The Role Of BACH1 On The Odontoblastic Differentiation Of Human Dental Pulp Stem Cells And The Underlying Mechanisms

Part Ⅰ Expression and distribution of BACH1 in human healthy dental pulp tissues Aim: To explore the expression and distribution of BACH1 in human healthy dental pulp tissues.Methods: Healthy human molars or premolars that met the inclusion criteria were collected.WB was conducted to detect the expression of BACH1 in human healthy dental pulp tissues.Immunohistochemical staining and tissue immunofluorescence staining were used after decalcification and sectioning to detect the distribution characteristics of BACH1 in human healthy dental pulp tissues.Results: The results of WB showed that BACH1 was expressed in human healthy dental pulp tissues.The staining results demonstrated that BACH1 was positively expressed in human healthy pulp tissues,and the expression was significantly enhanced in the odontoblastic layer than in the cell rich zone.Conclusion: BACH1 is widely distributed in healthy human dental pulp tissues and highly expressed in the odontoblast layer,suggesting that BACH1 may be involved in the dentin secretion and maintenance of dentin-pulp complex homeostasis.Part Ⅱ Expression patterns and upstream regulation of BACH1 during the odontoblastic differentiation of human dental pulp stem cells(h DPSCs)in vitroAim: To investigate the modulation and regulators of BACH1 expression during the odontoblastic differentiation of h DPSCs in vitro.Methods: Primary cells were isolated and cultured,and the cell type was identified as h DPSCs using cell immunofluorescence staining,flow cytometry and multi-directional induction of differentiation.The odonto/osteogenic differentiation of h DPSCs was induced in vitro,and was confirmed by detecting ALP activity,expression of mineralization markers RUNX2,DMP1 and DSPP,and calcium deposit.The expression levels of total BACH1 protein,nuclear protein and m RNA were measured by WB and RT-q PCR.Cellular immunofluorescence staining was performed to observe the change of subcellular distribution of BACH1 in h DPSCs during the differentiation process.The mode of regulation of post-translational modification of BACH1 was explored by specific inhibitors.Results: The odontoblastic differentiation of h DPSCs was induced in vitro,during which the total BACH1 protein decreased initially and then recovered gradually,while m RNA levels did not change significantly.Cellular immunofluorescence and WB results showed a significant downregulation in BACH1 nucleoprotein in the early stage of the differentiation process,followed by a gradual increase,although BACH1 nuclear protein levels were still slightly lower on day 14 than on day 0.The nuclear export of BACH1 was inhibited by the p38 MAPK inhibitor SB203580 and CRM1 inhibitor LMB.Meanwhile,the use of SB203580 inhibited the reduction of cytoplasmic CRM1 in response to odonto/osteogenic induction medium(OM)stimulation.The use of MG132,inhibited the reduction of total BACH1 protein.Conclusion: The decreased level of BACH1 total protein is due to proteasomal degradation and nuclear export of BACH1 is mediated by p38 MAPK/CRM1 axis during the differentiation of h DPSCs into odontoblastic-like cells in vitro.Part Ⅲ Effects of BACH1 on the proliferation,migration and odontoblastic differentiation of h DPSCs in vitroAims: To study the role of BACH1 downregulation in the proliferation,migration and odontoblastic differentiation of h DPSCs.Methods: The h DPSCs were transfected with a lentiviral vector to construct BACH1-knockdown h DPSCs(LV-sh BACH1 group).RT-q PCR,WB and observation of GFP were used to verify the knockdown efficiency.Cell proliferation viability was detected using CCK-8 and Ed U assay.The cell cycle was discerned by PI staining.Cell migration ability was determined by scratch assay and Transwell assay.ALP staining and activity were performed on day 7 after OM treatment;m RNA and protein expression of odontoblastic-related factors RUNX2,DMP1 and DSPP were measured on day 14;ARS staining assay was conducted on day 21 to assess the degree of the odontoblastic differentiation of h DPSCs.Results: The results of CCK-8 displayed that the proliferation viability of the LV-sh BACH1 group was decreased,and the percentage of Ed U-positive cells in the LV-sh BACH1 group was lower than that in the control group.The BACH1-knockdown group showed a lower proportion of S and G2/M phases and a higher proportion of G0/G1 phases.The percentage of closed wound area in the LV-sh BACH1 group was lower than that in the control group from 12 hours,and the wound width was significantly wider at 24 hours.In the LV-sh BACH1 group,ALP staining and ALP activity were decreased on day 7,RUNX2,DMP1 and DSPP expression was attenuated on day 14,and calcium nodule formation was reduced on day 21.Conclusion: Downregulation of BACH1 inhibits cell proliferation,induces cell cycle arrest,weakens migration and reduces the odontoblastic differentiation potential of h DPSCs,suggesting that BACH1 is an important regulator of the proliferation,migration and odontoblastic differentiation of h DPSCs in vitro.Part Ⅳ The molecular mechanism by which BACH1 functions in the odontoblastic differentiation of h DPSCsAims: To explore the potential mechanism of inhibition of odontoblastic differentiation of h DPSCs induced by BACH1 knockdown.Methods: h DPSCs from three different individuals were stably transfected with lentivirus to construct BACH1-knockdown h DPSCs(LV-sh BACH1 group)and a control group containing an empty vector(LV-NC)for OM induction.After 3 days of induction,total RNA was extracted from the samples and RNA-seq was performed.Screen out the different expression genes according to the corresponding filter criteria.Based on the results of previous studies and bioinformatics analysis,the possible signaling pathways involved in the differentiation of BACH1 into odontoblastic-like cells were excavated and then validated by a specific inhibitor or activator.To investigate the role of related pathways,the activator or inhibitor of related pathways was administrated.The methods of monitoring the extent of odontoblastic differentiation are the same as before.Result: As the expression of HO-1 was increased in the LV-sh BACH1 group,the HO-1inhibitor Sn PP was added.In the LV-sh BACH1+Sn PP group,all tests related to odontoblastic differentiation showed diminished results.Bioinformatics analysis showed significant enrichment of the Wnt/β-Catenin signaling pathway with the peak of enrichment score at the bottom and the gene set was enriched below,which means the gene set was inhibited in the LVsh BACH1 group.Enrichment results for promoter regions and transcription factor binding sites of all genes contained LEF1.After 1 day of mineralization induction,cellular immunofluorescence displayed that β-Catenin nuclear import was reduced in the LV-sh BACH1 group.After 3 days of mineralization induction,results by WB and RT-q PCR showed that the m RNA levels of some Wnt/β-Catenin signaling pathway related genes were reduced in the LVsh BACH1 group,and total protein levels,nuclear protein level and activation level of β-Catenin were decreased as well.With the addition of Li Cl,a Wnt/β-Catenin signaling pathway activator,the LV-sh BACH1+ Li Cl group demonstrated partial restoration of ALP activity on day 7.Subsequent m RNA levels of mineralization markers and the results of ARS staining assay displayed the same trend.Conclusion: BACH1 regulates odontoblastic differentiation of h DPSCs not dependent on HO-1 but related to the Wnt/β-Catenin signaling pathway.These findings shed new light on the function of BACH1 in the odontoblastic differentiation of h DPSCs,but additional research is required to fully characterize the role of BACH1 in the regenerative capacity of h DPSCs.