CN114042163B - Application of ALDH3A1/IL-17 shaft in preparation of drug for treating intractable ulcer - Google Patents

Abstract

The invention relates to an application of ALDH3A1/IL-17 axis in preparing a medicament for treating ulcer which is difficult to heal. The invention utilizes proteomics combined with biological information analysis and combined experiment verification to find that the protein expression of ALDH3A1 in the wound surface of db/db diabetic mouse is reduced; the overexpression of the HaCaT cell ALDH3A1, the proliferation of the cell is increased, the inflammatory infiltration is reduced, and the IL-17 pathway is obviously inhibited. The invention suggests that ALDH3A1 can improve chronic inflammation, promote cell proliferation and heal wound surfaces by inhibiting a downstream IL-17 signal path, and a targeted ALDH3A1/IL-17 axis can be used as a new treatment method for treating refractory ulcer, post-injury chronic inflammation, infection and other types of wounds in the future.

Description

Application of ALDH3A1/IL-17 shaft in preparation of drug for treating intractable ulcer

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of ALDH3A1/IL-17 axis in preparation of a drug for treating intractable ulcer.

Background

The skin is the organ which most contacts with the environment, is directly exposed in the surrounding environment, has the functions of feeling, immune protection and the like, and is easy to be injured and infected. The ability of diabetes, vascular disease and elderly patients to heal wounds is impaired. Diabetic Ulcers (DUs) are a serious complication of diabetes, often found on the feet of patients, and are also known as diabetic feet. The disability rate and the recurrence rate are extremely high, and the morbidity is increased year by year along with the increase of the prevalence rate of the diabetes. Clinical studies have shown that by controlling blood glucose levels and controlling wound infection, DUs still takes a long time to heal, and the wound is often deep and large, with high surgical difficulty and treatment risk. The diabetic ulcer wound is difficult to heal, the expense is the most expensive of all the complications of diabetes, and the medical expense is only second to surgical operation. Patients with long-term diabetes often have vascular lesions and complications of peripheral neuritis. The sensation at the toe of such patients is often unconscious due to neuritis, and therefore often collides with bleeding and is still unknown. Such wounds at the toe or lower extremities, together with lesions in peripheral blood vessels and abnormal collagen, are not easily healed and often develop into chronic ulcers. The main pathological manifestations of DU are: re-epithelialization process is blocked; chronic inflammation and impaired angiogenesis; neuropathy.

The specific mechanism of DU healing difficulty is not clear at present, and related treatment strategies are under study, and Keratinocytes (KC) play an important role in wound healing by producing cytokines and chemokines to interact with a variety of cell types such as fibroblasts and endothelial cells. Promoting keratinocyte-mediated epithelialization process and relieving chronic inflammation are important for overcoming refractory wound defects.

Acetaldehyde dehydrogenases (ALDHs) are NADP dependent metabolic enzymes with a wide range of biological activities including cellular homeostasis and control of stem cells, as well as detoxification of endogenous/exogenous alcohols and aldehydes, etc., especially 4-Hydroxynonanal (HNE). Acetaldehyde dehydrogenase 3A1 (ALDH 3 A1) is an important member of the ALDH superfamily and can oxidize a variety of endogenously and exogenously derived aldehydes to the corresponding carboxylic acids. Can regulate the proliferation, differentiation, survival and response to oxidative stress of normal and tumor cells. It is widely found in normal tissues (basal skin, bronchial mucosal cells, human cornea) and some solid tumors (non-small cell lung cancer (NSCLC), melanoma, stomach, breast), and is often overexpressed in the esophageal mucosa, small salivary glands, nasal epithelium, tonsils, and oral epithelium. In recent years, ALDH3A1 has been shown to play an important role in tumor survival, metastasis and drug resistance (Fan et al, 2021), and has a clear function in the process of stem cell formation, mechanistically involved in the expansion and differentiation of cancer stem cells. In the a549 and NCTC 2544 cell regeneration models induced to proliferate, protein expression of ALDH3A1 was significantly increased during proliferation (Oraldi et al, 2011). However, the function of ALDH3A1 in diabetic ulcers remains to be elucidated further.

IL-17 secreting cells are present in inflammatory processes in a variety of human patients with inflammatory and autoimmune diseases, including psoriasis, inflammatory bowel disease, rheumatoid arthritis, type 1 diabetes, multiple sclerosis and periodontitis, and are involved in the pathogenesis of the disease. The over-activated IL-17 is related to the susceptibility of autoimmune diseases, on one hand, the over-activated IL-17 can induce the expression of some factors related to tissue repair so as to accelerate the recovery of the organism, on the other hand, the IL-17 can also induce the expression of inflammatory factors and chemotactic factors so as to recruit more immune cells to the inflammatory site to exacerbate the inflammatory reaction of the organism, and the overhigh IL-17 level plays a worsening role in the pathological development of the disease.

Currently, only platelet derived growth factor (PDGF-BB) is approved for wound healing by the U.S. FDA, but is expensive and often unacceptable to patients. Although other growth factor preparations (EGF and bFGF) have certain curative effect, the growth factor preparations are easy to degrade, inconvenient to store medicines, easy to cause hypertrophic scars and the like, so that the popularization and the application of the growth factor preparations are limited; some new excipients are reported to have a certain carcinogenic risk. Therefore, it is very necessary to develop new drugs for treating diabetic ulcers.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides application of an ALDH3A1/IL-17 axis in preparing a drug for treating intractable ulcer.

In a first aspect, there is provided the use of ALDH3A1 or a upregulator for the preparation of a medicament for the treatment of chronic inflammation following a refractory ulcer or injury.

In a preferred embodiment, the refractory ulcer is a diabetic ulcer.

In another aspect of the present invention, a pharmaceutical composition for preventing and treating diabetic ulcers is provided, wherein the pharmaceutical composition comprises acetaldehyde dehydrogenase 3A1 (ALDH 3 A1) or a upregulating agent thereof as an active ingredient, and further comprises a pharmaceutically acceptable carrier.

In a preferred embodiment, the pharmaceutical composition is in the form of an external preparation or an internal preparation.

More preferably, the dosage form of the pharmaceutical composition is a patch, a paste, an ointment, a gel, a film coating agent, a cataplasm, a spray, a capsule, a granule, a tablet, a pill, an oral liquid or an injection.

In another aspect of the invention, the application of acetaldehyde dehydrogenase 3A1 (ALDH 3A 1) as a target point in screening and preparing medicines for preventing and treating diabetic ulcer and preventing and treating diabetic ulcer wound inflammation is provided.

In another aspect of the invention, the ALDH3A1/IL-17 axis is provided as a target for screening drugs for preventing and treating diabetic ulcers or relieving diabetic ulcer wound inflammation.

The invention has the advantages that:

1. the invention discovers that the ALDH3A1 protein expression at the skin lesion of a db/db diabetic mouse ulcer model is remarkably reduced by combining proteomics with bioinformation analysis and combining experimental verification, and prompts that the ALDH3A1 is possibly closely related to the onset of diabetic ulcer and is expected to be used as a new target for treating diabetic ulcer. At present, platelet-derived growth factor (PDGF-BB) is expensive in price, has potential carcinogenic risks after long-term use, and has limited curative effects on Fibroblast Growth Factor (FGF) and Epidermal Growth Factor (EGF). The development of new clinical therapeutic drugs is not slow.

2. The ALDH3A1 in HaCaT cells is over-expressed by adopting a lentivirus transfection technology, and the cell proliferation is obviously enhanced, and the inflammatory infiltration is reduced. The ALDH3A1 plays a key role in the biological process of HaCaT cells, represents a new molecular characteristic in DU pathology, and is a promising target point for treating intractable DU wounds.

3. Through the verification of a transcription profile analysis and an experiment, the downstream IL-17 inflammation signal channel is obviously inhibited after the ALDH3A1 is over-expressed, and the ALDH3A1/IL-17 axis has a new effect in refractory DU.

Overall, the present results demonstrate that targeting the ALDH3A1/IL-17 axis can be a novel therapeutic approach for the future treatment of refractory ulcers, post-injury chronic inflammation, infection and other types of wounds.

Drawings

FIG. 1 map of ALDH3A1 overexpression vector backbone plasmid.

Figure 2 animal experimental lots.

Figure 3 animal experimental treatment protocol.

FIG. 4 a-FIG. 4c. DUs proteomics combined with bioinformation analysis of ALDH3A1 protein in the wound surface. FIG. 4a.db/m and db/db groups clustering heatmap of differential protein expression levels for back ulcers. Figure 4b kegg assay. FIG. 4c. Bioinformatic analysis focuses on ALDH3A1. Figure 4d elisa confirmed the presence of low expression of ALDH3A1 in DUs wounds. * p <0.05.

Figure 5 aldh3a1 can promote HaCaT cell proliferation and ameliorate inflammation. and a, successfully constructing an ALDH3A1 overexpression HaCaT cell model by Western blot verification. CCK-8 cell proliferation capacity was measured at the indicated times. qPCR quantification of IL-6 and IL-1. Beta. Expression. * P <0.001, p <0.01, p <0.05.

FIG. 6 a-FIG. 6c transcriptional profiling of ALDH3A1 overexpressing HaCaT cells. FIG. 6A heatmap of all differentially expressed mRNAs in 6a.ALDH3A1 overexpression (n = 3) and normal HaCaT cells (n = 3) mRNA-seq data adjusted to p-value ≦ 0.05, | Log ₂ F-C | > 2. The red dots represent relatively highly expressed protein-encoding genes, while the blue dots represent low expressed gene expressions. Figure 6b. Go enrichment (top 30) histogram. Figure 6c.kegg pathway (top 20) scattergram, IL-17 signaling pathway was found to change significantly.

FIG. 7.QPCR assay for Il-17, ccl17, S100a7, S100a8, S100a9 expression changes in normal and ALDH3A1 overexpressing HaCaT cells. * P <0.001, P <0.01, P <0.05.

Detailed Description

The present inventors have conducted extensive and intensive studies and found that ALDH3A1/IL-17 axis is an important target for intractable DU.

ALDH3A1、IL-17

Acetaldehyde Dehydrogenase 3family Member A1 (Aldehydehydrogenase 3family Member A1, ALDH3A1) is a protein coding gene. ALDH3A1, located on human, mouse and rat chromosomes 17, 11 and 10, respectively. ALDH3A1 belongs to the second stage of drug metabolizing enzymes and is highly expressed in the stomach, lungs, keratinocytes and cornea. The protein size of the ALDH3A1 gene is 50395Da. Diseases associated with ALDH3A1 include conjunctival degeneration and paranoid schizophrenia. ALDHs play an important role in the detoxification of alcohol-based aldehydes, and are involved in the metabolism of corticosteroids, biogenic amines, neurotransmitters and lipid peroxidation.

The protein encoded by Interleukin 17 (Interleukin 17A, IL-17) is a pro-inflammatory cytokine produced by active T cells. The IL-17A-mediated downstream pathway induces the production of inflammatory molecules, chemotherapeutics, antimicrobial peptides, and engineered proteins. The encoded protein has a key effect on host defense, cell trafficking, immunomodulation and tissue repair and plays a key role in inducing innate immune defense.

In the present invention, the ALDH3A1, IL-17 used may be naturally occurring, for example, it may be isolated or purified from a mammal. In addition, the ALDH3A1 and IL-17 can also be artificially prepared, for example, recombinant ALDH3A1 and IL-17 can be produced according to the conventional genetic engineering recombination technology. Preferably, the present invention can employ recombinant ALDH3A1, IL-17.

Any suitable ALDH3A1, IL-17 may be used in the present invention. The ALDH3A1 and the IL-17 comprise full-length ALDH3A1 or bioactive fragments thereof. The preferred ALDH3A1 amino acid sequence is NM-001135168.1; preferably the amino acid sequence of IL-17 is NM-002190.3.

The amino acid sequences of ALDH3A1 and IL-17 formed by substitution, deletion or addition of one or more amino acid residues are also included in the present invention. ALDH3A1, IL-17 or biologically active fragments thereof comprise a substituted sequence of conserved amino acids which does not affect the activity or retains a portion of the activity. Appropriate substitutions of amino acids are well known in the art and can be readily made and ensure that the biological activity of the resulting molecule is not altered. These techniques allow one skilled in the art to recognize that, in general, altering a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity. See Watson, molecular Biology of The Gene, fourth edition, 1987, the Benjamin/Cummings Pub. Co. P224.

Any biologically active fragment of ALDH3A1 or IL-17 can be used in the present invention. As used herein, a biologically active fragment of ALDH3A1 or IL-17 is meant to be a polypeptide that retains all or part of the function of the full length of ALDH3A1 or IL-17. Preferably, the bioactive fragment maintains at least 50% of the activity of full-length ALDH3A1 and IL-17. Under more preferred conditions, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of full-length ALDH3A1, IL-17.

The invention can also use modified or improved ALDH3A1, IL-17, for example, can use modified or improved ALDH3A1, IL-17 in order to promote its half-life, effectiveness, metabolism, and/or protein potency. The modified or modified ALDH3A1, IL-17 may be a conjugate of ALDH3A1, IL-17, or it may comprise substituted or artificial amino acids. The modified or improved ALDH3A1 and IL-17 have a smaller common point with the naturally existing ALDH3A1 and IL-17, but can also relieve the diabetic ulcer wound or relieve the inflammation of the diabetic ulcer wound, and can not bring other adverse effects or toxicity. That is, any variant that does not affect the biological activity of ALDH3A1, IL-17 can be used in the present invention.

The corresponding nucleotide coding sequence can be conveniently obtained according to the amino acid sequences of ALDH3A1 and IL-17.

Use of

The invention provides application of ALDH3A1 in preparing a medicament for preventing and treating diabetic ulcer.

Diabetic Ulcers (DUs) are a serious complication in the middle and late stages of diabetes, and are frequently found in the feet of patients, so they are also called Diabetic feet. Clinical studies have shown that by controlling blood glucose levels and controlling wound infection, DUs still requires a long time to heal, and the wound is often deep and large, with extremely high surgical difficulty and treatment risk. It is reported that about 6.3% of people worldwide have diabetes mellitus, and about 1 patient amputates due to DUs every 30 seconds, medical expenses of diabetes patients in 2008 are as much as $ 9.1 million, expenses caused by difficulty in healing of diabetic ulcer wounds are the most expensive of all complications of diabetes mellitus, and medical expenses are only after surgery. Patients with long-term diabetes often have vascular lesions and complications of peripheral neuritis. The sensation at the toe of such patients is often unconscious due to neuritis, and therefore often collides with bleeding and is still unknown. Such wounds at the toe or lower extremities, together with lesions in peripheral blood vessels and abnormal collagen, are not easily healed and often develop into chronic ulcers. The main pathological manifestations of DU are: re-epithelialization process is hindered; chronic inflammation and impaired angiogenesis; neuropathy.

Pharmaceutical composition

The pharmaceutical compositions of the invention may contain an active agent as described herein and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are generally safe and non-toxic and include various excipients and diluents, and the like. The term refers to such pharmaceutical carriers: they are not necessary active ingredients per se and are not excessively toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack pub. Co. N.J.1991). Pharmaceutically acceptable carriers in the compositions may comprise liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as emulsifiers, fillers, binders, wetting agents, disintegrants, penetration enhancers, colorants, cosolvents and the like may also be present in these carriers. Above, the emulsifier is such as acetylated monoglyceride, acetylated diglyceride, sucrose ester, sorbitol ester, soybean lecithin, lauric monoglyceride, propylene glycol fatty ester, calcium stearoyl lactylate, diacetyl tartaric acid, glyceryl monostearate, modified soybean lecithin, etc. Such as magnesium stearate, microcrystalline cellulose, lactose, milk sugar, high molecular weight polyethylene glycols, and the like. Such as starch, mannitol, silicic acid, dextrin, calcium hydrogen phosphate, cellulose, etc. Such as carboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, gum arabic, starch slurry, hydroxypropyl starch, modified starch, pregelatinized starch, dextrin, microcrystalline cellulose, polyvinyl pyrrolidone mucilage, gelatin mucilage. Such as glycerol, etc. The disintegrating agent is agar, calcium carbonate, potato starch, tapioca starch, alginic acid, hydroxypropyl starch, modified starch, sodium carboxymethyl starch, microcrystalline cellulose, guar gum, xanthan gum, etc. Such as menthol, laurocapram, borneol, and the like. The colorant may be a plant colorant, an animal colorant, or a microbial colorant, such as beet red, turmeric, chlorophyll, shellac, cochineal, red yeast colorant, and the like. Such as beta-cyclodextrin, maltodextrin, tween, ethanol, span, sodium dodecyl sulfate, propylene glycol, polyethylene glycol, glycerol, etc. However, it will be appreciated by those skilled in the art that the pharmaceutically acceptable carriers useful in the present invention are not limited to the above-mentioned types.

Dosage forms

The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and may be any dosage form suitable for external use, including, but not limited to, a patch, a paste ointment, a gel, a film coating agent, or a cataplasm. It can also be any dosage form suitable for internal use, including but not limited to capsules, granules, tablets, pills, oral liquid or injection, etc.

Method of treatment

The invention provides a method for preventing and treating diabetic ulcer or diabetic ulcer wound inflammation, which comprises the steps of administering ALDH3A1 or an up-regulator of ALDH3A1 to a subject in need thereof; the amount administered is a therapeutically effective amount and can be determined based on the age, weight, sex, kind and severity of the disease of the individual. The subject may be a mammal, in particular a human, mouse, rabbit, pig, sheep, dog, etc. The method of administration is conventional in the art, e.g., painting, application, etc., and may be adjusted for different agents.

The following detailed description of the present invention is provided in connection with the accompanying drawings.

Example 1

1. Method and material

1. Cell culture and transfection

The human keratinocyte Cell line HaCaT is available from Cell Lines Service, eppelheim, eleumer, germany. Cells were cultured in DMEM-high sugar (4.5 g/L, gibco, life Technologies, calif. Calsbad) with 10% (v/v) fetal bovine serum (HyClone, logan, UT, USA) and 1% (v/v) streptomycin-penicillin solution (10000U/mL, gibco, life Technologies), with or without 20ng/mL bFGF (basic fibroblast growth factor, thermoFisher Science, # 13256029), incubated at 37 ℃ at constant temperature.

HaCaT cells with ALDH3A1 overexpression are constructed by using a lentivirus transfection system. 1.7. Mu.g of ALDH3A1 overexpression vector (backbone plasmid pLVX-IRES-Puro (Catalog No.632183, clontech, US), backbone plasmid map see FIG. 1, ALDH3A1 sequence directionally cloned into vector backbone as shown in SEQ ID NO:7 with insertion sites EcoRI/NotI) or CMV-MCS-3FLAG-SV 40-neomycin, 1.13. Mu.g of pCMV. DELTA.8.91 and 0.57. Mu.g of pMD.g were transfected with DNAFect transfection reagent (109120002, harOLife, shanghai, china). Cells were infected with lentivirus in the presence of 8. Mu.g/ml polybren (109120002, harolife, shanghai, china). After 48h of transduction, green fluorescence was observed to indicate transduction efficiency.

Western blot analysis

Western blot analysis was performed. The main antibodies used for western blot analysis are anti-ALDH 3A1 (1, 15578-1-AP, proteintech) antibody and anti- β -actin (1, 1000, 14395-1-AP, proteintech) antibody. Beta-actin served as an endogenous control.

3. Real-time quantitative polymerase chain reaction (qPCR)

Total mRNA was extracted from HaCaT cells using standard TRIzol protocol. By 2 ^-ΔΔCT The method analyzes the related quantitative data, and expresses the difference fold and blank control as the expression level of mRNA. The detailed information of the primers is shown in Table 1.

TABLE 1.QPCR primers

4. Transcript mapping assay

mRNA microarray analysis was performed. Total mRNA was extracted from HaCaT cells with or without ALDH3A1 overexpression using the RNeasy Mini Kit (Cat #74106, qiagen) according to the manufacturer's protocol. RNA integrity was assessed using an Agilent bioanalyzer 4200 (Agilent Technologies, nta Clara, CA, US). A cDNA library was constructed using TruSeq chain mRNA LT sample preparation kit (RS-122-2103, illumina). RNA sequencing was performed by Shanghai biochip, inc.

According to p value <0.05 and | Log ₂ F-C | > is more than or equal to 2, differential mRNA in ALDH3A1 overexpression cells and normal HaCaT cells is identified, and GO and KEGG analysis is carried out.

5. Cell counting kit-8 (CCK-8) detection

CCK-8 (Dojindo, tokyo, japan) was used to assess the proliferation of HaCaT cells according to the manufacturer's instructions. Briefly, cells were seeded in 96-well plates at a density of 2000 cells/100. Mu.L in triplicate, and CCK-8 solution was added at the indicated time points to measure absorbance at 450 nm. The absorbance values were normalized to match those of control cells. Data from 3 independent experiments were taken as mean standard deviation.

6. Mouse model and treatment

db/m (normal) and db/db (diabetic) male mice (6-7 weeks old) weighing 22-24 g were provided by Suzhou Kavens laboratory animals Co., ltd. (license number: 202101610; license number: SCXK (Su) 2016-0010; suzhou, china) (FIG. 2). Animals were housed at standard temperature of 23 + -2 deg.C with 5 mice per cage and treated for 12h light/12 h dark on the SPF scale. Animals were free to obtain a high fat or standard diet and water. Mice were anesthetized with isoflurane prior to molding of the ulcer model, followed by removal of 4cm x 4cm of hair from the back and making 4 circular wounds 6mm wide and 2mm deep with a punch, and the experiment was performed under sterile conditions.

7. Enzyme-linked immunosorbent assay

We examined ALDH3A1 protein expression in sera of groups of mice on day 9 after wound formation. The kit was purchased from institute of bioengineering, jiancheng (Nanjing, china).

8. Statistical analysis

Experimental data were analyzed using GraphPad Prism 8, all data expressed as mean ± standard error (s.e.m.). Differences between groups were compared using T-test or anova. The values for p ≦ 0.05 and p ≦ 0.01 are considered significant or very significant differences, respectively.

2. Results

1.ALDH3A1 protein in DUs wound has low expression

In order to research the pathological mechanism of diabetic ulcer wound surface delayed healing, db/db diabetic mice are used as research objects, a DU model (figure 3) is formed by punching, proteomics analysis is carried out on the normal wound surface in comparison with 9 days after the formation of wounds, a healing core molecule ALDH3A1 is screened out by combining with biological information analysis, and the experimental results prove that: ALDH3A1 expression was significantly down-regulated in DUs mouse skin lesions (fig. 4 a-4 d), suggesting that DU healing failure may be due in part to endogenous ALDH3A1 deficiency.

ALDH3A1 promotes HaCaT cell proliferation and reduces inflammation

We used lentiviruses to up-regulate ALDH3A1 expression in HaCaT cells (a in fig. 5). Next, we compared the effect of ALDH3A1 overexpression treated HaCaT cells with bFGF treated HaCaT cells by CCK-8 assay. CCK-8 assays showed that ALDH3A1 overexpressing cells proliferated significantly more at 48h and 72h than bFGF treated cells (b in FIG. 5). The above results demonstrate that ALDH3A1 helps to promote re-epithelialization of HaCaT cells. To better understand the significance of ALDH3A1 in inflammation regulation, three groups of proinflammatory cytokines IL-6 and IL-1 beta were tested. The results showed that the ALDH3A1 overexpressing cells expressed IL-6, IL-1. Beta. 2 times lower than normal cells and that ALDH3A1 had better anti-inflammatory effect than bFGF (FIG. 5, c). Taken together, these results indicate that ALDH3A1 can overcome the wound healing deficiencies of DU in vitro by reducing the inflammatory index and promoting KC proliferation.

Transcript profiling of ALDH3A1 overexpressing HaCaT cells

To further determine the role of ALDH3A1 in wound repair, we examined the transcriptional profiles of ALDH3 A1-overexpressing HaCaT cells and normal HaCaT cells and compared them based on RNA sequencing. After preliminary quality control of the sequencing data, a total of 20030 genes were retained, of which 343 genes were identified as DE mRNAs and 74 genes (21.57%) were up-regulated and 269 genes (78.43%) were down-regulated (fig. 6 a-6 c). This result suggests that the mRNAs expression state of ALDH3A1 over-expressed cells is very different from that of normal cells, and ALDH3A1 may promote wound healing by regulating the expression of DE mRNA.

In addition, we performed GO and KEGG pathway enrichment analyses. The down-regulated differential mRNA-related top 10 GO term is mainly associated with MHC class II protein complexes, cypermethrin-envelope endocytic vesicle membranes, endoplasmic reticulum luminal-side components, endocytic vesicle membranes, interferon-gamma ray-mediated signaling pathways, peptide antigen binding, endoplasmic reticulum to golgi trafficking vesicle membranes, T cell co-stimulation, sulfur compound metabolic processes, antigen processing and presentation of foreign peptide antigens (fig. 6 b). The up-regulated differential mRNA's first 10 th position is mainly associated with pregnancy, extracellular domain, metal ion binding, transcriptional control-template, DNA binding, plasma membrane composition, extracellular space, zinc ion binding, transcription, DNA template, cytoplasm in women (FIG. 6 b). These results indicate that overexpression of ALDH3A1 in HaCaT cells up-regulates DE mRNAs involved in cell growth proliferation, which is beneficial for tissue reconstruction, while it down-regulates DE mRNAs involved in immune responses, which are closely related to inflammatory mechanisms and skin function.

KEGG pathway analysis (TOP 5) showed that the relevant differential mrnas were mainly associated with s.aureus infection, antigen processing and presentation, type I diabetes, IL-17 signaling pathway, rheumatoid arthritis (fig. 6 c). Interestingly, these signaling pathways show a close correlation with diabetes and are particularly associated with inflammation.

IL-17 Signal Path is ALDH3A1 downstream Effect Path, exacerbating DU deficiency

Notably, the differential mRNA resulting in downregulation after ALDH3A1 overexpression in HaCaT cells significantly enriched the IL-17 signaling pathway (fig. 6 c), suggesting that ALDH3A1 may ameliorate DU deficiency by inhibiting the IL-17 signaling pathway. qPCR experiments also demonstrated that ALDH3A1 overexpression resulted in reduced IL-17 pathway-associated signal expression (FIG. 7). These results indicate that ALDH3A1 may improve the wound healing phenotype by inhibiting the IL-17 signaling pathway.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> Hospital for skin diseases of Shanghai city

Application of <120> ALDH3A1/IL-17 axis in preparation of drug for treating refractory ulcer

<130> /

<160> 17

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caaattgtgg agaagctcaa gaagtcactg aaagagttct acggggaaga tgctaagaaa 840

tcccgggact atggaagaat cattagtgcc cggcacttcc agagggtgat gggcctgatt 900

gagggccaga aggtggctta tgggggcacc ggggatgccg ccactcgcta catagccccc 960

accatcctca cggacgtgga cccccagtcc ccggtgatgc aagaggagat cttcgggcct 1020

gtgctgccca tcgtgtgcgt gcgcagcctg gaggaggcca tccagttcat caaccagcgt 1080

gagaagcccc tggccctcta catgttctcc agcaacgaca aggtgattaa gaagatgatt 1140

gcagagacat ccagtggtgg ggtggcggcc aacgatgtca tcgtccacat caccttgcac 1200

tctctgccct tcgggggcgt ggggaacagc ggcatgggat cctaccatgg caagaagagc 1260

ttcgagactt tctctcaccg ccgctcttgc ctggtgaggc ctctgatgaa tgatgaaggc 1320

ctgaaggtca gatacccccc gagcccggcc aagatgaccc agcactga 1368

Claims (4)

1. Use of ALDH3A1 in the manufacture of a medicament for the treatment of a refractory ulcer, said refractory ulcer being a diabetic ulcer.
2. 2. The pharmaceutical composition for preventing and treating the diabetic ulcer is characterized by taking ALDH3A1 as an active ingredient and further comprising a pharmaceutically acceptable carrier.
3. 3. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is in the form of an external preparation or an internal preparation.
4. 4. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition is in the form of a patch, a paste, an ointment, a gel, a film, a spray, a capsule, a granule, a tablet, a pill, an oral liquid, or an injection.

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