Study Of The Influence Of ERK5on Nucleus Pulposus Cells Apoptosis Induced By Fluid Shear Stress

Objective: To investigate the involvement of ERK5signaling pathway in theapoptosis response of nucleus pulpusos (NP) cells to fluid shear stress（FSS）.Methods: Cultured primary nucleus pulpusoscells by using enzyme digestiontechnique. Different level shear stress （0、6、12、18、24dyn�'cm-2）was appliedto nucleus pulposus cells at different time points（0、15、30、45、60、90min）byself-made parallel-plate flow chamber.1.Stained the F-actin of cells withFITC-phalloidin, and observed the fluorescence of F-actin under confocal laserscanning microscope (CLSM).2. Quantitative real time PCR technique was used todetecte the gene expression of ERK5.In order to review the state of ERK5after FSSstimulation,Western Blot was performanced.3.Using Quantitative real time PCRtechnique detected the gene expression of BAD, Bax, Bcl-2and Caspase3after shearstress stimulation. Inhibit active of ERK5with BIX02188, then stimulate cells withexcessive shear stress(18dyn�'cm-2×60min). Western Blot methods were used tostudy the apoptosis assosciated protein（BAD、Bcl-2、Bax and Caspase3） expressionrespectively.Results:1.NP cells were subjected to different levels FSS for different duration, theresult showed that F-actin filaments became more and more abundant and thicker,which result in reconstruction of the cytoskeleton. Beyond a certain range of FSS(＞12dyn�'cm-2or＞45min),the reorganization of cytoskeleton tended to slow down.When FSS came to24dyn�'cm-2×45min or12dyn�'cm-2×90min, F-actinfilaments became breakdown and the cytoskeleton collapse.2. qRT-PCR result showedthat FSS up-regulated gene expression of ERK5in NP cells in a strength-and time-dependent manner. As enhancement of strength and extension of duration（＞12dyn�'cm-2or＞45min）, the gene expression of ERK5became slow down.WesternBlotresult were in conformity with this result.3. Low fluid shear stress（＜12dyn�'cm-2or＜45min） play a anti-apoptosis role by decreasing the gene expression ofanti-apoptosis factor, such as BAD、Bax and Caspase3, but has not significant effecton the Bcl-2. Subjected to over-loading FSS(＞12dyn�'cm-2or＞45min),NP cells’gene expression of the BAD、Bax、Caspase3increased, while the Bcl-2geneexpression decreased, leaded to the apoptosis of NP cells.4.BIX02188had asignificant inhibitory effect on the activation of ERK5. Excessive fluid shear stress（18dyn�'cm-2×60min）up-regulate BAD、Bax、Caspase3protein expression in NPcells. When inhibited the ERK5signal pathway, this effect was improved.Conclusion:1.Within a certain range of FSS(≤12dyn�'cm-2or≤45min)，thecytoskeleton of NP cells reorganized obviously. Over12dyn�'cm-2or45min,thereorganization of cytoskeleton tended to slow down.2. FSS promoted the activation ofERK5in a strength-and time-dependent manner. Appropriate FSS（＜12dyn�'cm-2or＜45min）improved the activated level of ERK5significant. When the strength andduration increased, this effect had no significant different between each other.3. LowFSS may help NP cells survival by activating ERK5signal pathway. Due to the FSS’activation on the ERK5was limited and the protection of ERK5was finite, excessive FSS would promote apoptosis of NP cells.ERK5signaling path mediated apoptosis ofNP cells induced by FSS.