| Objective:To study the effects of Tongguanwan's drug-containing serum on the expression of PPAR-? and TRAF6 genes in the lipopolysaccharide(LPS)-induced RAW264.7 cell inflammation model,and to explore the mechanism of Tongguanwan's treatment of tubal inflammatory obstructive infertility.The clinical application and research of Tongguan Pill provide experimental basis.Methods:1.Preparation of drug-containing serum: 10 SPF Wistar female rats were randomly divided into an experimental group and a control group,with 5 rats in each group.The experimental group was administered according to the previous high-dose group,and the control group was given a corresponding dose of normal saline for 3days,2 times a day,and after 1 hour of the last gavage,blood was collected through the abdominal aorta,inactivated,and sterilized.,Separately pack and store in-20?environment for future use.2.RAW264.7 cell culture: Inoculate mouse RAW264.7 cells in a medium containing 10% DMEM + 10% FBS + 1% penicillin solution and place them in an incubator at 37°C and 5% CO2 After the culture is stable,take logarithmic phase cells for subsequent experiments.3.Detection of RAW264.7 cell proliferation rate: Randomly growing RAW264.7cells into 9 groups:(1)normal control group(10% FBS),(2)drug-free serum group(5%),(3)drug-free Serum group(10%),(4)drug-free serum group(20%),(5)drug-free serum group(40%),(6)medicine-containing serum group(5%),(7)medicine-containing serum group(10%),(8)medicine-containing Serum group(20%),(9)drug-containing serum group(40%),the proliferation rate of RAW264.7cells was detected by CCK-8 method.4.Establishment of LPS-induced inflammation model of RAW264.7 cells:RAW264.7 cells with logarithmic growth were selected,and a blank control group and an LPS induction group were set.The induction group was cultured with LPS at a concentration of 100ng/ml,and the control group was added with correspondingvolume After incubation in PBS DMEM medium at 37°C and 5% CO2 saturation humidity for 24 h,cells were collected and tumor necrosis factor ?(TNF-?)and interleukin-1?(IL-1?)inflammatory factors were detected by ELISA kit To determine the success of modeling.5.Real-time PCR method to detect the expression of PPAR-?and TRAF6 genes in RAW264.7 cells: use normal RAW264.7 cells to set blank control group A,and randomly divide the RAW264.7 cells after successful modeling into B,C,D,E group,group A(blank control group): normal RAW264.7 cells + fetal bovine serum 10%;group B(drug-free serum group): RAW264.7 cells + drug-free serum after successful modeling(10%);Group C(PPAR-? agonist group): RAW264.7cells + 20umol/L pioglitazone after successful modeling;Group D(NF-?B blocker group): RAW264 after successful modeling.7 cells + 20umol/L NF-?B blocker(SN50);Group E(Tongguanwan drug-containing serum group): RAW264.7 cells +Tongguanwan drug-containing serum(10%)after successful modeling.Six duplicate wells were set in each group,and the expression of PPAR-? and TRAF6 genes in RAW264.7 cells was detected by Real-time PCR method.Result:1.Morphological observation of mouse RAW264.7 cellsThe RAW264.7 cells that grew well after cultivation were normal under an inverted microscope,resembling a sphere,with a small number of cells deformed and projecting small angles.2.The CCK-8 method was used to detect the effect of Tongguan Pill-containing serum on the proliferation rate of RAW264.7 cellsWhen the serum concentration of Tongguan Pill is 10%,the proliferation rate of RAW264.7 cells is the highest.Observed by inverted microscope,it can be seen that the cells are intact and spherical,indicating that the cells are not damaged.3.Morphological observation of RAW264.7 cells after LPSLPS(100ng/ml)was induced for 24 h,and the cell morphology was observed under a microscope.The cells changed obviously,and more pseudopods were extended,indicating successful modeling.4.LPS-induced RAW264.7 cell inflammation model of TNF-?,IL-1? content5.LPS(100ng/ml)acts on RAW264.7 cells,and the content of TNF-? and IL-1?in the cell supernatant is detected by ELISA.Compared with the normal group,the content of TNF-? and IL-1? in the LPS group increased significantly.There were significant differences in the contents of TNF-? and IL-1? between the two groups,and the differences were statistically significant(P ? 0.01),indicating successfulmodeling.6.The effect of Tongguan Pills-containing serum on the expression of PPAR-?and TRAF6 in LPS-induced RAW264.7 cell inflammation model(1)Differences in gene expression of PPAR-? in RAW264.7 cellsComparing the blank control group with the PPAR-? agonist group,there was no significant difference in the expression of PPAR-? gene,and the difference was not statistically significant(P=0.148 ? 0.05);the blank control group and the drug-free serum group and Tongguan Pill contained drugs Comparing the serum group and the NF-?B blocker group,the expression of PPAR-? gene in the blank control group was significantly higher than that of the other three groups,and the difference was statistically significant(P?0.01).The drug-free serum group was compared with the NF-?B blocker group,PPAR-? agonist group,and Tongguanwan drug-containing serum group.The expression of PPAR-? gene in the drug-free serum group was significantly lower than that of the other three groups.Significant statistical significance(P?0.01).Compared with the NF-?B blocker group,the PPAR-? agonist group had significantly higher PPAR-? gene expression than the NF-?B blocker group,and the difference was statistically significant(P?0.01);Compared with the PPAR-?agonist group and the drug-containing serum group of Tongguan Pill,the expression of PPAR-? gene in the PPAR-? agonist group was slightly higher than that of the drug-containing serum group of the Tongguanwan,the difference was statistically significant(P ? 0.05).Compared with the NF-?B blocker group and the drug-containing serum group of Tongguan Pill,the expression of PPAR-? gene in the NF-?B blocker group was significantly lower than that of the drug-containing serum group of Tongguanwan,the difference was statistically significant(P ?0.01).(2)Differences in gene expression of TRAF6 in RAW264.7 cellsThe blank control group was compared with the drug-free serum group,PPAR-?agonist group,NF-KB blocker group and Tongguanwan drug-containing serum group.The expression of TRAF6 gene in the blank control group was significantly lower than that of the other four groups.Significant statistical significance(P ? 0.01).Comparing the drug-free serum group with the PPAR-? agonist group,NF-?B blocker group and Tongguanwan drug-containing serum group,the expression of TRAF6 gene in the drug-free serum group was significantly higher than that of the other three groups,and the difference was statistically significant Significance(P ? 0.01).Compared with the NF-?B blocker group and PPAR-? agonist group,the Tongguanwan drug-containing serum group showed lower TRAF6 gene expressionthan the NF-?B blocker group and higher than PPAR-? agonist group,the difference was statistically significant(P?0.05);NF-?B blocker group compared with PPAR-?agonist group,NF-?B blocker group TRAF6 gene expression was significantly higher than PPAR-The difference in the gamma agonist group was statistically significant(P?0.01).In conclusion:1.In the RAW264.7 cell inflammation model induced by LPS,the expression of PPAR-? gene in the drug-containing serum group of Tongguan Pill is significantly higher than that of the drug-free serum group,while the expression of PPAR-? in the drug-containing serum group of Tongguan Pill is slightly lower than that of PPARThe ?-agonist group,and the Tongguan Wan-containing serum group and PPAR-?agonist group had significantly higher PPAR-? expression than the NF-?B blocker group,suggesting that Tongguan Wan may inhibit NF by activating PPAR-?-KB inflammation signaling pathway plays an anti-inflammatory and bacteriostatic effect,and is one of the mechanisms of Tongguanwan in the treatment of SOI.2.In the model of LPS-induced RAW264.7 cell inflammation,the expression of TRAF6 gene in the drug-containing serum group of Tongguan Pill was significantly lower than that in the drug-free serum group and NF-?B blocker group,and slightly higher than that of the PPAR-? agonist group.It is suggested that Tongguan Pill may play an anti-inflammatory and bacteriostatic role by inhibiting the expression of TRAF6 gene and NF-?B inflammation signaling pathway,so as to achieve the purpose of treating infertility due to inflammatory obstruction of fallopian tube.Kind of mechanism. |
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