The Effect Of Tongguan Pill On PPAR-? And TRAF-6 Protein In Macrophage Inflammation Model

Objective: The RAW264.7 inflammatory model of mouse macrophage was established by lipopolysaccharide.The effect of Tongguan Pill drug-containing serum on TLR2 pathway related adaptor protein TRAF-6 and PPAR-?/NF-?B pathway PPAR-? protein was studied.It was further confirmed that Tongguan Pill could inhibit activation of TLR2/My D88/NF-?B signal pathway and up-regulate PPAR-?,thus achieving the therapeutic effect on salpingitis obstructive infertility.Methods:1.Ten healthy female rats were randomly divided into experimental group(n= 5)and control group(n = 5).The experimental group was fed with high-dose suspension of Tongguanwan,while the control group was fed with the same amount of normal saline.Blood samples were collected after 3 days of gastric perfusion to prepare serum,which were respectively drug-containing serum group and drug-free serum group.2.RAW264.7 was randomly divided into 9 groups: normal culture group(macrophage RAW264.7),drug-free serum group(5%,10%,20%,40%)and drug-containing serum group(5%,10%,20%,40%).The optimal drug-containing serum concentration was determined to be 10% by CCK8 method.3.RAW264.7 was randomly divided into normal control group and LPS model group.LPS model group was induced with 100ng/ml LPS,while normal control group was not treated.Its morphology was observed 24 hours later,and TNF-? and IL-1B were detected by ELISA to determine the success of modeling.4.Mouse macrophages after successful modeling were randomly divided into 4 groups: group B,group C,group D and group E.Compared with group A of blank control group,the groups were as follows: group A: blank control group(normal macrophages +10% fetal bovine serum);Group B:drug-free serum group(10% drug-free serum);Group C: PPAR-? agonist group(20umol/L pioglitazone);Group D: NF-?B blocker group(20umol/L NF-?B blocker SN50);Group E: drug-containing serum group(10% drug-containing serum);5.Finally,the expression of PPAR-? and TRAF-6 proteins was detected by Western Blot.Results:1.According to the calculation formula of increment rate,the macrophage increment rate of 10% drug-containing serum concentration is significantly higher than that of the other 8 groups(normal culture group,drug-free serum group 5%,drug-free serum group 20%,drug-free serum group 40%,drug-containing serum group 5%,drug-containing serum group 20%,drug-containing serum group 40%),and its increment rate is 118.3%.2.Macrophages in LPS model group showed irregular shape,accompanied by a large number of pseudopods and a large number of vacuoles in the cells.The expression of TNF-? and IL-1B in LPS model group was significantly higher than that in normal control group(P<0.05).3.At the test level of ? = 0.05,there are differences among blank control group a,drug-free serum group b,PPAR-? agonist group c,NF-?B blocker group d and drug-containing serum group e(p < 0.05).The expression of PPAR-? protein in each group is as follows: a > c > e > d > b(p < 0.05);TRAF-6 protein was expressed in each group as follows: b > d > e > c > a(p < 0.05).Conclusion:1.10% blood drug concentration is the best drug concentration.2.A mouse macrophage inflammatory model was successfully established with100ng/ml lipopolysaccharide.3.Drug-containing serum can up-regulate PPAR-? and reduce the expression of key linker protein TRAF-6 on TLR2 pathway,thus inhibiting NF-?B transcription and achieving anti-inflammatory effect;Its anti-inflammatory effect is stronger than NF-?B blocker and slightly weaker than PPAR-? agonist.