Effects Of 1, 25-dihydroxyvitamin D3 On The Function Of Neutrophils Derived From Type 2 Diabetes With Periodontitis

Objective: The aim of this research was to explore the effects of1,25-dihydroxyvitamin D3 on the function of neutrophils derived from type 2 diabetes with periodontitis, include chemotaxis, apoptosis and other functions. In order to provide the theoretical and experimental foundation for the development and treatment of type 2 diabetes with periodontitis.Methods:1. Forty-four patients of type 2 diabetes with periodontitis were selected as the study subjects, in strict accordance with the inclusion criteria. The experiments were divided into 1,25-dihydroxyvitamin D3 non intervention group and1,25-dihydroxyvitamin D3 intervention group, according to the different concentrations of 1,25-dihydroxyvitamin D3 were divided into the following three groups:10-6、10-8、10-10 mol/L intervention group.2. Neutrophils were isolated by density gradient centrifugation from type 2diabetes with periodontitis peripheral blood. The purity of neutrophil was identificated by Wright-Giesma staining and the cell viability was tested by trypan blue exclusion.3. Different concentrations of 1,25-dihydroxyvitamin D3 intervention neutrophils for 24 hours, then the effect of neutrophil chemotaxis was assayed by transwell system; the apoptosis rate of PMN and ROS generation were detected by flow cytometry(FCM); concentrations of tumor necrosis factor-α 、 interleukin-1β 、interleukin-6 and neutrophil elastase in supernatants were tested by enzyme-linked immunosorbent assay(ELISA).Results:1. We successfully obtained neutrophils derived from type 2 diabetes with periodontitis peripheral blood. The purity of neutrophil was up to 96 % or more and the cell viability reached 98 %.2. Different concentrations of 1,25-dihydroxyvitamin D3 effect on neutrophilsfor 24 hours, Then, using fMLP-induced neutrophils chemotaxis for 2 hours.Compared to 1,25(OH)2D3 non intervention group, 10-6 、 10-8 mol/L intervention group PMN chemotaxis function were increased significantly(P<0.05). However,there were no significant differences between 1,25(OH)2D3 non intervention group and 10-10 mol/L intervention group(P>0.05). And 10-6mol/L、10-8 mol/L、10-10 mol/L intervention group multiple comparison discovered that 10-6 mol/L intervention group>10-8 mol/L intervention group>10-10mol/L intervention group. There were significant differences(P<0.05).3. Different concentrations of 1,25-dihydroxyvitamin D3 intervention neutrophils for 24 hours, flow cytometry analysis showed that 1,25(OH)2D3 intervention group neutrophil apoptosis rate increased significantly(P<0.05). 1,25(OH)2D3 intervention group multiple comparison discovered that 10-8 mol/L intervention group>10-6 mol/L intervention group>10-10mol/L intervention group. There were significant differences(P<0.05). Compared to 1,25(OH)2D3 non intervention group, the 1,25(OH)2D3intervention group ROS generation decreased, except for 10-6 mol/L 1,25(OH)2D3intervention group. There were significant differences(P<0.05).4. ELISA results showed that 1,25(OH)2D3 intervention group tumor necrosis factor-α、interleukin-1β、interleukin-6 content were depressed(P<0.05). 1,25(OH)2D3intervention group multiple comparison discovered that 10-8 mol/L intervention group<10-6 mol/L intervention group, 10-8 mol/L intervention group<10-10mol/L intervention group. There were significant differences(P<0.05). However, there were no significant differences between 10-6mol/L intervention group and 10-10mol/L intervention group(P>0.05).Conclusion:The above experimental results showed that the certain concentration of1,25-dihydroxyvitamin D3 on type 2 diabetes with periodontitis peripheral blood neutrophil chemotaxis, apoptosis function had a certain impact. Finally, we hypothesized that 1,25-dihydroxyvitamin D3 as an immunomodulator, played an immune regulatory role in the occurrence and development of type 2 diabetes with periodontitis, but concrete mechanism research are still to be further clarification.