The Influence And Mechanism Of Leukaemia Inhibitory Factor On Cell Apoptosis And Extracellular Matrix Metabolismtie Degenerative Nucleus Pulposus Cells

Background:Due to the high morbidity,high rate of disability and high medical costs,low back pain(LBP)is increasingly recognized as a severe public health problem.It is now generally accepted that intervertebral disc degeneration(IDD)is the main pathogenesis of LBP.IDD is characterized with obvious changes in central nucleus pulposus(NP)tissue,where there is increased cell apoptosis and extracellular matrix(ECM)degradation.Thus,it represents a therapeutic strategy for IDD by decreasing NP cells(NPCs)apoptosis and increasing ECM expression.Leukemia inhibitory factor(LIF)is an important multifunctional cytokine.Our previous study demonstrated that LIF played an important role in regulating ECM metabolism in chondrocytes.Since NPCs and chondrocytes share similar cell phenotypes,in this study we put forward a novel hypothesis that LIF played a crucial role in regulating apoptosis and ECM metabolism of NPCs.Objective:To investigate the effects and mechanism of LIF on cell apoptosis and ECM metabolism of NPCs by both in vivo and in vitro study.Method:The rabbit IDD model was firstly established by acupuncture,then the degenerative degree of IDD was evaluated by magnetic resonance(MRI).The rabbits were executed to obtain intervertebral disc tissue at the corresponding time,which was used to detect ECM synthesis by safranine O/ fast green staining,and tissue protein expression of LIF was detected by western blot.Human degenerative NPCs were isolated and cultured,which were incubated with different concentrations of recombinant human LIF(rhLIF 0,10,20,50,100 ng /ml).Then alcian blue staining,CCK-8 test and Annexin V/PI flow cytometry were performed to preliminarily investigate the effects of LIF on cell apoptosis and ECM metabolism in degenerative NPCs.To further investigate the molecular mechanism of LIF on ECM expression,degenerative NPCs were stimulated by different concentration of LIF for different treatment periods.Then,the main components of ECM,aggrecan and type II collagen(COL2)were evaluated by western blot.Moreover,the key metabolic enzymes of ECM,MMP-3 and TIMP-1 were also detected.Moreover,the related Akt and MAPK signal pathway were detected.In order to further verify the influence of MAPK signal pathway on ECM expression,the specific inhibitor PD98059 was added to co-culture with NPCs,then the aggrecan and COL2 protein expressions were detected.Lastly,the rhLIF and rhLIF+ PD98059 were directly injected into intervertebral disc of rabbit IDD model to observe the therapeutic effect for deferring the degenerative process of intervertebral disc.Result:Following a success in establishing a rabbit IDD model,MRI scaning showed that water contents in intervertebral discs of the modeling animals decreased as time going on.Meanwhile,safranine O-fast green staining indicated that ECM degradation was aggravated with time,too.Western blot further demonstrated that protein expression of LIF increased in degenerative intervertebral discs.In vitro study showed that rhLIF treatment increased ECM synthesis in degenerative NPCs,and it strengthened cell vitality and reduced cell apoptosis too.These results preliminarily suggested that LIF played an important role in promoting ECM synthesis and prohibiting apoptosis in degenerative NPCs.Further study demonstrated that LIF significantly increased aggrecan and type II collagen contents.Simultaneously,LIF inhibited MMP-3 expression but increased TIMP-1 expression.These data suggested that LIF had significant influence on ECM metabolic enzymes.Mechanically,with the finding that aggrecan and type II collagen expressions elevated by LIF was significantly slacked down by PD98059,it could be concluded that LIF increased ECM synthesis by activating ERK1/2 pathway.Lastly,in vivo interventional study showed that rhLIF effectively delay the degenerative process of intervertebral disc,and this protective effect of LIF was markedly weakened by PD98059.Coclusion:LIF is highly expressed in NPCs from degenerative NP tissues and it inhibits NPCs apoptosis and promotes ECM synthesis.The MAPK-ERK1/2 signal pathway isinvolved in the increasing ECM synthesis effect regulated by LIF.Therefore,LIF has the potential for being a novel target for the treatment of IDD.