Expression Of AIM2,NLPR3,Caspase-1 And IL-1? In Nucleus Pulposus And Endplate Of Intervertebral Disc Tissue And Its Pathological Significance In Low Back Pain Patients With Modic Changes

Objective: To investigate the expression of AIM2,NLRP3,Caspase-1 and IL-1? in nucleus pulposus and endplate of lumbar disc tissue in low back pain patients with Modic Changes.Furthermore,to explore the role of AIM2,NLRP3,Caspase-1 and IL-1? in the pathogenesis of Modic Changes and lumbar intervertebral disc degeneration.Methods: 16 patients with low back pain and lumbar intervertebral disc degeneration were admitted in affiliated hospital between October 2015 and June 2016,patients with Modic changes were put into experimental group(8 cases)and those without modic changes were put into control group(8 cases)based on the MRI findings before surgery.We collected endplate and nucleus pulposus tissue from the 16 patients during surgery and employed hematoxylin and eosin staining to identify the intervertebral disc cells and evaluate cell density and degeneration.Immunohistochemistry,Reverse transcription-polymerase chain reaction and Western Blot were performed on endplate and nucleus pulposus tissues to investigate the expression of AIM2,NLRP3 inflammasome and its downstream molecules Caspase-1,IL-1?.Data analyses were performed using SPSS 19.0 statistical software.Statistical graphing were done using GraphPad Prism 6.0 software.All values were expressed as mean±SD.The single samples Kolmogorov-Smirnov test was taken as nomal test.Statistical comparisons between two groups were performed by independent sample t-test.The Pearson correlation analysis was used to analyse the correlation.P < 0.05 were considered statistically significant.Results:1.H.E staining:Nucleus pulposus cells in control group were mainly chondrocytic cells and fibroblasts which appeared round and flat oval,respectively.The cells had a larger nucleus comparing with the experimental group and showed "nuclear agglomeration".The matrix took the form of non-uniform wave.The cells of nucleus pulposus in the experimental group exhibited neovascularization,and the cell density was significantly lower than the control group.Endplate cells in the control group were mainly chondrocyte,which located in the cartilage lacuna,there is space between cells and the cartilage cysts.The cell nucleus had an oval shape and small size.The matrix presented an uniform distribution.Cartilage endplate in the experimental group showed similar neovascularization,the endplate was fissured.The space between the cells and cartilage cysts was prominent.Cell density was lower than in the control group.2.Immunohistochemistry staining:we observed a stronger positive expression of AIM2,NLRP3,Caspase-1,IL-1? in the experimental groups in the cytoplasm and nucleus of both nucleus pulposus and endplate cells.In nucleus pulposus and endplate cells of the control group,AIM2 showed moderately positive and negative staining,respectively,NLRP3 staining were moderately positive and positive,respectively.Caspase-1 showed positive and negative staining,respectively while IL-1? showed moderately positive in both cells.3.RNA extraction:we extracted RNA by liquid nitrogen grinding,glass homogenizer and plastic grinding rod,the corresponding concentrations were(37.44±35.93)ng/?l,(208.0±41.24)ng/?l and(320.7±70.22)ng/?l,respectively.The OD260/280 value were(1.657±0.1272),(1.463±0.1265)and(1.830±0.04262),respectively.The OD260/230 were(0.1346±0.08283),(0.4386±0.2653)and(0.6662±0.2512),respectively.No band was observed when we test the RNA integrity after isolating RNA by glass homogenizer and liquid nitrogen grinding.We saw three bright band of 5S,18 S,28S when we extract RNA from the fresh tissue by plastic grinding rod.4.RT-PCR and Western Blot:The mRNA and protein expression levels of AIM2,NLRP3 inflammasome and its downstream molecules Caspase-1,IL-1? showed an increase in the endplate and the nucleus pulposus in the experimental group with Modic changes compared with the control group without Modic changes.The difference was statistically significant(P<0.05 and P<0.01,respectively).Within each group,in comparison with mRNA or protein expression levels of AIM2,NLRP3,Caspase-1,IL-1? in the endplate,expression levels of these four proteins in the nucleus pulposus didn't showed a significant increase(P>0.05).5.Pearson correlation analysis of the average relative intensity values of NLRP3,AIM2 protein showed that AIM2 had a positive correlation with NLRP3(r=0.993,P=0.000,P < 0.01).The result also showed a positive correlation between AIM2 protein and Caspase-1 protein(r=0.993,P=0.000,P<0.01)and between AIM2 protein and IL-1? protein(r=0.988,P=0.000,P<0.01).Conclusion:1.The RNA of nucleus pulposus and endplate in fresh intervertebral disc tissue were extracted successfully by plastic grinding rod.This method provided us RNA of high concentration,ideal purity and integrated ingredient and therefore guaranteed the following molecular biology experiments.2.The expression level of AIM2,NLRP3,Caspase-1,IL-1? were significantly increased in the endplate and nucleus pulposus within Modic-changed group compared with the other group.The expression levels of AIM2 and NLRP3 were positively correlated.All of these results indicated that AIM2 and NLRP3 signaling pathways jointly promoted the pathogenesis of Modic changes.3.Within each group,in comparison with mRNA and protein levels of AIM2,NLRP3,Caspase-1,IL-1? in the endplate,the levels of these four proteins in the nucleus pulposus didn't showed a significant difference,which indicated that both AIM2 and NLRP3 contributed to the occurrence and development of modic changes.4.The discovery of AIM2,NLRP3 inflammasome may provide new therapeutic targets for Modic changes or intervertebral disc degeneration.