| Objective:Based on the CaN/NFAT signaling pathway,explore the effect of Xuefu Zhuyu Decoction on human umbilical vein endothelial cells after injury,explore the effect of Xuefu Zhuyu Decoction on the pathological changes of rat carotid artery injury model,the signaling pathway markers and inflammatory factors Related effects,clarify the possible mechanism of Xuefu Zhuyu Decoction in the treatment of vascular intimal injury,and enrich the therapeutic scope and pharmacological mechanism of Xuefu Zhuyu Decoction.Methods:1.Effects of Xuefu Zhuyu decoction on human umbilical vein endothelial cells after injury through CaN/NFAT signaling pathway.Human umbilical vein endothelial cells were cultured in BEGM medium.The experimental drug was Xuefu Zhuyu Decoction freeze-dried powder,which was formulated with serum-free medium BEGM into 100mg/ml drug-containing medium.The medicine-containing culture medium is thoroughly mixed on a vortex apparatus,sterilized and filtered with a 0.22?m filter membrane,and then placed in an autoclaved EP tube,and then refrigerated at 4?until use.The effect of Xuefu Zhuyu Decoction with different concentrations on HUVECs cell proliferation was tested by MTS experiment.Divided into normal group,drug-containing medium group?concentrations 2.5?g/ml,6.25?g/ml,12.5?g/ml,25?g/ml,37.5?g/ml,50?g/ml?,cultured for 24h and 48h,respectively Check the OD value.Cell scratch test was used to detect the effect of Xuefu Zhuyu Decoction on the migration and repair of HUVECs cells.They were divided into control group,Xuefu Zhuyu Decoction 12.5?g/ml group,Xuefu Zhuyu Decoction 25?g/ml group,and Xuefu Zhuyu Decoction.50?g/ml group.The 6-well plate was sealed,placed in a 37?,5%CO2incubator,and photographed under the microscope at 0,24,and 48 hours?×10?.Real-time PCR was used to detect the expression of CaN/NFAT-related factors at 24 h and 48 h after physical injury in HUVECs cells.2.In vivo study on regulation of Xuefu Zhuyu decoction by CaN/NFAT signaling pathway after balloon injury of common carotid artery in rats.Experiment 1:72 healthy SD rats were divided into 6 groups,sham operation group,model group,tacrolimus group,Xuefu Zhuyu Decoction high-dose group,Xuefu Zhuyu Decoction medium-dose group,Xuefu Zhuyu Decoction low-dose group.In the sham-operated group,only the external carotid artery was ligated,and no balloon dilatation injury was performed.The remaining groups of rats underwent balloon injury to cause damage to the rat carotid artery.All rats were given the drug for 28 days.The rat carotid artery was stained with HE to observe the pathological changes in each group,and the serum of each group was collected to detect the serum MCP-1 by ELISA.Experiment 2:The carotid artery tissues of rats were collected,and the CaN content was detected by immunohistochemistry.The expression levels of CaN,NFATc2,NFATc3,IL-2,TNF-?,COX2,and S100A4 mRNA in the carotid artery of each group were detected by Real-time PCR The protein expression levels of CaN,NFATc2,NFATc3,IL-2,TNF-?,COX2,and S100A4 in the carotid artery of each group were detected by Western blot method.Results:1.Effects of Xuefu Zhuyu Decoction on Human Umbilical Vein Endothelial Cells After CaN/NFAT Signal PathwayThe results of MTS cell proliferation experiments showed that the concentrations of6.25?g/ml,12.5?g/ml,and 25?g/ml of Xuefu Zhuyu Decoction at 24h and 48h can promote cell proliferation.By 48h,the performance of Xuefu Zhuyu Decoction in 50?g/ml group It can inhibit cell proliferation.The results of cell scratch test showed that at 24h and48h,Xuefu Zhuyu Decoction had the effects of promoting cell migration and repair at concentrations of 12.5?g/ml and 25?g/ml,and the 50?g/ml group had the effect of inhibiting cell migration at 48h.Real-time PCR results showed that the administration of Xuefu Zhuyu Decoction increased the expression of CaN/NFAT-related factors for 24 hours,and the expression of related factors decreased after 48 hours of administration,but there was no statistical difference.2.In vivo study on regulation of Xuefu Zhuyu decoction by CaN/NFAT signaling pathway after balloon injury of common carotid artery in rats.Experiment 1:The results of carotid hematoxylin and eosin staining showed that:compared with the model group,the high-dose group of Xuefu Zhuyu decoction had a very small amount of angiogenic intima,which was extremely thin,with a small amount of inflammatory cell infiltration,disordered arrangement of smooth muscle cells,and significantly improved and alleviated the uneven elastic fiber board compared with the model group.The results of serum MCP-1 showed that compared with the sham operation group,the levels of serum MCP-1 in rats in each group were extremely significant(**P<0.01).There was no significant difference in serum content between the tacrolimus group and the middle-dose Chinese medicine group?P>0.05?,and the content of the high-dose Chinese medicine group was lower than that of the tacrolimus group(&&P<0.01).Experiment 2:Immunohistochemical detection of CaN expression in rat carotid artery tissue sections.Brown yellow particles were rarely seen in the sham operation group,the endometrium was significantly thickened in the model group,CaN expression was positive,and several brown yellow particles were visible;tacrolimus group,low-dose Chinese medicine group,middle-dose Chinese medicine group,and high-dose Chinese medicine The brown-yellow granules in the group were significantly reduced compared with the model group,suggesting that the expression of CaN in the common carotid artery tissue of the rats decreased after drug intervention.The area ratio of CaN-positive areas in the model group was significantly higher than that in the sham operation group(##P<0.01);the area ratio of CaN-positive areas in the common carotid artery tissue of each drug intervention group was significantly lower than that of the model group(**P<0.01).Effects of Xuefu Zhuyu Decoction on CaN/NFAT signaling pathway-related factors in common carotid artery tissue of rats:Compared with the sham operation group,the gene and protein expression levels of CaN/NFAT signaling pathway marker molecules CaN,NFATc2 and NFATc3 in the model group were significantly up-regulated?P<0.01?,and the gene and protein expression levels of the marker molecules were significantly down-regulated in the high-dose group of Xuefu Zhuyu Decoction compared with the model group?P<0.01?.Effects of Xuefu Zhuyu Decoction on inflammation-related factors in common carotid artery of rats after balloon injury:Compared with the sham group,the expression levels of genes and proteins of inflammatory factors IL-2,TNF-?and COX2 in the model group were significantly upregulated(**P<0.01).Compared with the model group,the expression levels of genes and proteins of marker molecules were significantly down-regulated in the high-dose group of Xuefu Zhuyu decoction(##P<0.01).Conclusion:1.Xuefu Zhuyu decoction promoted the proliferation and migration of HUVECs cells at a certain concentration of dose,while it inhibited the proliferation and migration of HUVECs cells as the concentration increased,and the inhibition was manifested with the extension of drug action time.2.Xuefu Zhuyu Decoction can promote the CaN/NFAT signal pathway in the early stage after HUVECs injury,and the inhibition trend will be reflected with the prolonged action of the drug.3.Xuefu Zhuyu Decoction can effectively improve the intimal proliferation of common carotid artery in rats after balloon injury,and provide a basis for the study of restenosis.4.Xuefu Zhuyu Decoction can reduce the levels of IL-2,TNF-?,and COX2 in the common carotid arteries of rats,reduce the content of MCP-1 in serum,and have an anti-inflammatory effect.5.Xuefu Zhuyu Decoction can inhibit the expression of CaN,NFATc2,NFATc3 in the common carotid arteries of rats,suggesting that Xuefu Zhuyu Decoction can inhibit the intimal over-proliferation through the CaN/NFAT signal pathway to prevent restenosis. |
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