Effects Of17β-estradiol On Mouse Lung Fibroblasts

Objective To investigate the effects of17β-estradiol(17β-E2) could induce the expression ofCaveolin-1protein of mouse lung fibroblasts, and the extracellular signal-regulated kinase(ERK)signaltransductionpathway, and ifreducing theexpressionoftypeI, IIIcollagen,inhibiting fibrosisornottoprovideatheoreticalbasisforfurtherexperiments.Methods Cryopreserved mouse fibroblasts cells were woke in37℃water within1minute,thencultured in RPMI-1640which contains10%serum,1%dual anti in incubator of5%CO2and37℃. Toobserve morphologicalchangesofmouse fibroblastsby invertedphasecontrast microscopeafter24h,3d,5d.Themousealveolar macrophagecellswerestimulatedbySiO2withtheconcentrationof20mg/L,50mg/L and100mg/L,which produced the supernatant preserved on-20℃environment.According to the concentration of SiO2,17β-E2and the intervention time of17β-E2ofthree different factors and three levels of them,orthogonal design was used to divide them into9groupsand MTT proliferationto determinatethebest concentrationand timeof17β-E2;Based ontheresultsofthe MTT cellproliferation,dividing them into5groups:Controlgroup,SiO2group,SiO2+10-8mol/L17β-E2group(Low concentration of17β-E2group),SiO2+10-7mol/L17β-E2group(the higherconcentration of17β-E2group) and SiO62+10-mol/L17β-E2group(the highest concentration of17β-E2group);Cellproliferation was measuredby MTT method.Cell cycle and apoptosis were evaluatedby flow cytometry.The concentration change of calcium ion were measured by Laser scanningconfocal microscope.Intracellular Caveolin-1,ERK,type I,III collagen were measured byimmunohistochemistry.Results1Morphological changes:observed the nascent woke mouse fibroblasts under theinverted phase contrast microscope at the ratio of10×10, cells were round and highly refractive,suspending in liquid culture.After nearly24hwerebeginning to adherent walls, cellswerefusiform,occasionallyintriangleorpolygon.About3days later,cellswereaccounting for70%to80%oftheareaofthebottleoftheculture.And thencancover thefullculturebottlewallabout5days, withanobvious decrease of the refractive index.The density looks like "paving stone", then can besubcultured.2Orthogonal design:The results of thevarianceanalysis showed that:differentconcentrations of17β-E2,SiO2and17β-E2intervention time all had significant effectson cellproliferation of pre experimental differences(P<0.05);Further analysis the orthogonal design inrange analysis,the results showed: the most important factor was the concentration of SiO2,followed by the concentration of17β-E2,and the thirdly is the intervention time of17β-E2.And thebest pairing factors were as follows: the concentration ofSiO2and17β-E2was20mg/L,10-6mol/L respectively,17β-E2intervention time was24h.3Cell proliferation:Compared with the controlgroup,after adding the supernatant which stimulated by SiO2,the absorbance value was increasedsignificantly in SiO2group(P<0.05);After using the intervention17β-E2,the absorbance value wasdecreased significantly.And with the concentration of17β-E2increasing, the absorbance valuedecreased significantly(P<0.05).4Cell cycle:After adding the supernatant which stimulated bySiO2,the proportion of G2and S phase cells were increased significantly in SiO2group,while theproportion of G1phase cells were decreased obviously(P<0.05).After using the intervention17β-E2,the proportion of G2and S phase cells were decreased significantly and G1phase cells wereincreased obviously.And with the concentration of17β-E2increasing, the proportion of cells in G1phase increased significantly, and the proportion S and G2phase cells was significantlyreduced(P<0.05).5Cell apoptosis:Compared with the control group,mouse lung fibroblastapoptosis was significantly lower in SiO2group(P<0.05).Compared with SiO2group, theproportion of apoptotic cells in the late stage apoptotic were increasing(P<0.05).And with theincrease of concentration of estradiol, the percentage of apoptosis cells increased moreobviously(P<0.05).6Immunohistochemical method:Compared with the control group,theintracellular ERK,the expression of I and III type collagen were largely increasing in SiO2group(P<0.05).After adding17β-E2,Compared with SiO2group,the expression of IntracellularCaveolin-1were largely increasing,while the expression of I and III type collagen weredecreasing(P<0.05).And increased with the concentration of estradiol, the more obviousexpression of Caveolin-1,and the reduced expression of ERK, I and III collagen were moreobvious(P<0.05).7Confocal laser light:Compared with the control group,Higher fluorescenceintensity of SiO2group,and with a stronger fluorescence(P<0.05).After adding17β-E2,cells’fluorescence intensity of intervention group was decreased significantly than SiO2group’s(P<0.05).Compared with SiO2group,as17β-E2concentration increased,thecells’fluorescence intensity were reducing obviously, showing weak fluorescenceexpression(P<0.05),but stillhigherwhencomparedwiththeblankcontrolgroup(P<0.05).Conclusion117β-E2could block mouse lung fibroblast cells fromG1phase toS and G2phaseand inhibit the proliferation of the cells stimulated by SiO2, and induce their apoptosis.217β-E2could induce the expression of caveolin-1,and inhibit the expression of ERK protein, I and IIIcollagen.317β-E2could inhibit theincreaseofintracellular calciumionconcentrationwhichcausedinmouselung fibroblastsinducedbySiO2,andreducetheconcentrationofcalciumion.