| Objective:To study the pharmacodynamics of ruanjian qingmai granules on lower limb ischemic mice.In animal experiments,the blood flow test of ruanjian qingmai granules o n lower limb ischemic mice and the expression levels of VEGF,CD31 and PDGF were studied by making an experimental model of lower limb ischemic mice.The protective effect of ruanjian qingmai granule on hypoxic injury of vascular endothelial cells was studied by the preparation of drug serum containing ruanjian qingmai granule.Methods:1.Experiments on animals:C57BL/6 male mice were randomly divided into normal group and model group,model group with surgical ligation lower limb ischemic mice model was established,after the success of the model the random divided into soft firm qing pulse particle high,medium and low three dose groups,respectively by gastric gavage soft firm clear veins,0.185 g·kg-1·d-1,0.370 g·kg-1·d-1,and 0.740g·kg-1·d-1 solution,1 times a day,for 28 d give medicine grain of angiogenesis.The blood flow was measured by laser speckle flow imager.The expression levels of VEGF and CD31 in mice were observed by immunofluorescence staining.VEGF and PDGF in serum were detected by ELISA.2.Cell experiment:SD rats were randomly divided into control group and Ruanjianqingmai Granule low,medium and high dose groups.0.9%Na Cl solution and 0.275 g kg-1 d-1,0.550 g kg-1 d-1,1.100 g kg-1 D-1 Ruanjianqingmai Granule solution were intragastrically administered twice a day for 10 Cardiac blood was collected after the last administration to prepare drug-containing serum.Human umbilical vein endothelial cells?HUVECs?were divided into blank,model and low,medium and high dose groups of Ruanjianqingmai granules.The blank group was cultured in a complete medium without drug-containing serum and placed in a conventional environment?21%O 2?;the model group cells were added to the prepared drug-containing serum of the control group,and the cells of each dose group of Ruanjian Qingmai Granule were added to the prepared drug-containing serum of the corresponding groups respectively.Except for the blank group,the other groups were cultured in incubator containing 95%N2+5%CO2 to establish hypoxic injury model.MTT assay was used to detect cell viability;Hochest33342 and Annexin V-FITC/PI staining were used to detect cell apoptosis;flow cytometry was used to detect mitochondrial membrane potential?MMP?after Rho123 staining;and th e expression levels of TL1A and Caspase-3 were detected by PCR and Western blot,respectively.Results:Experiments on animals:1.After the general feed was fed and the ischemic model of lower limbs was established after the operation,the drug was continuously administered until the 28th day after the successful modeling,and the lower limb blood flow of the ischemic mice was bserved.Compared with the model group,newborn collateral circulation can be formed in the low,medium and high dose groups of ruanjian qingmai granules.?2?The expression of vascular endothelial growth factor in ischemic mice of lower limbs:Compared with normal group,the expression of vascular endothelial growth factor in model group was less?P<0.01?;Compared with model group,Ruanjianqingmai Granule was better in low dose group than model group?P<0.05?,with dose dependence,and the middle and high dose groups of Ruanjianqingmai Granule were significantly higher than model group?P<0.01?.?3?The expression of CD31,a new marker of angiogenesis:Compared with the normal group,the expression of CD31 in the blood vessels of the model group was lower than that of the normal group?P<0.05?;Compared with the model group,the middle and high doses of Ruanjianqingmai Granule were better than those of the model group?P<0.05?,while the expression of CD31 in the low dose group was not significantly different from that of the model group,but the middle and high doses of Ruanjianqingmai Granule were better than those of the model group.The expression was significantly higher than that of model group?P<0.01?.?4?Serum expression of vascular endothelial growth factor:Compared with the normal group,the expression of vascular endothelial growth factor in the model group was lower than that in the normal group?P<0.05?;the expression of vascular endothelial growth factor in the model group and Ruanjianqingmai Granule at low,medium and high doses showed an increasing trend?P<0.05,P<0.01?;and the expression of vascular endothelial growth factor in the model group and Ruanjianqingmai Granule at medium and high doses was significantly higher than that in the Low dose group?P<0.01?.?5?The expression of PDGF in serum:Compared with the normal group,the expression of PDGF in the model group was lower than that in the normal group?P<0.05?,and the expression of PDGF in the middle,low and high dose groups increased gradually with the change of dose,and the expression of PDGF in the middle and high dose groups was better than that in the low dose group?P<0.01?.Cell experiment:?1?After 48 hours of hypoxia induction,the cell viability of HUVECs in the model group was significantly lower than that in the blank group?P<0.01?;compared with the model group,the cell viability in the medium and high dose groups of Ruanjianqingmai Granules was significantly higher?P<0.01?;and the cell viability in the medium and high dose groups was significantly higher than that in the low dose group?P<0.01?.?2?After 24 hours of hypoxia induction,the nuclear pyknosis and apoptotic bodies appeared in the model group,and the apoptotic rate was significantly higher than that in the blank group?P<0.01?;the nuclear pyknosis and apoptotic bodies in the Ruanjian Qingmai Granule groups were significantly reduced,and the apoptotic rate in the model group was significantly lower than that in the low dose group?P<0.05,P<0.01?;and the apoptotic rate in the middle and high dose groups was significantly lower than that in the low dose group?P<0.01?;?3?Compared with the blank group,the cell MMP in the model group was significantly lower?P<0.01?;compared with the model group,the cell MMP in each dose group of Ruanjianqingmai Granule was significantly higher?P<0.05,P<0.01?;and the cell MMP in the medium and high dose group was significantly better than that in the low dose group?P<0.01?.?4?Compared with the blank group,the expression levels of TL1A and Caspase-3 in the model group were significantly higher?P<0.01?;compared with the model group,the expression levels of TL1A and Caspase-3 in each dose group of Ruanjian Qingmai Granule were significantly lower?P<0.05,P<0.01?;and the expression levels of TL1A and Caspase-3 in the medium and high dose groups were significantly lower than those in the low dose group?P<0.01?.Conclusion:Ruanjian qingmai granule can form a protective mechanism against hypoxia in ischemic mice.It can promote the expression of VEGF and CD31,promote the expression of VEGF and PDGF,promote the angiogenesis of lower limbs,and establish collateral circulation.The drug-containing serum of ruanjian qingmai granules can inhibit the apoptosis of HUVECs cells induced by hypoxia by stabilizing MMP,and the expression of its action mechanism may be related to the inhibition of the expression of TL1A and caspase-3. |
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