| Objective Diabetes is easy to cause multiple organ diseases in the body.Periodontitis is one of the complications.The more severe the inflammation of periodontitis,the more unfavorable it is for glycemic control.In this study,the SD rat model was established to explore the effect of periodontitis on pancreatic autophagy in type 2 diabetic rats,and regulation of classical autophagy signaling pathway PI3K/Akt/m TOR by autophagy inhibitor 3-MA and autophagy activator rapamycin(Rapa),Whether periodontitis induces excessive autophagy of islet cells in type 2 diabetic rats through the PI3K/Akt/m TOR signaling pathway,It lays a new theoretical and experimental basis for the clinical treatment of diabetes with periodontitis.Methods 1.100 SPF 6-week-old SD male rats were randomly divided into four groups.The experimental groups were as follows:(1)control group(C group,10);(2)diabetes model group(DM group,40);(3)periodontitis group(CP group,10);(4)periodontitis with diabetes model group(DM+P group,40);C group,CP group were fed with normal feed,DM group and DM+P group were fed with high fat and high sugar fed for 6 weeks,1%streptozocm(STZ)35 mg/kg was intraperitoneally injected,and the fasting blood glucose≥11.1 mmol/L was used as the model standard for type 2 diabetic rats.After the type 2diabetes model was established,the periodontitis mode was induced.The CP group and the DM+P group were ligated with the silk of the bilateral maxillary first and second molar teeth for 8 weeks.During the experiment,the fasting blood glucose and body weight of rats in each group were monitored regularly.At the 14 th weekend,10 rats in each group were anesthetized and sacrificed.(1)The pathological changes of maxillary alveolar bone and pancreas were observed by HE staining;(2)The alveolar bone resorption was observed by micro-CT;(3)ELISA was used to detect the expression of serum TNF-α,IL-1β,IL-6;(4)RT-q PCR was used to detect the expression of insulin secretion-related genes in pancreatic tissues: glucose stimulation(GCK),uncoupling protein-2(UCP-2),glucose transporter-2(GLUT-2)and autophagy-related genes LC3 II,Beclin1;(5)Western Blot detection of autophagy-related proteins LC3II/LC3 I,Beclin1 protein expression.2.At the end of the 14 th week,after the establishment of the periodontitis model,the ligation thread was removed in the DM+P group,and the PI3K/Akt/m TOR signaling pathway was regulated by the autophagy activator Rapa and the autophagy inhibitor 3-MA respectively in the DM group and the DM+P group.The experimental groups were as follows:(1)DM group(2)DM+3-MA group(3)DM+Rapa group(4)DM+P group(5)DM+P+3-MA group(6)DM+P+Rapa group(n=10 per group),DM group and DM+P group were not treated,DM+3-MA group and DM+P+3-MA group were intraperitoneally injected with 1.5mg /(kg · d)3-MA;DM+Rapa group and DM+P+Rapa group were intraperitoneally injected 1.0mg /(kg·d)rapamycin,after 4 weeks of continuous injection,the rats in each group were anesthetized and sacrificed,and the pancreatic tissues were taken for related detection.The expression of PI3K/Akt/m TOR signaling pathway-related proteins and autophagy-related proteins were detected by Western Blot.The expression of insulin secretion-related genes in pancreatic tissue was detected by RT-q PCR.Results(1)Body weight change: 6 weeks after high-fat and high-sugar feeding,there was no significant difference in body weight between the CP group and the C group(P > 0.05),and the weight of the DM group and the DM+P group were increased(P > 0.05);At the14 th week,after the periodontitis model was established,compared with the C group,the body weight of the CP group showed a downward trend(P>0.05),and the weight of the DM group and the DM+P group were decreased,and the decrease of the DM+P group was more significant(P < 0.05).(2)Fasting blood glucose changes: One week after STZ injection,the fasting blood glucose of DM group and DM+P group were higher than that of C group(P <0.05);At the 14 th week,the periodontitis model was successful,and compared with the C group,there was no significant difference in the CP group(P>0.05),The fasting blood glucose in the DM group and the DM+P group was significantly higher(P<0.05),and the DM+P group was higher than the DM group(P<0.05).(3)Micro-CT showed that compared with group C,alveolar bone resorption was more obvious in the CP group and DM+P group,root bifurcation was exposed,and DM+P group was the most serious.This indicates that the animal model of periodontitis has been successfully established.(4)HE staining of the maxillary alveolar bone showed that compared with the C group,the periodontal pockets of the CP group and the DM+P group were formed,the collagen fibers were destroyed,the epithelium migrated to the roots,and the alveolar bone was absorbed.The DM+P group was the most obvious.Further verification of the establishment of an animal model of periodontitis was successful.(5)HE staining results showed that compared with group C,pancreatic tissue of DM group and DM+P group showed different degrees of vacuolar degeneration,loose islet cells,inflammatory cell infiltration,and islet volume decreased,and DM+P the pathological changes in the group were more obvious.(6)ELISA results showed that compared with group C,the expression levels of IL-6,TNF-α and IL-1β in DM group,CP group and DM+P group were increased(P <0.05);compared with DM group,the expression of inflammatory cytokines in DM+P group and CP group were increased(P <0.05),and the DM+P group was higher than that in CP group(P <0.05).(7)RT-qPCR results showed that compared with group C,the relative expression of GCK and GLUT-2 m RNA in CP group was decreased(P>0.05),and the relative expression of DM+P group and DM group were decreased(P<0.05).The DM+P group was lower than the DM group(P<0.05).Compared with group C,the expression of UCP-2,Beclin1,and LC3 II in CP group had no significant difference(P > 0.05).The expression of genes in DM+P group and DM group were increased(P < 0.05).And DM+P group was higher than the DM group(P < 0.05).(8)Western Blot results showed that compared with group C,the autophagy protein Beclin1,LC3II/LC3 I in CP group was increased(P>0.05),the expression in DM group and DM+P group were increased(P<0.05),and the expression of DM+P group was higher than the DM group(P<0.05)Regulation of PI3K/Akt/m TOR signaling pathway:(1)Western Blot results showed that compared with DM group and DM+P group,PI3K/Akt/m TOR pathway related protein PI3 K,p-Akt/Akt,p-m TOR/m TOR and autophagic protein Beclin1,LC3II/LC3 I expression were lower in DM+3-MA group and DM+P+3-MA group(P<0.05),The expression of pathway protein in DM+P+3-MA group was lower than that in DM+3-MA group(P<0.05),but the expression of autophagy protein was higher(P<0.05).Compared with DM group and DM+P group,the expression of PI3 K,p-Akt/Akt and autophagic protein Beclin1,LC3II/LC3 I in DM+Rapa group and DM+P+Rapa group were increased(P<0.05),while the expression of p-m TOR/m TOR was decreased(P<0.05);the expression of pathway protein in DM+P+Rapa group was lower than that in DM+Rapa group(P<0.05),but the expression of autophagy protein was increased(P<0.05).(2)RT-q PCR results showed that compared with DM+3-MA group and DM+Rapa group,the expression of insulin secretion related genes GCK,GLUT-2 were decreased(P<0.05),and UCP-2 was increased(P<0.05)in DM+P+3-MA group and DM+P+Rapa group.Conclusion1.Periodontitis may promote the increase of serum inflammatory cytokines in IL-6,TNF-α,and IL-1β in type 2 diabetic rats.2.Periodontitis may aggravate the function of insulin secretion in islet cells of type 2diabetic rats.3.Periodontitis may promote excessive autophagy of islet cells in type 2 diabetic rats.4.Periodontitis may inhibit the excessive autophagy of islet cells in type 2 diabetic rats by inhibiting the activity of PI3K/Akt/m TOR signaling pathway and accelerate the destruction of insulin secretion from islet cells of type 2 diabetic rats. |
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