CN115068496B - Complex of DNA tetrahedral framework nucleic acid and typha neoglycoside and application thereof in preparing medicine for treating acute kidney injury - Google Patents
Complex of DNA tetrahedral framework nucleic acid and typha neoglycoside and application thereof in preparing medicine for treating acute kidney injury Download PDFInfo
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Abstract
The invention provides a new application of typhin in treating acute kidney injury, and discloses a compound of DNA tetrahedron framework nucleic acid and typhin, wherein the DNA tetrahedron framework nucleic acid and typhin have the functions of synergistically reducing apoptosis of epithelial cells of proximal tubular of renal cortex, reducing necrosis of renal tissue and recovering renal function, can prevent and treat renal fibrosis, prevent acute kidney injury from developing to chronic kidney disease, and provide a new choice for clinic.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a compound of DNA tetrahedral framework nucleic acid and typha neoglycoside and application thereof in preparing a medicine for treating acute kidney injury.
Background
Acute Kidney Injury (AKI) is one of the highest global syndromes, a detrimental global problem, and is closely related to Chronic Kidney Disease (CKD) and hospital mortality in various areas. Mortality in hospitalized patients with acute kidney injury is typically 10-20% and ICU patients are 44.7% -53%. Also, even mild impairment of kidney function can lead to poor prognosis and affect long-term kidney function. The risk of developing chronic kidney disease increases 6-fold in patients with acute kidney injury, and the risk of developing early stage chronic kidney disease to end stage chronic renal failure increases 28-fold. Acute kidney injury is a risk factor for the development of chronic kidney disease as well as a trigger for the progression of chronic kidney disease. There are many clinical studies currently directed to acute kidney injury, however, there is currently no specific and non-nephrotoxic treatment available to reverse acute kidney injury at an early stage to improve prognosis. Thus, there is an urgent need to develop novel and effective methods of treating acute kidney injury.
Pollen of typha is called as "pollen typhae", is a widely distributed aquatic plant, and has pharmacological effects of reducing blood lipid, resisting atherosclerosis and blood coagulation, protecting myocardial ischemia, relieving pain, etc. Flavonoid typhonine (Typhaneoside, typ, CAS: 104472-68-6) is the main extract of pollen Typhae, and has good antioxidant stress effect in heart ischemia reperfusion injury. However, the effect of typha neoglycoside on acute kidney injury has not been reported yet. Meanwhile, as with most traditional Chinese medicine monomers, typha-neoside is limited by poor water solubility, low systemic bioavailability and instability in physiological media, so that the treatment effect is severely restricted.
The DNA nano structure has the characteristics of natural biocompatibility, stable structure and high bioavailability, and has become a popular research subject in recent years. Among them, DNA tetrahedral framework nucleic acids (tFNAs) can be assembled from four single-stranded DNAs (ssdnas) by annealing, are easy to prepare and excellent in stability, and are suitable for a wide range of applications. Notably, tFNAs can carry species oligonucleotide drugs such as antisense oligonucleotides (ASOs), small interfering RNAs (sirnas), micrornas (mirnas), antimicrobial peptides and other polypeptides, as well as resveratrol, curcumin and other small molecule drugs, thanks to the double-stranded structure and chemical properties of the DNA material. Based on the negative surface charge and unique tetrahedral framework, tFNAs can exhibit excellent cell entry effects through the mediation of the pit proteins, and can increase the accumulation of drugs carried by tFNAs in cells. Meanwhile, chinese patent CN112587542a previously disclosed the therapeutic effect of tFNA on acute kidney injury. The pharmacological actions of the compound of tFNA and typha neoglycoside are still to be further researched.
Disclosure of Invention
The invention aims to provide a complex of DNA tetrahedral framework nucleic acid and typha neoside, which can treat acute kidney injury.
The invention provides application of typha saponin in preparing a medicament for treating acute kidney injury.
The invention also provides a compound which is formed by compositing DNA tetrahedron framework nucleic acid and typha neoside; the DNA tetrahedron framework nucleic acid is formed by base complementary pairing of 4 single-stranded DNA molecules with sequences shown as SEQ ID NO. 1-4 respectively.
Further, the content of typha glycoside in the compound is 10-70 wt%.
Further, the DNA tetrahedron framework nucleic acid and typha neoglycoside mixed solution are stirred and ultrafiltered to prepare the DNA tetrahedron framework nucleic acid-typha neoglycoside mixed solution;
preferably, the molar ratio of the DNA tetrahedral framework nucleic acid to typha neoglycoside is 1 (80-200); more preferably, the molar ratio of the DNA tetrahedral framework nucleic acid to typha neoglycoside is 1:200.
The invention also provides a preparation method of the compound, which comprises the following steps:
(1) Preparing a DNA tetrahedron framework nucleic acid solution and a typha neoglycoside solution respectively;
(2) Adding typha neoglycoside solution into DNA tetrahedral framework nucleic acid solution, stirring, and ultrafiltering to obtain the final product; preferably, the stirring is at 20-30 ℃ for 5-7 hours.
Further, the solution of the DNA tetrahedral framework nucleic acid is prepared by the following method: adding 4 single-stranded DNA molecules into TM buffer, maintaining at a temperature sufficient to denature the DNA molecules for at least 10min, and then reducing the temperature to 2-8 ℃ for at least 20min;
preferably, 4 single stranded DNA molecules are added to TM buffer, and the mixture is left at 95℃for 10min, and the temperature is lowered to 4℃for 20min.
The invention also provides application of the compound in preparing a medicament for treating acute kidney injury. Further, the above-mentioned drugs are drugs for reducing necrosis of kidney tissue and/or restoring kidney function.
Further, the above-mentioned drug is a drug for reducing apoptosis of epithelial cells of proximal tubular of renal cortex.
Further, the above-mentioned drugs are drugs for preventing the development of acute kidney injury into chronic kidney disease; preferably a medicament for the prevention and treatment of renal fibrosis.
The invention has the beneficial effects that: the invention provides a new application of typhin in treating acute kidney injury, and discloses a compound of DNA tetrahedron framework nucleic acid and typhin, wherein the DNA tetrahedron framework nucleic acid and typhin have the functions of synergistically reducing apoptosis of epithelial cells of proximal tubular of renal cortex, reducing necrosis of renal tissue and recovering renal function, can prevent and treat renal fibrosis, prevent acute kidney injury from developing to chronic kidney disease, and provide a new choice for clinic.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
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FIG. 1 is a representation of synthesis and entry of tFNA-Typ. (a) schematic of the synthesis of tFNA and TTC. (b) PAGE images of tFNA and TTC. (c) Statistical analysis of GelRed (λex=312 nM) at tFNAs (250 nM) or TTCs (tFNA-OD values,) p <0.0001vs Gel-Red, # # p <0.0001vs tFNAs (n=5), (d, e) the sizes of the typh, tFNA and TTCs were 2.4170 ± 0.1417nM, 11.77± 1.909nM and 16.93±3.727nM, respectively, zeta (zeta) potentials of-0.338±2.28mV, -2.51±3.7mV, -9.74±3.71mV. (f) absorption peaks of TTCs (tFNAs to 262nM, type p to 378 nM), (g) transmission electron microscope images of TTCs (h) the confocal microscope TTCs were observed after 6h, red, blue: nuclear cell, blue: cell scaffold and small cell of the formula of the mfj after 6h, respectively, blue: cell scaffold of the TTCs were observed after 6 th and small cell of the formula of the NAc.
FIG. 2 shows the anti-apoptotic capacity of tFNA-Typ in vitro. (a) I/R injury resulted in a schematic representation of apoptosis of tubular epithelial cells. (b) The effect of analyzed tFNAs, types and TTCs on cell viability of healthy and I/rhk-2 cells was determined by CCK-8 (n=3). (c) determining apoptosis of each group of cells using flow cytometry: control, I/R, I/R+tFNAs, I/R+Typ and I/R+TTC. (d) The apoptosis statistics of group (c) include early apoptosis rate and late apoptosis rate of cells. (e) Expression of cytochrome C in HK-2 after treatment with tFNas, typ and TTC was examined using confocal microscopy and Western blotting. Quantification of related proteins calculated by ImageJ (n=3). (f) Cofocal microscopy and Western blotting were used to detect Caspase-3 expression in tFNAs, typ and TTC treated HK-2. Quantification of related proteins calculated by ImageJ (n=3). Statistical analysis: * p <0.05, < p <0.01, < p <0.001 (: control, #: I/R, @: tFNAs, &: type).
FIG. 3 shows that tFNA-Typ restored kidney function and prevented tubular injury. (a) creating a schematic representation of murine I/R injury AKI model. (b) Biodistribution of TTC in healthy and AKI mice and renal fluorescence distribution from AKI mice. (c) TTC ability to target the renal tubules. Green: tubular kidney; red: cy5-TTC; blue: and (3) cell nucleus. (d, e) evaluation of AKI histopathological lesions by PAS staining and HE staining. Black arrows indicate the disappearance of brush border, red arrows indicate the exfoliated cells, and red asterisks indicate the transparent tube. (f) Kim-1 labeled damaged tubular. Red: kim-1; green: tubular kidney; blue: and (3) cell nucleus. (g) blood creatinine and urea nitrogen levels in five groups of mice: control, I/R, I/R+tFNAs, I/R+Typ and I/R+TTC. (h, i) the renal tubular injury score was calculated on ten random HE or PAS stained tissue sections and the results for each mouse were averaged (n=5). (j) Semi-quantitative analysis of Kim-1 relative fluorescence intensity (n=5). Statistical analysis: * p <0.05, < p <0.01, < p <0.001 (: control, #: I/R, @: tFNAs, &: type).
FIG. 4 shows that tFNA-Typ reduces apoptotic cells, helping to restore kidney function. TUNEL assay was performed on five groups of apoptotic cells: control, I/R, I/R+tFNAs, I/R+Typ and I/R+TTC. Green TUNEL; blue: and (3) cell nucleus.
FIG. 5 shows that tFNA-Typ can prevent the progression of acute kidney injury to chronic kidney disease. (a) WB and immunofluorescent stained Col I protein levels. Red: col I; blue: a cell nucleus; green: and a cytoskeleton. WB relative quantification of proteins calculated by ImageJ (n=3). (b) WB and immunofluorescent stained α -SMA protein levels. Red: alpha-SMA; blue: a cell nucleus; green: and a cytoskeleton. WB relative quantification of proteins calculated by ImageJ (n=3). Statistical analysis: * p <0.05, < p <0.01, < p <0.001 (: control, #: I/R, @: tFNAs, &: type).
FIG. 6 shows the standard curve of Typ and the encapsulation efficiency of tFNA-Typ, drug loading.
Detailed Description
The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.
EXAMPLE 1 Synthesis and characterization of the inventive Complex tFNA-Typ
As shown in the schematic diagram of FIG. 1a, the synthesis of tFNA-Typ is divided into two steps.
(1) tFNA synthesis: four sequence-specific single-stranded DNA (ssDNA) (Sangon, shanghai, china) were added to equimolar amounts of Tris-HCl and MgCl 2 In a composed TM buffer, then heated (95 ℃ C., 10 min) and cooled (4 ℃ C., 20 min), i.e., self-assembled to form tFNA.
Specific sequence of four single strands
(2) Compounding of Typ and tFNA: different concentrations of Typ (20. Mu.M, 40. Mu.M, 80. Mu.M, 160. Mu.M, 240. Mu.M, 320. Mu.M, 600. Mu.M, 900. Mu.M) were mixed with 250nM tFNA in equal volumes and stirred for 6 hours. Then, residual ssDNA and Typ were removed by ultrafiltration (30 kDa molecular weight membrane, millipore, USA), to obtain tFNA-Typ with different Typ contents.
The following experiments prove the beneficial effects of the invention.
Experimental example 1 characterization of the inventive Complex
1. Experimental method
Successful synthesis of tFNA-type was confirmed by polyacrylamide gel electrophoresis (PAGE), transmission Electron Microscopy (TEM), atomic Force Microscopy (AFM) and showed its size and shape. Dynamic light scattering DLS (Nano ZS, malvern, england) was used to measure particle size and Zeta potential of tFNA and tFNA-Typ. Absorbance curves for tFNA, type and tFNA-type were plotted with an ultra-differential spectrophotometer. GelRed fluorescence was detected by a Varioskan LUX microplate reader (Thermo Scientific, USA). The calculation formulas of the encapsulation efficiency and the drug loading rate are as follows:
2. experimental results
First, tFNA self-assembled from four equimolar amounts of ssDNA and successful synthesis was detected by PAGE (fig. 1 b). Subsequently, tFNA was complexed with type by stirring for 6 h. Successful synthesis of tFNA-Typ was also verified in PAGE (FIG. 1 b), wherein the position of tFNA-Typ was shown to shift up relative to tFNA. The Typ can interact with DNA through an intercalating binding mode. Thus, when tFNA and tFNA-Typ are stained with GelRed dye in PAGE, gelRed bound to nucleic acid is replaced by Typ. To further verify this hypothesis, a GelRed dye competition assay was applied. Briefly, fluorescence was stimulated when tFNA was incubated with 1x GelRed for 20min, whereas tFNA-type was incubated with GelRed, which decreased fluorescence intensity (fig. 1 c). This means that the location of the interaction of Typ with tFNA is the same double-stranded DNA double-helical groove region as GelRed.
In addition, the sizes and zeta potential of tFNA and tFNA-Typ were examined by DLS, and successful synthesis of tFNA-Typ was also confirmed. In FIG. 1d, the size of tFNA was 11.77.+ -. 1.91nm, the size of Typ was 2.417.+ -. 0.142nm, and the size of tFNA-Typ was 16.93.+ -. 3.73nm. the zeta potential of tFNA was-2.51.+ -. 3.70mV, the zeta potential of Typ was-0.338.+ -. 2.280mV, and the zeta potential of tFNA-Typ was-9.74.+ -. 3.71mV (FIG. 1 e). These differences indicate that the new nanoparticle tFNA-type has been produced and is relatively stable compared to tFNA. In addition, the spectrum in FIG. 1f shows that the characteristic absorption peaks of tFNA-Typ are close to tFNA (260 nm) and Typ (260, 385 nm), which indicates that Typ is successfully carried by tFNA without byproducts. To characterize the shape and size of tFNA-Typ, AFM and TEM were performed (FIG. 1g, h). AFM images showed tFNA-Typ of about 20nm. The same results were obtained from the TEM images, and triangular structures were observed by TEM. Taken together, these phenomena suggest that we have for the first time constructed a novel nano-drug system, tFNA-typh, in which the typh binds to tFNA via an intercalating binding mode.
Furthermore, we tested the encapsulation efficiency and drug loading in the Typ to tFNAs by applying different feed ratios of the Typ and tFNAs (FIG. 6). As the encapsulation efficiency (encapsulation efficiency, EE) decreases, the drug loading (loading efficiency, LE) increases. We selected the ratio of EE to LE at the intersection where tFNas concentration was 250nM and Typ concentration was 50. Mu.M, and the ratio of EE to LE was about 50% and LE was about 34%. In the subsequent experiments, the corresponding feed ratios (10. Mu.g tFNAs: 5.51. Mu.g Typ; i.e., molar ratio 1:200) were used.
Experimental example 2, cy 5-labeled cellular uptake of tFNA and tFNA-Typ
1. Experimental method
To examine the cell entry properties of tFNA and tFNA-Typ, HK-2 cells were first treated with Cy 5-labeled tFNA and tFNA-Typ for 4 hours or 6 hours. Cells were then collected and washed 3 times with PBS. Finally, the proportion of cells with fluorescence Cy5 to all cells was obtained by flow cytometry (CytoFLEX, beckman Coulter inc., brea, USA). In addition, images of Cy 5-labeled tFNA and tFNA-Typ dispersed in cells were obtained by confocal microscopy (N-SIM, nikon, tokyo, japan).
2. Experimental results
Several studies have demonstrated that tFNA is an excellent vector with excellent cell entry properties, which is also one of its most significant advantages. We examined the entry of Cy 5-labeled tFNA and tFNA-Typ into HK-2 cells using flow cytometry and confocal microscopy. According to the results of flow cytometry (FIG. 1 i), it was detected that the cell fractions of both tFNA-Typ and tFNA fluorescence reached more than 95%, with no statistical difference between tFNA and tFNA-Typ. Similar results were obtained from the image of the confocal laser microscope (FIG. 1 j), and Cy 5-labeled tFNA and tFNA-Typ were observed to be widely distributed in the cytoplasm of the cells. This phenomenon suggests that the addition of Typ does not interfere with the excellent cell entry properties of tFNA, which helps tFNA and Typ perform biological functions. Taken together, these results indicate that tFNA is a very promising carrier in the tFNA-type nanodrug delivery system, which can improve the stability and release of the type and maintain excellent cell entry.
Experimental example 3, anti-apoptosis ability of tFNA-Typ in vitro
1. Experimental method
1.1 cell viability after HK-2 cell treatment
Human renal cortex proximal tubular epithelial cells HK-2 cells were cultured in complete medium containing high glucose Dulbecco's modified Eagle's medium, 10% fetal bovine serum (FBS, hyClone, logan, USA) and 1% penicillin-streptomycin solution (HyClone, logan, USA). From previous studies and modeling exploration, a hypoxia/reoxygenation model was adopted: HK-2 cells were seeded in 96-well plates (8000 cells/well). Is cultured in a serum-free sugar-free medium in a low-oxygen incubator for 6 hours (1%O) 2 ，5％CO 2 ，94％N 2 ) The culture was continued for 24 hours in the complete medium of reoxygenation. Cells were reoxygenated while culturing cells for 24h with different feed ratios of tFNA-type prepared in example 1 (feed molar ratio tFNA: type=1:80, 1:120,1:160,1:200, concentration of tFNA 250nM according to previous study), i/R groups without drug addition, control groups were cultured in complete medium at normal oxygen concentration. Cells were then incubated in 10% CCK-8 solution (Keygen Biotech) for 1 hour at 37 ℃. Cell viability was measured by OD at 450 nm.
To verify the effect of tFNA-Typ, typ and tFNA treatment on hypoxia/reoxygenation induced activity of HK-2 cells, cells were cultured for 6 hours prior to culturing in a hypoxia incubator (1%O 2 ，5％CO 2 ，94％N 2 ) Reoxygenation was performed for an additional 24 hours and tFNA (250 nM), type (50 μm), tFNA-type (tFNA: 250nM; typ: 50. Mu.M, without ultrafiltration). Cell viability assays were performed as described above.
1.2 apoptosis assay
Flow cytometry was used to detect apoptosis and HK-2 cells were seeded on 6-well plates. After various treatments we digested cells with EDTA-free trypsin and washed twice with PBS. Apoptosis was detected by flow cytometry by PI and annexin V staining (Millipore Guava, MA, USA).
1.3 Western blot analysis
After different treatments, the samples were rinsed with PBS and treated with protein extraction reagent (KeyGen Biotech, south kyo, china) to extract the proteins. The purified samples were then mixed with loading buffer (beyotidme, shanghai, china) and heated at 100 ℃ for 10 minutes. The target proteins were separated by SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes. Thereafter, PVDF membranes were blocked with blocking solution (Beyotime, shanghai, china) at room temperature for 20 minutes and overnight at 4℃with a solution comprising anti-Caspase 3 primary antibodies (1:1000; abcam, cambridge, UK), anti-Cytocochrome C primary antibodies (1:1000; CST, boston, U.S.) and anti- β -Actin primary antibodies (1:1000; CST, boston, U.S.). After washing with TBST, the membranes were incubated with secondary antibodies (1:5000; beyotidme, shanghai, china) for 1 hour. Finally, protein bands on the membrane were detected by a chemiluminescent detection system (Bio-Rad, hercules, USA).
1.4 immunofluorescent staining
To further observe protein expression, immunofluorescent staining was performed. After treatment as described previously, the samples were fixed in cold 4% paraformaldehyde for 20 minutes followed by treatment with 0.5% triton X-100 for 10 minutes. The samples were then blocked in 5% goat serum for 1 hour and incubated with the target primary antibody overnight at 4 ℃. After 3 washes on the following day, the samples were incubated with secondary antibodies (1:500; invitrogen, carlsbad, USA) for 1 hour. The nuclei were then stained with DAPI and the cytoskeleton with phalloidin. Finally, all samples were observed using a confocal laser microscope (a1rmp+, nikon, tokyo, japan).
2. Experimental results: fig. 2a is a schematic representation of apoptosis of tubular epithelial cells of acute kidney injury. Proximal tubular epithelial cells undergo apoptosis, leading to tubular failure and damage to the blood-urine barrier, which in turn leads to reduced Glomerular Filtration Rate (GFR) and impaired renal function. Thus, studies have demonstrated that reducing proximal tubular epithelial cell apoptosis is an effective method of treating acute kidney injury. To verify the anti-apoptotic properties of tFNA-type we applied a hypoxia/reoxygenation cell model as an in vitro model. First, we performed a cell viability assay to ensure biosafety of tFNA-type, a precondition for cell experiments. As shown in FIG. 2b, the tFNA-Typ group promoted the viability of HK-2 cells, especially compared to the I/R group. This result demonstrates good biosafety of tFNA-type. Notably, promotion of cell viability by tFNA-type exhibited some concentration dose dependence. Specifically, when the concentration of tFNA is 250nM and the concentration of Typ is 40-50. Mu.M, the cell viability of the prepared complex is optimal, and the two concentrations have no statistical difference. Furthermore, we measured the apoptosis of cells treated with different drugs, as shown in fig. 2c, tFNA-type can significantly reduce early and late apoptosis of cells compared with other groups, and has significant statistical significance, and tFNA and type show synergistic effect.
Under normal physiological conditions, most of cytochrome c exists in the inner mitochondrial membrane and participates in ATP synthesis, and when mitochondria are damaged by oxidative stress, the permeability of the outer mitochondrial membrane is increased, and cytochrome c is dissociated into cytoplasm to promote Caspase-3 up-regulation so as to cause apoptosis. The present study investigated the expression of cytochrome c and Caspase-3 by Western blot analysis and immunofluorescent staining, as shown in FIGS. 2d, e, f, tFNA-Typ significantly down-regulated the expression of cytochrome c and Caspase-3 compared to the other groups. The results show that tFNA-Typ has better anti-apoptotic properties than pure tFNA and Typ, and consistent with the previous results, tFNA and Typ show synergistic effect.
Experimental example 4, tFNA-Typ restored renal function and prevented tubular injury
A model of Acute Kidney Injury (AKI) of ischemic reperfusion (I/R) of mice was established, and animal experiments were approved by the university of Sichuan ethical committee. We selected 6-8 week male C57 mice, first isolating the two kidneys and clamping the bilateral renal pedicles for 30 minutes of ischemia. Reperfusion after 30 minutes, intravenous injection of 100. Mu.L of physiological saline, an equal dose of 100. Mu.L of tFNAs, typ or tFNA-Typ (feed ratio 1:200) solution, finally, mice were sacrificed after 24 hours and serum was collected to measure serum creatinine (SCr) and serum urea nitrogen (BUN), and the heart, lung, spleen, liver, kidney were hematoxylin and eosin (H & E) stained, the kidney was PAS stained, and tissue immunofluorescence stained KIM-1, TUNEL stained (FIG. 3 a).
Kidney targeting ability is the basis of AKI treatment. To obtain the biodistribution of tFNA-type in healthy and AKI mice, cy 5-labeled tFNA-type was intravenously injected and detected by IVIS Spectrum imaging system (PerkinElmer). As shown in fig. 3b, fluorescence of AKI group was mainly distributed in liver and kidney, fluorescence was significantly increased and maintained for longer time compared to healthy group, indicating that tFNA-type was rapidly eliminated by healthy kidney but highly absorbed by AKI kidney. The harvested kidneys showed that 2h fluorescence peaked after tFNA-type injection. Proximal tubular injury is the most pronounced manifestation of acute kidney injury. To investigate the tubular targeting ability of tFNA-Typ, we labeled the tubular with LTL and tFNA-Typ with Cy 5. Fluorescence of Cy5 and LTL were highly overlapping (FIG. 3 c), validating the tubular targeting ability of tFNA-Typ. Injury to the tubules may induce dysfunction of the tubules, and decrease GFR, thereby decreasing renal function. Thus, accumulation of tFNA-Typ in the kidney tubules suggests that tFNA-Typ is a potent potential drug for AKI treatment.
To obtain therapeutic effects of tFNA-Typ, I/R injured AKI model mice were divided into four groups, and saline, tFNA, typ and tFNA-Typ were injected, respectively. After 24H we collected serum and examined serum creatinine (SCr) and Blood Urea Nitrogen (BUN) (fig. 3 g), the tFNA-type group was significantly reduced, followed by assessment of AKI histopathological lesions by H & E staining (fig. 3 d) and PAS staining (fig. 3E). To evaluate the tubular injury score, 10 random tissue sections per mouse were evaluated on H & E or PAS staining and semi-quantitative scoring was performed as follows: 0, no damage; 1, <25%;2, 25 to 50%;3, 50 to 75%;4, >75%. The results were averaged for each mouse (fig. 3h, i). Healthy groups showed normal kidney structure. Necrosis was observed in the AKI group, and the clear and exfoliated cells filled the tubules. Focal necrosis was observed in the tFNAs and type groups, while the tFNA-type treated group had significantly reduced lesions and good morphology at the proximal tubule brush border. We further labeled damaged tubules with KIM-1 (fig. 3 f), with little KIM-1 fluorescence in the healthy and tFNA-type treatment group, consistent with the tubular damage score (fig. 3 j).
Our results show that tFNA-Typ can be distributed in the kidney, especially AKI injury kidney, can target the tubular, can obviously reduce tubular injury, and has good treatment effect on acute kidney injury.
tFNA-Typ reduces apoptotic cells, contributing to the restoration of renal function.
AKI-induced oxidative stress typically results in apoptotic cell death. TUNEL assay was performed to measure apoptotic cells in AKI-damaged kidneys. Apoptotic cells were significantly reduced in the tFNAs, type and tFNA-type groups compared to the AKI group. the tFNA-type group still showed the lowest apoptotic cells, indicating the best anti-apoptotic effect of tFNA-type (fig. 4).
Experimental example 5, tFNA-Typ prevented the progression of acute kidney injury to chronic kidney disease
1. Experimental method
1.1 Western blot analysis
HK-2 cells were inoculated in 6-well plates, I/R group, tFNAs group, typ group, TTC group were cultured for 6 hours in a serum-free sugar-free medium in a low-oxygen incubator (1%O) 2 ，5％CO 2 ，94％N 2 ) The culture was continued for 24 hours in the complete medium of reoxygenation. Cell reoxygenation while tFNAs (250 nM), typ (50. Mu.M), TTC (tFNAs-Typ (feed ratio 250 nM-50. Mu.M) were incubated for 24h without drug addition in the I/R group, control group was incubated in complete medium at normal oxygen concentration, the samples were rinsed with PBS and treated with protein extraction reagent (KeyGen Biotech, nanjing, china) to extract the proteins, the purified samples were then mixed with loading buffer (Beyotide, shanghai, china) and heated at 100℃for 10min after separation of the target proteins by SDS-PAGE gel and transfer onto polyvinylidene difluoride (PVDF) membranes, the PVDF membranes were in the chamberBlocking with blocking solution (Beyotime, shanghai, china) for 20min at temperature and overnight at 4℃included anti- α -SMA primary antibodies (ab 124964,1:1000, abcam), anti-Col I primary antibodies (EPR 7785,1:1000, abcam), anti-GAPDH primary antibodies (2118, 1:1000, CST), anti- β -Actin primary antibodies (4970, 1:1000, CST). After washing with TBST, the membranes were incubated with secondary antibodies (1:5000; beyotidme, shanghai, china) for 1 hour. Finally, protein bands on the membrane were detected by a chemiluminescent detection system (Bio-Rad, hercules, USA).
1.2 immunofluorescent staining
To further observe protein expression, immunofluorescent staining was performed. After treatment as described previously, the samples were fixed in cold 4% paraformaldehyde for 20 minutes followed by treatment with 0.5% triton X-100 for 10 minutes. The samples were then blocked in 5% goat serum for 1 hour and incubated with the target primary antibody overnight at 4 ℃. After 3 washes on the following day, the samples were incubated with secondary antibodies (1:500; invitrogen, carlsbad, USA) for 1 hour. The nuclei were then stained with DAPI and the cytoskeleton with phalloidin. Finally, all samples were observed using a confocal laser microscope (a1rmp+, nikon, tokyo, japan).
2. Experimental results: incomplete repair after AKI has been shown to cause renal fibrosis, a pathological accumulation of extracellular matrix (ECM) proteins, principally collagen. The results show (FIGS. 5a,5 b) that protein levels of α -SMA and Col I and WB and immunofluorescent staining were down-regulated after tFNA-Typ treatment, indicating anti-fibrotic capacity of tFNA-Typ. This suggests that tFNA-type may reduce collagen deposition by inhibiting the expression of regulatory fibrosis factors (α -SMA and Col in cells), interrupting the progression of chronic kidney disease.
In conclusion, the invention provides a new application of typha neoglycoside in treating acute kidney injury, and discloses a DNA tetrahedron framework nucleic acid and typha neoglycoside compound, wherein the DNA tetrahedron framework nucleic acid and typha neoglycoside have the effects of synergistically reducing apoptosis of renal cortex proximal tubular epithelial cells, reducing necrosis of renal tissue and recovering renal function, can prevent and treat renal fibrosis, prevent acute kidney injury from developing to chronic kidney disease, and provide a new choice for clinic.
SEQUENCE LISTING
<110> university of Sichuan
<120> complex of DNA tetrahedral framework nucleic acid and typha neoglycoside and preparation of medicine for treating acute kidney injury
Use of the same
<130> GYKH1118-2022P0115470CC
<160> 4
<170> PatentIn version 3.5
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acatgcgagg gtccaatacc gacgattaca gcttgctaca cgattcagac ttaggaatgt 60
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acggtattgg accctcgcat gactcaactg cctggtgata cgaggatggg catgctcttc 60
ccg 63
Claims (6)
1. A compound for treating acute kidney injury is characterized in that the compound is formed by compositing DNA tetrahedron framework nucleic acid and typha neoside; the DNA tetrahedron framework nucleic acid is formed by base complementary pairing of 4 single-stranded DNA molecules with sequences shown as SEQ ID NO. 1-4 respectively; the content of typha neoglycoside in the complex is 34wt%;
the compound is prepared by stirring and ultrafiltering a mixed solution of DNA tetrahedral framework nucleic acid and typha neoglycoside, wherein the stirring is performed at 20-30 ℃ for 5-7 hours; the tetrahedral nucleic acid frame is prepared by adding 4 single-stranded DNA molecules into TM buffer, standing at 95deg.C for 10min, and cooling to 4deg.C for 20min.
2. The complex of claim 1, wherein the DNA tetrahedral framework nucleic acid and typha neoglycoside are fed in a molar ratio of 1 (80 to 200).
3. The complex of claim 2, wherein the DNA tetrahedral framework nucleic acid and typha neoglycoside are fed in a molar ratio of 1:200.
4. A method for preparing a composite according to any one of claims 1 to 3, comprising the steps of:
(1) Preparing a DNA tetrahedron framework nucleic acid solution and a typha neoglycoside solution respectively;
(2) Adding typha neoglycoside solution into DNA tetrahedral framework nucleic acid solution, stirring, and ultrafiltering.
5. The method of claim 4, wherein the solution of DNA tetrahedral framework nucleic acid is prepared by: 4 single-stranded DNA molecules were added to TM buffer, and the mixture was kept at 95℃for 10min, and then the temperature was lowered to 4℃for 20min.
6. Use of a complex according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment of acute kidney injury.
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