A Study About Mechanisms Of Action Of BMP2on Skin Aginging Involving Estrogen Alteration

ObjectiveTo study the effects of estrogen on structure of skin, we observe the histopathological changes of skin and explore the underlying mechanisms.Methods30female (aged6weeks)Wistar rats were pachased and randomly divided into three groups:the model group(the operated group was overiectomied, n=10), the Sham-operated group (the sham-operated group was unovariectomied, n=10) and overiectomied+estrogen injection group (OEI, n=10). Bilateral ovaries were cut off in the model group, the rats were prohibited water before operation, the peritoneal injection with6%chloral hydrate(0.5ml/100mg body weight), to rope their four limbs after the anesthesia, to poodle in the two side of spinal column and the empennage of the twelfth rib, then skin degerming with2%iodine and deiodinating with75%alcohol. The vertical incision was done about1～1.5cm lateral to the posterior median line, then to anatomize skin, muscle, parietal peritoneaum, and get into the peritonealcavity inorder, found out ovaries and performed the ovafiectomy. The procedure of the sham-operated group was similar to that of the model group, and only some fatty tissue around the ovaries was cut off without ovariectomy. After performing the ovafiectomy,0.05mg/d estradiol was given through subcutaneous injection in overiectomied+estrogen injection group. After breeding6weeks, the rats were anesthetizing respectively. The medial and lateral parts were prepared for sampling of routine HE staining and for sampling of the immunohistochemistry, respectively. The serum level of estradiol was detected by radioimmunoassay. The expression of BMP-2proteins associated with apoptosis were determined by immunohistochemistry for the epithelial cells of the skin. The histology and ultrastructure of skin were observed with optic microscopy and transmission electron microscopy. All data mentioned above were dealt with SPSS17.0.Results1. Level of serum estradiol in the model groups were significantly decreased compared with that of the sham-operated groups (P<0.05).2. In the model group6weeks after the operation there were such pathologic changes in the Sweat gland as thinning of the duct wall, expanded duct lumen, cell atrophy in all layers of the duct wall, fibrosis around the ducts, and some abnormal acinar structures, suggesting that the acinar structures were in atrophy state, manifesting disordered cytoarchitecture, shrinking cells, diminished cytoplasm, and clustered cellular nuclei.3. Using Picric-sirius red polarized light analysis of skin collagen deposition, analysis of rat skin in the Ⅰ collagen content, Ⅲ collagen content and Ⅰ/Ⅲ ratios, After6weeks of operation, Ⅰ type collagen content, Ⅲ type collagen content and Ⅰ/Ⅲ ratios was lower than those in the sham operation group (p<0.05), in the estrogen increased group, Ⅰ type collagen content, Ⅲ type collagen content and Ⅰ/Ⅲ ratios was significantly higher than that in the sham operation group (p<0.05).4. The positive cells labeled with BMP-2in the model groups was significantly lower than that of the sham-operated groups6weeks after the operation (P<0.05).5. The relationship between estradiol levels of serum and BMP-2positive cells were negtively corlerated(correlation coefficient r=-0.9600, P=0.0400).ConclusionIn the model group6weeks after the operation some structures in the epithelial cells of skin have been disordered, showing astate of atrophy, I type collagen content, III type collagen content and Ⅰ/Ⅲ ratios was significantly higher. Expression of BMP-2in the model group6weeks after the operation,estrogen levels are lower, resulting in dysfunction in skin and in pathogenesis of skin aging. Effects of estrogen on expression of bone morphogenetic protein-2in dermal fibroblastsObjectiveTo explore the mechanism of estrogen on delaying skin aging by observing the expression of bone morphogenetic protein-2in dermal fibroblasts in vitro.MethodsThe skin specimens, washed blood and stains thoroughly with antibiotic liquid PBS, cut skin tissue, clean skin specimens with0.25%trypsin digestion after4degrees14h, separation of epidermal and dermal tissue, abandoned to the epidermis, the remainder of the dermal tissue with1%collagenase liquid MEM37℃,5%CO2incubation box digestive4h isolated from fibroblasts, collect and washing the resulting cells, new separation of the fibroblast density of approximately4×104/mL, join MEM with10%fetal bovine serum culture medium, when cultured fibroblasts grown dense monolayer, the cell density of approximately16×104/mL, inl:3ratio of passage, in the incubator to cultured48H, to be adherent cell growth to the cell density of about12x104/mL, used in the experiment. Then the cells were divided into4groups:group first to estrogen stimulation group:respectively to the3flask adding different concentrations of estrogen (1×10-1mol/L,1×10-2mol/L,1×10-3mol/L) in serum free culture medium to culture MEM24h; second Noggin: antagonistic group respectively3added to cultured with different concentration of BMP2antagonist noggin (Noggin at concentration of10,20,30g/ml) in serum free culture medium to culture MEM24h; third groups to estrogen stimulation+noggin antagonistic groups:respectively to the3culture added with different concentrations of estrogen estrogen (final concentration1×10-1mol/L,1×10-2mol/L,1×10-3mol/L respectively)+with different concentration of Noggin (Noggin at concentration of10,20,30g/mL) in serum free culture medium to culture MEM24h; fourth groups: normal control group without any intervention by in vitro culture skin fibroblast cells. For each group of cultured skin fibroblasts, for drugs, which extract the total RNA, by means of the reverse transcription reaction to obtain cDNA, and performed in vitro amplification, the amplification products were agar gel electrophoresis, and according to the band brightness to judge the fibroblasts in the expression level of BMP-2. Transmission electron microscopic observation of different concentration of estrogen treatment group for fibroblast morphology in rats.ResultsSkin fibroblast proliferates well in vitro. Growth curve showed latency phase, index number growth phase and platform phase.we also found latency phase was shorter, about0to2clays, then index number growth phase. After about5days fibroblast cells proliferates stable. Agar gum electrophoresis showed, there was no significant differences in the level of BMP-2mRNA between the low-dose group and the sham-operated group. However, the level of BMP2mRNA was lower in the high-dose group compared to sham-operated group. There was significant differences(P<0.05).Under the transmission electron microscope in the model groups6weeks after the operation the basal cell size of the acinar epithelium of the skin was shrinked, their cytoplasm diminished, shape of their nuclei became irregular, nuclear chromatinwas condensed and concentrated near the nuclear membrane,the nucleus border was clear,the gaps of nuclear membrane were varied. The ultrastructural pathology of transitional cells mainly showed changes in mitochondrial swelling, damaging in mitochondrial cristae, vacuolization, cloudiness, and myelin figures in their cytoplasm.ConclusionEstrogen at high concentration exhibites inhibitory effects on mRNA expression of bone morphogenetic proteins, but low concentration not.