| OBJECTION Abnormal vascular smooth muscle cells (VSMCs) proliferation has a fundamental role in the pathogenesis of multiple cardiovascular diseases. Inhibition of the proliferation may be in favor of the prevention and treatment of the related diseases. The pharmacological effects of panaxynol, such as vasodilation, hypotension, antithrombogenesis and antiatherosclerosis, were well testified by experiments in vitro. However the effect of panaxynol on VSMCs proliferation is not known. The study was to examine the effect of panaxynol on fetal bovine serum (FBS)-induced proliferation of primary cultured Sprague-Dawley rat aortic smooth muscle cells (RASMCs) and the possible pathway. METHOD The 5～15th generation primary cultured RASMCs were used in the study. Cells were seeded at a concentration of 0.1～1×105 cells/well in plates and induced into synchronization by incubation with serum-free(0.5％FBS) dulbecco's modified eagle medium(DMEM) for 48h. Quiescent RASMCs were incubated respectively in 10% FBS DMEM as control group, 10% FBS DMEM supplemented with 0.1% dimethyl sulfide(DMSO) as vehicle group and 10% FBS DMEM supplemented with panaxynol(at concentration of 1,3,9×10-6 mol·L-1) as panaxynol groups. 1. Cell counting and double time. Cell viability was examined with trypan blue exclusion test and the cell number was counted at 0, 24, 48, 72h after the incubation using a hemocytometer. The results were shown with growth curve and the cell double times were calculated. 2. MTT (Methyl thiazolyl Tetrazolium) assay was employed to measure the activity of succinate dehydrogenase (SDH) of RASMCs mitochondrion. Quiescent cells were grouped and treated respectively for 72h and incubated subsequently with MTT solution supplemented for 4h. The absorbance value was measured with ELISA plate reader to reflect the activity of SDH. 3. The effect of panaxynol on DNA synthesis was studied by 3H-thymidine (3H-TdR) incorporation assay. Liquid scintillation counter was used to quantify CPM. 4. Measurement of lactate dehydrogenase (LDH) activity of the supernatant was employed to clarify the influence of panaxynol on RASMCs membrane function. Cells were grouped and incubated separately for 72h before the supernatant was collected and the absorbance value was measured to calculate the LDH activity. 5. Cell cycle progression was also analyzed by flow cytometry. Quiescent cells were incubated for 72 h respectively and measured by flow cytometry to estimate the percentage of cells in every phase of the cell cycle. The DNA density of single RASMCs was reflected by fluorecence intensity of PI-DNA complex. The proliferation ratio was evaluated. RESULT 1. Panaxynol significantly inhibited the increase of cell number(3×10-6 mol·L-1,9×10-6 mol·L-1, P＜0.05～0.01,vs control group)in a concentration-dependant fashion. Cell double times were prolonged(3×10-6 mol·L-1,9×10-6 mol·L-1, P＜0.05～0.01,vs control group). There was no loss founded in cell viability treated with panaxynol by typan blue exclusion test. There was no difference beween DMSO and control group in cell number and viability. 2. The absorbance value was decreased by panaxynol (3×10-6 mol·L -1, 9×10-6 mol·L -1, P＜0.05～0.01,vs control group),which indicates panaxynol may inhibit the activity of SDH in RASMCs mitochondrion. 3. Panaxynol significantly lowered CPM and impaired the incorporation of 3H-TdR (3×10-6 mol· L-1,9×10-6 mol·L-1, P＜0.01,vs control group).This result suggests the inhibitory effect of panaxynol on DNA synthesis in RASMCs. 4. The activity of LDH in supernatant was insignificantly lowered or not changed by panaxynol. 5. Flow cytometry showed that the proliferation ratio of RASMCs was significantly decreased by panaxynol(3×10-6 mol· L-1,9×10-6 mol·L-1,P＜0.05～0.01) accompanied with decreased percentage of cells in G2/M phase(3×10-6 mol· L-1,9×10-6 mol·L-1,P＜0.01)and increased percentage in G0/G1 phase (9×10-6 mol·L-1,P＜0.05). No apoptosis peak was discovered in all groups. CONCLUSION Panaxynol... |
