PI3K/AKT/mTOR Pathway Regulates The LPS-mediated Autophagy In High Glucose Status Of Mouse Insulinoma βtc6 Cell Line

Objective:There are few reports on the impact of periodontitis on diabetes.Recent studies have shown that autophagy plays an important role in the development of diabetes.The purpose of this study was to explore whether the periodontitis dominant bacteria P.gingivalis-derived LPS could silence the autophagy classic signaling pathway phosphatidylinositol 3-kinase/protein kinase B/rapamycin target protein(PI3K/AKT/m TOR)induces autophagy of islet β cell and promotes the development of diabetes,thereby elucidating the molecular mechanism of periodontitis promoting the development of diabetes.Methods: Mouse insulinoma βtc6 cell were cultured in vitro and stimulated with glucose and LPS at different concentrations alone or in combination for 12 h.Set up the Control group,LPS group,glucose group(Glu group)and LPS+Glu group.In addition,the autophagy inhibitor 3MA(5μmo/L)and the autophagy activator rapamycin(10mmol/L)(Rapa)were added to the PI3K/AKT/m TOR signaling pathway to intervene,including the Control group and three activation groups(Rapa+LPS group,Rapa+Glu group,Rapa+LPS+Glu group)and three inhibition groups(3MA+LPS group,3MA+Glu group,3MA+LPS+Glu group).1.(1)CCK-8 was used to detect the effects of different stimuli on the proliferation of mouse insulinoma βtc6 cell;(2)ELISA kit was used to detect insulin concentration in the supernatant.2.(1)Annexin V-FITC/PI combined with flow cytometry was usd to detect the intracellular reactive oxygen accumulation in mouse insulinoma βtc6 cell in each group;(2)Transmission electron microscope was usd to observe the number of autophagosomes and morphology of mitochondria;(3)RT-PCR and WB methods were used to detect the expressions of Becline1,p62,LC3II/LC3Ⅰ,PI3 K,p-PI3 K,AKT,p-AKT,m TOR and p-m TOR in mouse insulinoma βtc6 cell.Results: 1.(1)The ELISA test results showed that compared with the Control group,insulin secretion increased when glucose concentrations were 1.38mmol/L,5.50mmol/L,and 11.00mmol/L(P<0.05).When the glucose concentration was further increased to50mmol/L,the insulin secretion was similar to that in the Control group,and there was no statistically significant(P>0.05);(2)CCK-8 showed that high concentration of glucose inhibited the proliferation of mouse insulinoma βtc6 cell,glucose combined with LPS stimulation significantly inhibited the proliferation of mouse insulinoma βtc6 cell.2.(1)The intracellular ROS accumulation in the Glu group,LPS group,and Glu+LPS group was increased compared to the Control group,and the Glu+LPS group was increased compared to the LPS group and the Glu group(P<0.05).(2)TEM observation showed that compared with the control group,the mitochondrial structure of the Glu group,the LPS group and the Glu+LPS group was more severely destroyed,and the number of autophagosomes increased.(3)Compared with the Control group,the expression of autophagy-related genes LC3 II and Becline1 increased in the Glu group,LPS group,and Glu+LPS group(P<0.05),but the expression trend of p62 was opposite(P<0.05).3.(1)Compared with the Control group,the expression of Becline1 protein in the3MA+Glu group,3MA+LPS group,and 3MA+Glu+LPS group was decreased compared with the Control group(P<0.05).Compared with 3MA+ Glu group and 3MA+LPS group,the expression of Becline1 protein in the 3MA+Glu+LPS group was decreased(P<0.05);The expression of p62 protein showed the opposite trend(P<0.05);Compared with the Control group,the p-PI3K/PI3 K ratio decreased in the 3MA+Glu group,3MA+LPS group,and 3MA+LPS+Glu group(P<0.05).Compared with the3MA+Glu+LPS group,the p-PI3K/PI3 K ratios of the 3MA+Glu group and the 3MA+LPS group increased(P<0.05);Compared with the Control group,the p-AKT/AKT ratio in the Rapa+Glu group,Rapa+LPS group,and Rapa+Glu+LPS group was decreased(P<0.05).The p-m TOR/m TOR ratio trend was consistent with the p-PI3K/PI3 K ratio(P<0.05).(2)In the autophagy activation group,the expression of Becline1 protein was increased in the Rapa+Glu group,Rapa+LPS group,Rapa+Glu+LPS group,and Rapa+Glu+LPS group compared with the Control group.Compared with the Rapa+Glu+LPS group,the expression of Becline1 protein in the Rapa+Glu group and the Rapa+LPS group was decreased(P<0.05);The trend of p62 protein expression was opposite to Becline1(P<0.05).The trend of p-PI3K/PI3 K and p-m TOR/m TOR ratios was opposite to those of the autophagy inhibition group(P<0.05).Conclusion:1.Different microenvironments could regulate insulin secretion ability of mouse insulinoma βtc6 cell;2.LPS could promote ROS aggregation and induce excessive autophagy of mouse insulinoma βtc6 cell in high glucose state;3.The PI3K/AKT/m TOR signaling pathway couold regulate LPS-induced excessive autophagy of mouse insulinoma βtc6 cell in high glucose state and inhibit its insulin secretion function.