| Objective:To investigate the anti-viral effect and mechanisms of yingeshuangjieji (YGJ) oninfluenza A virus (H1N1)[IAV (H1N1)] in vitro and vivo.Methods:This experiment, YGJ as an experimental drug, ribavirin as a positive controldrug, researched on the anti-IAV (H1N1) effect of drug in vitro and vivo, tocomprehensive assess the anti-IAV (H1N1) effect and mechanisms of YGJ. Specificmethods were as follows:1The concentration of1g/ml YGJ liquid was condensed by taking appropriateprocess, depending on various ingredients and physicochemical properties of herbal.2Experimental study on anti-IAV (H1N1) of YGJ in vitro:Madin-Darby canine kidney (MDCK) cells as study object in vitro.2.1Hemagglutinin titer of IAV (H1N1) was detected by hemagglutination test. Themedian tissue culture infective dosage（TCID50） of suited hemagglutinin titer inMDCK cells was detected by Cytopathic Effect (CPE).2.2The survival rate of the cells of YGJ and Ribavirin on MDCK cells weredetected by using MTT．The maximum non-toxic concentration (TC0) and mediantoxic concentration (TC50) of YGJ and Ribavirin were calculated with Probitregression．2.3The impact of YGJ on IAV (H1N1) induced MDCK cell apoptosis were investigated to assess the anti-virus activities of YGJ. Cell apoptosis of YGJ (fourconcentration groups:50mg/ml,25mg/ml,12.5mg/ml,6.25mg/ml) and Ribavirin(four concentration groups:10mg/ml,5mg/ml,2.5mg/ml,1.25mg/ml) were detectedusing flow cytometry by double-labeling with Annexin V and PI．3Experimental study on anti-IAV (H1N1) of YGJ in vivo:90Kunming mice were randomly divided into six groups with15mice in eachgroup. There were normal control group, virus control group, group with ribavirin,group with low-dose YGJ, group with high-dose YGJ, group with preventive YGJtreatment．Normal control group excluded, all mice in the other groups were infectedwith intranasal IAV (H1N1) FM1strain to establish the experimental animal modelsof influenza virus pneumonia.3.1The general condition (such as weight, activity, food-intake, etc.) of the mice andthe pathological changes of the lung tissue were observed. The impact of YGJ onIAV (H1N1)-induced pneumonia was quantitative assessed by measuring the lungindex and Inhibition rate of the mice.3.2NK cell activities in the spleens of mice were detected by MTT assay. Theregulation of YGJ on the immune function of mice infected with IAV (H1N1) wasassessed to investigate the mechanisms on the anti-viral effect of YGJ.Results:1In this experiment, the TCID50of IAV (H1N1)(hemagglutinin titer of1：1024) was10-5.08in MDCK cells．The TC50and TC0of YGJ were167.12mg/ml,4.13mg/ml,respectively. The TC50and TC0of ribavirin were21.925mg/ml,0.541mg/ml,respectively.2The results of flow cytometry by double-labeling with Annexin V and PI showedthat the apoptosis rate of virus control group was the highest, and there werestatistically significant compared with normal control group (P<0.01) and other druggroup (P<0.01).The apoptotic rates of YGJ (four concentration groups:50mg/ml, 25mg/ml,12.5mg/ml,6.25mg/ml) were significantly decreased as compared withvirus control group [18.30±1.71(%)、27.00±1.35(%)、36.97±1.21(%)、45.20±2.19(%), VS55.43±2.48(%), respectively; P<0.01]. The apoptotic inhibition rate ofYGJ (four concentration groups:50mg/ml,25mg/ml,12.5mg/ml,6.25mg/ml) were72.29、55.35、35.94、19.92(%), respectively. Inhibition rate increased with theincreasing concentration of YGJ.The apoptotic rates of ribavirin (four concentrationgroups:10mg/ml、5mg/ml、2.5mg/ml、1.25mg/ml) were significantly decreased ascompared with virus control group [19.17±1.70、24.73±2.15、32.53±2.55、40.17±1.54, VS55.43±2.48(%), respectively; P<0.01]. The apoptotic inhibition rate ofribavirin (four concentration groups:10mg/ml、5mg/ml、2.5mg/ml、1.25mg/ml) were70.60、59.77、44.59、29.71(%), respectively. Inhibition rate increased with theincreasing concentration of ribavirin.3The lung index of virus control group was the highest, and there were statisticallysignificant as compared with normal control group (P<0.01) and other drug group(P<0.01). The lung index of YGJ treatment groups (low-dose group, high-dose group,preventive group) were decreased as compared with virus control group [2.08±0.39、1.15±0.17、1.65±0.15, VS2.47±0.32（%）, respectively; P<0.01]. The lungindex of YGJ high-dose group was slightly lower than ribavirin group (P<0.05), andthere is statistically significant differences.4The NK cell activity of virus control group was the lowest, and there werestatistically significant as compared with normal control group (P<0.01) and otherdrug group (P<0.01). The NK cell activity of YGJ treatment groups (low-dose group,high-dose group, preventive group) were increased as compared with virus controlgroup [36.35±0.92、50.16±0.86、38.46±0.98, VS29.81±1.53（%）, respectively;P<0.01]. The NK cell activity of YGJ high-dose group was slightly higher thanribavirin group (P<0.01), and there is statistically significant differences. Conclusion:1In this experiment, IAV (H1N1) can induce MDCK cell apoptosis. YGJ can inhibitIAV (H1N1) induced MDCK cell apoptosis, and the apoptosis inhibition rateincreased with the increasing concentration of YGJ. Compared with ribavirin, theeffect of YGJ suppressing cell apoptosis infected with IAV (H1N1) is similar.2In this experiment, IAV (H1N1) can induce viral pneumonia in mice. YGJ couldeffectively decrease the lung index of mice infected with IAV (H1N1), ease thegeneral symptoms of viral pneumonia, and markedly inhibit the lung consolidationof lung inflammation. Compared with ribavirin, the effect of YGJ suppressing lunginflammation injury is similar.3In this experiment, IAV (H1N1) can significantly inhibit the NK cell activity ofmice infected with IAV (H1N1). YGJ can increase the NK cell activity of mice, andenhance the immune function on the anti-viral effect of mice. Compared withribavirin, the effect of YGJ improving the NK cell activity of mice infected with IAV(H1N1) is similar. |
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