Application of acetoacetic acid in inhibition of skin fibrosis

Abstract

The invention relates to application of acetoacetic acid in inhibiting skin fibrosis and a method for inhibiting skin fibroblast fibrosis. The method comprises the following steps: S1, aseptically acquiring skin tissues of a human body; s2, cutting the adipose tissue into small blocks, mixing the small blocks with collagenase I, and digesting in an incubator with the temperature of 37 DEG C and 5% CO2; s3, after enzymolysis is completed, performing centrifugal treatment and washing, and transferring the cells into a culture bottle containing a culture medium for further culture; s4, when the cells are cultured to 80% of fusion degree, transferring the cells into a culture medium containing acetoacetic acid for culture to obtain cultured skin fibroblasts; and S5, detecting and cryopreserving. According to the application, acetoacetic acid is used for inhibiting synthesis of collagen and reducing deposition of extracellular matrix, so that development of skin fibrosis can be slowed down or prevented.

Description

Technical Field

[0001] The present application relates to the field of pharmaceutical chemistry, and in particular to the application of acetoacetic acid in inhibiting skin fibrosis and a method for inhibiting skin fibroblast fibrosis. Background Art

[0002] Cutaneous fibrosis is a disease that affects the skin and its underlying tissues, characterized by abnormal thickening and hardening of the skin and connective tissue. This pathological condition not only affects the patient's appearance, but can also lead to severe functional impairment and chronic pain. Cutaneous fibrosis can be localized, such as keloids, or systemic, such as systemic sclerosis. Currently, the treatment of cutaneous fibrosis remains a clinical challenge, and new treatments are needed to improve patients' quality of life.

[0003] The causes of skin fibrosis are diverse, including genetics, environmental factors, autoimmune diseases and unknown factors. The pathological mechanism involves inflammation, abnormal deposition of extracellular matrix, especially excessive synthesis and deposition of collagen, which lead to changes in skin structure, loss of elasticity and flexibility, and eventually the formation of hardened plaques. The core feature of skin fibrosis is the abnormal accumulation of extracellular matrix, especially collagen, which causes the skin to become thicker and harder. In normal skin, collagen and other matrix components maintain a dynamic balance, with both synthesis and degradation. However, in the fibrotic state, this balance is broken, resulting in excessive synthesis and insufficient degradation of collagen.

[0004] Fibroblasts are the main cell type that synthesizes and secretes collagen in the skin. During the process of skin fibrosis, these cells are abnormally activated and transformed into "activated fibroblasts" with higher synthesis capacity. These activated fibroblasts are able to synthesize collagen in large quantities and change the surrounding extracellular matrix environment. Multiple signaling pathways are involved in the occurrence of skin fibrosis, including the transforming growth factor-β (TGF-β) signaling pathway. TGF-β is a key fibrosis regulator that can promote the proliferation and activation of fibroblasts and stimulate the synthesis of collagen. During the fibrosis process, the TGF-β signaling pathway is often abnormally activated, which can lead to excessive collagen deposition and other fibrotic characteristics. Chronic inflammation is an important factor in many fibrotic diseases. In the early stages of skin fibrosis, inflammatory cells such as macrophages and T cells are recruited to the damaged area. These inflammatory cells release various cytokines and growth factors, which further stimulate the activation of fibroblasts and the synthesis of collagen. Oxidative stress during inflammation can also damage normal tissues and aggravate the process of fibrosis.

[0005] Current treatments for skin fibrosis include medication, physical therapy, laser therapy, and surgery. Medication usually includes immunosuppressants, anti-inflammatory drugs, and anti-fibrotic drugs. However, these treatments often have limited effects and may be accompanied by significant side effects. Physical therapy and laser therapy can provide some symptom relief, but usually require long-term and repeated treatments. Although surgical treatment can remove fibrotic tissue, there is a risk of recurrence and postoperative complications. Summary of the invention

[0006] The present application proposes the use of acetoacetic acid in inhibiting skin fibrosis. The chemical formula of the acetoacetic acid is CH 3 COCH 2 COOH.

[0007] The present application also proposes the use of acetoacetic acid in the preparation of a drug for skin fibrosis, wherein the drug is an injection, a spray, or a drop.

[0008] Wherein, the concentration of the acetoacetic acid is 0.1-10 mM.

[0009] The present application also proposes a method for promoting osteogenic differentiation of adipose stem cells to solve the problem of treating skin fibrosis in the prior art.

[0010] The present application proposes a method for inhibiting skin fibroblast fibrosis, comprising the following steps:

[0011] S1: Aseptically obtain human skin tissue;

[0012] S2: The adipose tissue was cut into small pieces, mixed with collagenase I, and incubated at 37°C and 5% CO 2 Digestion was performed in an incubator.

[0013] S3: After enzymatic hydrolysis, the cells are centrifuged and washed, and then transferred to a culture flask containing culture medium for further culture;

[0014] S4: When the cells are cultured to 80% confluence, they are transferred to a culture medium containing acetoacetate to obtain cultured skin fibroblasts;

[0015] S5: Detection and freezing.

[0016] Wherein, the digestion time in step S2 is 4-6 hours.

[0017] Wherein, the centrifugation condition in step S3 is 1000-1500 rpm, and the centrifugation time is 5-10 minutes.

[0018] The cell culture in step S3 and step S4 is carried out in an incubator, which is a 37°C constant temperature incubator containing CO2 The gas concentration is 5%.

[0019] The culture medium used for the cell culture in step S3 and step S4 is a high-glucose DMEM culture medium, and the high-glucose DMEM culture medium contains 10% fetal bovine serum and 1% penicillin / streptomycin by mass.

[0020] The present application obtains cultured skin fibroblasts by transferring the cells to a culture medium containing acetoacetate when the cells reach 80% confluence, and uses acetoacetate to inhibit collagen synthesis and reduce the deposition of extracellular matrix, thereby possibly slowing down or preventing the development of skin fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] By reading the detailed description below with reference to the accompanying drawings, the above and other purposes, features and advantages of the exemplary embodiments of the present disclosure will become readily understood. In the accompanying drawings, several embodiments of the present disclosure are shown in an exemplary and non-limiting manner, and the same or corresponding reference numerals represent the same or corresponding parts, wherein:

[0022] Figure 1 is a graph showing that acetoacetate treatment inhibits the expression of TGF-β-induced fibrosis genes in the examples of the present application;

[0023] Figure 2 is a graph showing that acetoacetate treatment inhibits the expression of inflammatory genes induced by TGF-β in the examples of the present application;

[0024] Figure 3 This is a graph showing that acetoacetic acid treatment inhibits BLM-induced mouse skin fibrosis gene expression in the examples of the present application;

[0025] Figure 4 This is a graph showing that acetoacetic acid treatment inhibits BLM-induced mouse skin inflammatory gene expression in the examples of this application;

[0026] Figure 5 The results of HE staining in the examples of the present application show that acetoacetic acid treatment inhibits BLM-induced mouse skin fibrosis;

[0027] Figure 6 This is the result of Masson staining in the examples of the present application showing that acetoacetic acid treatment inhibits BLM-induced collagen deposition in mice. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present disclosure to clearly and completely describe the technical solutions in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present disclosure.

[0029] The present application provides an application of acetoacetic acid in inhibiting skin fibrosis, wherein the chemical formula of the acetoacetic acid is CH 3 COCH 2 COOH.

[0030] The embodiment of the present application provides the use of acetoacetic acid in the preparation of a drug for skin fibrosis, wherein the drug is one of an injection, a spray, and drops.

[0031] In the application of acetoacetic acid in preparing a medicine for skin fibrosis, acetoacetic acid is used as an active ingredient, and the concentration of acetoacetic acid is 0.1-10 mM.

[0032] The method for inhibiting skin fibroblast fibrosis proposed in the present application specifically comprises the following steps:

[0033] S1: Aseptically obtain human skin tissue;

[0034] Skin tissue is generally obtained from normal skin tissue samples during plastic surgery, and the acquisition process needs to be carried out in a sterile environment to reduce the risk of infection.

[0035] S2: The adipose tissue was cut into small pieces, mixed with collagenase I, and incubated at 37°C and 5% CO 2 Digestion was performed in an incubator.

[0036] The obtained skin tissue needs to be cleaned with PBS buffer to remove blood and impurities in the skin tissue. After cleaning, it is cut into small pieces, mixed with collagenase I, and incubated at 37°C and 5% CO. 2 Digestion was performed in an incubator at 4°C for 4-6 hours.

[0037] S3: After enzymatic hydrolysis, the cells are centrifuged and washed, and then transferred to a culture flask containing culture medium for further culture;

[0038] The digested adipose tissue is filtered using sterile gauze, the cells in the filtrate are collected, and the cell suspension is washed with PBS buffer and centrifuged to remove the supernatant. The centrifugal speed is 1000-1500 rpm and the centrifugal time is 5-10 minutes. The corresponding parameters can be selected according to the actual experimental conditions and are not limited here.

[0039] The precipitated cells were resuspended in a culture medium, wherein the culture medium used was a high-glucose DMEM culture medium, to which 10% by mass of fetal bovine serum and 1% of penicillin / streptomycin were added, and then the cells were inoculated into a cell culture flask.

[0040] Place the culture flask in a 37°C, 5% CO 2 The cells were cultured in an incubator, the cell growth was observed every day, the culture medium was changed regularly to remove unattached cells and residual collagenase, and when the cells reached 80% confluence, they were digested with 0.25% trypsin-EDTA solution, and then the cells were passaged or used for subsequent experiments.

[0041] S4: When the cells are cultured to 80% confluence, they are transferred to a culture medium containing acetoacetate to obtain cultured skin fibroblasts;

[0042] The concentration of acetoacetic acid in the culture medium may be 0.1-10 mM. The concentration of acetoacetic acid used in the treatment in this example is 1 mM.

[0043] In order to further observe the anti-fibrotic effect of acetoacetate on TGF-β-induced human skin fibroblasts (HSF), the examples of the present application also added acetoacetate solution (concentration of 1 mM) to the culture medium containing TGF-β, so that the cells were exposed to TGF-β and acetoacetate at the same time.

[0044] S5: Detection and freezing.

[0045] The cells are subjected to biological efficacy testing, cell differentiation ability testing, etc., and the qualified adipose stem cells are added to a freezing tube, and freezing solution is added thereto. After being mixed and shaken evenly, they are frozen in a refrigerator and transferred to liquid nitrogen for long-term storage. The detection process and the freezing process can be carried out simultaneously to improve processing efficiency.

[0046] Test Example 1

[0047] The human skin fibroblasts in this test example were divided into three groups, namely a blank control group (Ctrl), a comparison group (TGF-β), and an experimental group (TGF-β+Acac).

[0048] In the blank control group (Ctrl), cells were treated with trypsin-EDTA solution, subcultured, and seeded into 6-well plates, with approximately 2 × 10 cells per well. 5 The cells were cultured until they were completely adherent for 48 hours.

[0049] The cell culture steps in the control group (TGF-β) were the same as those in the blank control group, except that TGF-β was contained in the culture medium.

[0050] The cell culture steps in the experimental group (TGF-β+Acac) were the same as those in the blank control group, except that the culture medium contained TGF-β and acetoacetate, wherein the concentration of acetoacetate was 1 mM.

[0051] Extract Total RNA, take out the sample to be tested from the refrigerator, add chloroform and shake evenly, then let it stand, centrifuge, extract RNA, dissolve RNA, measure its concentration and perform reverse transcription reaction.

[0052] RNA concentration determination steps: (1) Select the nucleic acid ID on the main interface, and the computer and instrument will automatically realize online; (2) Dissolve 1μL RNA in the solution (RNase-free water) on the test bench, click test, and then select the sample type in the upper right corner and select RNA-40; (3) Enter the sample name in the sample ID position; (4) Mix the samples, extract 1μL of the spot on the test bench, and measure again. (5) Save the data according to the report.

[0053] Reverse transcription reaction steps:

[0054] (1) Prepare RT reaction solution on ice

[0055]

[0056] (2) Reverse transcription reaction conditions:

[0057]

[0058] (3) RT-PCR

[0059] Real Time PCR reaction system (prepared on ice), related primers are shown in Table 1.

[0060]

[0061] Reaction conditions

[0062]

[0063] Table 1. Primer sequences for quantitative PCR of fibrosis genes

[0064]

[0065] Figure 1This is a graph showing that acetoacetic acid treatment inhibits the expression of TGF-β-induced fibrosis genes in the examples of this application. Under the stimulation of transforming growth factor TGF-β, fibroblasts will enhance the expression of fibrosis-related proteins, such as collagen 1α1 chain (Col1a1), fibronectin (Fibronectin), α-smooth muscle actin (α-SMA) and vimentin (Vimentin). However, when acetoacetic acid was added to the culture medium, the expression levels of the above-mentioned marker proteins decreased significantly. The results show that acetoacetic acid has a significant effect on inhibiting fibrogenesis in fibroblasts, such as Figure 1 As shown, the results were further confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and Col1a1, Fibronectin, α-SMA, and Vimentin were downregulated by 2.1, 2.2, 1.6, and 1.4 times, respectively.

[0066] Figure 2 is a graph showing that acetoacetic acid treatment inhibits the expression of inflammatory genes induced by TGF-β in the examples of the present application. In this test example, it was also observed that TGF-β can induce the expression of proinflammatory cytokines such as interleukin 6 (IL6), interleukin 1β (IL1-β) and tumor necrosis factor α (TNF-α). When acetoacetic acid was added to the culture medium, the expression levels of the above proinflammatory cytokines also decreased significantly, such as Figure 2 As shown, IL6, IL1-β and TNF-α were downregulated by 1.8, 1.8 and 1.6 times, respectively. The above results indicate that acetoacetate can not only inhibit the cell fibrosis process, but also has anti-inflammatory potential.

[0067] Test Example 2

[0068] In order to further verify the potential therapeutic effect of acetoacetic acid, this test case used a Babic mouse model for in vivo experiments. The mice in this test case were divided into three groups, namely a blank control group (Ctrl), a comparison group (BLM), and an experimental group (BLM+Acac). The experimental period was four weeks.

[0069] The back hair of 10-week-old BALB / c mice (2 cm × 2 cm) was shaved, and then a 1 cm × 1 cm square was drawn on the shaved skin. The mice in the blank control group (Ctrl) received subcutaneous injection of phosphate buffered saline (PBS), the mice in the comparison group (BLM) received subcutaneous injection of 1 mg / ml bleomycin, and the mice in the experimental group (BLM+Acac) received subcutaneous injection of 6 ug / ml acetoacetic acid and 1 mg / ml bleomycin. It should be noted that the injection amount of bleomycin is related to the weight of the experimental mice. The ratio of the injection amount of bleomycin to the weight of the mice in the comparison group and the experimental group is 5 mg / kg.

[0070] The injections of the two agents were kept one hour apart. Mice were housed individually and received the designated treatment every two days for a total of 28 days of treatment before being euthanized. At the end of the 28-day treatment period, mice were sacrificed and half of the fibrotic skin tissue was collected for in situ PCR analysis, while the other half was preserved for histological examination.

[0071] like Figure 3 and Figure 4 As shown, the application of acetoacetate significantly inhibited the increase in mRNA levels of fibrosis and inflammation-related markers induced by bleomycin, which was consistent with the results of in vitro experiments.

[0072] The mouse skin tissue samples were stained with HE and Masson staining, and the tissue structure and fibrosis degree of the skin tissue were further observed. Figure 5 and Figure 6 As shown, at the histological level, the skin of mice injected with acetoacetate showed significant improvements in the bleomycin-induced fibrotic response, including a reduction in dermal thickness, atrophy of subcutaneous adipose tissue, and an increase in collagen matrix deposition. The above histological changes further confirmed the effectiveness of acetoacetate in alleviating skin fibrosis.

[0073] The present application obtains cultured skin fibroblasts by transferring the cells to a culture medium containing acetoacetate when the cells reach 80% confluence, and uses acetoacetate to inhibit collagen synthesis and reduce the deposition of extracellular matrix, thereby possibly slowing down or preventing the development of skin fibrosis.

[0074] The present application further effectively inhibits abnormal collagen deposition, alleviates skin hardening, and improves skin function by topically applying a skin treatment agent containing acetoacetic acid.

[0075] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0076] Although the preferred embodiments of the present application have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present application.

[0077] Finally, it should be noted that, in this application, relational terms such as first and second, etc., are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article including the elements.

[0078] Specific examples are used in this application to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core idea of ​​this application. At the same time, for those skilled in the art, according to the idea of ​​this application, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as a limitation on this application.

Claims

1. Application of acetoacetic acid in inhibiting skin fibrosis, wherein the chemical formula of the acetoacetic acid is CH3COCH2COOH.

2. Use of acetoacetic acid in the preparation of a drug for skin fibrosis, wherein the drug is an injection, a spray, or a drop.

3. The use of acetoacetic acid in the preparation of a medicine for skin fibrosis according to claim 2, characterized in that: The concentration of the acetoacetic acid is 0.1-10 mM.

4. A method for inhibiting fibrosis of skin fibroblasts, characterized in that: The following steps are involved: S1: Aseptically obtain human skin tissue; S2: cutting the adipose tissue into small pieces, mixing with collagenase I, and digesting in an incubator at 37° C. and 5% CO 2 ; S3: After enzymatic hydrolysis, the cells are centrifuged and washed, and then transferred to a culture flask containing culture medium for further culture; S4: When the cells are cultured to 80% confluence, they are transferred to a culture medium containing acetoacetate to obtain cultured skin fibroblasts; S5: Detection and freezing.

5. The method for inhibiting fibrosis of skin fibroblasts according to claim 4, characterized in that: The digestion time in step S2 is 4-6 hours.

6. The method for inhibiting fibrosis of skin fibroblasts according to claim 4, characterized in that: The centrifugation condition in step S3 is 1000-1500 rpm, and the centrifugation time is 5-10 minutes.

7. The method for inhibiting fibrosis of skin fibroblasts according to claim 4, characterized in that: The cell culture in step S3 and step S4 is carried out in an incubator, which is a 37° C. constant temperature incubator with a CO 2 gas concentration of 5%.

8. The method for inhibiting fibrosis of skin fibroblasts according to claim 7, characterized in that: The culture medium used for the cell culture in step S3 and step S4 is a high-glucose DMEM culture medium, and the high-glucose DMEM culture medium contains 10% fetal bovine serum and 1% penicillin / streptomycin by mass.

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