Effect Of Astragalus Polysaccharide Combined With Paclitaxel On Proliferation And Autophagy Of Gastric Cancer BGC823 Cells And Related Mechanisms

Gastric cancer（GC）accounts for 6%of global cancers and is currently threatening the health of people worldwide.Paclitaxel（PTX）is a well-known first-line chemotherapeutic anticancer drug,but its long-term application has certain drug resistance and toxic side effects.Therefore,it is necessary to find adjuvant chemotherapeutic drugs with definite efficacy and reduced tumor resistance and drug toxic side effects.Astragalus polysaccharide（APS）has various effects such as anti-apoptotic,anti-inflammatory and anti-tumor,and can significantly improve the effect of chemotherapy drugs and reduce toxicity.However,whether APS can enhance the anti-gastric cancer effect of PTX and its molecular mechanism have not been elucidated.In this paper,we selected gastric cancer BGC823cell line as the target of our study,and investigated the effects of APS and PTX combination on the activity,migration,invasion and apoptosis levels of BGC823 cells and the relationship with cell autophagy,and conducted a preliminary study on the mechanism of synergistic effect of APS in combination with PTX.Firstly,the effect of different concentrations of APS,PTX and the combination of drugs on the viability of BGC823 cells was detected by MTT method,and the inhibitory effect of the drugs on cell viability was analyzed;the CI value was calculated based on the inhibition rate by using Compu Syn software,and then the interrelationship between the two drugs was studied based on the CI value and the optimal combination concentration was sought;the effects of APS,PTX and the combination of drugs on cell migration and invasion ability were detected by scratch and Transwell cell staining assays.The effects of APS,PTX and the combination of drugs on cell migration and invasion ability were detected by scratch and Transwell cell assays,respectively;the changes of apoptosis level by APS,PTX and the combination of drugs were observed by Hoechst staining;the morphological changes of each group of cells were observed by scanning electron microscopy（SEM）;the relative expression of LC3 protein was detected by immunofluorescence method;The expression of intracellular proteins related to autophagy and apoptosis was detected by Western blot.The results showed that the cell proliferation was inhibited by APS and PTX,and the rate of cell inhibition increased with increasing dose,and the inhibitory effect of the combined administration of APS and PTX was more significant than that of the drug alone（P<0.05）,APS group(10 mg·m L^-1),PTX group(10μg·m L^-1)and combined group(APS 10 mg·m L^-1+PTX 10μg·m L^-1)according to CI values.The scratch results showed that APS,PTX and the combination drug slowed down the healing of the cell scratch gap and significantly inhibited the migration of cells（P<0.05,P<0.01）.Transwell analysis showed that the number of cells in the experimental groups was significantly lower,indicating that APS,PTX and their combination groups inhibited the invasion of cells,but the effect of the combination group was more significant（P<0.01）.The Hoechst results showed that the nuclear concentration increased significantly and the bright blue fluorescence signal increased gradually after the action of APS,PTX and the combined group on gastric cancer BGC823 cells,indicating that apoptosis occurred in the cells,and the effect of apoptosis was more significant in the combined group（P<0.05,P<0.01）.The cell surface microvilli were abundant and the cell morphology was intact in the control group under scanning electron microscopy,while the cell surface morphology and structure were damaged in each drug group,and the cell morphology was more obviously changed in the combined group.Western blot experiments revealed that the expression of Bax,a marker protein of pro-apoptosis,increased and the level of Bcl-2,a marker protein of anti-apoptosis,decreased after drug treatment compared with the control group,while the level of Beclin-1,a protein related to autophagy,increased.The levels of Becline-1,a protein related to autophagy,were upregulated,the level of p62 was decreased,and the ratio of LC3Ⅱto LC3Ⅰwas increased,and the changes in the expression levels of each protein were more significant in the combined group（P<0.05,P<0.01）.In summary,APS,PTX and the combination can effectively reduce gastric cancer cell viability,migration and invasiveness,promote apoptosis and induce autophagy.the potential mechanism by which APS combined with PTX can exert synergistic effects may be through attenuating the expression of Bcl-2 and p62 proteins and upregulating the protein expression levels of Bax,Becline-1 and LC3Ⅱ/LC3Ⅰ.