The Research Of Signal Pathway For BNIP3Activated Nucleus Pulposus Cell Death In Intervertebral Disc Degeneration

IntroductionIntervertebral disc degeneration disease has become a worldwide health problem, andcost huge economic burden. During the degeneration, the intervertebral discs exhibitextensively histomorphological changes such as the nucleus pulposus cells death, lamellaedisorganizing of the annulus fibrosus, thinning and calcification of the cartilaginous endplates.At the same time, the nutrients supply such as oxygen and glucose also decreasedsignificantly and even disappeared. The increasing death of nucleus pulposus cells duringdegeneration coincides with the decrease in nutrients supply to the disc, implying that the fallin nutrients supply might be one of the factors involved in the disappearance of NP cells fromhuman discs.Bcl-2/adenovirus E1B19-kDa-interacting protein3(BNIP3) is a member of a uniquefamily of death-inducing mitochondrial proteins, which has a single Bcl-2homology3(BH3)domain and is a pro-death factor. Normally, BNIP3is expressed at low levels in a variety ofcells, where it is primarily localized to the cytosol. Expression of BNIP3is increased underhypoxic conditions and translocated from cytosol to mitochondria, and cause cell death.Apoptosis-inducing factor (AIF), one of the apoptosis-related proteins, is known to be a keyfactor in caspase-independent cell death. It is anchored to the outer face of the mitochondrialinner membrane. In response to apoptotic stimuli, AIF is released into the cytosol andtranslocates into the nucleus where it induces chromatin condensation and large-scale DNAfragmentation (～50kb) in a caspase3-independent way.Here we have tested the hypothesis that BNIP3activates a caspase3-independent NPcell death in nutrients deprivation.ObjectiveThis study was to create a cell culture model in nutrients deprivation condition, anddemenstrate the involvement of nutrient-sensitive protein, BNIP3in the death pathway of nucleus pulposus cell. Furthermore, inhibition of BNIP3expression during the intervertebraldisc degeneration can postpone or even block the nucleus pulposus cell death, thereby toprevent the disease and to provide a new theoretical base for the biological treatment of thedegenerative intervertebral disc disease.Methods1The identification of the nucleus pulposus cellsHealthy SD rat caudal disc nucleus pulposus cells were cultured in DMEM/F12medium with10%fetal bovine serum. Cell biological features were researched by invertedphase contrast microscope,light microscope,electron microscope,cell vitality assay and cellsstaining.2The expression of BNIP3in nucleus pulposus cells cultured in nutrientsdeprivation conditionCells isolated from rat caudal disc were cultured under two different oxygen, glucoseand serum concentrations for up to72hours. Interactions between two differentconcentrations were examined by cell vitality assay,mitochondrial membrane potential(Δψm)test and apoptosis detect. The expression and location of BNIP3and AIF were tested bywestern blot and immunofluorescence staining.3The culture of nucleus pulposus cells in nutrients deprivation condition withBNIP3overexpression or RNAiCells isolated from rat caudal disc were cultured under nutrients deprivation conditionwith BNIP3overexpression or RNAi for up to72hours. Interactions between two differentcondition were examined by cell vitality assay and apoptosis detect. The expression andlocation of BNIP3and AIF were tested by western blot and immunofluorescence staining.4Animal experimentsNeedle puncture injury disc to establish intervertebral disc degenerative model, and afterthe operation for0,2,4and8weeks, the discs were observed by MRI,histological stain,BNIP3IHC and gene express essay, and respectively compared the results with untreateddegenerative disc and normal disc to identify whether the BNIP3has correlation with the discdegeneration.Result1The biological characteristic of nucleus pulposus cells 1.1The rat caudal disc cells:1,Observing by inverted phase contrast microscopeshowed that the primary NPCs adherenced in5days, and growed exponentially in6～8days,finally subcultured in17days. With the times of passage increased, however, the phenotypeof the NPCs changed from polygon to long fusiform;2,The vitality assay showed that therewas about95%～97%NPCs survived just before isolated from intervertebral disc, and duringthe cultured in primary,fisrt generation and second generation, there were respectively about98%～100%,100%and75%～80%;3,The toluidine blue stain of the NPCs was stronglypositive,and HE stain showed clearly the cell nucleus and cytoplasm. The type II collagenimmunocytochemical stain and safranin O stain showed positive.4,It was observed bytransmission electron microscope that there were many glycogen particles and lesschondrosomes.1.2Because its resource is widespread and easy to culture and amplification, the P1nucleus pulposus cell is the most suitable for the following research.2Nutrients deprivation induced BNIP3expression and AIF translocationAs a result, nutrients deprivation induced a time-dependent increase of BNIP3expression correlating with the cell death response. The AIF was also detected correlated withthe increase of BNIP3expression. The western blot also showed that the total amount of AIFdid not vary with culture times to nutrients deprivation, but AIF in the mitochondrial fractiondecreased with exposure times to nutrients deprivation and was detected in nuclear fractionafter nutrients deprivation for48hours and accumulated with increased times of nutrientsdeprivation, suggesting that the released AIF from mitochondria was translocated to nuclear.3cells with BNIP3overexpression or BNIP3RNAi3.1By overexpressed BNIP3, NP cells showed significantly increased cell death duringnutrients deprivation, however, cells showed a good tolerant with nutrients deprivation byBNIP3RNAi;3.2The results of WB for AIF showed that:1,the overexpression of BNIP3couldaccelerate the translocation of AIF from mitochondria to nucleus;2,inhibition of BNIP3expression could decrease not only amount but also the speed of translocation of AIF, but itcould not block the translocation which imply that BNIP3was one of factors inducing the AIFtranslocation.4Correlationship between disc degeneration and BNIP3expresssion 4.1We successfully established the disc degeneration model in rabbit which wasdemonstrated by histological assay and MRI detection.4.2Annulus needle puncture induced decreases in NP signal intensity on T2-weightedMRI of the experimental discs compared with the control. Degenerative grades of thepunctured discs were significantly higher than those of controls and showed an intermediateintensity of grade2~3at2weeks, and rapidly increased during the experiment, reaching tograde4~5at8weeks.The mRNA-quantification in the NP cells of experimental discs demonstratedsignificant BNIP3gene up-regulation of2.0±0.1-fold at2weeks,2.8±0.3-fold at4weeksand4.2±0.2-fold at8weeks, compared with the control discs. BNIP3mRNA expressionshowed significantly high correlations with disc degeneration grades in MRI. MRI grade:rho=0.92, p<0.01.4.3The NP cells of experimental discs at0week showed a negative reactivity to BNIP3immunohistochemical staining, however, the NP cells of experimental discs demonstratedobviously positive staining for BNIP3at2weeks,4weeks and8weeks, which also showedsignificantly high correlations with disc degeneration histological grades. Histological grade:rho=0.926, p<0.01.Conclusion1. The nutrients deprivation can induce upregulation of BNIP3, and promote thetranslocation of AIF from mitochondria to nucleus which causes caspase-independent celldeath;2. Overexpression of BNIP3can significantly increase the nucleus pulposus cell deathwhile inhibition of BNIP3can not significantly decrease the nucleus pulposus cell death,which implied that BNIP3was involved in the cell death but not the only factor.3. Animal experiments demonstrated that the expression of BNIP3was correlative withthe disc degeneration; inhibition of BNIP3expression may relieve the degeneration but notstop.