If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
1 Zixiang Jian and Yao Li contributed equally to this work.
Zixiang Jian
Footnotes
1 Zixiang Jian and Yao Li contributed equally to this work.
Affiliations
College of Stomatology, Chongqing Medical University, Chongqing, ChinaChongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing, ChinaChongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing, China
1 Zixiang Jian and Yao Li contributed equally to this work.
Yao Li
Footnotes
1 Zixiang Jian and Yao Li contributed equally to this work.
Affiliations
NMPA Key Laboratory for Dental Materials, National Engineering Laboratory for Digital and Material Technology of Stomatology, Department of Geriatric Dentistry, Peking University School and Hospital of Stomatology, Peking, China
College of Stomatology, Chongqing Medical University, Chongqing, ChinaChongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing, ChinaChongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing, China
College of Stomatology, Chongqing Medical University, Chongqing, ChinaChongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing, ChinaChongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing, China
College of Stomatology, Chongqing Medical University, Chongqing, ChinaChongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing, ChinaChongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing, China
Corresponding author at: Department of Orthodontics, Stomatological Hospital of Chongqing Medical University, 426 North of Songshi Avenue, Chongqing, China 401147.
College of Stomatology, Chongqing Medical University, Chongqing, ChinaChongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing, ChinaChongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing, China
Corresponding author at: Department of Orthodontics, Stomatological Hospital of Chongqing Medical University, 426 North of Songshi Avenue, Chongqing, China 401147.
College of Stomatology, Chongqing Medical University, Chongqing, ChinaChongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing, ChinaChongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing, China
The goal of the work described here was to determine if low-intensity pulsed ultrasound (LIPUS) has an anti-inflammatory effect on lipopolysaccharide (LPS)–induced inflammation in periodontal ligament cells (PDLCs). The mechanism underlying this effect remains to be explored and is likely related to PDLC apoptosis regulated by Yes-associated protein (YAP) and autophagy.
Methods
To verify this hypothesis, we used a rat model of periodontitis and primary human PDLCs. We examined alveolar bone resorption in rats and apoptosis, autophagy and YAP activity in LPS-treated PDLCs with and without application of LIPUS by cellular immunofluorescence, transmission electron microscopy and Western blotting. Then, siRNA transfection was used to decrease YAP expression to confirm the regulatory role of YAP in the anti-apoptotic effect of LIPUS on PDLCs.
Discussion
We found that LIPUS attenuated alveolar bone resorption in rats and this was accompanied by YAP activation. LIPUS inhibited hPDLC apoptosis by YAP activation, and promoted autophagic degradation to help autophagy completion. These effects were reversed after YAP expression was blocked.
Conclusion
LIPUS attenuates PDLC apoptosis by activating Yes-associated protein-regulated autophagy.
Periodontitis is a chronic inflammatory disease caused by plaque and is characterized by local inflammation of periodontal tissue, which often leads to the destruction and loss of alveolar bone and the attached periodontal tissue. To date, there is no effective way to completely regenerate periodontal tissue once alveolar bone resorption occurs [
]. Around the tooth root is a self-renewal system known as the periodontal ligament (PDL), which contains stem cell-like periodontal ligament cells (PDLCs). An increasing number of studies have confirmed that PDLCs play a key role in maintaining the dynamic balance of periodontal tissue and defect repair [
In the inflammatory microenvironment, the biological behaviors of PDLCs, such as proliferation, apoptosis and differentiation, differ from the normal situation. Studies have indicated that periodontal pathogens and their main toxin, lipopolysaccharide (LPS), can induce a local immune response in periodontal tissue, resulting in the production of reactive oxygen species and other harmful substances in host cells to promote apoptosis [
]. Autophagy is a highly conserved process of degradation and recovery of cellular components in eukaryotic cells and has been proven to affect LPS-induced apoptosis [
Impaired autophagy in microglia aggravates dopaminergic neurodegeneration by regulating NLRP3 inflammasome activation in experimental models of Parkinson's disease.
]. However, studies have reported increased expression of autophagy-related proteins in PDLCs in periodontal disease, and the increase in autophagic activity in PDLCs contributes to anti-inflammatory effects [
Given that the inflammatory microenvironment affects apoptosis, anti-inflammatory measures that could decrease PDLC apoptosis would be beneficial for PDL repair [
]. Low-intensity pulsed ultrasound (LIPUS) is a non-invasive technique that transforms acoustic signals into mechanical signals and has an effect on cells in local lesions [
]. Previous studies have confirmed that LIPUS can induce bone tissue repair and regeneration by enhancing cell proliferation and osteogenic differentiation [
Low-intensity pulsed ultrasound induces osteogenic differentiation of human periodontal ligament cells through activation of bone morphogenetic protein-smad signaling.
]. In the LPS-induced inflammatory microenvironment, LIPUS has been proven to inhibit the expression of inflammatory factors and enhance the osteogenic differentiation of human PDLCs (hPDLCs) [
Low-intensity pulsed ultrasound upregulates osteogenesis under inflammatory conditions in periodontal ligament stem cells through unfolded protein response.
LIPUS inhibited the expression of inflammatory factors and promoted the osteogenic differentiation capacity of hPDLCs by inhibiting the NF-κB signaling pathway.
]. However, the mechanism underlying the anti-inflammatory effect of LIPUS remains to be explored.
Combined with the enhanced autophagic activity of PDLCs in the inflammatory state, the anti-inflammatory effect of LIPUS may be mediated by alterations in the autophagic activity of cells. During this process, the transformation of LIPUS physical signals to activate changes in biochemical signals in cells may require a "rheostat" cytokine. In recent years, related studies on cell biomechanics have revealed that the transcriptional coactivator Yes-associated protein (YAP) is an essential factor in transcription and phenotypic changes caused by biophysical signal-induced microenvironmental changes. Through changes in its intracellular localization, YAP regulates the expression of genes related to various biological behaviors in cells [
]. When physical signals in the cellular microenvironment are detected, YAP undergoes nuclear translocation and activates downstream metabolic signaling pathways [
To explore whether YAP is involved in the molecular mechanism by which LIPUS regulates hPDLC apoptosis in an inflammatory microenvironment, we used LPS to induce hPDLC inflammation. The findings of these experiments will further reveal the mechanism underlying the anti-inflammatory effect of LIPUS and provide a research basis for the clinical use of LIPUS for periodontal tissue regeneration.
Methods
The experiments were conducted according to protocols approved by the Ethics Committee of the Affiliated Stomatological Hospital of Chongqing Medical University. All teeth were donated by healthy volunteers aged 10 to 20 y with informed consent for “orthodontic extraction treatment” with items about PDLC separation for cell culture. All experiments described here were constructed in accordance with the ARRIVE (Animal Research Reporting In Vivo Experiments) guidelines.
Animals and models
Eight‐week‐old (200 ± 20 g) male Sprague–Dawley rats were obtained from the Animal Centre of Chongqing Medical University. They were used for models of periodontal disease (PD) and LIPUS treatment.
Fifteen rats were randomly divided into three groups: the control group, the LPS group (with PD) and the LPS + LIPUS (L+L) group. After anesthesia, the 0.020 stainless-steel ligature was inserted and fixed around the upper first molars of the LPS and L+L groups for 28 d. One hundred microliters of LPS (10 μg/mL) was injected at 2-d intervals, four times in total, with inhaled gas anesthesia (2% isoflurane in O2, 2 L/min). On the 29th day, the rats in the L+L group received another 28 days of LIPUS (90 mW/cm2, 1.5 mHz, 30 min) treatment according to a previous study [
Low-intensity pulsed ultrasound upregulates osteogenesis under inflammatory conditions in periodontal ligament stem cells through unfolded protein response.
]. All rats were killed on the 59th day by CO2 inhalation before the maxillae were recovered (Fig. 1A). After photographs were taken, the maxillae were fixed overnight at 4°C in 4% paraformaldehyde (Affymetrix, Cleveland, OH, USA).
Figure 1LIPUS treatment alleviated alveolar bone resorption in rats and was accompanied by YAP activation. (A) Schematic of the experimental timeline for PD induction and LIPUS treatment. (B) Photos of samples among different groups (red circles indicate gingival recession). (C) Cross-sections and 3-D reconstruction images of maxillae among groups (green lines indicate CEJ, and yellow lines indicate ABC). (D) Graphs depicting the measurement values from cross-sections in (C). (E) Representative hematoxylin and eosin–stained sections with marked line of the CEJ and ABC. (F) Graphs depicting the measurement values of CEJ–ABC distance in (E). (G) Representative immunohistochemical staining results for YAP in PDL of 100 × 100 μm range among different groups. (H) Graphs depicting the percentages of YAP+ cells, with YAP protein translocation to the nucleus, among different groups. (I) Representative immunohistochemical staining results for p-YAP in PDL of 100 × 100 μm range among different groups. (J) Graphs depicting the IOD value of p-YAP among groups. *p < 0.05, **p < 0.01, ***p < 0.001 between the linked groups (n = 5 for every group). ABC, alveolar bone crest; CEJ, cemento-enamel junction; Con, control; IOD, integrated option density; LIPUS, low-intensity pulsed ultrasound; PD, periodontal disease; p-YAP, phosphor-YAP; YAP, Yes-associated protein.
The maxillae were scanned with a micro-computed tomography (μ‐CT) system (μCT‐80; Scanco Medical AG, Bassersdorf, Switzerland) at a resolution of 10 μm, voltage of 80 kV and current of 500 mA. Three‐dimensional images were reconstructed using Scanco software (Version X). The distance from the cemento-enamel junction (CEJ) of the first molar to the non-discrete alveolar bone crest (ABC) between the first and second molars was measured with an average of 10 equally spaced scan cross-sections for every sample. All measurements were performed by one investigator in a blinded protocol.
Slide staining and analysis
The maxillae were soaked in 14% ethylenediaminetetraacetic acid (EDTA, pH 7.4) for 8 wk. After dehydration and embedding in paraffin, the demineralized maxillary tissues were cut into 5-μm-thick sections from sagittal aspects of molars. Subsequently, five slides of each sample were stained with hematoxylin and eosin (H&E).
For immunohistochemistry and immunofluorescence staining, the paraffin sections were first dewaxed, rehydrated and treated in 0.01 M sodium citrate buffer for 15 min in 95°C water for antigen retrieval. Then 5% bovine serum albumin (BSA) were applied to the sections for 1 h, followed by primary rabbit monoclonal anti‐YAP (1:100; Zhengneng, Chengdu, China) and anti-p-YAP (S127, 1:100; Cell Signaling Technology, Danvers, MA, USA) antibody incubation at 4°C overnight. After being washed, sections were incubated with secondary antibody for 1 h at room temperature, followed by color precipitation with a diaminobenzidine substrate kit (Zhongshan Biotechnology, Beijing, China). YAP cells in PDL with a nuclear positive signal were counted as positive, which indicates YAP protein translocation to the nucleus when active. The average percentages of CEJ-ABC and YAP+ cells and integrated option density (IOD) of p-YAP on every sample were measured by one blinded investigator with ImageJ software.
Cell culture and groups
Human PDL was separated from the premolar or third molar of orthodontic patients from our maxillofacial surgery clinic. PDL was digested with 0.3 mg/ml collagenase type I (Sigma, St. Louis, MO, USA) for 30 min. The digested PDLCs were collected and transferred to a new flask with fresh α-MEM medium (HyClone; GE Healthcare Life Sciences, Waukesha, WI, USA) of 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Milwaukee, WI, USA). The primary cells were cultured at 37°C in a 5% CO2 incubator and then passaged or cryopreserved when the cells were approximately 80% confluent.
Periodontal ligament cells at passages 3 to 5 with positive cell viability were used for further experiments. The irradiation intensity of LIPUS was 90 mW/cm2, and the exposure procedures were those used in our previous study [
]. The concentrations of chemicals used to treat hPDLCs were as follows: LPS (1 or 10 µg/ml, HyClone, Logan, UT, USA). The LIPUS group was treated with LIPUS at 90 mW/cm2 for 30 min at 37°C for 3 successive days in an ultrasonic transmitter (Chongqing Medical University, Chongqing, China). LPS group cells were cultured with LPS, and L+L group cells were treated with both LIPUS irradiation and LPS. The cells in the L+L group that were transfected with small interfering RNA (siRNA) constituted the L+L+siRNA group. Those without any special treatment served as the control group.
Flow cytometry
For apoptosis assessment, cells were digested with 0.25% trypsin and washed twice with phosphate-buffered saline (PBS). The cells were stained with an annexin V-fluorescein isothiocyanate (FITC)/propidium Apoptosis Detection Kit (BD, East Rutherford, NJ, USA) after centrifugation. Apoptotic cells were identified and analyzed by flow cytometry (BD Influx).
For cell cycle analysis, the cell pellets were fixed with 70% alcohol at 4°C and frozen at –20°C overnight. Then, the cells were incubated with 100 μL/tube RNase at 37°C for 30 min before propidium iodide staining detection dye (Beyotime, Shanghai, China) was added to the tubes. The cell DNA content of each group was assessed by flow cytometry (BD Influx) and analysis with related software. The S‐phase fraction (SPF) and DNA proliferation index (PI) of the total cells were calculated according to the formulas SPF (%) = S (G0/G1 + S + G2/M) × 100% and PI (%) = (S + G2/M)/(G0/G1 + S + G2/M) × 100%.
TUNEL staining
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed using an in situ cell death detection kit (TUNEL POD). Briefly, cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.1% Triton X-100 (Beyotime, Shanghai, China) for 5 min before being stained with TUNEL reaction mix (Beyotime, Shanghai, China) for 60 min according to the manufacturer's instructions. An inverted fluorescence microscope (Nikon, Japan) was used to acquire fluorescence images.
siRNA transfection
Control siRNA and YAP siRNA were synthesized by Shenggong Company, Ltd. (Shanghai, China) and transfected into hPDLCs with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Three sequences of YAP siRNA were detected with reverse transcription polymerase chain reaction (RT-PCR) and Western blotting for knockdown efficiency in cells. The sequences of YAP siRNA were as follows: –955 (5′- GGUCAGAGAUACUUCUUAATT-3′ and 5′- UUAAGAAGUAUCUCUGACCTT-3′), –1557 (5′- CCUUAACAGUGGCACCUAUTT-3′ and 5′-AUAGGUGCCACUGUUAAGGTT-3′), –1719 (5′-CCGUUUUCCCAGACUACCUUTT-3′ and 5′-AAGGUAGUCUGGGAAACGGTT-3′). The scrambled negative control siRNA (5′- UUCUCCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′) was also included.
Western blot
Total protein extracts were separated and subsequently transferred to a polyvinylidene fluoride membrane. The transferred membranes were blocked with 5% BSA in PBS for 2 h at room temperature and then incubated with anti-caspase 3 (Zhengneng, Chengdu, China), anti-cleaved caspase 3 (Zhengneng, Chengdu, China), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Zhengneng), anti-YAP (1:1000; Zhengneng), anti-p-YAP (S127) (1:1000, CST, Danvers, MA, USA), anti-LC3 (1:1000, CST) and anti-P62 (1:1000, CST) at 4°C. Subsequently, the membranes were washed with 20% Tween-20 in PBS and probed with goat anti‐rabbit IgG H&L secondary antibodies (1:10000, CST) for 2 h. A hypersensitive ECL chemiluminescence kit (Beyotime) was used to help detect immunoreactive bands (Bio-Rad, Hercules, CA, USA).
Cellular immunofluorescence
Cells of different groups were fixed with 4% PFA for 15 min at 4°C and permeabilized with 0.3% Triton X-100 for 5 min. After being washed with PBS, the cells were blocked in 5% bovine serum albumin for 30 min. Then, the cells were immunostained using a primary antibody against YAP (1:100, CST) at 4°C overnight. The Cy3 fluorescent secondary antibody (1:400, Bioss, Beijing, China) was added to incubated cells at 37°C for 1 h. Finally, the cells were mounted using mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) after washing with PBS. Images were obtained using an inverted fluorescence microscope.
Transmission electron microscopy
The cells were washed and digested with 0.25% trypsin for 1 min, harvested and centrifuged (3000g) for 10 min in a high-speed centrifuge, fixed with 4% glutaraldehyde (diluted in PBS, pH 7.2), dehydrated and embedded for sectioning. Ultrathin sections were stained with uranyl acetate (30 min) and lead citrate (10 min) and observed with a Hitachi-7500 transmission electron microscope (Hitachi Co., Ibaraki, Japan) with an accelerating voltage of 100 kV.
Autophagic flux was measured with an Autophagy mRFP–GFP–LC3 reporter system (Hanheng, Shanghai, China) according to the manufacturer's instructions. Cells were plated on laser confocal dishes and infected with monomeric red fluorescent protein (mRFP)–green fluorescent protein (GFP)–light chain 3 (LC3) lentivirus with a multiplicity of infection of 30 when fused at approximately 50% for 8 h. Then, hPDLCs were subjected to different treatments based on the experimental design. After permeabilization with 0.3% Triton X-100 for 60 min, DAPI was applied to the medium for 1 h to label the nucleus. Confocal imaging was performed with 667-nm emission for mRFP, 543-nm emission for green fluorescent protein (GFP) and 405-nm emission for DAPI. The yellow puncta (merged RFP and GFP puncta) in the merged channel represent autophagosomes, which indicate the presence of autophagy. The merged channel of red puncta indicates autolysosomes, the GFP of which was quenched after LCII degradation, and these red puncta are indicators of autophagy completion.
Data analysis
Each experiment was repeated three times. SPSS 23.0 software (IBM, Armonk, NY, USA) was used for data analysis. Data are expressed as the mean ± standard deviation (SD). Statistical comparisons were performed using factorial analysis of variance (ANOVA) followed by multiple comparisons using the Student–Newman–Keuls test. Values of p < 0.05 were considered to indicate statistical significance.
Results
LIPUS treatment alleviated alveolar bone resorption in rats and was accompanied by YAP activation
The maxillae of the LPS group exhibited more gingival recession than those of the L+L group and even had root bifurcation exposure. Compared with the LPS groups, the L+L group exhibited alleviated alveolar bone resorption, as reflected by CEJ–ABC distance measurement using μCT scanning and H&E staining. The level of alveolar bone resorption was approximately 1.5 times larger in the LPS group than in the L+L group. Less YAP expression and a lower percentage of YAP+ cells were found in PDLs from the LPS group compared with the control group. Robust YAP expression and translocation to the nucleus were clearly observed in the L+L group, with greater than 50% positive cells, and was accompanied by decreased p-YAP expression (Fig. 1).
LIPUS attenuated apoptosis in LPS-treated hPDLCs
As the main inflammatory factor in periodontitis, LPS was used to induce hPDLC inflammation. The effect of LPS on hPDLCs was dose dependent. Proliferation of hPDLCs was not notably affected by different concentrations of LPS (Fig. S1, online only). hPDLC apoptosis decreased in response to lower concentrations of LPS and increased in response to high concentrations of LPS, as indicated by flow cytometry (Fig. S2, online only). The LPS group had more TUNEL+ apoptotic cells and higher caspase-3 activity than the control group (Fig. 2). However, apoptotic cell numbers and caspase-3 activity were decreased in the LIPUS group. LIPUS attenuated hPDLC apoptosis in the context of inflammation, as indicated by fewer apoptotic cells and lower caspase-3 activity in the L+L group than in the LPS group (Fig. 2). Notably, the total percentage of apoptotic cells (late plus early apoptotic cells) was approximately 6% in the L+L group, which was much lower than that in the LPS group (approximately 14%).
Figure 2LIPUS attenuated apoptosis in LPS-treated hPDLCs. (A) TUNEL (with green fluorescence to indicate the apoptotic cells) and DAPI (with blue fluorescence to indicate nucleus of all cells) staining, together with their merged and magnified images in hPDLCs among different groups. (B) After being processed with LPS and/or LIPUS, hPDLCs were stained with Annexin V–FITC/PI and analyzed with flow cytometry. Cells appearing in Q2 are late apoptotic cells, and cells in Q3 experienced early apoptosis. (C) Quantitative results of the flow cytometry study in (B) on early and late apoptotic cell numbers among groups. (D) Western blot results of caspase-3 and cleaved caspase-3 expression among groups. (E) Quantitative results of Western blot in (D). **p < 0.01, ***p < 0.001 between the linked groups (n = 3 for every group). Con, control group; DAPI, 4′,6-diamidino-2-phenylindole; FITC/PI, fluorescein isothiocyanate/propidium iodide; PI, hPDLCs, human periodontal ligament cells; LIPUS, low-intensity pulsed ultrasound; LPS, lipopolysaccharide group; L+L, LIPUS + LPS group; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.
Low-intensity pulsed ultrasound stimulated YAP activity, as indicated by increased YAP and decreased p-YAP protein expression. Furthermore, the LIPUS group had more cells with YAP nuclear localization (shown as red fluorescence overlapping with blue fluorescence in the nucleus) than the control group (Fig. 3). Positive YAP fluorescence was barely observed, and expression of p-YAP, which indicated YAP inactivity, was increased in the LPS group compared with the control group (Fig. 3). However, when LIPUS was applied to LPS-treated hPDLCs, the protein level of YAP increased while that of p-YAP decreased in the L+L group, and nuclear YAP localization increased compared with that in the LPS group (Fig. 3).
Figure 3LIPUS rescued YAP activity in LPS-treated hPDLCs. (A) YAP (with red fluorescence to indicate positive cells) and DAPI (with blue fluorescence to indicate the nucleus of all cells) staining together with their merged and magnified images in hPDLCs among different groups. Note that more intensive red fluorescence exists in the LIPUS and L+L groups. Meanwhile, the overlapping red and blue fluorescence in the nucleus could easily be observed after LIPUS was applied to hPDLCs. (B) Western blot results of YAP and phospho-YAP (Y127) expression among groups. Notably, more YAP and less p-YAP were expressed after LIPUS treatment. (C) Quantitative results of total YAP expression among groups. (D) Quantitative difference in the expression of p-YAP compared with that of YAP. **p < 0.01, ***p < 0.001 between the linked groups (n = 3 for every group). Con, control; DAPI, 4′,6-diamidino-2-phenylindole; hPDLCs, human periodontal ligament cells; LIPUS, low-intensity pulsed ultrasound; L+L, LIPUS + LPS group; LPS, lipopolysaccharide; p-YAP, phosphor-YAP; YAP, Yes-associated protein.
LIPUS promoted autophagic activity completion in LPS-treated hPDLCs
An increased number of autophagosomes were observed by transmission electron microscopy (TEM) in the LPS group, accompanied by increased protein expression of LC3Ⅱ in hPDLCs. The protein level of p62 increased, and more cells with combined green and red fluorescence (indicated by yellow dots) were detected in the LPS group than in the control group. These results indicated that early autophagy was activated in LPS-induced hPDLCs but did not progress to the later stage when lysosomes fused; thus, autophagic activity was not complete (Fig. 4). Compared with that in the control group, autophagy in the LIPUS group was less active, and the expression of related proteins declined (Fig. 4). When LIPUS was applied to LPS-treated hPDLCs, more autolysosomes were observed. These autolysosomes were fused autophagosomes and lysosomes, which revealed as the monolayer in TEM. As more autolysosomes (red fluorescent dots) were present and less p62 was expressed in the L+L group than in the LPS group (Fig. 4), LIPUS promoted autophagy completion in the LPS-treated hPDLCs.
Figure 4LIPUS promoted autophagy completion in LPS-treated hPDLCs. (A) After being processed with LPS and/or LIPUS, hPDLCs were subjected to TEM for autophagic vacuole detection. The yellow arrows indicate autophagic vacuoles (magnification: 30,000 ×). Note that more autophagosomes with double membranes appeared in the LPS group, while the number of single membrane vacuoles, which are supposed to be autolysosomes, increased in the L+L group. (B) After transfection with mRFP-GFP-LC3 double-labeled adenovirus, autophagic flux was detected with confocal imaging in different groups. Note that increased autophagosomes with yellow dots appeared in the LPS group, while more red dots, which indicated autolysosomes, were found in the L+L group. (C) Western blot results of LC3 and p62 expression among groups. (D) Quantitative results of LC3Ⅱ expression among groups. (E) Quantitative results of p62 expression among groups. *p < 0.05, **p < 0.01, ***p < 0.001 between the linked groups (n = 3 for every group). Con, control group; GFP, green fluorescent protein; hPDLCs, human periodontal ligament cells; LC3, light chain 3; LIPUS, low-intensity pulsed ultrasound; L+L, LPS + LIPUS group; LPS, lipopolysaccharide; RFP, monomeric red fluorescent protein; TEM, transmission electron microscopy.
YAP silencing exacerbated apoptosis in LPS-treated hPDLCs after LIPUS treatment
We silenced YAP expression in hPDLCs by siRNA transfection. Transfection reduced the mRNA and protein levels of YAP (Fig. S3, online only). An increased number of TUNEL+ cells and increased caspase-3 activity were observed in the LPS-treated hPDLCs. This effect was reversed by the application of LIPUS to these cells. An increased number of apoptotic cells and relatively high expression of cleaved caspase-3 were observed after YAP silencing in the L+L-treated hPDLCs (Fig. 5). This result indicates that knocking down YAP expression exacerbated apoptosis in LPS-treated hPDLCs, but this effect was attenuated by LIPUS.
Figure 5YAP silencing exacerbated apoptosis in LPS-treated hPDLCs after LIPUS treatment: (A) TUNEL and DAPI staining, together with their merged and magnified images in hPDLCs among different groups. Notice that more apoptotic cells appeared after YAP expression was inhibited. (B) Western blot results of caspase-3 and cleaved caspase-3 expression among groups. (C) Quantitative results of Western blot in (B). ***p < 0.001 between the linked groups (n = 3 for every group). Con, control group; DAPI, 4′,6-diamidino-2-phenylindole; hPDLCs, human periodontal ligament cells; LIPUS, low-intensity pulsed ultrasound; LPS, lipopolysaccharide; L+L, LIPUS + LPS group; siYAP, small interfering Yes-associated protein; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.
YAP silencing inhibited autophagy completion in LPS-treated hPDLCs after LIPUS treatment
After YAP expression was knocked down in hPDLCs, the number of autophagosomes increased in the L+L+siYAP group and was comparable to that in the LPS group. More cells with combined green and red fluorescence (yellow dots) were detected after YAP silencing in the L+L hPDLCs, and LC3Ⅱ and p62 protein levels were substantially increased compared with those in the LIPUS group (Fig. 6). These results indicated a similar state of autophagic activity in the LPS group, in which autophagy was probably initially activated but did not progress through the later stage when lysosomes fused to form autolysosomes. Thus, YAP silencing inhibited autophagy completion in the L+L group.
Figure 6YAP silencing inhibited autophagy completion in LPS-treated hPDLCs after LIPUS treatment. (A) Autophagic flux was detected with mRFP-GFP-LC3 double-labeled adenovirus transfected among different groups. Note that increased autophagosomes with yellow dots appeared after YAP expression was inhibited. (B) Western blot results of LC3 and p62 expression among groups. (C) Quantitative results of LC3Ⅱ expression among groups. (D) Quantitative results of p62 expression among groups. *p < 0.05, **p < 0.01, ***p < 0.001 between the linked groups (n = 3 for every group). Con, control group; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; hPDLCs, human periodontal ligament cells; LC3, light chain 3; LIPUS, low-intensity pulsed ultrasound; LPS, lipopolysaccharide; L+L, LIPUS + LPS group; mRFP, monomeric red fluorescent protein; siYAP, small interfering Yes-associated protein; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.
Lipopolysaccharide is a common stimulatory factor that affects cell activity and is used to imitate the inflammatory microenvironment of periodontal tissue. A previous study reported that low concentrations of LPS could promote the proliferation of splenocytes and skin fibroblasts in vitro [
TLR4 activation by lipopolysaccharide and Streptococcus mutans induces differential regulation of proliferation and migration in human dental pulp stem cells.
]. These controversial results may be caused by factors related to the origin of LPS, the kinds and activity of target cells, the processing periods and the incubation environment of the cells. The irradiation intensity of LIPUS we used was low and was described in our previous study [
Low-intensity pulsed ultrasound upregulates osteogenesis under inflammatory conditions in periodontal ligament stem cells through unfolded protein response.
LIPUS inhibited the expression of inflammatory factors and promoted the osteogenic differentiation capacity of hPDLCs by inhibiting the NF-κB signaling pathway.
]. High-intensity pulsed ultrasound led to apoptosis in endothelial cells. A study with LIPUS at 30 mW/cm2 reported apoptotic inhibition in rat mesenchymal stem cells after the establishment of an inflammatory microenvironment [
Effects of low-intensity pulsed ultrasound (LIPUS)-pretreated human amnion-derived mesenchymal stem cell (hAD-MSC) transplantation on primary ovarian insufficiency in rats.
]. Our results first revealed that LIPUS rescued YAP activity in hPDLCs, which was decreased in the inflammatory microenvironment, resulting in increased YAP expression and nuclear translocation after treatment. This effect of LIPUS on YAP activation has been proven by other studies. A study on umbilical vein endothelial cells reported that both YAP and phosphorylated YAP levels increased after LIPUS treatment to promote angiogenesis [
]. However, another study found that YAP expression increased and p-YAP expression decreased during LIPUS-mediated protection of retinal ganglion cells from apoptosis [
]. A previous study reported formation of the YAP/TEAD complex after YAP nuclear translocation, which enhanced cell proliferation and prevented apoptosis [
]. Thus, LIPUS-induced apoptotic inhibition in LPS-treated hPDLCs was probably regulated by YAP activation, with YAP nuclear translocation and p-YAP expression decreased.
Overexpression of YAP has been observed in several studies related to inflammation. YAP was activated in LPS-induced inflammation in vascular endothelial cells [
]. Another study of inflammatory bowel disease reported that the overexpression of gp130, a co-receptor for the cytokine interleukin-6, activated YAP expression to regulate tissue growth and regeneration, thus preventing mucosal erosion [
]. Similar to the role of YAP as a mechanical signal transducer, the different downstream signals in response to occlusal trauma in these studies and LIPUS in our experiments may be the reason for the different effects caused by YAP activation in different contexts. As different phosphorylation sites of YAP exert various effects on the same tissue or cell by activating different downstream signals [
], further studies on downstream reactions are needed to reveal the internal mechanism of YAP expression under inflammatory conditions.
The relationship between autophagy and periodontitis has been a concern of researchers in recent years. Increased levels of autophagy-related proteins are expressed in PDLCs from patients with periodontitis [
]. The autophagic activity of PDLCs was increased by LPS, and the increase in autophagic activity in PDLCs contributed to the anti-inflammatory effect in vitro [
]. Autophagosomes are a typical structure associated with autophagy and are generated by the conversion of LC3-I to LC3-Ⅱ. However, it has been reported that the degradation rather than the formation of autophagosomes completes the autophagic process [
]. We used TEM to investigate autophagosome changes and a double-fluorescence system to identify the generation and disappearance of LC3-Ⅱ. The results revealed that LIPUS alleviated PDLC apoptosis by promoting the degradation of autophagosomes. A similar effect on cell apoptotic inhibition regulated by autophagic activation has been observed in other cells or tissues [
]. However, when autophagic activation in a cell exceeds its tolerance, excessive degradation of cellular contents may also promote apoptosis in some circumstances [
A study on cell contact inhibition reported that cell survival was regulated by the YAP/autophagy axis, and YAP deactivated the formation of impaired autophagosomes [
]. Here, we determined that YAP regulates autophagy activity in PDLCs.
Periodontitis involves different responses of multiple cell populations in periodontal tissue. The tolerance of distinct cells to inflammatory stimuli and the response to the same intensity of LIPUS may differ, which poses challenges in the clinical application of LIPUS. More systematic and comprehensive mechanistic research is needed to ensure the effectiveness and safety of this technology for clinical treatment in the future.
Conclusions
Low-intensity pulsed ultrasound inhibited hPDLC apoptosis by YAP activation and promoted autophagic degradation to help complete autophagy. These effects were reversed after YAP expression was blocked.
Conflict of interest
The authors declare no competing interests.
Acknowledgments
This work was supported by the Natural Science Foundation of China (Grant 81771082) and Postdoctoral Research Foundation of China (Grant 2018M640902). Special support was received for the Postdoctoral Program of Chongqing (Grant XmT2018079) and the Chongqing Graduate Tutor Team (Grant dstd201903).
Data availability statement
Data are available on request to Yao He (e-mail: [email protected]).
Impaired autophagy in microglia aggravates dopaminergic neurodegeneration by regulating NLRP3 inflammasome activation in experimental models of Parkinson's disease.
Low-intensity pulsed ultrasound induces osteogenic differentiation of human periodontal ligament cells through activation of bone morphogenetic protein-smad signaling.
Low-intensity pulsed ultrasound upregulates osteogenesis under inflammatory conditions in periodontal ligament stem cells through unfolded protein response.
LIPUS inhibited the expression of inflammatory factors and promoted the osteogenic differentiation capacity of hPDLCs by inhibiting the NF-κB signaling pathway.
TLR4 activation by lipopolysaccharide and Streptococcus mutans induces differential regulation of proliferation and migration in human dental pulp stem cells.
Effects of low-intensity pulsed ultrasound (LIPUS)-pretreated human amnion-derived mesenchymal stem cell (hAD-MSC) transplantation on primary ovarian insufficiency in rats.
00021-2&id=gr1.jpg){kind=link}
00021-2&id=gr2.jpg){kind=link}
00021-2&id=gr3.jpg){kind=link}
00021-2&id=gr4.jpg){kind=link}
00021-2&id=gr5.jpg){kind=link}
00021-2&id=gr6.jpg){kind=link}