Anti-Inflammatory Effect And Osteogenesis Of Extracts From Blaps Rynchopetera Fairmaire Experimental Periodontitis In Rats

ObjectivesBy establishing the rat experimental periodontitis model and applying the oral membrane of the Blaps rynchopetera Fairmaire,we observed the inhibition of inflammation and the regeneration of alveolar bone.This study provided a research basis for searching the new drugs which can treat periodontitis and promote periodontal tissue regeneration.Materials and Methods1.The oral membranes used in the experiment(matrix film and drug film containing the extract component of Blaps rynchopetera Fairmaire)were fabricated.2.Fifty-three adult female Sprague-Dawley rats(220 g-250 g)were randomly divided into seven groups,which were Negative Control group(non-ligation),a vehicle group,a Positive Control group(Minocycline Hydrochloride Ointment,MHO),and an En AI-L((low dose of active site I),En AI-H(high dose of active site I),En AII-L(low dose of active site II),En AII-H(high dose of active site II).The 2-0 ligature was placed around the left maxillary second molar on the 47 female SD rats.The rat periodontitis model was established two weeks after the operation,and the administration is started.The experimental rats were sacrificed two weeks after the administration,and the bilateral maxilla were collected(to ensure that three molars were intact,and the gingival tissue was retained).The bone tissue was preserved in Formalin,the gingival tissue was stored at low temperature in liquid nitrogen.3.Morphological measurements(linear distances between the Cemento-enamel junction(CEJ)to the Alveolar Bone Crest(ABC))were performed using a stereomicroscope.A total of 18 sites in the medial,central and distal of the palatal and buccal sides of the three molars were selected for measurement and statistical analysis.4.q RT-PCR was used to detect the m RNA levels of inflammation-related genes IL-6,IL-1β,MMP-2,MMP-9 and osteogenic related genes ALP,OPG,RANKL.5.Micro-CT scanning: the image of the Maxillary bone sample was scanned,and the two-dimensional linear analysis was performed.The root length(RLs,mm)between CEJ and apical end of the palatal root was measured.The two-dimensional linear data were analyzed to evaluate and calculate the percentage of sagittal residual bone mass.The ROI of Total volume(TV),Trabecular Bone Volume(BV),Trabecular Bone Surface(BS)and Bone Mineral Density(BMD)were obtained after 3d Volume analysis using Mimics17.0 software.Bone Volume Fraction(BVF),Trabecular Number(Tb.N),Trabecular Thickness(Tb.Th),and Trabecular Separation(Tb.Sp)were calculated by the formula.Results1.Preparation of buccal film for Blaps rynchopetera Fairmaire: After screening by orthogonal experiment,1.5gof polyvinyl alcohol,0.5 g of sodium carboxyl methyl cellulose-Na and 6 g of glycerin was be incorporated in the buccal film.2.Pre-experiment of rat periodontitis model: statistical analysis results showed that the CEJ-ABC distance of experimental periodontitis rats in the model group was significantly increased two weeks after the model establishment(P<0.01),so the experimental periodontitis model in rats of two weeks after the model establishment was selected as the experimental model.3.Morphological measurement: The distance between the cementoenamel junction to the alveolar bone crest was detected by stereotype.The Vehicle group was shown to present a higher CEJ-ABC distance than in the MHO group and En A group(P< 0.01).4.q RT-PCR:IL-6 results showed that when compared with Vehicle group,MHO,En AI-L and En AI-H groups were significantly reduced(P< 0.05).Results of IL-1β showed that compared with Vehicle group,MHO,En AII-L and En AII-H groups were significantly reduced(P< 0.01).Results of mmp-2 showed that compared with Vehicle group,MHO group and four drug delivery groups were significantly reduced(P< 0.01).Results of mmp-9 showed that compared with Vehicle group,MHO group and four drug delivery groups were significantly reduced(P< 0.01).There was no difference in m RNA expression levels of the four genes related to inflammation between the MHO group and the high-low dose En AI group and the high-low dose En AII group.The results of ALP showed that when compared with Vehicle group,the MHO,En AII-L and En AII-H groups all showed an increase(P< 0.01),while when compared with MHO group,En AI-L,En AI-H and En AII-L all showed a decrease(P< 0.01).OPG expression results showed that compared with Vehicle group,En AI-H and En AII-H groups showed a decrease(P< 0.05),while compared with MHO group,the results showed no significant difference.Results of RANKL showed that compared with Vehicle group,MHO,En AI-L and En AII-H groups all showed a decrease(P< 0.01),while compared with MHO group,En AI-H and En AII-L showed an increase(P< 0.01).5.Micro-CT scanning: Two-dimensional linear analysis results: the percentage of residual bone in the sagittal plane showed that the residual bone in the alveolar bone of the MHO group and the administration groups was greater than that of the matrix group(P<0.01),and there was no difference between the MHO and the administration group.Results of three-dimensional volume analysis: BMD results showed that compared with the Vehicle,the MHO,En AI-H,En AII-L and En AII-H groups all showed a decrease,in which the En AI-H and En AII-H groups showed a significant decrease(P< 0.01),while there was no difference between the MHO group and the administration group.Bone volume fraction(BVF)results showed that,compared with the Vehicle group,the positive drug and the four administration groups showed increased(P< 0.01),while there was no difference between the MHO group and the administration group.The number of bone trabeculae(Tb.N)showed that there was no difference between the MHO group and the administration group,and no difference between the MHO group and Vehicle group.Bone trabecular thickness(Tb.Th)showed increased in the En AII-H group compared with Vehicle group(P< 0.05)and increased in the En AII-L and En AII-H groups compared with the positive group(P< 0.01).Trabecular bone separation degree(Tb.Sp)results showed that,compared with the Vehicle group,En AII-L and En AII-H groups showed a decrease(P< 0.01),and compared with the MHO group,the En AI-L and En AI-H groups showed an increase(P< 0.01).Conclusion1.Thebuccalfilm agent containing Blaps rynchopetera Fairmaire was prepared by quality evaluation of preparation and prescription screening,which can preliminarily meet the application of pharmacological and pharmacodynamic experiments in experimental rats.2.The Blaps rynchopetera Fairmaire has a certain anti-inflammatory effect on the periodontal tissues of the rat periodontitis model and has certain osteogenic effects on the periodontal bone defect.