KR20210116851A - A Abti-Inflammation Material H2O2-Induced from Membrane-Free Stem Cell - Google Patents

Abstract

The present invention relates to a composition for treating anti-inflammation induced by hydrogen peroxide of a membrane-free stem cell extract. The configuration thereof is characterized by using an adipose stem cell-derived membrane-free stem cell extract at a concentration of 0.5 μg/mL. The present invention has a protective effect on oxidative stress due to H_2O_2 in HPLF cells of human adipose tissue-derived membrane-free stem cell extract, and when treating the membrane-free stem cell extract to HPLF cells induced by oxidative damage with H_2O_2, cell viability increases, thereby having an effect of protecting cells from oxidative stress.

Description

Composition for the treatment of anti-inflammatory induced by hydrogen peroxide of amacrine stem cell extract {A Abti-Inflammation Material H2O2-Induced from Membrane-Free Stem Cell}

The present invention relates to a composition for anti-inflammatory treatment, and more particularly, a substance extracted from stem cells isolated from adipocytes is induced by hydrogen peroxide of amacrine stem cell extract, which can treat inflammation caused by stress of hydrogen peroxide, etc. It relates to a composition for the treatment of anti-inflammatory drugs.

Periodontitis is the most common oral disease and destroys periodontal ligaments and gingiva, and in severe cases causes tooth loss. It is known that oxidative stress caused by inflammatory bacteria is the main cause of the occurrence of such periodontal inflammation. H2O2 is one of the intermediate metabolites generated during biological metabolism, but is one of the representative substances that induce oxidative stress in cells when excessively produced.

Oxidative stress caused by H2O2 plays an important role in the pathogenesis of periodontal disease, and in particular, human periodontal ligament fibroblasts (HPLFs) release inflammatory factors such as inflammatory cytokines and chemokines to induce inflammatory responses, It is known to cause periodontitis by causing cell morphology and cell death.

Therefore, research on the treatment of periodontitis by improving the inflammatory response is being actively conducted.

Cells that make up organs, skin, bones, and cartilage of the human body maintain homeostasis in the body through apoptosis and cell production. Cells that newly induce cell production are called stem cells.

Adult stem cells (adult stem cells), a type of stem cells, are known to have excellent regenerative function and excellent self-renewal ability. In addition, it has been reported that adult stem cells have the ability to be multi-differentiated into other tissues, and not only help normal repair and recovery when tissue is damaged, but also have anti-inflammatory action. Therefore, adult stem cells are being actively used to treat various diseases such as dementia, heart disease, diabetes, and skin improvement.

Patent Registration No. 10-2082745

It is an object of the present invention to provide a composition for treating anti-inflammatory drugs induced by hydrogen peroxide of amacrine stem cell extracts by confirming the periodontitis protective effect against H2O2-induced oxidative damage using amacrine stem cells.

In order to achieve the above object, the composition for anti-inflammatory treatment induced by hydrogen peroxide of the adipose stem cell extract of the present invention uses an adipose stem cell-derived amacrine stem cell extract at a concentration of 0.5 μg/mL. characterized.

After the adipose stem cells are separated and purified from adipose tissue, the adipose stem cells are initially cultured in an incubator under a condition of 5% CO2 at 37°C, and subculture is repeated 6 to 10 times, and the medium is After removal, it is characterized in that the extract is obtained after removing the cell membrane of the stem cells.

The composition for anti-inflammatory treatment induced by hydrogen peroxide of the amacrine stem cell extract according to the present invention has the following effects.

H2O2 induced oxidative stress protection effect in HPLF cells derived from human adipose tissue, and cell viability increases when treating HPLF cells inducing oxidative damage with H2O2. It has the effect of protecting cells from oxidative stress.

1 is a graph showing the cell viability when the composition for the treatment of anti-inflammatory induced by hydrogen peroxide of the present invention amacrine stem cell extract is applied by concentration.
Figure 2 is a graph showing the measurement of the expression of NF-κB, iNOS and IL-6 proteins, which are inflammation-related factors, when the composition for treating anti-inflammatory drugs induced by hydrogen peroxide of the present invention amacrine stem cell extract is applied by concentration.
Figure 3 is to confirm the apoptosis protection mechanism for the case of applying the composition for the treatment of anti-inflammatory induced by hydrogen peroxide of the present invention of the present invention by concentration, caspase-9, -3, PARP protein expression was measured. A graph showing the results.

Hereinafter, a preferred embodiment of the composition for treating anti-inflammatory induced by hydrogen peroxide of the amacrine stem cell extract according to the present invention will be described in detail with reference to the accompanying drawings.

For the composition for treating anti-inflammatory drugs induced by hydrogen peroxide of the amacrine stem cell extract of the present invention, it is preferable to use an adipose stem cell-derived adipose stem cell extract at a concentration of 0.5 μg/mL.

And, after the adipose stem cells are separated and purified from the adipose tissue, the adipose stem cells are initially cultured in an incubator at 37 ° C. and 5% CO2 condition, and subculture is repeated 6 to 10 times, After removing the medium, after removing the cell membrane of the stem cells, the extract can be obtained and used.

Hereinafter, the anti-inflammatory experiment of the composition for the treatment of anti-inflammatory induced by hydrogen peroxide of the amacrine stem cell extract of the present invention will be described in detail.

1. Experimental Materials

H2O2 was purchased from Junsei Chemical Co (Tokyo, Japan), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) was obtained from Sigma Chemical Co (St Louis). , MO, USA) and used in the experiment. The RIPA buffer used for protein extraction and western blot analysis was from Elpis Biotech (Daejeon, Korea), the protease inhibitor cocktail was from Calbio Chem (Novabiochem, La Jolla, CA), and the primary and secondary antibodies were Cell Signaling Technology (Beverly , MA, USA) and Santa Cruz Biotechnology (Santa Cruz, MA, USA) were purchased and used.

2. Manufacture of membraneless stem cell extract

The amucosal stem cell extract used in this test was provided by Tistem Co., Ltd. Membraneless stem cell extract is a stem cell component extract, obtained by separating and culturing stem cells from human adipose tissue and then removing the cell membrane. Adipose tissue, which is a raw material, was obtained by obtaining a consent form for local donation from women in their 20s who had no abnormalities through blood tests and had a BMI of 25 to 299. The blood tests performed were hepatitis B virus, hepatitis C virus, human immunodeficiency virus, human T lymphotrophic virus, parvovirus B19, cytomegalovirus, Abstein-Barr virus, and syphilis creponema. The final product, amacrine stem cells, has been tested for safety by a Good Laboratory Practice accredited institution and confirmed that it is a non-toxic substance. After separating and purifying adipose stem cells from the adipose tissue obtained through the above process, initial culture was performed in an incubator at 37°C and 5% CO2 conditions. After checking the degree of cell growth, subculture was performed 6 to 10 times. repeated. After removing the medium and obtaining a certain amount of stem cells, the cell membrane was peeled off using a physical method such as ultrasound, and the cell membrane fragments were removed using a method such as a filter to obtain a membraneless stem cell extract. The aqueous membrane-free stem cell extract was lyophilized to form a powder formulation and stored at 5 ± 2°C.

3. Cell Types and Reagents

Human periodontal ligament fibroblasts (hereafter, HPLF) cells used in this experiment were purchased from ScienCell (Carlsbad, CA, USA) and used in the experiment. For culture, fetal bovine serum (FBS), fibroblast growth supplement, penicillin/streptomycin solution, fibroblast medium medium, and trypsin-ethylenediaminetetraacetic acid (EDTA) solution were purchased from ScienCell (Carlsbad, CA, USA) and used.

4. Cell Culture

HPLF cells were cultured in a fibroblast medium containing 2% FBS, 1% fibroblast growth supplement, and 1% penicillin/streptomycin solution at 37°C and 5% CO2 incubator. Cultured cells are cultured while changing the culture medium once every 1 to 3 days, washed with phosphate buffered saline (PBS, pH 74) under 80-90% confluence, and then adhered cells are separated with 025% trypsin EDTA mixture. After centrifugation at 1,000 rpm for 5 minutes, the suspension was used for the experiment while subcultured.

5. Cell Viability Measurement

To determine the cytoprotective effect against oxidative damage, cell viability was measured by MTT assay. HPLF cells were seeded in 96 well plates at 5×104 cells/mL and incubated at 37° C. for 24 hours. When the cells are well adhered, the membrane-free stem cell extract (0025, 005, 01,025, 05 μg/mL) is treated at each concentration and cultured for 4 hours, then treated with H2O2 (100 μM) and incubated for 24 hours, followed by 5 mg /mL of MTT solution was injected into each well.

After treatment with the MTT solution, the resulting formazan crystals were dissolved in DMSO after culture at 37° C. for 4 hours, left at room temperature for 30 minutes, and absorbance was measured at 540 nm. In the case of the control group, the experiment was carried out by treating only H2O2 (100 μM) without treatment with a membrane-free stem cell extract.

6 Western blot analysis

Inflammation and apoptosis-related protein expression was determined by western blot analysis. After adding RIPA buffer to the cultured cells and centrifuging at 4°C and 12,000 rpm for 30 minutes, protein quantification was performed using the supernatant. Each sample was electrophoresed on 8-10% sodium dodecyl sulphate-polyacrlamide (SDS-PAGE) gel and transferred to the membrane. The protein-attached membrane was blocked with 5% skim milk at room temperature for 50 minutes, and then primary antibody [nuclear factor kappa light chain enhancer of activated B cells (NF-κB, 1:1000); inducible nitric oxide synthase (iNOS 1:1000); caspase-9 (1:1000); caspase-3 (1:1000); Poly (ADP-ribose) polymerase (PARP, 1:1000); interleukine-6 (IL-6, 1:200); B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax, 1:100); Bcl-2(1:200)] was reacted overnight at 4°C, washed with PBS-T, and mixed with a secondary antibody [Anti-rabbit and anti-mouse IgG HRP-linked antibody (1:1000)] at room temperature for 1 time was reacted. After reacting with an enhanced chemiluminescence (ECL) solution, protein expression was confirmed using a Chemilium inescence image system (Davinch-ChemiTM).

7. Statistical analysis

All experimental results were analyzed by analysis of variance (ANOVA) through the Statistical Package for the Social Sciences (SPSS, Chicago, USA) program. did.

8. Effect of membraneless stem cell extract on cell survival from H2O2 damage

After each concentration of non-membranous stem cell extracts were treated on HPLF cells (0025 - 1 μg/mL) and cultured for 24 hours, MTT assay was used to determine the cytotoxicity of the non-membrane stem cell extracts.

was reviewed. As a result, as shown in Table 1, up to 05 μg/mL of the membraneless stem cell extract showed a cell viability of more than 95%, indicating that there was no cytotoxicity.

Treatment (μg/ml) Cell viability (%) 0 100.00±1.07 0.025 100.53±2.99 0.05 93.02±1.46 0.1 91.79±1.59 0.25 90.85±2.32 0.5 95.41±1.54

In order to confirm the cytoprotective effect of the membraneless stem cell extract against oxidative damage caused by H2O2, oxidative damage was induced by treating HPLF cells with 100 μM H2O2, and then the membraneless stem cell extract by concentration within the range of no cytotoxicity. was treated to measure cell viability. As shown in FIG. 1 , in the case of the control group treated with H2O2 alone in the HPLF cells, the cell viability was 6150% compared to 100% of the normal group, confirming the cell damage caused by the H2O2 treatment.

On the other hand, it was confirmed that the cell viability increased in the group treated with amacrine stem cell extract compared to the control group, which is considered to have a protective effect against cell damage caused by H2O2.

9. Inflammation improvement effect of amucosal stem cell extract

The inflammatory response is one of the initial responses of the body's immune system to infection or stimuli, and oxidative damage caused by H2O2 activates the NF-κB pathway to cause an inflammatory response. NF-κB is composed of two subunits, p50 and p65, to form a heterodimer structure, and exists in an inactivated form by binding with inhibition of nuclear transcription factor-κB (IκB). When activated by external stimuli such as oxidative damage, IkB is dissociated and NF-κB subunits move into the nucleus, inducing the transcription of genes such as inflammation-related mediators and cytokines. Such overexpressed inflammatory mediators and cytokines induce genetic mutations and cell damage.

HPLF cells treated with H2O2 are widely used as an in vitro model for periodontal inflammation. Therefore, in this study, in order to confirm the inflammatory protective mechanism against H2O2 of the amacrine stem cell extract in HPLF cells, the expression of NF-κB, iNOS and IL-6 proteins, which are inflammation-related factors, was measured in FIG. 2 . In the control group treated with only H2O2, the expression of inflammation-related proteins was significantly increased compared to the normal group untreated, and it was found that the inflammatory response was induced by H2O2. On the other hand, it was confirmed that the expression of these proteins was suppressed when compared to the control group when the membraneless stem cell extract was treated. In particular, it was confirmed that the lowest expression was observed at a concentration of 05 μg/mL of the amenorrhea stem cell extract. It is thought that the amacrine stem cell extract showed an inflammatory response protective effect by regulating inflammatory mediators and inflammatory cytokines through the regulation of the NF-κB pathway activated by H2O2.

According to domestic and foreign studies, adipose tissue-derived stem cells have been reported to have anti-inflammatory effects by inhibiting pro-inflammation factor, regulating NF-κB pathway activation, and inhibiting nitric oxide production. In addition, adipose tissue-derived amucosal stem cell extract improved the pain accompanying inflammation in the joints, ligaments, tendons and muscles caused by golf by anti-inflammatory and regenerative functions, and also improved the inflammatory response of skin cells due to oxidative damage. did it Therefore, it is considered that the adipose tissue-derived amucosal stem cell extract has a protective effect against the inflammatory reaction caused by oxidative damage.

10. Mechanism of protection against apoptosis of a membrane-free stem cell extract

Oxidative damage caused by excessive intracellular H2O2 reduces the mitochondrial membrane potential and activates caspase that mediates cell death and DNA and protein degradation by mitochondrial dysfunction. Upon oxidative damage, mitochondria release cytochrome-c, which induces cell nuclear condensation through activation of caspase-9, caspase-3, and PARP, leading to apoptosis. As shown in FIG. 3 in this study, in order to confirm the apoptosis protective mechanism of the adipose-derived amacrine stem cell extract in HPLF cells induced with oxidative damage with H2O2, the expression of caspase-9, -3, and PARP proteins was measured. Compared with the normal group untreated, the control group treated with H2O2 significantly increased the protein expression ratio of cleaved caspase-9/caspase-9, cleaved caspase-3/caspase-3, and cleaved PARP/PARP to activate caspase It was confirmed that apoptosis was induced through On the other hand, it was confirmed that the expression of cleaved caspase-9, -3, and PARP proteins was decreased in the group treated with the membrane-free stem cell extract compared to the control group. In addition, according to a previous study, adipose tissue-derived stem cells exhibited apoptosis protective effect by regulating caspase-9 and -3 activation in cisplatin-induced apoptosis-induced tongue squamous cells. Therefore, it is thought that adipose tissue-derived acellular stem cell extracts have apoptotic protective effect against oxidative damage through reduced caspase activation.

The Bcl family is one of the representative mechanisms involved in apoptosis. Overexpression of the pro-apoptotic factor, Bax, induces apoptosis by increasing the production of cytochrome-c, and the anti-apoptotic factor, Bcl-2, induces apoptosis. It is known to play a role in protecting apoptosis by reducing production. As a result of measuring the apoptosis protective effect by measuring the protein expression of the Bax/Bcl-2 ratio, a significant increase in protein expression was confirmed in the control group compared to the normal group. On the other hand, when the membrane-free stem cell extract was treated, the adipose-derived membrane-free stem cell extract significantly down-regulated the expression of apoptosis-related protein compared to the control group at a concentration of 01,05 μg/mL, thereby regulating the expression ratio of Bax and Bcl-2 proteins. It is thought to have an apoptosis protective effect through Chiu et al. reported that adipose tissue-derived stem cells were effective in inhibiting apoptosis through inhibition of Bad protein, a pro-apoptotic factor in apoptosis-induced tongue squamous cells. This has been reported In addition, the adipose tissue-derived stem cell extract was confirmed to have a cytoprotective effect through the regulation of the p53/Foxo pathway against amyloid beta-induced apoptosis. Therefore, it is thought that the adipose tissue-derived stem cell extract exhibits apoptotic protective effect through various pathways such as caspase activation and Bcl family regulation.

As such, those skilled in the art to which the present invention pertains will understand that the above-described technical configuration of the present invention may be implemented in other specific forms without changing the technical spirit or essential characteristics of the present invention.

Therefore, the embodiments described above are to be understood as illustrative and not restrictive in all respects, and the scope of the present invention is indicated by the following claims rather than the above detailed description, and the meaning and scope of the claims and their equivalents It should be construed as being included in the scope of the present invention all changes or modifications derived from it.

Claims (2)

A composition for the treatment of anti-inflammatory treatment induced by hydrogen peroxide of a membrane-free stem cell extract, characterized in that the extract of the adipose stem cell-derived adipose stem cell is used at a concentration of 0.5 μg/mL. The method of claim 1,
The amacrine stem cells are
After separating and purifying adipose stem cells from adipose tissue, after initial culture at 37° C. in an incubator under 5% CO2 conditions, subculture is repeated 6 to 10 times, and after removing the medium, the stem cells A composition for treating anti-inflammatory treatment induced by hydrogen peroxide of a membrane-free stem cell extract, characterized in that the extract is obtained after removing the cell membrane.

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