Application of cyanidin-3-O-glucoside in preparation of medicine for treating diseases caused by tsRNA expression disorder

Abstract

The invention discloses an application of cyanidin-3-O-glucoside in preparation of a medicine for treating diseases caused by tsRNA (tsRNA) expression disorder. It is found through tests that cyanidin-3-O-glucoside can achieve the effect of improving the relative expression quantity disorder of tsRNA. The invention provides a new drug choice for treating diseases caused by tsRNA expression disorder, and also provides a new application of cyanidin-3-O-glucoside.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, relates to pharmaceutical uses of cyanidin-3-O-glucoside, and particularly relates to the use of cyanidin-3-O-glucoside in preparing drugs for treating diseases caused by tsRNA expression disorders. Background Art

[0002] Small non-coding RNA (sncRNA) is a small non-coding RNA transcribed from DNA but not translated into protein, and plays an important role in cells and organisms. SncRNA includes tRNA-derived small RNA (tsRNA), microRNA, piwi-interacting RNA, small nucleolar RNA and rRNA-derived small RNA.

[0003] The occurrence of metabolic diseases, nervous system diseases, cancer and other diseases is related to the disorder of tsRNA expression. Tyr and tsRNA Arg The reduction of is potentially related to the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and affects vascular cognitive impairment. Paternal insulin resistance induced by a high-fat diet can cause lipid metabolism disorders in offspring, and abnormally expressed tsRNA is inherited to offspring through the fertilization process, inducing metabolic diseases in offspring. Epigallocatechin gallate regulates the ferroptosis pathway by downregulating tsRNA-13502 to treat non-small cell lung cancer.

[0004] Cyanidin-3-O-glucoside (C3G) is the most easily accessible and most abundant anthocyanin in nature. It has a typical skeleton of flavonoid compounds and is a polyhydroxyl and methoxy derivative of 2-phenylbenzopyran structure or flavonoid salt. Anthocyanins are widely distributed in dark fruits, vegetables and grains such as purple grapes, blueberries, purple corns and black beans. Black bean husk extracts rich in C3G can lower blood sugar, relieve liver damage, improve blood lipid levels and reduce obesity-induced inflammation by mediating specific microRNA and non-coding long RNA expression profiles. C3G has a good regulatory effect on metabolic disorders caused by a high-fat and high-fructose diet, but its effect on tsRNA has not been reported. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides the use of cyanidin-3-O-glucoside in the preparation of a drug for treating diseases caused by tsRNA expression disorder.

[0006] The first object of the present invention is to provide the use of cyanidin-3-O-glucoside in the preparation of drugs for treating diseases caused by tsRNA expression disorders.

[0007] According to further features of the use of the present invention, the disease caused by disordered tsRNA expression is a metabolic disease, a nervous system disease, or a cancer caused by abnormal relative expression of tsRNA.

[0008] Preferably, the abnormal relative expression level of tsRNA is an increased relative expression level of tsRNA.

[0009] The second object of the present invention is to provide a drug for treating diseases caused by disordered tsRNA expression.

[0010] The drug contains cyanidin-3-O-glucoside (C3G).

[0011] Preferably, the medicament further contains a pharmaceutically acceptable excipient.

[0012] More preferably, the drug can be prepared into different dosage forms, such as tablets, powders, pills, capsules, granules, drop pills, oral liquids, etc.

[0013] The present invention uses an appropriate dose of cyanidin-3-O-glucoside to study the effect of disordered tsRNA expression in mouse sperm, and uses sncRNA microarray technology to analyze the relative expression changes of tsRNA. The present invention found that C3G intervention significantly improved 10 tsRNAs with upregulated relative expression, indicating that cyanidin-3-O-glucoside has the potential to improve tsRNA expression disorder.

[0014] The experimental results of the present invention show that cyanidin-3-O-glucoside significantly down-regulated the abnormal increase in the relative expression levels of 10 tsRNAs, and has the function of improving the abnormal expression of tsRNA, thereby providing a new drug option for treating diseases caused by tsRNA expression disorders and broadening the application of cyanidin-3-O-glucoside. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the animal experiment design in Examples 1 and 2.

[0016] Figure 2 This is the composition of the mouse sperm non-coding small RNA in Example 1.

[0017] Figure 3It is the differential fold change of sncRNA and tsRNA by C3G intervention in Example 1. Among them, A is the differential fold change of different types of sncRNA in the Model-vs-Control group, B is the differential fold change of different types of sncRNA in the C3G-vs-Model group, C is the differential fold change of different types of sncRNA in the Met-vs-Model group, D is the differential fold change of different types of tsRNA in the Model-vs-Control group, E is the differential fold change of different types of tsRNA in the C3G-vs-Model group, and F is the differential fold change of different types of tsRNA in the Met-vs-Model group.

[0018] Figure 4 The number of sncRNA and tsRNA significantly changed under the intervention of HFHF diet and C3G in Example 2. Wherein, A is the number of sncRNA significantly changed under the intervention of HFHF diet and C3G, and B is the number of tsRNA significantly changed under the intervention of HFHF diet and C3G.

[0019] Figure 5 The changes in tsRNA content, types and multiple differences under HFHF diet and C3G intervention in Example 2. Among them, A is the log10 logarithm of the relative expression of tsRNA in the Model group, B is the log2 logarithm of the multiple difference in the Model-vs-Control group, C is the log2 logarithm of the multiple difference in the C3G-vs-Model group, D is the log2 logarithm of the multiple difference in the Met-vs-Model group, and E is the percentage of isotopic decoding tsRNA types.

[0020] Figure 6 This is the cluster heat map of high-fat and high-fructose sensitive tsRNA in Example 2.

[0021] Figure 7 This is a dendrogram of the tsRNAs that changed significantly in Example 2.

[0022] Figure 8 This is the tsRNA sequence that was significantly changed under the intervention of C3G and Met in Example 2. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0024] Unless otherwise specified, the reagents and materials used in the following examples were purchased from commercial channels.

[0025] 1. Preparation and purification of C3G

[0026] Cyanidin-3-O-glucoside (C3G) is extracted and purified from black bean husk and mulberry resources. The raw materials can be black bean husk and mulberry dried fruit (References: [1] Li Xusheng, Jiang Xinwei, Shan Xiaoyu, et al. Medium pressure liquid chromatography preparation and structural identification of black bean husk anthocyanins [J]. Food and Fermentation Industries, 2018, 44(06): 51-57. DOI: 10.13995 / j.cnki.11-1802 / ts.015859.; [2] Ran Guojing, Jiang Xinwei, Li Haoyi, et al. Rapid preparation of cyanidin-3-glucoside monomer from mulberry using medium pressure preparative liquid chromatography [J]. Food Science, 2019, 40(03): 94-100.). The purified C3G has a purity of 97% at 280nm and is used in the following experiments.

[0027] 2. Establishment of a high-fat, high-fructose diet model

[0028] Twenty-eight 6-week-old C57 / BL6J male mice were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. Figure 1 As shown, mice were randomly divided into four groups (n=7) according to the weight stratification method: a normal diet and saline group (control group, Control), a high fat and high fructose (HFHF) diet and saline group (model group, Model), a high fat and high fructose diet and C3G intervention group (C3G), and a high fat and high fructose diet and metformin (Met) intervention group (Met). During the 4-week modeling phase, the Control group received a normal diet (10 kcal% normal feed) and normal drinking water, and the other three groups received a high fat and high fructose diet (10% fructose water and 60 kcal% high fat feed). In the subsequent 8-week intervention phase, the C3G group received C3G (100 mg / kg·bw) gavage intervention, the Met group received Met (200 mg / kg·bw) gavage intervention, and the Control group and the Model group were gavaged with an equal amount of saline.

[0029] 3. Sperm RNA Pretreatment

[0030] After the intervention, the epididymal tails of mice in each group were placed in 1 mL of normal saline and released at 37°C. The sperm suspension was transferred to a new 1.5 mL EP tube and centrifuged at 2665 × g for 3 min at room temperature. The precipitate was washed with 1 mL of dd H2O and centrifuged again. 500 μL of somatic cell lysis buffer was added, the two tubes were combined, resuspended at room temperature, passed through a 40 μm cell sieve, and transferred to a new 1.5 mL EP tube.

[0031] 4. sncRNA Microarray

[0032] The sncRNA microarray was completed by Shanghai Kangcheng Biological Co., Ltd. The specific steps are as follows.

[0033] (1) RNA purification

[0034] Total RNA from mouse sperm was extracted using Trizol reagent. RNA concentration was determined using Nanodrop ND-1000 spectrophotometer, and RNA integrity was determined using Bioanalyzer 2100 and denaturing gel electrophoresis.

[0035] (2) RNA labeling

[0036] ① Dephosphorylation: 100 ng of total RNA was dephosphorylated at 37°C for 40 min with 3 units of T4 polynucleotide kinase to remove (P) and (cP) chemical groups from the 3' end of RNA to form a 3-OH end. The reaction was terminated at 70°C for 5 min and immediately cooled to 0°C.

[0037] ② Denaturation: Add 7 μL DMSO, heat to 100°C for 3 min to denature RNA, and immediately cool to 0°C;

[0038] ③Add labeling: RNA end labeling was performed by adding ligase buffer, BSA, 50 mM pCp-Cy3 and 15 units of T4 RNA ligase to a 28 μL reaction system at 16°C overnight.

[0039] (3) Array hybridization

[0040] Add 22.5 μL of 2× hybridization buffer to the above labeling reaction and mix to obtain 45 μL of the system. Heat at 100°C for 5 min and immediately cool to 0°C. Mix 45 μL of labeled samples on the microarray and hybridize at 55°C for 20 h. Wash the slides in 6× saline sodium citrate buffer (SSC) containing 0.005% Triton X-102 at room temperature for 10 min, and then wash in 0.1× SSC containing 0.005% Triton X-102 for 5 min. The slides were scanned on an Agilent G2505C chip scanner.

[0041] (4) Results analysis

[0042] The acquired array images were analyzed using Agilent Feature Extraction software (version 11.0.1.1). The raw fluorescence intensities were log2 transformed and quantile normalized. After normalization, probe signals with at least 3 present (P) or marginal (M) QC flags in 12 samples were retained. Multiple probes from the same sncRNA were averaged and integrated into one RNA level. The differentially expressed sncRNAs between the two groups were determined by screening the fold difference (Fold change>1.5 or Fold change<0.67) and p value (p<0.05).

[0043] 5. Data processing and result analysis methods

[0044] The pie chart was drawn in Excel, the dendrogram was drawn in Origin 2021, and the cluster heat map was drawn on the Oebio Cloud platform (https: / / cloud.oebiotech.com). The remaining bar graphs, scatter plots, and bar graphs were drawn in GraphPad Prism8.0. The data were compared pairwise using the t-test (Unpaired t-test).

[0045] Example 1: Effect of C3G intervention on sncRNA induced by HFHF diet

[0046] 1. Composition of mouse sperm sncRNA

[0047] Mouse sperm sncRNA microarray detected a total of 6185 non-coding small RNAs, including 1944 miRNAs, 992 precursor miRNAs (pre-miRNAs), 249 tRNAs, 1267 snoRNAs, and 1733 tsRNAs ( Figure 2 ). Among them, miRNA and tsRNA have the highest content, accounting for 31% and 28% respectively. According to the different cleavage sites, tsRNA can be further subdivided into 3'tiRNA, 5'tiRNA, tRF1, 3'tRF and 5'tRF. 5'tiRNA and 5'tRF are the most abundant types of tsRNA, accounting for 9.8% and 9.7% of all sncRNAs respectively.

[0048] 2. Differential fold changes of sncRNA and tsRNA after C3G intervention

[0049] The fold difference is the fold change in expression level between two groups. The pre-miRNA and tsRNA in mouse sperm showed an overall increase in fold difference under the HFHF diet. After C3G intervention, the overall fold difference of tsRNA showed a downward trend ( Figure 3 AC). Most miRNAs and snoRNAs showed a decrease in fold difference under HFHF diet, an increase in fold difference under C3G intervention, and an increase in fold difference under Met intervention ( Figure 3 AC upper pie chart). Compared with the control diet, the HFHF diet resulted in 59% of tsRNA fold differences increased; after C3G intervention, 63% of tsRNA fold differences decreased; after Met intervention, 55% of tsRNA fold differences decreased ( Figure 3 AC pie chart above).

[0050] Compared with the Control group, the HFHF diet increased the overall fold difference of sperm 3'tiRNA and 3'tRF in the Model group mice, and reduced the expression of 5'tRF; compared with the Model group, C3G intervention reduced the overall fold difference of 3'tiRNA and 3'tRF, and Met intervention reduced the overall fold difference of 3'tiRNA and 3'tRF, and increased the fold difference of 5'tRF. The overall fold difference regulation trend of tsRNA by C3G intervention and Met intervention was similar ( Figure 3 DF).

[0051] Example 2: Regulatory Effects of C3G Intervention on tsRNAs Significantly Altered by HFHF Diet

[0052] 1. Regulatory effects of C3G intervention on sncRNAs and tsRNAs that were significantly altered by HFHF diet

[0053] Compared with the control group (Model-vs-Control), HFHF diet led to a significant upregulation of 47 sncRNAs and a significant downregulation of 54 sncRNAs. Compared with the model group (C3G-vs-Model), 31 sncRNAs were significantly upregulated and 42 sncRNAs were significantly downregulated under C3G intervention. Compared with the model group (Met-vs-Model), 75 sncRNAs were significantly upregulated and 120 sncRNAs were significantly downregulated under Met intervention ( Figure 4A). Compared with the control group (Model-vs-Control), the HFHF diet resulted in 17 tsRNAs that were significantly upregulated and 15 tsRNAs that were significantly downregulated. Compared with the model group (C3G-vs-Model), 3 tsRNAs were significantly upregulated and 37 tsRNAs were significantly downregulated under C3G intervention. Compared with the model group (Met-vs-Model), 8 tsRNAs were significantly upregulated and 29 tsRNAs were significantly downregulated under Met intervention ( Figure 4 B) Most of the tsRNAs that changed significantly were 5' tiRNA and 5' tRF.

[0054] tRNAs that carry the same amino acid but have different anticodons are called isoacceptors (e.g., tRNA Cys ); tRNAs that carry the same amino acid and have the same anticodon but have different sequences (there are amino acid differences elsewhere) are isodecoders (tRNA CysGCA ). According to the total expression of tsRNA produced by the same isodecoder in the Model group, the expression of tsRNA in mouse sperm changed to varying degrees under the HFHF diet ( Figure 5 The first and second groups). Under the intervention of C3G and Met, the tsRNA produced by the tRNAs that are isodecoders to each other showed a trend opposite to that after the HFHF diet, indicating that C3G and Met intervention has a tendency to improve the disorder of tsRNA expression caused by the HFHF diet ( Figure 5 In addition, mitochondrial tRNA (such as tRNA mtSerTGA The expression level of tsRNA produced by ) in the Model group was higher than that of tRNA derived from the nucleus (such as tRNA CysGCA ) produces lower tsRNA, mainly distributed in Figure 5 The lower half of the first group. Most of the tsRNAs produced by isodecoders are 5'tRF and 5'tiRNA, followed by 3'tiRNA, while tRF1 is the least ( Figure 5 of the fifth group).

[0055] Cluster analysis was performed based on the tsRNAs with significant expression changes in the Model group under the HFHF diet. The expression levels of 17 tsRNAs were significantly upregulated under the HFHF diet, and the expression levels of 15 tsRNAs were significantly downregulated under the HFHF diet ( Figure 6). For tsRNA significantly upregulated by HFHF, both C3G and Met interventions had good improvement effects; for tsRNA significantly downregulated by HFHF, Met intervention had a more obvious effect, while C3G intervention had a large individual difference. Compared with downregulated tsRNA, HFHF diet more obviously led to upregulation of tsRNA expression, potentially indicating that a high-fat and high-fructose diet may stimulate the sperm microenvironment of mice epididymis and cause upregulation of HFHF-sensitive tsRNA expression.

[0056] 2. Effects of C3G intervention on tsRNA processing pathway

[0057] Some tsRNAs that were significantly changed by HFHF only differed in a few nucleotides, such as 5'tiRNA-35-MetCAT-1 and 5'tiRNA-35-MetCAT-2. To verify whether the tsRNAs that were significantly changed by HFHF diet came from the same processing pathway, we performed an unsupervised cluster analysis of tsRNA expression based on Euclidean distance. The results showed that tsRNAs of the same isodecoder and type were close to each other ( Figure 7 AC). In addition, there was no obvious grouping of tsRNAs derived from mitochondria and tsRNAs derived from nuclei in terms of expression, indicating that HFHF-sensitive tsRNAs, tsRNAs significantly changed by C3G intervention, and tsRNAs significantly changed by Met intervention may come from multiple processing pathways such as the nucleus, cytoplasm, or mitochondria.

[0058] 3. Regulatory effect of C3G intervention on HFHF-sensitive tsRNA

[0059] HFHF diet induced significant changes in the expression levels of 32 tsRNAs in mouse sperm, and differential analysis is needed to further explore the regulatory effect of C3G intervention on tsRNA expression disorders.

[0060] The results showed that 10 tsRNAs were significantly upregulated under HFHF diet and significantly downregulated under C3G intervention; 5 tsRNAs were significantly upregulated under HFHF diet and significantly downregulated under Met intervention; 3 tsRNAs were significantly downregulated under HFHF diet and significantly upregulated under Met intervention. C3G and Met intervention significantly improved the increased expression of 5'tiRNA-35-AlaTGC-7, 5'tiRNA-35-MetCAT-1, 5'tiRNA-36-IleGAT-1, mt-tRF5-18-MetCAT, and 5'tiRNA-35-AlaTGC-6 caused by HFHF diet (see Figure 8 ).

[0061] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of cyanidin-3-O-glucoside in the preparation of drugs for treating diseases caused by tsRNA expression disorders.

2. The use according to claim 1, characterized in that: The diseases caused by tsRNA expression disorder are metabolic diseases, nervous system diseases and cancers caused by abnormal relative expression of tsRNA.

3. The use according to claim 2, characterized in that: The abnormal relative expression of tsRNA refers to an increase in the relative expression of tsRNA.

4. A drug for treating diseases caused by disordered tsRNA expression, characterized in that: The drug contains cyanidin-3-O-glucoside.

5. The drug according to claim 4, characterized in that: The medicament further contains a pharmaceutically acceptable excipient.

6. The drug according to claim 4, characterized in that The drug is prepared into a dosage form selected from the following: tablets, powders, pills, capsules, granules, drop pills, and oral liquid.

Images