CN117323438A - Application of tetrahedron framework nucleic acid-curcumin complex in preparation of medicine for preventing and/or treating diabetic osteoporosis - Google Patents
Application of tetrahedron framework nucleic acid-curcumin complex in preparation of medicine for preventing and/or treating diabetic osteoporosis Download PDFInfo
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Abstract
The invention provides application of tetrahedron framework nucleic acid-curcumin complex in preparing a medicament for preventing and/or treating diabetic osteoporosis, and belongs to the field of medicines. The tetrahedral framework nucleic acid-curcumin compound is prepared from tetrahedral framework nucleic acid and curcumin serving as raw materials, wherein the molar ratio of the tetrahedral framework nucleic acid to the curcumin is 1: (50-800). The tetrahedron framework nucleic acid-curcumin compound improves the water solubility of the natural product curcumin, the stability in physiological body fluid and the in-vivo utilization efficiency, and solves the defects of poor water solubility, low bioavailability, unstable physiological medium and the like of the curcumin. In the obtained compound, curcumin and tetrahedron framework nucleic acid exert a synergistic effect, target acting on NRF2, inhibit iron death of bone marrow mesenchymal stem cells under a diabetic bone microenvironment, exert the effects of reducing blood sugar and promoting bone, can be used for preventing and/or treating diabetic osteoporosis, and has good application prospect.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to application of a tetrahedron framework nucleic acid-curcumin complex in preparing a medicine for preventing and/or treating diabetic osteoporosis.
Background
Diabetic osteoporosis (Diabetic osteoporosis, DOP) is a major cause of concurrent bone mass reduction and destruction of bone tissue microstructure in diabetics and is susceptible to fracture, and is currently considered as a systemic metabolic bone disease. 2021, nat Rev Endocrinol, reported that the fracture risk of diabetics is increased by 32% compared with those without diabetes, and the mortality rate is increased by 1.23 times compared with those with simple type 2 diabetes (T2 DM), bringing a great deal of morbidity and mortality to patients, and causing a great medical burden. In the last decade, the development of changes in the pathology of bone diseases caused by long-term diabetes has been increasing, and 2022, lancet Diabetes Endocrinol, reported that one of the key causes of brittle fracture due to changes in the bone microenvironment is iron death in bone marrow mesenchymal stem cells (bone mesenchymal stem cells, BMSCs), more specifically, the increase in iron death will inhibit the expression of the osteogenic transcription factor osterix (OSX; also called SP 7) and Runt-related transcription factor 2 (RUNX 2), disrupting the balance of osteogenic commitment and differentiation of BMSCs, thereby negatively affecting bone homeostasis. Thus, targeting iron death is a new strategy for treating diabetic osteoporosis.
Iron death is a recently discovered form of apoptosis, distinct from traditional apoptosis, autophagy and pyrodeath, characterized by iron-dependent accumulation and lipid peroxidation. Glutathione peroxidase 4 (GPX 4) is an antioxidant enzyme negatively regulated by endoplasmic reticulum stress (endoplasmic reticulum, ER), which can scavenge excess lipid peroxide, a key upstream regulator of iron death. However, in the diabetic microenvironment, the intracellular GPX4 protein content and activity is significantly reduced, leading to Reactive Oxygen Species (ROS) and Fe 2+ Excessive accumulation of lipid hydroperoxides and iron-death related proteins. Recent studies have shown that iron death inhibitors are directly applied in DOP mouse modelsferrostatin-1 (Fer-1) can reduce bone loss, indicating that iron death is closely related to DOP pathogenesis, and is a potential therapeutic target. Nuclear factor E2-associated factor 2 (NRF 2) can inhibit iron death by up-regulating GPX4 expression. Therefore, the design and development of a powerful GPX4 agonist based on NRF2 can effectively eliminate ROS and regulate lipid peroxidation, inhibit iron death and increase bone formation, and has important significance in relieving diabetes and osteoporosis.
Curcumin is a polyphenol derived from the medicinal plant Curcuma longa and has been reported to activate NRF2. In addition, curcumin has pharmacological actions such as anti-inflammatory, antioxidant, hypolipidemic and osteoclast generation inhibiting in vitro, and has been widely studied. Unfortunately, the circulation time of curcumin is short, repeated administration increases toxic and side effects, and absorption rate and bioavailability are low. Along with the rapid development of the nano technology, the nano material has good biological safety, stability and multifunctional design, and has wide application prospect in the biomedical field. However, iron death nano-inhibitors remain blank in the area of treatment of diabetic osteoporosis.
Tetrahedral framework nucleic acid (tFNA) is self-assembled by temperature-regulated four single-stranded DNA (ssDNA) molecules, capable of freely crossing cell membranes. Has good biocompatibility, safety, editability and stability, and is a DNA nano material with wide application potential. The application No. 202211183683.X provides a drug delivery system for loading curcumin with tetrahedral framework nucleic acid, which enhances the water solubility of curcumin and improves the stability and bioavailability of curcumin, and can be used for preventing or treating radioactive oral mucositis. However, it is not clear whether the tetrahedral framework nucleic acid curcumin-carrying drug delivery system can be well used for treating diabetic osteoporosis, and further research is required.
Disclosure of Invention
The invention aims to provide the application of tetrahedral framework nucleic acid-curcumin complex in preparing a medicament for preventing and/or treating diabetic osteoporosis.
The invention provides application of tetrahedron framework nucleic acid-curcumin complex in preparing a medicament for preventing and/or treating diabetic osteoporosis; the tetrahedral framework nucleic acid-curcumin compound is prepared from tetrahedral framework nucleic acid and curcumin serving as raw materials, wherein the molar ratio of the tetrahedral framework nucleic acid to the curcumin is 1: (50-800).
Further, the medicine is a medicine for inhibiting the death of bone marrow mesenchymal stem cell iron under the micro-environment of diabetic bone.
Further, the drug is a drug that targets NRF2 binding.
Further, the medicament is a medicament for reducing blood sugar and/or promoting bone formation.
Further, the molar ratio of tetrahedral framework nucleic acid to curcumin is 1:100.
further, the tetrahedral framework nucleic acid is formed by base complementary pairing of four DNA single strands, and the sequences of the four DNA single strands are shown in SEQ ID NO. 1-4.
Further, the preparation method of the tetrahedral framework nucleic acid comprises the following steps: after four DNA single strands are equimolar dissolved in TM buffer, the mixture is maintained for 5 to 15 minutes at the temperature of between 85 and 105 ℃ and then maintained for 10 to 30 minutes at the temperature of between 2 and 8 ℃;
preferably, the method for preparing the tetrahedral framework nucleic acid comprises the steps of: four DNA single strands were equimolar dissolved in TM buffer, and maintained at 95℃for 10min and then at 4℃for 20min.
Further, the concentration of Tris-HCl in the TM buffer is 10mM, mgCl 2 Concentration 50mM, pH 8.0; the concentration of the four DNA single strands was 1. Mu.M.
Further, the preparation method of the tetrahedral framework nucleic acid-curcumin complex comprises the following steps:
incubating curcumin and tetrahedral framework nucleic acid according to a molar ratio to obtain the recombinant strain;
preferably, the temperature of the incubation is 20-40 ℃ and the incubation time is 1-8 hours.
Further, the medicament is an injection preparation.
Compared with the prior art, the invention has the beneficial effects that:
the tetrahedron framework nucleic acid-curcumin compound prepared by the invention improves the water solubility of the natural product curcumin, the stability in physiological body fluid and the in-vivo utilization efficiency, and solves the defects of poor water solubility, low bioavailability, unstable physiological medium and the like of the curcumin. In the obtained compound, curcumin and tetrahedron framework nucleic acid exert a synergistic effect, target acting on NRF2, inhibit iron death of bone marrow mesenchymal stem cells under a diabetic bone microenvironment, exert the effects of reducing blood sugar and promoting bone, can be used for preventing and/or treating diabetic osteoporosis, and has good application prospect.
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.
Drawings
FIG. 1 shows the results of synthesis characterization of tetrahedral framework nucleic acid-curcumin complex (tFNA-Cur): a is a synthetic pattern diagram of tFNA-Cur; b is a dissolved image of tFNA, cur and tFNA-Cur; c is a result diagram of verifying successful synthesis of tFNA by PAGE; d is a result diagram of successful synthesis of the tFNA-Cur verified by PAGE; e is the absorption spectrum of tFNA, cur and tFNA-Cur; f is the particle size of tFNA, cur and tFNA-Cur and the Zeta potential result; g is an AFM detection result of tFNA-Cur; h is the encapsulation efficiency of tFNA-Cur; i is a drug release curve of tFNA-Cur; j is 6h of cell entering capacity of Cur and tFNA-Cur; k is the 12h cell entering capacity of Cur and tFNA-Cur; l is the serum stability gel electrophoresis result of tFNA and tFNA-Cur; m is the serum stability statistics of tFNA and tFNA-Cur.
FIG. 2 is a graph showing the effect of tetrahedral framework nucleic acid-curcumin complex (tFNA-Cur) on the osteogenic capacity of BMSCs in a diabetic microenvironment: a is the result of detecting the influence of different concentrations of tFNA on the activity of BMSCs cells by CCK-8; b is the result of CCK-8 detection of the influence of Cur with different concentrations on BMSCs cell activity; c is the result of detecting the influence of different concentrations of tFNA-Cur on the activity of BMSCs cells by CCK-8; d is the result of detecting the influence of AGEs with different concentrations on the activity of BMSCs cells by CCK-8; e is an alkaline phosphatase (ALP) staining test result; f is alizarin red staining detection result; g is the quantitative statistic of alkaline phosphatase (ALP); h is the quantitative statistical result of alizarin red; i is the expression result of RT-qPCR detection osteogenic gene Alp; j is the expression result of RT-qPCR detection osteogenic gene Runx 2; k is the expression result of the osteogenic gene Osx detected by RT-qPCR; l is the expression result of the RT-qPCR detection osteogenic gene Opn; m is the expression result of WB detection osteogenic proteins ALP, RUNX2, OSX and OPN; n is the statistical result of the expression of WB detection osteogenic proteins ALP, RUNX2, OSX and OPN; o is the expression result of the bone formation protein ALP detected by immunofluorescence; p is the result of immunofluorescence detection of the expression of the osteogenic protein RUNX 2; q is the result of immunofluorescence detection of the expression of osteogenic protein OSX.
FIG. 3 is a graph showing the results of activation of the NRF2/GPX4 pathway by tetrahedral framework nucleic acid-curcumin complex (tFNA-Cur) to inhibit iron death: a and E are Reactive Oxygen Species (ROS) fluorescence detection results and statistical results; b and F are fluorescence detection results and statistical results of Mitochondrial Membrane Potential (MMP); c is G, which is the fluorescence detection result and the statistical result of intracellular ferrous ions (Ferroorange); d is a TEM detection mitochondrial morphology change result; h is the expression result of RT-qPCR detection gene Gpx 4; i is the expression result of RT-qPCR detection gene Acsl 4; j is the expression result of RT-qPCR detection gene Nrf 2; k and L are the expression and statistical results of WB detection proteins GPX4, ACSL4, NRF2 and KEAP 1; m is the expression result of the immunofluorescence detection protein GPX 4; n is the expression result of immunofluorescence detection protein ACSL 4; o is the expression result of immunofluorescence detection protein NRF 2; p is a binding site for molecular docking mimicking curcumin (Cur) and NRF2.
FIG. 4 shows the therapeutic effect of tetrahedron framework nucleic acid-curcumin complex (tFNA-Cur) on diabetic osteoporosis: a is a pattern diagram of tFNA-Cur treatment DOP; b is the result of HE section of pancreatic tissue; c and E are femur micro-CT reconstruction and quantitative analysis results; d and F are tibia micro-CT reconstruction and quantitative analysis results; g is the staining result of femur and tibia HE; h is the femur and tibia Masson staining results.
FIG. 5 is a graph showing the results of activation of the NRF2/GPX4 pathway in vivo by tetrahedral framework nucleic acid-curcumin complex (tFNA-Cur) to inhibit iron death and promote bone formation of BMSCs: a and D are immunofluorescence assays for tibial tissue protein ALP expression; b and E are immunofluorescence detection of tibial tissue protein GPX4 expression; c and F are immunofluorescence assays for tibial tissue protein NRF2 expression.
Detailed Description
The materials and equipment used in the embodiments of the present invention are all known products and are obtained by purchasing commercially available products.
Curcumin (Curcumin, cur) is a commercial product with purity of more than 98%.
Example 1 preparation of tetrahedral framework nucleic acid-curcumin complex: tFNA-Cur
The synthetic route for preparing tetrahedral framework nucleic acid-curcumin complexes is shown in fig. 1A.
1. Preparation of tetrahedral framework nucleic acid (tFNA)
The four DNA strands (S1, S2, S3, S4) shown in Table 1 were added to a TM buffer (10 mM Tris-HCl,50mM MgCl) at an equimolar ratio 2 pH 8.0), the final concentration of the four DNA single strands was 1. Mu.M, and the reaction mixture was heated to 95℃for 10min, and then rapidly cooled to 4℃for 20min, to give a tetrahedral framework nucleic acid designated as tFNA.
TABLE 1 sequence of four DNA single strands
2. Preparation of tetrahedral framework nucleic acid-curcumin (tFNA-Cur) Complex
Curcumin was dissolved in cell culture grade DMSO to give a curcumin solution at a concentration of 10mM, 2. Mu.l of curcumin solution (at a concentration of 10 mM) was added to 200. Mu.l of tFNA solution (at a concentration of 1000 nM) (molar ratio of tetrahedral framework nucleic acid to curcumin 1:100), incubated for 3 hours at room temperature with shaking, and then centrifuged with an ultrafiltration centrifuge tube having a molecular weight cut-off of 30kDa to remove residual free curcumin, to give tetrahedral framework nucleic acid-curcumin complex (tFNA-Cur).
3. Tetrahedral framework nucleic acids and related characterization of tetrahedral framework nucleic acid-curcumin complexes
Successful synthesis of tFNA and tFNA-Cur was verified using polyacrylamide gel electrophoresis (PAGE); absorption spectra of tFNA, cur and tFNA-Cur were analyzed using a super-microscopic spectrophotometer (UV 5 Nano, mettler Toledo, switzerland); particle sizes and Zeta potentials of tFNA, cur and tFNA-Cur were measured using a Malvern Zetasizer nano-particle size analyzer (Malvern, UK); three-dimensional nanostructures of tFNA-Cur were observed by Transmission Electron Microscopy (TEM) and atomic particle microscopy.
Tetrahedral framework nucleic acid-curcumin complexes were prepared according to the preparation method described above at molar ratios of 1:50,1:100,1:200,1:400,1:800, respectively (tetrahedral framework nucleic acid concentration 1000nM, 200. Mu.l; curcumin concentration 10mM, 1. Mu.l, 2. Mu.l, 4. Mu.l, 8. Mu.l, 16. Mu.l), and the encapsulation efficiency of the complexes prepared at each curcumin concentration was examined.
Release and stability of tFNA-Cur: PBS (pH 7.4,0.01 mol/L) was used as medium and dialysis bags (30 kDa; solarbio, beijing, china) were divided into an external liquid (30 ml) and an internal liquid (3 ml). Equal molar concentrations of Cur (200. Mu.l, 20. Mu.M) and tFNA-Cur (200. Mu.l, for the preparation of tFNA-Cur, the molar ratio of tetrahedral framework nucleic acid to curcumin was 1:100, and the concentration of curcumin was 20. Mu.M) were dissolved in an internal fluid at 37℃with continuous stirring (150 rpm), and the OD of curcumin released in the external liquid was determined using a ultramicrospectrophotometer at different time points. The stability results were analyzed by agarose gel electrophoresis with different times (0 h,2h,4h,6h,8h,10h,12 h) of incubation of tFNA and tFNA-Cur with 2% or 10% serum, respectively, and finally images of tFNA and tFNA-Cur were taken using an ultraviolet exposure device (Bio-Rad, hercules, USA).
Uptake of Cur and tFNA-Cur by BMSCs: BMSCs were inoculated into confocal dishes (10000 cells) and treated with Cur and tFNA-Cur for 6 and 12 hours. Images taken by Cur and tNNA-Cur cells were taken using a confocal microscope (Olympus, tokyo, japan).
As can be seen from fig. 1A: the tFNA-Cur is synthesized by a two-step method, and the synthesis method is simple and convenient; according to the visual image shown in fig. 1B, curcumin has poor water solubility, tFNA-Cur has good water solubility, so that the water solubility of Cur can be remarkably improved, and the problem that the poor water solubility of Cur limits clinical application is solved.
FIGS. 1C, 1D and 1E illustrate the successful synthesis of tFNA and tFNA-Cur.
FIGS. 1F and 1G illustrate that the prepared tFNA-Cur has a particle size of 40nm, which is between 1 and 100nm, meets the definition of nano-drugs, eliminates the limitation of special biological barriers on the action of the drugs, such as blood brain barrier, blood eye barrier, cell biological membrane barrier and the like, is absorbed by tissues and cells in a pinocytosis way after entering the body through blood circulation, and improves the bioavailability; and tFNA-Cur is negatively charged, so that the biological side effect is small.
FIGS. 1H and 1I illustrate that the tFNA-Cur complex prepared when the molar ratio of tetrahedral framework nucleic acid to curcumin was 1:50,1:100 and 1:200, respectively (tetrahedral framework nucleic acid concentration was 1000nM, 200. Mu.l; curcumin concentration was 10mM, 1. Mu.l, 2. Mu.l and 4. Mu.l), was more efficient in loading tFNA-Cur, 91.8259%, 84.2881% and 82.226%, respectively, and the molar ratio of tetrahedral framework nucleic acid to curcumin was 1:100 was subsequently selected for further investigation in combination with the results of FIG. 3C. The tFNA-Cur compound prepared by the invention is slowly released in a physiological environment and continuously plays a biological role.
FIGS. 1J and 1K illustrate that cells have strong cell entry capacity for tFNA-Cur, have better bioavailability than free Cur, and can more effectively exert the biological effect of Cur.
As can be seen from fig. 1L and 1M: the tFNA-Cur improves the stability of Cur in physiological fluids.
The beneficial effects of the present invention are demonstrated by specific test examples below.
Test example 1 tetrahedral framework nucleic acid-curcumin Complex (tFNA-Cur) promotes osteogenic ability of BMSCs in diabetic microenvironment
1. Experimental method
Effect of different concentrations of tFNA, cur and tFNA-Cur on BMSCs activity: BMSCs were seeded in 96-well plates at 20000 cells/well density, 37℃at 5% CO 2 After 24 hours of culture, tFNA, cur and tFNA-Cur with different concentrations are added for further culture for 12 hours, or AGEs with different concentrations are added for further culture for 24 hours, and then CCK8 is added for cell activity detection. tFNA and tFNA-Cur were prepared as described in example 1, with a molar ratio of tetrahedral framework nucleic acid to curcumin of 1:100 (tetrahedral framework nucleic acid concentration 1000nM, 200. Mu.l; curcumin concentration 10mM, 2. Mu.l).
The glycosylated end products (advanced glycation end products, AGEs) are formed by non-enzymatic saccharification of ketoses such as glucose, fructose and glucose-6-phosphate with protein amino groups, which are irreversibly formed. Diabetes, inflammation, aging and other diseases can accelerate the generation and accumulation of AGEs in the body; accumulation of AGEs can exacerbate diabetes, inflammation, aging, etc., thereby forming a vicious circle, ultimately leading to the occurrence of various diabetic chronic complications. Treatment of BMSCs with AGEs simulates the diabetic bone microenvironment in vitro.
Alkaline phosphatase (ALP) and alizarin red staining: BMSCs were seeded in 6-well plates at 200000 cells/well density, 37℃at 5% CO 2 BMSCs were pretreated with tFNA-Cur (molar ratio of tetrahedron framework nucleic acid to curcumin 1:100) prepared in example 1 for 24 hours of incubation, followed by replacement to osteogenic medium, while BMSCs were immobilized with 150. Mu.g/mLAGEs for 7 days, 4% paraformaldehyde (4 ℃,20 min) and stained with ALP stain (C3250S, beyotime, china) for 10 minutes at 37 ℃. After the same treatment for 14 days, the cells were incubated with alizarin red dye solution for 5 minutes at room temperature. ALP activity and mineralized nodules were observed under an optical microscope and photographed.
Real-time fluorescent quantitative RT-PCR analysis: BMSCs were seeded in 6-well plates at 200000 cells/well density, 37℃at 5% CO 2 The BMSCs were pretreated with tFNA-Cur (molar ratio of tetrahedral framework nucleic acid to curcumin 1:100) prepared in example 1 for 24 hours of incubation, followed by replacement with osteogenic medium, while being treated with 150. Mu.g/mLAGEs for 7 days. Reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate the transcript levels of Alp, runx2, osx and Opn. Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, MA, USA). UsingcDNA synthesis was performed by Premix Ex Taq II (Perfect Real Time kit; takara, dalian, china). Use of +.o in Q7>Green I master mix (ABIQuantum studio 7,Thermo Fisher,USA) measures target genes. Use 2 -ΔΔCT The results were analyzed by the relative quantification method, and beta-action was used as a control gene. All experiments were repeated three times.
Western blot analysis (WB): BMSCs were treated with RT-PCR. BMSCs were lysed using a cell protein extraction reagent (KEYGEN Biotech, nanjing, china) and then mixed with loading buffer at a ratio of 4:1 (v/v) and then boiled for 5 minutes. Proteins were separated using 10% SDS-PAGE and subsequently transferred to PVDF membrane. Then blocked with blocking buffer (Thermo Fisher Scientific, MA, USA) for 10min, incubated against ALP (1:500, hua ' an organism, zhejiang, china), against RUNX2 (1:500, hua ' an organism, zhejiang, china), against OSX (1:500, hua ' an organism, zhejiang, china), against OPN (1:1000, abcam, cambridge, UK) overnight at 4 ℃. The following day, after washing with TBST, the strips were incubated with secondary anti-rabbit antibodies (1:3000, BEYOTIME, shanghai, china) for 1 hour. The results were visualized and quantitatively analyzed using ECL chemiluminescent detection system (Bio-Rad, hercules, calif., USA).
Immunofluorescence (IF) staining: BMSCs were inoculated into confocal dishes at a density of 50000 cells/well, 37℃and 5% CO 2 The BMSCs were pretreated with tFNA-Cur (molar ratio of tetrahedral framework nucleic acid to curcumin 1:100) prepared in example 1 for 24 hours of incubation, followed by replacement with osteogenic medium, while being treated with 150. Mu.g/mLAGEs for 4 days. BMSCs were immobilized using 4% paraformaldehyde solution (4 ℃,25 min), perforated on cell membranes with 0.5% Triton X-100 (RT, 20 min), blocked with 5% sheep serum (37 ℃,20 min). They were then incubated with osteogenic related antibodies (ALP, RUNX2 and OSX) overnight at 4 ℃. The next day, BMSCs were incubated with secondary anti-rabbit antibodies (1:200, invitrogen, carlsbad, USA) for 1 hour at 37 ℃. Ghost for cytoskeletonPen cyclic peptide (FITC) staining (37 ℃,20 min) and nuclei were stained with DAPI (37 ℃,10 min). Images were taken using a confocal microscope (Olympus, tokyo, japan).
2. Experimental results
FIGS. 2A-2D show the effect of various drugs at various concentrations on cell activity, and the experimental results demonstrate that tFNA and Cur present a concentration inhibition on BMSCs, with optimal cell activity when Cur is 20. Mu.M and tFNA is 200 nM. The complex tFNA-Cur prepared with Cur of 20. Mu.M and tFNA of 200nM was selected for further experiments. The AGEs have concentration dependence on BMSCs, the low-concentration AGEs promote the proliferation of the BMSCs, the high-concentration AGEs inhibit the proliferation of the BMSCs, when the concentration of the AGEs is 150 mug/ml, the BMSCs have good activity, and 150 mug/ml AGEs are selected for subsequent experiments. In fig. 2C, when tFNA: when Cur is 1:100, the concentration of tFNA for preparing tFNA-Cur is 200nM, and Cur is 20 mu M, which is the optimal concentration ratio, and the cell activity is optimal.
FIGS. 2E and 2G show alkaline phosphatase (ALP) staining results, FIGS. 2F and 2H show alizarin red staining results, and from the results of FIGS. 2E-2H, it can be seen that 150 μg/ml AGEs treated BMSCs simulate a diabetic microenvironment with reduced osteogenic capacity and reduced calcium nodule formation; after the tFNA, cur and tFNA-Cur pretreatment, the osteogenesis ability and calcium nodule formation of BMSCs are reversed, wherein the effect of the tFNA-Cur treatment group is obviously better than that of the pure tFNA and Cur group.
FIGS. 2I-2L are results of RT-qPCR detection of the expression of osteogenic genes Alp, runx2, osx and Opn, which indicate that in the diabetes microenvironment, BMSCs osteogenic gene expression is significantly inhibited, tFNA, cur and tFNA-Cur are pretreated to reverse the osteogenic gene expression, wherein the tFNA-Cur treatment group contributes to the best effect of bone gene expression, cur is inferior, and tFNA group is also reversed to a certain extent.
FIGS. 2M and 2N show the results of WB assay for the expression of the osteogenic proteins ALP, RUNX2, OSX and OPN, which demonstrate reduced osteogenic protein expression in the diabetic microenvironment; after pretreatment of tFNA, cur and tFNA-Cur, the expression of ALP, RUNX2, OSX and OPN was reversed, wherein the effect of the tFNA-Cur treatment group was significantly better than that of the pure tFNA and Cur groups.
FIGS. 2O, 2P and 2Q show that the results of immunofluorescence detection on the expression of the osteogenic proteins ALP, RUNX2 and OSX are consistent with the results of WB detection on the expression of the proteins, and the results show that tFNA-Cur can significantly reverse the expression of the osteogenic proteins inhibited in the microenvironment of diabetes mellitus, and the effects are superior to those of the pure tFNA and Cur groups.
The results in FIG. 2 show that after tFNA-Cur pretreatment, the impaired osteogenesis of BMSCs in the diabetic microenvironment was reversed, the expression of osteogenesis-related genes and proteins ALP, RUNX2, OSX and OPN increased, calcium nodule formation increased, and the effect was significantly better than that of pure tFNA and free Cur.
Test example 2 tetrahedral framework nucleic acid-curcumin complex (tFNA-Cur) activating NRF2/GPX4 pathway to inhibit iron death
1. Experimental method
Reactive Oxygen Species (ROS) and Mitochondrial Membrane Potential (MMP) level detection: BMSCs were first pretreated with tFNA-Cur (in the preparation of tFNA-Cur, the molar ratio of tetrahedral framework nucleic acid to curcumin was 1:100, cur was 20. Mu.M, tFNA was 200 nM) for 12 hours, followed by 150. Mu.g/mLAGEs for 24 hours. Subsequently, the cells were incubated with Hoechst 33342 (1X, C1028, beyotime, china) for 10 minutes and with DCFH-DA (10. Mu.M, S0033S, beyotime, china) for 20 minutes to detect ROS levels. In addition, they were incubated with rhodamine 123 (1 x, c 20088 s, beyotime, china) for 20 minutes to assess MMP levels. After washing with PBS, images of ROS and MMP were obtained using confocal microscopy.
Fe 2+ Detection (FerroOrange): using Fe 2+ Probe (1. Mu.M, MKBio, mx 4559) for assessment of intracellular Fe 2+ Horizontal. After the same treatment as described above, hoechst 33342 was incubated for 10 minutes and Ferroorange was incubated for 20 minutes. Subsequently, fluorescence images of BMSCs were captured using a confocal microscope.
Mitochondrial morphology changes were observed by transmission electron microscopy: after the above treatment, BMSCs were fixed with 3% glutaraldehyde solution at 4℃for 16 hours. Subsequently, BMSCs were dehydrated using acetone, embedded in Epon812, sectioned (60-90 nm), and stained with uranium acetate and lead citrate. Finally, mitochondrial morphology was examined and captured for each treatment group using a JEM-1400FLASH transmission electron microscope.
GPX4, ACSL4, NRF2 and KEAP1 genes and protein level detection: BMSCs were first pretreated with tFNA-Cur (in the preparation of tFNA-Cur, the molar ratio of tetrahedral framework nucleic acid to curcumin was 1:100, cur was 20. Mu.M, tFNA was 200 nM) for 12 hours, followed by 150. Mu.g/mLAGEs for 24 hours. The detection method is the same as that of test example 1.
Cur and NRF2 molecular docking: NRF2 prediction structures are generated by Alphafold. The protonated state of the small molecule is set to ph=7.4 and Cur is extended to a 3D structure using Open Babel. A series of preparations of receptor proteins and ligands were made using the AutoDock tool (ADT 3). The docking box was generated through an autoprid program, followed by molecular docking using an autopock Vina (1.2.0). The optimal binding conformation is selected for interaction analysis. Finally, a protein-ligand interaction map was generated by PyMOL. NRF2 protein is represented as a cartoon model in dark blue, ligand as a club model in cyan, and their binding sites as club structures in magenta. The nonpolar hydrogen atoms are omitted. Hydrogen bonding, ionic interactions, hydrophobic interactions are depicted as yellow, magenta and green dashed lines, respectively.
2. Experimental results
FIGS. 3A and 3E show Reactive Oxygen Species (ROS) detection results, FIGS. 3B and 3F show Mitochondrial Membrane Potential (MMP), and FIGS. 3C and 3G show intracellular ferrous ion (Ferroorange) detection results, from which 150 μg/ml AGEs treat BMSCs to produce excessive ROS, mitochondrial membrane permeability change, intracellular Fe in a diabetic-mimetic microenvironment 2+ Excessive accumulation. After tFNA, cur and tFNA-Cur pretreatment, the method can remove the excessively accumulated ROS and Fe 2+ The effects of the tFNA-Cur group are superior to those of the pure tFNA and free Cur.
Fig. 3D is a TEM examination of mitochondrial morphology changes, which demonstrates mitochondrial atrophy, dense aggregation and mitochondrial cristae reduction in the diabetic microenvironment. the pretreatment of tFNA, cur and tFNA-Cur can reverse mitochondrial change, and the tFNA-Cur group has the best effect.
Fig. 3H-3J show the results of expression of genes Gpx4, acsl4, nrf2 in sequence, and the experimental results show that the expression of iron death related genes Gpx4, acsl4, nrf2 is significantly inhibited in the diabetes microenvironment, and after the pretreatment of tFNA, cur and tFNA-Cur, the gene expression level is reversed, wherein the effect of the tFNA-Cur group is superior to that of the simple tFNA and free Cur.
FIGS. 3K and 3L show the results of WB detection proteins GPX4, ACSL4, NRF2 and KEAP1, which demonstrate that the expression of iron death-related proteins GPX4, ACSL4 and NRF2 is significantly inhibited in the diabetic microenvironment, and the protein expression levels are reversed after pretreatment of tFNA, cur and tFNA-Cur, wherein the effects of the tFNA-Cur group are superior to those of the pure tFNA and free Cur.
FIGS. 3M, 3N and 3O show the results of immunofluorescence detection of the expression of proteins GPX4, ACSL4 and NRF2, which are consistent with the experimental results and WB results.
Fig. 3P is a molecular docking mimicking the binding sites of curcumin (Cur) and NRF2, which results demonstrate that Cur and NRF2 directly form multiple sets of interactions, such as Asp408 of NRF2 forming hydrogen bonds with Cur. Under the action of the interaction forces, the binding energy of the protein small molecule complex is-7.1 kcal/mol, the whole performance is excellent, and Cur activates NRF2 to play a biological role.
The results of fig. 3 show that: BMSCs increased iron death in a diabetic microenvironment simulated by 150 μg/mL AGEs treated BMSCs, and was mainly represented by: lipid peroxidation (increased ROS expression), fe 2+ Increased levels, inhibited GPX4 expression, altered mitochondrial morphology (mitochondrial atrophy, increased membrane permeability, loss of cristae). Through molecular docking simulation, the Cur in the tFNA-Cur forms a hydrogen bond with Asp408 of NRF2, the binding energy is-7.1 Kcal/mol, and NRF2 is a target protein of an iron death inhibition pathway, so that the synthesized tFNA-Cur can inhibit iron death in a targeted manner. Experimental results also demonstrate that after tFNA-Cur pretreatment, cur activates NRF2 expression, up-regulating GPX4, thereby inhibiting iron death. More exciting, the effect of tFNA-Cur on inhibiting iron death is significantly better than that of free Cur.
Test example 3 treatment of diabetic osteoporosis with tetrahedral framework nucleic acid-curcumin complex (tFNA-Cur)
1. Experimental method
Animal experiment: all animal experiments were approved by the animal ethics committee of the oral hospital, university Hua Xi, sichuan. Male C57BL/6J mice, 4 weeks old, were purchased from Jiuzhikang (Nanjing, china) and raised under pathogen-free conditions of 55% + -5% humidity and 24+ -2deg.C. First, mice were randomly divided into five groups: control, DOP, dop+tfna, dop+curcumin and dop+tfna-Cur (n=6). Throughout the experiment, control mice were fed a normal diet (10% kcal from fat), while model and treatment groups were fed a high-fat diet (HFD) (60% kcal from fat). After four weeks, all groups except the control group were intraperitoneally injected with Streptozotocin (STZ) (35 mg/kg) for 7 days to induce diabetes. Control mice received citrate buffer injection. Subsequently, mice with high blood glucose levels (11.1 mmol/L) accompanied by polyphagia, polydipsia and diuresis were considered diabetic mice for subsequent experiments. The treatment group was intraperitoneally injected with tFNA (1. Mu.M, 200. Mu.L), curcumin (40. Mu.M, 200. Mu.L) or tFNA-Cur (tFNA: 1. Mu.M, cur: 40. Mu.M, 200. Mu.L) three times per week for 8 weeks when preparing tFNA-Cur at a concentration of 1. Mu.M and Cur at a concentration of 40. Mu.M. In addition, the control group and DOP group were injected with physiological saline (0.9%, 200. Mu.L). Body weight and fasting blood glucose levels were monitored every two weeks. Finally, pentobarbital anesthetized mice to euthanasia, and long bones (femur and tibia), blood, pancreas, heart, liver, spleen, lung, kidney, and other tissues were collected for subsequent experiments.
Micro-CT analysis: the femur and tibia were scanned using a SCANCO medical microcomputer tomography 50 (70 kv,200 ua, 300ms,10 μm). A region of interest (ROI) of the femur and tibia was determined at a position 2mm below the epiphysis.
Bone tissue and serum AGEs examination: the blood sample was left at 4℃for 30min and then centrifuged at 3000rpm for 10min to obtain serum. The levels of AGEs in serum and bone samples were determined using a mouse AGEs ELISA kit (yww-20124, shanghai, china).
Histological analysis (H & E, masson and IF): specimens from different treatment groups were fixed with 4% paraformaldehyde for 72 hours. After decalcification for one month, the bones were dehydrated, embedded in paraffin, and sectioned (3 μm thick). The sections were then stained with H & E, masson and IF to observe tissue morphology, structural components and protein expression.
2. Experimental results
FIG. 4B shows the results of HE sections of pancreatic tissue, and FIG. 4B shows that islets of the model group of diabetes osteoporosis are destroyed and inflammatory cells infiltrate; the tFNA, cur and tFNA-Cur treatment groups were reversed, with tFNA-Cur treatment being most effective.
FIGS. 4C and 4E are results of femur micro-CT reconstruction and quantitative analysis, from which it is known that the mice of the modeling group develop severe osteoporosis, bone trabecular absorption, fracture; the tFNA, cur and tFNA-Cur treatment groups reversed bone destruction, with tFNA-Cur treatment being most effective.
Fig. 4D and 4F show results of tibial micro-CT reconstruction and quantitative analysis, where the trend of the tibial experimental results is consistent with that of femur, and the tFNA-Cur treatment group can significantly reduce osteoporosis caused by diabetes and increase bone formation.
Fig. 4G shows the results of femur and tibia HE staining, and fig. 4H shows the results of femur and tibia Masson staining, from which it is known that the modeled group of mice bone trabeculae absorb, break, and bone marrow cavity fat increases; the tFNA, cur and tFNA-Cur treatment groups were able to reverse bone destruction and reduce bone marrow cavity fat, with the tFNA-Cur group being most effective.
FIGS. 5A and 5D are results of immunofluorescence detection of ALP expression of tibial tissue protein, which demonstrate that osteoblast protein expression was significantly reduced in modular mice and a large amount of bone marrow cavity fat was accumulated; after treatment of tFNA, cur and tFNA-Cur, the expression of the osteogenic protein in the body is improved, wherein the tFNA-Cur group has the best effect.
FIGS. 5B and 5E are results of immunofluorescence detection of tibial tissue protein GPX4 expression, which demonstrate that GPX4 protein expression is significantly reduced in a model mouse; tFNA, cur and tFNA-Cur treated with the composition can reverse iron death in the microenvironment of diabetes, and the tFNA-Cur has the best effect.
FIGS. 5C and 5F show the results of immunofluorescence detection of NRF2 expression of tibial tissue protein, which demonstrate that Cur and tFNA-Cur significantly activate NRF2 protein expression, and that tFNA-Cur has an effect superior to that of free Cur.
The results of fig. 4 and 5 confirm that the tFNA-Cur of the present invention achieves excellent effects in the treatment of diabetic osteoporosis, and verify that the therapeutic effects of tFNA-Cur are significantly superior to those of tFNA alone and free Cur by targeted activation of NRF2/GPX4 pathway to inhibit iron death, promote osteogenesis of BMSCs, and increase bone formation in vitro.
In conclusion, the tetrahedron framework nucleic acid-curcumin compound is prepared, the compound improves the water solubility of the natural product curcumin, the stability in physiological body fluid and the in-vivo utilization efficiency, and overcomes the defects of poor water solubility, low bioavailability, unstable physiological medium and the like of the curcumin. In the obtained compound, curcumin and tetrahedron framework nucleic acid exert a synergistic effect, target acting on NRF2, inhibit iron death of bone marrow mesenchymal stem cells under a diabetic bone microenvironment, exert the effects of reducing blood sugar and promoting bone, can be used for preventing and/or treating diabetic osteoporosis, and has good application prospect.
Claims (10)
1. Use of a tetrahedral framework nucleic acid-curcumin complex for the preparation of a medicament for the prevention and/or treatment of diabetic osteoporosis; the tetrahedral framework nucleic acid-curcumin compound is prepared from tetrahedral framework nucleic acid and curcumin serving as raw materials, wherein the molar ratio of the tetrahedral framework nucleic acid to the curcumin is 1: (50-800).
2. Use according to claim 1, characterized in that: the medicine is used for inhibiting the death of bone marrow mesenchymal stem cell iron under the microenvironment of diabetic bones.
3. Use according to claim 2, characterized in that: the drug is a drug which is targeted to bind NRF2.
4. Use according to claim 1, characterized in that: the medicine is a medicine for reducing blood sugar and/or promoting bone formation.
5. Use according to any one of claims 1 to 4, characterized in that: the molar ratio of the tetrahedral framework nucleic acid to curcumin is 1:200.
6. use according to any one of claims 1 to 4, characterized in that: the tetrahedral framework nucleic acid is formed by four DNA single chains through base complementation pairing, and the sequences of the four DNA single chains are shown in SEQ ID NO. 1-4.
7. Use according to claim 6, characterized in that: the preparation method of the tetrahedral framework nucleic acid comprises the following steps: after four DNA single strands are equimolar dissolved in TM buffer, the mixture is maintained for 5 to 15 minutes at the temperature of between 85 and 105 ℃ and then maintained for 10 to 30 minutes at the temperature of between 2 and 8 ℃;
preferably, the method for preparing the tetrahedral framework nucleic acid comprises the steps of: four DNA single strands were equimolar dissolved in TM buffer, and maintained at 95℃for 10min and then at 4℃for 20min.
8. Use according to claim 7, characterized in that: the concentration of Tris-HCl in the TM buffer solution is 10mM, mgCl 2 Concentration 50mM, pH 8.0; the concentration of the four DNA single strands was 1. Mu.M.
9. Use according to any one of claims 1 to 4, characterized in that: the preparation method of the tetrahedral framework nucleic acid-curcumin complex comprises the following steps:
incubating curcumin and tetrahedral framework nucleic acid according to a molar ratio to obtain the recombinant strain;
preferably, the temperature of the incubation is 20-40 ℃ and the incubation time is 1-8 hours.
10. Use according to any one of claims 1 to 4, characterized in that: the medicine is an injection preparation.
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