| Objective:Based on the TCM theory of "lung and large intestine facing and inside",the molecular mechanism of "lung and intestine" of influenza and the regulatory effect of Maxingshigan Decoction were studied from the TLR2/ 4-myd88 signaling pathway,to explore the effect mechanism of this prescription against influenza virus.Methods:1.Effect of Maxingshigan Decoction on the TLR2/ 4-myd88 signaling pathway of lung macrophages and intestinal macrophages stimulated by influenza virus(In vitro experiment)(1)MTT assay was used to detect the effect of rat drug-containing serum on the activity of lung macrophages and intestinal macrophages cultured for 24 h and 48 h in mice.(2)The levels of il-1,il-8 and TNF-α/β in supernatant of Maxingshigan Decoction containing drug serum for 24 h and 48 h of influenza virus infection were detected by ELISA.(3)The expression levels of TLR2 and TLR4 in lung and intestinal macrophages infected with influenza virus after 24 h were detected by using immunohistochemical method.2.Effect of Maxingshigan decoction on TLR2/ 4-myd88 signaling pathway in lung and intestinal tissues of mice infected with influenza virus(In vivo experiment)(1)An animal model of influenza virus pulmonary infection was established by nasal inoculation.Body weight was measured after 3 days and 7 days of gavage,and organs were taken to calculate the body weight inhibition rate,lung index and intestinal propulsion rate.(2)The expression levels of TLR2,TLR4,MyD88 and NF-κB in lung and colon tissues were detected by immunohistochemistry and western blotting.(3)Body weight and lung index,intestinal propulsion rate and lung index,lung index and lung tissue(TLR2,TLR4,MyD88,NF-κB),lung index and intestinal tissue(TLR2,TLR4,MyD88,NF-κB),lung tissue and intestinal tissue(TLR2,TLR4,MyD88,NF-κB)were analyzed.Results:1.Effect of Maxingshigan decoction on the TLR2/4-myd88 signaling pathway of lung macrophages and intestinal macrophages stimulated by influenza virus(In vitro experiment)(1)The results of MTT assay showed that the blank serum of rats with concentrations of 40%,20%,10% and 5% had no toxicity compared with the 10%fetal bovine serum group.Compared with the corresponding concentrations of fetal bovine serum and rat blank serum control group,rat drug-containing serum with concentrations of 40%,20%,10% and 5% acted for 24 h and 48 h without toxic effects on pulmonary macrophages and intestinal macrophages.(2)ELISA results showed that,compared with the normal group,the contents of il-1,il-8 and TNF in the supernatant of lung macrophages infected by influenza virus increased significantly after 24h(P<0.05).Compared with the viral model group,the contents of il-1,il-8 and TNF in the supernatant of lung macrophages infected by influenza virus decreased significantly after 24h(P<0.05).Compared with the normal group,the contents of il-8 and TNF in the supernatant of intestinal macrophages infected with influenza virus increased significantly after 24h(P<0.05).Compared with the viral model group,the contents of il-8 and TNF in the supernatant of intestinal macrophages infected with influenza virus decreased significantly after 24h(P<0.05).There was no significant change in the indicators detected by ELISA at the time point of 48 h,so the subsequent time point of 24 h was adopted for cell immunohistochemistry.(3)Results of cell immunohistochemistry showed that the expression levels of TLR2 and TLR4 proteins in lung macrophages infected with influenza virus were significantly increased after 24 h compared with the normal group(P<0.05).Compared with the viral model group,the expression levels of TLR2 and TLR4 proteins in lung macrophages were decreased after 24 h intervention of serum containing maxingshigan soup(P<0.05).Compared with normal group,the influenza virus infection,intestinal macrophages after 24 h TLR2 and TLR4 protein expression level was significantly increased(P<0.05),compared with model group virus,24 h im Ma Xing Shi sweet soup drug-containing serum intervention macrophages TLR4 protein expression level decreased significantly(P<0.05),while TLR2 protein expression level had no significant change(P<0.05).2.Effect of maxingshigan decoction on TLR2/ 4-myd88 signaling pathway in lung and intestinal tissues of mice infected with influenza virus(In vivo experiment)(1)The weight of mice infected with influenza virus was significantly decreased(P < 0.05)after 3 days of administration of maxingshigan decoction compared with the normal group,and significantly increased(P<0.05),compared with the viral model group,with a weight inhibition rate of 25.37%.After 7 days of administration,compared with the normal group,the weight of the virus model group was significantly reduced(P<0.05);compared with the virus model group,the weight of each ma xing shi gan tang group was significantly increased(P<0.05),and the weight inhibition rate was 30.91%.(2)Lung index of maxingshigan decoction in mice infected with influenza virus showed that lung index of the virus model group was significantly increased(P<0.05),compared with that of the normal group after 3 days of administration,while lung index of the virus model group was significantly decreased(P<0.05),and the inhibition rate of lung index was 41.9%.After 7 days of administration,lung index of the virus model group was significantly increased(P<0.05),compared with that of the normal group,and lung index of the ma xing shi gan tang group was not significantly decreased compared with that of the virus model group(P<0.05).(3)The intestinal propulsion rate of mice infected with influenza virus by maxingshigan decoction showed that: after 3 days of administration,the intestinal propulsion rate of the virus model group was significantly lower than that of the normal group(P<0.05),and that of the virus model group was significantly higher than that of the virus model group(P<0.05).After 7 days of administration,there was no significant difference between the groups(P<0.05).(4)Organization immunohistochemical results show:(1)for 3 days,compared with normal group,the influenza virus infection in mice after TLR2 and TLR4 lung tissue,the nf-kappa B protein expression level was significantly increased(P<0.05),compared with model group virus,Ma Xing Shi sweet soup in the lung tissue of mice after intervention TLR2 and TLR4,the NF-κB protein expression level decreased significantly(P<0.05);After 7 days of administration,the expression level of MyD88 protein in the lung tissues of mice infected with influenza virus was significantly increased(P<0.05),compared with that of the normal group.Compared with the virus model group,the expression level of MyD88 protein in the lung tissues of mice was significantly decreased after the intervention of maxingshigan decoction(P<0.05).(2)after 3 days of administration,the expression levels of TLR4,MyD88 and NF-κB in colon tissues of mice infected with influenza virus were significantly different from those of the normal group(P<0.05).Compared with the virus model group,the expression levels of TLR4,MyD88 and NF-κB in colon tissues of mice were regulated by maxingshigan decoction(P<0.05).After 7 days of administration,there was no significant change in the expression levels of TLR2,TLR4,MyD88 and NF-κB protein in colon tissues.(5)The testing results of western blot method: for 3 days,compared with normal group,the influenza virus infection in mice after TLR2 and TLR4 in organization of lung,colon,MyD88,nf-kappa B protein expression level was significantly increased(P<0.05),compared with model group virus,Ma Xing Shi sweet soup in the lung,colon tissue of mice after intervention TLR2 and TLR4,MyD88,NF-κB protein expression level decreased significantly(P<0.05).After 7 days of administration,there was no significant difference between the groups(P>0.05).(6)Correlation analysis of animal body weight,lung index,intestinal propulsion rate and immune imprinting results showed that there was a linear correlation between body weight and lung index of mice infected with influenza virus(P<0.05).There was a linear correlation between intestinal propulsion rate and lung index in mice infected with influenza virus(P<0.05).There was a linear correlation between the lung index and the expression levels of TLR2,TLR4,MyD88 and NF-κB protein.There was a linear correlation between lung index and colonic tissue TLR2,TLR4,MyD88 and NF-κB(P<0.05).After influenza virus infection in mice,there was a linear correlation between TLR2 protein expression in lung and intestinal tissues(P<0.05).After influenza virus infection in mice,there was a linear correlation between TLR4 protein expression levels in lung and intestinal tissues(P<0.05).There was a linear correlation between MyD88 protein expression in lung and intestinal tissues of mice infected with influenza virus(P<0.05).There was a linear correlation between NF-κB protein expression and lung tissue level(P<0.05).There was a linear correlation between the intestinal propulsion rate and the expression levels of TLR2,TLR4,MyD88 and NF-κB protein(P<0.05).Conclusion:1.Maxingshigan decoction can significantly regulate the contents of il-1,il-8and TNF-α in the supernatant of pulmonary macrophages after 24 h of influenza virus infection and the contents of il-8 and TNF-β in the supernatant of intestinal macrophages after 24 h of influenza virus infection.2.Maxingshigan decoction can significantly regulate the expression levels of TLR2 and TLR4 in lung macrophages and intestinal macrophages after 24 h of influenza virus infection in mice.3.Maxingshigan decoction can significantly regulate the body weight of mice infected with influenza virus for 3 days and 7 days;Maxingshigan decoction can significantly regulate the lung index and intestinal propulsion rate of mice after 3 days of influenza virus infection.4.Maxingshigan decoction can significantly regulate the expression levels of TLR2,TLR4,MyD88 and NF-κB protein in lung and intestinal tissues of mice infected with influenza virus after 3 days.5.The TLR2/ 4-myd88 signaling pathway is one of the many pathways involved in the pathological transmission of "lung and intestinal diseases".maxingshigan decoction can regulate the target index of the TLR2/ 4-myd88 signaling pathway after influenza virus infection. |
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