Use of camellia sinensis outer vesicles for preparing anti-inflammatory drugs

Abstract

The present application relates to the technical field of molecular biology, more particularly to the application of camellia sinensis var. assamica exovesicles in the preparation of anti-inflammatory drugs, and a preparation method of the camellia sinensis var. assamica exovesicles, comprising the following steps: collecting juice after fresh camellia sinensis var. assamica slices are juiced, sequentially centrifuging, and collecting the supernatant of the third time, centrifuging at 120000g-140000g for 50-60min to obtain a crude extract; adding the crude extract into a gradient concentration sucrose solution, centrifuging, collecting the solution of the band between 30%-45%, and then centrifuging at 150000g-160000g for 50-60min to obtain the camellia sinensis var. assamica exovesicles. The camellia sinensis var. assamica exovesicles prepared by the method of the present application meet the standards and are stable in quality. The present application also obtains miRNAs related to anti-inflammatory treatment in the camellia sinensis var. assamica exovesicles.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically, to the application of *Tea dwarf tea* exovesicles in the preparation of anti-inflammatory 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] Ardisia japonica, the dried whole herb of the plant Ardisia japonica Herb (family Myrsinaceae), possesses the effects of resolving phlegm and relieving cough, clearing heat and dampness, and promoting blood circulation and removing blood stasis. It is commonly used to treat chronic and acute coughs, wheezing with excessive phlegm, damp-heat jaundice, amenorrhea due to blood stasis, rheumatic pain, and traumatic injuries. Ardisia japonica has a pungent and slightly bitter taste, is neutral in nature, and enters the lung and liver meridians. Its pungent taste promotes circulation and disperses, while its slightly bitter taste drains and dries. Entering the lung meridian, it resolves phlegm and relieves cough; entering the liver meridian, it promotes blood circulation and removes blood stasis. Modern research shows that Ardisia japonica contains various medicinal components, such as bergenin (ardisin), kaempferol, quercetin, ardisin, and succinylquinone. These components have significant antitussive, expectorant, anti-inflammatory, analgesic, antibacterial, antiviral, hepatoprotective, and anti-fibrotic effects. For example, *Tea japonica* has inhibitory effects on *Staphylococcus aureus*, *Streptococcus pneumoniae*, and influenza virus. Its water and alcohol extracts significantly inhibit xylene-induced ear swelling and acetic acid-induced writhing in mice. *Tea japonica* can also be used as an adjunct treatment for rheumatic pain, traumatic injuries, and other ailments. Through its effects of promoting blood circulation, removing blood stasis, and relieving dampness and urination, it improves local blood circulation and reduces inflammation and pain. However, the uses of the exovesicles of *Tea japonica* have not yet been studied using existing technology. Summary of the Invention

[0005] The purpose of this invention is to provide the application of *Tea dwarf tea* exovesicles in the preparation of anti-inflammatory drugs.

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

[0007] The application of *Tea glomerata* exovesicles in the preparation of anti-inflammatory drugs, the preparation method of *Tea glomerata* exovesicles includes the following steps:

[0008] Fresh dwarf tea slices were juiced and the juice was collected. The juice was then centrifuged sequentially at 200g-300g for 20-30 min, 3000g-3500g for 30-40 min, and 5000g-6000g for 50-60 min. The supernatant from the third centrifugation was collected and centrifuged at 120000g-140000g for 50-60 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 150000g-160000g for 50-60 min. The solution containing bands between 30% and 45% was collected and centrifuged again at 150000g-160000g for 50-60 min to obtain the dwarf tea exovesicles.

[0009] The dwarf tea exovesicles prepared by this invention have good biosafety, biocompatibility, biotargeting and excellent anti-inflammatory effects, and the therapeutic effect is better than that of an equal dose of dwarf tea 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 dwarf tea exovesicles is 30-150 nm.

[0016] 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.

[0017] The sequence of miRNA PC-5p-570173_4 (SEQ ID NO.5) is: GAG ...

[0018] Based on the same inventive concept, the present invention also claims protection for the use of the miRNA in the preparation of anti-inflammatory agents.

[0019] The newly discovered miRNA, PC-5p-570173_4 (gene sequence: GAGAGAGAGAGAGAGAGAGAGAGAGAGAG), is significantly enriched in the following pathways: plant hormone signal transduction and phosphatidylinositol signaling system, inositol phosphate metabolism, and glycosaminoglycan degradation.

[0020] This invention relates to the design of a newly discovered PC-5p-570173_4 inhibitor. The above-mentioned miRNA PC-5p-570173_4 inhibitor demonstrates that miRNA PC-5p-570173_4 can significantly alleviate LPS-induced reductions in the mRNA expression levels of inflammatory factors IL-6 and TNF-α in RAW264.7 cells by 58% and 68%, respectively (P<0.01), verifying that the miRNA PC-5p-570173_4 is suitable for anti-inflammatory targeted therapy.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] 1. The inventors have discovered that the method of this invention can produce dwarf tea vesicles that meet standards and have stable quality.

[0023] 2. This invention has identified miRNAs in the outer vesicles of *Tea glomerata* that are associated with anti-inflammatory treatment. Therefore, the outer vesicles of *Tea glomerata* extracted by the method described in this invention can provide insights for new anti-inflammatory drugs in clinical drug therapy. Attached Figure Description

[0024] Figure 1 The characterization results of the external vesicles of *Tea pyrenoidosa*, among which, Figure 1 Image A is a transmission electron microscope image depicting the morphology of external vesicles in *Tea dwarf tea*. Figure 1 B is a diagram showing the RNA composition of the exovesicles of *Tea dwarf tea* analyzed by agarose gel electrophoresis.

[0025] Figure 2 The results of the hemolysis experiment on the outer vesicles of *Tea glomerata* were compared with those of the positive control. ** P<0.01, * P<0.05;

[0026] Figure 3 The figure shows the effects of dwarf tea extract, crude dwarf tea exoves, and dwarf tea exoves on RAW264.7 cells and LPS-induced RAW264.7 cell viability. Figure 3 A represents the effect of dwarf tea glycosides, crude dwarf tea exoves, and dwarf tea exoves on the activity of RAW264.7 cells. Figure 3 B represents the effects of dwarf tea extract, crude dwarf tea exoves, and dwarf tea exoves on LPS-induced RAW264.7 cell activity.

[0027] Figure 4 The results of RT-qPCR experiments on LPS-induced inflammatory factors in RAW264.7 cells are shown in the external vesicles of *Tea glomerata*. Figure 4 A represents the effect of *Tea dwarf tea* exovesicles on the inflammatory factor IL-6. Figure 4 B represents the effect of *Tea dwarf tea* exovesicles on TNF-α;

[0028] Figure 5 These are the sequencing results of extravesicular miRNAs from *Tea dwarf tea*, among which... Figure 5 A represents the number and length distribution of miRNAs obtained from miRNA sequencing; Figure 5 B represents the results of KEGG enrichment analysis of 53 newly discovered miRNAs;

[0029] Figure 6 The effects of newly discovered miRNAs (PC-5p-570173_4, PC-3p-81257_21, PC-5p-1255157_3) mimics and inhibitors from the outer vesicles of *Tea pygmaea* on LPS-induced RAW264.7 cell viability; among which, Figure 6 A represents the effect of various miRNAs on LPS-induced RAW264.7 cell viability. Figure 6 B represents the effect of miRNA PC-5p-570173_4 mimics and inhibitors on LPS-induced RAW264.7 cell viability;

[0030] Figure 7 A bar graph showing the effects of mimics and inhibitors of newly discovered miRNAs (PC-5p-570173_4, PC-3p-81257_21, PC-5p-1255157_3) from the exovesicles of *Tea pygmaea* on LPS-induced inflammatory factors in RAW264.7 cells; among which, Figure 7A represents the effect of mimics and inhibitors of various miRNAs on LPS-induced inflammatory factor IL-6 in RAW264.7 cells. Figure 7 B represents the effect of mimics and inhibitors of various miRNAs on LPS-induced inflammatory factor TNF-α in RAW264.7 cells.

[0031] 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

[0032] 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.

[0033] Example 1

[0034] Preparation and characterization analysis of Ardisia japonica-derived extracellular vesicles (AJEVs)

[0035] A method for isolating extracellular vesicles from *Tea gloriosa*, the preparation and identification steps of which are as follows:

[0036] 1. Isolation of the outer vesicles of *Tea dwarf tea*

[0037] (1) After washing the fresh tea leaves, slice them and add 1 part water to extract the juice. Filter the juice through gauze and collect the juice. After extracting the juice from the fresh tea leaves, centrifuge at 200×g for 20 min, 3000×g for 30 min, and 6000×g for 50 min. Collect the supernatant and centrifuge at 120,000×g for 60 min. Collect the precipitate, resuspend it in PBS and place it in a centrifuge tube to obtain the crude extract of tea leaves vesicles after resuspension. Use a long needle to add 8%, 30%, 45% and 60% sucrose solutions from the bottom of the centrifuge tube to prepare a discontinuous sucrose density gradient solution. Finally, the resuspended crude extract of *Tea dwarf tea* vesicles was added to the resuspension in an 8% sucrose solution layer and centrifuged at 150,000×g for 60 min. The bands between 30% and 45% were collected and transferred to a new ultracentrifuge tube. The sucrose was removed by centrifugation at 150,000×g for 60 min, thus obtaining *Tea dwarf tea* vesicles. The centrifugation temperature for all steps was 3-10℃.

[0038] (2) Centrifuge the filtrate of the dwarf tea obtained in step (1) at 4°C, centrifuge at 200 ×g for 20 min to remove large residues; centrifuge at 3000 ×g for 30 min to remove small residues; centrifuge at 6000 ×g for 50 min to remove small plant fragments, and retain the supernatant.

[0039] (3) At 4℃, the supernatant was centrifuged at 120,000×g for 60min, the precipitate was collected, and the precipitate was resuspended with PBS to obtain the crude tea vesicles.

[0040] 2. Purification of the exovesicles of *Tea dwarf tea*

[0041] (1) Prepare sucrose solutions with mass fractions of 8%, 30%, 45% and 60% using ultrapure water;

[0042] (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 *Tetracentron sinense* exovesicles to the uppermost layer of the sucrose density gradient solution, which is the 8% sucrose solution layer.

[0043] (3) Centrifuge at 150,000 g for 60 min at 4℃, collect the band between 30% and 45% and transfer it to a new ultracentrifuge tube, centrifuge at 150,000 g for 60 min to remove sucrose, resuspend the precipitate with PBS to obtain dwarf tea exovesicles (AJEVs).

[0044] 3. Characterization analysis of the external vesicles of *Tea dwarf tea*

[0045] a. Electron microscopy analysis

[0046] The vesicle particles of *Tea dwarf tea* were fixed and examined by transmission electron microscopy using standard procedures. The resulting transmission electron micrographs are shown below. Figure 1 As shown in Figure A, electron microscopy results show that there are round or elliptical vesicle structures, which are uniform in size and intact, with diameters mainly between 30-150 nm, indicating that the extravesicles of *Tea dwarf tea* were successfully extracted.

[0047] b. RNA component analysis

[0048] The exovesicles of *Tea glomerata* were resuspended in PBS, and exosomal RNA was extracted using an exosomal 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 B.

[0049] 4. Safety of the outer vesicles of Dwarf Tea

[0050] A 2% chicken erythrocyte suspension was prepared and incubated for 1 h with different concentrations of AJEVs (0 µg / mg, 10 µg / mg, 30 µg / mg, 50 µg / mg, 100 µg / mg, 500 µg / mg, 1000 µg / mg). Positive and negative precipitates (PBS) were used. 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%. Results are as follows: Figure 2 As shown in Table 1, the positive control group was treated with pure water, and the red blood cells in this group lysed, defined as a hemolysis rate of 100%. Compared with pure water, the hemolysis rate of each concentration of *Tea dwarf tea* vesicles was less than 10%, indicating that *Tea dwarf tea* vesicles have good biocompatibility.

[0051] Table 1. Hemolytic properties of *Tea dwarf tea* vesicles at different concentrations

[0052]

[0053] The results showed that vesicles of *Tea dwarf tea* that met the required standards and were of stable quality had been successfully extracted. Furthermore, these vesicles exhibited good biocompatibility.

[0054] Example 2

[0055] The extravesicles of *Tea dwarf tea* act on an LPS-induced inflammatory damage model in mouse mononuclear macrophage leukemia cells (RAW264.7).

[0056] This invention, through cell experiments, discovered that the exovesicles of *Tea dwarf tea* have an anti-LPS-induced damaging effect on mouse mononuclear macrophage leukemia cells (RAW264.7). The verification process is as follows:

[0057] 1. Cell viability detection

[0058] a. Effects of Bergenin, crude *Tea dwarf tea* exoves, and *Tea dwarf tea* exoves on RAW264.7 cell viability.

[0059] (1) Extraction of dwarf tea: The same amount of dwarf tea as the extract of the outer vesicles of dwarf tea was crushed, and extracted twice by reflux with 80% ethanol. The filtrates were combined, concentrated, dissolved in water, extracted with ethyl acetate, separated and purified by silica gel column chromatography, and finally concentrated and dried under reduced pressure to obtain dwarf tea.

[0060] (2) Crude extract of tea vesicles: The extraction method is the same as that of tea vesicles, but no purification is performed;

[0061] (3) Exovesicles of dwarf tea: Extract exovesicles of dwarf tea according to the above method;

[0062] (4) According to 4×10 4 RAW264.7 cells were seeded into 96-well plates at 100 μl per well. After the cells adhered, RAW264.7 cells were treated with 30 μg / mL of chloranthus var. ...

[0063] (5) CCK8 assay: After 24 h of intervention in RAW264.7 cells, the supernatant was discarded, and 100 μl of CCK8 working solution (CCK8 reagent: basal culture medium = 1:10) was added. After 1 h of reaction, the cell viability was measured at 450 nm using a microplate reader. The cell viability of each group was calculated and statistically analyzed. The results are as follows: Figure 3 As shown in Figure A, compared with the control group, 30 μg / mL of catechin, crude catechin exoves, and catechin exoves had no significant effect on the activity of RAW264.7 cells (P>0.05).

[0064] b. Effects of dwarf tea extract, crude dwarf tea exoves, and dwarf tea exoves on LPS-induced inflammatory damage and cell viability in RAW264.7 cells.

[0065] (1) Extract dwarf tea extract, crude dwarf tea outer capsules, and dwarf tea outer capsules according to the above method;

[0066] (2) Seed RAW264.7 cells into 96-well plates as described above;

[0067] (3) After the cells adhered to the wall, RAW264.7 cells were treated with 1 μg / mL LPS. After 24 h, RAW264.7 cells were treated with 30 μg / mL dwarf tea extract, crude dwarf tea exoves, and dwarf tea exoves, respectively.

[0068] (4) CCK8 assay: After 24 h of intervention with chlortetracycline in RAW264.7 cells, the supernatant was discarded, and 100 μl of CCK8 working solution (CCK8 reagent: basal culture medium = 1:10) 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 3 As shown in B, compared with the LPS model group, 30 μg / mL of dwarf tea glycosides, crude dwarf tea exoves, and dwarf tea exoves all increased the activity of RAW264.7 cells to varying degrees (P<0.05).

[0069] 2. RT-qPCR experiment

[0070] (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;

[0071] (2) RNA concentration measurement: Take 1 µL of RNA and measure the RNA concentration using a spectrophotometer;

[0072] (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);

[0073] (4) PCR of IL-6, TNF-α and internal control genes: PCR system (1 μL upstream primer, 1 μL downstream primer, 1 μL cDNA, 7 μL enzyme-free water, 10 μL PCR amplification reagent) was prepared according to the PCR amplification kit (NovoScript, E099-01A) and amplified. Primer sequences are shown in Table 2. The reaction conditions were: 95 °C pre-denaturation for 30 s, 95 °C denaturation for 10 s, 60 °C annealing extension for 30 s, 40 cycles. GAPDH was used as the internal control. －ΔΔCt The relative expression of relevant genes was calculated using a statistical method, and the results were analyzed. Figure 4 As shown, where, Figure 4 A represents the effect of *Tea dwarf tea* exovesicles on the expression of the inflammatory factor IL-6. Figure 4 B represents the effect of AJEVs on TNF-α expression. Compared with the model group, AJEVs downregulated the expression of inflammatory factors IL-6 and TNF-α (P<0.05).

[0074] Table 2 Primer sequences for PCR amplification

[0075]

[0076] The results showed that the exovesicles of *Tea gloriosa* could be efficiently taken up by RAW264.7 cells. At the same concentration of *Tea gloriosa* extract (30 μg / mL), the cell viability of the exovesicle group was 141.4%, the crude exovesicle group was 91.3%, and the catechin group was 87.2%. The exovesicles of *Tea gloriosa* showed the most significant anti-LPS inflammatory effect and the most significant inhibitory effects on cell damage and the expression of inflammatory cytokines.

[0077] Example 3

[0078] Sequencing and enrichment analysis of extravesicular miRNAs from *Tea dwarf tea*

[0079] 1. Extraction of vesicles from *Tea glomerata*: Same as above.

[0080] 2. RNA extraction and quality testing:

[0081] RNA was extracted from the exosomes of *Tea glomerata* using an exosome RNA purification kit (Hangzhou Xinjing Bioreactor Development Co., Ltd., Cat. No. 5202050, batch number: 20210410). 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.

[0082] 3. Library Construction:

[0083] 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.

[0084] 4. Sequencing:

[0085] The constructed library was subjected to high-throughput sequencing using the Illumina platform to obtain a large amount of miRNA sequence data.

[0086] 5. Data Analysis:

[0087] The raw sequencing data underwent quality control to remove low-quality sequences and adapter sequences, and was then compared with a plant reference genome or miRNA database to identify known miRNAs and predict new miRNAs. Results are as follows: Figure 5 As shown in A, a total of 455 miRNA sequences were detected, among which the miRNAs (PC-5p-570173_4, PC-3p-81257_21, PC-5p-1255157_3) were novel miRNAs (Table 3).

[0088] Table 3 miRNA sequences

[0089]

[0090] 6. KEGG enrichment analysis of novel miRNAs from the outer vesicles of *Tea dwarf tea*

[0091] (1) miRNA target gene prediction: GSTAr (v1.0) was used to predict the target genes of new miRNAs. GSTAr used RNAplex software to predict the complementary pairing relationship between miRNAs and target genes, and the minimum free energy was calculated based on the thermodynamic structure to predict the optimal base pairing relationship.

[0092] (2) KEGG enrichment analysis: KEGG pathway enrichment analysis was performed on the predicted miRNA target genes, and a total of 122 signaling pathways were enriched. The top 20 signaling pathways were visualized. Figure 5 (B) These enrichment pathways indicate that the newly discovered miRNAs in the extravesicles of *Tea glomerata* exert their effects primarily through multiple metabolic pathways and signal transduction mechanisms. Among these, plant hormone signal transduction and the phosphatidylinositol signaling system play important roles in regulating inflammatory responses; while inositol phosphate metabolism and glycosaminoglycan degradation regulate inflammation and tissue repair by affecting cell signal transduction and extracellular matrix stability.

[0093] Example 4

[0094] miRNA (PC-5p-570173_4, PC-3p-81257_21, PC-5p-1255157_3) inhibitors and mimics in an LPS-induced mouse mononuclear macrophage leukemia cell (RAW264.7) inflammatory damage model.

[0095] This invention, through cell experiments, discovered that the extravesicular miRNA of *Tea dwarf tea* (PC-5p-570173_4) has a good anti-LPS-induced damage effect on mouse mononuclear macrophage leukemia cells (RAW264.7). The verification process is as follows:

[0096] 1. Cell viability assay: Effect of miRNA (PC-5p-570173_4, PC-3p-81257_21, PC-5p-1255157_3) transfection assay on RAW264.7 cell viability

[0097] (1) The mimics and inhibitors of miRNAs PC-5p-570173_4, PC-3p-81257_21, and PC-5p-1255157_3 were synthesized at Sangon Biotech (Shanghai) Co., Ltd. The sequences are shown in Table 4.

[0098] Table 4. Sequences of miRNA mimics and inhibitors

[0099]

[0100] 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.

[0101] (2) According to 4×10 4 RAW264.7 cells were seeded into 96-well plates at 100 μl / mL. After the cells adhered, RAW264.7 cells were transfected with miRNA Inhibitor and miRNA mimics, respectively. Four hours later, RAW264.7 cells were treated with 1 μg / mL LPS.

[0102] (3) CCK8 assay: After LPS intervention of RAW264.7 cells for 24 h, the supernatant was discarded, and 100 μl of CCK8 working solution (CCK8 reagent: basal culture medium = 1:10) 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 6 As shown, where, Figure 6 A represents the effect of various miRNA mimics on LPS-induced RAW264.7 cell viability. Figure 6 B represents the effect of the inhibitor and mimics of miRNA PC-5p-570173_4 on LPS-induced RAW264.7 cell activity. Compared with the model group, miRNA PC-5p-570173_4 showed the most significant anti-LPS inflammatory damage effect.

[0103] 2. RT-qPCR experiment

[0104] (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;

[0105] (2) RNA concentration measurement: Take 1 µL of RNA and measure the RNA concentration using a spectrophotometer;

[0106] (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);

[0107] (4) PCR was performed on 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. Figure 7 As shown, where, Figure 7 A represents the effect of mimics and inhibitors of various miRNAs on LPS-induced inflammatory factor IL-6 in RAW264.7 cells. Figure 7 B represents the effect of mimics and inhibitors of various miRNAs on LPS-induced inflammatory factor TNF-α in RAW264.7 cells. Compared with the control group and miRNAs (PC-3p-81257_21, PC-5p-1255157_3), miRNA (PC-5p-570173_4) showed better inhibition of damage and inflammatory cytokine expression.

[0108] The results showed that the extravesicular miRNA of *Tea dwarf tea* (PC-5p-570173_4) had the effects of inhibiting cell damage and inhibiting the expression of inflammatory cytokines.

[0109] 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. A miRNA, characterized in that, Its sequence is shown in SEQ ID NO.

5.

2. The application of the miRNA according to claim 1 in the preparation of anti-inflammatory agents.

3. The application of a type of *Tea dwarf tea* exovesicle in the preparation of anti-inflammatory drugs, characterized in that, The vesicles of *Tea dwarf tea* comprise the miRNA described in claim 1; The preparation method of the dwarf tea outer vesicle includes the following steps: Fresh dwarf tea slices were juiced and the juice was collected. The juice was then centrifuged sequentially at 200g-300g for 20-30 min, 3000g-3500g for 30-40 min, and 5000g-6000g for 50-60 min. The supernatant from the third centrifugation was collected and centrifuged at 120000g-140000g for 50-60 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 150000g-160000g for 50-60 min. The solution containing bands between 30% and 45% was collected and centrifuged again at 150000g-160000g for 50-60 min to obtain the dwarf tea exovesicles.

4. The application according to claim 3, 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.

5. The application according to claim 3, 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.

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

7. The application according to any one of claims 3-6, characterized in that, The diameter of the external vesicles of the dwarf tea is 30-150 nm.

Images