Use of miR-184-3p in preparation of a medicine for improving repair of diabetes-related tissue damage

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses application of miR-184-3p in preparation of a medicine for improving repair of diabetes-related tissue damage. The miR-184-3p has a nucleotide sequence as shown in SEQ ID NO. 1. Research finds that miR-184-3p can regulate anti-inflammatory factors of a diabetes wound surface and play a role in wound repair, down-regulate expression of an inflammatory factor IFN-gamma, and up-regulate expression levels of anti-inflammatory cytokines IL-4 and IL-13. In particular, for regulating excessive inflammatory reactions of a diabetes wound surface and promoting wound healing, miR-184-3p can be used as an active ingredient to develop a diabetes wound anti-inflammatory or a medicine preparation for promoting repair of related tissue damage, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the technical field of biomedicine, and in particular relates to the application of miR-184-3p in the preparation of drugs that improve the repair of diabetes-related tissue damage. Background Technology

[0002] Skin wound repair is a complex process involving the coordinated action of multiple cells to regulate inflammation, proliferation, and remodeling. The transition from early inflammation to the subsequent proliferative phase is crucial for healing. Once the skin is injured, the body immediately initiates a cascade of precisely regulated wound repair processes to restore the skin's barrier function and maintain homeostasis as quickly as possible. However, the regeneration process of injured skin is easily affected by underlying diseases and the external environment, leading to chronic inflammation, poor wound healing, or slow healing. Diabetes is a chronic metabolic disease affecting multiple systems throughout the body, and its prevalence is increasing annually. Diabetic wounds are characterized by prolonged inflammation and poor angiogenesis. The chronic inflammatory state in diabetic patients hinders the normal healing process, resulting in delayed wound closure and an increased risk of infection.

[0003] miRNAs are a class of 18-25 nt single-stranded endogenous non-coding RNA molecules that are widely involved in the physiological or pathological processes of cell proliferation, differentiation, aging, apoptosis, and various diseases. Current research indicates that many specific miRNAs are enriched in EPSC-derived exosomes, including miR-16, let-7a, miR-425-5p, and miR-142-3p. These specific RNAs reduce scar formation by inhibiting myofibroblast differentiation by reducing TGF-β1 expression in dermal fibroblasts. Furthermore, studies have shown that miRNAs may also play an important role in the pathogenesis of skin wound healing and chronic inflammatory wounds; however, there are currently no reports on the use of miR-184-3p for wound repair therapy. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this application proposes the use of miR-184-3p in the preparation of drugs or formulations for promoting tissue damage repair, making it a promising candidate for use in novel drug formulations for the treatment of skin, nerve, and blood vessel repair in chronic wounds.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides the application of miRNA in the preparation of drugs or preparations for promoting tissue damage repair, which can promote wound healing. The miRNA is miR-184-3p, and its nucleotide sequence is shown in SEQ ID NO.1, specifically TGGACGGAGAACTGATAAGGGT.

[0007] Preferably, the tissue damage includes skin trauma, burns, chronic wound healing, radiation ulcers, skin cell repair and regeneration, etc.

[0008] More preferably, the tissue damage is diabetes-related tissue damage.

[0009] Secondly, the present invention provides the application of a reagent that promotes miR-184-3p expression or enhances miR-184-3p function in the preparation of drugs that promote tissue damage repair.

[0010] Preferably, the tissue damage is diabetes-related tissue damage.

[0011] Preferably, the reagent includes the chemically modified form of miR-184-3p, AgomiR-184-3p, a miR-184-3p analog, or a carrier carrying miR-184-3p.

[0012] Thirdly, the present invention provides a pharmaceutical composition for promoting tissue damage repair, the pharmaceutical composition comprising miR-184-3p or an agent that promotes miR-184-3p expression or enhances miR-184-3p function, and pharmaceutically acceptable excipients, wherein the miR-184-3p nucleotide sequence is shown in SEQ ID NO.1.

[0013] Preferably, the reagent includes the chemically modified form of miR-184-3p, AgomiR-184-3p, a miR-184-3p analog, or a carrier carrying miR-184-3p.

[0014] Preferably, the pharmaceutically acceptable excipient is selected from one or more of solvents, solubilizers, wetting agents, antioxidants, antibacterial agents, chelating agents, and surfactants.

[0015] Fourthly, the present invention provides the application of the above-mentioned miR-184-3p in the preparation of anti-inflammatory drugs, wherein the nucleotide sequence of miR-184-3p is shown in SEQ ID NO.1.

[0016] Preferably, anti-inflammatory refers to the use of this term to regulate excessive inflammatory response in diabetic wounds.

[0017] Preferably, the anti-inflammatory effect refers to upregulating the expression levels of cytokines IL-4 and IL-13 and downregulating the expression level of the pro-inflammatory factor IFN-γ.

[0018] Compared with the prior art, the beneficial effects of this application are as follows:

[0019] This invention provides the application of miR-184-3p in promoting tissue repair, particularly significantly increasing the proliferation, migration, and scratch repair capacity of keratinocytes under high glucose conditions. It also alleviates wound inflammation by downregulating the expression of the inflammatory cytokine IFN-γ and upregulating the expression of anti-inflammatory cytokines IL-4 and IL-13, offering a new avenue for non-cellular therapy during the inflammatory phase. Furthermore, the miR-184-3p mimic treatment model validated its effect in promoting diabetic wound healing, demonstrating promising application prospects and value. Attached Figure Description

[0020] Figure 1 The relative expression level of miR-184-3p in keratinocytes transfected with miR-184-3p mimic was measured under negative control (NC), normal glucose (NG, 6 mM), or high glucose (HG, 30 mM) culture conditions (a) and the (OD) value reflecting cell proliferation (b); the number of HUVECs that migrated (c) and the scratch repair rate (d) in each group were also measured. Values ​​are mean ± SEM (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0021] Figure 2 This is a wound healing curve over time for each group in a diabetic mouse model.

[0022] Figure 3 The results show the expression levels of pro-inflammatory cytokines IFN-γ and anti-inflammatory cytokines IL-4 and IL-13 in wound tissue. Detailed Implementation

[0023] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0025] Unless otherwise specified, miR-184-3p and other microRNAs listed in this invention are known microRNA types in the art. Those skilled in the art are capable of obtaining molecular biological information related to miR-184-3p, such as sequences, from conventional databases. The nucleotide sequence of miR-184-3p used in this invention is TGGACGGAGAACTGATAAGGGT. Furthermore, to facilitate in vitro and in vivo experiments, this invention selects the commonly used molecular biological tool miR-184-3p mimic to mimic the endogenous miR-184-3p in organisms, thereby enhancing the function of endogenous miR-184-3p. The miR-184-3p mimic can be purchased commercially available according to experimental needs, or it can be designed and synthesized based on the miR-184-3p sequence. The miR-184-3p mimic and negative control miR-NCmimic used in this invention are both commercially available (purchased from Guangzhou Ruibo Biotechnology Co., Ltd.) and used according to the product instructions.

[0026] Example 1: Effects of miR-184-3p on cell proliferation, migration, and scratch repair in tissue repair-related cells.

[0027] 1. Cell proliferation experiment

[0028] The experimental method for detecting cell proliferation in this invention uses the CCK-8 assay. Keratinocytes are seeded in 96-well plates (5 × 10⁶ cells / well). 3 Cells were cultured continuously for 4 hours after cell adhesion, and then transfected according to the Rivia transfection reagent instructions. Two groups were selected: a miR-184-3p upregulation group (miR-184-3p mimic group) and a blank control group (miR-NC mimic group). 48 hours after transfection, the original culture medium was discarded, and 100 μL of medium to CCK-8 reagent was added to each well at a ratio of 9:1. The culture plates were then incubated in a cell culture incubator for another 2 hours. OD values ​​were measured using a multi-mode microplate reader, and the data are expressed as Relative OD Value (OD value of experimental group / OD value of control group).

[0029] The results are as follows Figure 1 As shown in Figure a, this invention established normal glucose (NG, 6mM) and high glucose (HG, 30mM) culture conditions, and transfected the miR-184-3p expression group (miR-184-3p mimic group) and the blank control group (miR-NC mimic group), respectively. The miR-184-3p content in keratinocytes of each group was detected using qRT-PCR. Compared with the NC transfected group, the miR-184-3p content in the miR-184-3p mimic transfected group was significantly increased (see Figure a). Figure 1 a).

[0030] CCK-8 results indicate that ( Figure 1 (b) The OD value of the HG+NC group was significantly lower than that of the NG+NC group, indicating that cell proliferation in the HG+NC group was significantly inhibited under high glucose conditions. Compared with the NG+NC group, the OD value of the NG+miR-184-3p mimic group was slightly increased, but the difference was not significant. However, the OD value of the HG+miR-184-3p mimic group was significantly higher than that of the HG+NC group, indicating that upregulation of miR-184-3p can promote the proliferation of keratinocytes, especially under high glucose conditions.

[0031] 2. Cell migration experiment

[0032] This invention employs the Trans-well assay to detect cell migration ability. A Trans-well plate with an 8 μm pore size is inserted into a 24-well plate (lower chamber) as the upper chamber. Keratinocytes are then introduced at a cell density of 1 × 10⁻⁶ cells / well. 4 After seeding cells into the upper chamber and culturing for 4 hours, transfection was performed according to the Rivia transfection reagent instructions. Two groups were selected: a miR-184-3p expression upregulation group (miR-184-3p mimic group) and a blank control group (miR-NC mimic group). Fresh medium containing 10% FBS was then added to the lower chamber of the 24-well plate. Cells were incubated at 37°C for 24 hours. After incubation, cells were washed with sterile PBS, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet for 20 minutes. Finally, the upper chamber was washed with PBS to remove non-migrating cells, retaining only those that had migrated to the lower chamber. Migration was recorded using an inverted microscope, and the number of migrating cells was calculated using ImageJ software for statistical analysis.

[0033] Trans-well experimental results show (e.g.) Figure 1 c) Cell migration in the HG+NC group was significantly lower than that in the NG+NC group, indicating that cell migration in the HG+NC group was significantly inhibited under high glucose conditions. Cell migration in the NG+miR-184-3p mimic group was significantly increased compared to the NG+NC group, and cell migration in the HG+miR-184-3p mimic group was significantly increased compared to the HG+NC group, suggesting that upregulation of miR-184-3p can promote keratinocyte migration.

[0034] 3. Cell scratch assay

[0035] Experimental methods: Keratinocytes were cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% antibiotics (100 U / mL penicillin, 100 U / mL streptomycin) at 37°C and 5% CO2 humidity. Keratinocytes were seeded into 24-well plates (2 × 10⁶ cells / well). 5( / well) When the cell density reached 60%, transfection was performed according to the Rivia transfection reagent instructions, with the miR-184-3p expression upregulated group (miR-184-3p mimic group) and the blank control group (miR-NC mimic group). When keratinocytes formed a confluent monolayer, the cell monolayer was scratched with a 200μL sterile pipette tip to simulate a wound. After washing away cell debris with PBS, 500μL of serum-free medium was added to the transfected group and the blank group, and they were incubated at 37°C and 5% CO2 for 24 hours.

[0036] The results are as follows Figure 1 As shown in Figure d, upregulation of miR-184-3p expression significantly promotes scratch repair in keratinocytes. In summary, compared with NC treatment under NG culture conditions, HG culture conditions inhibited cell proliferation, migration, and scratch repair capabilities. Transfection with miR-184-3p mimic enhanced cell proliferation, migration, and scratch repair capabilities, and these effects were more pronounced under HG culture conditions than under NG culture conditions. miR-184-3p molecules can significantly improve the biological functions of keratinocytes.

[0037] Example 2: The effect of miR-184-3p on promoting the repair of diabetic wounds

[0038] This invention utilizes 6-8 week old male C57BL / 6 mice, weighing approximately 22g, to establish a diabetic mouse model using intraperitoneal injection of streptozotocin (STZ). Mice were fasted for 12 hours prior to each injection. STZ powder was weighed according to the number and weight of the mice, and a 2μg / μL STZ injection solution was prepared using citrate buffer (pH 4.2-4.5). The injection dose used in this experiment was 50μg / g, administered once daily for 5 consecutive days. The volume of STZ injection was calculated based on the weight of each mouse, and the corresponding volume was drawn up using a sterile syringe for intraperitoneal injection. Feeding was resumed immediately after injection. One week after all injections were completed, fasting blood glucose was measured using the tail-cutting blood sampling method. A fasting blood glucose level greater than 16.7 mmol / L indicated successful establishment of the diabetic mouse model.

[0039] All mice included in the diabetic group had fasting blood glucose levels greater than 16.7 mmol / L and were prepared for skin wound experiments. All experimental procedures followed the relevant international regulations outlined in the Guidelines for the Care and Use of Laboratory Animals. This study was approved by the institution's animal ethics committee for use in animal experiments. Before the start of the formal experiments, the mice were acclimatized for two weeks and then grouped according to the experimental purpose, with at least 6 mice in each group. Incisions were prepared using electric scissors and 70% ethanol. The mice were fed normally during wound creation and treatment. This experiment was conducted in two batches: (1) The first batch consisted of control mice, divided into the AgomiR-184-3p injection group (Ctrl miR-184-3p mimic) and the NC (miR-NC mimic) group (Ctrl NC); (2) The second batch consisted of diabetic model mice, divided into the AgomiR-184-3p injection group (DM miR-184-3p mimic) and the NC (miR-NC mimic) group (DM NC). 5 μL of 50 mmM injection solution was injected subcutaneously at six equidistant points 5 mm from the wound edge, with intervention occurring every other day. Wound healing progress was monitored every two days, and the wound diameter was measured in Photoshop to calculate the closure rate. Mice were euthanized humanely, and wound tissue was collected and preserved according to experimental requirements. The expression of cytokines L-4, L-13, and IFN-γ was detected by qRT-PCR.

[0040] The results are as follows Figure 2 First, we evaluated the gross healing rate of the wounds. Wound healing curves showed that, in both normal mice and diabetic model mice, the AgomiR-184-3p treatment group had a significantly higher healing rate compared to the NC group. Specifically, the wound size in the diabetic group treated with AgomiR-184-3p was smaller than in other groups at all time points, indicating significantly reduced damage and promoted wound healing. The wound repair rate reached 89.8% after 7 days of treatment, which was significantly better than the Ctrl miR-184-3p mimic group.

[0041] Under normal circumstances, a proper inflammatory response is beneficial for pathogen elimination and tissue repair, while an excessive inflammatory response may lead to tissue damage. Results from detecting the expression of inflammatory factor markers during wound healing (see...) Figure 3 Compared with the control group, the expression levels of pro-inflammatory factor IFN-γ and anti-inflammatory cytokines IL-4 and IL-13 were increased in the AgomiR-184-3p injection group in the diabetic group, which effectively reduced the excessive inflammatory response of diabetic wounds, thus enabling the wound healing of diabetic patients to be completed in an orderly and rapid manner.

[0042] Obviously, the above embodiments of the present invention are merely examples to clearly illustrate the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. The application of miR-184-3p in the preparation of a drug for promoting the repair of diabetic wounds, characterized in that, The nucleotide sequence of miR-184-3p is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The miR-184-3p can inhibit excessive inflammatory response in diabetic skin wounds, upregulate the expression levels of cytokines IL-4 and IL-13, and downregulate the expression level of pro-inflammatory factor IFN-γ.

3. The use of a pharmaceutical composition in the preparation of a medicament for promoting the repair of diabetic wounds, characterized in that, The pharmaceutical composition comprises miR-184-3p or a carrier carrying miR-184-3p, and pharmaceutically acceptable excipients, wherein the nucleotide sequence of miR-184-3p is shown in SEQ ID NO.

1.

4. The application according to claim 3, characterized in that, The pharmaceutically acceptable excipients are selected from one or more of the following: solvents, solubilizers, wetting agents, antioxidants, antibacterial agents, chelating agents, and surfactants.

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