| Objective:To analyze the material basis of hematoxylin efficacy by network pharmacology,and to predict its targets related to the treatment of vascular restenosis,which was verified by experimental study.The model of vascular smooth muscle cell injury induced by TNF-? and the rat model of vascular restenosis were established.the anti-vascular restenosis effect and mechanism of hematoxylin extract were observed by intervention with hematoxylin extract.Methods:The first part: network pharmacological study on the effect of hematoxylin extract on vascular restenosis: the material basis and mechanism of hematoxylin extract intervention in vascular restenosis were studied by network pharmacology.The main active components and potential therapeutic targets were calculated.the second part: the effect of hematoxylin extract on the proliferation and migration of vascular smooth muscle cells in vitro.Vascular smooth muscle cells(VSMCs)were divided into 5 groups:blank group,model group,PPAR agonist group,PPAR inhibitor group and hematoxylin group.Vascular smooth muscle cells(VSMCs)were treated with TNF-? for 12 h to induce injury of vascular smooth muscle cells(VSMCs).The cells in the blank group were cultured normally for 24 hours,and the cells in the model group were cultured for 12 hours after the model was replicated.PPAR agonist group,PPAR inhibitor group and hematoxylin group were treated with PPAR agonist,PPAR inhibitor and hematoxylin extract for 12 hours.The morphological changes of cells in each group were observed,cell proliferation was observed by MTT assay,apoptosis was observed by flow cytometry,IL-2,IL-4 level in supernatant was detected by Elisa method,and the expression of PPAR-? and NFAT-4 protein was detected by Western-blot method.The third part: the effect of hematoxylin extract on vascular restenosis and its molecular mechanism.In this experiment,50 SD rats were randomly divided into 5 groups: 10 rats as blank group and 10 rats as sham operation group.The model of femoral artery restenosis was established by wire contact in 20 rats,and then divided into model group(n = 10)and hematoxylin group(n = 10).The other 10 rats were treated with the same method for 1 month in advance.after 28 days of modeling,the rats in each group were given intragastrically for 28 days.At the end of gavage,HE staining was used to detect the morphology of blood vessels.The contents of PPAR-? and NFAT-4 protein in vascular smooth muscle cells were detected by immunohistochemistry,and the levels of IL-2,IL-4,TNF-? and IFN-? in serum of rats were detected by Elisa method.The mRNA and protein expressions of PPAR-? and NFAT-4 in the diseased femoral artery were detected by RT-PCR and Western-blot.Results:1.Network pharmacology studies have shown that the anti-vascular restenosis mechanism of hematoxylin extract may play an anti-vascular restenosis effect by affecting PPAR network related factors.2.Apoptosis and proliferation results showed that TNF-? significantly increased vascular smooth muscle cell proliferation,late apoptosis and total apoptotic rate compared with the blank group,compared with the model group,hematoxylin group and PPAR activation.The agent group can inhibit the proliferation of vascular smooth muscle cells,reduce the late apoptosis and total apoptotic rate,the difference was statistically significant(P<0.05).3.Cell supernatant Elisa results showed: Compared with the blank group,the IL-2 and IL-4 levels in the model group were increased,the difference was statistically significant(P<0.05);compared with the model group,the hematoxylin group and PPAR The levels of IL-2 and IL-4 in the activator group were decreased,and the difference was statistically significant(P<0.05).4.The results of Western Blot showed that the expression of PPAR-? and NFAT-4 protein in the model group was significantly higher than that in theblank group(P<0.05).Compared with the model group,the hematoxylin hematoxylin group and PPAR were activated.The expression of PPAR-?protein and the expression of NFAT-4 protein were decreased in the agent group,and the difference was statistically significant(P<0.05).5.The results of pathological morphology of rat femoral artery showed that the endothelial cells and subendothelial cells in the model group were edema degeneration,the middle smooth muscle cells were disordered,the blood vessels were obviously narrowed,the endothelial cells in the hematoxylin group and the prevention group were slightly swollen,and the middle smooth muscle cells were arranged.Disorders,mild stenosis of the blood vessels,compared with the model group,pathological damage was alleviated;the lesions in the prevention group were further alleviated than those in the hematoxylin group.6.The results of femoral artery immunohistochemistry showed that there was a small amount of PPAR-? expression in the middle layer of normal blood vessels,and the expression of PPAR-? was significantly increased in the model group after vascular injury.The expression of PPAR-? in the neointimal,hematoxylin group The expression of PPAR-? was increased in the prophylactic and prophylactic groups,and a large amount of PPAR-?expression was observed in the neointimal.7.Serum Elisa results showed that the levels of IL-2,IL-4,TNF-? and IFN-? in the model group were significantly higher than those in the blank group(P<0.05).Comparing,the levels of IL-2,IL-4,TNF-? and IFN-? in the hematoxylin group and the prevention group were significantly lower(P<0.05).Compared with the hematoxylin group,the IL-2 in the prevention group.IL-4,TNF-? and IFN-? levels were decreased,and the difference was statistically significant(P<0.05).8.The results of Western Blot in the femoral artery of rats showed that the PPAR-? and NFAT-4 protein levels in the model group were significantly higher than those in the blank group(P<0.05).Compared with the model group,the hematoxylin group and The expression of PPAR-? protein was increased and the content of NFAT-4 protein was decreased in the prevention group(P<0.05).Compared with the hematoxylin group,the expression of PPAR-? protein was increased in the prevention group,NFAT-4 protein.The content was decreased,and the difference was statistically significant(P<0.05).9.The results of Real Time PCR of rat femoral artery showed that the levels of PPAR-? and NFAT-4 in the model group were significantly higher than those in the blank group(P<0.05).Compared with the model group,the hematoxylin group and The PPAR-? mRNA content and NFAT-4 mRNAcontent in the prevention group were significantly lower(P<0.05).Compared with the hematoxylin group,the PPAR-? mRNA content and NFAT-4 mRNA content in the prevention group were decreased.Statistically significant(P <0.05).Conclusion:1.Through the network pharmacology study,it is found that the anti-restenosis mechanism of hematoxylin extract may be through the influence of PPAR network-related factors.2.Hematoxylin extract can decrease the proliferation rate of vascular smooth muscle cells induced by TNF-?,reduce the apoptosis rate and decrease the content of IL-2,IL-4 in the supernatant of vascular smooth muscle cells.3.Hematoxylin extract could improve the pathological injury of diseased femoral artery in vascular restenosis model rats,and decrease the levels of serum IL-2,IL-4,TNF-? and IFN-? in vascular restenosis model rats.4.Hematoxylin extract can down-regulate the expression of PPAR-? and NFAT-4 in vascular restenosis model rats and TNF-?-induced vascular smooth muscle cell injury model,which may be one of the mechanisms of prevention and treatment of vascular restenosis. |
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