Activating Liver X Receptor β Promotes Neuronal Differentiation Of Dental Pulp Stem Cells In Normal And Inflammatory Environments

Background and objectivesDental pulp stem cells(DPSCs)belong to the oral and maxillofacial mesenchymal stem cells,and have been widely used in multi-lineage cellular differentiation in vitro because of their potential to repair irreversibly damaged or diseased tissue and organs.Studies confirmed DPSCs’ expression of neural lineage markers,such as Nestin,β-Ⅲ-tubulin(TUJ1),neuronucleoprotein,etc,and demonstrated the potential of DPSCs for stem cell transplantation after neuronal differentiation in vitro,which aims to treat neurodegenerative diseases.The injected cells for stem cell transplantation should preferentially possess two characteristics:improving neuronal survival and efficacy,and modifying stem cell niches by inflammation regulation,thus promoting nerve regeneration in pathological conditions.However,researchers have not reached a standard in vitro treatment protocol before stem cell transplantation.Liver X receptors,including LXRα and LXRβ,are mainly responsible for regulating cholesterol homeostasis,lipogenesis and inflammation.LXRβ is highly expressed in nervous tissues,and its participation in metabolic activities also affects the survival and physiological activities of dopaminergic neurons.In addition,LXRs regulate inflammation and immune response,improve the survival rate of human adipose-derived stem cells in inflammatory microenvironment.Only a few studies have conducted on the application of LXRβ activation pathways in the induced differentiation of stem cells.The effects of suitable ligands and their dosages on neurotropic differentiation have not been clarified,and the additional effects of inflammatory microenvironment on stem cell characteristics remains to be examined.The purpose of this study is to determine whether the activation of LXRβ is involved in the regulation of neuronal differentiation of DPSCs in vitro and in inflammatory environment,and to screen out the ligands and dosages that achieve the best efficiency of neuronal differentiation and anti-inflammation.We hope to provide theoretical references for the combined treatment of neuro-regeneration and anti-inflammation based on DPSCs.Methods1.Expression and activation of LXRβ in human dental pulpImmunohistofluorescence staining was used to observe the expression of LXRβ in human pulp tissue.Human dental pulp cells were isolated and cultured,which were used for detecting their osteogenic differentiation,lipogenic differentiation potential and mesenchymal stem cell related surface markers.After DPSCs were treated with LXRs agonists GW3965 and T0901317 at gradient doses(0.5/1/5/10 μM),the cell viability was detected by CCK-8 kit,and the activation of LXRβ in each group was detected by qRT-PCR after 3,7,and 14 days of coaxing DPSCs in culture.2.Activation of LXRβ affect neural differentiation of DPSCsDPSCs were divided into control group,N-A group(neurobasal culture),and neurobasal medium treated with GW3965 and T0901317(each with 0.5/1/5/10 μM),Gene and protein levels of neuronal markers were detected by qRT-PCR and Western blot after 3,7 and 14 days of culture.3.Activation of LXRβ in LPS-induced inflammatory environment and its regulation of neuronal differentiationAfter 6 days of induction of DPSCs in the same group as above,LPS was added to stimulate the inflammatory environment for 24 hours.Gene level of LXRβ,inflammatory factors and neuronal markers in each group were detected by qRT-PCR.4.Konckdown of LXRβ regulates neuronal differentiation in an LPS-induced inflammatory environmentLXRβ knockdown lentivirus was used to infect LPS-stimulated DPSCs with the optimal multiplicity of infection.qRT-PCR was used to detect the efficiency of reducing LXRP gene expression and gene levels of inflammatory factors and neuronal markers after 72 hours.Results1.LXRβ was positively stained in adult pulp tissue.Monoclonal cultured human DPSCs have multidirectional differentiation potential.0.5/1/5/10 μM GW3965 and T0901317 showed no toxicity to human DPSCs,and activated intracellular LXRβ in a dose-dependent manner.2.GW3965 and T0901317 could significantly up-regulate gene and protein levels of most neuronal markers,and the up-regulation trend was mostly consistent with the activation level of LXRβ.3.With LPS-induced inflammation,LXRβ was dose-dependently activated by mediumhigh doses of GW3965 and T0901317 at 5 μM/10 μM.Inflammation had no significant effect on the activation level of LXRβ.Medium and high doses of GW3965 and T0901317 partially rescued the up-regulation of inflammatory cytokines in LPS-stimulated DPSCs.The activation of LXRβ can still promote the neuronal differentiation of DPSCs under inflammatory conditions,and 10 μM high doses of GW3965 and T0901317 can partially rescued gene levels of neuron markers down-regulated by inflammation.4.LXRβ expression of DPSCs was down-regulated by 80.24%on average after lentivirus infection,inflammation level was significantly increased after LPS treatment,and gene levels of neuronal markers showed a partial downward trend.Conclusions1.LXRβ expresses in human dental pulp tissue,and human DPSCs can be dosedependent activated by LXRs agonists GW3965 and T0901317,which can promote the neuronal differentiation of dental pulp stem cells under normal and inflammatory conditions,as well as down-regulate levels of inflammatory factors.2.GW3965 with 5 μM and 10 μM achieved optimal level of LXRβ activation in DPSCs,which promoted neuronal differentiation and inhibited inflammation after 7 days cells culture.