CN112587544B - Use of DNA tetrahedral framework nucleic acid in the preparation of drugs for treating fibrotic diseases - Google Patents
Use of DNA tetrahedral framework nucleic acid in the preparation of drugs for treating fibrotic diseases Download PDFInfo
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
本发明公开了DNA四面体框架核酸在制备治疗纤维化疾病的药物中的用途，属于纤维化疾病药物领域。本发明的原理是：DNA四面体框架核酸可通过抑制细胞外基质生成和细胞上皮间质转化，抑制细胞纤维化的进程，该原理不同于现有的纤维化疾病药物。加之DNA四面体框架核酸的生物相容性高，对人体没有明显副作用。将DNA四面体框架核酸用于制备治疗纤维化疾病的药物将具有十分良好的应用前景。
The invention discloses the use of DNA tetrahedral frame nucleic acid in preparing medicines for treating fibrosis diseases, and belongs to the field of medicines for fibrosis diseases. The principle of the present invention is: DNA tetrahedral framework nucleic acid can inhibit the process of cell fibrosis by inhibiting the formation of extracellular matrix and cell epithelial-mesenchymal transition, and the principle is different from the existing fibrotic disease drugs. In addition, the biocompatibility of DNA tetrahedral framework nucleic acid is high, and there is no obvious side effect on the human body. The use of DNA tetrahedral framework nucleic acid for the preparation of drugs for the treatment of fibrotic diseases will have very good application prospects.
Description
Technical Field
The invention belongs to the field of medicaments for treating fibrotic diseases.
Background
Fibrotic diseases are a common problem affecting multiple organs and body systems, leading to dysfunction and even death of the patient. Many human organs and systems may be involved in fibrotic diseases, including lung, kidney, heart, liver and skin. Tissue fibrosis usually occurs after chemical stimulation, e.g., alcohol-induced liver fibrosis, bleomycin-induced lung fibrosis, silica dust-induced silicosis. In addition to chemical irritation, severe trauma, surgery or injury can also lead to tissue fibrosis, such as hypertrophic scarring of the skin and infertility caused by endometrial fibrosis.
For the treatment of fibrosis diseases, no specific medicine can reverse fibrosis at present. For the treatment of common pulmonary fibrosis, recognized and recommended anti-fibrosis drugs such as pirfenidone (pharmacological action is anti-inflammatory and anti-fibrosis, and antioxidation), nintedanib (pharmacological action multi-target tyrosine kinase inhibitor for blocking proliferation, migration and transformation of fibroblast) and various traditional Chinese medicines have side effects of different degrees. When fibrosis develops to the terminal stage, an adopted means is organ transplantation to improve the life quality of a patient, but the problems of high operation difficulty, uncertain survival rate after transplantation, high cost and the like exist.
DNA Tetrahedra (TDN), also known as DNA Tetrahedral Framework Nucleic Acids (TFNAs), is a tetrahedral structure formed by base complementary pairing of single strands of DNA (usually 4). According to previous studies by the inventors, TFNAs may be used to promote cell proliferation, migration and differentiation, and thus, TFNAs are applied to spinal injuries, osteoarthritis and other traumatic diseases. In addition, TFNAs can be used as a carrier, can enhance the cell delivery of oligonucleotides and chemotherapeutic drugs, and has great potential in antitumor treatment. More importantly, the TFNAs can be used as a novel nucleic acid nano material, can regulate the generation of inflammatory factors, active oxygen and the like, and has good anti-inflammatory and anti-oxidation effects.
The application of TFNAs in the treatment of fibrotic diseases is not known at present.
Disclosure of Invention
The invention aims to solve the problems that: provides the application of DNA tetrahedral framework nucleic acid in preparing medicine for treating fibrosis diseases.
The technical scheme of the invention is as follows:
use of a DNA tetrahedral framework nucleic acid in the manufacture of a medicament for the treatment of fibrotic disease.
As for the use as described above, the fibrotic disease is a fibrotic disease of the lung.
The DNA tetrahedral framework nucleic acid is formed by base complementary pairing of single-stranded DNA molecules with sequences shown in SEQ ID NO. 1-4.
As previously mentioned, the cellular fibrosis is induced by TGF-. beta.1.
The use as described hereinbefore, the medicament is a medicament for inhibiting extracellular matrix production.
As previously mentioned, the medicament is one which inhibits the expression of cellular type I collagen and/or fibronectin.
The use as described hereinbefore, the medicament is a medicament for inhibiting epithelial-to-mesenchymal transition.
As used herein, the term "agent" refers to an agent that inhibits the expression of alpha-smooth muscle actin.
A medicament for treating fibrotic diseases, said medicament comprising a DNA tetrahedral framework nucleic acid as an active ingredient.
As the medicine, the DNA tetrahedral framework nucleic acid is formed by base complementary pairing of single-stranded DNA molecules with sequences shown as SEQ ID NO. 1-4.
The inventor finds that the DNA tetrahedral framework nucleic acid has the function of inhibiting cell fibrosis, and the application of the DNA tetrahedral framework nucleic acid disclosed by the invention in preparing the medicament for treating the fibrotic diseases, particularly the pulmonary fibrotic diseases, provides a new direction for treating the fibrotic diseases; in addition, due to the biocompatibility of the DNA tetrahedral framework nucleic acid, the prepared medicine does not cause obvious side effect theoretically and is safer than the existing medicine.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is the synthesis and characterization of DNA Tetrahedral Framework Nucleic Acids (TFNAs): a is a schematic diagram of the synthetic principle of TFNAs; b is TFNAs and the results of High Performance Capillary Electrophoresis (HPCE) of four single strands (legend is from top to bottom, molecular size Marker, S1, S2, S3, S4, S1+ S2, S1+ S2+ S3, TFNAs); c is the gel electrophoresis picture of TFNAs and four single chains. Lanes: 1 is DNA marker, 2 is S1, 3 is S2, 4 is S3, and 5 is TFNAs; d is a transmission electron microscopy image with red labeled DNA tetrahedra and blue labeled agglomerates.
FIG. 2 shows the uptake of TFNAs by RLE-6TN cells: a is the detection result of flow cell entrance; b is Cy5 labeled TFNAs fluorescence detection and tracing graph.
FIG. 3 is a graph showing the active oxygen detection of LPS-induced RLE-6TN cells by TFNAs: a, fluorescence confocal detection map; b, flow cytometric mapping; c, D, flow detection result analysis chart.
FIG. 4 is a graph of expression of fibrosis-associated type I Collagen (Collagen I) gene protein levels: a, immunofluorescence detection map; b, Western blot; c, western blot quantification map; d, fluorescent quantitative PCR detection graph; microscale, 25 μm.
FIG. 5 is a graph showing the expression of the levels of Fibronectin (Fibronectin) gene protein associated with fibrosis: a A, immunofluorescence detection map; b, Western blot; c, western blot quantification map; d, fluorescent quantitative PCR detection graph; microscale, 25 μm.
FIG. 6 is a graph showing the expression of the protein level of the marker protein alpha-smooth muscle actin (alpha-SMA) gene for the fibrosis-related epithelial-mesenchymal transition EMT process: a, immunofluorescence detection map; b, Western blot; c, western blot quantification map; d, fluorescent quantitative PCR detection graph; microscale, 25 μm.
Detailed Description
Example 1 preparation and characterization of TFNAs
1. Synthesis of
Four DNA single strands (S1, S2, S3 and S4) are dissolved in TM Buffer (10mM Tris-HCl, 50mM MgCl2, pH 8.0), the four DNA single strands are fully mixed, rapidly heated to 95 ℃ for 10 minutes, then rapidly cooled to 4 ℃ for more than 20 minutes, and self-assembly process is carried out on the four single strands in the system according to the base complementary pairing principle under the temperature control, thus obtaining the DNA tetrahedral framework nucleic acid.
The four single-stranded sequences (5 '→ 3') are as follows:
S1：
S2：
S3：
S4：
2. identification
The results of the identification are shown in FIG. 1. In FIG. 1, A is TFNAsSchematic diagram of the synthetic principle of (1); b is TFNAsAnd four single-stranded High Performance Capillary Electrophoresis (HPCE) results; c is the gel electrophoresis picture of TFNAs and four single chains. Lanes: 1 is DNA marker, 2 is S1, 3 is S2, 4 is S3, and 5 is TFNAs; d is a transmission electron microscopy image with red labeled DNA tetrahedra and blue labeled agglomerates.
It can be seen that TFNAs were successfully synthesized.
The advantageous effects of the present invention will be further described below in the form of experimental examples.
Experimental example 1 cell uptake experiment
The experiment detects the cell entry of the TFNAs and verifies the good cell entry performance of the TFNAs.
Cy5 fluorescent substance is hung on an S1 chain to synthesize Cy5-TFNAs, RLE-6TN cells (an alveolar epithelial cell) are cultured in a 10% fetal bovine serum DMEM culture medium for 24 hours, the culture medium is changed to Cy5-TFNAs with the final concentration of 125nM and 250nM respectively, and the cell uptake is detected by flow cytometry and immunofluorescence technology after 6 hours and 10 hours of culture to detect the cell entry condition of the Cy 5-TFNAs.
(1) Flow cytometry
The method comprises the following steps:
a. RLE-6TN cell suspension was inoculated into a 6-well plate and pre-incubated in an incubator for 24 hours (37 ℃ C., 5% CO 2).
b. Cy5-TFNAs was added at 125nM and 250nM, respectively, and incubated in an incubator for 6 hours and 12 hours (37 ℃, 5% CO2), respectively.
c. Digesting cells, collecting cell suspension, centrifuging at 1000r/min for 5min, resuspending with PBS, repeating for three times, and detecting on a computer.
As a result: after 6 hours of culture, the amount of TFNAs entering cells is less, and the amount of TFNAs entering cells is more at 10 hours, and the result shows that the TFNAs can be taken up by alveolar epithelium to exert subsequent biological effects of entering cells.
(2) Fluorescent tracing technique
The method comprises the following steps:
a. the confocal dish was inoculated with a suspension of RLE-6TN cells and pre-incubated in an incubator for 24 hours (37 ℃ C., 5% CO 2).
b. Cy5-TFNAs was added at a concentration of 250nM and incubated in an incubator for 12 hours (37 ℃, 5% CO 2).
c. The medium was aspirated off, washed three times with PBS, 5 minutes each; fixing with 4 wt% paraformaldehyde for 25 min, removing paraformaldehyde by suction, and washing with PBS for 5min three times; then processing with phalloidin (FITC marker), keeping out of the sun for 10-30 minutes, sucking off phalloidin, washing with PBS for three times, 5 minutes each time; then treating with DAPI, keeping out of the sun for 10 minutes, removing DAPI by suction, and washing with PBS for three times, 5 minutes each time; sealing with 10 wt% glycerol, protecting from light, storing at 4 deg.C, and detecting on machine.
As a result: as shown in FIG. 2, the RLE-6TN cells took up more TFNAs, which were mainly distributed in the cytoplasm after entering the cells.
And (4) conclusion: this experimental example shows that TFNAs can be efficiently taken up by cells.
Experimental example 2 TFNAs reduce cellular reactive oxygen species production
The experiment detects the effect of TFNAs on active oxygen generated by LPS induced cells, and verifies the antioxidation effect of the TFNAs.
(1) Cell grouping process
Negative control group
LPS-induced group: 1 μ g/ml LPS
TFNAs treatment group 1: mu.g/ml LPS +125nM TFNAs (added simultaneously)
TFNAs treatment group 2: mu.g/ml LPS +250nM TFNAs (added simultaneously)
(2) Fluorescent tracing technique
The method comprises the following steps:
a. RLE-6TN cell suspension is inoculated in a confocal cuvette, and is pre-cultured for 24 hours in an incubator (the basic culture medium is a DMEM high-sugar medium containing 10% fetal bovine serum at 37 ℃ and 5% CO 2).
b. After the cells are attached to the wall, the serum is reduced to 1% in a gradient manner, and corresponding detection is carried out after the cells are correspondingly treated for 12 hours according to the groups.
c. The medium was aspirated off, washed three times with PBS, 5 minutes each; fixing with 4 wt% paraformaldehyde for 25 min, removing paraformaldehyde by suction, and washing with PBS for 5min three times; then processing with phalloidin (FITC marker), keeping out of the sun for 10-30 minutes, sucking off phalloidin, washing with PBS for three times, 5 minutes each time; then treating with DAPI, keeping out of the sun for 10 minutes, removing DAPI by suction, and washing with PBS for three times, 5 minutes each time; sealing with 10 wt% glycerol, protecting from light, storing at 4 deg.C, and detecting on machine.
(3) Flow cytometry
The method comprises the following steps:
a. the 6-well plate was inoculated with RLE-6TN cell suspension and pre-incubated in an incubator for 24 hours (37 ℃ C., 5% CO)2)。
b. After the cells are attached to the wall, the serum is reduced to 1% in a gradient manner, and corresponding detection is carried out after the cells are correspondingly treated for 12 hours according to the groups.
c. Digesting cells, collecting cell suspension, centrifuging at 1000r/min for 5min, resuspending in PBS, repeating for three times, and detecting on a computer in a dark place.
Results and conclusions: as shown in FIG. 3, the present experimental example shows that TFNAs can reduce the active oxygen production of RLE-6TN induced by LPS, and TFNAs with a concentration of 250nM has better effect than 125 nM.
Experimental example 3 TFNAs inhibits expression of fibrosis-associated Gene protein
Although the mechanism of fibrosis is not fully elucidated, overproduction of extracellular matrix ECM is considered to be an important factor in fibrotic diseases, and ECM accumulation reduces tissue motility, leading to loss of normal function of organ tissues. Therefore, reducing the production of extracellular matrix ECM is an important tool in the prevention and treatment of fibrotic diseases.
TGF-beta 1 induced extracellular matrix production is a well-established in vitro cell model of fibrosis, and in order to verify the influence of tetrahedral framework nano-nucleic acid TFNAs on the extracellular matrix ECM, the experiment utilizes TFNAs to interfere with the expression of typical extracellular matrix molecules induced by TGF-beta 1, including the expression of the gene and protein levels of Collagen I (Collagen I) and Fibronectin (Fibronectin).
1. Experimental methods
(1) Cell grouping process
Negative control group
TGF-. beta.1 Induction group: 5 ng/mlTGF-beta 1
TFNAs treatment group 1: 5ng/ml TGF-. beta.1 +125nM TFNAs (added simultaneously)
TFNAs treatment group 2: 5ng/ml TGF-. beta.1 +250nM TFNAs (added simultaneously)
The treatment time was 24/48 hours
(2) Immunofluorescence technique
A. Inoculating cell suspension (RLE-6TN cells) into a confocal dish, placing in an incubator, and culturing for 24 hours (the basic culture medium is DMEM high-sugar medium containing 10% fetal calf serum, 37 ℃, 5% CO2)
And B.after 24 hours of cell adherence, performing gradient serum reduction to 1%, and performing corresponding detection after 24/48 hours of corresponding treatment according to the above groups.
C. The medium was aspirated off and washed 3 times with PBS for 5 minutes each;
after fixing with 4% paraformaldehyde for 25 minutes, removing paraformaldehyde by suction, washing with PBS for 3 times, 5 minutes each time;
d.0.5% Triton-100 for 20-25 min, removing Triton-100 by suction, washing 3 times with PBS, 5min each time;
E. treating sheep serum for 1 hr, sucking out sheep serum, washing with PBS for 3 times, each for 5 min;
F. primary antibody (anti-Collagen I, Fibronectin antibody) treatment was performed at 4 ℃ overnight. The next day, rewarming at 37 ℃ for 0.5 hour, recovering the primary antibody, washing 3 times with PBS, 5 minutes each time. Treating the secondary antibody carrying fluorescence at 37 ℃ for 1 hour in a dark place, absorbing the secondary antibody, and washing with PBS for 3 times for 5 minutes each time;
G. treating phalloidin in dark for 10-30 min, removing phalloidin, washing with PBS for 3 times (5 min each);
DAPI treatment, protected from light for 10 min, blotted off DAPI, washed 3 times with PBS for 5min each. Sealing with 10% glycerol, and storing at 4 deg.C in dark. And (6) performing detection on the machine.
(3) Western blotting method
a. Inoculating cell suspension (RLE-6TN cells) into a confocal dish, placing in an incubator, and culturing for 24 hours (the basic culture medium is DMEM high-sugar medium containing 10% fetal calf serum, 37 ℃, 5% CO2)
And b, after 24 hours of cell adherence, carrying out gradient serum reduction to 1%, and carrying out corresponding detection after 24/48 hours of corresponding treatment according to the above groups.
c. Extracting the whole protein of all samples, and detecting the changes of Collagen I and Fibronectin through Western blot.
(4) Real-time quantitative fluorescent PCR experiment
a. RLE-6TN cells were seeded in 6-well plates and the plates were pre-incubated in an incubator for 24 hours (37 ℃ C., 5% CO 2).
b. TFNAs were added at concentrations of 125nM and 250nM, respectively, and incubated in an incubator for 24 hours (37 ℃, 5% CO 2).
c. Extracting RNA of each group, obtaining cDNA through a reverse transcription kit, and detecting the expression of genes related to fibrosis by using a dye method through fluorescent quantitative PCR: collagen I and Fibronectin.
2. Results and conclusions
As shown in FIGS. 4 and 5, RLE-6TN cells expressed increased amounts of Collagen type I (Collagen I) and Fibronectin (Fibronectin) under the induction of TGF-beta 1, indicating that the in vitro cell model of fibrosis was successfully established. The interference treatment of TFNAs can reduce the expression of two extracellular matrix molecules from the gene and protein level, and shows that TFNAs have certain inhibition effect on the generation of extracellular matrix molecules related to fibrosis, especially on lung cells.
Experimental example 4 TFNAs inhibits epithelial-mesenchymal transition process
In addition to important extracellular matrix ECM production, epithelial-mesenchymal transition (EMT, meaning epithelial to mesenchymal transition) induced by the TGF- β family is also an important factor in the development of fibrosis. TGF-beta is a powerful fibrosis promoting cytokine, induces epithelial-mesenchymal transition, transforms epithelial cells into mesenchymal cells, further increases ECM secretion and accumulation, causes irreversible damage to normal tissue structure, and accelerates development of fibrosis diseases.
In order to study whether TFNAs can regulate the TGF-beta 1-induced epithelial-mesenchymal transition EMT process and thus relieve further development of fibrosis, the experiment detects the expression of the epithelial-mesenchymal transition cell marker protein alpha-smooth muscle actin (alpha-SMA) on the basis of the experimental method of experimental example 3.
Results are shown in FIG. 6, where alpha-SMA expression was enhanced after 24 hours of treatment with TGF-beta 1, whereas alpha-SMA expression was lower in RLE-6TN cells after treatment with TGF-beta 1 in combination with TFNAs, compared to TGF-beta 1 alone.
And (4) conclusion: the experimental result shows that TFNAs can inhibit the TGF-beta 1 induced EMT process, particularly has very definite effect on lung cells, and can be used as a medicament for treating lung fibrosis diseases.
In conclusion, the TFNAs can inhibit extracellular matrix related molecule production and epithelial-mesenchymal transition process induced by TGF-beta 1, and further inhibit cell fibrosis, so that the TFNAs are expected to be applied to preparation of medicines for treating fibrotic diseases (particularly pulmonary fibrotic diseases).
SEQUENCE LISTING
<110> Sichuan university
Use of <120> DNA tetrahedral framework nucleic acid in preparation of medicine for treating fibrosis disease
<130> GYKH1118-2020P019469CC
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 63
<212> DNA
<213> Artificial sequence
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atttatcacc cgccatagta gacgtatcac caggcagttg agacgaacat tcctaagtct 60
gaa 63
<210> 2
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<212> DNA
<213> Artificial sequence
<400> 2
acatgcgagg gtccaatacc gacgattaca gcttgctaca cgattcagac ttaggaatgt 60
tcg 63
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acggtattgg accctcgcat gactcaactg cctggtgata cgaggatggg catgctcttc 60
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| CN114569628A (en) * | 2022-03-11 | 2022-06-03 | 四川大学 | Use of DNA tetrahedral framework nano-nucleic acid in cosmetology |
| CN115957209B (en) * | 2022-05-13 | 2024-09-17 | 四川大学 | Sichuan citrinin-tetrahedron framework nucleic acid compound and preparation method and application thereof |
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| CN114984032B (en) * | 2022-06-27 | 2023-07-07 | 四川大学 | DNA tetrahedron framework nucleic acid-chlorogenic acid complex and its use in the preparation of medicines for treating liver fibrosis |
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| CN118320116B (en) * | 2024-04-18 | 2024-12-20 | 四川大学 | Pirfenidone nanocomposite based on tetrahedral framework nucleic acid and preparation method and application thereof |
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| Anti-inflammatory and Antioxidative Effects of Tetrahedral DNA;Qi Zhang等;《ACS Applied Materials & Interfaces》;20180104;第10卷;3421-3430 * |
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