ABSTRACT
Objective To explore the influence of Rapamycin (RAPA) on apoptosis of acute lymphoblastic leukemia EU-4 cells induced by Methotrexate (MTX).Methods EU-4 cells were pretreated 0.5 h with 5 pμg/L,10 μg/L,25 μg/L,50 μg/L and 100 μg/L RAPA.RAPA untreated group was set up as control group.The cells were collected and the expression of LC-3 was assayed by using Western blot.The apoptosis of EU-4 cells was detected by using flow cytometry (FCM).The effects of different concentrations of RAPA pretreatment on autophagy and apoptosis of EU-4 cells were observed,and the pretreatment concentration of RAPA was determined.EU-4 cells were divided into RAPA pretreatment group and untreated group.The cells were treated with 0.05 μmol/L,0.10 pμmol/L and 0.20 μmol/L MTX for 24 h,respectively.The apoptotic rate was detected by using FCM.Western blot was performed to test the expression of LC-3 protein,while the absorbance(A562) was measured by using microplate reader,and the protein concentration in the sample was calculated according to the standard curve.Results The apoptotic rates of EU-4 cells in the control group and the different concentrations of PAPA pretreatment group showed that the control group,5 μg/L,10 μg/L,25 μg/L,50 μg/L and 100 μg/L RAPA were (7.51 ±0.32)％,(7.33 ±0.41)％,(7.71 ± 0.51) ％,(7.63 ± 0.38) ％,(7.80 ± 0.43) ％ and (16.66 ± 0.87) ％,respectively.The apoptotic rates of EU-4 cells in the 5 μg/L,10 μg/L,25 μg/L and 50 μg/L PAPA pretreatment group had no significant difference from those in the control group(t =0.427,-0.417,-0.297,-3.561,all P ＞ 0.05).The apoptotic rate of EU-4 cells in 100 μg/L PAPA pretreatment group was significantly higher than that in control group,and the difference was significant (t =-28.815,P ＜ 0.01).Combined with the results of Western blot and FCM,50 μg/L was used as the pretreatment of PAPA.The apoptosis rates of EU-4 cells in PAPA pretreatment group were (50.23 ± 2.11) ％,(66.88 ± 2.89) ％ and (73.11 ± 2.67) ％ after treated with 0.05 μmol/L,0.10 μmol/L and 0.20 μmol/L MTX for 24 h,respectively.The apoptotic rates of EU-4 cells in PAPA unpretreatment group were (22.53 ± 1.67) ％,(42.82 ± 2.26) ％ and (53.22 ± 1.93)％ after treated with 0.05 μmol/L,0.10 μmol/L and 0.20 μmol/L MTX for 24 h,respectively.The apoptotic rates of EU-4 cells in RAPA pretreatment group were significantly higher than those in untreated group in the same concentration of MTX treatment after 24 h,and the differences were significant(t =12.693,66.148,14.429;all P ＜ 0.01).Conclusion With RAPA pretreatment,relative low dose MTX can induce a great deal of acute lymphoblastic leukemia EU-4 cells apoptosis.
ABSTRACT
Objective To investigate the effects of angelica solution on chronic hypoxic pulmonary hypertension in rats.Methods Thirty SD rats were randomized into normoxic group,hypoxic group and angelica solution-protected group.The model of rat chronic hypoxic pulmonary hypertension was made by method of isobaric hypoxia.Angelica solution were injected before hypoxia,while the other two groups were injected normal saline.After 28d of hypoxia,pulmonary artery pressure were measured.Expressions of proliferating cell nuclear antigen (PCNA) and inducible nitric oxide synthase (iNOS) in pulmonary artery were detected by immunohistochemical staining.The index of wall thickness of rat pulmonary arteriole-percentage of the wall area in the total vascular area(wA%) were measured by a computerized image analyzer.Results The mean pulmonary artery pressure (mPAP) of normoxic group,hypoxic group and angelica solution-protected group were10.50±1.90,35.36±9.11,18.32±2.30 (mm Hg);wA% of the three groups were 52.71±5.16,82.38±8.43,64.58±9.54 (%),mPAP and wA% were significantly higher in the hypoxic group than those in the normoxic group (P<0.01) and angelica solution-protected group (P<0.01).PCNA expression of normoxic group,hypoxic group and angelica solution-protected group were 3.15±1.10,24.50±5.72,12.67±3.46 (%).The PCNA expression in the pulmonary artery was significantly higher in the hypoxic group than those in the normoxic group (P<0.01) and in the angelica solution-protected group (P<0.01).iNOS expression of normoxic group,hypoxic group and angelica solution-protected group were 2.13±1.01,17.33±3.53,37.50±7.04 (%).iNOS expression in the pulmonary artery was higher in the hypoxic group than those in normoxic group (P<0.01),and angelica significantly increased iNOS expression in comparison with the normoxic and hypoxic groups (P<0.01).Conclusion Angelica solution alleviates chronically hypoxia induced pulmonary hypertension in rats by inhibiting the espression of PCNA in pulmonary artery and up-regulating the expression of iNOS.