Wnt4 has been shown to promote the recovery of odontogenic differentiation of dental pulp stem cells under inflammatory conditions, but its role in inflammation and apoptosis of pulpitis remains to be elucidated. Lipopolysaccharide (LPS) (10 µg/ml) was applied to treat the human dental pulp cells (HDPCs) for 24 h. Western blotting measured the expressions of inflammatory cytokines and apoptosis-related proteins. Cell apoptosis was measured by flow cytometry. The level of Wnt4 was evaluated by reverse transcription-quantitative PCR and western blotting. The results indicated that LPS could promote inflammatory response and apoptosis in HDPCs and downregulated Wnt4 expression was found in LPS-HDPCs. Overexpression of Wnt4 ameliorated cell inflammatory response and apoptosis, presented by reduced expressions of IL-8, IL-6, TNF-α, IL-1β, Bax, cleaved-caspase 3 and enhanced Bcl-2 expression as well as decreased apoptosis rate. Moreover, overexpression of Wnt4 reduced the phosphorylation levels of IKK2, IκBα and p65 proteins upregulated by LPS. Finally, overexpression of IKK2 reversed the effects of Wnt4 on inflammation and apoptosis of LPS-HDPCs and NF-κB inhibitor reversed the effect of IKK2 overexpression in LPS-HDPCs. Wnt4 inhibited LPS-triggered inflammation and apoptosis in HDPCs via regulating the IKK/NF-κB signaling pathway, which provided a new viewpoint for understanding the pathological mechanism of pulpitis.
Pulpitis is one of the most common dental pulp diseases, which brings a great burden to the life of afflicted individuals (
Wnts belong to a highly conserved family of secreted growth factors, which couple to various receptors, thereby activating different downstream pathways which classify as either canonical or noncanonical signaling pathways (
The NF-κB pathway mediates inflammation, immunity and cell survival (
Based on the aforementioned studies, it was hypothesized that Wnt4 serves an anti-inflammatory and anti-apoptotic role in pulpitis through the NF-κB signaling pathway. The present study investigated the effect and underlying mechanisms of Wnt4 on the inflammatory levels and apoptosis of lipopolysaccharide (LPS)-induced human dental pulp cells (LPS-HDPCs), which might provide a novel therapy target for pulpitis. In addition, exploring effective treatment target therapeutic targets combined with advanced materials such as polyvinylidene fluoride/barium titanate composites (
Normal human impacted third molars free of carious lesions and oral infection were obtained from patients in Anhui Medical College after informed consent was collected from each patient. HDPCs were isolated from the dental pulp tissues of non-decayed third molars. In brief, human dental pulp tissue obtained from sectioned teeth was removed aseptically and minced into small pieces. The pulp was treated with 3 mg/ml collagenase type I solution for 0.5 h at 37˚C. The digested tissues were cultured in DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.), 300 mg/ml L-glutamine (Thermo Fisher Scientific, Inc.) and 100 U/ml antibiotics (Thermo Fisher Scientific, Inc.) in an incubator at 37˚C with 5% CO2; 3 to 5 passages were used for the experiments. For LPS stimulation, LPS (10 µg/ml) was administrated for 24 h as reported previously (
HDPCs were cultured in an 8-well slide for 24 h. Then cells were fixed with 4% paraformaldehyde at room temperature for 15 min, permeabilized with 0.5% Triton X-100 (Thermo Fisher Scientific, Inc.) and blocked with goat serum (cat. no. SL038; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 20 and 30 min, respectively. Then primary anti-keratin (cat. no. 13063; 1:200; CST), anti-vimentin (cat. no. 5741; 1:600; CST) antibodies and anti-p-p65 (cat. no. 3033; 1:800l; CST) were applied to incubate HDPCs at 4˚C overnight. Next, the cells were cultured in secondary antibody goat against rabbit IgG (cat. no. A32731; 1:200; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. DAPI (1:1,000) was applied for nuclei staining. The images were obtained using a confocal microscope (Leica Microsystems GmbH) (
Cells were harvested and lysed a lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.). Protein concentrations were measured using a BCA kit (Thermo Fisher Scientific, Inc.). Proteins (25 µg per lane) were separated using 10% SDS-PAGE and transferred onto a PVDF membrane (BD Biosciences). The membrane was blocked with 5% skimmed milk at room temperature for 60 min and incubated with following primary antibodies: IL-8 (cat. no. ab289967; 1:1,200; Abcam), IL-6 (cat. no. ab233706; 1:1,000; Abcam), TNF-α (cat. no. ab183218; 1:1,000; Abcam), IL-1β (cat. no. ab254360; 1:1,000; Abcam), cleaved-caspase-3 (cat. no. ab32042; 1:800; Abcam), caspase-3 (cat. no. ab32351; 1:3,000; Abcam), Bax (cat. no. 2774S; 1:1,000; CST), Bcl-2 (cat. no. 15071; 1:1,000; CST), Wnt4 (cat. no. ab277798; 1:800, Abcam), p-IKK2 (cat. no. 2694; 1:1,000; CST), IKK (cat. no. ab124957; 1:1,000; Abcam), p-p65 (cat. no. 3033; 1:1,000; CST), p65 (cat. no. 8242; 1:1,000; CST), p-IκBα (cat. no. 2859; 1:1,000; CST), IκBα (cat. no. 9242; 1:1,000; CST), β-actin (cat. no. 4970; 1:5,000; CST), GAPDH (cat. no. 5174; 1:5,000; CST) at 4˚C overnight. Then, the HRP-conjugated secondary antibody (goat against rabbit IgG (cat. no. 7074; 1:2,000; CST) and goat against mouse IgG (cat. no. 96714; 1:3,000; CST) was administrated to the cells at room temperature for 1 h. The results were detected using an ECL kit (Beyotime Institute of Biotechnology) and quantitated using ImageJ (National Institutes of Health) (
Treated HDPCs were collected, washed and resuspended with binding buffer (PBS with 5% FBS). Subsequently, HDPCs were incubated with FITC annexin V at room temperature for 0.5 h and washed using PBS. Propidium iodide (PI; 50 mg/ml) was used to culture HDPCs at room temperature for 5 min in the dark. Analysis was performed using flow cytometry by a Cytomics FC500 flow cytometer (Beckman Coulter, Inc.) and Cytomics CXP software version 2.2 (Beckman Coulter, Inc.). Untreated HDPCs were used as the control (
HDPCs were seeded into six-well plate (1x106 cells/well) and total RNA was extracted using TRIzol® according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.) after cells were treated with LPS or transfected for 48 h. Reverse transcription was executed with PrimeScript RT reagent kit (Takara Bio, Inc.) at 42˚C for 40 min and 85˚C for 5 min and 4˚C for 60 min according to the manufacturer's protocol. RT-qPCR was conducted with SYBR-Green I Master Mix (Roche Diagnostics) via LightCycler 480 (Roche Diagnostics) according to the manufacturer's protocols. Three parallel spaces were conducted for each sample and experiment was performed three times. PCR amplification conditions were: 95˚C for 10 min, followed by 40 cycles of denaturation at 95˚C for 15 sec and annealing 60˚C for 1 min and extension 72˚C for 20 sec. The relative changes were calculated using the 2-ΔΔCq method (
The Wnt4 overexpression plasmids (pcDNA-Wnt4) were constructed using pcDNA3.1 vector by Shanghai GenePharma Co., Ltd. and empty plasmids were used as normal control (NC). The constitutive activator of IKK2 (pCMV-IKK2EE) constructed using pCMV-MCS vector (
All experiments were conducted three times and the data were presented as mean ± standard deviation. Analysis used GraphPad Prism 8.0 via one-way ANOVA followed by a Bonferroni test or an unpaired Student's t-test. P<0.05 was considered statistically significant.
Morphological observation identified that HDPCs of the third generation were fusiform or polygon (
The expression of Wnt4 in HDPCs was detected. In
It has been found that Wnt4 inhibits the NF-kB pathway in the bone disease mouse model (
To investigate whether IKK/NF-κB pathway was involved in the regulation of Wnt4 on inflammation and apoptosis in LPS-HDPCs, pcDNA-Wnt4 or negative control and pCMV-IKK2EE which could mimic the IKK2 loop to activate the downstream NF-κB signaling pathway (
To further explore the effect of the IKK/NF-κB pathway on the inflammatory and apoptosis of HDPCs overexpressing of Wnt4. NF-κB inhibitor (BAY11-7082) was used to treat HDPCs co-transfected with OE-Wnt4 and pCMV-IKK2EE. The results showed that BAY11-7082 decreased the expression levels of IL-8, IL-6, TNF-α and IL-1β in the LPS + OE-Wnt4 + pCMV-IKK2EE + BAY group compared with the LPS + OE-Wnt4 + pCMV-IKK2EE + DMSO group (
Pulpitis is a physiological response to bacterial infections, physical and chemical injuries (
LPS is an important inducer of pulpitis. Administration of LPS to dentinal surfaces can trigger pulpal inflammatory responses in mice (
Wnt4 is reported as a nonclassical Wnt signaling pathway component to exert crucial roles in physiological and pathological conditions (
NF-κB is a transcription factor that controls the inflammatory response, cell cycle and apoptosis (
In conclusion, Wnt4 could inhibit inflammation and apoptosis by hindering the activation of the IKK2/NF-κB pathway in LPS-HDPCs, which might provide a potential therapeutic target in pulpitis.
Not applicable.
The datasets generated and/or used during the present study are available from the corresponding author upon reasonable request.
All authors contributed to the conception and design of the study. GW, TM and JX performed the experiments, data collection and analysis and wrote the manuscript. CN conceived and designed the experiments and revised the manuscript. CN and GW confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
The present study was approved by the Ethics Committee of Anhui Medical College (approval no. 2022-LLBG-001) and obeyed the principles of the Declaration of Helsinki. Informed consent was obtained from all participants.
Not applicable.
The authors declare that they have no competing interests.
Effects of LPS on inflammation and apoptosis in HDPCs. The HDPCs were treated with LPS for 24 h. (A) The morphology of HDPCs was observed under an optical microscope. Scale bar, 100 µm. (B) Expressions of vimentin and keratin in HDPCs were measured by immunofluorescence. Scale bar, 100 µm/30 µm. (C) The expressions of inflammatory factors and (D) apoptosis-related proteins were detected by western blotting. (E) Flow cytometry was applied to measure cell apoptosis. (F) Reverse transcription-quantitative PCR and (G) western blotting was used to detect the expression of Wnt4. Data represent the mean values ± standard deviation of three experiments. **P<0.01. LPS, lipopolysaccharide; HDPCs, human dental pulp cells.
Overexpression of Wnt4 inhibits inflammation and apoptosis in LPS-induced HDPCs. pcDNA-Wnt4 or pcDNA-NC plasmid was transfected into LPS-HDPCs. (A) Reverse transcription-quantitative PCR and (B) western blotting were applied to detect the expression of Wnt4 after transfection with pcDNA-Wnt4 or pcDNA-NC in LPS-HDPCs. The expressions of (C) inflammatory factors and (D) apoptosis-related proteins were detected by western blotting. (E) Flow cytometry was applied to measure cell apoptosis. Data represent the mean values ± standard deviation of three experiments. **P<0.01, ***P<0.001, ****P<0.0001. LPS, lipopolysaccharide; HDPCs, human dental pulp cells; OE-Wnt4, overexpression of Wnt4; OE-NC, negative control; p-, phosphorylated.
Wnt4 suppresses the activation of IKK/NF-κB pathway in LPS-induced HDPCs. (A) Western blotting and (B) immunofluorescence staining were used to analyze the activation of IKK/NF-κB pathway. Data represent the mean values ± standard deviation of three experiments. **P<0.01, ***P<0.001, ****P<0.0001. LPS, lipopolysaccharide; HDPCs, human dental pulp cells; p-, phosphorylated; OE-Wnt4, overexpression of Wnt4; OE-NC, negative control.
Wnt4 inhibits inflammation and apoptosis through the IKK/NF-κB pathway. pcDNA-Wnt4 or negative control and pCMV-IKK2EE or negative control plasmids were transfected in LPS-HDPCs. (A) Western blotting and (B) immunofluorescence staining were used to measure protein levels of the IKK/NF-κB pathway. The expressions of (C) inflammatory factors and (D) apoptosis-related proteins were detected by western blot. (E) Flow cytometry was applied to measure cell apoptosis. Data represent the mean values ± standard deviation of three experiments. **P<0.01, ***P<0.001, ****P<0.0001. LPS, lipopolysaccharide; HDPCs, human dental pulp cells; OE-Wnt4, overexpression of Wnt4, OE-NC, negative control.
NF-κB participates in the effect of Wnt4 on LPS-induced HDPCs. The expressions of (A) inflammatory factors and (B) apoptosis-related proteins were detected by western blot. (C) Flow cytometry was applied to measure cell apoptosis. Data represent the mean values ± standard deviation of three experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. LPS, lipopolysaccharide; HDPCs, human dental pulp cells; OE-Wnt4, overexpression of Wnt4, OE-NC, negative control.
Schematic model for the molecular mechanism of Wnt4 in LPS-induced HDPCs. LPS, lipopolysaccharide; HDPCs, human dental pulp cells.