Experimental Study About Total Saponins Of Paris Polyphylla Var.yunnanensis On Salivary Adenoid Cystic Carcinoma ACC-83 Cell Proliferation And Mechanism

Objectives:To investigate the proliferation inhibition and mechanism of total saponins of Paris polyphylla var.Yunnanensis on salivary adenoid cystic carcinoma ACC-83 cell,in order to provide a theoretical basis for the treatment of salivary adenoid cystic carcinoma using Paris polyphylla var.Yunnanensis.Methods:1 MTT assay was used to evaluate the cell growth activity and drug screening,respectively,from the leaves of the plant root,extraction and purification and root,fibrous root extract obtained,select the most valuable medicinal extract and the appropriate cell plate density.2 In vitro,cell culture was performed.the proliferation of ACC-83 cells who were treated with different concentrations(5、10、20、40、60、80、100 μg·mL-1)of total saponins of Paris polyphylla var.Yunnanensis was observed using CCK-8 assay,and Calculate the IC50.3 It was divided into four groups:the low,the middle,the high concentration group and the negative control group,respectively,the experimental group after 24 hours after drug action,the apoptosis of ACC-83 cells was observed using flow cytometry,and the Annexin V-FITC/PI marker.4 The expression of macrophage migration inhibitory factor MIF and CD74 protein was detected using Western blot.5 The expression of macrophage migration inhibitory factor MIF and CD74 mRNA was detected by RT-PCR.6 Immunocytochemistry assay to detect the expression of MIF,CD74 and Ki-67 in ACC-83 cells.7 Statistical analysis was carried out with SPSS22.0.Results:1 The cell morphology were observed by inverted microscope showed that ACC-83 cells grew well,uniform distribution,the shape of polygon and vary in size.The cell growth curve showed that the cell growth activity was better when the cell density was 3～4×104/ml.the effects of four kinds of drugs had different degrees of inhibition of cell growth,physical refining of Paris root was more sensitive to ACC-83 cells,the inhibition rate was 97.34%,the extract refined post experiment design will all use of Paris root.2 CCK8 assay showed that,the different concentration of the total saponins of Paris polyphylla var.Yunnanensis can inhibit the growth of ACC-83 cells in different degree,and there is a dose-effect relationship,comparison between each drug concentration group,with significant difference(P<0.05),calculate the IC50 value of 37.956ug/ml.3 With Annexin V-FITC/PI labeling and analysis by flow cytometry,showed that each experimental group were different levels of apoptosis,The apoptosis rates of low,middle and high concentration groups were 3.29%±1.18%,60.23%±4.26%and 81.51%±5.11%,the apoptosis rate compared between the experimental group and negative control group(2.60+0.569)%,there was significant difference between the middle and high drug concentration group apoptosis rate(P<0.05),but the low concentration group had no significant difference.4 The expression of MIF and CD74 protein was detected by Western blot show that the experimental group of the expression quantity of MIF and CD74 gray value decreased,compared with the negative control group,high concentration group MIF gray value decreased significantly,there were significant differences(P<0.05),in the middle and high drug concentration group CD74 gray value decreased significantly,there are differences significant(P<0.05).5 RT-PCR detection of MIF,CD74 mRNA relative expression amount was found:The relative expression level of MIF mRNA in the high concentration group was 0.45±0.09,Compared with the control group,the expression was decreased,the difference was significant(P<0.05).The relative expression level of CD74 mRNA in the high concentration group was 0.56±0.008,Compared with the control group,the expression was decreased,the difference was significant(P<0.05).There was no significant difference in the other groups.6 The expression of MIF and CD74 in salivary adenoid cystic carcinoma cell line ACC-83,MIF and CD74 was detected by immunohistochemistry,It was mainly localized in cytoplasm,There was also a small amount of nuclear expression.Conclusions:Total saponins of Paris polyphylla var.Yunnanensis has obviously inhibitory effect on cell proliferation of ACC-83 cells,inhibition of the MIF and the CD74 expression and,promotion of apoptosis.