Inhibitory Effect And Mechanism Of Rhizoma Reed Polysaccharide On Proliferation Of Non-small Cell Lung Cancer A549 Cells By Inducing Autophagy And Apoptosis

OBJECTS:To establish an in vitro and in vivo activity screening model of non-small cell lung cancer(NSCLC)cell line A549,and to explore the effect and mechanism of Rhizoma Reed Polysaccharide on the proliferation of A549 cells.METHODS:Rhizoma Reed Polysaccharide,we used in this research,was prepared in the preliminary experiment.The effects of Rhizoma Reed Polysaccharideon on the proliferation of A549 cells were investigated by A549cells model in vitro and A549 cells transplantation tumor model in vivo.(1)We established a screening model for the activity of NSCLC in vitro by culturing A549 in vitro.CCK-8 cell activity kit was used to detect the effect of400?g/mL,200?g/mL,100?g/mL,50?g/mL,25?g/mL Rhizoma Reed Polysaccharide and 4?mol/mL of Sorafenib on the activity of A549 cells after24,48 and 72 hours of treatment.Flow cytometry was used to detect the effect of 100?g/mL Rhizoma Reed Polysaccharide on the apoptosis of A549 cells after 48 hours.qRT-PCCR was used to detect the effect of 100?g/mL Rhizoma Reed Polysaccharide on the mRNA expression of Bax,Bcl-2,Caspase-3.The expression of LC-3,AKT,p-AKT,PI3K,p-PI3K,Bax,Bcl-2 and Caspase-3 in A549 cells was detected after 100?g/mL Rhizoma Reed Polysaccharide treatemt 48 hours.(2)The effect of 100 mg/mL Rhizoma Reed Polysaccharide on the growth of tumors in nude mice was observed by subaxillary injection of A549 cells.We used immune-histochemistry to observe the expression of apoptotic proteins Bax,Bcl-2 and Caspase-3 in tumour tissues of A549 cells.RESULTS:Rhizoma Reed Polysaccharide 100?g/mL inhibited the proliferation of A549 cells more than 30%after treatment with 48h and 72h,and the other doses also inhibited the proliferation of A549 cells.The results showed that Rhizoma Reed Polysaccharide 100?g/mL inhibited the proliferation of A549 cells significantly.Compared with the control group,the effects of 100?g/mL Rhizoma Reed Polysaccharide and 4?mol/mL Sorafenib on A549 cells significantly increased the proportion of early apoptosis(Q2quadrant)and late apoptosis(Q3 quadrant),and the apoptosis rate(Q2 and Q3quadrant sum)significantly increased(P<0.05),the flow cytometry result demonstrated suggesting that Rhizoma Reed Polysaccharide could promote A549 cells apoptosis.The qRT-PCR results showed that the expression of apoptotic proteins Bax and Casepes-3 mRNA increased significantly(P<0.05)and the expression of Bcl-2 decreased significantly(P<0.05)in Rhizoma Reed Polysaccharide group.The protein expression of AKT and PI3K were detected after the treatment of 100?g/mL Rhizoma Reed Polysaccharide and 4?mol/mL Sorafenib with 48 hours.Compared with the control group,the LC-3 II/I,p-AKT/AKT,p-PI3K/PI3K ratio of the Rhizoma Reed Polysaccharide group and the Sorafenib group increased significantly(P<0.05);Bax and Casepes-3mRNA increased significantly(P<0.05)and the expression of Bcl-2 decreased significantly(P<0.05)(2)Tumor mass began to grow in the axilla of nude mice7 days after subaxillary injection with A549 cells.On the 13th day,the volume of tumors in 18 mice reached 90 mm~3.The success rate of the model was 90%.Then the mice were divided into three groups according to the volume of tumors in mice.The mice were given 100 mg/kg Rhizoma Reed Polysaccharide and 2 mg/kg Cisplatin.On the 18th day,compared with the model group,the inhibition rates of tumors in the Cisplatin group and the polysaccharide group were 39%and 36%respectively,and the Rhizoma Reed Polysaccharide could significantly inhibit the growth of A549 cells in the tumor-bearing mice.Immuno-histochemical results showed that the expression of apoptotic proteins Bax and Casepes-3 in the polysaccharide and Cisplatin group increased significantly(P<0.05)and the expression of Bcl-2 protein decreased significantly(P<0.05).These results suggest that the Rhizoma Reed Polysaccharide could promote the apoptosis of A549 cell transplanted tumor model mice.CONCLUSION:In this study,the A549 cells were intervened by the Rhizoma Reed Polysaccharide,and then the apoptotic and autophagy-related proteins were detected by flow cytometry and Western blot.The results showed that Rhizoma Reed Polysaccharide could inhibit the proliferation of non-cellular lung cancer A549 cells by activating PI3K-AKT signaling pathway to promote autophagy of A549 cells,upregulating Bcl-2 and Caspase-3 proteins to promote apoptosis of A549 cells.It indicates that Rhizoma Reed Polysaccharidecan inhibit proliferation of A549 cells by activating autophagy and promoting apoptosis,suggesting that Rhizoma Reed Polysaccharidemay could be a potential drug for the treatment of non-small cell lung cancer.