results were observed and photographed under a fluorescence microscope. Figure 6 As shown in the figure, the cell nucleus is stained blue by DAPI, PKH67-labeled SMEVs are green, the cytoskeleton is stained red by Phalloidine, and merge is the merged image. The figure demonstrates that the tanshinone exovesicles can be effectively taken up by HUVEC cells.

[0088] 2. Cell viability detection

[0089] a. Effects of Tanshinone exovesicles on HUVEC cell viability

[0090] (1) According to 4×10 4HUVEC cells were seeded into 96-well plates at 100 μl per well. After the cells adhered, different concentrations of Tanshinone exovesicles were applied to the HUVEC cells.

[0091] (2) CCK8 detection: After 24 h of intervention with HUVEC cells by *Salvia miltiorrhiza* exovesicles, the supernatant was discarded, and 100 μl of CCK8 working solution (CCK8 reagent: basal culture medium = 1:9) was added. After 1 h of reaction, the cell viability was measured at 450 nm using a microplate reader, and the cell viability of each group was calculated and statistically analyzed. The results are as follows: Figure 7 As shown, compared with the normal group, the different concentrations of tanshinone vesicles had no significant effect on the activity of HUVEC cells (P>0.05).

[0092] b. Effects of Tanshinone exovesicles on LPS-induced inflammatory damage in HUVEC cells

[0093] (1) HUVEC cells were seeded into 96-well plates using the method described above;

[0094] (2) After the cells adhered to the wall, HUVEC cells were treated with 1 μg / mL LPS. After 24 h, different concentrations of Tanshinone exovesicles were used to treat LPS-induced HUVEC cells.

[0095] (3) CCK8 detection: After 24 h of LPS-induced HUVEC cells were treated with tanshinone exovesicles, the supernatant was discarded, and 100 μl of CCK8 working solution (CCK8 reagent: basal culture medium = 1:9) was added. After 1 h of reaction, the cell viability was measured at 450 nm using an ELISA reader, and the cell viability of each group was calculated and statistically analyzed. The results are as follows: Figure 7 As shown, compared with the LPS group, all concentrations of Tanshinone exovesicles could increase the activity of LPS-induced HUVEC cells to varying degrees (P<0.05), with the 60 μg / mL concentration showing the most significant increase.

[0096] c. Effects of different tanshinone exovesicle extracts on the cell viability of LPS-induced inflammatory damage in HUVEC cells

[0097] (1) Extraction of crude salvia miltiorrhiza vesicles: The extraction method is the same as that of salvia miltiorrhiza vesicles, but purification is not performed.

[0098] (2) Extraction of Tanshinone IIA: An equal amount of Tanshinone extracted from the outer vesicles was pulverized and passed through a 40-mesh sieve. Five times the volume of 95% ethanol aqueous solution was added, and the mixture was soaked at room temperature in the dark for 48 hours (stirring every 6 hours). The mixture was filtered, and the residue was extracted once more with 95% ethanol aqueous solution. The filtrates were combined, and the filtrate was rotary evaporated at 60°C until no alcohol odor remained (approximately 1 / 5 volume remained, 20-30 mL / 100 g raw material), yielding a brownish-red extract. The extract was dispersed in 200 mL of distilled water, and ethyl acetate was added at a 1:1 volume ratio. The mixture was shaken thoroughly for 10 minutes. After standing and separating into layers, the ethyl acetate phase was collected. The aqueous phase was extracted twice more with ethyl acetate (100 mL ethyl acetate each time). The ethyl acetate phases were combined and passed through a silica gel column. A 200-300 mesh silica gel column (20 times the sample volume) was used, and the column was dry-packed (column diameter to height ratio 1:10). The ethyl acetate phase was mixed with 1.5 times silica gel (15 g / 10 g extract), dried, and eluted with petroleum ether:ethyl acetate (20:1, v / v) for 3 BV to remove nonpolar impurities. The elution was then switched to petroleum ether:ethyl acetate (10:1, v / v), and the red target fraction (tanshinone IIA main spot) with Rf≈0.5 was collected by TLC monitoring. The target fractions were combined and concentrated under reduced pressure at 40 °C. The target fraction was then dissolved in methanol-water (8:2) and cooled to crystallize, or further purified by semi-preparative HPLC (C18 column, methanol:water = 85:15). The crystals were dried under vacuum at 50 °C for 6 hours to obtain orange-red needle-like crystals, which were tanshinone IIA.

[0099] (3) Extraction of salvianolic acid B: The same amount of salvia miltiorrhiza extracted as the vesicles of salvia miltiorrhiza was crushed and extracted twice by reflux with 70% ethanol (1:10 material-liquid ratio, 1.5 hours each time). The filtrates were combined and concentrated under reduced pressure until there was no alcohol taste. The pH was adjusted to 2-3 with hydrochloric acid, and the mixture was allowed to stand for 12 hours. The precipitate was collected by centrifugation, washed with water and freeze-dried to obtain crude salvianolic acid B extract. Finally, it was purified by macroporous resin (eluted with 30% ethanol), concentrated and dried to obtain salvianolic acid B.

[0100] (4) Seed HUVEC cells into 96-well plates;

[0101] (5) After the cells adhered to the wall, HUVEC cells were treated with 1 μg / mL LPS. After 24 h, HUVEC cells treated with LPS were treated with different extracts of 60 μg / mL.

[0102] (6) CCK8 assay: After 24 h of LPS-treated HUVEC cells treated with different Danshen extracts, the supernatant was discarded, and 100 μl of CCK8 working solution (CCK8 reagent: basal culture medium = 1:9) was added. After 1 h of reaction, the cell viability was measured at 450 nm using a microplate reader, and the cell viability of each group was calculated and statistically analyzed. The results are as follows: Figure 8As shown, compared with the LPS group, different Tanshinone extracts at 60 μg / mL all increased the activity of LPS-induced HUVEC cells (P<0.05), but the improvement effect was significantly different compared with purified Tanshinone vesicles.

[0103] The results showed that the external vesicles of Salvia miltiorrhiza could be efficiently taken up by HUVEC cells. Among the same concentration of Salvia miltiorrhiza extract (60 μg / mL), the cell activity of the external vesicle group was 131.7%, the cell activity of the crude external vesicle group was 78.48%, the cell activity of the tanshinone IIA group was 93.87%, and the cell activity of the salvianolic acid B group was 107%. The external vesicles of Salvia miltiorrhiza showed the most significant anti-LPS inflammatory damage effect.

[0104] Example 3: Tanshinone exovesicles acting on an LPS-induced inflammatory injury model of human umbilical vein endothelial cells (HUEVC).

[0105] 1. Scratch test

[0106] (1) HUVEC cells were seeded in 6-well plates and incubated in a 5% CO2 incubator at 37°C;

[0107] (2) LPS intervention and Danshen vesicle treatment: HUVEC cells were subjected to LPS intervention and Danshen vesicle treatment according to the above method;

[0108] (3) After treating the external vesicles of Salvia miltiorrhiza for 24 hours, use a pipette tip to make a horizontal line mark on the back as close as possible to the ruler. The pipette tip should be vertical and not tilted. After washing with PBS 2-3 times, add low serum (2%) culture medium for culture.

[0109] (4) Place in a 37℃ 5% CO2 incubator and incubate. Take samples and photographs at 0 and 24 hours. Results are as follows. Figure 9 As shown, SMEVs can restore the migration ability of HUVECs (P<0.05).

[0110] 2. RT-qPCR experiment

[0111] (1) Extraction of total RNA from cells: After collecting cells from each group, add 500 µL of TRIzol, pipette, lyse on ice for 5 min, add 100 µL of chloroform, mix well, incubate at room temperature for 5 min, and centrifuge at 10,000×g for 10 min. After removing the upper layer of colorless aqueous RNA, add 250 µL of isopropanol, mix well, and incubate at -20 ℃ for 2 h. Centrifuge at 10,000×g for 10 min, remove the supernatant, add 500 µL of 75% cold ethanol, centrifuge at 5,000×g for 5 min twice. After drying for 10 min, add 30 µL of RNase-free water and mix well;

[0112] (2) RNA concentration measurement: Take 1 µL of RNA and measure the RNA concentration using a spectrophotometer;

[0113] (3) Reverse transcription to synthesize cDNA: Reverse transcription of each RNA sample to synthesize cDNA according to the operation steps of the reverse transcription kit (NovoScript, E047-01B);

[0114] (4) PCR was performed on IL-1β, IL-6, TNF-α and the internal reference gene: A PCR system was prepared (1 μL upstream primer, 1 μL downstream primer, 1 μL cDNA, 7 μL enzyme-free water, 10 μL PCR amplification reagent) and amplified. The reaction conditions were: 95 °C pre-denaturation for 30 s, 95 °C denaturation for 10 s, 60 °C annealing extension for 30 s, for 40 cycles. GAPDH was used as the internal reference. －ΔΔCt The relative expression of relevant genes was calculated using a statistical method, and the results were analyzed. The results are as follows: Figure 10 As shown, compared with the model group, SMEV can significantly downregulate the expression of inflammatory factors IL-1β, IL-6 and TNF-α (P<0.05).

[0115] The results showed that the external vesicles of Danshen have the effects of repairing blood vessel migration and inhibiting the expression of inflammatory cytokines.

[0116] Example 4: Effects of Tanshinone exovesicles on an LPS-induced mouse lung injury model

[0117] Animal experiments verified that the external vesicles of *Salvia miltiorrhiza* have a therapeutic effect on an LPS-induced acute pneumonia model in mice. The verification process is as follows:

[0118] 1. Animal preparation and model construction

[0119] (1) Animal preparation: C57 mice, weighing 16-18g, half male and half female. SPF grade adaptive breeding. Animal identification was done by ear tagging. The mice were caged, 6 mice / cage, with bedding changed every 2 days, temperature 22±2℃, 12-hour light and dark cycle, and relative humidity 55±15%.

[0120] (2) Model establishment: After 3 days of adaptive feeding, mice were randomly divided into three groups (n=6 in each group): control group, LPS group, and SMEV group. The SMEV group and LPS group were given 25 mg / kg of SMEVs by gavage once a day for 3 consecutive days; 24 hours after administration on the 3rd day, LPS (5 mg / kg) was administered via nasal route to stimulate the mice to establish the model, and all mice were sacrificed 24 hours later.

[0121] 2. Evaluation of its effect on pneumonia / acute pneumonia

[0122] a. Body weight, organ index

[0123] Collect mouse samples from each group and weigh them, such as Figure 11 As shown, where, Figure 11 A represents weight change over 3 days; Figure 11 B represents the body mass index on day 3; Figure 11 C represents the change in lung index. The results showed that compared with the normal group, the LPS group had a significantly lower body mass index (P<0.05) and a significantly higher lung index (P<0.05); compared with the LPS group, the SMEV group had a significantly higher body mass index (P<0.05) and a significantly lower lung index (P<0.05).

[0124] b. RT-qPCR experiment

[0125] (1) Lung tissue RNA extraction: Same as above;

[0126] (2) Reverse transcription to synthesize cDNA: Same as above:

[0127] (3) PCR was performed on IL-1β, IL-6, TNF-α and the internal reference gene: as before;

[0128] (4) Statistical analysis: The results are as follows Figure 12 As shown, compared with the normal group, the expression of IL-1β, IL-6 and TNF-α in the LPS group was significantly increased (P<0.05); compared with the LPS group, SMEV could downregulate the expression of inflammatory factors IL-1β, IL-6 and TNF-α (P<0.05).

[0129] c. HE diagnostic pathology results

[0130] Lung tissues from each group of mice were collected, and the pathological changes in the lung tissues were observed using HE staining. The results are as follows: Figure 13 As shown, in the normal group, the alveolar pores of the lung tissue of mice were intact, and clear capillary networks, a small number of erythrocytes, fibroblasts, and macrophages were visible in the alveolar septa. Compared with the normal group, the alveolar cavities of the LPS group mice were of varying sizes, with a large number of inflammatory cells infiltrating the alveolar cavities, significantly thickened alveolar septa, and obvious edema and hemorrhage in the pulmonary interstitium. The SMEV group could improve LPS-induced lung tissue damage, reduce pulmonary edema, and significantly reduce inflammatory cell infiltration.

[0131] d. Immunofluorescence detection of the expression of vascular endothelial marker protein CD31 and vascular endothelial barrier marker protein VE-cadherin in the lung tissue of mice in each group.

[0132] Lung tissue sections from each group of mice were baked at 60 ℃, dewaxed, and then dehydrated in a gradient of ethanol-water solutions (70wt%, 80wt%, 95wt%, anhydrous ethanol 1, and anhydrous ethanol 2, each soaked for 1 min), followed by washing with distilled water. Antigen retrieval was then performed on the lung tissue sections from each group of mice. Antigen retrieval was performed by microwave heating with pH 6.0 citrate buffer, followed by washing with PBS and blocking of endogenous enzymes with 3% H2O2. Diluted primary antibodies (CD31, Servicebio, GB113151; VE-cadherin, Servicebio, GB14013) were added, and the sections were incubated overnight at 4 ℃. The next day, the sections were washed with PBS, and fluorescent secondary antibodies (CY3-labeled goat anti-rabbit IgG, Servicebio; Alexa Fluor488-labeled goat anti-mouse IgG, Servicebio, GB25301) were added, and the sections were incubated at 37 ℃ in the dark for 1 h. After DAPI staining of the nuclei and washing with PBS, the slides were mounted and the expression of vascular endothelial marker protein CD31 (red) and vascular endothelial barrier marker protein VE-cadherin (green) was observed under a fluorescence microscope. The results are as follows: Figure 14 As shown, where Figure 14 A shows the fluorescence results of the expression of vascular endothelial marker protein CD31 and vascular endothelial barrier marker protein VE-cadherin in the lung tissue of mice in each group. Figure 14 B is a bar chart showing the quantitative analysis of the expression of vascular endothelial marker protein CD31 and vascular endothelial barrier marker protein VE-cadherin in the lung tissue of mice in each group. Compared with the normal group, the expression of CD31 and VE-cadherin in the lung tissue of mice in the LPS group was decreased, while the expression of CD31 and VE-cadherin in the lung tissue of mice in the SMEV group was increased compared with the LPS group. The results indicate that Tanshinone exovesicles can inhibit the expression of inflammatory cytokines and promote the repair of the vascular endothelial barrier in lung tissue in an LPS-induced acute pneumonia model, and have a therapeutic effect on LPS-induced acute pneumonia.

[0133] Example 5: Sequencing and Enrichment Analysis of Tanshinone Exovesicle miRNAs

[0134] 1. Extraction of salvia miltiorrhiza vesicles: Same as above.

[0135] 2. RNA extraction and quality testing:

[0136] Exosomal RNA purification kit (Hangzhou Xinjing Bioreactor Development Co., Ltd., Cat. No. 5202050, batch number: 20210410) was used to extract RNA from the exosomal vesicles of *Salvia miltiorrhiza*. The integrity, concentration, and purity of the RNA were detected by agarose gel electrophoresis, NanoDrop spectrophotometer, and Agilent 2100 bioanalyzer to ensure that the RNA quality met the requirements for subsequent sequencing.

[0137] 3. Library Construction:

[0138] Library construction was performed on qualified RNA samples, including ligation of 3' and 5' adapters for miRNAs, synthesis of cDNA through reverse transcription, followed by PCR amplification. Finally, the library was quality checked to ensure its validity.

[0139] 4. Sequencing:

[0140] The constructed library was subjected to high-throughput sequencing using platforms such as Illumina to obtain a large amount of miRNA sequence data.

[0141] 5. Data Analysis:

[0142] The raw sequencing data underwent quality control to remove low-quality sequences and adapter sequences. The data was then compared with plant reference genomes or miRNA databases to identify known miRNAs and predict new miRNAs. A total of 161 miRNA sequences were detected, including 29 newly discovered miRNAs, of which 5 were highly expressed.

[0143] Table 1. miRNA sequences

[0144]

[0145] Example 6

[0146] Among the five newly discovered miRNAs, miRNA PC-3p-8407_77 has a restorative effect on the inflammatory damage cell activity of HUVEC cells.

[0147] 1. Cell transfection of miRNA

[0148] (1) Five mimics and inhibitors of the newly discovered miRNAs were synthesized at Sangon Biotech (Shanghai) Co., Ltd. The sequences are shown in Table 2.

[0149] Table 2. Sequences of miRNA mimics and inhibitors

[0150]

[0151] The 'm' in the inhibitor represents 2'-O-methyl modification, which introduces a methyl group at the ribose position to enhance the binding affinity to the target miRNA and reduce off-target effects.

[0152] (2) HUVEC cells were incubated in a 5% CO2 incubator at 37°C until they were fused to 50%-60%;

[0153] (3) Preparation of working solutions for miRNA mimic and miRNA inhibitor: Take 20 μM of miRNA mimic and miRNA inhibitor stock solution and dilute it with culture medium containing serum. The mass of miRNA mimic and miRNA inhibitor in each well is 20 pmol. Shake gently to mix and set aside.

[0154] (4) Preparation of RNA transfection reagent-miRNA mimic / miRNAinhibitor complex: Prepare two DEPC-treated centrifuge tubes, add DMEM serum-free basal culture medium to each tube, then add miRNA mimic / miRNAinhibitor working solution (labeled tube A) and RNA transfection reagent (BBI, UK, E607402) (labeled tube B) to each tube, and gently shake to mix; then mix tube A and tube B, gently mix, and let stand at room temperature for 10 min to form siRNA-RNA complex. Add the complex to the well plate of cultured HUVEC cells and intervene for more than 24 h for experiments.

[0155] 2. RT-qPCR experiment

[0156] (1) Extraction of total RNA from cells: After collecting cells from each group, add 500 µL of TRIzol, pipette, lyse on ice for 5 min, add 100 µL of chloroform, mix well, incubate at room temperature for 5 min, and centrifuge at 10,000×g for 10 min. After removing the upper layer of colorless aqueous RNA, add 250 µL of isopropanol, mix well, and incubate at -20 ℃ for 2 h. Centrifuge at 10,000×g for 10 min, remove the supernatant, add 500 µL of 75% cold ethanol, centrifuge at 5,000×g for 5 min twice. After drying for 10 min, add 30 µL of RNase-free water and mix well;

[0157] (2) RNA concentration measurement: Take 1 µL of RNA and measure the RNA concentration using a spectrophotometer;

[0158] (3) Reverse transcription to synthesize cDNA: Reverse transcription of each RNA sample to synthesize cDNA according to the operation steps of the reverse transcription kit (NovoScript, E047-01B);

[0159] (4) PCR was performed on IL-1β, IL-6, TNF-α, and the internal reference gene: A PCR system was prepared (1 μL upstream primer, 1 μL downstream primer, 1 μL cDNA, 7 μL enzyme-free water, 10 μL PCR amplification reagent) and amplified. The reaction conditions were: 95 °C pre-denaturation for 30 s, 95 °C denaturation for 10 s, 60 °C annealing extension for 30 s, for 40 cycles. GAPDH was used as the internal reference. －ΔΔCtThe relative expression of relevant genes was calculated using a statistical method, and the results were analyzed. Figure 15 As shown, compared with the model group, the miRNA PC-3p-8407_77 mimic downregulated the expression of inflammatory factors IL-1β, IL-6, and TNF-α (P<0.05), while the miRNA PC-3p-8407_77 inhibitor significantly promoted the expression of inflammatory factors IL-1β, IL-6, and TNF-α (P<0.05). However, apart from miRNA PC-3p-8407_77, the other four miRNAs, although also highly expressed miRNAs, did not show significant effects from the mimic and inhibitory effects compared to the model group (P>0.05).

[0160] 3. Scratch test

[0161] (1) HUVEC cells were seeded in 6-well plates and incubated in a 5% CO2 incubator at 37°C;

[0162] (2) Transfection of miRNA mimics and inhibitors: The miRNA mimics and inhibitors were transfected into HUVEC cells according to the above method;

[0163] (3) LPS intervention: HUVEC cells were treated with LPS according to the above method.

[0164] (4) After LPS treatment for 24 h, use the pipette tip to make a vertical cut on the back of the pipette tip, with the pipette tip perpendicular to the ruler. The pipette tip should be vertical and not tilted. After washing with PBS 2-3 times, add low serum (2%) culture medium for culture.

[0165] (5) Place in a 37℃ 5% CO2 incubator for incubation. Take samples and photographs at 0 h and 24 h. Results are as follows. Figure 16 As shown, where, Figure 16 A shows the fluorescence results of the LPS-induced scratch assay on HUVEC cells using miRNA PC-3p-8407_77. Figure 16 B represents the quantitative analysis of the scratch wound healing results. Compared with the model group (9.33%), the miRNA mimic (15.21%) promoted scratch wound healing in HUVEC cells (P<0.05), while the miRNA inhibitor (7.24%) was slightly worse than the model group, but there was no significant difference between the miRNA and LPS compared with the model group (P>0.05), suggesting that miRNA can repair the migration ability of HUVECs (P<0.05).

[0166] The results show that: Figure 16As shown, among the five newly discovered highly expressed miRNAs, miRNA PC-3p-8407_77 has functions such as repairing angiogenesis and inhibiting the expression of inflammatory cytokines.

[0167] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. The application of tanshinone exovesicles in the preparation of drugs for treating acute pneumonia, characterized in that, The preparation method of the danshen external vesicles includes the following steps: Fresh Salvia miltiorrhiza slices were juiced and the juice was collected. The juice was then centrifuged sequentially at 3000g-3500g for 30-40 min, 8000g-9000g for 20-30 min, and 10000g-11000g for 20-30 min. The supernatant from the third centrifugation was collected and centrifuged at 200000g-220000g for 120-150 min. The precipitate was collected and resuspended to obtain a crude extract. The crude extract was added to a gradient concentration sucrose solution containing an 8% sucrose solution layer and centrifuged at 200000g-220000g for 120-150 min. The solution containing bands between 30% and 45% was collected and centrifuged again at 200000g-220000g for 120-150 min to obtain Salvia miltiorrhiza vesicles.

2. The application according to claim 1, characterized in that, The method for preparing the gradient concentration sucrose solution containing the 8% sucrose solution layer is as follows: 8%, 30%, 45% and 60% sucrose solutions are added sequentially from the bottom of a centrifuge tube to prepare the gradient concentration sucrose solution.

3. The application according to claim 1, characterized in that, The procedure for adding the crude extract to a gradient concentration sucrose solution containing an 8% sucrose solution layer is as follows: the crude extract is added to an 8% mass fraction sucrose solution layer of the gradient concentration sucrose solution.

4. The application according to claim 1, characterized in that, The centrifugation temperature for all steps was 3-10 ℃.

5. The application according to claim 1, characterized in that, The diameter of the external vesicles of the danshen is 30-150 nm.

6. The application according to any one of claims 1-5, characterized in that, The acute pneumonia mentioned is LPS-induced acute pneumonia.

7. A miRNA, characterized in that, Its sequence is shown in SEQ ID NO.

1.

8. The use of the miRNA according to claim 7 in the preparation of anti-inflammatory agents.

9. The application according to claim 8, characterized in that, The inflammation described is LPS-induced inflammation of human umbilical vein endothelial cells.

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