Effects Of Calcitriol On Autophagy And Inflammation Of Gingival Epithelial Cells In Rats

Objective:The aim of this study was to investigate the effects of calcitriol（1,25-dihydroxyvitamin D₃,1,25（OH）₂D₃,hereinafter referred to as 1,25D）on autophagy and inflammation of gingival epithelial cells in rats with periodontitis.And we want to further understand the role of autophagy in the regulation of inflammation and infection in rat periodontal tissues at 1,25D.Methods:Nine 8-week-old SD male rats weighing 220（±20 g）were selected.Nine rats were randomly divided into three groups（blank solvent group,low concentration group1,25D and high concentration group 1,25D）.Three rats in each group were fed in cages.The non-periodontitis side was defined as N side and the periodontitis side was P side.The periodontal tissues of rats were divided into A-N,A-P,B-N,B-P,C-N and C-P.Nine rats were anesthetized by intraperitoneal injection of 2%sodium pentobarbital（45mg/kg）.The gingival and root surfaces of the left upper and lower first molars of rats were separated,and then the neck of the teeth was ligated with orthodontic ligation wire.The steel wire is knotted on the buccal side and pushed as far as possible into the gingival sulcus.P.gingivalis was inoculated orally once every other day,five times from 1 week after ligation.The ligation wire was removed after 4 weeks.The solvent was prepared by propylene glycol:water:ethanol=60:30:10.Rats in groups A,B and C were injected with 0.2 ml of blank solvent,0.2 ml of solvent dissolved 0.2μg/kg（1,25D/rat weight）and 0.2 ml of solvent dissolved 2μg/kg（1,25D/rat weight）daily for one week.The rats were executed after administration.Bilateral upper and mandibular bones were cut and separated quickly,and fixed after rinsing with saline solution.We need to observe the following indicators:（1）gross specimen and HE staining to observe the inflammation of tissue;（2）immunohistochemical staining to detect the expression of BECN1,ATG7 and LL-37;（3）immunofluorescence to detect the expression of LC3;（4）immunohistochemical staining to detect the expression of IL-1β,IL-6.（5）methyl thymol blue（MTB）colorimetric method to determine serum calcium concentration in rats.Results:（1）In group A,B and C,the expression of A-P,B-P,C-P was higher than that of ATG7,BECN1 and LC3,LL-37 in group A-N,B-N and C-N,and the difference was statistically significant.（2）After 1,25D,the expression of ATG7 in gingival epithelial cells in B-P group and C-P group was higher than that in A-P group（P=0.004,P=0.0001）,and that in C-P group was higher than that in B-P group（P=0.003）;that in B-N group and C-N group was higher than that in A-N group（P=0.0001,P=0.001）,but there was no significant difference between C-N group and B-N group（P=0.074）.The expression of BECN1 in gingival epithelial cells in B-P group and C-P group was higher than that in A-P group（P=0.005,P=0.001）,but there was no significant difference between C-P group and B-P group（P=0.233）;the expression of BECN1 in B-N group and C-N group was higher than that in A-N group（P=0.0001,P=0.0001）,but there was no significant difference between C-N group and B-N group（P=0.051）.The expression of LC3 in gingival epithelial cells in B-P group and C-P group was higher than that in A-P group（P=0.003,P=0.0001）,and that in C-P group was higher than that in B-P group（P=0.006）;that in B-N group and C-N group was higher than that in A-N group（P=0.001,P=0.0001）,and that in C-N group was higher than that in B-N group（P=0.001）.The expression of LL-37 in B-P group and C-P group was higher than that in A-P group（P=0.0001,P=0.0001）,and that in C-P group was higher than that in B-P group（P=0.009）;that in B-N group and C-N group was higher than that in A-N group（P=0.003,P=0.0001）,and that in C-N group was higher than that in B-N group（P=0.018）.（3）Expression of IL-1beta and IL-6 in gingival epithelial cells A-P>B-P>C-P>A-N.（4）Serum calcium concentration increased by 0.16 mmol/L（P=0.012）in group B and 0.14 mmol/L（P=0.018）in group C,but there was no significant difference between group B and group C（P=0.286）.Conclusion:1.Experimental periodontitis promotes autophagy of rat gingival epithelial cells.2.1,25D promotes autophagy of rat gingival epithelial cells.3.1,25D can alleviate the production of pro-inflammatory cytokines in rat gingival epithelial cells to some extent,and the inhibitory effect of 1,25D on pro-inflammatory cytokines is concentration-dependent.