| Objective:(1)To explore the concentration and time of the protective effect of isoliquiritigenin on amiodarone-induced phlebitis through in vitro experiments.(2)To explore the effect of is isoliquiritigenin on phlebitis caused by amiodarone in vitro.(3)To explore the possible mechanism of the effectiveness of isoliquiritigenin.Methods:(1)Experiment 1:(1)Human umbilical vein endothelial cells were cultured for 12 h,24h,48 h and 72 h at different concentrations of isoliquiritigenin.The concentration and time of isoliquiritigenin.Without cell damage were screened.Set the concentration gradient(0,5 μmol/L,10 μmol/L,20 μmol/L,40 μmol/L,80 μmol/L,100 μmol/L).(2)After pretreatment of HUVEC in the concentration range of isoliquiritigenin without damage to cells for 1h,amiodarone(30 μmol/L)treated cells for 24 hours to create a model of phlebitis induced by amiodarone.The concentration and time of isoliquiritigenin without damage to amiodarone-induced phlebitis were screened.(2)Experiment 2: To explore the effect of isoliquiritigenin on amiodarone-induced phlebitis.According to the above experimental results,human umbilical vein endothelial cells were divided into three groups,NC(normal group)and AM(amiodarone group 30μmol/L treatment for 24 hours),ISL pretreatment group(isoliquiritigenin 10 μmol/L pretreatment for 1 hour and then amiodarone treatment for 24 hours).(1)The migration ability of cells in three groups at 0h,6h,12 h and 24 h was detected by scratch test.(2)The apoptosis rate of the three groups was detected by flow cytometry.(3)The total length and total number of branches of angiogenesis in the three groups at 2h,4h,6h and 24 h were detected in the angiogenic experiment.(4)Three groups of inflammatory factors(TNF-α m RNA,IL-1β m RNA,IL-6 m RNA,IL-10 m RNA)and(ICAM-1 m RNA,VCAM-1 m RNA).(5)The expression of proliferation-related gene and protein PCNA,apoptosis-related gene and protein Casepase-3,vascular growth factor-related gene and protein VEGF were detected by PCR and Western-blot respectively.(3)Experiment 3: To explore the possible mechanism of the protective effect of isoliquiritigenin on amiodarone-induced phlebitis.Western-blot was used to detect the expression of three groups of NF-κB signal pathway related protein NF-κBp65,p-NF-κBp65.Results:(1)(1)At 12 h,the concentration range of isoliquiritigenin did not significantly damage the cells.At 24 h,48h and 72h(5μmol/L,10μmol/L,20μmol/L)the concentration of isoliquiritigenin did not damage the cells.(2)Isoliquiritigenin can play the protective role of amiodarone-induced phlebitis after pretreatment with 10μmol/L for 1 hour and treatment with amiodarone for 24 hours.(2)(1)At 6h,there was no significant difference in cell migration rate between the isoliquiritigenin pretreatment group and the amiodarone group(P>0.05).The cell migration rate increased at 12 h and 24h(P<0.05).(2)Compared with the amiodarone group,the apoptosis rate of the cells pretreated with isoliquiritigenin decreased(P<0.05).(3)Compared with the amiodarone group,the total length and total branches of neovascularization increased with the increase of time(P<0.05).At 24 h,the blood vessels produced by cells in each group were scorched,the difference was not significant,and there was no statistical significance(P>0.05).(4)The inflammatory factor gene TNF-α,IL-1β,the expression of IL-6 was significantly decreased(P<0.05).The expression of anti-inflammatory factor gene(IL-10)was significantly increased(P<0.05).(5)The expression of proliferation-related gene and protein PCNA,apoptosis-related gene and protein Casepase-3,vascular growth factor-related gene and protein VEGF were detected by PCR and Western-blot respectively.(3)Compared with the amiodarone group,the expression of NF-κBp65 of the signal pathway related protein was not significant(P>0.05),and the expression of p-NF-κBp65was significantly decreased(P<0.05).Conclusion:(1)The concentration and time of the protective effect of isoliquiritigenin on amiodarone-induced phlebitis are:(10 μmol/L pretreatment for 1 hour).(2)The protective effect of isoliquiritigenin on amiodarone-induced phlebitis in vitro is mainly through promoting the proliferation,migration,angiogenesis and inhibiting the apoptosis of damaged endothelial cells.(3)The protective mechanism of isoliquiritigenin on phlebitis is partly through the down-regulation of NF-κB pathway works. |
