CN116251126A - Application of periodontal ligament stem cell-derived exosome in relieving cell aging and treating periodontitis - Google Patents

Abstract

The invention discloses application of exosomes derived from periodontal ligament stem cells in preparing a product for relieving cell aging and application of exosomes in preparing a medicament for treating periodontitis. The study of the invention proves that PDLSC-Exos can play an anti-aging role by transmitting miR-141-3p to down-regulate KEAP1 expression and activate NRF2 anti-oxidation pathway. The invention can provide a certain theoretical basis for the application of cell-free therapy in preventing and treating the periodontitis of diabetes, and the research of the invention proves that the PDLSC-Exos is hopeful to be used as a novel bioactive medicament for resisting cell aging, and has better application prospect.

Description

Application of periodontal ligament stem cell-derived exosome in relieving cell aging and treating periodontitis

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of exosomes derived from periodontal ligament stem cells in relieving cell aging and treating periodontitis.

Background

Diabetes and its complications have been prevalent worldwide for the last decades. Periodontitis is the sixth complication of diabetes, a major cause of tooth loss in adults, and severely affects the health and quality of life of patients. However, the treatment of diabetic periodontitis has had limited clinical success, but the development of tissue engineering and stem cell studies provides new opportunities for the repair of periodontal tissue damage. Human periodontal ligament stem cells (pdcscs), also known as undifferentiated Mesenchymal Stem Cells (MSCs), are widely recognized as ideal seed cells for periodontal tissue regeneration. According to previous reports, a variety of factors affect the biological activity of pdcscs, with hyperglycemia being one of the most common risk factors.

Cell senescence is a permanently arrested state of the cell cycle caused by internal or external factors. Hyperglycemia is a critical extracellular stress signal that triggers cellular senescence. Hyperglycemia causes excessive oxidative stress, shortening of telomeres, epigenetic changes, and mitochondrial dysfunction, thereby triggering MSCs senescence. Various factors lead to aging of pdcscs, such as induction of transforming growth factor-beta, long-term in vitro culture, and overexpression of insulin-like growth factor binding protein 5. However, there are still few reports of the effects of high sugar levels on the aging of pdcscs. The aging of PDLSC is accompanied by the reduction of proliferation potential, the impaired multipotent differentiation ability and the increased apoptosis, which is unfavorable for the repair and regeneration of periodontal defects. Thus, delaying cell senescence may be an emerging option for activating endogenous periodontal tissue repair mechanisms.

Exosomes (Exosomes) are membrane-derived lipid bilayer vesicles secreted by various cells, containing various cell-derived lipids, proteins and nucleic acids. Exosomes can transport various signaling molecule components and regulate receptor cells through intercellular communication. The stem cell-derived exosomes function similarly to the stem cells from which they are derived and may represent cell replacement for tissue regeneration and improve cellular senescence through a variety of pathways. For example, the exosomes derived from deer antler stem cells alleviate osteoarthritis by counteracting aging of intra-articular cells. In addition, exosomes produced by human embryonic stem cells stimulate the Kelch-like ECH-related protein 1 (KEAP 1) -nuclear factor red blood cell 2-related factor 2 (NRF 2) signaling pathway by transferring microRNA-200a (miR-200 a), alleviating endothelial cell senescence. Human periodontal ligament stem cell-derived exosomes (PDLSC-exos) are the major components of the paracrine factors of PDLSC, with a wide range of biological activities, including treatment of bone abnormalities associated with periodontitis, stimulation of proliferation, angiogenesis, and immunomodulation. However, the function and underlying mechanisms of PDLSC-Exos in cell senescence are not yet defined.

According to the oxidative stress theory of aging, enhancing the body's antioxidant defenses may be a therapeutic option to delay aging and extend life. The KEAP1-NRF2 pathway is one of the most important cellular antioxidant systems, which controls the expression of many antioxidant enzymes. Previous studies have shown that NRF2 activity is reduced during aging, and that activation of NRF2 signaling can treat aging-related diseases or prevent cellular aging and slow down the aging process. Activation of NRF2 can prevent cell senescence of mesenchymal stem cells, fibroblasts and dendritic cells, reduce the burden of cardiovascular disease in aged people, and reverse progression of age-related fibrotic disease. Thus, activation of NRF2 may be very important for improving aging and its related diseases. The mesenchymal stem cell-derived exosomes (MSC-Exos) have been reported to have excellent antioxidant properties, for example, to protect against oxidative stress-induced skin damage by modulating the NRF2 antioxidant defense system.

Disclosure of Invention

The invention researches the aging of PDLSC under high sugar microenvironment and the action and potential mechanism of PDLSC-Exos for relieving cell aging. The influence of the high sugar microenvironment on the aging of the PDLSCs is evaluated by separating the PDLSCs from periodontal ligament, and the ability of the PDLSC-Exos to reduce oxidative stress and improve cell aging is studied, and the molecular mechanism of the PDLSC-Exos to improve the aging of the PDLSC is verified. Based on the above, the invention provides the following technical scheme:

use of periodontal ligament stem cell-derived exosomes in the preparation of a product for alleviating cellular senescence.

The cell aging relieving is to relieve periodontal ligament stem cell aging.

The cell aging relieving is to relieve periodontal ligament stem cell aging under high-sugar microenvironment.

The exosomes derived from periodontal ligament stem cells alleviate high sugar-induced aging of pdcscs by transmitting miR-141-3p to activate KEAP1-NRF2 signaling pathway.

The invention also provides application of the periodontal ligament stem cell-derived exosome in preparing medicines for treating periodontitis.

The periodontitis is diabetic periodontitis.

In the above technical solution, the periodontal ligament stem cells are human periodontal ligament stem cells.

In the technical scheme, the product or the medicine consists of an active ingredient and pharmaceutically acceptable auxiliary materials, wherein the active ingredient is an exosome derived from periodontal ligament stem cells.

In the technical scheme, the exosome preparation method comprises the steps of collecting serum-free culture medium of the cultured young periodontal ligament stem cells, and extracting the exosome by adopting a low-temperature ultracentrifugation method.

In the above technical scheme, the exosome preparation method comprises the following steps: culturing young periodontal ligament stem cells in a complete culture medium, changing the culture medium into a serum-free culture medium when the cell density reaches more than 60%, continuously culturing for 44-52h, and collecting cell supernatant to obtain supernatant 1; subjecting the obtained supernatant 1 to differential centrifugation to obtain a supernatant 2; and (3) ultracentrifugating the obtained supernatant 2, and removing the supernatant to obtain the exosomes.

The invention researches the aging of PDLSC under high-sugar microenvironment and the action and potential mechanism of exosomes (PDLSC-Exos) derived from human periodontal ligament stem cells for relieving cell aging. The cells were evaluated for senescence by isolating and extracting PDLSCs and PDLSC-Exos, and then treating PDLSCs with high sugar (25 mM) medium. The role of PDLSC-Exos in cell senescence was further explored by co-culturing PDLSC-Exos with senescent PDLSCs, and differences in the levels of oxidative stress of cells after treatment with PDLSC-Exo were examined. Next, we studied whether PDLSC-Exos ameliorates cellular senescence by restoring the balance of oxidative stress signals and explored potential molecular pathways. We found that high sugar induced premature aging of PDLSCs, whereas PDLSC-Exos treatment restored the viability of senescent cells. When the PDLSC-Exos is co-treated with the nuclear factor erythroid 2-related factor 2 (NRF 2) inhibitor ML385, the effect of PDLSC-Exos on restoring cell viability is significantly inhibited, indicating that the therapeutic effect of PDLSC-Exos is dependent on the activation of NRF 2. Further studies showed that microRNA-141-3p (miR-141-3 p) was expressed at relatively high levels in PDLSC-Exos and promoted PDLSC-Exos mediated aging cell rejuvenation by down-regulating Kelch-like ECH-related protein 1 (negative regulator of KEAP1, NRF2 expression). Our study shows that PDLSC-Exos alleviates high sugar-induced aging of PDLSCs by transmitting miR-141-3p to activate KEAP1-NRF2 signaling pathway. Based on this study, PDLSC-Exos may behave similarly to their parent PDLSCs and have a significant impact on cell senescence by delivering their encapsulated bioactive chemicals to target cells. This suggests that our PDLSC-Exos as a new candidate for cell-free treatment may be a potential anti-aging formulation, hopefully playing a role in the treatment of diabetic periodontitis.

Drawings

FIG. 1 is a feature of human PDLSCs and PDLSC-Exos; isolation and passaging of a-D human pdcscs, cells exhibiting typical spindle-like morphology; e calcified nodorubin red staining proves that the PDCSCs have osteogenic differentiation potential; f oil red O staining shows that PDLSCs have adipogenic differentiation potential; g colony formation experiments show the proliferation potential of PDLSCs; A-G scale bar, 100 μm; h TEM observation shows that PDLSC-Exos is in a typical cup shape, with a scale of 100nm; i WB shows that PDLSC-Exos express exosome marker proteins; JNTA shows particle size and concentration of PDLSC-Exos.

FIG. 2 shows that high sugar induces senescence in human PDLSCs; SA-beta-gal staining after 14 days of A high sugar treatment, the proportion of SA-beta-gal positive cells (blue) is obviously increased, and the proportion scale is 100 mu m; b quantifying the percentage of SA- β -gal positive PDLSCs in each group; c Hoechst 33258 staining to observe apoptosis, and the concentration of apoptotic nuclei (white arrow) is visible, with a scale of 100 μm; d IF staining measures p16 expression (green), scale 100 μm; quantitative analysis of E-FWB detection of cell senescence-associated protein expression (p 53 and p 21); g the expression of SASP genes (IL-6 and IL-8) was quantified using qRT-PCR.

FIG. 3 shows impaired glucose-induced aging PDLSCs function; A-B adopts a CCK-8 test and a colony formation test to evaluate the proliferation capacity of the PDCSCs, and the scale is 100 mu m; cell migration was detected by wound healing experiments C-D and Transwell experiments E-F, with a scale of 100 μm; cell osteogenesis ability was evaluated by ALP staining and activity assay 7 days after induction of G-H PDLSCs, scale bar 100 μm; I-J uses alizarin red staining and quantification to evaluate cellular calcium deposition, scale bar 100 μm; K-L oil red O staining and quantification show that all three groups of cells have the capacity of differentiating towards adipocytes, with a scale of 100 μm.

FIG. 4 shows that PDCSCs-Exos improved high sugar-induced aging of human PDCSCs; aging PDLSCs ingest Dill-labeled exosomes at a scale of 100 μm; b SA-beta-gal staining cell image, scale bar 100 μm; quantification of the percentage of SA- β -gal positive pdcscs in each group C; d IF detects p16 expression (green), scale bar 100 μm; E-FWB detects and quantifies expression of senescence-associated proteins p53 and p 21; g the expression of SASP genes (IL-6 and IL-8) was detected using qRT-PCR.

FIG. 5 shows that PDLSCs-Exos can restore high sugar-induced dysfunction of aging PDLSCs; A-B adopts a CCK-8 method and a colony formation experiment to detect the proliferation capacity of PDLSCs, and the scale is 100 mu m; cell migration was assessed by wound healing C-D and Transwell experiments E-F, scale bar 100 μm; G-HALP staining/activity assay, scale bar 100 μm; I-J alizarin red staining and mineralization nodule quantification, and a proportion scale is 100 mu m; K-L oil red O staining quantitative determination of three groups of cells adipogenic differentiation capacity, the scale is 100 μm.

FIG. 6 shows that PDLSC-Exos reduces oxidative stress and increases the activity of the endogenous NRF2 antioxidant system; A-B intracellular ROS levels were assessed by measuring DCFH-DA fluorescence, scale bar 100 μm; C-D detects cellular oxidative stress levels by detecting MDA levels and SOD activity; total NRF2, nuclear NRF2, KEAP1, HO-1, NQO1 protein expression levels of each group of E-F; g IF staining showed localization of NRF2 (green), scale 100 μm.

FIG. 7 shows that PDLSC-Exos rejuvenates aged human PDLSCs by activating NRF 2; a-B WB analysis of each group for total NRF2, nuclear NRF2, HO-1 and NQO1 levels; c IF staining detects NRF2 expression and localization (green), scale bar 100 μm; D-E stained cells with SA- β -gal, and the percentage of SA- β -gal positive cells was calculated, scale bar 100 μm; F-G WB detects and quantifies expression of senescence-associated proteins (p 53 and p 21); h IF staining showed p16 expression, scale bar 100 μm; i, detecting the SASP gene (IL-6, IL-8) expression level by adopting qRT-PCR; j, detecting and quantifying the ROS level of the cells by DCFH-DA staining, wherein the scale is 100 mu m; L-M measures MDA levels and SOD activity.

FIG. 8 shows that PDLSC-Exos enhances the biological function of senescent PDLSCs in an NRF2 dependent manner. A cell proliferation was detected with CCK-8. B cell colony formation assessed by colony formation experiments. Scale bar, 100 μm. Cell migration was assessed using wound healing experiments C-D and Transwell experiments E-F. Scale bar, 100 μm. After G-H osteogenesis induction for 7d, PDLSC osteogenesis was assessed using ALP staining and activity assays. Scale bar, 100 μm. Alizarin red staining 21 days after I-J osteoinduction and quantification, assessment of cellular calcium deposition. Scale bar, 100 μm. K-L oil red O staining method for detecting cell adipogenic differentiation. Scale bar, 100 μm.

FIG. 9 shows that PDLSC-Exos delivered miR-141-3p activates NRF2 signal by decreasing KEAP1 expression; a predicts Venn diagram of miRNAs targeting KEAP1 gene (hsa-miR-141-3 p, hsa-miR-200a-3 p); b qRT-PCR (B qRT-polymerase chain reaction) detection of the change of the expression level of miRNAs of human PDLSCs before and after high sugar treatment; cqRT-PCR detects the expression of selected miRNAs in PDLSC-Exos. D, detecting the expression of miR-141-3p by adopting qRT-PCR; E-F uses dual luciferase reporter gene experiments to verify the targeting relationship between miR-141-3p and KEAP 1; g adopts qRT-PCR analysis to determine the expression level of miR-141-3p in the aging PDLSCs treated by PDLSC-Exos; H-I was assayed for total NRF2, nuclear NRF2, KEAP1, HO-1 and NQO1 protein expression levels using WB and quantitatively analyzed using ImageJ software; j NRF2 was localized by IF staining, scale bar 100 μm.

FIG. 10 shows that exosome miR-141-3p from PDCSCs is critical for delaying high sugar-induced PDLSC senescence; A-B SA-beta-gal staining image and percentage of SA-beta-gal positive cells, scale bar 100 μm; C-D analysis of expression levels of p53 and p21 with WB; e IF staining detects p16 expression, scale bar 100 μm; relative levels of SASP genes IL-6 and IL-8; g detects the level of the ROS in the cells by DCFH-DA staining and quantifies the level, and the scale is 100 mu m; I-J assesses the level of oxidative stress in cells by measuring MDA levels and SOD activity.

FIG. 11 shows that PDLSC-Exos restores cell biological function by modulating miR-141-3p/KEAP1/NRF2 axes in senescent PDLSCs; A-B cell proliferation was assessed using CCK-8 and colony formation experiments, scale bar 100 μm; cell migration was detected by wound healing C-D and Transwell E-F experiments, scale bar 100 μm; G-H alkaline phosphatase staining and activity determination, with a scale of 100 μm; I-J adopts alizarin red staining method to observe and measure calcium deposition, and the proportion scale is 100 μm; k oil red O dyeing, the proportion is 100 μm; quantification of L oil red O staining.

Detailed Description

The invention is further illustrated, but is not limited, by the following examples.

The experimental methods in the following examples are conventional methods unless otherwise specified.

Example 1

1. Materials and methods

1.1 cell culture and identification

After informed consent was obtained for all patients, pdcs were extracted from healthy wisdom teeth or orthodontic teeth of young patients. The freshly extracted teeth were rinsed with Phosphate Buffered Saline (PBS), and 1/3 of the central root area was scraped to collect periodontal ligament tissue. Next, the cells were digested with collagenase type I solution (3 mg/mL) (Sigma-Aldrich, st.Louis, MO, USA) at 37℃for 15 minutes, then 5mL of alpha-MEM medium was added to supplement penicillin-streptomycin solution (1%) and fetal bovine serum (FBS; gibco, grand Island, N.Y., USA) (10%). Subsequently, the tissue in the flask was incubated at 37℃with 5% CO ₂ Is cultured in a wet incubator. After observing that the cells migrate out of the tissue, the culture medium is replaced, subcultured, and the third generation cells are taken for subsequent cell identification. The multidirectional differentiation potential and colony forming ability of the PDLSCs are verified through alizarin red staining, oil red O staining and colony forming experiments.

1.1.1 alizarin Red staining

Cells were seeded in 6-well plates (5X 10) ⁵ Cells/wells). After the cell growth density reached 80%, osteogenic induction medium was added, and medium was changed every 2 days. Alizing 21 days after induction, alizarin red staining (Biyun Tian Biotechnology institute, shanghai, china) was performed. With hexadecyl chloridePyridine dissolves calcified nodules and absorbance is measured at 562 nm.

1.1.2 oil Red O staining

Cells were seeded in 6-well plates (5X 10) ⁵ Cells/wells). When the cell density reached 80%, the medium was changed to lipid-forming medium. After 14 days of continuous culture, staining with oil red O. The stained lipid droplets were dissolved in isopropanol and absorbance was measured at 520 nm. The Soy Co., ltd (Beijing, china) produced a kit for use in the experiment.

1.1.3 colony formation assay

Cells were in 60mm dishes (800 cells/dish). After 14 days of continuous culture, cells were fixed with 4% Paraformaldehyde (PFA) and then stained with 0.1% crystal violet. After the staining was completed, excess dye was rinsed off and cell colony formation was observed under a microscope. Wherein a cell aggregate of more than 50 cells is considered a colony.

1.2 Effect of high sugar on PDLSC aging

Second generation pdcs from the same donor were continuously cultured in high sugar medium (HG group, 25mM glucose) to explore the effect of high sugar environment on PDLSC senescence. The medium was changed every 2 days to reach passage at 80% confluence. The control group was incubated in high concentration mannitol (HM group, osmolality control, 5.5mM glucose+19.5 mM mannitol) or normal glucose medium (NG group, 5.5mM glucose). Aging-associated beta-galactosidase (SA-beta-gal) activity was monitored at various time intervals (7, 14, 21 and 28 days) for each group of cells to determine the time at which high sugar induced cell aging. The HG group was stained by Hoechst 33258 at various time points (7, 14, 21, 28 days) to observe the apoptotic morphology, to distinguish apoptosis from cell senescence. In subsequent studies, we used groups of cells cultured continuously for 14 days. Cell senescence was assessed by detecting expression of senescence-associated proteins (p 53, p21 and p 16) by western immunoblotting (WB) technique or Immunofluorescence (IF) staining. Expression of senescence-associated secretory phenotypes (SASP) interleukin 6 (IL-6) and interleukin 8 (IL-8) was analyzed using real-time fluorescent quantitative PCR (qRT-PCR).

Next, we examined cell proliferation, migration and multipotency to assess changes in cellular biological function following high sugar-induced aging. Cell proliferation was assessed using a cell counting kit-8 (CCK-8) and a colony formation assay (see section "colony formation assay"); performing wound healing and Transwell experiments to detect cell migration; alkaline phosphatase (ALP), alizarin red staining (see "alizarin red staining" section), oil red O staining (see "oil red O staining" section), and the cells were examined for their ability to differentiate in multiple directions.

1.2.1SA-beta-gal staining

SA- β -gal activity was identified for each group of cells using SA- β -gal staining kit. SA-beta-gal positive cells were visualized as blue under the mirror. 3 fields of view (at least 100 cells per field of view) were randomly selected from each sample, and the percentage of SA- β -gal positive cells was calculated as (SA- β -gal positive cells/total cells in field) ×100%. The kit used in this study was manufactured by Biyun Tian Biotechnology research all of the company Limited (Shanghai, china).

1.2.2Hoechst 33258 dyeing

Apoptosis of high sugar induction group PDCSCs was detected using an apoptosis Hoechst 33258 staining kit. Cells were fixed at appropriate time intervals and stained with Hoechst 33258 staining solution. The nuclear status was observed under a fluorescence microscope. The kit used in this experiment was produced by Biyun Biotechnology institute (Shanghai) Inc.

1.2.3 immunofluorescent staining

Each group of cells was cultured in 24-well plates, and when the cell density reached about 70%, it was fixed with 4% PFA. Cells were then permeabilized with 0.1% Triton X-100 for 10 min and blocked with goat serum (Biyun Biotechnology institute, shanghai, china) for 30 min. Cells were incubated with primary antibody overnight at 4 ℃ and then with secondary antibody for 1 hour at room temperature. Then stained with 4', 6-diamino-2-phenylindole (DAPI). The cells were observed with a fluorescence microscope. Antibodies used in this experiment are listed in table 1.

TABLE 1 list of antibodies used in this study

1.2.4 Western immunoblotting

Total and nuclear proteins were extracted using RIPA strong lysate (containing protease and phosphatase inhibitors) and nuclear and cytoplasmic protein extraction kit (bi yun biotechnology institute, shanghai, china), respectively. Protein concentration was determined using BCA kit. Protein samples were separated by sodium dodecyl sulfate on polyacrylamide gel electrophoresis. Proteins were transferred to PVDF membranes and then blocked with 5% skim milk for two hours at room temperature. The protein bands were incubated with primary antibody overnight at 4 ℃ and then with secondary antibody for 1 hour at room temperature. Finally, proteins were detected using enhanced chemiluminescent reagents (Biosharp, syndesmosis, china). Spectral density analysis was performed with ImageJ software. The internal reference for total cellular protein quantification in this experiment was GAPDH, and the internal reference for nucleoprotein quantification was histone H3. The secondary antibody is goat anti-rabbit IgG secondary antibody (1:5000,Affinity Biosciences, jiangsu, china). The antibodies used in this experiment are shown in table 1.

1.2.5qRT-PCR analysis of mRNA levels

Total RNA from cells was extracted with TRIzol and cDNA was synthesized using a reverse transcription kit. Using SYBR Premix Ex Taq ^TM II, real-time quantitative polymerase chain reaction is carried out. The SASP major factors IL-6, IL-8 were tested for expression. The internal control is GAPDH. Takara (Tokyo, japan) produced the kit used in the present study, and the primer sequences for detecting each gene sequence are listed in Table 2.

TABLE 2 mRNA primers for qRT-PCR.

The PCR amplification procedure of qRT-PCR was: pre-denaturation at 95 ℃ for 30 seconds; 95℃for 5 seconds, 60℃for 30 seconds, 40 cycles.

1.2.6CCK-8 detection

CCK-8 detection Using the CCK-8 kit (ApexBio Technology, MA, USA). Cells were seeded in 96-well plates (1X 10) ³ Cells/well), 10. Mu.L of CCK-8 assay and 100. Mu.L of medium were added to each well and incubated at 37℃for 2 hours. Absorbance values (OD values) were measured at 450nm using a microplate reader.

1.2.7 wound healing experiments

Cells were seeded in 6-well plates. The cell monolayer was scored with a 200 μl pipette. The optical microscope was photographed at predetermined time intervals (0 hours, 24 hours and 48 hours). Calculations are performed from the initial scratch area to determine the change in scratch area over time and the proportion of wound healing. Wound healing rate (%) = (0 hour scratch area-specific time point scratch area)/0 hour scratch area×100.

1.2.8Transwell migration experiments

Each group of cells (4X 10) ⁴ cells/well) were inoculated into the upper well of a Transwell 24-well plate, and medium containing 10% exosome-free FBS (750. Mu.L/well) was added to the lower well. Cells were incubated for 24 hours, then stored with 4% pfa, and stained with 0.1% crystal violet. Then, cells on the inner surface of the membrane were removed, and an image of the stained cells was taken with an inverted microscope. The crystal violet stained cells were washed with 33% acetic acid and the absorbance of the solution was measured at OD 570 nm.

1.2.9ALP staining and Activity determination

The cells were grown at 5X 10 ⁵ Is seeded in 6-well plates. After the cell density reached 80%, the medium was replaced with osteoinductive medium. ALP staining was performed one week after osteoinduction culture. Alkaline phosphatase level was quantitatively determined using alkaline phosphatase assay kit from the Biyun institute of biotechnology (Shanghai, china).

1.3 extraction and identification of exosomes

Extracting exosomes by adopting a low-temperature ultracentrifugation method. Young pdcscs were cultured in complete medium. When the cell density reached 70%, the medium was changed to serum-free medium, and the culture was continued for 48 hours, and then the cell supernatant was collected. The conditioned medium obtained (i.e., the supernatant obtained above) was centrifuged at 300 Xg for 15 minutes, the supernatant was taken and then centrifuged again at 3,000 Xg for 15 minutes, and the obtained supernatant was centrifuged at 100,000 Xg twice for 70 minutes each. After removal of the supernatant, the exosome-containing particles were resuspended in cold PBS and stored at-80 ℃ until used in subsequent experiments. In experiments involving exosomes, the same volume of PBS was used as a control.

Exosome concentration was determined using BCA assay kit. The profile of PDLSC-Exos was characterized using Transmission Electron Microscopy (TEM). The particle size and concentration of exosomes were assessed using Nanoparticle Tracking Analysis (NTA). WB is used to recognize exosome markers such as CD9, CD63, CD81, TSG101 and calnexin (see "western immunoblotting" section).

1.4 Effect of PDLSC-Exos treatment on aging PDLSCs

We first determined whether PDLSC-Exos could be taken up by senescent PDLSCs. Aged PDLSCs were treated with 25, 50 and 100 μg/mL PDLSC-Exos for 72h to determine the appropriate PDLSC-Exo treatment concentration. The response of high sugar-induced cellular senescence to PDLSC-Exo treatment was assessed by SA- β -gal staining (see section "SA- β -gal staining"). Subsequently, we selected 50 μg/mL of PDLSC-Exos to be co-incubated with aged PDLSCs for 72h (HG-Exos group), and the control group of PDLSCs received the same volume of PBS (HG group) or normal glucose (NG group, without high sugar treatment). Likewise, senescence-associated protein expression assays employ WB (see "Western immunoblotting" section) or IF staining (see "immunofluorescent staining" section). In addition, qRT-PCR was used to assess the expression of both SASP genes (see "qRT-PCR analysis of mRNA level" section). We examined cell proliferation, migration and multipotency to determine changes in cellular biological function (see section "influence of 1.2 high sugar on PDLSC senescence").

1.4.1 exocrine endocytosis experiments

Exosomes were labeled with the fluorescent dye Dil (red) to assess the absorption of PDLSC-Exos by senescent PDLSCs. Briefly, 2. Mu.LDil was added to the isolated exosomes (1 mL, 100. Mu.g/mL protein concentration) at a final concentration of 10. Mu.M. Dil-labeled exosomes were re-centrifuged after incubation at 37℃for 30 min. Aged pdcscs were incubated with the labeled exosomes for 24h at 37 ℃. Next, the cells were fixed, the nuclei were stained with DAPI, and uptake of senescent pdcs into exosomes was observed with an inverted fluorescence microscope. The reagents used in this study were developed by the Biyun Biotechnology institute (Shanghai) Inc.

1.5NRF2 effects on PDLSC-Exos mediated regeneration of aging PDLSCs

Before and after treatment of senescent pdcs with PDLSC-Exo, we measured ROS levels, malondialdehyde (MDA) content, and superoxide dismutase (SOD) activity to assess cellular oxidative stress. We used WB technology to identify NRF2 and the expression levels of the target proteins NADPH quinone oxidoreductase 1 (NQO 1) and heme oxygenase 1 (HO-1) in cells to examine whether NRF2 is involved in PDLSC-Exos mediated regeneration of senescent PDLSCs (see "Western blotting" section). Furthermore, NRF2 expression and intracellular distribution were detected by IF staining (see "immunofluorescent staining" section).

The high sugar-induced aging PDCSCs were cultured in the following groups to investigate the effect of NRF2 activation on PDLSC-Exo mediated recovery of aging PDCSCs (1) HG group (PBS treatment), (2) HG-Exos group (50. Mu.g/mL PDLSC-Exo treatment), (3) HG-Exos-ML385 (MedChem Express, monmouth Junction, NJ, USA) group (50. Mu.g/mL PDLSC-Exos and 5. Mu.M ML 385). Also, the level and location detection method of NRF2 in each group is as described above. Cell senescence, oxidative stress and biological function were then examined to assess the role of ML385 processing in PDLSC-Exos mediated restoration of senescent cell viability.

1.5.1 oxidative stress level determination

Intracellular ROS levels were measured using the ROS assay kit according to the manufacturer's instructions. Briefly, cells were added with 10. Mu. Mol/L DCFH-DA probe and incubated at 37℃for 20min. Fluorescence intensity was analyzed with a fluorescence microscope and a microplate reader and ROS formation was assessed. To determine MDA levels and SOD activity, cells were lysed and cell supernatants were collected. MDA levels and SOD activity were determined using commercial kits according to the manufacturer's instructions. The kit used in this experiment was produced by Biyun Biotechnology institute (Shanghai) Inc.

1.6 role of MicroRNA-141-3p (miR-141-3 p)/KEAP 1/NRF2 axis in PDLSC-Exos mediated senescent cell regeneration

The expression level of KEAP1 (an NRF2 negative regulatory protein) in senescent pdcs and its expression change after PDLSC-Exos treatment were examined by WB (see "western blot" section). We then detected mirnas that specifically target KEAP1 to modulate NRF2 activity. The expression levels of the two miRNAs (miR-141-3 p and miR-200a-3 p) screened in the early stage are detected in normal and aged PDLSCs and PDLSC-Exos by using qRT-PCR technology. The cells were then tested for miR-141-3p expression following treatment of senescent PDCSCs with PDLSC-Exo. And verifying the targeting relationship between KEAP1 and miR-141-3p through a double-luciferase reporter gene experiment. We used miR-141-3p inhibitors to down-regulate the expression level of miR-141-3p in PDLSC-Exos to verify the molecular mechanism of PDLSC-Exos in regulating NRF2 signaling pathway. The high sugar-induced aging PDCSCs were then divided into three groups, (1) HG group (PBS treatment), (2) HG-NCI-Exos group (50. Mu.g/mLNCI-Exo treatment, the PDCSCs exosomes transfected with the Inhibitor negative control), (3) HG-141I-Exos group (50. Mu.g/mL 141I-Exo treatment, the PDCSCs exosomes transfected with miR-141-3p Inhibitor). Subsequent experiments were performed according to the above classification. Protein expression in the NRF2 pathway was identified by WB (see "western immunoblot" section) and IF staining (see "immunofluorescent staining" section). Furthermore, we also assessed the senescence, oxidative stress levels and biological functions of each group of cells.

1.6.1MiRNA screening

Candidate mirnas targeting KEAP1 were screened based on cross-filtering of miRNA databases (miRWalk, starBase, miRDB, miRcode and TargetScan) using Venn plots. In addition, the keywords "micrornas, cellular senescence, periodontal ligament stem cells, periodontal disease, oxidative stress, mesenchymal stem cells, KEAP1-NRF2" were searched in PubMed to determine whether these previously screened mirnas were expressed in periodontal tissues, involved in cellular senescence, oxidative stress and periodontal disease.

1.6.2qRT-PCR detection of miRNA levels

Total exosome RNA was extracted using BIOG Exosome RNAEasy Kit and miRNA was reverse transcribed using BIOG miRNAPolyA+RT Kit. qRT-PCR was performed using the BIOG miRNA PolyA SYBR qPCR kit. And detecting the expression of the target genes miR-141-3p and miR-200a-3p by adopting qRT-PCR. The internal control of miRNA analysis was U6. The kit used in this experiment was prepared by Changzhou Baidai biotechnology Co., ltd (China), and the sequences of the primers are shown in Table 3.

TABLE 3 miRNA primers for qRT-PCR.

PCR amplification procedure for qRT-PCR: activating Taq:95 ℃ for 2min;

PCR cycle: 95 ℃ for 10s and 60 ℃ for 30s;40 cycles;

dissolution profile: 95 ℃ for 15s;60 ℃ for 1min;95 ℃ for 15s;1 cycle.

1.6.3 double luciferase reporter Gene experiments

The 3' UTR of KEAP1 mRNA was searched in the China national center for Biotechnology information database, and the sequence was generated and cloned downstream of the luciferase minigene of the pmirGLO vector (luciferase reporter vector). The 3' UTR of the putative miR-141-3p target sequence was altered using a site-directed mutagenesis kit (Umibio, shanghai, china). Each product was sequenced. miR-141-3p sequences were also determined and synthesized. Day before transfection, HEK293 cells were seeded into 48-well plates (1X 10) ⁵ Cells/wells). Transfection was performed using Lipofectamine 2000 (Invitrogen, carlsbad, calif., USA) according to the manufacturer's instructions. Cells were transfected with pmirGLO luciferase expression construct containing the KEAP1 gene 3' UTR, pRL-TK Renilla luciferase vector (Promega, madison, wis., USA) and miRNA negative control (Ambion, austin, TX, USA). Luciferase activity was assessed 48 hours after transfection using the Promega company (Madison, wis., USA) dual luciferase reporter kit and normalized to Renilla luciferase activity.

1.6.4 transfection miRNA inhibitors

PDLSCs were transfected with 100nM miR-141-3p Inhibitor (Ribo Bio, guangzhou, china) and Inhibitor negative controls, respectively, using the Lipofectamine RNAiMAX (Thermo Fisher Scientific, waltham, mass., USA) kit according to the reagent manufacturer's instructions. Transfected cells were cultured in serum-free medium for 48 hours. Exosomes were isolated from the cell culture supernatant using the methods described previously.

1.7 statistical analysis

At least three separate experiments were performed using pdcscs from the same donor. Statistical analysis was performed using GraphPad Prism9 software. All results are expressed as mean ± Standard Deviation (SD). The Student t test was used to examine whether the differences between groups were statistically significant. Multiple comparisons were performed using analysis of variance (ANOVA). Pdcscs from at least three independent donors were reused for each experiment. A statistical difference is considered when p <0.05, p <0.01, p <0.001, or p < 0.0001.

2 results

2.1 characterization of PDLSCs and PDLSC-Exos

Pdcscs were derived from young healthy teeth (fig. 1A). Primary cells grew from the center of the tissue to the periphery (fig. 1B). After passaging, the cells grew actively, in a typical fibroblast-like morphology, in a swirl arrangement (FIGS. 1C and D). Alizarin red and oil red O staining demonstrated that pdcscs possess osteogenic (fig. 1E) and adipogenic (fig. 1F) differentiation potential. Cell dryness was identified by colony formation experiments (fig. 1G).

PDLSC-Exos were extracted from young PDLSCs supernatant and further characterized by TEM, WB and NTA. TEM analysis revealed that PDLSC-Exos has a typical cup-like morphology (FIG. 1H). WB showed that PDLSC-Exos expressed exosome specific markers CD9, CD81, CD63 and TSG101, but not calnexin (FIG. 1I). According to NTA data, the concentration of PDLSC-Exos was 3.9X10 ¹⁰ The average particle size was 113.8nm per mL (FIG. 1J). These results indicate that the PDLSC-Exos we extract has the essential characteristics of exosomes.

2.2 high sugar Induction of early senescence by PDCSCs

After 14 days of high sugar treatment, the number of SA- β -gal positive cells was significantly increased compared to NG and HM groups (fig. 2A and B), but no significant apoptosis was observed (fig. 2C). Therefore, we selected 14 days of induction as the time point for high sugar treatment of PDLSCs in the subsequent experiments. After high sugar induction, the levels of senescence-associated proteins (p 53, p21 and p 16) (FIG. 2D-F) and the two main indicators of SASP (IL-6 and IL-8) (FIG. 2G) were also significantly elevated. Based on these results, continued culture in high sugar media can induce premature aging of the pdcscs. The results of the cell biology function experiments showed that proliferation, migration, osteogenesis and adipogenic differentiation of the high sugar-induced aged pdcscs were reduced (fig. 3). Based on these results, continued culture in high sugar media can induce premature aging of the pdcscs.

2.3PDLSC-Exos to alleviate high sugar-induced aging of PDLSCs

To further determine the effect of PDLSC-Exos in delaying cell senescence, we first tested the ability of PDLSC-Exos to internalize using Dill-labeled exosomes. Red fluorescence was observed around the nuclei of senescent pdlcs co-cultured with exosomes, indicating the presence of Dill-labeled PDLSC-Exos (fig. 4A); the results indicate that PDLSC-Exos enters senescent PDLSCs by endocytosis and is dispersed in the cytoplasm. According to the SA- β -gal staining results, the treatment with PDLSC-Exo significantly reduced the SA- β -gal activity of senescent PDLSCs and was concentration dependent (FIGS. 4B and C), especially when the PDLSC-Exos concentration reached 50 μg/mL, the SA- β -gal positive staining rate was significantly reduced for the HG-Exos group, similar to the NG group. Thus, we used 50 μg/mL of PDLSC-Exos in the subsequent experiments, and the other groups used equal amounts of PBS as placebo. In addition, PDLSC-Exos down-regulated the expression levels of p53, p21 and p16 in senescent PDLSCs (FIGS. 4D-F), and IL-6 and IL-8 (FIG. 4G).

Since the biological function of pdcs decreases during aging, we next studied the effect of PDLSC-Exos treatment on the biological properties of high sugar-induced aging pdcs. The results of the CCK-8 assay (FIG. 5A) and the colony formation assay (FIG. 5B) indicate that incubation with PDLSC-Exos can restore high sugar-induced proliferation of senescent PDLSCs. Both wound healing (FIGS. 5C and D) and Transwell experiments (FIGS. 5E and F) showed that PDLSC-Exo treatment can improve the reduction in migration potential of senescent PDLSCs. In addition, incubation of aged pdcscs with PDLSC-Exos improved osteogenesis (fig. 5G-J) and adipogenic differentiation (fig. 5K and L) of cells. Taken together, these findings demonstrate that PDLSC-Exo treatment reduces the senescent phenotype of PDLSCs and restores viability of aged PDLSCs to levels approximately comparable to young cells.

2.4PDLSC-Exos rejuvenating senescent PDLSCs by activating NRF2

The most common type of stress that causes cellular senescence in vitro is oxidative stress. We first examined ROS levels in pdcscs to determine the mechanism by which PDLSC-Exos exerts anti-aging effects. As shown in fig. 6A and B, intracellular ROS levels were significantly increased in high sugar-induced senescent pdcs, but this effect was almost completely diminished after incubation with PDLSC-Exos. We also measured MDA levels and SOD activity and found that HG MDA levels were significantly higher than NG (fig. 6C) whereas the antioxidant activity of SOD was significantly lower than NG (fig. 6D). These changes in aging pdcs were largely reversed after treatment with pdcsc-Exos. Thus, PDLSC-Exos can reduce oxidative stress in aging PDLSCs. NRF2 plays a key role in regulating redox and metabolic homeostasis, oxidative stress and other cytoprotective reactions. Subsequently, we studied the anti-aging mechanism of PDLSC-Exos against aging PDLSCs by assessing whether PDLSC-Exos might promote NRF2 nuclear translocation and activate its downstream signaling pathway. According to our findings, aged pdcs showed lower levels of NRF2 signal-associated protein (NRF 2, NQO1 and HO-1) expression. The PDLSC-Exo treatment can restore NRF2 signal-related protein expression levels in senescent cells (fig. 6E and F), promoting NRF2 nuclear translocation (fig. 6G).

Aged pdcs were incubated with NRF2 specific inhibitors ML385 and PDLSC-Exos to further determine if PDLSC-Exos delayed aging by up-regulating NRF 2. The results demonstrate that ML385 can prevent PDLSC-Exos mediated upregulation of NRF2 (fig. 7A and B) and nuclear translocation (fig. 7C). In ML 385-treated senescent PDCS, PDLSC-Exos did not decrease SA- β -gal activity (FIGS. 7D and E) or expression levels of senescence-associated proteins (FIGS. 7F-H) and SASP genes (FIG. 7I). In addition, ML385 significantly inhibited PDLSC-Exos-mediated oxidative stress reduction (FIG. 7J-M). An improvement in the functional phenotype of senescent pdcs is associated with NRF2 signaling pathways. The improvement in the functional phenotype of senile pdcscs is associated with NRF2 signaling pathway. After addition of ML385, the ability of PDLSC-Exos to improve biological functions such as proliferation, migration and differentiation of senescent PDLSCs cells was significantly inhibited (fig. 8). Based on these results, PDLSC-Exos inhibited PDLSC senescence and delayed the senescence phenotype by promoting NRF2 accumulation and nuclear translocation and decreasing ROS levels.

2.5PDLSC-Exos activation of NRF2 by delivery of miR-141-3p down-regulates KEAP1 expression

In subsequent experiments we tried to get more information about the underlying mechanism of NRF2 activation. KEAP1 negatively regulates NRF2 activity; thus, we examined the expression of KEAP1 protein. The expression of KEAP1 in aged pdcs increased after PDLSC-Exo treatment, but its expression level decreased after PDLSC-Exos treatment (fig. 6E and F), suggesting that PDLSC-Exos may activate NRF2 signal by inhibiting KEAP1 expression. According to previous reports, exosome miRNAs may be delivered into recipient cells by posttranscriptional control of host gene expression, thereby altering their function. By looking at the relevant literature and performing comprehensive bioinformatics analysis, we found that miR-141-3p and miR-200a-3p were associated with periodontal disease and regulated NRF2 activity by targeting KEAP1 expression (fig. 9A). We examined the expression levels of these two miRNAs in PDLSCs and PDLSC-Exos to further verify the molecular mechanism by which PDLSC-Exos modulates the NRF2 signaling pathway. Notably, miR-141-3p expression was significantly down-regulated in high sugar-induced senescent PDLSCs, while expression was significantly enriched in PDLSC-Exos (fig. 9B and C). After incubation with PDLSC-Exos, miR-141-3p expression was significantly increased in senescent PDLSCs (FIG. 9D), consistent with the down-regulation of KEAP1 expression. The detection result of the double luciferase reporter gene shows that cells co-transfected with miR-KEAP1-WT and miR-141-3p micrometers show that the activity of the luciferase reporter gene is remarkably reduced. In cells co-transfected with miR-141-3p micrometers and pmiR-KEAP1-MUT, no decrease in luciferase activity was observed (FIGS. 9E and F). These results reveal a clear targeting relationship between miR-141-3p and KEAP 1.

Then, we down-regulated miR-141-3p levels in PDLSC-Exos (FIG. 9G) and co-cultured them with senescent PDLSCs to investigate miR-141-3p function in PDLSC-Exos and improve high-sugar induced senescence of PDLSCs. Knock-down of miR-141-3p almost completely reversed the effect of PDLSC-Exos down-regulation of KEAP1 expression and up-regulation of NRF2 expression (FIG. 9H-J). In addition, the down-regulation of miR-141-3p blocked the restoration of the senescent phenotype (SA- β -gal activity, senescence-associated protein expression, and SASP levels) and biological functions (proliferation, migration, and multipotency) of senescent pdcs (fig. 10) (fig. 11). These findings indicate that miR-141-3p is one of the main mediators of PDLSC-Exos activating NRF2 regeneration of senescent PDLSCs by inhibiting KEAP1 expression. Namely, PDLSC-Exos decreased KEAP1 expression and activated NRF2 antioxidant pathway by transferring miR-141-3p into senescent PDLSC, thereby preventing high sugar-induced PDLSC senescence.

In summary, high levels of glucose induce premature aging of the pdcs, while PDLSC-Exos promotes regeneration of aged pdcs. PDLSC-Exos may exert an anti-aging effect by transmitting miR-141-3p to down-regulate KEAP1 expression and activate the NRF2 antioxidant pathway. The research can provide a certain theoretical basis for the application of cell-free therapy in preventing and treating the periodontitis of diabetes, and the research proves that the PDLSC-Exos is hopeful to be used as a novel bioactive medicament for resisting cell aging, and has a good application prospect.

Claims (10)

1. Use of periodontal ligament stem cell-derived exosomes in the preparation of a product for alleviating cellular senescence.

2. The use according to claim 1, characterized in that: the cell aging relieving is to relieve periodontal ligament stem cell aging.

3. The use according to claim 2, characterized in that: the cell aging relieving is to relieve periodontal ligament stem cell aging under high-sugar microenvironment.

4. A use according to claim 3, characterized in that: the exosomes derived from periodontal ligament stem cells alleviate high sugar-induced aging of pdcscs by transmitting miR-141-3p to activate KEAP1-NRF2 signaling pathway.

5. Application of exosomes derived from periodontal ligament stem cells in preparing medicines for treating periodontitis.

6. The use according to claim 5, characterized in that: the periodontitis is diabetic periodontitis.

7. Use according to any one of claims 1 to 6, characterized in that: the periodontal ligament stem cells are human periodontal ligament stem cells.

8. Use according to any one of claims 1 to 6, characterized in that: the product or the medicine consists of an active ingredient and pharmaceutically acceptable auxiliary materials, wherein the active ingredient is an exosome derived from periodontal ligament stem cells.

9. Use according to any one of claims 1 to 6, characterized in that: the exosome preparation method is to collect serum-free culture medium of cultured young periodontal ligament stem cells and extract exosome by adopting a low-temperature ultracentrifugation method.

10. The use according to claim 9, characterized in that: the preparation method of the exosome comprises the following steps: culturing young periodontal ligament stem cells in a complete culture medium, changing the culture medium into a serum-free culture medium when the cell density reaches more than 60%, continuously culturing for 44-52h, and collecting cell supernatant to obtain supernatant 1; subjecting the obtained supernatant 1 to differential centrifugation to obtain a supernatant 2; and (3) ultracentrifugating the obtained supernatant 2, and removing the supernatant to obtain the exosomes.

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