| Objective:The aim of this study is to study the impact of hIAPP on the development of diabetic periodontitis,as well as to investigate the underlying mechanisms behind the destruction of local periodontal tissue in patients with type 2 diabetes.This research endeavors to offer novel insights into the early detection and treatment of diabetic periodontitis.Method:1.Mouse gingival fibroblasts were obtained from the primary culture of the gingival tissue.Mouse gingival fibroblasts were divided into six groups:Control(5.5 mM Dglucose),rIAPP(5.5 mM D-glucose+10 μM rIAPP),hIAPP(5.5 mM D-glucose+10 μM hIAPP),High glucose(40 mM D-glucose),High glucose+rIAPP(40 mM D-glucose+10μM rIAPP),and High glucose+hIAPP(40 mM D-glucose+10 μM hIAPP).The proliferation and apoptosis of gingival fibroblasts in each group were measured by CCk-8 assay to study the effects of high glucose and hIAPP intervention on gingival fibroblasts,and RT-qPCR and immunofluorescence staining were served to investigate the potential molecular mechanisms involved in this process.2.C57BL/6 mice(n=36)were randomly assigned to two groups:the T2DM group,which was subjected to a high-fat and high-glucose diet for 6 weeks followed by intraperitoneal injection of Streptozotocin in dose of 30 mg/kg for 5 days,and the normoglycemic group.All mice with fasting blood glucose level higher than 11.1mmol/L was marked as successfully constructed mice model for further studies.Mice were divided into normoglycemia-saline,normoglycemia-hIAPP,normoglycemia-rIAPP,T2DM-saline,T2DM-hIAPP,and T2DM-rIAPP group according to the type of reagents injected into the gingiva of mice.Hematoxylin-eosin staining and methylene blue staining were used to observe the changes of the pathomorphology of periodontal tissue and alveolar bone absorption.Additionally,RT-qPCR was served to identify changes in the mRNA levels of NLRP3,Caspase-1,and IL-1β,in ordr to investigate the underlying mechanism of hyperglycemia and hIAPP intervention in the progression of diabetic periodontitis.Results:1.Gingival fibroblasts were successfully isolated and cultured by tissue explant,which was easy to operate.2.Both high glucose and hIAPP could inhibited the proliferation activity of gingival fibroblasts,and the 40 mM-hIAPP Group had the most inhibition capability on cell proliferation.3.The intervention of high glucose and hIAPP resulted in an increase in the mRNA expression levels of NLRP3 and caspase-1 in mouse gingival fibroblasts when compared to the normal control group.The highest expression levels of NLRP3 and Caspase-1 were observed in the 40 mM-hIAPP intervention groupwith around 2.57 folds and 1.99 folds,respectively,higher than that in the control,and there were significant differences in the expression levels among the groups(P<0.05).Additionally,the intervention of high glucose and hIAPP led to an increase in the average mRNA expression levels of IL-1β in mouse gingival fibroblasts when compared to the normal control group.The highest expression levels of IL-1β were observed in the 40 mM-hIAPP intervention group with around 8.09 folds more than control,and there were significant differences in the expression levels among the groups,except the 5.5 mM-hIAPP group(P<0.05).4.The intervention of high glucose and hIAPP was found to cause a notable increase in the protein expression levels of NLRP3 and Caspase-1 in mouse gingival fibroblasts,when compared to the normal control group.In particular,the 40 mM-hIAPP intervention group exhibited the highest expression levels,with significant differences observed among the groups(P<0.05).5.The combination effects of high-fat diet and STZ contributed to the induction of type 2 diabetes.6.Hyperglycemia and deposition of hIAPP was found in significant changes in the histological morphology of periodontal tissues in mice,including redness and swelling of the gingiva,disorganization of periodontal ligament fibers,root migration of the junctional epithelium,and bone resorption at the alveolar crest.In comparison to normoglycemic mice,the degree of periodontal tissue destruction was more pronounced than that in the T2DM-hIAPP intervention group.This is characterized by dark red gingiva,disrupted arrangement of the periodontal ligament,noticeable root migration of the junctional epithelium,and inflammatory cell infiltration in the epithelial lamina propria.Additionally,there was a decrease in the alveolar crest and full exposure of the cervical region.7.The intervention of high glucose and hIAPP resulted in a higher expression of caspase-1 at the mRNA level in the gingival tissues of mice compared to the normal control group.Under the intervention of high glucose and hIAPP,the highest expression of caspase-1 was detected in T2DM-hIAPP group with around 66.77 folds more than control.An elevated level of NLRP3 and IL-1β mRNA were detected as well under the intervention of high glucose and hIAPP in the gingival tissues of mice compared to the normal control group,and the expression of NLRP3 and IL-1β was the highest in the T2DM-hIAPP intervention group,338.05 folds and 33 folds over control,respectively,there were significant differences in expression among the groups,except for the normoglycemia-hIAPP group(P<0.05).Conclusions:1.The proliferative activity of gingival fibroblasts was impeded by both a high glucose environment and hIAPP.The inhibitory effect of these factors was more pronounced when they were simultaneously introduced.2.The present of hyperglycemia and hIAPP deposition could result in histological alterations in periodontal tissues,leading to an increase in alveolar bone resorption in mice.The extent of periodontal tissue destruction was more pronounced in mice subjected to hIAPP and high glucose treatment,and the combined effect of hIAPP and high glucose was synergistic.3.The expression of NLRP3 inflammasome in mouse gingival fibroblasts and periodontal tissues could be triggered by hIAPP and high glucose intervention,which in turn activated the NLRP3-Caspase-1-IL-1β pathway,ultimately resulting in the onset of periodontitis. |
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