Effects Of National Herbal Medicine Dicliptera Chinensis Polysaccharides On Post-irradiation Growth Activity And Proliferative Repair Of Rat Submandibular Gland Cells

Objective To investigate the proliferation and repair of rat submandibular gland?SMG?cells exposure to dicliptera chinensis polysaccharides?DCPs?after irradiation conditions,and to investigate whether DCPs can enhance the ability of repairing radioactive DSBs and provide experimental basis for the development of new radiation drugs.Method Dicliptera chinensis polysaccharides were obtained with water-extraction and alcohol precipitation method.Albumen was eliminated with proteinase hydrolization method.Pigments were decolored by H₂O₂.The total sugar content of DCPs was determined by phenol-sulfate acid method.Homogeneous polysaccharide was further purified from crude polysaccharides by DEAE-cellulose?OH-?ion-exchange chromatography.The primary rat SMG cells were obtained by mechanical enzyme digestion.⁶⁰Co?-rays were used to irradiate SMG cells 0,5,8 and 10 Gy to establish a radiation injury model and to intervene with different concentrations of DCPs.SMG Cells were randomly divided into five groups:blank control group?BC group?,radiation injury group?8Gy exposure dose,IR group?,low-dose polysaccharide intervention group?concentration 40?g/ml+8Gy exposure dose,DCPs-L group?,mid-dose polysaccharide intervention group?concentration 60?g/ml+8Gy exposure dose,DCPs-M group?,and high-dose polysaccharide intervention group?concentration 80?g/ml+8Gy exposure dose,DCPs-H group?.The CCK-8method was used to detect the inhibition rate of SMG cells in each group.The rate of apoptosis and cell cycle were detected by flow cytometry.The expression of MRN complex?Mre11/Rad50/Nbs1?was detected by Western blot.Results In this experiment,The polysaccharide content of the DCPs extracted was 90.5%.Most of the normal SMG cells cultured in vitro are polygonal or triangular,and a few are long shuttle-shaped.A small number of fibroblast-like spindle cells are scattered around the tissue block.After passage every 7-10 days,the first generation of SMG cells have good morphology and strong proliferation ability.It could be passaged to the third generation of cells.The viability of the third generation of cells decreased significantly,and the number of apoptotic cells increased.More floating cell debris can be observed under the microscope.CCK-8 results showed that after irradiation,the survival rate of SMG cells decreased with the increase of irradiation dose,with the increase of time after the irradiation,it first decreased and then gradually recovered.DCPs can increase the proliferation of SMG cells after irradiation,and the cell growth activity is positively correlated with the concentration of polysaccharides.Flow cytometry analysis of SMG cell apoptosis showed that DCPs could significantly reduce the total apoptosis rate after irradiation,especially the early apoptosis rate.Cell cycle results showed that DCPs can effectively reduce the number of cells in S?G2 phase after irradiation and alleviate S?G2 block.Western Blot results showed that the expression of Mre11,Rad50,and Nbs1 in the radiation-injured group was decreased,and the expression of Mre11,Rad50,and Nbs1 in the different concentrations of the polysaccharide was increased as the drug concentration increased.Conclusion Ionizing radiation causes SMG cell double-strand DNA breaks,S?G2 arrest,and inhibition of cell growth.The anti-radiation effect of DCPs is exerted by reducing the proportion of SMG apoptosis after irradiation and alleviating the S and G2 arrest of SMG cells.DCPs can also up-regulate the expression of three subunits of the MRN complex,thereby promoting DSBs repair function to reduce the damage caused by ionizing radiation.DCPs has a preliminary function of promoting repair of cell damage caused by ionizing radiation.It is of great value to study the dicliptera chinensis polysaccharides as a research target and discover its anti-radiation medicine.