The Effect Of Tongguan Pill-containing Serum On The Expression Of Phosphorylated IRAK4 And IKKs Protein In A Macrophage Inflammation Model

Objective:By studying the effect of serum containing tongguan pills on the expression levels of IRAK4 and IKKs protein in the inflammation model of macrophages,the mechanism of Tongguan pills in the treatment of salping obstruent infertility was clarified from the protein molecular level,which provided a practical and reliable experimental basis for the prevention and treatment of salping obstruent infertility by TCM.Methods:1.Ten non-pregnant female SPF Wistar rats were randomly divided into experimental group and control group.Rats in the experimental group were given tongguan pill suspension by gavage,and rats in the control group were given the same amount of normal saline by gavage.After 3 days of continuous gavage,blood was collected from the abdominal aorta and serum was prepared.2.RAW264.7 cells were divided into 9 groups: normal culture group,drug-free serum group(5%,10%,20%,40%),and drug-containing serum group(5%,10%,20%,40%).10% fetal bovine serum was added to the normal culture group,and 5%,10%,20% and 40% drug-free serum was added to the normal culture group,and 5%,10%,20% and 40% drug-free serum was added to the normal culture group.3.RAW264.7 cells were divided into the model group and the non-model group.In the model group,100ng/ml lipopolysaccharide was added to the medium,and the same amount of PBS buffer was added to the medium of the non-model group.After 24 hours of culture,the contents of TNF-α and IL-1β inflammatory factors in the two groups were detected by ELISA to determine whether 100ng/ml lipopolysaccharide could successfully produce a macrophage inflammatory model.4.RAW264.7 cells were divided into three groups: blank control group,PPARagonist group,NF-B blocker group,non-drug serum group and drug serum group.Normal RAW264.7 cells and 10% FBS were added to the blank control group.RAW264.7 cells and 20umol/L NF-B blocker SN50 were added to nf-kb blocker group.RAW264.7 cells and 20umol/L pioglitazone were added to PPAR-agonist group.In the drug-free serum group,RAW264.7 cells and 10% drug-free serum were added after modeling.RAW264.7 cells and 10% drug serum were added to the drug serum group.Supernatant was collected 24 hours after culture,and the expressions of phosphorylated IRAK4 and IKKs proteins in each group were determined by Western Blot.Results:1.Comparison of RAW264.7 cell viability in each groupCompared with the normal culture group,there was no significant change in RAW264.7 cell activity between the 5% non-drug serum group and the 20% non-drug serum group(P > 0.05).The activity of RAW264.7 cells in 10% non-drug serum group,5%drug serum group,10% drug serum group and 20% drug serum group increased significantly(P < 0.05).The activity of RAW 264.7 cells in the 40% non-drug serum group and the 40% drug-containing serum group was significantly decreased(P <0.05).Compared with the 10% drug-containing serum group,the activity of RAW 264.7cells in the 5% drug-containing serum group,20% drug-containing serum group and40% drug-free serum group was significantly decreased(P < 0.05).2.Comparison of levels of TNF-α and il-1 protein in RAW 264.7 cells in each groupCompared with the non-modeling group,the contents of TNF-and IL-1β in the modeling group increased significantly,with statistically significant difference(P <0.01).3.Comparison of the relative expression levels of phosphorylated IRAK4 and phosphorylated IKKs in RAW 264.7 cells in each groupCompared with the blank control group,phosphorylation of IRAK4 and IKKs increased in RAW 264.7 cells in the non-drug containing serum group,the non-drug containing serum group,the NF-κB blocker group,the PPAR-γ control agonist group and the drug containing serum group(P < 0.05).Compared with the non-drug containing serum group,the phosphorylation of IRAK4 and IKKs in RAW 264.7 cells in the NF-κB blocker group,PPAR-γ agonist group and the drug containing serum group were all decreased(P < 0.05).Conclusion: 1.The optimal serum concentration of RAW264.7 cells was 10%.2.100ng/ml LPS was able to successfully produce macrophage inflammation model.3.The mechanism of tongguan pill in treating salping obstruent infertility is that tongguan pill can inhibit the activation of TLR2/My D88/NF-κB signaling pathway by inhibiting the expression of IRAK4 and IKKs proteins phosphorylated by the "key elements" in the TLR2/My D88/NF-κB signaling pathway,reduce the expression of inflammatory factors and inflammatory mediators,and achieve the purpose of inhibiting the inflammatory reaction of fallopian tubes.