The Effects Of Prednisolone Acetate On Rats With Radiation-induced Oral Mucositis And The Expression Of β-defensins In The Oral Epithelium

Objective : 1.To establish a model of radiation-induced oral mucositis（RTOM）in rats,to provide an animal model basis for further research.2.To observe the influence of prednisolone acetate on the expression ofβ-defensins（rBD2,rBD3）and MAPKp38,NF-κBp65,IL-1β,IL-6 and TNF-α in the oral epithelium of rats with RTOM,to further discuss possible mechanisms that prednisolone acetate may effect β-defensins.Methods:1.45 SD rats were selected and divided into 9 groups,8 groups of them were received an irradiation of 35 Gy dose of⁶⁰Coγ-rays,while the 9th group was a blank control group.The condition of the oral mucosal was observed and recorded every day,and the rats who refused to eat was gavaged with liquid food.at the 1^st,3^rd,5^th,7^th,10 th,14^th,21^st and 28^th day after the irradiation,one of experimental group was selected and the rats of this group were sacrificed.The blank control group of rats were sacrificed on the 28 th day.Tongue tissue of the dead rats were harvested and stained with hematoxylin eosin.2.Thirty SD rats with RTOM were randomly selected and divided into prednisolone acetate treatment group（PA group）and saline NS group（NS group）in the initial stage of inflammation（5 days after radiation）.0.1ml（0.1mg）prednisolone acetate injection was sprayed 3 times a day the tongue of rats in the PA group for 3 days.And 0.1ml（0.1mg）physiological saline was also sprayed 3times a day on the tongue of rats in the NS group for 3 days.Five rats of each group were sacrificed on the 1^st,4^th,and 7^th day after all the spray finished,the tongue tissue of the rats was obtained by incision.The expression of rBD2,rBD3,MAPKp38,NF-κBp65,IL-1β,IL-6 and TNF-α in epithelium were detected by Immunohistochemical technique and the average optical density was analyzed by IPP6.0 software.SPSS20.0 software was used to analyzed the data.Variance analysis of the factorial design was used to compare the differences of mean Optical Density of the detection between the PA group and NS group,and two sample T-test was used to analyzed the difference of mean Optical Density of the detection between the PA group and NS group.one-way ANOVA was used to analyze the intra-group comparison and the relationship among the expressions of detection factors was expressed by the pearson product difference correlation coefficient.Results:1、The establishment and observation of the non-invasive model of radiation-induced oral mucositis（RTOM）in rats clinically,5 to 6 days after irradiation,redness and swelling of oral mucosa along with small ulcers were observed.In the 7^th to 8^th day of after irradiation,small ulcers gradually merged into large ones which lasted to the 13 th day.From the 14 th day of radiation,the ulcers began to became smaller.They were found to be completely healed within21 st to 28 th days after irradiation.The H&E staining showed that a large number of inflammatory cells appeared in the submucosal layer after 5 days of irradiation,but the epithelial lamina was observed to be intact.Fragmentation and exfoliation of epithelial layer was observed only at the 7^thto 10 th day ofirradiation.New blood capillaries and granulation tissue could be observed at the bottom of the ulceration at the 14 th day of irradiation whereas at the 21 st and28^th day,the process of re-epithelialization was noted to have started and the basal layer cells were arranged closely.2 、the effects of prednisolone acetate on rats with radiation-induced oral mucositis and the expression of β-defensins in the oral epithelium.2.1 Effects of prednisolone acetate on RTOM:The first day after the end of the spray,large area of ulcers in the tongue of NS group was observed,In the PA group,ulcers in the tongue were also obsered,but the area was smaller than those of NS group.On the 4^th day,the tongue ulcers still lasted in the NS group meanwhile the tongue ulcers began to became smaller in the PA group.On the 7^th day,tongue ulcers in the NS group were observed to begin to became smaller while most of the ulcers in the PA group healed.2.2 rBD2:Immunohistochemical results showed that the stain of rBD2 was observered in the cytoplasm of the epithelial cells.The results of variance analysis of the factorial design indicated that the optical density value of immunohistochemical staining was significantly different between the PA group and the NS group（F = 7.392,P = 0.012）.There was a significant difference between the expression of rBD2 at different points of time within the same group（F = 7.016,P = 0.004）,and no interaction effect was found between group and time（F = 2.312,P = 0.121）.Results of two sample T-test showed that the optical density value of rBD2 in PA group at the 7^th day after all the treatment was significantly lower than that in NS group（P = 0.001）.One-way ANOVA results indicated that the optical density value of rBD2 in the NS group decreased first and then increased with time passing（P = 0.001）.2.3 rBD3:Immunohistochemical results showed that rBD3 expression isobservered in the cytoplasm of the epithelial cells.The variance analysis of the factorial design results indicated that the optical density value of immunohistochemical staining was significantly different between the PA group and the NS group（F = 49.586,P < 0.001）.There was a significant difference between the expression of rBD3 at different points of time within the same group（F = 86.777,P < 0.001）,and interaction effect exists between group and time(F = 76.⁶⁰4,P < 0.001).Results of two sample T-test showed that the optical density value of rBD3 in PA group at the 4^th and 7^th day after the end of the treatment was significantly lower than that in NS group（4d: P = 0.007,7d: P= 0.018）.One-way ANOVA results indicated that the optical density value of rBD3 decreased with time（PA group: P<0.001,NS group: P<0.001）.2.4 MAPKp38:Immunohistochemical results showed that MAPKp38 expression is observered in the cytoplasm of the epithelial cells.The variance analysis of the factorial design results indicated that the optical density value of immunohistochemical staining was significantly different between the PA group and the NS group（F = 9.081,P = 0.006）.There was a significant difference between the expression of MAPKp38 at different points of time within the same group（F = 45.062,P<0.001）,and interaction effect was found between group and time（F = 38.152,P<0.001）.Results of two sample T-test showed that the optical density value of MAPKp38 in each PA group and NS group was significantly different.The optical density value of MAPKp38 in PA group at the 1^st day was significantly higher than that in NS group（P <0.001）,while which at the 4^th and 7^th day lower than that in NS group（4d: P = 0.040,7d: P<0.001）.One-way ANOVA results indicated that in PA group and NS group,the optical density value of MAPKp38 decreased first and then increased（PA group:P<0.001,NS group: P<0.001）.2.5 NF-κBp65 : Immunohistochemical results showed that NF-κBp65expression is observered in cell nuclei and partial cytoplasmthe of the epithelial cells.The variance analysis of the factorial design results indicated that the optical density value of immunohistochemical staining was significantly different between the PA group and the NS group（F = 5.680,P = 0.025）.There was a significant difference between the expression of NF-κBp65 at different points of time within the same group（F = 138.259,P<0.001）,and interaction effect was found between group and time（F = 41.801,P<0.001）.Results of two sample T-test showed that the optical density value of NF-κBp65 in PA group at the 1^st day was significantly higher than that in NS group（P = 0.001）,while the optical density value of NF-κBp65 expression at the 4^th and 7^th day lower than that in NS group（4d: P = 0.021,7d: P<0.001）.One-way ANOVA results indicated that in the PA group,the optical density value of NF-κBp65decreased with time（P<0.001）,while the expression of NF-κBp65 in the NS group decreased first and then increased（P<0.001）.2.6 IL-1β :Immunohistochemical results showed that IL-1β expression is observered in the cytoplasm of the epithelial cells.The variance analysis of the factorial design results indicated that the optical density value of immunohistochemical staining was significantly different between the PA group and the NS group（F = 5.014,P = 0.035）.There was a significant difference between the expression of IL-1β at different points of time within the same group（F = 149.023,P<0.001）,and interaction effect exists between group and time（F = 5.251,P = 0.013）.Results of two sample T-test showed that the optical density value of IL-1β in PA group at the 7^th day expressed significantly lower than that in NS group（P<0.001）.One-way ANOVA results indicated that the optical density value of IL-1β decreased with time（PA group: P<0.001,NSgroup: P<0.001）.2.7 IL-6:Immunohistochemical results showed that IL-6 expression is observered in the cytoplasm of the epithelial cells.The variance analysis of the factorial design results indicated that the optical density value of immunohistochemical staining of IL-6 was significantly different between the PA group and the NS group（F = 21.821,P<0.001）.There was no significant difference between the expression of IL-6 at different points of time within the same group（F = 1.655,P = 0.212）,and interaction effect exists between group and time（F = 8.831,P = 0.001）.Results of two sample T-test showed that the optical density value of IL-6 in PA group at the 4^th and 7^th day expressed significantly lower than that in NS group（4d: P = 0.009,7d: P < 0.001）.One-way ANOVA results indicated that the optical density value of IL-6 in the PA group decreased with time（P = 0.002）.2.8 TNF-α:Immunohistochemical results showed that TNF-α expression is observered in the cytoplasm of the epithelial cells.The variance analysis of the factorial design results indicated that there was no significant difference between the optical density value of immunohistochemical staining of TNF-α in PA group and the NS group（F = 0.989,P = 0.330）.There was significant difference between the expression of TNF-α at different points of time within the same group（F = 4.322,P = 0.025）,and no interaction effect exists between group and time（F = 1.401,P = 0.266）.Results of two sample T-test showed that the optical density value of TNF-α in each PA group and NS group showed no significant difference.One-way ANOVA results indicated that the optical density value of TNF-α in the NS group decreased with time（P<0.001）.3 、 The relationship between β defensins（rBD2,rBD3）and its related signaling pathways（MAPKp38,NF-κBp65）and inflammatory cytokines（IL-1β,IL-6,TNF-α）The expression of rBD2 was positively correlated with that of MAPKp38（r= 0.554,P = 0.002）,NF-κBp65（r = 0.508,P = 0.004）,IL-1β（r = 0.552,P =0.002）and IL-6（r = 0.427,P = 0.018）,while no significant correlation was observed between rBD2 and TNF-α（r = 0.278,P = 0.137）.The expression of rBD3 was positively correlated with that of NF-κBp65（r = 0.383,P = 0.037）,IL-1β（r = 0.538,P = 0.002）,IL-6（r = 0.503,P = 0.005）,and MAPKp38（r =0.554,P = 0.002）,while no significant correlation was observed between rBD3 and TNF-α（r = 0.140,P = 0.462）.Conclusions: The model of radiation-induced oral mucositis in rats was established by irradiation with 35 Gy ⁶⁰Coγ-rays.In this model,prednisolone acetate can decrease the expression of rBD2,rBD3 and MAPKp38,NF-κBp65,IL-1β and IL-6.The inhibitory effect of prednisolone acetate on rBD2 and rBD3 may be related to MAPKp38 and NF-κBp65.