Effect Of Pannexin3 On Inflammatory Regulation And Differentiation Of Human Dental Pulp-derived Stem Cells

Part ?:Panx3 inhibits TNF-a-induced-inflammatory response by suppressing NF-?B signaling pathway in Human dental pulp-derived stem cellsAims:The aim of this study is to demonstrate the role and the potential mechanism of Panx3 in inflammatory response.Materials and Methods:Collection of normal human tooth and inflamed pulpitis,induction of rat pulpitis and collection of samples,each sample was used for H&E and immunohistochemical staining.In the particular experiments,cells were treated with recombinant human TNF-a or pretreated with BAY 11-7082 or MG 132 before TNF-a stimulation for the given time.And then the mRNA and protein expression of Panx3 was tested using qRT-PCR and Western blot.Panx3 overexpression and knockdown were constructed by lentiviral system,and the infection efficiency was identified using qRT-PCR and Western blot.The inflammatory cytokines IL-6 and IL-1? mRNA and protein expression were tested by qRT-PCR and ELISA following TNF-a stimulaton.Dual-luciferase assay,Western blot and immunofluorescence were prformed for NF-?B transcription factor.BAY 11-7082 was used to reverse the effect of Panx3 inhibition on inflammatory response.Results:Immunohistochemical staining showed that Panx3 levels were diminished in inflamed human and rat dental pulp tissues.Panx3 expression was significantly downregulated in hDPSCs following TNF-a challenge in a concentration-denpendent way.Such decrease could be reversed by MG 132,a proteasome inhibitor.Unlike MG 132,BAY 11-7082,a NF-?B inhibitor,even reinforced the inhibitory effect of TNF-a.TNF-a-induced IL-1? and IL-6 were significantly lessened when Panx3 was overexpressed in hDPSCs.Conversely,Panx3 knockdown exacerbated the expression of pro-inflammatory cytokines.Moreover,Panx3 participated in dental pulp inflammation in a NF-?B-dependent manner.Conclusion:In the present study,we explored that inflammatory stimulation leads to Panx3 dysfunction via proteasome pathway,which may result in positive regulation of inflammatory response through excessive activation of NF-?B.Meanwhile,NF-?B pathway also acts in a balancing manner during Panx3 degradation indicating that Panx3 knockdown aggravated TNF-a-induced inflammatory response via activating NF-kB signaling pathway.Part ?:The effect and mechanism of Panx3 on mineralizaition of hDPSCs.Aims:We aim to demonstrate the role and mechanism of Panx3 in the commitment of hDPSCs.Materials and methods:qRT-PCR and Western blot were used to test the mRNA and protein expression of Panx3 at different time points during odonto/osteogenic commitment.CCK-8 assay was performed for assessing the effect of Panx3 on the proliferation of hDPSCs.To elucidate how Panx3 regulates odonto/osteogenic differentiation of hDPSCs,we conducted a series of analyses,including qRT-PCR,western blot,ALP activity,ALP staining,or alizarin red s staining,at indicated time points.Western blot analysis,dual-luciferase reporter,qRT-PCR and immunofluorescence analysis were performed to determine the role of Panx3 on Wnt/p-catenin signaling pathway.qRT-PCR and Western blot were used to determine the expression of mineralization-related markers following LiCl stimulation.Western blot analysis,immunofluorescence analysis and dual-luciferase reporter were used to detect the effect of Wnt/?-catenin on Panx3.Western blot analysis was performed to detect the protein level of p-ERKl/2 and ERK1/2.U0126 was usted to determine the mechanism involved in Panx3-upregulated dentinogenesis and osteogenesis of hDPSCs.Results:Panx3 expression was significantly enhanced at day 4 than that at day 0 but decreased at day 7.Overexpressing Panx3 in hDPSCs was sufficient to enhance odonto/osteogenic differentiation,whereas the depletion of Panx3 resulted in a decline of odonto/osteogenic potential.Panx3 could interact with the Wnt/?-catenin signaling pathway,forming a negative feedback loop between Panx3 with Wnt/p-catenin.However,promotion of odonto/osteogenic differentiation by Panx3 was not influenced by Wnt/p-catenin.Moreover,we demonstrated that Panx3 promotes ERK signaling pathway and disruption of the ERK1/2 suppresses the promotive effect of Panx3 overexpression.Conclusion:We undercored the bi-directional interaction between Panx3 and Wnt/?-catenin.Meanwhile,Panx3 plays an essential role in the regulation of odonto/osteogenic differentiation by activating the ERK signaling pathways.Part ?:Panx3-modified hDPSCs increased the rat cranial critical-sized bone defect.Aims:This study aims to investigate the repair ability of Panx3-modified hDPSCs loaded on ?-TCP for rat cranial critical-sized bone defect.Materials and methods:hDPSCs were seeded on ?-TCP scaffold for 24h,and then the cells were stained with phalloidin.Construction of the rat cranial critical-sized bone defect(diameter:5mm).All of the 24 critical-sized defectsfrom twelve rats were divided into four groups:blank group(n=6),?-TCP group(n=6),hDPSCs/Plvx-?-TCP group(n=6),and hDPSCs/Panx3-?-TCP group(n=6).All rats were sacrificed 8 weeks postoperation,the calvarial bones were harvested.All samples were tested using micro-CT,HE staining,Masson staining,TRAP staningand double immunofluorescence staining.Results:hDPSCs could be clearly seen to be uniformly distributed in the P-TCP scaffold,and were of spindle shape.Micro-CT and histological analysis showed that hDPSCs/Panx3-?-TCP group displayed more bone formation than hDPSCs/Plvx-?-TCP at 8 week spost-operation even though the defect was not completely restored.A small quantity of newly formed bone was detected in the ?-TCP only group,and almost no newly formed bone was detected in the blank group.TRAP staining showed thatosteoclasts were presented in the border of the newly formed bone.Double immunofluorescence staining of OCN and GFP showed that both host cells and donor cells participated in newly bone formation.Conclusion:Panx3 overexpression hDPSCs enhanced in vivo bone formation.