Study On The Role Of Wnt Pathway In The Strain Aggravating Periodontal Tissue Destruction

Objectives: To culture the human periodontal ligament fibroblasts(h PDLFs)in vitro,and we analyzed the expression of m RNA and protein of the canonical Wnt pathway molecule β-catenin,non-canonical Wnt pathway molecules Wnt5 a,the receptor tyrosine kinase-like orphan receptor(Ror2),and the key regulators of bone formation—Runt-related transcription factor 2(Runx2)of h PDLFs under endotoxin of Porphyromonas gingivalis(P.g LPS)and the uniaxial cyclic tensile strain(2500u,0.5Hz),We’d like to make a preliminary exploration on the possible role of Wnt pathway in the mechanism of bone resorption in the synergistic effect of inflammation and strain,and to provide a theoretical basis for further exploration of the occlusal trauma aggravating the inflammatory periodontal tissue destruction.Methods: 1.Approved by the Ethics Committee of the Stomatological Hospital of Tianjin Medical University(TMUh MEC2018104),we chosen the 12-16 year-old healthy bicuspids that needed to be removed for orthodontics in the Department of Maxillofacial Surgery,and we cultured h PDLFs by enzymatic digestion.The cells was identified by immunohistochemistry.2.RT-PCR was used to detect the expression of β-catenin,Runx2,Wnt5 a and Ror2 m RNA in h PDLFs and inflammatory h PDLFs under mechanical microenvironment,and we observed when the peak or trough of m RNA expression of the target molecule occurs,that is,the optimal intervention duration;RT-PCR and Western blot were used to detect the expression of β-catenin,Runx2,Wnt5 a and Ror2 in P.g LPS group(group P),uniaxial cyclic tensile strain group(group S)and synergistic effect group(group P+S)under the optimal intervention duration.Results: 1.The immunohistochemistry showed that the cells cultured in vitro were stained with anti-vimentin staining(+)and anti-keratin staining(-),proving to be PDLFs.2.RT-PCR showed at the three time points selected in this experiment,that is from 0.5h to 2h to 4h,the expression of β-catenin,Runx2,Wnt5 a and Ror2 m RNA had a fold line change over time both in the strain group and the P.g LPS+strain group,and there is a peak or grain value at 2h,that is the optimal intervention duration.The results of RT-PCR and Western blot showed that when stretched for 2h,the expression of h PDLFs β-catenin in group P increased(PCR:P<0.05;WB:P>0.05),Runx2 decreased(P<0.05),and Wnt5 a,Ror2 increased(P<0.05)compared with the group C;in group S,the expression of β-catenin,Runx2,Wnt5 a and Ror2 increased(P<0.05);in group P+S,the expression of β-catenin and Runx2 decreased(P<0.05),as well as Wnt5a(PCR:P<0.05;WB:P>0.05),yet Ror2 increased(P<0.05).Compared with group P,the expression of β-catenin,Runx2 and Wnt5 a in group P+S decreased(P<0.05),yet Ror2 increased(P<0.05).Conclusion: 1.We selected healthy bicuspids,and cultured h PDLFs by enzyme digestion.A large number of experimental cells with high purity are obtained in a short time.The cells were metabolic,responded well to interventions,and satisfies the need of the experiment.The aseptic operations、strict selection、proper scraping and the control of the digestion time are the key points to culture the h PDLFs.2.In h PDLFs treated with or without P.g LPS,the expression of Wnt pathway-related molecules including β-catenin,Runx2,Wnt5 a and Ror2 m RNA all reflected a fold change over time indicating that in the two micro-environments,Wnt pathways may have a certain role in the reaction to strain.The tensile strain activated Wnt/β-catenin pathway and Wnt5a/Ror2 pathway,and P.g LPS also activated the Wnt5a/Ror2 pathway,however the change of Runx2 were opposite,indicating the activation of Wnt pathway in different microenvironments may cause the opposite effect.When the two synergistically,Ror2-related non-canonical Wnt pathways was activated,while the canonical Wnt pathway may be inhibited,and Runx2 expression was the lowest,which may be related to the fact that mechanical strain aggravated bone destruction of inflammatory periodontal tissues.It provided a theoretical basis for occlusal trauma aggravating bone resorption in patients with uncontrolled periodontitis.