Table Method. "lungs Is Often Insufficient Argument Anti-influenza Virus Infection In The Tlr Signaling Pathway Time-effect Relationship Study

ObjectiveThrough establishing young and mature mice model with mouse influenza virus (IV) infection and changes of TLR4-NF-κB P65 signaling pathway in lungs of mice infected with influenza virus to discuss the situation of young mice and mature mice against influenza virus susceptibility and Lung often inadequate mechanism under animal experiments. Compare the classic cold-pungent diaphoresis relieving exterior syndrome prescription Yinqiaosan and consolidating superficies prescription Yupingfeng, discuss the role of the two methods in the prevention of influenza virus infection through the effects of the protection of the two methods in chick embryo study infected with Influenza virus and the activation of TLR4-NF-κB P65 signaling pathway in the lung tissues of mice. And thus provide experimental basis for the Chinese medicine to protect children from influenza virus infections. Obtained the finger printings of classical cold-pungent diaphoresis prescription yinqiaosan by high efficiency liquid chromatography.Materials and Methods:1. Using influenza A virus A/FM1/47 to infect mice .The mice were divided into two groups: young mice and mature mice, observed their general status, survival, the differences of lung lesions, calculated lung index, applied RT-PCR method to detect IV-RNA viral load differences at two different time points in lung tissues of mice , which was to provide experimental verification for Lung often inadequate theory mechanism.2. Immunohistochemistry and RT-PCR was applied to detect the expressions of lung TLR4 and NF-κB P65 protein and mRNA at different time points in young mice and mature rat, comparing the changes of the TLR4-NF-κB P65 signaling pathway after young mice and mature mice infected with influenza virus, as providing experimental evidence for the"Lung often inadequate"theory mechanism.3. Used Hemagglutination test to observe the role of direct inhibition IV effects with different concentrations of Yinqiaosan extract, Yupingfeng particles at different time points in vitro; studied different concentrations of Yinqiaosan extract and Yupingfeng particles on IV infection prevention and protective effects of chick embryo.4. Used Immunohistochemical methods to detect TLR4, NF-κB P65 protein expression in lungs of mice in each group administered at different time points with Yinqiaosan extract, Yupingfeng particle and particle of both.5. Using RT-PCR method to detect TLR4, NF-κB P65mRNA expression lungs of mice in each group administered at different time points with Yinqiaosan extract, Yupingfeng particle and particle of both.6. The Yinqiaosan and nine herbs finger printings were established by the high efficiency liquid chromatography and analysis the distribution features and focuses of finger printing capyluses of Yinqiaosan. Simultaneous determination of five components and discuss the relationship of finger printing capyluses.Result:1. Inoculated with influenza virus, the mature model group basically maintained normal conditions, young model group had worse general states, and normal mice had better ones. 14 days after inoculation, the normal group all survived. Most died in young model group, and death reached the peak at the 5th day. The normal group and the mature model group showed significantly difference (P <0.05). Mature model group and normal group showed no significant difference (P> 0.05).)2. The appearance of lung lesions showed: The normal lung tissue of mice at different times showed no pathological changes in the appearance of the whole lung field; young model group showed the worst changes, at each time point the normal group and model group mature showed significant differences (P <0.05), the 5th day the young model group showed the most significant appearances of disease; mature model group at different time points compared with the normal group had no significant difference (P> 0.05), and there was a significant group difference compared with young model group(P <0.05).)3. Lung index determination results: lung indexes of young and mature normal mice groups at different time points were the lowest. Young model group were the highest and the comparisons with the normal group and the mature model group were significantly different (P <0.05), lung infection on day 5 reached the highest index. Mature model group and normal group showed no significant difference (P> 0.05), and there was a significant group difference compared with young model group(P <0.05).)4. The semi-quantitative determination results of IV-RNA in lung tissue at different time points : In normal lung tissue of mice at each time point did not detected any virus nucleic acid of IV, young model group detected at each time point a large number of viral nucleic acid, at day 5 there were the largest viral nucleic acid as 2.1238±0.2774, In the model groups at different time points in young and mature model groups, there was a significant difference (P <0.05). Mature model group at all time points the expression of viral nucleic acid showed only traces, and there was a significant group difference compared with young model group(P <0.05), no significant difference compared with the normal group.(P> 0.05).)5. TLR4 protein expression in lung tissues: the young normal group and mature normal group were able to detect small amounts of TLR4 protein expression, and compared at each time point, protein expressions had no significantly differences (P> 0.05).Young model group showed the strongest expression of TLR4 protein, and the expression increased gradually over time, peaked at day 5; young model group compared with normal group and the mature model group showing significant differences (P <0.05).TLR4 protein expressions in mature model group were higher than the normal group but lower than the young model group.6. Lung tissue NF-κB P65 protein expression: the normal group and mature young normal group were able to detect small amounts of NF-κB P65 protein expression, and compared at each time point, protein expression was not significantly different (P> 0.05).In young model group, the NF-κB P65 protein expression was the strongest, and the expression increased gradually over time, each time point compared with the previous point in time showing a significant difference (P <0.05); NF-κB P65 protein expressed more than the normal group in the mature model group but lower than the young model group (compared with young normal group and model group, P <0.05), expression of mature model group at each time point there was no significant difference (P> 0.05).7. TLR4 mRNA expression in lung tissue: the young normal group and mature normal group were able to detect small amounts of TLR4 mRNA expression, and compare each time points, mRNA expression was not significantly different (P> 0.05).Young model group showed the strongest expression of TLR4 mRNA, and expression gradually increased with the progression in the first 5 days to reach the peak, TLR4 mRNA and the internal reference intensity ratio was 1.8351±0.0110; TLR4 mRNA of young model group was significantly higher than the normal group and the mature model group, with significant difference (P <0.05).TLR4mRNA in mature model group was higher than the normal group but lower than the young model group.8. Lung tissue NF-κB P65 mRNA expression: the young normal group and mature normal group were able to detect small amounts of NF-κB P65 mRNA expression,at each day point 1,3,5 and 7, the comparisons of mRNA expressions were not significantly different (P> 0.05).In young model group, the NF-κB P65 mRNA expression was the strongest, and gradually increased with the progression of expression; compared with the normal group and the mature model group, there were no significant differences (P <0.05).In mature model group, NF-κB P65 mRNA expression was higher than the normal group but lower than the young model group.9. The effects of medicines directly inhibiting the virus in vitro: Yinqiaosan extract, Yupingfeng particles can directly inhibit the different levels of activity of influenza A virus FM1, the sustainable time lasted from 1h to 24h, the medicine showed significant effect from 8 to 18 hours after the effects wore off gradually after that; Yinqiaosan Extract showed a role of direct inhibition of viruses in 8h when the strongest inhibition titers up to 16 times; Yupingfeng particle took strongest inhibition effects in vitro of IV in 8 -18h when the strongest inhibition titers up to 8 times; Yinqiaosan extract and Yupingfeng particles at different time points compared with western medicine ribavirin showed no advantages.10. Comparison of Yinqiaosan extract and Yupingfeng particles on IV infection prevention and treatment of chick embryo : When Yinqiaosan extract and Yupingfeng particle concentrations were at 400g/ml, either prophylaxis or treatment , blood clotting virus titer significantly decreased than the control group, there were significant differences (P <0.05); When combined together, the hemagglutination titer virus control were significantly lower, with significant difference (P <0.05); Comparing the groups with one anther showed that combined group showed lower blood clotting, but the factors of HI titer between the groups had no significant difference (P> 0.05).11. The lung TLR4, NF-κB P65 protein expression in different groups: The TLR4 protein expression were lower than ribavirin group in each TCM groups, with significant difference (P <0.05).At the same time point, there were significant differences between each TCM groups (P <0.05), the group using double relieving exterior syndrome methods showed the lowest TLR4 expression. At each time point, the TLR4 protein expression in TCM groups were higher than the normal group, but significantly lower than the model group, there were significantly differences compared with model group and normal group (P <0.05).Within the TCM groups at all time points, TLR4 expression were significantly different (P <0.05), the group using double relieving exterior syndrome methods showed the lowest TLR4 expression.Yinqiaosan extraction group and the group using double relieving exterior syndrome methods at each time point the expression of NF-κB P65 protein were lower than ribavirin group, with significant difference (P <0.05).Yupingfeng particle group at day 1 and 3, protein expressed lower than ribavirin group, with significant difference (P <0.05),day 5 and 7, protein expressed similar to ribavirin, with no significant difference(P> 0.05).The TCN groups at all time points, NF-κB P65 protein expression were significantly different (P <0.05), the group using double relieving exterior syndrome methods showed the lowest expression at all time points.12. The lung TLR4, NF-κB P65mRNA expression in different groups: Yinqiaosan extract group showed higher TLR4mRNA expression than ribavirin group at day 1 and 5, with significant difference (P <0.05), similar at day 3 and 7, with no significant difference (P> 0.05).Yupingfeng particle group was higher than ribavirin group at day 1 and 3, with significant difference (P <0.05), similar at day 5 and 7, with no significant difference (P> 0.05).the group using double relieving exterior syndrome methods showed lower expression than ribavirin group, with significant difference (P <0.05).The TCM group at all time points had significant differences in expression (P <0.05), the group using double relieving exterior syndrome methods had the lowest expression at all time points.Ribavirin group and the TCM groups at all time points showed lower NF-κB P65 mRNA expression compared with model group, higher than the normal group, with significant difference (P <0.05).Yinqiaosan extract group and other TCM groups at all time points were lower than the ribavirin group, with significant difference (P <0.05).InTCM groups the group using double relieving exterior syndrome methods was the lowest compared with other groups ,with significant difference (P <0.05).13. Finger printings of different extraction artworks of Yinqiaosan have 26 fingerprint capylus, which belong to 9 single herbs.Acorrding to the quantity of chromatographic peak groups, Fructus arctii,Flos Lonicerae,Forsythia Suspense Radix Glycyrrhizae,and Spica Schizonepetae were the main herbs.Conclusion:1. With the general states observed in mice, lung pathology and the IV-RNA semi-quantitative determination in lung tissue, in young rats can be successfully shaped as the IV infection model in mice while mature rats can not, which can support the Lung often inadequate theory.2. TLR4-NF-κB P65 signaling pathway of lung tissue of young mice and mature rat after IV infection activated in varying degrees, young rat TLR4, NF-κB P65 expression was significantly increased while mature rats increased slightly, with significant difference; the over activation of TLR4-NF-κB P65 signaling pathway may be one lung immune pathological mechanism of serious injury in the young rat, which can provide evidence for the"Lung often inadequate"theory.3. Different concentrations of Yinqiaosan extracts and Yupingfeng particles have good effects on the prevention and treatment of chick embryo infected with IV. its role in the prevention and treatment decreases with lower drug concentrations; combination of Yinqiaosan extract and Yupingfeng particles has a slightly better effect than use either alone, but has no significant difference, And the delivery sequence showed no significant effects on the HA titer factor.4. Yinqiaosan extracts and Yupingfeng particles have good effects on the prevention and treatment of chick embryo infected with IV as ribavirin did, but shows no clear advantage over the former one.5. Yinqiaosan extract group, Yupingfeng particle group and the group using double relieving exterior syndrome methods can at different levels block TLR4 and NF-κB P65 protein over expression and inhibit TLR4 and NF-κB P65mRNA over expression at different time points.6. Yinqiaosan extract group, Yupingfeng particle group and the group using double relieving exterior syndrome methods showed higher effects on the inhibit of TLR4 and NF-κB P65mRNA over expression in the disease process at most of the time points than ribavirin group, and the group using double relieving exterior syndrome methods was the strongest.7. Cold-pungent diaphoresis ,consolidating superficies and double relieving superficies methods are related to the prevention and treatment of mice infected with influenza virus and the inhibition of lung TLR4-NF-κB P65 signaling pathway over-activation.8. Simultaneous determination of five components in the classic yinqiaosan was a simple and reasonably with well reproducibility. the finger printing of classic yinqiaosan complex has 26 generous fingerprint capyluses belong to the single herbs in the formula; The relative retention time and peak area ratio of generous capyluses could being as one of the quality control of formula.