| Objective:With a view To further investigate the effectiveness and mechanism of Chinese medicine for influenza,this study established infected model mice of H1N1 influenza virus FM1 strain,using Ganghuo anti-influenza Soup which is characteristic of the south of the five ridges,to observe the effects of Ganghuo anti-influenza Soup on viral infected mice during the experimental study,the variation of the lung index,death-protection?virus replication ratio in lung tissue,arterial blood gas analysis,viral load?cytokine levels in serum and inflammatory cell count and classification in bronchoalveolar lavage fluid(BALF)from the aspect of cytology,molecular biology and pathology.The study has explored the mechanism of Ganghuo anti-influenza Soup on anti-influenza in order to provide effective and reliable theoretical foundation for traditional Chinese treatment for influenza.Methods:Experiment 1 Determination of LD50 in mice infected with influenza virus FM1 strainThere were ninety-one KM mice,weighing 13.0-15.Og,half male and half female.The KM mice were randomly divided into 7 groups,9 mice in each group.Chose 7 virus titer of influenza virus liquid and get mice in each group infected with one kind of virus titer of influenza virus through nasal infection with 4 drops about 0.05ml under ether mild anesthesia.Death and survival was observed indicators except which died within 24h.Daily mortality of mice were observed and recorded until 15 days after infection so far.Calculate the median lethal dose of the virus by using the Reed-Muench method after the data Processing.Experiment 2 Research on death of protection of mice infected with influenza virusThere were fifty KM mice,weighing 13.0-15.Og,half male and half female.KM mice were randomly divided into five groups on average,namely normal group,Ribavirin group,Ganghuo anti-influenza Soup(large and small)dose group,10 mice per group.Except the normal group,each group was under ether mild anesthesia infected with influenza virus with 101d50 of flu virus droplets with nasal infection,while normal group were with the same amount of saline nose drops.Since infection on the day of the continuous observation for 15 days,mice daily observation records of deaths,and calculate the mortality and death of protection.Four hours after infection each group began to be administrated separately and the dosage of each group were 0.2mL/10g.Continuous observation had been made for general condition,activity on mice,mental condition,wheezing,coughing,shiny fur,weight changes of each mice and deaths were observe and recorded,and calculation the mortality and death of protection.Experiment 3 Mechanism of pharmacodynamic actions of Ganghuo anti-influenza Soup against mice infected with influenza virus1 There were thirty KM mice,weighing 13.0-15.0g,half male and half female.KM mice were randomly divided into five groups on average,Normal group,Ribavirin group,Ganghuo anti-influenza Soup(large and small dose)group,and there were six mice in each group.Except the normal group,each group was under ether mild anesthesia in mice infected with influenza virus with 10 1d50 of flu virus droplets nasal infection,normal group was with the same amount of saline nasal drops.Four hours after infection each group began to be administered separately and the dosage of each group were 0.2mL/10g.Administered for 5 consecutive days and begun to experiment.2 Determination of lung index and lung wet-dry weight ratio of mice infected with with influenza virus.Half an hour after the last administration,the mice were drawn blood from eyeball and were killed after weighing,supine position fixed in mice board.Opened the chest and removed the whole lung after alcohol disinfection.visual inspection and recording of lung lesions.Weighed wet weight after abstersion with physiological saline and siphoning off residual brine with a clean filter paper.And Weighed dry weight after dried in an oven at 60?for 72h.At last calculate lung index,inhibition rate of lung index and wet to dry weight ratio.3 The changes of lung morphology of mice infected with influenza virus.Half an hour after the last administration,the mice were drawn blood from eyeball and were killed after weighing,supine position fixed in mice board.Opened the chest and removed the right lung after alcohol disinfection.Visual inspection and recording of lung lesions.Abstersion the lung with physiological saline and siphoning off residual brine with a clean filter paper.Fixed the lung in 4%polyformaldehyde solution with 5ml of EP tube for 24 hours and then used it for HE staining.4 The Effect of Ganghuo anti-influenza Soup on blood gas analysis of mice infected with influenza virus.Half an hour after the last administration,the mice were drawn blood from eyeball and were killed after weighing,supine position fixed in mice board.Cut the skin and muscle of abdomenby by using scissor after alcohol disinfection,then the abdominal aorta was separated.0.4ml heparin anti-coagulating blood were taken by blood taking needle.Syringe needle was inserted into the small rubber stopper immediately after extraction and gently rubbing syringe to make heparin thoroughly mixed with the blood.Determination of blood gas immediately by automatic blood gasanalyzer.5 Effect on the viral load of mice infected with influenza virus.Half an hour after the last administration,the mice were drawn blood from eyeball and killed after weighing,with supine position fixed in mice board.Opened the chest and removed the left lung after alcohol disinfection.Visual inspection and recording of lung lesions and absterge the lung with physiological saline and siphoning off residual brine with a clean filter paper.Tissue samples of left lung of mice were taken to measure the levels of virus.6 Effect on cytokine levels in serum and inflammatory cell count and classification in bronchoalveolar lavage fluid(BALF)of mice infected with influenza virus.Half an hour after the last administration,blood samples were collected from ophthalmic venous plexus.Determination of serum IL-6,IL-10,IL-12,IFN-?,TNF-?,MCP-1 content were made with CBA kit methods.Observation was taken on inflammatory cell count and classification in bronchoalveolar lavage fluid(BALF).Results:Experiment 1 Determination of LD50 in mice infected with influenza virus FM1 strainCounted the number of deaths in each group of mice after observed 15 days and Calculate the median lethal dose of the virus by using the Reed-Muench method.The result was each 0.05ml virus solution containing 10-2.57 concentration of virus.Experiment 2 Research on death of protection of mice infected with influenza virusThere were no death on Normal group and Ribavirin group.However there were nine mice died in model group,that begun at the fourth day and peak at the sixth day.Ganghuo anti-influenza Soup large dose group had six survived mice,the average living days compared to model group,the chi-square test,P<0.05,has the obvious death protection.Ganghuo anti-influenza Soup small dose group had survived four mice,but the average living days wasn't significant difference compared with model group,it had tendency to prolong the survival time.Experiment 3 Mechanism of pharmacodynamic actions of Ganghuo anti-influenza Soup against mice infected with influenza virus1 Determination of lung index and lung wet-dry weight ratio of mice infected with influenza virus.Lung index and lung wet-dry weight ratio of mice markedly increased after infected with Influenza A virus.Lung index and lung wet-dry weight ratio of Ribavirin group reduced obviously compared with model group(P<0.01).Ganghuo anti-influenza Soup large dose group could reduce lung index obviously.Ganghuo anti-influenza Soup small dose group also had reduce lung index,but it wasn't significant difference compared with model group.Lung wet-dry weight ratio of Ganghuo anti-influenza Soup(large and small dose)group reduced obviously compared with normal and model group(P<0.01).It showed that Ganghuo anti-influenza Soup could inhibited the production of inflammatory mediators and oozing.2 The changes of lung morphology of mice infected with influenza virus.The lung cells and alveolar septum of normal group was in good condition.Inflammatory cell infiltration could not be found in the lung cells and there was few lymphocytes in it.In the model group,thickening of alveolar walls and alveolar septum,the lung tissue of mice with inflammatory cell infiltration,dilation of alveolar capillaries,a large number of lymphocyte can be seen and formation of lung consolidation.Ribavirin group restored alveolar walls and alveolar septum and reduced inflammatory cell infiltration obviously.Ganghuo anti-influenza Soup(large and small dose)group recovered the morphological structure of lung tissue basically and had reduced inflammatory cells compared with model group.3 The effect of Ganghuo anti-influenza Soup on blood gas analysis of mice infected with influenza virus.The blood gas analyses showed that virus infected can elevate carbon dioxide partial pressure,reduce partial pressure of oxygen and sulfur dioxidearterial.Carbon dioxide partial pressure were reduced,partial pressure of oxygen and sulfur dioxidearterial were elevated in the mice of the Ribavirin group significantly compared with the normal and model group(P<0.01,F<0.05).Ganghuo anti-influenza Soup large dose group could elevate partial pressure of oxygen and sulfur dioxidearterial,reduce carbon dioxide partial pressure,showing a remarkable change compared with the normal and model group(P<0.01,P<0.05)while the small dose group could not.4 Effect on the viral load of mice infected with influenza virus.Levels of virus of lung tissue increase significantly after infected with virus.Viral load of Ribavirin group decreased significantly compared with model group(P<0.01);Ganghuo anti-influenza Soup large dose group also could decreased the viral load significantly(P<0.01);Viral load of Ganghuo anti-influenza Soup small dose decreased but not significantly,there was no statistical difference compared with the model group.The result suggested that Ganghuo anti-influenza Soup(large and small dose)groupcan inhibit influenza virus replication in the lung tissue,and the large dose was superior to the small dose.5 Effect on cytokine levels in serum and inflammatory cell count and classification in bronchoalveolar lavage fluid(BALF)of mice infected with influenza virus.Ribavirin could lower pro-inflammatory cytokine levels,improve anti-inflammatory cytokine,reduced leukocyte count,lymphocyte percentage,neutrophils percentage and increased the percentage of macrophages,showing a remarkable change compared with the normal and model group(P<0.01,P<0.05).Ganghuo anti-influenza Soup(large and small dose)group could lower IL-6,TNF-?,MCP-1 and other pro-inflammatory cytokine levels,improve anti-inflgammatory cytokine IL-10,IFN-? levels,showing a remarkable change compared with the normal group(P<0.01,P<0.05),except IL-12 levels.Compared with the model group,every kind of cytokines had significant changes except IL-6 after using Ganghuo anti-influenza Soup small dose and IL-12 after using Ganghuo anti-influenza Soup(large and small dose),with a statistical significance(P<0.01,P<0.05).Ganghuo anti-influenza Soup large dose group could reduce bronchoalveolar lavage fluid leukocyte count,lymphocyte percentage,neutrophils percentage of infected with virus strain mice significantly(P<0.01,P<0.05),increased the percentage of macrophages(P<0.05);Ganghuo anti-influenza Soup small dose group also could reduce bronchoalveolar lavage fluid leukocyte count and neutrophils percentage of infected with virus strain mice significantly(P<0.01),reduced lymphocyte percentage and increased the percentage of macrophages.However there were no statistical difference.Conclusion:1 Ganghuo anti-influenza Soup could improve living condition and quality of Life of influenza virus infected mice,and prolong the survival time of influenza virus infected mice.Ganghuo anti-influenza Soup large dose group was superior to the small dose group.2 Ganghuo anti-influenza Soup could inhibite the production of inflammatory mediators and oozing,reduce lung index and lung wet-dry weight ratio,attenuate lung edema,restore respiration function,relieve pathological injury of lung tissue of influenza virus infected mice.Ganghuo anti-influenza Soup large dose group was superior to the small dose one.3 Ganghuo anti-influenza Soup could inhibit influenza virus replication in the lung tissue directly,decrease viral load significantly of influenza virus infected mice.Ganghuo anti-influenza Soup large dose group was superior to the small dose one.4 Ganghuo anti-influenza Soup could regulate the proinflammatory cytokines and anti-inflammatory cytokine of influenza virus infected mice.By promoting the secretion of IL-10,IFN-? and other anti-inflammatory cytokines and inhibiting the synthesis and secretion of IL-6,IL-10,TNF-a,MCP-1 and other pro-inflammatory cytokines,Ganghuo anti-influenza Soup could modulate the immune mechanisms of injury in mice infected with influenza virus.At the same time,Ganghuo anti-influenza Soup may regulate leukocyte classification and counting,scale inhibition of influenza virus production and had exudation caused by inflammatory mediators in the lungs of mice,relieved the production of inflammatory mediators and oozing caused by infection of influenza virus as to achieve its anti-virus action.The ideal anti-viral effect concentration was high-dose. |
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