| Estrogen is an endogenous active compound which is necessary for women's physiological and psychological health. From the beginning of the menopause to the post-menopause, with the decreasing level of estrogen, women may have some near-term, metaphase and long-term diseases of menopause. Therefore, estrogen supplementary therapy becomes an important area that can maintain the women's health. But estrogen has some untoward effects that may increase the endometrial hyperplasia and then lead to cancer. As a result, now people use Hormone Replacement Therapy (HRT) which applies estrogen and progestogen together. However, the HRT still have some adverse effects. The progestogen not only can not rectify the estrogen's adverse reactions, but also it may impair the estrogen's protection to the cardiovascular system. By now, new therapy is in uninterrupted explore. Raloxifene is a selective estrogen receptor modulator (SERM). It can antagonist the estrogen acceptors in lacteal gland and hystera tissue. However, it is an excitomotor to bone tissue and blood fat, and people found it had some estrogen-like effects on cardiovascular system. We can infer the results of using the two drugs together: When the two drugs are used together, the advantageous effects of these two drugs can be enhanced, and the adverse effects of them can be weakened by each other.By now, there are not many studies on the new methods of HRT. We did not find any report about substituting the progestogen with raloxifene. In this paper, we adpted raloxifene to replace progestogen, and use aspirin to assist them, then we detected their effects on the proliferation of vascular smooth muscle cells. The drugs' effects on the expression of ICAM-1, PAI-1 and ET-1 in vascular endothelial cells were also observed. We hope to study the effects of this new project at cell and molecular levels.Part 1 Effects of aspirin on the proliferation of VSMC and themechanisms involved Methods: 1. The inhibit effects on VSMC were measured by MTT assay.The cells of log phase growth were seeded in 96 micro wells plate. When the cells grew to 80% confluence, we used the culture solution (DMEM) without phenol red and serum and cultured the cells for 24 hours. Then the cells were synchronized at G0 phase. We changed the culture solution with DMEM that contains 2% FCS and added aspirin. The final concentrations of the aspirin were 10-4mol/L, 10-3mol/L, 2×10-3mol/,5×10-3mol/L, we also set up a control group. The cells were cultured for another 24 hours and 20 ul MTT (0.5g/L) was added into each well. Then the cells were incubated for 4 hours at 37°C. The liquid was removed and 150 ul DMSO was added into each well to release the blue formazan reduction product. The plate was shaken slightly for 10 minutes in order to make the precipitation dissolve completely. Then the plate was placed into the Bio-rad microplate reader. The optical density of the samples was measured at 570nm.2. Detect the PCNA protein expression in VSMC treated by aspirin with immunocytochemistry.The cells of exponential phase were seeded in 6 wells plate with the concentration of 105/well. The cover glasses were placed in the wells in advance. When the cell grew to 80% confluence, the medium were replaced by DMEM which contained no serum and phenol red and the cells were cultured for 24 hours. Then we used DMEM with 2% FCS to culture the cells and added aspirin in the medium. The final concentrations of aspirin were the same as MTT assay. The medium was discarded after 24 hours. The slides were washed by PBS for three times and PFA was added on the cells. We washed the slides with PBS for three times. Then the slides were incubated in goat serum at room temperature for 30 minutes. The first antibody was added then the slides were put in a wet box and incubated for 12 hours. Then the second antibody was added followed the introduction of the kit. The slides were colored and re-stained. Five different fields of visions on a slide were selected under 100× microscopic. Then the positive cells were counted. We applied x2 analysis, Fisher's Exact Test to analyze the results. P<0.05 was considered as significant difference.3. Cell cycle analysis is performed by flow cytometry.The cells were seeded in 6 wells plate (105/well) and cultured until 80% confluence. Then the medium was replace by DMEM contained no serum and cultured for 24 hours. The medium was substituted by DMEM with 2% FCS and aspirin was added. The final concentrations were the same as the MTT assay. The plate was incubated at 37°C for 24 hours. The cells were harvested (about 106/group). The cells were fixed with 70% ethanol and incubated at 4°C for one night. Then the cells were stained by PI and the cell cycle was detected by flow cytometry. The results were analyzed with Modtif.4. The expression of PCNA mRNA was quantified by RT-PCRThe cells were treated with different concentrations of aspirin (10-3mol/L> 2×10-3mol/L,5×10-3mol/Land control group) for 24 hours. Followed by the instruction of the kit, total RNA was extracted by Trizol. The purity and concentration of the RNA were quantified with the ultraviolet spectrophotometer. In our experiments, we chose β-actin as inner reference. The cDNA was synthesized followed by the introduction of the kit. The PCR products were electrophoresised in 1.5% agarose gel contained 0.5ug/ul EB. Then the bands were analyzed by Bio-rad system. The expression of PCNA mRNA was adjusted by the expression of β-actin and the means the PCNA expression were calculated by PCNA/β-actin. Statistical treatment: Each sample was quantified for three times and the experimental resultswere reported as mean ± S.D ((?)±s). Then we applied SPSS 10.0 to analyze the datawith t test, analysis of variance and x2 test.Results:1. MTT assay demonstrated that aspirin was able to inhibit the proliferation of VSMC significantly. The inhibition ratios of each group were 4.9%,15.5%, 16.2%, 31.5%.2. The immunocytochemistry results showed that the 5×10-3mol/L group was stained slightly. However, the control group, 10-3mol/L and 2×10-3mol/L group were stained deeper. The picture were analyzed by software and the results indicated that the expression of PCNA in 5×10-3mol/L group was lower than control group (P＜0.05). But no significant difference was observed among 10-3mol/L, 2×10-3mol/Lgroup and control group.3. The results of the flow cytometry showed that the cells proliferation was arrested at S phase.4. After VSMC treated by 5×10-3mol/L aspirin, the expression of PCNA mRNA was lower than the control group.Conclusion:In this paper, we verified that 5×10-3mol/L aspirin could inhibit the proliferation of VSMC and the PCNA expression in the cells. It indicated that this inhibition effect may act by inhibiting the expression of PCNA. As a result, it made the cell cycle of VSMC stopped at S phase. And aspirin's inhibition effects may be act as early as mRNA level. Part 2 Single drug's effects on the expressions of ET-1, PAI-1and ICAM-1 in endothelial cells Methods:The endothelial cells of log phase were seeded in 6 wells plate and cultured until 70-80% confluence. Then the cells were cultured in DMEM which contains 1% serum and no phenol red. Different drugs were added and the cells were incubated for 24 hours. Total RNA was extracted followed the instruction of Trizol kit. Then mRNA of each group was used to synthesize cDNA. The mRNA expression of ET-1, PAI-1 and ICAM-1 in the endothelial cells were detected. The groups of the three drugs were:Aspirin: 5×10-3mol/L, 2×10-3mol/L, 10-3mol/L, control group. Estrogen: 10-9mol/L, 10-8mol/L, 10-7mol/L, control group Raloxifene: 10-8mol/L, 10-7mol/L, 10-6mol/L, control group. Results:5×10-3mol/L aspirin inhibited the expression of PAI mRNA.10-7mol/L and 10-6mol/L estrogen inhibited the expression of ET-1, and 10-6mol/L estrogen inhibited the expression of ICAM-1. 10-6mol/L raloxifene inhibited the expression of ET-1 and ICAM-1. Conclusion:Higher concentration of estrogen and raloxifene inhibited the expression of ET-1 and ICAM-1, aspirin inhibited the expression of PAI-1. The three genes are known as their danger to cardiovascular system. Consequently, the results provide theoretical evidence for the substitution of raloxifene with progestogen.
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