Study On Inflammatory Factor-mediated Apoptosis And Mechanotransduction By Integrin α5β1in Degenerative Intervertebral Disc

Part1The establishment of intervertebral disc degeneration model inrat tails and imaging histological changesObjective: To establish a new type of mechanics induced intervertebral discdegeneration model in a rat tail, and detect reliability of the model by MRI and histology.Methods: Twenty-four Sprague-Dawley rats were randomly divided into controlgroup and experimental group. Each group contained12rats. The experimental group wasabdominal anesthesiaed with3.6%chloral hydrate (10ml/Kg). After the success ofanesthesia, the apparatus were applied to the7th and10th caudal vertebrae of theexperimental group rats, while the control group rats wwere not installed. Not only all ofthe rats before surgery but also four rats selected randomly from each group after surgery2,4and8weeks, which were taken the MRI examination and measured Co7~8, Co8~9andCo9~10nucleus pulposus T2WI MEANS value. Adopted the MRI evaluation criteria toevaluate the degree of degeneration.After the MRI examination, the rats were killedimmediately by excessive anesthesia. The intervertebral disc tissue of Co7~8, Co8~9andCo9~10was removed, fixed with formaldehyde, HE stained and detected collagen throughimmunohistochemical.Results: MRI examination: in the control group, the T1WI imaging signal wasbetween moderate and low; however, the T2WI imaging signal in Co8~9nucleus pulposusshowed high.In the experimental group,2weeks after surgery, the signal of Co8~9nucleuspulposus reduced, MEANS value also decreased;4weeks after surgery, the space of the intervertebral bodies narrowed, the signal of Co8~9nucleus pulposus decreasedsignificantly, MEANS value also decreased significantly. There was significantstatistically between the MEANS value of Co8~9nucleus pulposus of control group andexperimental group at2,4, and8weeks after surgery (P<0.05). HE staining: In the controlgroup, quite a few nucleus pulposus cells arranged in uniform; annulus layers arranged inneat rows. In the experimental group,2weeks after surgery, nucleus pulposus cellsdecreased, annulus fibrosus disordered. Four weeks after surgery, nucleus pulposusdecreased furtherly, and fibroplastic cells came up. At8weeks, fibrous cartilage tissuealmost entirely replaced nucleus pulposus tissue, the arrangement of the annulus fibrosusbecomed disorganized, and the boundary was unclear. Immunohistochemistry: In theCo~89of control group, the staining type Ⅰ and type II collagen arranged like mesh. TheCo8~9of experimental group, type II collagen staining concentration decreased and thenetwork structure was destructed at4,8weeks, compared with the control group.Conclusions: The first time in the country to build the model of external fixationstress intervertebral disc degeneration and for the first time using MRI observations on therat tailintervertebral disc degeneration. Compared with similar foreign models, the externalfixator four segment disc degeneration model eliminates the nutritional factors of discdegeneration and provide a good model for studying the mechanism and treatmentofintervertebral disc degeneration. Part2IL-1β, MMP-2expression in the intervertebral degenerated disctissue and p38-mediated apoptosis of nucleus pulposus cellsObjective: To investigate the alteration and effect of MMP-2, IL-1β in the process ofdisc degeneration in rats, and their relationships with p38-mediated nucleus pulposusapoptosis. Methods: HE staining: Four weeks after surgery, five rats were randomly selectedfrom the two groups, sacrificed by excessive anesthesia. Co8~9discs (separation of thenucleus pulposus and annulus fibrosus) were removed and then placed in the formaldehydesolution24h. After decalcification, the specimens were dehydrated, transparent, dip wax,embedded, and sliced. The postoperative cell apoptosis were detected by TUNEL: fourweeks after surgery, five rats were randomly selected from each group. Co8~9nucleuspulposus and annulus fibrosus were removed and TUNEL marked cell apoptosis.Quantitative Real-time PCR detected IL-1β, MMP-2and Collagen II gene: fifty rats ineach group were randomly selected. Co8~9discs (separation of the nucleus pulposus andannulus fibrosus) were removed, repeatedly washed with PBS and immediately placed inTrizol solution used for the fluorescence quantitative RT-PCR detection. Western Blottingdetection of p38protein and its phosphorylation level: four weeks after surgery,5rats wererandomly selected from each group. Co8~9nucleus pulposus and annulus fibrosus wereremoved and intracellular p38protein and its phosphorylation level were detected byWestern blotting.Results: In the experimental group, Co8~9nucleus pulposus cell density significantlyincreased, while extracellular matrix components decreased, and the annulus fibrosusdisodred. Nucleus pulposus cells were obvious apoptosis. The apoptosis index of theexperimental group was up to0.29, and the apoptosis index between the two groups wasstatistically different (P<0.05). IL-1β mRNA level of the nucleus pulposus in theexperimental group compared with the control group was increased by2.63±0.33(P<0.05),and IL-1βmRNA level in annulus fibrous was increased by8.50±0.16(P <0.05); while theMMP-2mRNA level of nucleus pulposus compared with the control group was increasedby8.87±0.24in the experimental group (P<0.05). Compared with the control group,Collagen II in the nucleus pulposus and annulus fibrous were decreased to0.15±0.09and0.44±0.17(P<0.05). The p38protein content of nucleus pulposus tissue in theexperimental group compared with the control group had no significant difference. P38phosphorylation level was significantly increased. P-p38/β-actin ratio was only0.12±0.02 in the control group, while0.63±0.05in the experimental group. There has significantdifference between the two groups (P<0.05). The p38protein phosphorylation level ofintervertebral disc degeneration rose.Conclusions: Cell apoptosis increased significantly and extracellular matrixcomponents reduced significantly in intervertebral degeneration disc. IL-1β in dics tissueof the experimental group was significantly higher than the control group and furtherconfirmed that IL-1β participate in disc degeneration process. In addition, IL-1β expressionlevel was correlated with MMP-2, indicating that IL-1β and MMP-2were interacted in theprocess of disc degeneration, and jointly promoted tissue degeneration. It was the first timeconfirmed that p38MAPK phosphorylation level significantly increased in the degeneratednucleus pulposus cells in the country, which induced cell apoptosis. Part3The expression and mechanotransduction of integrin α5β1indegenerative discObjective: To explore the possible mechanisms of mechanical signaling transductionin a mechanics induced intervertebral disc degeneration model after dying integrin α5β1expression and detecting the change of its downstream signaling molecules.Methods: Integrin α5localization and quantitative immunohistochemical quantitativeimmunohistochemical detection: four weeks passed, three rats were randomly selected ineach group, sacrificed by excessive anesthesia, and quickly removed Co8~9disc tissue(separation of the nucleus pulposus and annulus fibrosus) placed in informaldehydesolution24h. Paraffin sections, using immunohistochemical methods to detect thelocalization and quantitative expression of integrin α5, were observed under lightmicroscope. Immunofluorescence detectionof the located expression of integrin α5on thecell surfaces: Two rats were randomly extracted from both control group (Mock group) and experimental group (IVDD group) after4weeks, excessive anesthesia executed,quickly removed Co8~9disc tissue (separation of the nucleus pulpos-us and annulusfibrosus), fixed, rinsed, incubated dropping specificprimary antibody, secondary antibodies,rinsed, DAPI-stained nucleus, mounting with nuclearglycerol phosphate buffer, observingunder a fluorescence microscope. Flow cytometry to detect cell surface integrin α5β1expression quantitatively: Fifteen rats were bandomly selected from Mock group andIVDD group respectively after suegery4weeks, excessive anesthesia executed, quicklyremove the Co8~9disc tissue (separation of the nucleus pulposus and annulus fibrosus),and as far as possible with ophthalmic scissors cut it into pieces and to the mushysuspension, adding0.25%trypsin and0.2%collagenaseamount of digestive tissuesuspension, filtered to a single cell suspension through nylon mesh. After centrifugation,50ml buffer added, resuspended cells were counted with Trypan blue (Trypan Blue)detecting cell activity necessarily. Indirect marker of an anti-integrin α5antibody joined,and adding the FITC secondary antibody, incubated on ice, cell staining. QuantitativeReal-time PCR detection of integrin α5β1gene: Five rats were randomly selected fromMock group and IVDD group respectively after suegery4weeks, excessive anesthesiaexecuted, quickly removed Co8~9disc tissue (separation of the nucleus pulposus andannulus fibrosus). Total cellular RNA was extracted by using TRIzol, with oligo dT to beprimer, reverse transcription into cDNA. Real-time quantitative PCR was put into use byusing SYBR green. Western Blotting detected FAK phosphorylation level: Five rats wererandomly selected from each group respectively after surgery4weeks, to remove Co8~9nucleus pulposus and annulus fibrosus. Western blot detected intracellular FAK and itsphosphorylation level.Results: Immunohistochemical staining showed that nucleus pulposus cell surfacehad a great number of integrin α5expression in the Mock group, distributing uniform;while in the IVDD group, the nucleus pulposus cells scattered, with cell surface staininguneven. Immunofluorescence detection the expression of integrin α5in the nucleuspulposus and annulus tissue, by pressure, the fluorescence intensity of the protein in the cell membrane of the nucleus pulposus and annulus declined. Flow cytometry detectioncell surface integrin α5β1quantitative expression of nucleus pulposus and annulus tissuecells in the IVDD group, flow cytometry mean fluorescence intensity decreased, comparedwith the Mock group significant difference (P<0.05). Integrin α5mRNA level in theexperimental group, nucleus pulposus mild improved compared with the control group,integrin α5of fibrous ring mild declined; the experimental group, integrin β1mRNA levelof the nucleus pulposus and the fibrous ring mildly increasd compared with the controlgroup. But the level of mRNA in each group was not statistically different (P>0.05). Theexperimental group of nucleus pulposus content of FAK protein expression compared withthe control group, no significant difference. FAK protein phosphorylation level: p-FAK/β-actin ratio was0.15±0.07(MOCK group);0.42±0.11(IVDD group), a significantdifference (P<0.05) between them.Conclusions: It was the fist time detected that integrin α5expression of nucleuspulposus decreased in the rats degenerative disc. Resulted from mechanical role, it wasfound that FAK (integrin downstream signaling molecules) phosphorylation level rosesignificantly, to prove the activity of integrin increased.