| Purpose: To study the preparation of Yiqihuoxue compound serum and the culture and identification of human umbilical vein endothelial cells,and to observe the effect of serum containing Yiqihuoxue compound on toll-like receptor 4 and its downstream signals,and Effects of LOX-1,TNF-a,ICAM-1.Study the mechanism and target of Yiqi Huoxue Recipe from the aspects of protein and gene,and provide theoretical experimental basis and new ideas for prevention and treatment of traditional Chinese medicine.Material and method:1.20 New Zealand white rabbits were randomly divided into blank group and Chinese medicine middle,low and high concentration groups.The number of each group was 5,totaling 4 groups,and the benefits were low,medium and high.Qiqihuoxue compound and normal saline were intragastrically administered once a day for 7 days.After 2 hours of the last intragastric administration,cardiac blood collection,centrifugation,and supernatant removal were performed,and the same group of drug-containing serum was mixed,and then sterile filtered using a 0.22 μm disposable filter,and placed at-80 ℃ to leave.use.2.Fresh umbilical cord for healthy infants,placed in a sterile environment,isolated by trypsinization to obtain HUVECs,subcultured at 5% CO2,37 ℃,and with factor VIII-related antigen Identification.3.Stimulate HUVECs with LPS(1μg/ml),activate TLR4 and downstream signal transduction pathway,and intervene with Yiqihuoxue compound serum containing spirulina and atorvastatin at low,medium and high concentrations,and set blank control.Groups and model groups were harvested after 24 h.The expression of TLR4,MyD88,TRAF-6,TRAM,TRIF and NF-KB mRNA and protein were detected by Real time PCR and Western blot.4.Stimulate HUVECs with LPS(1μg/ml),activate LOX-1,TNF-a and ICAM-1,and then intervene with Yiqi Huoxue Compound medicated serum with low,medium and high concentrations and atorvastatin.A blank control group and a model group were set,and the cells were collected after 24 hours.The mRNA and protein expressions of LOX-1,TNF-a and ICAM-1 were detected by Real time PCR and Western blot,respectively.5.Statistical processing: All data were statistically analyzed by SPSS22.0 statistical software,and expressed as mean ± standard deviation(X(-)±S).Each group was compared with each other using one-way analysis of variance(one-way)ANOVA),P < 0.05 was considered statistically significant.Results:1.The drug-containing serum is light yellow and clear liquid after centrifugation,and about 20-25 ml of serum can be obtained per 50 ml of blood.A total of 4 groups of serum were obtained,namely,Yiqihuoxue compound high concentration group containing drug serum,medium concentration group containing drug serum,low concentration group containing drug serum and blank control group serum.2.The cells cultured to the fused state were arranged in a single layer of "paving stone",and most of the cells were positive for the factor VIII-related antigen by immunofluorescence staining with the factor VIII-related antigen.3.After 24 hours of LPS stimulation,the expression of TLR4 and downstream signal transduction pathways in the model group,MyD88,TRAF-6 and NF-κB mRNA and protein were significantly increased(compared with the blank control group P<0.01);After intervention,the TLR4,MyD88,TRAF-6 and NF-κB mRNA and protein were significantly decreased in each treatment group,while the Western medicine control group had a significant effect(compared with the model group P<0.01);the three Chinese medicine groups were all on TLR4 and MyD88.The expression of TRAF-6 and NF-κB mRNA and protein were significantly inhibited(P<0.01 or P<0.05 compared with the model group),and the high concentration group was the best in the three concentration groups.4.After 24 hours of LPS stimulation,the expression of TRAM and TRIF mRNA and protein in the TLR4 downstream signal transduction pathway was significantly increased in the model group(compared with the blank control group P<0.01);through the drug intervention,the TRAM of each treatment group There was no significant decrease in TRIF mRNA and protein expression(P>0.05 vs.model group).5 After 24 hours of LPS stimulation,the expression of LOX-1,TNF-α,ICAM-1 mRNA and protein in the model group was significantly increased(compared with the blank control group P<0.01);the cells in each treatment group were treated with drugs.-1,TNF-α,ICAM-1 mRNA and protein expression were significantly decreased,of which Western medicine control group had significant effect(P<0.01 compared with model group);3 Chinese medicine groups were all on LOX-1,TNF-α,ICAM-1 mRNA and protein expression were significantly inhibited(P<0.01 or P<0.05 compared with the model group),and the high concentration group was the best in the three concentration groups.Conclusion:1.Obtaining drug-containing serum with different drug concentrations,in order to provide raw materials for further in vitro experimental research.2 Using trypsin digestion to obtain EC in human umbilical vein,according to human factor VIII-related antigen immunohistochemistry,it was proved that the experiment obtained EC.3 LPS can activate the expression of MyD88,TRAF-6 and NF-KB mRNA and protein in TLR4 and its downstream MyD88-dependent signal transduction pathway,after 24 hours of intervention,Yiqi Huoxue Recipe could inhibit the expression of TLR4,MyD88,TRAF-6 and NF-KB mRNA and protein,it is suggested that Yiqi Huoxue Recipe can inhibit the expression of mRNA and protein of TLR4 and its downstream MyD88-dependent signal transduction pathway.4 Yiqi Huoxue Recipe contains no significant inhibitory effect on TRAM and TRIF mRNA and protein expression,indicating that Yiqi Huoxue Recipe has no inhibitory effect on MyD88-independent signal transduction pathway.5 Yiqi Huoxue Recipe can inhibit the adhesion of LOX-1,TNF-α and ICAM-1 mRNA and protein,and reduce the adhesion of monocytes to VEC,which may be one of the mechanisms of AS prevention and treatment.This suppression effect needs to be expressed after the concentration reaches a certain value.6 LPS can activate the expression of LOX-1,TNF-α and ICAM-1 mRNA and protein.After 24 hours of intervention,Yiqi Huoxue Recipe can prevent the appearance of the three,and reduce the adhesion of monocytes to VEC.One of the mechanisms for the prevention and treatment of AS.This suppression effect needs to be expressed after the concentration reaches a certain value. |
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