Effects Of Raloxifene On The Expression Of Proliferation-related Genes In Rats’ VSMCs

Objective To investigate the inhibitory effect of Raloxifene and Estradiol valerate on the proliferation of vascular smooth muscle cells (VSMCs) and to explore the protective effect of raloxifene on cardiovascular system on the level of mRNA, we measured the mRNA expressions of VSMC-marker (MYOCD, SRF), estrogen receptor ESRs (ESRa,β and GPER), and apoptotic-related Caspase-3in rat VSMCs which were treated with different concentration of raloxifene or estradiol valerate, with the hope to provide the basis for the prevention and treatment of cardiovascular disease in postmenopausal women.Method (1) VSMCs were harvested rapidly from adult rats thoracic aorta with its inner and outer layers removed under aseptic condition, and cultured in DMEM with20%FBS performanced by explant technique and trypsin digestion. Growth curve of VSMCs were determinated by the method of cytometry.(2) The influence of raloxifene and estradiol valerate on VSMCs’proliferation was measured by MTT assay through observing optical density (OD) value of VSMCs.(3) Expressions of MYOCD, SRF, ESR, GPER, Caspase-3in VSMCs were measured by real-time quantitative PCR after treating VSMCs with different dose of E2and RAL for24h.Results (1) Primary VSMCs were seen growing out of tissue-pieces about7or10days after explanting in DMEM with20%FBS and approached confluent approximately2or3weeks. VSMCs were fusiform, stellate or irregular shape, and typical "hill-valley" growth pattern could be observed in subculture cells.(2) Growth curve of VSMCs resembled "S" shape. The number of the cells approximately remained steady on the1st day after passage, and increased marginally on2nd and3rd day. VSMCs entered the logarithm grow period with the number of cells multiplied rapidly after incubation time. The platform period lasted for2or3days that cell proliferated slowly.(3) Compared with control group, the OD value was reduced in groups treated by E2and RAL, suggesting that the proliferation of VSMCs could be inhibited by both E2and RAL, and displayed concentration dependency.(4) The mRNA expression of MYOCD, SRF was evidently lower than the control group (P<0.05)in groups stimulated by E2and RAL. Between different groups, the expression gradually increased with decreasing concentration(P<0.05).(5) The mRNA expression of ESR, GPER was evidently higher than the control group (P<0.05) in groups stimulated by E2and RAL. Between different groups, the expression gradually increased with increasing concentration(P<0.05).(6) The mRNA expression of Caspase-3was higher than control group, and increased with the concentration of E2and RAL, suggesting that the proliferation of VSMCs could be inhibited by E2and RAL.Conclusions (1) Explant technique has the advantages of easy to operate, economy, and could obtain the highly purified VSMCs with good structure and function.(2) Like estradiol valerate, raloxifene can inhibit the proliferation, and possibly stimulate apoptosis of VSMCs through the activation of caspase-3pathway.(3) The effect of raloxifene on VSMCs is mediated by estrogen related receptor.(4) Raloxifene could postpone the formation of arterial plaque and take the place of estrogen for estrogen replacement therapy to prevent and treat cardiovascular disease in postmenopausal women.