| Object:The objective of the study was to elucidate the effect of Anzi mixture on Toll-like receptor 2(TLR2)and Toll-ike receptor 4(TLR4)and the Tumornecrosis factor-?(TNF-?)and TLR4/myeloid differentiation factor 88(MyD88)/nuclear factor-?B(NF-?B)signaling pathway in maternal-fetal interface with antiphospholipid antibodies(APA)and to explore the therapeutic mechanism from the expression of each factor protein and mRNA in the signaling pathway,in order to provide a new theoretical and experimental basis for the treatment of APA positive abortion.To establish the HPLC fingerprint of Anzi mixture and make the basis for exploring the spectrum-activity relationship and the pharmacodynamic material basis of TLR4/MyD88/NF-?B signaling pathway.Methods:1.Efeect of Anzi mixture with single dose on TLR2,TLR4,TNF-a:Female BALB/c mice were randomly divided into blank control group,model group,aspirin group(positive control,0.0195g/kg)and Anzi mixture single dose group(37.7g/kg),6 mice in each group.Except the blank control group,other groups of mice were immuned with human ?2-glycoprotein I(?2GP I)to establish APA positive abortion model.From the first day of pregnancy,treatment groups were given relevant medicine intragastrically,and blank control group and model group were given constant volume of distilled water intragastrically,once a day,for consecutive 9d.To investigate fetal mice and placenta weight and calculate the embryo absorption rate.The levels of ACA,anti-?2GP I antibody and TNF-? in peripheral blood of mice were detected by ELISA.Real-time quantitative PCR was used to determine TLR2 and TLR4 mRNA levels in placental tissue.The expression of TLR2 and TLR4 protein in placental tissue was detected by immunohistochemistry.2.Effect of multi-dose of Anzi mixture on TLR4/MyD88/NF-?B signaling pathway.Female BALB/c mice were randomly divided into blank control group,model group,aspirin group(positive control,0.0195g/kg)and Anzi mixture low-dose,medium-dose and high-dose groups(37.7,75.4,150.8g/kg).Each group of 10 mice.Except for blank control group,other groups were given human ?2-glycoprotein I as derivant to establish APA positive abortion model.From the first day of pregnancy,treatment groups were given relevant medicine intragastrically,and blank control group and model group were given constant volume of distilled water intragastrically,once a day,for consecutive 9d.The expressions of TLR4,MD2,MyD88,NF-?B mRNA and protein in placenta were measured by real-time fluorescence quantitative polymerase chain reaction(RT-PCR)and immunohistochemistry.3.Establishment of HPLC Fingerprint of Anzi mixture:HPLC was applied with a column of YMC-Pack ODS-A(4.6mm×250mm,5?m).The mobile phase consisted of formaldehyde and 0.01%formaldehyde solution with gradient elution.The flow rate was 1.0mLˇmin-1.The column temperature was 30? and wavelength detection was 254nm.Datas were analyzed by The Similarity Calculation Software of Fingerprint Chromatography of Traditional Chinese Medicine(2004A)?Results:1.The mRNA and protein levels of TLR2?TLR4 in placenta tissue of mice from model group were significantly increased compared to that of mice from blank control group(P<0.01 or P<0.001).Compared with the model group,Anzi mixture group could increase the weight of fetal mice and placenta(P<0.05 or P<0.01)and significantly reduced embryo resorption rate and decreased the concentration of ACA(P<0.05).Compared with the model group,concentration of the anti-?2GP I antibody and TNF-?were decreased(P<0.05 or P<0.001),at the same time mRNA level of TLR2 and TLR4 were down-regulated and TLR2 and TLR4 proteins were in lower expression in both Anzi mixture and the aspirin group.(P<0.05 or P<0.01).Compared with the aspirin group,the decline of anti-?2GP I antibody concentration in Anzi mixture group was more effective(P<0.01).2.Compared with blank control group,mRNA and protein expression of TLR4,MD2,MyD88 and NF-?B in placental tissue were increased markedly in the model group(P<0.01).Compared with model group,mRNA and protein expression of TLR4,MD2 and MyD88 in aspirin group and Anzi mixture low-dose and medium-dose groups were decreased significantly as well as the protein expression of TLR4 in Anzi mixture high-dose group and the protein expression of NF-?B in all medicine groups(P<0.05 or P<0.01).mRNA expression of TLR4 and MD2 and the protein expression of MD2 and MyD88 in Anzi mixture low-dose groups were lower than those in aspirin group(P<0.05 or P<0.01).3.Fingerprint of 10 batches of Anzi mixture was established and 20 peaks were regarded as the common peaks.The peaks in fingerprint of 10 patch of Anzi mixture were well separated,which reaches the standard of fingerprint.Conclusions:Anzi mixture can inhibit upstream key promoter TLR2 and TLR4,which are in the TLR2 and TLR4 signal transduction pathway and inhibits TLR4/MyD88/NF-?B signaling pathway in maternal-fetal interface of APA positive miscarriage mice,reduces the release of inflammatory factor,which may be one of its action mechanisms.At the same time the best dose is the clinical dose of normal use.The HPLC fingerprint of Anzi mixture are established,and the method is reliable and simple. |
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