The Effect Of Dark Plum And Licorice Mixture On Apoptosis Of Rat Submandibular Gland Cells With Radiation-induced Damage

Object:Radiation xerostomia occurs in patients with head and neck tumors due to salivary gland injury after radiotherapy.Radiation xerostomia affects the diet,conversation and even causes local oral infection and pain,these problems hinder the rehabilitation and reduce the quality of life of patients.Clinical studies have found that using dark plum and licorice spray(main ingredients:dark plum and licorice)can promote saliva secretion and improve xerostomia.Basic research found that Dark plum and Licorice Mixture can reduce the apoptosis rate of rat submandibular gland cells with radiation-induced damage.One of the main components of dark plum is citric acid,which is often used as acid stimulation to stimulate saliva secretion.In this study,we established model(primary SD rat.Submandibular gland(SMG)cell with radiation-induced damage),and compared the effects of Dark plum and Licorice Mixture and citric acid on submandibular gland cells with radiation-induced damage,so as to provide basis for clinical application of Dark plum and Licorice Mixture.Methods:To establish an in vitro culture system of SD rat submandibular gland cells and observe cell characteristics.MTT assay was used to detect the proliferation activity of different.concentrations of citric acid on submandibular gland cells with radiation-induced damage,and the optimum concentration of citric acid was determined.Flow cytometry was used to detect the effect of citric acid on apoptosis rate of injured model cells.MTT assay was used to detect the toxicity of Dark plum and Licorice Mixture and citric acid on submandibular gland cells.The effect of Dark plum and Licorice Mixture and citric acid on the morphology of submanndibular gland cells after radiation-induced damage was observed and cmpared under microscope.Western-blot technique was used to detect the expression levels of apoptosis proteins bax and bcl-2,and the effects of Dark plum and Licorice Mixture and citric acid on apoptosis of submandibular gland cells in radiation-induced damage rats were compared and explored.Results:1.Under a phase contrast inverted microscope,it was observed that the newly obtained and inoculated submandibular gland cells were round and spherical in suspension state,with clear and smooth outline and annular halo.They were dispersed in the culture solution.After 24 hours of inoculation,the cells slowly adhered to the wall and grew,and then the inclusion was expanded.Flat polygons were visible.The number of cells increased and fused into pieces,forming "paving stones" and "goose soft stones".2.The survival rate of submandibular gland cells changed after 4 different concentrations of citric acid were used to interfere with the radiation-induced damage model cells,among which the survival rate of submandibular gland cells in 10ug/mL citric acid group was significantly improved(P<0.05).3.When 10ug/mL citric acid was used to interfere with radiation-induced damage submandibular gland cells,the apoptosis rate did not change significantly(P>0.05).4.Ebony licorice group(50ug/mL,the best concentration obtained from previous studies)and citric acid group(10ug/mL)had no significant effect on the survival rate of normal submandibular gland cells(P>0.05).5.Morphological observation after 24 hours of modeling showed that the cells in normal group were c.losely arranged,with flat polygon and clear boundary,in the shape of "paving stone" and "goose soft stone".In the model group,the cells contracted,the boundary was unclear,the dead cells increased,the cell spacing increased,and the density decreased.Compared with the model group,the number of cells in Dark plum and Licorice Mixture group and citric acid group increased,the cell spacing decreased and the density increased.6.Compared with the normal group,the expression of Bax/Bcl-2 protein in the model group increased significantly(P<0.05).Compared with the model group,Bax/Bcl-2 protein expression in 50ug/mL Dark plum and Licorice Mixture group was significantly reduced(P<0.05).Compare d with the model group,the expression of Baxi/Bcl-2 protein in lOug/mL citric acid group did not change significantly(P>0.05).Conclusion:1.Dark plum and Licorice Mixture may inhibit apoptosis of radiation-induced damage submandibular gland cells by adjusting Bax/Bc1-2 balance.2.Current research shows that 10ug/mL citric acid can promote prolifer ation of r at submandibular gland cells with radiation-induced damage.3.10ug/mL citric acid has no effect on the apoptosis rate of radiation-induced damage cells,and no effect on the apoptosis rate may be due to no effect on the expr ession of apoptosis protein Bax/Bc1-2.