| Objective:To investigate the material basis,potential targets and possible mechanismes of Ganluxiaodu formula in the treatment of Mycoplasma pneumoniae pneumonia(MPP)in children.Method:1.Based on TCMSP database and BATMAN-TCM database,the active ingredients of Ganluxiaodu formula were searched and potential targets were predicted;Based on Drug Bank database and Gene Cards database,the potential targets of MPPS in children were identified.Cytoscape software was used to construct the drug-disease interaction network.GO functional enrichment analysis and KEGG pathway enrichment analysis were conducted based on David database.2.35 rats were randomly divided into blank serum group,Ganluxiaodu formula serum group,Decomposed formula 1 serum group,Decomposed formula2 serum group and azithromycin serum group,and were administered continuously for 7 days to prepare drug-containing serum.The effects of different serum containing drugs on the viability of NR8383 cells were detected by CCK-8 method.The cells were divided into control group,model group,Ganluxiaodu formula group,Decomposed formula 1 group,Decomposed formula 2 group and azithromycin group.Except the control group,all the cells were treated with MP bacterial solution at the concentration of 10~6～10~7CCU/ml for 4 hours to establish MP infection model,and then the corresponding drug-containing serum was added and incubated for 24 hours to collect cells and supernatant.mRNA and protein expressions of pathway in each group were detected by qPCR and WB.The expressions of macrophage polarization were detected by qPCR.The expression levels of Inflammatory factor were determined by ELISA.Result:1.46 potential targets such as TNF,IL1B,and 93 signaling pathways such as NF-κB,PI3K/Akt were identified in the network pharmacological study.2.CCK-8 showed that only medicated serum with concentration of5μl/100μl had no difference in cell survival rate compared with blank serum,so serum with this concentration was selected for subsequent experiments.q-PCR showed that mRNA expressions of TLR4,MyD88 and NF-κB-p50in model group were higher than those in control group(P<0.01).Compared with model group,mRNA expressions of TLR4 and NF-κB-p50 had no statistical difference(P>0.05),while mRNA expressions of TLR4,MyD88 and NF-κB-p50in supplemental group were decreased(P<0.01 or 0.05).The mRNA expression of MyD88 in Ganluxiaodu formula group was higher than that in azithromycin group(P<0.05),and the mRNA expression of TLR4,MyD88 and NF-κB-p50 in Docomposed group 1 was higher than that in Docomposed group 2(P<0.01).The mRNA expressions of PI3K and Akt in model group were lower than those in control group(P<0.01 or 0.05).The mRNA expressions of PI3K and Akt in all treatment groups were not statistically significant compared with those in model group(P>0.05).WB showed that the protein expressions of TLR4,MyD88 and NF-κB-p50 in model group were higher than those in control group(P<0.01).Compared with model group,the protein expressions of MyD88 and NF-κB-p50 in Decomposed 1 group had no statistical difference(P>0.05),but the protein expressions of TLR4,MyD88 and NF-κB-p50 in treatment groups were decreased(P<0.01 or 0.05).The protein expressions of TLR4 and NF-κB-p50 in Ganluxiaodu formula group were higher than those in azithromycin group(P<0.01 or 0.05).The protein expressions of TLR4,MyD88and NF-κB-p50 in Decomposed 1 group were higher than those in Decomposed2 group(P<0.01 or 0.05).q-PCR showed that mRNA expressions of i NOs and CD86 in model group were higher than those in control group(P<0.01).Compared with model group,there was no significant difference in i NOs mRNA expression in Decomposed 1 group(P>0.05),but the mRNA expression of i NOs and CD86 in treatment groups decreased(P<0.01).The mRNA expression of CD86 in Ganluxiaodu formula group was higher than that in azithromycin group(P<0.01).The mRNA expressions of i NOs and CD86 in Decomposed 1 group were higher than those in Decomposed 2 group(P<0.05 or 0.01).The mRNA expressions of Arg-1 and CD206 in the model group were lower than those in the control group(P<0.01),but there was no statistical difference between the model group and treatment groups(P>0.05).Elisa showed that the expressions of TNF-αand IL-1βin model group were higher than those in control group(P<0.01).Compared with model group,there was no statistical significance in the expression of TNF-αin decomposed 1 group(P>0.05),but the expressions of TNF-αand IL-1βin treatment groups were decreased(P<0.01 or 0.05).The expressions of TNF-αand IL-1βin decomposed 1group were higher than those in decomposed 2 group(P<0.01).Conclusion:The M1-type polarization and the expression levels of TNF-αand IL-1βof NR8383 cells after MP infection could be inhibited by the drug-containing serum of Ganluxiaodu formula and its decomposed formula.The mechanism may be related to the inhibition of TLR4/MyD88/NF-κB pathway.The effect of azithromycin group was better than that of Ganluxiaodu formula group,and the effect of decomposed formula 2 group was better than that of decomposed formula 1 group. |
PDF Full Text Request