MicroRNA derived from a decoction of a traditional Chinese medicine formula of Rehmannia glutinosa and Alpinia japonica, method for producing the same, and use thereof

Abstract

The present invention discloses microRNA derived from Forsythia-Astragalus Root Combined Chinese Herbal Decoction, its preparation method and use, the microRNA is selected from miRNAs whose nucleotide sequences are shown in SEQ ID NO.1-15, preferably QQ_159 whose nucleotide sequence is shown in SEQ ID NO.14, including artificially synthesized QQ_159, plant QQ_159, precursor form of QQ_159 or mature form of QQ_159. The present invention extracts functional plant microRNA or extract containing the microRNA from Forsythia-Astragalus Root Combined Chinese Herbal Decoction, and proves through experiments that the microRNA QQ_159 derived from Forsythia-Astragalus Root Combined Chinese Herbal Decoction has a certain inhibitory effect and therapeutic effect on viral influenza and pneumonia caused by novel coronavirus infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to microRNAs derived from a decoction of a traditional Chinese medicine formula of taro and aloe, a method for producing the same, and uses thereof.

Background Art

[0002] Influenza (abbreviated as "flu") is an acute respiratory disease that commonly affects mammals and poultry. It is caused by the influenza virus, is highly contagious, has a rapid transmission rate, usually causes simple respiratory infections, and includes clinical symptoms such as cough, fever, muscle pain, chills, and sweating. The discomfort lasts for 2 to 8 days, and usually has a rapid onset. Some patients, especially the elderly, infants, and other patients with chronic diseases, are prone to develop viral or secondary pneumonia. In severe cases, it is accompanied by respiratory distress and multiple organ failure. Treatment with anti-influenza drugs is currently one of the common means to combat influenza, and the prescription is generally determined according to the degree of infection of the patient and the physical condition of the patient. Although the influenza virus is one of the most deeply studied pathogens, because the influenza virus is very prone to mutation, conventional control and treatment plans need to be continuously improved. The development of new drugs against viral influenza is strongly demanded in current society and is also very important for the health of the people.

[0003] The novel coronavirus (abbreviated as the novel corona) is a type of novel virus that has spread widely in the past two years. It is mainly transmitted through the respiratory route of droplets, and recently it has been further discovered that it may be transmitted through aerosols and contact with objects. It has extremely strong infectivity and a very fast transmission speed. The clinical symptoms are mainly fever, fatigue, dry cough, anosmia / hyposmia. In severe cases, it is accompanied by severe pneumonia with dyspnea, tachypnea, and hypoxemia, and may also cause other complications. At present, there is no specific therapeutic drug for the novel coronavirus, so it has spread widely around the world. Therefore, in response to the drug demand for infectious disease prevention and control, developing new drugs specifically targeting the novel coronavirus is an important means to combat the novel coronavirus and ensure the safety of the lives of the people.

[0004] MicroRNAs (abbreviated as miRNAs) are small endogenous non-coding RNAs. Usually, miRNA-encoding genes are transcribed by RNA polymerase II to produce primary transcripts, which are processed into small RNAs of about 21 nucleotides by the RNaseIII endonucleases Drosha and Dicer. miRNAs mediate post-transcriptional gene silencing through binding to the coding region or the 3' and 5' untranslated regions of target mRNAs, play important roles in various physiological and pathological processes, and have promising prospects for applications in the medical field.

[0005] In recent years, the research on plant miRNAs has developed rapidly. The biological functions are mainly studied through up-regulation / down-regulation strategies. Many studies have revealed that they play important regulatory roles in aspects such as photosynthesis, nutrient homeostasis, growth and development, hormone signaling, and stress response. Furthermore, it has been confirmed that plant miRNAs can regulate animal gene expression as endogenous miRNAs. A large number of literatures have reported that traditional Chinese medicines play an active role in the prevention and treatment of influenza and pneumonia caused by the novel coronavirus. However, it is not clear which specific active ingredients they are. There are literatures reporting that traditional Chinese medicines contain miRNAs, but it is still unclear which miRNAs stably exist in traditional Chinese medicine decoctions and what functions these miRNAs will play when they enter the animal body.

Summary of the Invention

Problems to be Solved by the Invention

[0006] In view of the drawbacks of the prior art, the present invention provides microRNAs derived from a traditional Chinese medicine decoction of Allium macrostemon Bunge and Bupleurum chinense DC., a method for producing the same, and uses thereof. Functional plant microRNAs or extracts containing the microRNAs are extracted from the traditional Chinese medicine decoction of Allium macrostemon Bunge and Bupleurum chinense DC. Through experiments, it has been demonstrated that the microRNA QQ_159 derived from the traditional Chinese medicine decoction of Allium macrostemon Bunge and Bupleurum chinense DC. has a certain inhibitory effect and therapeutic effect against viral influenza and pneumonia caused by novel coronavirus infection.

Means for Solving the Problems

[0007] The present invention is achieved by the following technical solutions.

[0008] MicroRNAs derived from a Renkyo·Ougi compound traditional Chinese medicine decoction, wherein the microRNAs are selected from novel_mir7, novel_mir33, novel_mir35, novel_mir10, novel_mir20, novel_mir5, novel_mir36, novel_mir28, novel_mir31, ppt-miR894, novel_mir32, novel_mir21, pab-miR3711, QQ_159 or bdi-miR159a-3p, the nucleotide sequence of the novel_mir7 is shown in SEQ ID NO.1, the nucleotide sequence of the novel_mir33 is shown in SEQ ID NO.2, the nucleotide sequence of the novel_mir35 is shown in SEQ ID NO.3, the nucleotide sequence of the novel_mir10 is shown in SEQ ID NO.4, the nucleotide sequence of the novel_mir20 is shown in SEQ ID NO.5, the nucleotide sequence of the novel_mir5 is shown in SEQ ID NO.6, the nucleotide sequence of the novel_mir36 is shown in SEQ ID NO.7, the nucleotide sequence of the novel_mir28 is shown in SEQ ID NO.8, the nucleotide sequence of the novel_mir31 is shown in SEQ ID NO.9, the nucleotide sequence of the ppt-miR894 is shown in SEQ ID NO.10, the nucleotide sequence of the novel_mir32 is shown in SEQ ID NO.11, the nucleotide sequence of the novel_mir21 is shown in SEQ ID NO.12, the nucleotide sequence of the pab-miR3711 is shown in SEQ ID NO.13, the nucleotide sequence of the QQ_159 is shown in SEQ ID NO.14, The nucleotide sequence of the bdi-miR159a-3p is shown in SEQ ID NO.15.

[0009] Preferably, the microRNA is QQ_159 including artificially synthesized QQ_159, plant QQ_159, the precursor form of QQ_159 or the mature form of QQ_159.

[0010] A method for producing a microRNA derived from a decoction of a compound Chinese herbal medicine of Lepidium apetalum Willd. and Angelica acutiloba Kitag., Put 300 mL of the decoction of the compound Chinese herbal medicine of Lepidium apetalum Willd. and Angelica acutiloba Kitag. into 50 mL enzyme removal centrifuge tubes respectively, centrifuge at 2000 rpm to remove impurities, aspirate 10 mL of the supernatant for each tube and place it on ice, add 30 mL of TRIzolls at a ratio of 3 times the volume of the decoction, shake vigorously to make it uniform, then let it stand at room temperature for 10 min, and then add 6 mL of chloroform, shake vigorously to make it uniform, and let it stand for 10 min in step 1); Centrifuge at 10000 g for 10 min at 4 °C and collect the supernatant in step 2); Add an equal volume of isopropanol and precipitate at -20 °C for 1 h, and then centrifuge at 12000 g for 15 min in step 3); Carefully discard the supernatant, add 5 mL of 75% ethanol to wash the precipitate, and centrifuge at 12000 g for 5 min at 4 °C in step 4); After centrifugation, carefully discard the ethanol, leave the precipitate, dry the precipitate, then add 200 μL of DEPC water at 65 °C to dissolve it, and measure the RNA concentration in step 5); Purify the miRNA using a commercially available miRNA isolation kit, pass 100 μg of RNA through each column, and dissolve it with 60 μL of DEPC water for each tube in step 6); Measure the concentration of the miRNA, lyophilize it for 8 h, and then store it at -80 °C in step 7).

[0011] Use of the microRNA QQ_159 derived from the decoction of the compound Chinese herbal medicine of Lepidium apetalum Willd. and Angelica acutiloba Kitag. in the manufacture of a drug for inhibiting influenza virus replication.

[0012] Use of microRNA QQ_159 derived from a decoction of a traditional Chinese medicine combination of Rheum palmatum and Angelica acutiloba in the manufacture of a drug for treating viral influenza.

[0013] Preferably, the influenza includes influenza B virus Victoria or H5N1 avian influenza.

[0014] Use of microRNA QQ_159 derived from a decoction of a traditional Chinese medicine combination of Rheum palmatum and Angelica acutiloba in the manufacture of a drug for inhibiting SARS-CoV-2 virus replication.

[0015] Use of microRNA QQ_159 derived from a decoction of a traditional Chinese medicine combination of Rheum palmatum and Angelica acutiloba in the manufacture of a drug for treating viral pneumonia caused by SARS-CoV-2 virus.

[0016] A pharmaceutical composition for inhibiting influenza virus replication and / or treating influenza, comprising the above microRNA QQ_159 and a pharmaceutically acceptable carrier.

[0017] A pharmaceutical composition for inhibiting SARS-CoV-2 virus replication and / or treating viral pneumonia caused by SARS-CoV-2 virus, comprising the above microRNA QQ_159 and a pharmaceutically acceptable carrier.

[0018] A method for in vitro inhibiting the replication of influenza B virus Victoria for non-therapeutic purposes, comprising contacting the above microRNA QQ_159 with cells infected with influenza B virus Victoria.

Advantages of the Invention

[0019] The beneficial effects of the present invention are as follows. The present invention provides an experimental method for extracting microRNA in traditional Chinese medicine decoctions. Using this method, 15 types of microRNA that stably exist in the Rhizoma Zingiberis Recens·Ramulus Cinnamomi compound traditional Chinese medicine preparation were extracted and identified for the first time. Among them, QQ_159 was demonstrated to have a certain inhibitory effect and therapeutic effect against viral influenza and pneumonia caused by novel coronavirus infection. The present invention not only obtains the natural active antiviral components of traditional Chinese medicine but also provides a potential nucleic acid medicine for the clinical treatment of pneumonia caused by influenza or novel coronavirus.

Brief Description of the Drawings

[0020]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Modes for Carrying Out the Invention

[0021] To clarify the technical problems to be solved, the technical solutions, and the advantages of the present invention more clearly, the following will be described in detail with reference to the drawings and specific examples.

[0022] Example 1 Extraction of MicroRNA from a Compound Chinese Herbal Medicine Decoction of Allium macrostemon Bunge and Curcuma aromatica Salisb. (1) 300 mL of the decoction of the compound Chinese herbal medicine (Allium macrostemon Bunge, Curcuma aromatica Salisb., Curcuma longa L., Rheum palmatum L., Alpinia officinarum Hance, Bupleurum falcatum L., Aucklandia lappa Decne., Pueraria lobata (Willd.) Ohwi, Paeonia lactiflora Pall., Glycyrrhiza glabra L.) was placed into 50 mL enzyme-removing centrifuge tubes respectively, and centrifuged at 2000 rpm to remove impurities. 10 mL of the supernatant was aspirated for each tube and placed on ice. 30 mL of TRIzol was added at a ratio of three times the volume of the decoction, shaken vigorously to make it uniform, and then left at room temperature for 10 min. Then, 6 mL of chloroform was added, shaken vigorously to make it uniform, and left standing for 10 min. (2) Centrifuged at 10000 g for 10 min at 4 °C, and the supernatant was collected. (3) An equal volume of isopropanol was added and precipitated at -20 °C for 1 h, and then centrifuged at 12000 g for 15 min. (4) The supernatant was carefully discarded, 5 mL of 75% ethanol was added to wash the precipitate, and centrifuged at 12000 g for 5 min at 4 °C. (5) After centrifugation, the ethanol was carefully discarded, leaving the precipitate. After drying the precipitate, 200 μL of DEPC water at 65 °C was added to dissolve it, and the RNA concentration was measured. (6) The miRNA was purified using a commercially available miRNA isolation kit (100 μg of RNA was passed through each column), and dissolved in 60 μL of DEPC water for each tube. (7) The concentration of miRNA was measured, freeze-dried for 8 h, and then stored at -80 °C. (8) Sequencing of the microRNA was performed using the BGISEQ-500 technology, and the sequencing results are shown in Table 1 below.

Table 1

[0023] Measurement of the effect of QQ_159 on the amount of influenza B virus in Madin-Darby canine kidney (MDCK) cells in Example 2 (1) MDCK cells were cultured in a 24-well culture plate. (2) While transfecting MDCK cells with 10 nM, 50 nM, or 100 nM of QQ_159a respectively using the commercially available transfection reagent riboFECT™ CP, MDCK cells were transfected with the same concentration of nonsense sequence as a control group. (3) The above cells were infected with influenza B virus of the Victoria lineage. (4) 24 h after virus infection, the supernatant was collected, centrifuged to combine the cells, RNA was extracted, and the virus amount was detected by real-time PCR.

[0024] As a result, as shown in Figure 1, compared with the control group, 100 nM QQ_159 could significantly inhibit the virus amount of influenza B virus of the Victoria lineage in MDCK cells.

[0025] Example 3 Therapeutic effect of QQ_159 on mice infected with H5N1 subtype avian influenza virus (1) 2 h after 6-week-old female BALB / c mice were infected with H5N1 subtype avian influenza virus, the drug was intraperitoneally injected. The grouping of animals and the dosage of the drug are shown in Table 2 below.

Table 2

[0026] As is clear from this experiment, the artificially synthesized QQ_159 and the mixed RNA extracted in one batch from the compound traditional Chinese medicine decoction of sea cucumber and iris have a certain therapeutic effect on H5N1 avian influenza virus, and it was found that the decrease in body weight was slowed down, the mortality rate was decreased, and the pulmonary inflammation was reduced.

[0027] Example 4 Examination of the inhibitory effect of QQ_159 on pneumonia caused by SARS-CoV-2 virus (1) SARS-CoV-2 was intranasally infected into 6 - 8-week-old hACE2 transgenic mice at 10 5 TCID 50 / mL. (2) It was administered by intraperitoneal injection (ip) for 5 days. It was administered 2 h after virus infection on the first day. The grouping of the experiments and the dosages of the drugs are shown in Table 3 below.

Table 3

[0028] As a result, as shown in Figure 6, when QQ_159 was injected at 120 pmol / mouse / day, it had a certain inhibitory effect on the inflammation of the lung tissues of hACE2 gene recombinant mice infected with SARS-CoV-2. From this, it was found that QQ_159 has a certain inhibitory effect on mouse pneumonia caused by SARS-CoV-2 infection.

[0029] The above are only preferred embodiments of the present invention. Still, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should be regarded as within the protection scope of the present invention.

Claims

1. A method for producing microRNA derived from a decoction of a traditional Chinese medicine formula of Rheum palmatum and Angelica acutiloba, comprising: The microRNA is QQ_159, and the nucleotide sequence of QQ_159 consists of SEQ ID NO. 14, Step 1): Put 300 mL of the traditional Chinese medicine formula of Rheum palmatum and Angelica acutiloba (Rhei Rhizoma et Radix, Rheum palmatum, Angelicae Sinensis Radix, Scutellariae Radix, Asari Radix et Rhizoma, Bupleuri Radix, Aucklandiae Radix, Poria, Paeoniae Radix Alba, Glycyrrhizae Radix et Rhizoma) into 50 mL enzyme-removing centrifuge tubes respectively, centrifuge at 2000 rpm to remove impurities, aspirate 10 mL of the supernatant for each tube and place it on ice, add 30 mL of TRIzol (registered trademark) at a ratio of 3 times the volume of the decoction, shake to make it uniform, then let it stand at room temperature for 10 min, and then add 6 mL of chloroform, shake to make it uniform and let it stand for 10 min; Step 2): Centrifuge at 10000 g for 10 min at 4 °C and collect the supernatant; Step 3): Add an equal volume of isopropanol and precipitate at -20 °C for 1 h, and then centrifuge at 12000 g for 15 min; Step 4): Discard the supernatant, add 5 mL of 75% ethanol to wash the precipitate, and centrifuge at 12000 g for 5 min at 4 °C; Step 5): After centrifugation, discard the ethanol, leave the precipitate, dry the precipitate, then add 200 μL of DEPC water at 65 °C to dissolve it and measure the RNA concentration; Step 6): Purify the miRNA using a commercially available miRNA isolation kit, pass 100 μg of RNA through each column, and dissolve it with 60 μL of DEPC water for each tube; Step 7): Measure the concentration of miRNA, lyophilize it for 8 h, and then store it at -80 °C. The manufacturing method is characterized by the above steps.

2. Use of microRNA in the manufacture of a drug for treating viral influenza, wherein the viral influenza includes influenza B Victoria or H5N1 avian influenza, the microRNA is QQ_159, and the nucleotide sequence of QQ_159 consists of SEQ ID NO.

14. Use of microRNA derived from a decoction of a traditional Chinese medicine formula of Rheum palmatum and Angelica acutiloba.

3. Use of microRNA in the manufacture of a drug for treating viral pneumonia caused by the SARS-CoV-2 virus, wherein the microRNA is QQ_159, and the nucleotide sequence of the QQ_159 consists of SEQ ID NO. 14, the use of microRNA derived from a decoction of a traditional Chinese medicine formula of taro and mugwort.

Images