Use of shikonin in preparation of drug for promoting tissue regeneration

Abstract

The use of shikonin in the preparation of a drug for promoting tissue regeneration. In a Danio rerio caudal fin excision experiment, the addition of shikonin in the treatment of a model can upregulate the expression level of a gene related to Danio rerio caudal fin regeneration, and increase the number of proliferating cells during Danio rerio caudal fin regeneration. The new use of shikonin for realizing tissue regeneration widens the application field of shikonin, and provides a reference basis for tissue regeneration.

Description

Application of shikonin in preparing medicine for promoting tissue regeneration Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of shikonin in the preparation of a medicine for promoting tissue regeneration. Background Art

[0002] In recent years, the zebrafish (Danio rerio), known for its remarkable regenerative capacity of its caudal fin, has been developed as a commonly used model for limb regeneration and wound repair. In this organism, the regeneration of several tissues, including the caudal fin, relies on a robust non-morphogenic regenerative process, typified by the formation of a blastema. This blastema, composed of highly proliferative cells, can regenerate and fully grow a lost limb within a few days. Consequently, the zebrafish caudal fin regeneration model is widely used for drug screening and efficacy evaluation related to limb regeneration and wound repair. While zebrafish caudal fin regeneration models cannot replace mammalian models for drug functional characterization, they can nonetheless provide a cost-effective bridge between cell-based assays and mammalian models.

[0003] Shikonin is a perennial herbaceous plant in the Boraginaceae family. Oils and ointments containing shikonin as the main ingredient can be used clinically to treat burns and as skin care products. Shikonin can also be used as a food additive. Dried shikonin extracts have been used in traditional Chinese medicine to treat various diseases, including inflammation and cancer. Its key active ingredient is the naphthoquinone shikonin. In recent years, shikonin has been shown to be effective in treating obesity, inhibiting the proliferation of human lung adenocarcinoma cells, inducing apoptosis and autophagy in human liver and pancreatic cancer cells, and reducing immune rejection. However, the effects and mechanisms of shikonin on tissue regeneration have not been reported.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide the application of shikonin in medicine for promoting tissue regeneration, broaden the application field of shikonin, and provide a theoretical basis for studying the regeneration of severed limbs.

[0006] To achieve the above objectives, the first aspect of the present invention provides the use of shikonin in the preparation of a medicament for promoting tissue regeneration.

[0007] In some embodiments of the present invention, the tissue comprises zebrafish tail fin tissue.

[0008] In some embodiments of the present invention, the drug upregulates the expression level of genes related to zebrafish tail fin regeneration.

[0009] In some embodiments of the present invention, the zebrafish tail fin regeneration-related genes include the lef1 gene, the msxb gene, and the shha gene.

[0010] In some embodiments of the present invention, the drug increases the number of proliferating cells during the regeneration of the zebrafish tail fin.

[0011] In the present invention, shikonin was purchased from Beijing Solebow Technology Co., Ltd. with a product number of SS8500 and a CAS number of 517-88-4. Wild-type (AB) adult zebrafish at 3 months post fertilization (mpf) were used as the material.

[0012] In the present invention, three-month-old, sexually mature wild-type zebrafish were selected and anesthetized and fixed using an anesthetic. The selected anesthetic was 0.22% by volume of Ethyl 3-aminobenzoate methanesulfonate (Sigma, E10521). Anesthesia and fixation ensured that the zebrafish were pain-free during surgery and remained fixed for subsequent procedures.

[0013] In some embodiments of the present invention, a sterile, sharp scalpel is used to remove half of the tail fin, ensuring a smooth incision. Following surgery, the fish are quickly transferred to a culture system for cultivation, and a freshly prepared 1 mg / mL shikonin stock solution is added to the culture tank. The final concentration of shikonin (Solarbio, SS8500) is 85 μg / L, and 4-7 adult wild-type zebrafish with tails removed are placed in each tank.

[0014] During the post-operative culture period, the animals were fed and cultured normally, and anesthetized and fixed with anesthetics 3 days post amputation (dpa) and 7 days post amputation, respectively. Subsequently, a Leica stereomicroscope (Leica M205FA) was used to photograph the phenotypic images of zebrafish tail fin regeneration at 3 and 7 dpa. The regeneration area at 3 and 7 dpa was then calculated using the evaluation formula: the ratio of regeneration area to total area (Number = tail fin tissue regeneration area / total area). The data were then statistically analyzed to assess whether shikonin could promote the regeneration of damaged tail fin tissue in adult wild-type zebrafish. The final experimental results showed that shikonin promoted the regeneration of zebrafish limbs.

[0015] A second aspect of the present invention provides a drug for promoting tissue regeneration, comprising shikonin.

[0016] In some embodiments of the present invention, shikonin is the sole active ingredient or one of the active ingredients of the drug.

[0017] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0018] In some embodiments of the present invention, the drug is a liquid preparation.

[0019] In some specific embodiments of the present invention, the shikonin is dissolved in a liquid solvent to prepare the drug, and the concentration of shikonin is 70-100 μg / L.

[0020] The present invention has the following beneficial effects: By constructing a zebrafish tail fin amputation model and adding shikonin to the model, it was found that shikonin can upregulate the expression levels of genes related to zebrafish tail fin regeneration, increase the number of proliferating cells during zebrafish tail fin regeneration, and have no effect on apoptotic cells. Shikonin was found to promote zebrafish tail fin tissue regeneration. This discovery of a new use of shikonin for tissue regeneration broadens the application field of shikonin.

[0021] The present invention promotes the regeneration of zebrafish severed limbs by targeting shikonin, providing a theoretical basis for a deeper understanding of the mechanism of action of shikonin and for the field of human limb injury repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly describes the necessary drawings. Obviously, the drawings described below only represent some embodiments of the present invention. Those skilled in the art can derive other relevant drawings based on these drawings without inventive effort.

[0023] FIG1 shows a calculation method for evaluating the regeneration degree of the adult zebrafish tail fin according to the present invention; wherein the double arrows indicate the amputation incision; the A1 area is the regenerated part; and the A2 area is the remaining part of the tail after amputation.

[0024] FIG2 is a diagram showing the caudal fin regeneration phenotype of the blank group and the shikonin-treated group of adult wild-type zebrafish after tail amputation at 3 dpa and 7 dpa in the present invention;

[0025] Among them, Figure 2 (a) represents the bright field phenotype image of the zebrafish with cut tails 3 days after breeding (3dpa); Figure 2 (b) represents the enlarged bright field phenotype image of the red area in Figure 2 (a); Figure 2 (c) represents the bright field phenotype image of the zebrafish with cut tails 7 days after breeding (7dpa); Figure 2 (d) represents the bright field phenotype image of the zebrafish with cut tails 3 days after treatment with shikonin (3dpa); Figure 2 (e) represents the enlarged bright field phenotype image of the red area in Figure 2 (d); Figure 2 (f) represents the bright field phenotype image of the zebrafish with cut tails 7 days after treatment with shikonin (7dpa).

[0026] FIG3 is a statistical diagram of the number values ​​during the regeneration process of the adult wild-type zebrafish tail fin excision in the present invention (n=7, mean±sem).

[0027] FIG4 shows the detection results of genes related to zebrafish blastema development.

[0028] FIG5 is a schematic diagram of the expression levels of zebrafish blastema development-related genes shown by in situ hybridization;

[0029] Among them, Figure 5 (a) is the expression level of gene lef1 at the bud base of the control group shown by in situ hybridization; Figure 5 (b) is the expression level of gene lef1 at the bud base of the shikonin-treated group shown by in situ hybridization; Figure 5 (c) is the expression level of gene msxb at the bud base of the control group shown by in situ hybridization; Figure 5 (d) is the expression level of gene msxb at the bud base of the shikonin-treated group shown by in situ hybridization; Figure 5 (e) is the expression level of gene shha at the bud base of the control group shown by in situ hybridization; Figure 5 (f) is the expression level of gene shha at the bud base of the shikonin-treated group shown by in situ hybridization.

[0030] FIG6 is a graph showing the number of proliferating cells in the control group and the shikonin-treated group developed with PCNA antibodies;

[0031] Among them, Figure 6 (a) is the number of cell nuclei at the blastema of the control group displayed by antibody staining DIPA; Figure 6 (b) is the number of proliferating cells at the blastema of the control group displayed by antibody staining PCNA; Figure 6 (c) is the superposition of the number of cell nuclei and the number of proliferating cells at the blastema of the control group displayed by antibody staining; Figure 6 (d) is a white light image of the number of proliferating cells at the blastema of the shikonin-treated group displayed by antibody staining DIPA; Figure 6 (f) is the number of proliferating cells at the blastema of the shikonin-treated group displayed by antibody staining PCNA; Figure 6 (g) is the superposition of the number of cell nuclei and the number of proliferating cells at the blastema of the shikonin-treated group displayed by antibody staining; Figure 6 (h) is a white light image of the number of proliferating cells at the blastema of the shikonin-treated group displayed by antibody staining.

[0032] FIG7 is a bar graph showing the number of proliferating cells during tail fin regeneration. DETAILED DESCRIPTION

[0033] The present invention is described in detail below in conjunction with specific examples, but should not be construed as limiting the present invention. The test methods in the following examples where specific conditions are not specified are usually performed under conventional conditions and are not described in detail because they do not relate to the invention.

[0034] Shikonin is a perennial herbaceous plant in the Boraginaceae family. Oils and ointments containing shikonin as the main ingredient can be used clinically to treat burns and as skin care products. Shikonin can also be used as a food additive. Dried shikonin extracts have been used in traditional Chinese medicine to treat various diseases, including inflammation and cancer. Its key active ingredient is the naphthoquinone shikonin. In recent years, shikonin has been shown to be effective in treating obesity, inhibiting the proliferation of human lung adenocarcinoma cells, inducing apoptosis and autophagy in human liver and pancreatic cancer cells, and reducing immune rejection. However, the effects and mechanisms of shikonin on tissue regeneration have not been reported.

[0035] The present invention provides the use of shikonin in the preparation of a drug for promoting tissue regeneration. By establishing a zebrafish tail fin tissue excision model and administering shikonin to the amputated zebrafish, the present invention found that shikonin upregulated the expression levels of genes associated with tail fin regeneration and increased the number of proliferating cells during tail fin regeneration. This invention is the first to discover a new use of shikonin for tissue regeneration, broadening its application and providing a reference for tissue regeneration.

[0036] Example 1 Construction of zebrafish tail fin tissue excision model

[0037] Wild-type (AB) adult zebrafish, 3 months post-fertilization (mpf), were used as materials. They were anesthetized and fixed with 0.22% anesthetic (Ethyl 3-aminobenzoate methanesulfonate, Sigma, E10521). Half of the caudal fin was then removed using a sterile sharp scalpel under a microscope, creating a smooth incision.

[0038] Example 2 Zebrafish tail fin tissue regeneration experiment

[0039] After the surgery, the fish were quickly transferred to the aquaculture system for culture. A freshly prepared 1 mg / L shikonin stock solution was added to the aquarium. The shikonin (Solarbio, SS8500, CAS number 517-88-4) was added to a final concentration of 85 μg / L. Four to seven adult wild-type zebrafish, tail-cut, were placed in each aquarium and fed normally. The fish were then anesthetized and fixed with anesthetic at 3 days post-amputation (dpa) and 7 days post-amputation. Images of the caudal fin regeneration phenotype of the zebrafish were captured using a Zeiss stereomicroscope at 3 and 7 days post-amputation.

[0040] Example 3 Evaluation of the degree of regeneration of zebrafish tail fin tissue

[0041] The formula for calculating the extent of zebrafish caudal fin regeneration is: Number = caudal fin tissue regeneration area / total area (as shown in Figure 1). The regeneration area at 3 and 7 days post-operatively was calculated and statistically analyzed. After statistical analysis, the ability of shikonin to promote regeneration of caudal fin tissue following excision injury in adult wild-type zebrafish was evaluated.

[0042] 1. qRT-PCR

[0043] The tail fins of zebrafish at 3 dpa in different groups were collected into different 1.5 mL EP tubes, with 8 tail fins in each tube. After rinsing with PBS buffer three times, total RNA was extracted using Trizol reagent (Invitrogen). Only samples with an RNA260 nm / 280 nm ratio between 1.8 and 2.0 were used. cDNA was synthesized using the cDNA reverse transcription reagent TransStart@Tip Green qPCR Supermix (AQ601-02). Real-time quantitative PCR detection of genes was performed using the ABI Step One+RT-PCR system. Primordial development-related genes (lef1, fgf20a, osn, mxsb, shha) were detected, and the primers were from (Cao et al., 2021. Calcineurin controls proximodistal blastema polarity in zebrafish fin regeneration. Proceedings of the National Academy of Sciences of the United States of America, 118(2), e2009539118.). β-actin was used as an internal reference for each gene, and the relative RNA expression was calculated using the 2 -ΔΔ Calculation by Ct method.

[0044] 2. In situ hybridization:

[0045] 1) Sample collection: The caudal fins of zebrafish from different groups at 2 dpa were collected into different 1.5 mL EP tubes, with 8 caudal fins in each tube.

[0046] 2) After rinsing with PBS buffer for 3 times, the samples were fixed with 4% PFA at 4°C and incubated on a shaker at 4°C overnight.

[0047] 3) Dehydration: Wash three times with 1× PBT, 1 mL each time for 5 minutes.

[0048] 4) The sample was then dehydrated and rinsed with 100% methanol for 5 times, with each rinse lasting 5 minutes and 1 mL. The sample was then stored at -20°C.

[0049] 5) Rehydration: Rinse the sample in the following solutions: 75% methanol-25% PBT 1 mL for 5 minutes, 50% methanol-50% PBT 1 mL for 5 minutes, 25% methanol-75% PBT 1 mL for 5 minutes, and finally rinse in 1× PBT 4 times, 5 minutes each time.

[0050] 6) Digestion: Dilute Proteinase K in 1x PBT to a working solution of 10 μg / mL. Digest the sample with 1000 μL per EP tube for 60 minutes. Shake gently on a horizontal shaker during digestion.

[0051] 7) Terminate digestion: Rinse quickly twice with 1×PBT, then rinse twice more: first for 1 minute, second for 5 minutes.

[0052] 8) Refixation: Samples digested with proteinase K should be fixed in 4% PFA for 20 minutes to prevent sample fragmentation. After refixation, rinse five times with 1× PBT (1 mL each, 5 minutes).

[0053] 9) Prehybridization: Add 200 μL of preheated Hyb buffer to each tube of embryos and incubate in a 68.5°C water bath for 2-5 hours.

[0054] 10) Hybridization: Preheat the hybridization solution (3 μL probe stock solution in 200 μL Hyb buffer) in a water bath for 10 minutes, then replace the pre-hybridization solution with hybridization solution and hybridize overnight in a constant temperature water bath at 67°C.

[0055] 11) Probe recovery: After overnight hybridization, the probe is recovered and can be reused about 3 times.

[0056] 12) Rinsing: Rinse with the following solutions preheated to 65°C in sequence (100% Hyb buffer, once for 10 minutes, once for 10 minutes with 25% 2×SSCT-75% Hyb buffer, once for 10 minutes with 50% 2×SSCT-50% Hyb buffer, once for 10 minutes with 75% 2×SSCT-25% Hyb buffer, once for 10 minutes with 2×SSCT, once for 10 minutes with 0.2×SSCT, four times for 5 minutes with 5×SSCT, then once for 5 minutes with 75% 0.2×SSCT-25% MABT at room temperature with 50% 0.2×SSCT-50% MABT, once for 5 minutes with 25% 0.2×SSCT-75% MABT, and once for 5 minutes with 100% MABT).

[0057] 13) Blocking: Add 1 ml of blocking solution to each tube, place on a horizontal shaker at 60 RPM, and block at room temperature for 2 hours.

[0058] 14) Antibody Incubation: Dilute anti-Dig-AP antibody at a 1:2000 dilution in blocking buffer to achieve the desired concentration. After removing the blocking buffer, add 200 μL of antibody to each tube and incubate overnight at 4°C with gentle shaking.

[0059] 15) Recover the antibodies and put them back to the fourth level: The recovered antibodies can be used repeatedly, but 10% of newly prepared antibodies should be added when used next time.

[0060] 16) Rinse: Rinse eight times for 15 minutes in 1× MABT. NTMT solution equilibration: Rinse three times for 5 minutes each in freshly prepared NTMT solution. After the third rinse, transfer the embryos from the centrifuge tube to a 24-well plate using a trimmed 1 mL pipette tip.

[0061] 17) Color Development: Add 500 μL of color development solution to each well of a 24-well plate. Wrap with tin foil and incubate at 37°C for color development. Observe color development under a microscope every 10 minutes until sufficient color is developed. Immediately remove the color development solution and rinse three times with 1× PBT. Add 1 mL of stop solution (Sangon Biotech, Cat. No. C520019) to stop color development to minimize background.

[0062] 18) Observation: Place the embryo in 500 μL of 80% glycerol-20% stop solution and photograph using a Zeiss stereomicroscope.

[0063] 3. PCNA antibody color development:

[0064] Day 1

[0065] 1) Sample collection: The caudal fins of zebrafish from different groups at 2 dpa were collected into different 1.5 mL EP tubes, with 8 caudal fins in each tube.

[0066] 2) After rinsing with PBS buffer for 3 times, the samples were fixed with 4% PFA at 4°C and incubated on a shaker at 4°C overnight.

[0067] the next day

[0068] 1) Rinse the sample with PBS three times, 5 minutes each time.

[0069] 2) Block with PBTN at 4°C for 2 hours.

[0070] 3) Add primary antibody: anti-H3P antibody (1:500) (Invitrogen). Dilute with PBTN according to the antibody manufacturer's instructions. Incubate overnight at 4°C.

[0071] Day 3

[0072] 1) Recover antibodies. Antibodies can be reused 3-5 times.

[0073] 2) Rinse the sample with PT 5 times, 45 minutes each time.

[0074] 3) Add secondary antibody: Dilute the secondary antibody (Dsred fluorescent protein; Invitrogen) 1:500 in PBTN, adding 200 μL to each tube. Incubate at room temperature for 2 hours, or overnight at 4°C (usually overnight). Protect from light during incubation (cover with tin foil) to prevent fluorescence quenching.

[0075] Day 4

[0076] 1) Remove the secondary antibody and rinse the sample with PT 5 times, 45 minutes each time.

[0077] 2) DAPI staining for 60 min.

[0078] 3) The samples were transferred into PT solution containing 80% glycerol. Antibody-stained tissues were imaged using a TCS Sp8 confocal microscope (Leica).

[0079] 4. DAPI staining

[0080] DAPI (Roche Diagnostics, Indianapolis, IN, USA) was diluted 1:2000 with PBTN and added to the samples, and incubated at 37°C for 2 h.

[0081] 5. Data Statistics

[0082] All statistical analyses were performed using GraphPad Prism version 7.0. Statistical significance was determined using the t-test. All data are expressed as mean ± standard deviation. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

[0083] 6. Experimental Results

[0084] As shown in (a)-(f) of Figure 2, shikonin (Sh) treatment can accelerate the regeneration of the tail fin of adult zebrafish.

[0085] As shown in Figure 3 , shikonin treatment can accelerate the regeneration of the tail fin of adult zebrafish.

[0086] As shown in FIG4 , shikonin treatment significantly upregulated the expression levels of genes related to zebrafish tail fin regeneration.

[0087] As shown in (a)-(f) of Figure 5 , shikonin treatment significantly upregulated the expression levels of genes related to zebrafish tail fin regeneration.

[0088] As shown in (a)-(h) of FIG6 and FIG7, shikonin treatment increased the number of proliferating cells during the regeneration of the zebrafish tail fin.

[0089] In summary, the present invention, by utilizing the zebrafish tail fin injury repair model, discovered that shikonin has a promoting effect on tail fin regeneration, provided a new application of shikonin, and provided a theoretical basis for studying the regeneration of severed limbs.

[0090] It should be noted that when numerical ranges are mentioned in the present invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints may be used. To avoid redundancy, the present invention describes preferred embodiments. However, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts. Therefore, the appended claims are intended to be interpreted as covering the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0091] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Application of shikonin in the preparation of drugs for promoting tissue regeneration.

2. The use according to claim 1, characterized in that The tissue comprises zebrafish tail fin tissue.

3. The use according to claim 2, characterized in that The drug upregulates the expression level of zebrafish tail fin regeneration-related genes.

4. The use according to claim 3, characterized in that The zebrafish tail fin regeneration-related genes include lef1 gene, msxb gene and shha gene.

5. The use according to claim 2, characterized in that The drug increases the number of proliferating cells during the regeneration of the zebrafish tail fin.

6. A drug for promoting tissue regeneration, characterized in that: The drug includes shikonin.

7. The drug according to claim 6, characterized in that Shikonin is the only active ingredient or one of the active ingredients of the medicine.

8. The drug according to claim 7, characterized in that The drug also includes pharmaceutically acceptable excipients.

9. The drug according to claim 8, characterized in that The medicine is a liquid preparation.

10. The drug according to claim 9, characterized in that The shikonin is dissolved in a liquid solvent to prepare the medicine, and the concentration of the shikonin is 70-100 μg / L.

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