| Objective:To investigate the antivirus mechanism of the Astragalus injection and the water decoction of Astragalus,the Raw264.7 cells were infected with the influenza virus(H1N1).The effects of the Astragalus injection and the water decoction of Astragalus on the the cell proliferation cycle,SOD activity and MDA level were discussed,as well as the expressions of protein of the vital genes in the TLR3 mediated signal pathway were detected by analytical technology in cellular biology and molecular biology.The related immune factors included TLR3,TAK1,TBK1,IRF3 and IFN-?.Methods:MTT method was used to investigate the cytotoxicity on Raw264.7 cells about the Astragalus injection and the water decoction of Astragalus and to calculate the concentration of the medicine in antivirus mechanism study;Flow cytometry was used to detect the effects of the Astragalus injection and the water decoction of Astragalus on the cell cycle of Raw264.7 which was infected with the influenza virus(H1N1);To observe the effects of the Astragalus injection and the water decoction of Astragalus on the SDO and MDA of influenza virus infected cells by measuring the content of SOD and MDA;The western blot were applied to study the effects of the Astragalus injection and the water decoction of Astragalus on the expressions of protein of TLR3,TAB1,TBK1,IRF3 and IFN-p,when Raw264.7 cells were infected with H1N1.Results:MTT results showed that the Astragalus injection and the water decoction of Astragalus about different concentrations had different effects on cell proliferation.When the cells were treated 24 h with medicine,the Astragalus injection began to show inhibitory effect on cell proliferation at the concentration of 400 mg/ml,and the inhibition rate of 400 mg/ml and 800 mg/ml concentrations was 2.58% and 47.43%,respectively.The water decoction of Astragalus showed the inhibitory effect on cell proliferation at the concentration of 50 mg/ml,and the inhibition rates of 50,100,200 and 400 mg/ml were 2.70%,17.20%,29.69% and79.57%,respectively.When the cells were treated 48 h with medicine,the inhibition rates of25,50,100,200,400 and 800 mg/ml of the Astragalus injection were 3.90%,15.90%,18.2%,19.20%,21.03% and 69.98%;The water decoction of Astragalus showed the inhibitory effect on cell proliferation at the concentration of 50mg/ml,and the inhibition rates of 50,100,200 and 400 mg/ml were 0.84%,10.67%,49.95% and 88.61%,respectively.When the cells were treated 72 h with medicine,the inhibition rates of 25,50,100,200,400 and 800 mg/ml of the Astragalus injection were 3.19%,19.88%,27.68%,34.92%,69.77% and 82.40% respectively;The inhibition rates of 12.5,25,50,100,200 and 400 mg/ml of the water decoction of Astragalus were 38.57%,58.92%,69.60%,66.60%,84.61% and 96.34%,respectively.The corresponding ICO,IC10 and IC25 of the medcine were calculated by SPSS Statistics 20 statistical software.The ICO of the Astragalus injection on Raw264.7 was 9.5 mg/ml,IC10 was 40 mg/ml,IC25 was 92 mg/ml;the ICO of the water decoction of Astragalus on Raw264.7 was 0.2 mg/ml,IC10 was 1.6 mg/ml,IC25 was 5 mg/ml;9.5 mg/ml concentration of Astragalus injection and 0.2 mg/ml concentration of the water decoction of Astragalus showed significant antiviral activity.Flow cytometry showed that influenza virus(H1N1)infection will affect the cell cycle of Raw264.7 cells,preventing cells transform to S phase and accumulating cells in G1 phase;compared with the virus control group,the Astragalus injection and the water decoction of Astragalus reduced the percentage of cells in G1 phase and increased the percentage of cells in S phase.The results of SOD and MDA showed that influenza virus(H1N1)could reduce the SOD activity and increase the lipid peroxide content of MDA in Raw264.7 cells;After the intervention,the Astragalus injection and the water decoction of Astragalus(0.2 mg/ml)can increase the SOD activity and decrease the content of MDA.Western Blot results showed that influenza virus(H1N1)could up-regulated the expressions of TLR3,TAK1,TBK1,IRF3 and IFN-? in Raw264.7 cells.After the medicine intervention,the Astragalus injection could down-regulated the expressions of TLR3,TAK1,TBK1,IRF3 and IFN-? at the concentration 9.5 mg/mL;the Astragalus injection could down-regulated the expressions of TLR3 and up-regulated the expressions of IFN-? at the concentration 40 mg/mL;the Astragalus injection could down-regulated the expressions of TLR3,TAK1,TBK1,IRF3 and up-regulated the expressions of IFN-? at the concentration 92mg/mL;the water decoction of Astragalus could down-regulated the expressions of TAK1,IFN-? and up-regulated the expressions of TLR3,TBK1,IRF3 at the concentration 0.2mg/ml;the water decoction of Astragalus could up-regulated the expressions of TLR3,TBK1,IRF3,IFN-? and down-regulated the expressions of TAK1 at the concentration 1.6 mg/ml;the water decoction of Astragalus could up-regulated the expressions of TLR3,TAK1,TBK1,IRF3 and IFN-? at the concentration 5 mg/ml.Conclusions:The Astragalus injection and the water decoction of Astragalus showed obvious anti influenza virus activity,they could improve the survival rate of Raw264.7 cells which were infected with influenza virus(H1N1).The antivirus mechanism of the medicine was to regulate the cell proliferation cycle and the content of SOD,MDA of cells which were infected with influenza virus.At the same time,the immune system was affected by regulating the expression of TLR3,TAK1,TBK1,IRF3 and IFN-? in the TLR3 mediated signaling pathway,thus exerting its antiviral effect in vitro. |
PDF Full Text Request