Cell culture and treatment
Primary HDPCs were collected from the caries-free third molars and the premolars that need to be extracted for orthodontics of healthy donors in maxillofacial surgery clinic of Peking University School of Stomatology (ethics approval number: PKUSSIRB-201732003). Cells were maintained in α-MEM medium (Gibco, USA) with 10% fetal calf serum (FBS) (Excell Bio, Australia) and 100U/ml penicillin and 100μg/ml streptomycin sulfate, and passages 3–5 were used . Cells were treated with recombinant EZH2(20ng/mL,abnova,USA) or the inhibitor of EZH2 EI1 (2uMol/L APExBIO,abnova,USA) for the given times. In the specified experiments, cells were pretreated with one of the following specific pathway inhibitors: BAY11-7082(an Iκ B phosphorylation inhibitor, 1 uM , Selleckchem , Houston, TX) or SB203580(a p38 MAPK inhibitor,20 uM, Cell Signaling Technology, Danvers, MA).
Characterization of HDPCs
The fourth-generation HDPC was inoculated in a 12-well plate and grown in slides. After the cells grew close to confluence, they were fixed with 4% paraformaldehyde. Primary antibodies included vimentin(1:1000; proteintech , USA) and cytokeratin 14 (1:1000; proteintech ,USA ). A standard immunohistochemistry kit (Zhongshanjinqiao , Beijing, China) was used for immunohistochemistry. After counterstaining with hematoxylin and dehydration, the expression of related antibodies was observed.
RNA Isolation and Reverse Transcription and Real-time PCR
Total RNA of the HDPCs was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA). RNA concentrations were measured using a Nanodrop Spectrophotometer (Thermo Fisher Scientiﬁc). Complementary DNA was synthesized from 1000 ng RNA using a PrimeScript RT kit (TaKaRa, Dalian, China). The produced complementary DNA was prepared as templates for the polymerase chain reaction using SYBR Premix Ex Taq (Takara) according to the manufacturer’s instruction. Conditions were applied as following: 50 °C for 2 min, then 95 °C for 10 min, followed by 40 cycles of 94 °C for 15 s and 60 °C for 1 min using an ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The Ct values obtained from different samples were compared using the 2–ΔΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal reference gene. .Primers were designed to generate products of less than 200 bp for efficient analysis and were as follows:MMP-1,5’-AGATTCTACATGCGCACAAATC-3’ (forward) and 5’-CCTTTGAAAAACCGGACTTCAT-3’(reverse);MMP-2,5’-CAACTACAACTTCTTCCCTCGCA-3’ (forward) and 5’-GGTCACATCGCTCCAGACTTG-3’ (reverse); MMP-3,5’-GAGGACACCAGCATGAACCT-3’ (forward) and 5’-CACCTCCAGAGTGTCGGAGT-3’ (reverse);MMP-8,5’- CCAACTATGCTTTCAGGGAAAC-3’ (forward) and 5’- GTTGGATAGGGTTGCTTGAAAG-3’ (reverse);MMP-10,5’-TTGCCCAGCAATACCTAGAAAA-3’ (forward) and 5’-GAACTTCTGCATTCCTTGGATT- 3’ (reverse);MMP-13,5’-GCACTTCCCACAGTGCCAT -3’(forward) and 5’-AGTTCTTCCCTTGATGGCCG-3’ (reverse) ;COL1A1,5’-GCTCGTGGAAATGATGGTGC-3’ (forward) and 5’-ACCCTGGGGACCTTCAGAG-3’ (reverse); GAPDH,5’-TCAACAGCGACCCACTC-3’ (forward) and 5’-GCTCTAGCCAAATTCGTTGTC-3’ (reverse).
Western Blot Analysis
The cells were lysed using RIPA buﬀer(Solarbio,China) supplemented with protease inhibitors (Solarbio,China). Cell homogenates were obtained by gently scraping the cells from each well, and the protein concentrations from the lysates were determined by using the BCA Protein Assay Kit (ThermoScientific, Rockford,IL). Prior to loading, total protein samples were denatured by heating at 95°C for 10min in 5x SDS-PAGE sample loading buﬀer (Applygen, China). 30μg of the protein sample were separated by SDS-polyacrylamide gel electrophoresis at 110V for 90min and transferred to 0.45-μm polyvinylidene ﬂuoride (PVDF) membranes at a constant current of 300mA for 60min. After blocking in 5% nonfat dry milk (Bioruler, China) at room temperature, the membranes were incubated with rabbit anti-phospho-p65 (catalogno.3033, Cell Signaling Technolog, Danvers,MA), rabbit anti -p65 (catalogno.8242, Cell Signaling Technolog, Danvers,MA), rabbit anti -phospho-p38 (catalogno. 4511, Cell Signaling Technolog, Danvers,MA), rabbit anti- p38(catalogno.9212, Cell Signaling Technolog, Danvers,MA) overnight at 4°C. The membrane was incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin(Proteintech,USA) based on the source of the corresponding primary antibody, and the immunoblots were detected using a Western enhanced chemiluminescence blotting kit (ECL, SOLIBRO, China).
Construction of Rat Experimental Pulp Infection Model
12 male 6-week-old SD rats (Weitong Lihua Company, China) were subdivided into 4 groups of 3 rats each.Rats were classified according to the sealing medicine: LPS(10mg/ml, Sigma, USA) for group1, LPS (10mg/ml) +EZH2 (20ng/mL) for group2, LPS (10mg/ml) +EI1(20ng/mL) for group3 and group4 was not operated on as the control . Animals were sacrificed 1, 3 days after surgery and compared to a control group. Under general intraperitoneal anaesthesia, all animals except the control group underwent pulp exposure on the occlusal face of the mandibular first molar using a 1/4‐size spherical bur at high speed. The gelatin sponge with sealing medicine is placed in the perforated hole, and the cavity was sealed with glass–ionomer (Fuji, GC Corporation, Tokyo, Japan). Upon decapitation, the jaws were immersed promptly in 4% formaldehyde, embedded in paraffin, and sectioned at 5-mm thickness.
Immunostaining was performed on formalin-fixed, paraffin-embedded tissue. For immunohistochemistry, paraffin sections were dewaxed in xylene, rehydrated with distilled water, and then subjected to antigen retrieval for 30 min at 95°C. The slides were subsequently incubated overnight at 4°C with rabbit anti-Collagen I antibody (1:400,bioss,Beijing,China).Slides were then treated with an anti-rabbit secondary antibody(Zhongshanjinqiao , Beijing, China)and developed using avidin-conjugated HRP with diaminobenzidine as a substrate (Zhongshanjinqiao , Beijing, China),followed by hematoxylin counter staining.
Results are presented as means ± SD of at least 3 independent biological experiments. For each experiment, we have at least 3 technical repeats. Significance was determined via one-way analysis of variance test. The difference was considered statistically significant at the P < 0.05 level.