Evaluation Of The Therapeutic Effect Of Recombinant Human Amelogenin On Periodontitis In SD Rats Exploration Of Its Anti-inflammatory Mechanism

Objective:Amelogenin(Am),as the main component of enamel matrix protein,has been proved to promote periodontal regeneration.Porcine amelogenin has been developed abroad as a drug for periodontal regeneration and has been put on the market.However,the extraction of porcine amelogenin is difficult and expensive,so the use of biotechnology to obtain recombinant amelogenin has become a new research trend in recent years.Our group has successfully constructed a prokaryotic expression system of human full-length amelogenin in the early stage,but the purification effect is not ideal,and its effect has not been evaluated in animal experiments.This topic uses the constructed human full-length amelogenin engineering bacteria for fermentation and expression,and optimizes the purification method to obtain recombinant human amelogenin with higher purity.The SD rat periodontitis model was constructed,and the recombinant human amelogenin was tested in vivo to verify its effect on the treatment of periodontitis at the animal level.Secondly,using LPS(lipopolysaccharide)to stimulate the construction of RAW264.7 cells and THP-1 cell inflammation models,at the cellular level,to explore whether recombinant human amelogenin has the effect of inhibiting inflammation,and preliminary exploration of its mechanism of action.Lay the foundation for the subsequent exploration of the mechanism of recombined amelogenin to promote periodontal regeneration.Methods and Results:1.The recombinant amelogenin expressed by bl21-pet-28a-am was purified by Ni column affinity chromatography and acetic acid method respectively,but the purification effect of the former was not ideal.Further optimization of acetic acid method showed that when the concentration of acetic acid was 0.5%,the treatment conditions were the whole bacteria broken,the temperature was 80 ℃ and the treatment time was 20 min,the purification effect was the best,and the purity of recombinant human amelogenin was more than 90%.2.1.0 ligation wire was used to ligate the tooth neck of SD rats,and the periodontitis model feed(diet2000)was used to successfully construct the rat periodontitis model.At the eighth week of modeling,the levels of TNF-α in serum were significantly lower than those in negative control group after administration of low concentration(0.315 mg / kg)and high concentration(3.15 mg / kg),which was also verified by immunohistochemistry of posterior periodontal tissue.The m RNA levels of IL-1 β and IL-6 in periodontal tissue also decreased after administration.Micro CT analysis showed that the alveolar bone loss of the treatment group was also lower than that of the control group,and the degree of inflammation of periodontal tissue of the treatment group was lower than that of the control group by HE staining.3.RAW264.7 cells and THP-1 cells were stimulated with 100 ng / ml and 200 ng/ ml LPS respectively to construct cellular inflammatory models.Two kinds of inflammatory cell models were pretreated with recombinant human amelogenin.Fluorescence microscopy showed that amelogenin could reduce the expression of no and ROS,and down regulate the expression of COX-2 and i NOS,the key proteins of ROS expression.RT q PCR showed that recombinant human amelogenin could down regulate the m RNA levels of IL-1 β,IL-6 and TNF-α.The above results indicate that recombinant human amelogenin has certain anti-inflammatory ability at the cellular level.Western blot showed that the recombinant human amelogenin could down regulate the expression of p-ikk α / β / IKK α,p-ik-α / IK-α,p-p65 / p65 in NF-κ B signaling pathway.The laser confocal experiment showed that the recombinant human amelogenin could inhibit p65 entry into the nucleus,indicating that the mechanism of inhibiting inflammation by recombinant human amelogenin may be achieved by inhibiting NF-κ B signaling pathway.Conclusion:In this study,the purification scheme of recombinant human amelogenin was optimized,and the protein with higher purity and concentration was obtained.Followup animal experiments show that recombinant human amelogenin has a better ability to promote periodontal regeneration and reduce the loss of alveolar bone.It is proved at the cellular level that recombinant human amelogenin has anti-inflammatory effects and can inhibit the NF-κB signaling pathway.It laid a good foundation for the recombinant human amelogenin to be used as a periodontitis treatment drug development.