Effects And Mechanism Of ROS-Nrf2 On Osteogenic Differentiation In Periodontal Ligament Stem Cells Under Cyclic Mechanical Stress

Background and objectiveThe biological basis in orthodontic tooth movement under the biomechanical stimuli is the alveolar bone remodeling.The alveolar bone remodeling in orthodontics involves boneforming and deposition at the tension side and bone-resorbing at the pressure side.Osteogenic differentiation stimulated by the mechanical stress in PDLSCs（periodontal ligament stem cells）at the tension side is the important process in the alveolar bone remodeling during the orthodontic tooth movement.However,some side effects,including the alveolar bone loss,the loss of attachment and gingival recession,may occur in orthodontics and cause tissue damage to the orthodontic patients.It is of great significance to study the molecular mechanisms of alveolar bone remodeling in orthodontics.It may be helpful to find the potential targets and develop the orthodontic therapeutic approaches for avoiding the side effects.Previous studies showed that cyclic mechanical stress could promote the production of reactive oxygen species（ROS）in many cell types,such as the retinal pigment epithelial cells,the alveolar epithelial cells and the endothelial cells.ROS played the vital role in the cell physiological functions,including the cell proliferation and differentiation.The excessive ROS production could cause oxidative damage of DNA,proteins,and lipids,which was associated with the pathophysiological mechanisms of aging,type Ⅱ diabetes,and neurodegenerative diseases.The production of ROS stimulated by the mechanical stress might activate mechanosensitive cytokines and the cellular signaling,participating in the physiological and pathological process.The excessive production of ROS could stimulate the osteoclast differentiation and bone resorption,while inhibit osteogenic differentiation and bone formation.The overproduction of ROS could lead to an imbalance between bone formation and bone resorption,and play a crucial role in bone diseases.The excessive accumulation of ROS and the decreased levels of antioxidants could be related to osteoporosis.The application of antioxidants could have the positive effects on promoting osteogenic differentiation,preventing bone loss and improving bone remodeling.Therefore,it was crucial to maintain the ROS level during osteogenic differentiation.Nuclear factor erythroid 2-related factor 2（Nrf2）is the pivotal transcriptional regulator that maintains the level of ROS in cells and participates in intracellular antioxidant response.Under the physiological state,Nrf2 binds to Kelch-like ECH-associated protein 1（Keapl）and is degraded by ubiquitination.In response to oxidative and chemical insults,the structures of Keap1 and Nrf2 are modified,leading to Nrf2 disassociation from Keapl and translocation to the nucleus.Subsequently,Nrf2 can be responsible for the antioxidant response and the cellular defense by regulating and promoting the antioxidant response elements（AREs）mediated expression of antioxidants,including heme oxygenase-1（HO-1）,catalase（CAT）and NADPH dehydrogenase quinone 1（NQO1）.Nrf2 is a mechanosensitive transcription factor.The mechanical stimuli,such as the shear stress,ultrasound and the mechanical stretch,can regulate the expression of Nrf2 and participate in the physiological activities and the pathological process of diseases.Previous studies confirmed that Nrf2,as the vital regulator in the antioxidant response,was involved in osteogenic differentiation.The Nrf2-deficient mice had the lower bone mass and bone strength owing to the imbalance between bone formation and bone resorption.It is not yet known that the effect of cyclic mechanical stress on ROS and Nrf2 in PDLSCs during osteogenic differentiation.And the molecular mechanisms of Nrf2 underlying this process have not been studied.Given the vital role of ROS and Nrf2 in the mechanical stress and osteogenic differentiation,the first aim of this study was to determine that the effect of cyclic mechanical stress on the ROS level in PDLSCs.And the second aim was to examine the role of Nrf2 in PDLSCs during osteogenic differentiation under cyclic mechanical stress.The third aim was to detect the effect of t-BHQ,the Nrf2 activator,on the osteogenic differentiation in orthodontic rats at the tension side during orthodontic tooth movement.And the fourth aim was to explore the molecular mechanisms of Nrf2 underlying this process via the tandem mass tag（TMT）-based liquid chromatography-tandem mass spectrometry（LC-MS/MS）technology.Investigating the mechanisms of osteogenic differentiation in PDLSCs under cyclic mechanical stress might have positive effects on the alveolar bone remodeling during orthodontic tooth movement.Materials and methods1.The culture and identification of PDLSCsPDLSCs were isolated and cultured from the human periodontal ligament（PDL）tissues.The surface markers in PDLSCs,including STRO-1,CD 146,CD34 and CD45,were examined by AccuriTM C6 flow cytometer.The multidirectional differentiation potential of PDLSCs was evaluated by alizarin red staining,oil red O staining and alcian blue staining,followed by the osteogenic,adipogenic or chondrogenic induction,respectively.2.The effect of 10%0.5 Hz cyclic mechanical stress on the osteogenic differentiation of PDLSCsCyclic mechanical stress with 10%deformation and 0.5 Hz was performed by Flexercell FX-5000 Strain Unit.The expression levels of osteogenic relative markers,including COL1（collagen type 1）,RUNX2（runt-related transcription factor 2）and OPN（osteopontin）in PDLSCs were examined and analyzed by qRT-PCR and Western blot experiments.And ALP（alkaline phosphatase）activity in PDLSCs was detected by NBT/BCIP staining kit and ALP assay kit.3.The effect of 10%0.5 Hz cyclic mechanical stress on the ROS generation in PDLSCsThe PDLSCs under cyclic mechanical stress incubated with DCFH-DA and DHE were examined and analyzed by BD Accuri^TM C6 flow cytometer.The hydrogen peroxide（H2O2）level in PDLSCs was measured by the hydrogen peroxide assay kit.4.The vital role of Nrf2 in the osteogenic differentiation of PDLSCs under cyclic mechanical stress（1）The effect of cyclic mechanical stress on the expression of Nrf2 and its downstream antioxidans in PDLSCsThe expressions of Nrf2 and its downstream antioxidants,including HO-1 and NQO1,in PDLSCs under cyclic mechanical stress were detected by qRT-PCR and Western blot experiments.The nuclear and cytoplasmic protein extraction kit was used to detect the Nrf2 protein levels in the cytoplasm and nuclei.（2）The effect of ROS generation on the Nrf2 expression under cyclic mechanical stressAccording to the CCK-8 experiments,2.5 mmol/L and 5 mmol/L N-acetylcysteine（NAC）were selected.The ROS level in PDLSCs with NAC treatment was analyzed by BD AccuriTM C6 flow cytometer.The expression levels of Nrf2,HO-1 and NQO1,in PDLSCs with NAC treatment under cyclic mechanical stress were detected by qRT-PCR and Western blot experiments.（3）The effect of siNrf2 on osteogenic differentiation in PDLSCs under cyclic mechanical stressPDLSCs were transfected with siNrf2 to knock down Nrf2 expression.The transfection efficiency of siNrf2 in PDLSCs was examined by qRT-PCR and Western blot experiments.The ROS level in siNrf2-transfected PDLSCs was analyzed by BD AccuriTM C6 flow cytometer.The effects of siNrf2 on osteogenic differentiation under cyclic mechanical stress were examined by qRT-PCR,Western blot and ALP staining and ALP activity analysis.（4）The effect of Nrf2 activator t-BHQ on osteogenic differentiation in PDLSCs under cyclic mechanical stressAccording to the results of CCK-8 experiments,10μmmol/L tert-butylhydroquinone（tBHQ）was selected.The expression levels of Nrf2,HO-1 and NQO1,in PDLSCs with t-BHQ treatment under cyclic mechanical stress were detected by qRT-PCR and Western blot experiments.The ROS level in PDLSCs with t-BHQ treatment was analyzed by BD AccuriTM C6 flow cytometer.The effects of t-BHQ on osteogenic differentiation in PDLSCs under cyclic mechanical stress were detected by qRT-PCR,Western blot and ALP staining and ALP activity analysis.5.The effect of the Nrf2 activator t-BHQ on the osteogenic differentiation in rats at the tension side during orthodontic tooth movement（1）Establishing the tooth movement modelMesial force of 25 g,was applied for mesial movement of the maxillary first molar using the maxillary incisors as the anchorage in rats.The rats were divided into the control group and t-BHQ treatment group.（2）The effect of Nrf2 activator t-BHQ on the expression levels of Nrf2 and HO-1 in orthodontic ratsThe expression levels of Nrf2 and HO-1 in the PDL around the distal to the cervical third of mesiobuccal root of the maxillary first molar were evaluated by immunohistochemistry（IHC）staining.（3）The effect of t-BHQ on osteogenic differentiation at the tension side in orthodontic ratsThe alveolar bone around distal to the cervical third of mesiobuccal root of the maxillary first molar in orthodontic rats was selected as the region of interest（ROI）for micro-CT（micro-computed tomography）analysis.We measured the microarchitectural parameters of the alveolar bone,including bone volume/total volume（BV/TV）;trabecular thickness（Tb.Th）;trabecular separation（Tb.Sp）,and structure model index（SMI）,in orthodontic rats by the CT Analyser software.The expression levels of ALP and COL1 in the PDL around the distal to the cervical third of mesiobuccal root of the maxillary first molar were evaluated by IHC staining.6.The mechanism of Nrf2 in osteogenic differentiation under cyclic mechanical stress through TMT-based LC-MS/MS analysisThe mechanism of Nrf2 in cyclic mechanical stress-stimulated osteogenic differentiation was explored by TMT-based LC-MS/MS analysis.The differentially expressed proteins in the control group and siNrf2 groups were quantified and selected.The gene ontology（GO）analysis,kyoto encyclopedia of genes and genomes（KEGG）analysis and the protein-protein interaction（PPI）analysis among the differentially expressed proteins were performed.Western blot,qRT-PCR,Co-IP（co-immunoprecipitation）experiments were carried out to explore the mechanism of Nrf2 in osteogenic differentiation under cyclic mechanical stress.Results1.The culture and identification of PDLSCsPDLSCs were obtained from the human PDL（periodontal ligament）tissues.The results of flow cytometry showed that PDLSCs positively expressed STRO-1 and CD 146,while negatively expressed CD34 and CD45.PDLSCs had the potential of multidirectional differentiation,as evidenced by the alizarin red staining,oil red O staining and alcian blue staining.2.10%0.5 Hz cyclic mechanical stress promoted the osteogenic differentiation of PDLSCs10%0.5 Hz cyclic mechanical stress promoted the expression levels of osteogenic relative markers,including COL1,RUNX2 and OPN,in PDLSCs.The results of ALP staining and ALP activity analysis showed that cyclic mechanical stress increased the ALP activity in PDLSCs.The above results indicated that 10%0.5 Hz cyclic mechanical stress promoted the osteogenic differentiation of PDLSCs.3.10%0.5 Hz cyclic mechanical stress up-regulated the ROS generation in PDLSCsThe level of ROS in PDLSCs under cyclic mechanical stress was increased at 1 h,peaking at 12 h,and then decreased.The level of O2·-（superoxide radical anion）was upregulated at 1 h,peaking at 3 h in PDLSCs under cyclic mechanical stress,and then maintained the steady level without significant difference,compared with the control group.The H2O2 level in PDLSCs was significantly increased at 12 h,24 h and 36 h under cyclic mechanical stress.The above results indicated that 10%0.5 Hz cyclic mechanical stress promoted the production of ROS,including O2·-and H2O2,in PDLSCs.4.The vital role of Nrf2 in the osteogenic differentiation of PDLSCs under cyclic mechanical stress（1）10%0.5 Hz cyclic mechanical stress increased the expression levels of Nrf2 and its downstream antioxidants in PDLSCs10%0.5 Hz cyclic mechanical stress increased the Nrf2 mRNA expression in PDLSCs.Western blot results showed that the Nrf2 protein levels in cytoplasm and nuclei were enhanced under cyclic mechanical stress.Cyclic mechanical stress increased the expression levels of HO-1 and NQO1 in PDLSCs.These results suggested that Nrf2 might play a role in the osteogenic differentiation of PDLSCs under cyclic mechanical stress.（2）The expression of Nrf2 was associated with the ROS generation under cyclic mechanical stressThe results of the flow cytometry showed that NAC reduced the ROS production in PDLSCs under cyclic mechanical stress.NAC inhibited the expression levels of Nrf2,HO-1 and NQO1 under cyclic mechanical stress.The results indicated that the ROS production might be involved in the cyclic mechanical stress-induced the expression of Nrf2.（3）siNrf2 inhibited the osteogenic differentiation of PDLSCs under cyclic mechanical stressInhibiting the Nrf2 expression enhanced the ROS production in PDLSCs under cyclic mechanical stress and inhibited the expression levels of osteogenic relative markers（COL1,RUNX2,OPN and ALP）,which was partially rescued by NAC treatment.The above results showed that Nrf2 played a vital role in the osteogenic differentiation of PDLSCs under cyclic mechanical stress,which might be associated with the ROS generation.（4）The Nrf2 activator t-BHQ promoted the osteogenic differentiation of PDLSCs under cyclic mechanical stressThe Nrf2 activator t-BHQ could increase the expression levels of Nrf2,HO-1 and NQO1,while restrain the ROS production in PDLSCs under cyclic mechanical stress.The Nrf2 activator t-BHQ could promote the expression levels of osteogenic relative markers（COL1,RUNX2,OPN and ALP）in PDLSCs.The above findings indicated that t-BHQ promoted the osteogenic differentiation of PDLSCs under cyclic mechanical stress,and Nrf2 played a positive role in promoting the osteogenic differentiation of PDLSCs.5.The Nrf2 activator t-BHQ promoted the osteogenic differentiation at the tension side during orthodontic tooth movement in rats（1）The Nrf2 activator t-BHQ promoted the expression levels of Nrf2 and HO-1 in orthodontic ratsThe Nrf2 activator t-BHQ could enhance the expression levels of Nrf2 and HO-1 in orthodontic rats at the tension side through IHC staining.（2）The Nrf2 activator t-BHQ promoted the osteogenic differentiation at the tension side in orthodontic ratsAccording to the results of micro-CT,t-BHQ played the positive role in improving the microarchitectural parameters of the alveolar bone,including BV/TV,Tb.Th,Tb.Sp and SMI,in orthodontic rats with t-BHQ treatment at the tension side.The up-regulated expression levels of the osteogenic relative markers（ALP and COL1）were observed in orthodontic rats with t-BHQ treatment at the tension side.Our data showed that the increased expression of Nrf2 by using t-BHQ was beneficial to the osteogenic differentiation at the tension side in orthodontic rats.6.Nrf2 participated in the osteogenic differentiation of PDLSCs under cyclic mechanical stress through the PI3K/Akt signaling pathwayAccording to the KEGG analysis,the PI3K（phosphatidylinositol 3-kinase）/Akt（protein kinase B）signaling pathway was selected for the further research.PDLSCs were treated with LY294002,the PI3K inhibitor.The Nrf2 expression was inhibited with LY294002 treatment under cyclic mechanical stress.LY294002 down-regulated the expression levels of osteogenic relative markers in PDLSCs exposed to cyclic mechanical stress.The results of Co-IP experiments showed that the protein-protein interaction between Akt and Nrf2 was found in PDLSCs under cyclic mechanical stress.The above data confirmed that the PI3K/Akt signaling pathway was associated with regulating Nrf2 during the osteogenic differentiation of PDLSCs exposed to cyclic mechanical stress.7.The involvement of Nrf2 in the osteogenic differentiation of PDLSCs under cyclic mechanical stress was associated with the protein-protein interaction between HO-1 and SOD2According to the STRING（the search tool for the retrieval of interacting genes/proteins）database,we found that the interaction between HO-1 and SOD2（superoxide dismutase 2）might be involved in the osteogenic differentiation of PDLSCs exposed to cyclic mechanical stress.The expression level of SOD2 in PDLSCs was increased during cyclic mechanical stress.The results of Co-IP experiments showed that the protein-protein interaction between HO-1 and SOD2 was observed in PDLSCs exposed to cyclic mechanical stress.These data showed that the involvement of Nrf2 in the osteogenic differentiation of PDLSCs under cyclic mechanical stress was associated with the interaction between HO-1 and SOD2.ConclusionsIn vivo and in vitro experiments confirmed the important role of Nrf2 in the osteogenic differentiation of PDLSCs under cyclic mechanical stress.The potential mechanism of Nrf2 involved in the osteogenic differentiation was explored by LC-MS/MS analysis.The main conclusions in this study were as follows:1.ROS-Nrf2 played the vital role in the osteogenic differentiation under cyclic mechanical stress in PDLSCs.2.The Nrf2 activator t-BHQ promoted the osteogenic differentiation at the tension side during orthodontic tooth movement in rats.3.The mechanisms of Nrf2 in the osteogenic differentiation of PDLSCs under cyclic mechanical stress were related to the PI3K/Akt pathway and the protein-protein interaction between HO-1 and SOD2.This study showed that Nrf2 had the positive effect on the osteogenic differentiation of PDLSCs under cyclic mechanical stress.Nrf2 might be a potential and promising target to improve the osteogenic differentiation and the alveolar bone remodeling during orthodontic treatment.