| This study intended to investigate the protective effects of Suppressing Frizzled-2 on hypoxia/reoxygenation BRL-3A Cell.In order to understand the possible mechanism we detected the Ca2+ concentration、Autophagy related proteins expression and apoptosis degree in normal BRL-3A cell or in BRL-3A cell after Hypoxia reoxygenation.The protective mechanism would provide new theoretical basis for treating human liver diseasePart OneObjective:This part of study intended to investigate the changing of expression of Wnt5a/Frizzled-2 pathway in BRL-3A cell after Hypoxia reoxygenation(H/R),and the influence of suppressing Frizzled-2 on apoptosis degree in BRL-3A Cell.Materials and methods:BRL-3a Cells were divided into H/R group、only plasmid H/R group(NC-H/R group)、Frizzled-2 gene silence and H/R group(Si-H/R group),group of normal cells(Cell group).After made H/R model,BRL-3a Cells were detected the expression of gene and protein of Wnt5a/Frizzled-2 by Q-PCR and West Blotting.Cell damage were accessed by detected LDH.The degree of apoptosis of each group were accessed by testing the Caspase-3 protein expression and flow cytometry instrument.Results:(1)The expressions of gene and protein ofWnt5a/Frizzled-2 of H/R group were higher than Cell group(P<0.05).And the expression of Caspase-3 protein in H/R group higher than its in Cell group(P<0.05);(2)The expression of gene and protein of Frizzled-2 of Si-H/R group was shorter than H/R group(P<0.05).The data of LDH and Caspase-3 protein in Si-H/R group shorter than its in H/R group(P<0.05);Flow cytometry show that the early apoptosis BRL-3a Cells rate in Si-H/R group were lower than it in H/R group.Conclusion:There are normal expression of Wnt5a/Frizzled-2 pathway in normal BRL-3A cell.After H/R processing,the expressions of gene and protein of Wnt5a/Frizzled-2 are significantly increased,at the same time,BRL-3a Cell damage and apoptosis have significantly increased too.After the Frizzled-2 gene is silenced,the increasing of BRL-3a Cell damage and apoptosis are suppressed significantly.So that,Frizzled-2 is play an important role in aggravating the damage and apoptosis of H/R BRL-3A cell.Part twoObjective:This part of study intended to investigate the influence of suppressing Frizzled-2 on intracellular Ca2+ concentration.Materials and methods:BRL-3a Cells were divided into H/R group、NC-H/R group、Si-H/R group and Cell group like previous study.After made H/R model,BRL-3a Cells were detected the Intracellular Ca2+ concentration.Results:The intracellular Ca2+ concentration of Si-H/R group was lower than H/R group(P<0.05)Part threeObjective:In this part of study,we want to know that the changing of Wnt5a/Frizzled-2 pathway in BRL-3A cell after H/R processing effect on autophagy related proteins expression.Materials and methods:BRL-3a Cells were divided into H/R group、NC-H/R group、Si-H/R group and Cell group like previous study.After made H/R model,BRL-3a Cells were detected the proteins expressions of Beclin-1,Atg7,LC3B I and LC3B Ⅱ,which are important autophagy related proteins,and observed autophagosome by Electron microscopic.Results:(1)The expressions of autophagy related proteins including Beclin-1 and Atg7 of H/R group were higher than Cell group(P<0.05),on the other hand,the LC3BII/I of H/R group was lower than it of H/R group.Conclusion:after H/R processing,the inhibition of Wnt5a/Frizzled-2 pathways can promote autophagy flux in BRL-3a Cells,which reduce cell damage and apoptosis.Part fourObjective:This part of study intended to investigate the effect of the ch anging of Wnt5a/Frizzled-2 pathway on Ca2+ concentration、Autophagy related proteins expression and apoptosis degree in BRL-3A Cell.Materials and met hods:BRL-3a Cells were divided into group of normal cells(Cell group)、only plasmid group(NC group)、Frizzled-2 gene silence group(Si group),Fri zzled-2 gene overexpression group(FRI group).Each group were detected th e expression of gene and protein of Wnt5a/Frizzled-2,intracellular Ca2+concentr ation,the proteins expressions of Beclin-1,Atg7,LC3B II/I and Caspase-3.R esults:(1)The expressions of gene and protein of Wnt5a/Frizzled-2 of FRI gr oup were higher than Cell group(P<0.05-0.001)(2)The expressions of autoph agy related proteins including Beclin-1 and Atg7 of FRI group were higher t han Cell group(P<0.05),and the LC3BII/I of FRI group was lower than it of Cell group(3)the data of Caspase-3 protein in FRI group higher than it in cell group(P<0.05).Conclusion:After eliminating the interference of H/R,t he up-regulation of Wnt5a/Frizzled-2 pathway could suppressed autophagy flux.On the contrary,the Inhibition of Wnt5a/Frizzled-2 pathway,although the ex pression of Beclin-1 and Atg7 protein down slightly,is able to promote autop hagy flux,which reduce the apoptosis degree. |
