Effect Of High Glucose On Extracellular Matrix Metabolism Of Rat Intervertebral Disc And Apoptosis Of Nucleus Pulposus Cells

Part1Background and purposeIntervertebral disc (IVD) degeneration is a major cause of low back pain that affects asignificant proportion of the population, and found to be associated with sciatica and discherniation. Although some investigations suggest that diabetes is involved in theprogression of IVD degeneration, the mechanisms connecting them are still uncertain. IVDdegeneration is typically characterized by a loss of extracellular matrix (ECM) comprisedlargely of two structurally distinct molecular components, collagen and aggrecan, wherebymatrix anabolism is decreased and matrix catabolism is increased. Matrixmetalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondins(ADAMTSs) are capable of degrading ECM, and can be inhibited by specific endogenoustissue inhibitors of metalloproteinases (TIMPs). During the process of IVD degeneration,MMPs and ADAMTSs are commonly up-regulated and TIMPs are down-regulated,resulting in increased ECM degradation. Loss and remodelling of ECM lead to theoccurrence of clefts and tears and eventually complete IVD structural failure. p38MAPKpathway potentially plays a vital role in mediating the molecular events responsible forenhanced ECM degradation in the IVD. It controls the expression of catabolic (MMPs,ADAMTSs) factors, and also influences the expression of anabolic (collagen and aggrecan)and anti-catabolic (TIMPs) factors. The aim of the present study was to determine thesignificance of diabetes on degradation of intervertebral disc (IVD) extracellular matrix.MethodsThe amount of glucose and sorbitol was analyzed. Western blot analyses were carriedout following standard procedures to examine AR levels, p38MAPK phosphorylation,MMPs, ADAMTSs and TIMPs levels. Crosslinked C-terminal neo-epitopes of type IIcollagen (CTX-II), an MMP-mediated degradation fragment of type II collagen, wasdetected by a competitive ELISA. The ELISA for the detection of the aggrecanase-deriveddegradation fragments of the N-terminal neoepitope374ARGSV combined two monoclonalantibodies in a sandwich ELISA system. The detection process of342FFGVG was similar tothe374ARGSV ELISA. ResultsDiabetic rats showed a significant increase in glucose and sorbitol contents in the IVD.The levels of aldose reductase (AR), p38and metalloproteinases, and degradation ofmetalloproteinase-derived aggrecan and type II collagen were increased, while tissueinhibitors of metalloproteinases levels were decreased in the IVD of diabetic rats. Thesechanges were markedly affected by inhibition of AR or p38.ConclusionEnhanced degradation of MMP-mediated type II collagen and aggrecan wasconfirmed in the IVD of diabetic rats. The beneficial effects of ARI on the degradationprocess were accompanied by inhibition of increased ARI activity and sorbitolaccumulation in the IVD, indicating that the polyol pathway played an important role inECM degradation in the IVD of diabetic rats. p38MAPK activation seemed to be anintermediate mechanism inducing ECM degradation in the IVD of diabetic rats byinfluencing the balance of MMPs/TIMPs. Part2Background and purposeIVD degeneration is typically characterized by a loss of extracellular matrix (ECM).Apoptosis of nucleus pulposus cells (NPCs) plays a central role in the development of IVDdegeneration. Although the events leading to IVD degeneration are not well understood,there is ample evidence that oxidative stress and stress-induced signaling pathways play arole in the development of IVD degeneration. Meanwhile, some investigations suggest thatthe progression of IVD degeneration is associated with diabetes and hyperglycemia.However, to our knowledge, whether hyperglycemia-induced oxidative stress is implicatedin the progression of IVD degeneration has not been investigated previously. In this study,we hypothesis that excessive ROS generation induced by elevations in glucose plays a keyrole in causing apoptosis of NPCs and abnormal expression of critical genes involved inthe metabolic balance of ECM by its ability to activate the stress-sensitive p38MAPKsignaling pathway.MethodsNPCs were cultured with various concentrations of glucose to detect cell viability andapoptosis. Cells cultured with high glucose (25mM) were untreated or pretreated withN-acetylcysteine or a p38MAPK inhibitor SB202190. Reactive oxygen species (ROS)production was evaluated. Activation of p38MAPK was measured by Western blot. Theexpression of ECM metabolism-related genes, including type II collagen, aggrecan,SRY-related high-mobility-group box9(Sox-9), matrix metalloproteinase3(MMP-3) andtissue inhibitor of metalloproteinase1(TIMP-1) was analyzed by RT-PCR.ResultsThe results indicated that high glucose reduced cell viability and induced apoptosis.High glucose resulted in increased ROS generation and p38MAPK activation. In addition,it negatively regulated the expression of type II collagen, aggrecan, Sox-9and TIMP-1,and positively regulated MMP-3expression. These results were changed by pretreatmentwith N-acetylcysteine or SB202190.Conclusion High glucose might promote apoptosis of NPCs, trigger ECM catabolic pathways andinhibit its anabolic activities, possibly through a p38MAPK-dependent oxidative stressmechanism.