Effects Of EPO On Cell Cycle,apoptosis And Osteogenic Differentiation In HPDLSCs Induced By AGEs And The Underneath Mechanism

Objective:The purposes of the study to examine the anti-oxidative effect of EPO on reducing AGEs-induced oxidative stress of periodontal ligament stem cell(PDLSCs)and provide a better understanding of the mechanism of these processes,to evaluate the effect of EPO on the cell cycle,apoptosis,inhibition of osteogenic differentiation in PDLSCs through p38-MAPK and Wnt/?-Catenin signaling pathway,to examine the effect of EPO on attenuation of diabetic periodontitis-associated bone loss in rats.Materials and Methods:(1)Activity of PDLSCs was identified using adipogenic and osteoplastic differentiation.The stemness of PDLSCs was characterized by the scanning of cell surface markers(CD34,CD44,CD45,CD90,CD105)through flow cytometric analysis.(2)Oxidative stress was evaluated by detecting intracellularreactive oxygen species(ROS)generation,malondialdehyde(MDA)level,and superoxide dismutase(SOD)activity.(3)Flow cytometry was used to detect the effects of AGEs intervention on cell cycle and apoptosis of PDLSCs as well as the reverse effect of EPO after 24 hours,and Westernblot detected the expressions of Bax,Bcl-2,caspase-3,caspase-9,p21,p53 and CyclinDl proteins.(4)Used GeneChip HTArray 2.0(Affymetrix,USA)chip screening which under the action of osteogenesis induced groups with pure osteogenesis induced PDLSCs has obvious expression differences of LncRNAs,and through cluster analysis,the difference of gene function analysis and the analysis of signal path analysis methods,such as preliminary discussion on which to PDLSCs osteogenesis mRNAs after induction and expression of LncRNAs spectrum difference,trying to build mRNAs with LncRNAs express network.(5)Realtime PCR and Westernblot were used to detect the expression of ALP,Runx2,Osterix and OCN after AGEs induction and EPO intervention for 7 and 14 days,alizarin red staining and quantitative analysis of the formation of mineralized nodules,as well as the expression of key proteins of p38 MAPK and Wnt/?-catenin signal pathway,including ?-catenin,CyclinD1,p-p38,p-GSK3?,p38 and GSK3?.(6)Rat models of diabetic periodontitis were established,and the expressions of RANKL-OPG,iNOS and TNF-beta in the periodontal tissues of rats were detected by hematoxylin-eosin staining and immunohistochemistry after EPO intervention.Results:(1)PDLSCs were cultured and purified by tissue block method.Osteogenesis and lipogenesis were successfully induced,and the positive expression rates of CD44,CD90 and CD 105 were all over 98%.Both CD45 and CD34 were negative(<1%).(2)After being induced by AGEs for 24 hours,ROS and MDA in periodontal stem cells increased and SOD decreased,while EPO could play a significant antioxidant role,reducing ROS and increasing SOD,significantly scavenging ability of oxygen free radicals in cells,and reducing MDA content in cells.(3)AGEs can significantly increase the number of PDLSCs cells in G1/G0 phase,reduce the number of G2/M phase cells,induce G1/G0 phase block,and inhibit the proliferation of PDLSCs cells.After the intervention of 50U/ml,100U/ml and 200U/ml EPO,the G1/G0 cycle arrest of PDLSCs was reversed,and the number of PDLSCs cells in G1/G0 phase was significantly reduced.With the increase of EPO concentration,the expression of p53 and p21 protein decreased gradually,and the expression of CyclinDl protein increased gradually,and the cell cycle tended to be normal.By changing the ratio of Bax/bcl-2 key proteins,AGEs can induce the downstream Caspase cascade effect and induce the apoptosis of PDLSCs.EPO can enhance the expression of bcl-2 protein in PDLSCs to resist the over-expression of Bax induced by AGEs,inhibit the Caspase cascade effect of bax-bcl-2 downstream,and weaken the apoptosis initiation(caspase-9)and apoptosis effect(caspase-3)signals induced by AGEs to reduce the apoptosis effect.(4)AGEs could significantly inhibit the osteogenic differentiation ability of PDLSCs,mainly showing that the expression levels of Runx2,OCN and Osterix after 14 days of osteogenic induction were significantly lower than those of the simple induction group,and alizarin red mineralized deposition staining was also significantly lower than that of the simple induction group.After the addition of 50U/ml EPO,we found that EPO could partially reverse the decline in the osteogenic differentiation ability of PDLSCs caused by AGEs,increase the expression of Runx2,OCN and Osterix osteogenic related proteins,and increase the mineralization deposition.The phosphorylation of p38 and GSK3 beta was blocked by the increase of AGEs concentration(0 micron/ml,10 micron/ml,50 micron/ml).With the increase of EPO concentration,the expression of beta-catenin and CyclinDl protein in PDLSCs was significantly increased,and the phosphorylation of p38 and GSK3 beta was increased,which played a certain protective effect on the osteogenic differentiation ability of PDLSCs.(5)Compared with normal control group in the periodontal tissue of rats,diabetes periodontitis OPG protein expression in rat periodontal tissue volume declined obviously,dyeing was significantly weakened,RANKL positive expression cells increased obviously,RANKL-OPG ratio increased significantly,iNOS was significantly positive,NO concentration increased significantly,TGF-?1 activity is restrained,osteoclast activity,bone resorption.After EPO intervention,OPG protein expression was significantly increased,staining degree was significantly enhanced,positive expression cells of RANKL were significantly reduced,staining degree was weakened,iNOS expression was significantly weakened,NO concentration was decreased,and TGF-?1 activity was increased.Conclusion:(1)EPO can antagonize the p53 pathway,remove the G0/G1 phase block of PDLSCs,significantly increase the proportion of PDLSCs in S and G2/M phases,maintain the normal cell cycle process,and promote cell proliferation.By changing the key protein ratio of Bax/bcl-2,AGEs can resist the Bax overexpression induced by AGEs,inhibit the Caspase cascade effect of bax-bcl-2 downstream,and weaken the apoptosis initiation(caspase-9)and apoptosis effect(caspase-3)signals induced by AGEs to reduce the apoptosis effect.)AGEs can induce oxidative stress injury of periodontal stem cells,mainly manifested as increased intracellular ROS and MDA,and decreased SOD.EPO can play a significant antioxidant role,partially reverses oxidative stress injury of PDLSCs induced by AGEs,and has a certain protective effect on antioxidant tissues.(2)EPO can promote the osteogenic differentiation of PDLSCs by activating Wnt/?-catenin signaling pathway,increase the expression of Osterix,OCN and Runx2,and increase the deposition of calcium salt to form mineralized nodules.EPO can partially reverse the reduced osteogenic differentiation ability of PDLSCs caused by AGEs and play a certain protective role.The main mechanism is that EPO simultaneously regulates the Wnt/P-catenin signaling pathway and p38-MAPK signaling pathway.(3)EPO can partially reverse alveolar bone absorption caused by high glucose and periodontal inflammation,and play a certain role in bone homeostasis maintenance and periodontal tissue protection.