Extraction of salvia miltiorrhiza miRNA and application in anti-monocyte inflammatory activation

Abstract

The application discloses extraction of a Danshen miRNA and application of the Danshen miRNA in resisting monocyte inflammatory activation, and the Danshen miRNA is miR2603, and the nucleotide sequence is shown as SEQ ID NO.1. The miR2603 in the Danshen is used to target a human TRAF2 gene, the latter is a key adapter protein of a MAPK signal path, mediates inflammatory signals downstream of receptors such as TNFR and TLR, and is closely related to inflammatory activation of monocyte macrophages, and plays an important role in promoting atherosclerosis.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically the extraction of a miRNA from Danshen and its application in combating inflammatory activation of mononuclear cells. Background Technology

[0002] Danshen (Salvia miltiorrhiza) is a traditional Chinese medicine that promotes blood circulation and removes blood stasis. It has multiple effects, including anti-platelet aggregation, potent anti-inflammatory effects, improvement of local microcirculation, and reduction of vascular endothelial damage. Current research on its pharmacological mechanism is mostly limited to one or a few active ingredients, and focuses primarily on the study of secondary metabolites, neglecting the activity of primary metabolites such as nucleic acids and proteins. This hinders a comprehensive understanding of the pharmacological mechanism of traditional Chinese medicine and limits the precise clinical application of Danshen.

[0003] Plant miRNAs possess 2'-O-methylated 3' ends, high cytosine and guanine (GC) content, and exosome packaging characteristics, exhibiting high stability even in harsh environments. Zhang et al.'s research was the first to demonstrate that rice plant-derived miR168a affects cholesterol transport in mice by inhibiting the expression of low-density lipoprotein receptor adaptor proteins. This reveals the mystery of how plant-derived miRNAs can cross species boundaries to enter the human body and stably exist in the digestive and circulatory systems, thereby exerting their effects in different target organs. This opens new directions for in-depth exploration of the pharmacological mechanisms of traditional Chinese medicine. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this application provides an extraction method for *Salvia miltiorrhiza* miRNA and its application in combating monocyte inflammatory activation. It utilizes *mtr-MIR2603-p3_2ss9AC17AC* (hereinafter referred to as miR2603) found in *Salvia miltiorrhiza* to target the human TRAF2 gene. TRAF2 is a... It is a key linker protein in the MAPK signaling pathway and is closely related to the inflammatory signaling downstream of receptors such as TNFR and TLR and the inflammatory activation of monocytes and macrophages.

[0005] Specifically, the technical solution is as follows: This application discloses a tanshinone miRNA, wherein the tanshinone miRNA is miR2603, and its nucleotide sequence is shown in SEQ ID NO.1.

[0006] This application also discloses the application of the above-mentioned Tanshinone miRNA in the preparation of anti-atherosclerotic drugs.

[0007] Furthermore, the target gene of miR2603 is the human TRAF2 gene, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] This application also discloses a method for extracting the above-mentioned miRNA from Tanshinone, comprising the following steps: S1: Take the Salvia miltiorrhiza herb, wash it, soak it in purified water, then decoct it, filter the decoction, and collect the filtrate. S2: Take the filtrate, add the extraction reagent, mix well, let stand, then centrifuge to take the supernatant, add chloroform to the supernatant, centrifuge again to take the supernatant, add isopropanol to the supernatant, centrifuge to obtain a yellow-brown precipitate, and wash the yellow-brown precipitate with ethanol. S3: The washed yellowish-brown precipitate was vacuum dried, then nuclease-free water was added, and after standing, tanshinone miRNA was obtained.

[0009] Further, the specific method of step S1 is as follows: take the Salvia miltiorrhiza medicinal material, wash it, add 8 times the amount of purified water to the Salvia miltiorrhiza medicinal material, soak for 20-40 minutes, decoct at 100℃ for 30 minutes to obtain the medicinal liquid, filter the medicinal liquid, add 8 times the amount of water to the residue, decoct at 100℃ for 30 minutes, filter, and combine the two filtrates.

[0010] Furthermore, in step S2, the specific method for taking the filtrate, adding the extraction reagent, mixing evenly, letting it stand, and then centrifuging to obtain the supernatant is as follows: take the filtrate, add Trizol lysis buffer, mix evenly, let it stand at room temperature for 4-7 minutes, and then centrifuge at 4°C and 12000 rpm for 5 minutes. The volume ratio of the filtrate to the Trizol lysis buffer is 1:2.

[0011] Furthermore, in step S2, the specific method for adding chloroform to the supernatant and centrifuging again to obtain the supernatant is as follows: take the supernatant and transfer it to a centrifuge tube containing 200 μL of chloroform in advance, shake to mix, let it stand at room temperature for 5 minutes, and then centrifuge at 4°C and 12000 rpm for 15 minutes.

[0012] Furthermore, in step S2, the specific method for adding isopropanol to the supernatant and centrifuging to obtain a yellowish-brown precipitate is as follows: add an equal volume of pre-cooled isopropanol to the supernatant, gently invert and mix, let stand at room temperature for 4-7 minutes, and then centrifuge at 4°C and 12000 rpm for 10 minutes to obtain a yellowish-brown precipitate.

[0013] Furthermore, in step S2, the specific method for washing the yellowish-brown precipitate with ethanol is as follows: rinse the yellowish-brown precipitate twice with 75% ethanol.

[0014] This application also discloses a pharmaceutical composition comprising the above-mentioned tanshinone miRNA.

[0015] The extraction of miRNA from Danshen and its application in combating inflammatory activation of monocytes described in this application, through analysis of the components extracted from Danshen, reveals the main nucleic acids involved in its anti-atherosclerotic effects. Specifically, miR2603 targets the human TRAF2 gene, mediating inflammatory signals downstream of receptors such as TNFR and TLR, and is closely related to the inflammatory activation of monocytes and macrophages, playing an important role in promoting atherosclerosis, thus providing theoretical support for the development of related drugs in the future. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a flowchart illustrating the steps of this application; Figure 2 This is a sequencing quality diagram of the miRNAs from Danshen in this application; Figure 3 This is a sequencing length diagram of the miRNAs from *Taishenia sambac* in this application; Figure 4 This is a diagram showing the GO analysis of the target genes of the miRNAs in Tanshinone in the human body in this application; Figure 5 This is a KEGG analysis diagram of the target genes of the miRNAs in Tanshinone in this application. Figure 6 This is a schematic diagram of the target human TRAF2 gene by Tanshinone miR2603 in this application; Figure 7 This is a diagram showing the results of the dual-luciferase assay using miR2603 in Tanshinone in this application. Figure 8 This is a diagram showing the experimental results of the presence of Tanshinone miRNA in rat plasma in this application; Figure 9 This is a diagram showing the results of the inhibition of the expression of the monocyte-macrophage target gene TRAF2 by Tanshinone miR2603 in this application; Figure 10 This is a graph showing the results of the inhibition of inflammatory molecular protein levels in monocytes and macrophages by Tanshinone miR2603 in this application; Figure 11 This is a graph showing the results of the inhibition of inflammatory molecule mRNA levels in monocytes and macrophages by Tanshinone miR2603 in this application; Figure 12 This is a graph showing the results of flow cytometry analysis of the regulation of mononuclear cell heterogeneity in AS patients by Danshen in this application. Detailed Implementation

[0018] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments.

[0019] Various embodiments of this application will be described more fully below. This application may have various embodiments, and adjustments and changes may be made therein. However, it should be understood that there is no intention to limit the various embodiments of this application to the specific embodiments disclosed herein, but rather this application should be understood to cover all modifications, equivalents, and / or alternatives falling within the spirit and scope of the various embodiments of this application.

[0020] In the following, the terms “comprising” or “may include” as used in the various embodiments of this application indicate the presence of the disclosed functions, operations, or elements, and do not limit the addition of one or more functions, operations, or elements. Furthermore, as used in the various embodiments of this application, the terms “comprising,” “having,” and their cognates are intended only to indicate a particular feature, number, step, operation, element, component, or combination of the foregoing, and should not be construed as primarily excluding the presence of one or more other features, numbers, steps, operations, elements, components, or combinations of the foregoing, or the possibility of adding one or more combinations of features, numbers, steps, operations, elements, components, or combinations of the foregoing.

[0021] In various embodiments of this application, the expression "or" or "at least one of A and / or B" includes any combination or all combinations of the words listed simultaneously. For example, the expression "A or B" or "at least one of A and / or B" may include A, may include B, or may include both A and B.

[0022] To better understand the technical solution of this application, the following describes the application through specific implementation methods.

[0023] 1. Extraction of miRNA from Tanshinone Please see Figure 1 This application discloses a method for extracting miRNA from Tanshinone, comprising the following steps: S1: Take the Salvia miltiorrhiza herb, wash it, and soak it in 8 times the amount of purified water for 20-40 minutes. Then decoct it at 100℃ for 30 minutes. Filter the decoction, add 8 times the amount of water to the dregs, decoct it at 100℃ for 30 minutes, filter it again, and combine the two filtrates.

[0024] S2: Mix the filtrate and extraction reagent at a volume ratio of 1:2, let stand for 4-7 minutes, then centrifuge at 12000 rpm for 5 minutes at 4℃. Remove the sample, carefully aspirate the supernatant and transfer it to a centrifuge tube pre-filled with chloroform. Shake to mix, let stand at room temperature for 5 minutes, then centrifuge again at 12000 rpm for 15 minutes at 4℃. Aspirate the supernatant and add an equal volume of isopropanol. Gently invert to mix, let stand at room temperature for 4-7 minutes, then centrifuge again at 12000 rpm for 10 minutes at 4℃ to obtain a yellowish-brown precipitate. Finally, rinse the yellowish-brown precipitate twice with 75% ethanol to remove impurities and isopropanol from the precipitate surface.

[0025] S3: After vacuum drying the washed, yellowish-brown precipitate, add nuclease-free water and let it stand for a period of time to obtain tanshinone miRNA.

[0026] 1.1 Take 100g of Salvia miltiorrhiza, wash it clean, add 800g of purified water to completely submerge the Salvia miltiorrhiza, soak for 30 minutes, then decoct at 100℃ for 30 minutes, filter the decoction, add 800g of water to the dregs, decoct at 100℃ for 30 minutes, filter again, and combine the two filtrates.

[0027] Add 1 mL of Trizol lysis buffer to 500 μL of the filtrate, mix well, and incubate at room temperature for 5 minutes. Then, centrifuge at 12,000 rpm for 5 minutes at 4°C. Remove the sample, carefully aspirate the supernatant, and transfer it to a 1.5 mL centrifuge tube containing 200 μL of chloroform. Vortex to mix, incubate at room temperature for 5 minutes, and then centrifuge at 12,000 rpm for 15 minutes at 4°C. Carefully aspirate approximately 350 μL of the supernatant, add 350 μL of pre-chilled isopropanol to the supernatant, gently invert to mix, and incubate at room temperature for 4 minutes. Avoid aspirating the mesophase when aspirating the supernatant. Then, centrifuge at 12,000 rpm for 10 minutes at 4°C. A yellowish-brown, gelatinous, transparent precipitate will be visible. Discard the supernatant, carefully rinse the bottom of the yellowish-brown gelatinous transparent precipitate with 500 μL of 75% ethanol, continue centrifuging at 7500 rpm for 5 minutes at 4°C, discard the supernatant, repeat the rinsing once with 500 μL of 75% ethanol, centrifuge briefly, and discard the supernatant.

[0028] The precipitate was vacuum dried for 5 minutes, and 20 μL of nuclease-free water was added. The mixture was then incubated for 10 minutes. Electrophoresis was performed on a 0.8% agarose gel at 160V for 10 minutes, which showed that a large number of small RNA fragments were present in the filtrate.

[0029] 1.2 Take 80g of Salvia miltiorrhiza, wash it clean, add 640g of purified water to completely submerge the Salvia miltiorrhiza, soak for 30 minutes, then decoct at 100℃ for 30 minutes, filter the decoction, add 640g of water to the dregs, decoct at 100℃ for 30 minutes, filter again, and combine the two filtrates.

[0030] Add 0.8 mL of Trizol lysis buffer to 400 μL of filtrate, mix well, and incubate at room temperature for 4 minutes. Then centrifuge at 12,000 rpm for 5 minutes at 4°C. Remove the sample, carefully aspirate the supernatant, and transfer it to a 1.5 mL centrifuge tube pre-filled with 160 μL of chloroform. Vortex to mix, incubate at room temperature for 5 minutes, and centrifuge at 12,000 rpm for 15 minutes at 4°C. Carefully aspirate approximately 280 μL of the supernatant, add an equal volume of pre-chilled isopropanol, gently invert to mix, and incubate at room temperature for 5 minutes. Avoid aspirating the mesophase when aspirating the supernatant. Then centrifuge at 12,000 rpm for 10 minutes at 4°C. A yellowish-brown, gelatinous, transparent precipitate will be visible. Discard the supernatant. Carefully rinse the bottom of the yellowish-brown, gelatinous, transparent precipitate with 500 μL of 75% ethanol. Continue centrifuging at 7500 rpm for 5 minutes at 4°C. Discard the supernatant, repeat the 75% ethanol rinsing once, centrifuge briefly, and discard the supernatant.

[0031] The precipitate was vacuum dried for 5 minutes, and 20 μL of nuclease-free water was added. The mixture was then incubated for 10 minutes. Electrophoresis was performed on a 0.8% agarose gel at 160V for 10 minutes, which showed that a large number of small RNA fragments were present in the filtrate.

[0032] 1.3 Take 150g of Salvia miltiorrhiza, wash it clean, add 1200g of purified water to completely submerge the Salvia miltiorrhiza, soak for 40 minutes, then decoct at 100℃ for 30 minutes, filter the decoction, add 1200g of water to the dregs, decoct at 100℃ for 30 minutes, filter again, and combine the two filtrates.

[0033] Add 750 μL of filtrate to 1.5 mL of Trizol lysis buffer, mix well, and incubate at room temperature for 7 minutes. Then, centrifuge at 12,000 rpm for 5 minutes at 4°C. Remove the sample, carefully aspirate the supernatant, and transfer it to a 1.5 mL centrifuge tube pre-filled with 300 μL of chloroform. Vortex to mix, incubate at room temperature for 5 minutes, and then centrifuge at 12,000 rpm for 15 minutes at 4°C. Carefully aspirate approximately 530 μL of the supernatant, add an equal volume of pre-chilled isopropanol, gently invert to mix, and incubate at room temperature for 7 minutes. Avoid aspirating the mesophase when aspirating the supernatant. Then, centrifuge at 12,000 rpm for 10 minutes at 4°C. A yellowish-brown, gelatinous, transparent precipitate will be visible. Discard the supernatant. Carefully rinse the bottom of the yellowish-brown, gelatinous, transparent precipitate with 500 μL of 75% ethanol. Continue centrifuging at 7500 rpm for 5 minutes at 4°C. Discard the supernatant, repeat the 75% ethanol rinsing once, centrifuge briefly, and discard the supernatant.

[0034] The precipitate was vacuum dried for 5 minutes, and 20 μL of nuclease-free water was added. The mixture was then incubated for 10 minutes. Electrophoresis was performed on a 0.8% agarose gel at 160V for 10 minutes, which showed that a large number of small RNA fragments were present in the filtrate.

[0035] 2. Danshen miRNA sequencing Small RNA sequencing libraries were prepared using the TruSeq Small RNA Sample Prep Kits. RNA extracted from the decoction of *Salvia miltiorrhiza* was then sequenced. The sequencing quality profile showed that the quality of the full-read bases was consistently above Q24, with the first 48 bp remaining in the high-quality range of Q34-Q37, indicating excellent data quality (e.g., ...). Figure 2 The length distribution plot shows that the unique sequences in the Salvia miltiorrhiza decoction samples are mainly concentrated in the 18-25 nt range, with the highest proportion at 22 nt, which is consistent with the typical miRNA length characteristics. The overall data can meet the requirements for subsequent miRNA omics analysis (e.g., Figure 3 A total of 333 miRNAs were detected in the total RNA extracted from the decoction of Salvia miltiorrhiza, of which 85 were highly expressed and 10 had more than 1,000 reads.

[0036] 3. Prediction of human gene targeting by Tanshinone miRNA The psRNATarget tool was used to predict the human target genes of miRNAs from Salvia miltiorrhiza, and GO and KEGG annotation analyses were performed on the target genes. The GO functional enrichment results showed that ( Figure 4 At the biological process level, target genes are mainly enriched in signal transduction, transcriptional regulation, and cell differentiation; suggesting that target genes are widely involved in core biological processes such as cell signal transduction and transcriptional regulation. KEGG pathway enrichment analysis results indicate that ( Figure 5 The target genes were significantly enriched in the PI3K-Akt signaling pathway, Ras signaling pathway, and cancer-related pathways, suggesting that these pathways may be key regulatory pathways for the biological effects mediated by the decoction of Danshen.

[0037] Among 85 highly expressed miRNAs, a newly discovered miR2603-p3_2ss9AC17AC (hereinafter referred to as miR2603), specific to *Salvia miltiorrhiza*, was identified. Its gene sequence is 5'-GUCCCUGCCCCUUUGUACA-3', as shown in SEQ ID NO.1. Based on predictions from TargetScan and miRanda bioinformatics software, nucleotides 86-104 of the 3'UTR of the human target gene TRAF2 (NM_021138) (UGUACAAGUGGGCAGGGGC, as shown in SEQ ID NO.2) contain a potential binding site for miR2603. Figure 6 ).

[0038] 4. miR2603 luciferase activity To verify the direct targeting effect of miR2603 on human TRAF2, recombinant plasmids pGL3-TRAF2-3'UTR-WT and pGL3-TRAF2-3'UTR-Mut were constructed based on the predicted binding sites. Simultaneously, miR2603 mimic or a negative control (NC mimic) was synthesized. The above plasmids and miR2603 mimic / NC mimic were co-transfected into 293T cells using Lipofectamine 2000 reagent, along with the René luciferase internal control plasmid (pRL-TK) to standardize transfection efficiency. Forty-eight hours after transfection, luciferase activity was measured according to the kit instructions. The results showed (…). Figure 7 293T cells were co-transfected with TRAF2-WT+NC mimic:pGL3-TRAF2-3'UTR-WT and NC mimic; TRAF2-WT+miR2603 mimic:pGL3-TRAF2-3'UTR-WT and miR2603 mimic; TRAF2-Mut+NC mimic:pGL3-TRAF2-3'UTR-Mut and NC mimic; and TRAF2-Mut+miR2603 mimic:pGL3-TRAF2-3'UTR-Mut and miR2603 mimic. Compared with NC mimic, the relative luciferase activity in 293T cells co-transfected with miR2603 mimic and pGL3-TRAF2-3'UTR-WT was significantly reduced, and the difference was highly statistically significant (P<0.001). The relative luciferase activity in cells co-transfected with miR2603 mimic and pGL3-TRAF2-3'UTR-Mut showed no significant difference compared to the control group (P>0.05). This result demonstrates that this site is a direct target for the interaction between miRNA2603 and TRAF2 mRNA.

[0039] 4.1 The sequence of TRAF2-3'UTR-WT (5'→3') (SEQ ID NO.3): Fragment length: 300 bp Enzyme restriction sites: 5' end XhoI (CTCGAG), 3' end NotI (GCGGCCGC), with protective bases added at both ends. Bold text: miR2603 bonding region CCGCTCGAGCTGCCCCCTACTGGTGTCTGGGGGTTGGGGGCAGCCAGGCACAGCCGGCTCACGGAGGGGCCACCACGCTGGGCCAGGGTCTCACTGTACAAGTGGGCAGGGGCCGCGCTTGGGCGCTTGGGAGGGTGTCGGCCTGCAGCC AAGTTCACTGTCACGGGGGAAGGAGCCACCAGCCAGTCCTCAGATTTCAGAGACTGCGGAGGGGCTTGGCAGACGGTCTTAGCCAAGGGCTGTGGTGGCATTGGCCGAGGGTCTTCGGGTGCTTCCCAGCACAAGCTGGCGGCCGCTTAA 4.2 Mutant Insertion Retrieval (Mut-UTR) The complementary core sequence “GCAGGGG” in the seed region was mutated to “CGTCCCC”, completely disrupting the miRNA binding ability. The remaining sequences were identical to the wild type.

[0040] 4.3 Both miR2603 and NC mimic sequences (universal negative control) have 2'-O-methylation modification at the 3' end to mimic the characteristics of plant miRNAs.

[0041] miR2603 mimic Justice Chain (SEQ ID NO.4): 5'-GUCCCUGCCCCUUUGUACA-3' miR2603 mimic antisense strand (SEQ ID NO.5): 5'-UGUACAAAGGGGCAGGGAC-3' NC mimic justice chain (SEQ ID NO.6): 5'-UCACAACCUCCUAGAAAGAGU-3' NC mimic antisense strand (SEQ ID NO.7): 5'-ACUCUUUCUAGGAGGUUGUGA-3' 5. miR2603 rat plasma experiment To test whether the Danshen-derived miR2603 could be stably present in rat plasma, 12 6-8 week old SD rats were randomly divided into an experimental group and a control group. The experimental group was administered Danshen decoction by gavage, while the control group was administered 0.9% saline by gavage, 1 mL twice daily. During this period, blood was collected from the orbital sinus to detect miR2603 expression. After one week of continuous gavage, blood was collected from the abdominal aorta under aseptic conditions. Serum was separated by centrifugation. To remove the influence of the original active components from the parent rat and better reflect the effect of miR2603, the centrifuged serum was inactivated by water bath at 56℃ for 30 minutes to prepare animal Danshen-containing serum. miRNAs were extracted from rat plasma according to the miRNeasy kit (No. 217184, Qiagen) manual, and miR2603 expression was detected using an ABI reverse transcription kit and a TaqMan kit, with endogenous animal miRNA (miR-16) used as an internal control. Results are as follows: Figure 8 As shown, high abundance of miR2603 was found in the plasma of rats in the danshen decoction gavage group, while no danshen-derived miRNA was detected in the plasma of rats in the saline gavage group.

[0042] 6. In vitro assay of miR2603 inhibiting inflammatory molecule expression in monocytes and macrophages Oxidized low-density lipoprotein (oxLDL) is a product of low-density lipoprotein oxidized by free radicals, and it plays a central role in the occurrence and development of atherosclerosis. To verify the effect of miR2603 on atherosclerosis and to verify whether the anti-inflammatory pharmacological effect of the traditional Chinese medicine Danshen (Salvia miltiorrhiza) is related to the miRNAs abundant in its decoction, synthesized miR2603mimic and NC mimic were transfected into THP-1 macrophages via liposomes. The in vitro cultured THP-1 macrophages were divided into 5 groups according to different treatments: conventional culture group (M0), oxLDL treatment group (oxLDL), oxLDL + Danshen-containing serum treatment group (oxLDL+Serum), oxLDL + miR2603 transfection group (oxLDL+miR2603), and oxLDL + NC mimic transfection group (oxLDL+NC).

[0043] Forty-eight hours after transfection, total RNA was extracted from cells and reverse transcribed into cDNA. Real-time quantitative PCR was performed to detect the expression of the TRAF2 gene and inflammatory factors in cells, and flow cytometry was used to detect the protein levels of inflammatory factors. The results showed that serum containing Danshen (Salvia miltiorrhiza) and miR2603 mimic could inhibit oxLDL-induced TRAF2 expression in macrophages. Figure 9M0: conventional culture group; oxLDL: oxLDL treatment group; oxLDL+Serum: oxLDL and Danshen-containing serum treatment group; oxLDL+miR2603: oxLDL treatment and miR2603 mimic transfection group; oxLDL+NC: oxLDL treatment and NC mimic transfection group) and the protein levels of inflammatory marker genes IL-1β and IL-6 ( Figure 10 ) and mRNA levels ( Figure 11 M0: conventional culture group; oxLDL: oxLDL treatment group; oxLDL+miR2603: oxLDL treatment and miR2603 mimic transfection group), inhibiting inflammatory activation of cells.

[0044] 7. Clinical trial of miR2603 for anti-atherosclerosis To verify whether miR2603 is the main anti-inflammatory component of Danshen in its anti-atherosclerotic effects, relevant clinical verification was conducted. According to the "Abdominal and Peripheral Vascular Color Doppler Diagnosis", an intima-media thickness (IMT) between 1.0 mm and 1.4 mm was defined as carotid intima-media thickening; an IMT ≥ 1.5 mm was defined as atherosclerotic plaque formation.

[0045] Inclusion criteria: Patients with carotid intima-media thickening of IMT ≥ 1.0 mm.

[0046] Exclusion criteria: Patients with carotid artery stenosis rate >70% or those with recent severe liver or kidney dysfunction, severe heart failure, infectious diseases, tumors, epilepsy, or under stress.

[0047] Sixty-two patients meeting the inclusion and exclusion criteria were selected from the hospital. One patient with carotid artery stenosis >70%, one patient with severe liver and kidney dysfunction, one patient with severe heart failure, three patients with cancer, one patient with epilepsy, and five patients under stress were excluded, for a total of 12 excluded patients. Patients were divided into a control group and an experimental group based on their clinical medication use. The control group received traditional statin therapy; the experimental group received a combination of Danshen (Salvia miltiorrhiza) and statins. Clinical data and blood samples were collected before and after treatment. Flow cytometry was used to detect peripheral blood mononuclear cell heterogeneity, and ELISA was used to detect the expression levels of serum inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and von Willebrand factor (VWF).

[0048] Table 1. Baseline characteristics of the experimental group and the control group before treatment Among them, SBP: systolic blood pressure; DBP: diastolic blood pressure; TC: total cholesterol; TG: triglycerides; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol; GLU: blood glucose; HbA1c: glycated hemoglobin; HCY: high homocysteine; UA: uric acid; BUN: blood urea nitrogen; MHR: monocyte-to-HDL cholesterol ratio; NHR: neutrophil-to-HDL cholesterol ratio; PLR: platelet-to-lymphocyte ratio; NLP: neutrophil-to-lymphocyte ratio; CPS: carotid plaque score.

[0049] Table 2. Baseline characteristics after treatment between the Danshen formula combined with statin group and the statin-only group. The results showed that, compared with the control group, the experimental group had a lower proportion of patients with high-risk plaques (carotid plaque burden score >3), better lipid control, and lower serum inflammatory factor levels (see Tables 1 and 2); flow cytometry analysis showed ( Figure 12 (Monocytes: mononuclear cells; Classical: classical mononuclear cells; Intermediate: intermediate mononuclear cells; Nonclassical: nonclassical mononuclear cells) The proportion of classical and intermediate mononuclear cells in the peripheral blood of AS patients in the Danshen combined treatment group was significantly lower than that in the statin treatment group, suggesting that Danshen may regulate monocyte heterogeneity, inhibit inflammatory response, and alleviate the progression of atherosclerosis, providing additional benefits to patients on top of statin treatment.

[0050] In summary, the miR2603 derived from the decoction of Danshen may be the main active ingredient in Danshen, which promotes the stability of AS plaques by inhibiting the expression of inflammatory molecules in monocytes and regulating monocyte heterogeneity.

[0051] The embodiments of this application have been described above with reference to the accompanying drawings. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims. All of these forms are within the protection scope of this application.

Claims

1. A tanshinone miRNA, characterized in that, The miRNA described is miR2603, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The application of the tanshinone miRNA according to claim 1 in the preparation of anti-atherosclerotic drugs.

3. The application of the tanshinone miRNA according to claim 2 in the preparation of anti-atherosclerotic drugs, characterized in that, The target gene of miR2603 is the human TRAF2 gene, the nucleotide sequence of which is shown in SEQ ID NO.

2.

4. The method for extracting miRNA from *Salvia miltiorrhiza* according to claim 1, characterized in that, Includes the following steps: S1: Take the Salvia miltiorrhiza herb, wash it, soak it in purified water, then decoct it, filter the decoction, and collect the filtrate. S2: Take the filtrate, add the extraction reagent, mix well, let stand, then centrifuge to take the supernatant, add chloroform to the supernatant, centrifuge again to take the supernatant, add isopropanol to the supernatant, centrifuge to obtain a yellow-brown precipitate, and wash the yellow-brown precipitate with ethanol. S3: The washed yellowish-brown precipitate was vacuum dried, then nuclease-free water was added, and after standing, tanshinone miRNA was obtained.

5. The method for extracting miRNA from *Salvia miltiorrhiza* according to claim 3, characterized in that, The specific method of step S1 is as follows: Take the Salvia miltiorrhiza medicinal material, wash it, add 8 times the amount of purified water to the Salvia miltiorrhiza medicinal material, soak for 20-40 minutes, decoct at 100℃ for 30 minutes to obtain the medicinal liquid, filter the medicinal liquid, add 8 times the amount of water to the residue, decoct at 100℃ for 30 minutes, filter, and combine the two filtrates.

6. The method for extracting miRNA from *Salvia miltiorrhiza* according to claim 3, characterized in that, In step S2, the specific method for taking the filtrate, adding the extraction reagent, mixing evenly, letting it stand, and then centrifuging to obtain the supernatant is as follows: take the filtrate, add Trizol lysis buffer, mix evenly, let it stand at room temperature for 4-7 minutes, and then centrifuge at 4°C and 12000 rpm for 5 minutes. The volume ratio of the filtrate to the Trizol lysis buffer is 1:

2.

7. The method for extracting miRNA from *Salvia miltiorrhiza* according to claim 3, characterized in that, In step S2, the specific method for adding chloroform to the supernatant and centrifuging again to obtain the supernatant is as follows: take the supernatant and transfer it to a centrifuge tube containing 200 μL of chloroform, shake to mix, let it stand at room temperature for 5 minutes, and then centrifuge at 4°C and 12000 rpm for 15 minutes.

8. The method for extracting miRNA from Tanshinone according to claim 3, characterized in that, In step S2, the specific method for adding isopropanol to the supernatant and centrifuging to obtain a yellowish-brown precipitate is as follows: add an equal volume of pre-cooled isopropanol to the supernatant, gently invert and mix, let stand at room temperature for 4-7 minutes, and then centrifuge at 4°C and 12000 rpm for 10 minutes to obtain a yellowish-brown precipitate.

9. The method for extracting miRNA from Tanshinone according to claim 3, characterized in that, In step S2, the specific method for washing the yellowish-brown precipitate with ethanol is as follows: rinse the yellowish-brown precipitate twice with 75% ethanol.

10. A pharmaceutical composition, characterized in that, Including the tanshinone miRNA as described in claim 1.

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