| Objective:The bidirectional relationship between chronic periodontitis(CP)and diabetes mellitus(DM)has been extensively studied.CP is a risk factor for vascular complications of DM.At present,the effect and mechanism of periodontal pathogen infection on diabetic-related vascular complications have not been clarified.Endothelial-mesenchymal transition(End MT)is closely related to the occurrence of diabetic vascular disease.In this study,we constructed a DM rat model with CP to observe the effects of periodontal infection on the carotid arteries of diabetic rats,and preliminary explored the role of End MT in CP and DM vascular lesions.Providing experimental basis for the prevention and treatment of vascular diseases in clinical diabetes patients with periodontitis.Methods:We randomized 24 SD rats into four groups: control,CP,DM,and DM + CP groups.DM rat model was established by high fat and high sugar diet combined with streptozotocin(STZ)intraperitoneal injection.Fasting blood glucose(FBG)and haemoglobin(Hb A1c)were monitored during modeling.The model of periodontitis was established by periodontal ligation combined with Porphyromonas gingivalis suspension.After 18 weeks,maxilla samples and common carotid artery and its bifurcation were collected.Micro-CT analysis,HE staining and Masson staining were performed to detect alveolar bone resorption.Detect the pathological changes of carotid artery by HE staining.The changes of End MT related markers were detected by immunofluorescence staining.Human umbilical vein endothelial cells(HUVECs)were stimulated by glucose(HG)and Porphyromonas gingivalis lipopolysaccharide(PG.-LPS)in vitro.The levels of inflammatory factors(IL-6,IL-1β and TNF-α)in the supernatant of HUVECs after different stimulation were compared by ELISA.The m RNA and protein levels of End MT related molecules were detected by real-time PCR and Western Blot.Results:1.After model establishment,DM group and DM+CP group showed symptoms of polydipsia,polyphagia,polyuria and weight loss,FBG≥ 7.8mmol /L,serum HbA1c content was higher than control group(P < 0.001).DM rat model was established.HE staining,Masson staining and micro-CT analysis results showed that: The resorption of alveolar bone in CP group and DM+CP group was the most serious in the DM+CP group.The distance between the cementoenamel junction and alveolar bone crest(CEJ-ABC)of the second molars in DM+CP group was increased(2.1107 ± 0.0086 mm)compared with that in the control group(P < 0.0001).CP model was established.2.HE staining results of carotid arteries of rats in each group showed that the intima of vessels in CP group was relatively intact,and the elastic fibers in the vascular wall were disorder.In DM group,carotid intima continuity was interrupted,and pathological changes were found in some medial smooth muscle cells.The vascular wall lesions in DM+CP group were the most serious,and elastic fibers were broken or even partially disappeared.Immunofluorescence staining showed that interleukin-6(IL-6)and vascular cell adhesion molecule-1(VCAM-1)expressions were increased in DM+CP group compared with the control group(P < 0.0001).3.Compared with control,DM and CP group,the expression of endothelial cell marker platelet-endothelial cell adhesion molecule(PECAM-1/CD31)was decreased(P<0.001)and mesenchymal marker α-smooth muscle actin(α-SMA)was up-regulated(P<0.001)in DM+CP group.The expression levels of End MT related transcription factors twist-related protein(TWIST)and zinc finger protein SNAI2(SLUG)were higher in DM+CP group(P<0.001).4.In vitro experiment results showed that Pg.-LPS or HG stimulation could increase the secretion of IL-6,IL-1β and TNF-α of HUVECs,and the difference was statistically significant(P < 0.05).Realtime PCR and Western Blot results showed that the expressions of CD31 and vascular endothelial cadherin(VE-cadherin)were significantly decreased after Pg.-LPS and HG combined stimulation,while the expressions of α-SMA and fibroblast specific protein 1 were increased(P < 0.05).Conclusion:Periodontitis exacerbates endothelial dysfunctions partly via End MT in STZinduced diabetes rats.Hyperglycemic inflammatory microenvironment can promote the phenotype change of HUVECs. |
