Cyclic heptapeptide FZ1 with activity of promoting repair of diabetic skin ulcers and application thereof

Abstract

The application discloses a cyclic heptapeptide FZ1 with activity of promoting repair of diabetic skin ulcers and application thereof. The amino acid sequence of the cyclic heptapeptide FZ1 is CKLSNDC. The application of the cyclic heptapeptide FZ1 is the application in preparation of a medicine for treating and promoting repair of diabetic skin ulcers. The cyclic heptapeptide FZ1 provided in the application exhibits good treatment effect on wound repair in vitro and in vivo, and has excellent activity of promoting tissue reparation of diabetic skin ulcers. In addition, the cyclic heptapeptide FZ1 only has 7 amino acids, has high activity and low synthesis cost, is one of the shortest repair active peptides in the world, provides a new way for research and development of chronic diabetic skin wound repair drugs, and provides a new direction for development of drugs for promoting skin wound treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a cyclic heptapeptide FZ1 with activity promoting the repair of diabetic skin ulcers and its application. Background Technology

[0002] In recent years, the incidence of diabetes has been increasing globally, affecting more than 400 million people worldwide, accounting for over 6% of the global population. In the United States alone, more than $30 billion is spent annually on the treatment and care of diabetes. Diabetes is a group of metabolic diseases characterized by elevated blood glucose levels. Long-term hyperglycemia leads to disorders in insulin secretion or action, causing damage and dysfunction to human tissues and organs (skin, eyes, kidneys, heart, nerves, and blood vessels, etc.). Approximately 19-34% of these cases develop into diabetic chronic skin wounds. Diabetic wounds are most commonly seen as skin ulcers on the legs or feet. The treatment and care of these wounds are characterized by high amputation rates, high recurrence rates, and high mortality rates, making them a major challenge for global healthcare systems.

[0003] Normal skin wound repair is a complex biological process requiring the joint participation of cells, extracellular matrix, and signaling pathways, encompassing four continuous and overlapping dynamically coordinated processes: hemostasis, inflammation, proliferation, and tissue remodeling. However, many factors interfere with wound healing (such as infection, inflammation, ischemia, and cellular senescence), leading to delayed or even non-healing wounds, resulting in chronic, difficult-to-heal wounds (such as diabetic wounds, venous or arterial diseases, vasculitis, or autoimmune diseases). Diabetic wounds involve complex pathophysiological processes, including hyperglycemia, skin barrier disruption, persistent inflammation, reactive oxygen species (ROS) accumulation, and impaired angiogenesis, all of which severely hinder wound healing. Current treatments for chronic diabetic wounds suffer from drawbacks such as long treatment cycles, high costs, and the potential for secondary damage to diabetic patients. Therefore, the discovery of novel lead molecules promoting chronic skin tissue regeneration and the exploration of their wound-healing molecular mechanisms are inevitable trends for future development.

[0004] Compared to small molecule compound drugs, peptide drugs have advantages such as natural origin, high specificity, and high activity, exhibiting characteristics such as lower toxicity, stronger targeting, and greater activity. Furthermore, the development of peptide drugs has a broad commercial market and has gradually become a "potential stock" that cannot be ignored in the field of drug development.

[0005] The present invention aims to provide a cyclic heptapeptide that exhibits good activity in promoting the repair of diabetic skin ulcers both in vivo and in vitro. Summary of the Invention

[0006] The first objective of this invention is to provide a cyclic heptapeptide FZ1 with activity promoting the repair of diabetic skin ulcers, and the second objective of this invention is to provide the application of the active cyclic heptapeptide FZ1.

[0007] The first objective of this invention is achieved by the cyclic heptapeptide having the amino acid sequence shown in SEQ ID No. 1 as CKLSNDC, and its structural formula as shown in formula (I):

[0008] Formula (I).

[0009] The second objective of this invention is achieved by using the cyclic heptapeptide FZ1 in the preparation of a drug that promotes the repair of diabetic skin ulcers.

[0010] The beneficial effects of this invention are as follows: The cyclic heptapeptide FZ1 provided by this invention exhibits good therapeutic effects on wound repair both in vivo and in vitro, and has excellent activity in promoting the regeneration of skin tissue in diabetic chronic diseases. Furthermore, this cyclic heptapeptide contains only 7 amino acids, making it easy to synthesize and inexpensive. It is one of the shortest repair-active peptides in the world, providing a new avenue for the development of drugs for repairing skin wounds in chronic diabetes and offering a new direction for the development of drugs that promote the treatment of skin wounds. Attached Figure Description

[0011] Figure 1 The structural formula of the cyclic heptapeptide FZ1 of this invention is shown below.

[0012] Figure 2 This is a diagram illustrating the scratch repair activity of the cyclic heptapeptide FZ1 of this invention in promoting human keratinocyte (HaCaT) keratinocyte repair.

[0013] Figure 3 This is a diagram illustrating the proliferative activity of the cyclic heptapeptide FZ1 of this invention in promoting the proliferation of human keratinocytes (HaCaT).

[0014] Figure 4 This is a diagram illustrating the human keratinocyte (HaCaT) migration-promoting activity of the cyclic heptapeptide FZ1 of this invention.

[0015] Figure 5 This is a diagram showing the activity of the cyclic heptapeptide FZ1 of the present invention in repairing skin damage across the entire dermis of type II diabetic mice. In this diagram, A represents the changes in skin wound repair across the entire dermis of type II diabetic mice, and B represents the quantification of skin wound repair across the entire dermis of type II diabetic mice. Detailed Implementation

[0016] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0017] This invention provides a cyclic heptapeptide FZ1 having the amino acid sequence shown in SEQ ID No. 1: CKLSNDC, and its structural formula is shown in formula (I):

[0018] Formula (I).

[0019] The present invention also provides the application of the cyclic heptapeptide FZ1 in the preparation of drugs that promote skin wound repair.

[0020] The skin trauma described is a chronic diabetic skin trauma.

[0021] The present invention further provides a pharmaceutical composition comprising the cyclic heptapeptide FZ1 as an active agent and its pharmaceutically effective carrier.

[0022] Example 1: Synthesis of FZ1, an active cyclic heptapeptide that promotes skin wound repair

[0023] The amino acid sequence of the cyclic heptapeptide FZ1 is CKLSNDC. The cyclic heptapeptide FZ1 was prepared by conventional chemical synthesis or gene expression.

[0024] The cyclic heptapeptide FZ1 of this invention was synthesized by Wuhan Baiyixin Biotechnology Co., Ltd. according to the designed cyclic heptapeptide structure.

[0025] Example 2: In vitro assay of the skin wound repair activity of cyclic heptapeptide FZ1

[0026] 1. In vitro keratinocyte scratch repair activity assay

[0027] After digestion and centrifugation of keratinocytes in the logarithmic growth phase, the cells were resuspended in DMEM / F12 medium containing 10% fetal bovine serum and adjusted to a 2×10⁻⁶ cell ratio. 5 500 μL / well was seeded into 24-well plates and cultured. When the keratinocytes reached confluence in the 24-well plates, a straight line was vertically drawn in the well using a 200 μL pipette tip, and 500 μL of PBS was gently added along the well wall for washing three times. 450 mL of fetal bovine serum-free DMEM / F12 medium was added, followed by treatment of the keratinocyte scratch with 50 μL of PBS (control), rh-bFGF (100 ng / mL, positive control), and FZ1 (1 nM) cyclic heptapeptide. The keratinocyte scratch repair was observed and photographed using an inverted microscope at 0 h, 12 h, and 24 h post-scratching. The experiment was repeated three times, with three wells per replicate. The scratch repair area was quantified using ImageJ software. The calculation formula was: Scratch repair rate (%) = (Initial cell-free area - Cell-free area at different time points) / Initial cell-free area × 100%

[0028] Result: As Figure 2 As shown, the cyclic heptapeptide FZ1 significantly promoted scratch repair in keratinocytes, with a repair rate of 94.60% after 24 h of FZ1 treatment. Furthermore, the repair activity of 1 nM (2.086 ng / mL) OA-RD17 was significantly higher than that of the positive control 100 ng / mL rh-bFGF (87.94%), indicating that the skin wound repair effect of cyclic heptapeptide FZ1 is equivalent to 49 times that of commercially synthesized rh-bFGF, demonstrating promising research and development potential.

[0029] 2. In vitro detection of keratinocyte proliferation activity

[0030] After digestion, the above-mentioned keratinocytes in the logarithmic growth phase were centrifuged at 1000 rpm for 5 min, resuspended in serum-free culture medium, and the cell density was adjusted to 5×10⁴ cells / cell. 3 Cells were seeded at 90 mL / well in 96-well plates. After 4 h of cell adhesion, 10 mL of different cyclic heptapeptide solutions were added to each well. After overnight incubation, 10 μL of MTS reagent was added to each well, and the cells were incubated for another 3 h in a 5% CO2 incubator at 37 ℃. The OD value of each well was then measured at 490 nm using a microplate reader, and the results were recorded and averaged. The absorbance of each well was controlled between 0.8 and 1.2. Each cell proliferation experiment was repeated at least three times, with six replicates per well. The data were calculated using the following formula and analyzed using GraphPad Prism software: Relative cell proliferation rate (%) = (mean control group OD490 - mean experimental group OD490) / (mean control group OD490) × 100%

[0031] Result: As Figure 3 As shown, at a concentration of 1 nM, the cyclic heptapeptide FZ1 exhibited significant keratinocyte proliferation-promoting activity.

[0032] 3. In vitro keratinocyte migration activity assay

[0033] After digesting and centrifuging keratinocytes in the logarithmic growth phase, the cells were resuspended in serum-free culture medium and the cell density was adjusted to 1×10⁻⁶. 5200 mL / well was seeded into the upper chamber of a trans-well plate. The upper chamber filter membrane had an 8 μm pore size. DMEM / F12 medium containing 10% fetal bovine serum was added to the 24-well plate. After the cells adhered to the upper chamber of the trans-well plate for 4 h, 20 μL of each group of cyclic heptapeptide solution was added to the upper chamber. After incubation at 37°C for 24 h with 5% CO2, the chamber was removed, washed 2-3 times with PBS, and the cells in the inner membrane of the chamber were gently wiped with a cotton swab moistened with PBS. The chamber was then fixed with 4% paraformaldehyde solution for 20 min and stained with crystal violet (1 mg / mL) for 20 min before being photographed under an inverted microscope. Five fields of view were randomly counted for each sample. After each group was photographed and recorded, the outer membrane of the small cell was immersed in 33% acetic acid solution to dissolve the crystal violet stain. After dissolving for 20 min, 100 μL / well was aspirated into a 96-well plate, and the OD value was detected at 570 nm using a microplate reader. Each group was repeated at least 3 times, with 3 replicate wells each time. The average OD570 value was analyzed and the cell migration rate was calculated using GraphPad Prism software.

[0034] Result: As Figure 4 A and Figure 4 As shown in B, the cyclic heptapeptide FZ1 can significantly promote keratinocyte migration.

[0035] In summary, the cyclic heptapeptide FZ1 exhibits good wound repair activity in vitro.

[0036] Example 3: Detection of the skin wound repair activity of cyclic heptapeptide FZ1 in type II diabetic mice

[0037] Experimental Methods: Full-Cortical Injury Experiment in Type II Diabetic Mice

[0038] A type 2 diabetes mouse model was established using streptozotocin. C57BL / 6 mice were anesthetized with 1% sodium pentobarbital (60 mg / kg), and the fur on their backs was removed. Two full-thickness wounds, each 10 mm in diameter, were created on both sides of the mouse's back using a biopsy puncture device. Mice were randomly divided into three groups: a control group (PBS), a positive control group (rh-bFGF, 100 ng / ml), and a cyclic heptapeptide group (FZ1, 1 nM). The mice were administered 20 µL twice daily, and wound changes were recorded by photographs every other day until day 14.

[0039] Result: As Figure 5 A and Figure 5As shown in Figure B, the cyclic heptapeptide FZ1 significantly promoted the repair of full-thickness skin wounds on the back of mice. After 7 days of treatment, the wound repair activity of cyclic heptapeptide FZ1 reached 83.6%, significantly higher than that of the blank control (58.4%) and rh-bFGF (74.4%). Notably, after 14 days of FZ1 treatment, the skin wound repair rate reached 99.1%, significantly higher than that of rh-bFGF (92.5%), demonstrating good therapeutic activity and research potential.

[0040] In summary, the wound repair peptide FZ1 provided by this invention has strong skin tissue regeneration activity both in vivo and in vitro, and has certain clinical application value.

Claims

1. Cyclic heptapeptide FZ1, with the amino acid sequence CKLSNDC, has the structural formula shown in formula (I): Equation (I).

2. The use of the cyclic heptapeptide FZ1 of claim 1 in the preparation of a drug that promotes skin wound repair.

3. The application according to claim 2, characterized in that, The skin trauma described is a chronic diabetic skin trauma.

4. A pharmaceutical composition comprising the cyclic heptapeptide FZ1 of claim 1 as an active agent and its pharmaceutically effective carrier.

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