| BackgroundAs an important pathological process,intervertebral disc degeneration(IVDD)plays an important role in influencing the morphology and normal physiological function of lumbar intervertebral disc,and eventually leads to the decline of its ability to bear compression load.As a multi factor disease,its etiology is still not completely clear,among which genetic tendency,age,lifestyle(obesity,smoking,depression)and non physiological mechanical load are all the reasons for its progress.Studies have shown that IVDD is' the main cause of low back pain(LBP),about 80%of adults have different degrees of low back pain symptoms,and become one of the important risk factors of disability in the world.It is estimated that the direct cost caused by LBP in the United States can be as high as US $90 billion per year.At the same time,the reduction of productivity caused by LBP poses a significant challenge to the overall socio-economic impact.Many studies have pointed out that the pathological changes in IVDD are related to intervertebral disc degeneration,and the degradation of extracellular matrix,inflammation and apoptosis are the main reasons.Extracellular matrix(ECM)plays an important role in the mechanical function of intervertebral disc.Type ? collagen and type ? collagen provide tensile strength,and proteoglycans participate in the formation of disc tissue structure.However,the ability of nucleus pulposus(NP)cells located in the central area of intervertebral disc to synthesize ECM components is reduced,and the secretion of ECM degradation molecules(such as matrix metalloproteinases)is significantly increased,which leads to the continuous decrease of water binding ability of intervertebral disc tissue,and eventually leads to structural and functional damage.Studies have confirmed that degeneration first occurs in the nucleus pulposus,and then the damage further extends to the annulus fibrosus(AF)in the outer region of the intervertebral disc,leading to the disappearance of the boundary between the two different tissues.In this process,cell loss and density reduction are an important reason for intervertebral disc degeneration,and further enhance the degradation of ECM.Most studies indicate that cell loss can be induced by programmed cell apoptosis,which is enhanced by cell aging,mechanical stress and other potential factors.In addition to ECM degradation and cell loss,inflammation also plays an important role in the occurrence and progression of IVDD,which has become an important factor to distinguish between asymptomatic and symptomatic disc degeneration.NP cells can release pro-inflammatory cytokines in the process of injury,and the expression of TNF-?,IL-6,IL-17 and IL-1? is the most significant.These cytokines can promote the degradation of matrix and activate host immune response,and eventually lead to the infiltration of immune cells in the nerve fibers,which leads to progressive degeneration,and nerve infiltration is the main cause of intervertebral disc degeneration pain.Most studies have confirmed that ECM degradation,apoptosis and inflammation are the markers of IVDD,and the three pathological reactions are interrelated and dependent on each other.Proinflammatory cytokines can lead to metabolic disorders by up regulating the expression of ECM degrading enzymes and reducing the structural components of ECM.At the same time,a large number of intrinsic degradation of ECM leads to the accumulation of ECM fragments outside the cells,and further stimulates the inflammatory response of NP cells as autoimmune antigens.In addition,the higher apoptosis rate,aging rate and lower ECM production ability in intervertebral disc tissue are closely related to inflammation.In recent years,it has been pointed out that the physiological changes of IVDD are closely related to non mechanical stress.At present,most researchers have found that mechanical stress,ECM degradation,cell loss and inflammatory response are interdependent in the occurrence and progress of IVDD,but there is still a lack of consensus on the specific mechanism of each injury process.At the early stage,non steroidal drug therapy and physical therapy are the conventional treatment of IVDD,and invasive surgery for spinal fusion or arthroplasty can be used as the second-line treatment which is difficult to relieve.Although the success rate of surgery is high at present,the limitations of high operation cost,postoperative pain and lower quality of life seriously affect the surgical effect.Therefore,exploring new treatment schemes to improve the treatment status has become a hot issue to be solved urgently.In recent years,the feasibility,safety and effectiveness of growth factor injection in the treatment of intervertebral disc degeneration have been widely studied,and show a good therapeutic prospect.Most studies have shown that growth factor therapy,as a treatment scheme to promote intervertebral disc tissue regeneration,can improve the recovery ability of IVD through typical combination of growth factors,and show the potential of regulating anabolism and inhibiting catabolism in vitro and animal models.Previous studies have pointed out that increasing ECM anabolism and reducing ECM catabolism are of great significance in maintaining the stability of ECM and alleviating the process of intervertebral disc degeneration.Therefore,growth factor therapy may be a new scheme for alleviating and treating IVDD.? type Collagen is an important component of ECM,mainly secreted by chondrocytes,and plays an important role in maintaining the stability of endplate cartilage.? Collagen type ? reflects the anabolic ability of chondrocytes to some extent.Matrix metalloproteinase(MMP),which degrades ? type collagen in ECM,is one of the key catabolic enzymes in endplate cartilage.Tissue inhibitor 4(TIMP-4)of MMPs is a specific inhibitor of MMPs,which can inhibit the metabolic regulation of MMP and reduce the decomposition level of ECM.Therefore,TIMP-4 can also be used as a key marker of chondrocyte anabolism.In recent years,fibroblast growth factor-2(FGF-2)is a member of fibroblast growth factor family,which can promote mitosis and cell proliferation,and is widely used in various tissue repair.Some studies have shown that the role of FGF in the skeletal system is initiated by binding with fibroblast growth factor receptor(FGFR),and it may have a certain therapeutic effect in alleviating intervertebral disc degeneration.Studies have also shown that the content of FGF-2 in patients with osteoarthritis(OA)is significantly higher than that in normal conditions,so the high content of FGF-2 may aggravate the degradation of cartilage.FGF-2 can promote the expression of MMP-13 and ADAMTS-5,and inhibit the synthesis and aggregation of proteoglycan by binding to FGFR1,thus accelerating cartilage degradation.Comprehensive analysis shows that FGF-2 plays a two-way role in human CHS,which can accelerate the occurrence of OA.However,some researchers found that FGF-2 knockout mice are more prone to OA than normal mice,and subcutaneous injection of FGF-2 can slow down this effect.All of the above studies indicate that FGF2 may play a unique role in delaying the pathological process and treatment of IVDD,but there are few reports on its specific mechanism.Therefore,this study aims to explore the mechanism and biological significance of FGF2 in improving the metabolism of intervertebral disc degeneration.ObjectiveIn this study,the expression level of FGF2 in cartilage endplate of different lumbar degenerative patients was observed,and the application effect of exogenous FGF2 on normal chondrocytes,degenerative chondrocytes was used to explore the regulatory role of FGF2 in the metabolism and catabolism of cartilage endplate cells.In this study,we further analyzed the regulation of FGF2 on the expression of Caspase-3 and Bcl-2 in the process of improving metabolic response,and explored the specific mechanism of FGF2 in promoting the proliferation and inhibiting apoptosis of cartilage endplate cells.To clarify the biological significance of FGF2 in alleviating the degeneration of cartilage endplate cells,and to provide basic experimental data for exploring the effective treatment of IVDD in the future.MethodsPart One Expression of FGF2 in endplate cartilage of patients with lumbar degenerative diseaseFrom March 2017 to November 2018,the medical records of 8 patients who underwent spinal fusion in the Department of orthopedics of our hospital were analyzed.According to the MRI imaging results,the patients were divided into mild IVDD group and severe IVDD group,with 4 patients in each group.All patients underwent MRI to observe the morphology of the diseased intervertebral discs,and the resected intervertebral discs were stained with HE.Real time quantitative polymerase chain reaction(RT qPCR)was used to detect the mRNA expression of FGF2,FGFR1,FGFR,MMP-13,TIMP-4 and type ? collagen.CCK-8 method was used to detect the proliferation and metabolic status of cartilage endplate cells after cultured in vitro for 0,24,48 and 72h.Part Two Effect of exogenous FGF2 on metabolism of normal endplate chondrocytesNormal human chondrocytes were used for resuscitation,culture and passage.Logarithmic growth phase cells were cultured with different concentrations(5ng/ml and 10NG/ml)of exogenous FGF2.At the same time,the blank control group was set without FGF2 protein treatment.Real time quantitative polymerase chain reaction(RT qPCR)was used to detect the mRNA expression of FGF2,FGFR1,FGFR,MMP-13,TIMP-4 and type ? collagen.The expression of type ? collagen was analyzed by immunofluorescence.CCK-8 method was used to detect the proliferation and metabolic status of cartilage endplate cells after cultured in vitro for 0,24,48 and 72h.Annexin V-FITC/PI double staining method was used to stain the cells at 0,12,24,48 and 72 h after culture,and the apoptosis level was analyzed by flow cytometry.Part Three Metabolic regulation and expression of Caspase-3 and Bcl-2 in cartilage endplate cells in vitroNormal human chondrocytes were selected for resuscitation,culture and passage.Logarithmic growth phase cells were selected to induce degenerative degeneration with IL-1?.Real time quantitative polymerase chain reaction(RT qPCR)was used to detect the mRNA expression of FGF2,FGFR1,FGFR,MMP-13,TIMP-4 and type ?collagen.The expression of type ? collagen was analyzed by immunofluorescence.CCK-8 method was used to detect the proliferation and metabolic status of cartilage endplate cells after cultured in vitro for 0,24,48 and 72h.The expression of Caspase-3 and Bcl-2 was detected by Western blot.Annexin V-FITC/PI double staining method was used to stain the cells at 0,12,24,48 and 72 h after culture,and the apoptosis level was analyzed by flow cytometry.Part Four Effect of exogenous FGF2 combined with FGFR1 blocker on delayed degeneration of endplate chondrocytesNormal human chondrocytes were selected for resuscitation,culture and passage.Logarithmic growth phase cells were selected to induce degenerative degeneration with IL-1?.The chondrocytes were divided into two groups:FGF2 group and FGF2+PD(FGFR1 inhibitor)group.Another blank group was selected as control group,in which FGF2 and PD proteins were not added.Real time quantitative polymerase chain reaction(RT qPCR)was used to detect the mRNA expression of FGF2,FGFR1,FGFR,MMP-13,TIMP-4 and type ? collagen.The expression of type ? collagen was analyzed by immunofluorescence.CCK-8 method was used to detect the proliferation and metabolic status of cartilage endplate cells after cultured in vitro for 0,24,48 and 72h.The expression of Caspase-3 and Bcl-2 was detected by Western blot.Annexin V-FITC/PI double staining method was used to stain the cells at 0,12,24,48 and 72 h after culture,and the apoptosis level was analyzed by flow cytometry.Results1.The height of adjacent vertebrae in severe degeneration was lower than that in mild degeneration,and the degree of moisture in MRI was lower than that in mild degeneration.He staining showed that cartilage endplate tissue was harder and more densely distributed than nucleus pulposus tissue,and the distribution of cartilage endplate cells was closer than nucleus pulposus cells.The expression levels of FGF2,FGFRl and MMP-3 in cartilage endplate cells with severe degeneration of intervertebral disc were significantly higher than those with mild degeneration of intervertebral disc(P<0.001),but there was no difference in the expression levels of FGFR3 between the two degenerative changes(P>0.05).The expression levels of type ? collagen and TIMP-4 in severe disc degeneration were lower than those in mild disc degeneration(P<0.05).2.After 72 hours of exogenous FGF2 protein treatment,there was no difference in the expression of FGF2 mRNA among the three groups(P>0.05);the levels of FGFR1 and FGFR3 in the FGF2 treatment group were significantly higher than those in the control group(P<0.05).There was no difference in the expression level of FGFR1 between 5ng/ml group and 10NG/ml group(P>0.05);the expression level of FGFR3 in 10NG/ml group was significantly higher than that in 5ng/ml group(P<0.001).After 72 hours of treatment with exogenous FGF2 protein,the expression level of TIMP-4 in the two groups was higher than that in the control group significantly,and the expression in the 10ng/ml group was more than that in the 5ng/ml group(P<0.05);there was no difference in the expression level of MMP-13 among the three groups(P>0.05).The expression level of type ? collagen in 10NG/ml group was significantly higher than that in control group and 5ng/ml group(P<0.001);there was no difference between control group and 5ng/ml group(P>0.05).The cell proliferation activity of the three groups increased with time at different time points(P<0.05).At the same time point,the proliferative activity of the three groups showed that:? there was no difference in the proliferative activity of the three groups at 0 h(P>0.05);?The proliferation level of 10NG/ml group was higher than that of 5ng/ml group and control group(P<0.05),but there was no difference between 5ng/ml group and control group(P>0.05);? At 24h,the proliferation level of 10NG/ml group and 5ng/ml group was higher than that of control group(P<0.05),but there was no difference between 10NG/ml group and 5ng/ml group(P>0.05);?After 48h and 72h of culture,the level of 10NG/ml group was higher than that of 5ng/ml group and control group,and the level of 5ng/ml group was significantly higher than that of control group(P<0.05).The apoptosis rates of the three groups were not all the same(P<0.05),and there was no difference among the three groups at Oh after culture(P>0.05);at 12h after culture,the apoptosis rate of 10NG/ml group was higher than that of 5ng/ml group and control group,and the apoptosis rate of 5ng/ml group was lower than that of control group(P<0.05);there was no difference among the three groups at 24h,48h and 72h after culture(P>0.05).3.The expression level of FGF2 was lower 24 hours after IL-1? treatment than that of the control group;however,the expression level of FGF2 was higher 48 and 72 hours after IL-1? treatment than that of the control group,and the level of FGF2 72 hours after IL-1? treatment was higher than that 48 hours after IL-1? treatment(P<0.05).At 24,48 and 72 h after IL-1? treatment,the content of FGFR1 in degenerative cells in the treatment group was higher than that in the control group,and the level of FGFR1 in the treatment group continued to increase with time(P<0.05).There was no difference in the expression level of degenerative cells between the treatment group and the control group 24 h after IL-1? treatment(P>0.05);the expression level of degenerative cells in the treatment group was lower than that in the control group 48 h and 72 h after IL-1? treatment;there was no difference in the level of FGFR3 between the treatment group 48 h and 72 h after IL-1?treatment(P<0.05).72 hours after IL-1? treatment,the level of type ? collagen in degenerative cells in the treatment group was lower than that in the control group(P<0.05).At the same time,the level of MMP-13 in IL-1? treated cells was significantly higher than that in the control group(P<0.001);there was no difference in the expression of TIMP-4 between the two groups(P>0.05).The proliferation activity of cells in the two groups was not the same at different time points within 72 hours,and the proliferation activity levels were continuously decreased(P<0.05),and there was no difference between the two groups at 0 hours(P>0.05);after 12,24,48 and 72 hours of culture,the proliferation activity of cells in the IL-1? treatment group was lower than that in the control group(P<0.05).The expression of Caspase-3 in IL-1? treated group was higher than that in control group,but the expression of Bcl-2 in IL-1? treated group was lower than that in control group(P<0.05).The apoptotic rate of IL-1? treatment group increased continuously,while that of control group decreased first and then increased slowly(P<0.05).The apoptotic rate of the two groups at the same time point was compared and analyzed.The results showed that:? there was no difference in the apoptotic rate of the two groups at 0 h after culture(P>0.05);?the apoptotic rate of the IL-1? treated group was higher than that of the control group at 12,24,48 and 72 h after culture(P<0.05).4.There was no difference in the expression level of FGF2 mRNA among the three groups(P>0.05),but the expression levels of FGFR1 and FGFR3 mRNA were not the same among the three groups(P<0.05).The expression level of FGFR1 in IL-1?+FGF2 treatment group was significantly higher than that in control group,and that in IL-1 ?+FGF2+PD group was significantly lower than that in control group and IL-1?+FGF2 group(P<0.05).The expression of FGFR3 in IL-1 ?+FGF2+PD group was significantly higher than that in IL-1 ?+FGF2 group and control group,and the expression level in IL-1 ?+FGF2 group was higher than that in control group(P<0.05).There was no difference in the expression of MMP-13,TIMP-4 and type ? collagen among the three groups(P<0.05).The expression of MMP-13 in IL-1 ?+FGF2 group was higher than that in IL-1 ?+FGF2+PD group and IL-1 ? group,and the expression level in IL-1 ?+FGF2+PD group was higher than that in IL-1 ? group(P<0.05).The expression of TIMP-4 in IL-1 ?+FGF2+PD group was higher than that in IL-1 ?+FGF2 group and IL-1 ? group,but there was no difference between IL-1 ?+FGF2 group and IL-1 ? group(P>0.05).The expression level of type ? collagen in IL-1?+FGF2 group was significantly lower than that in IL-1 ?+FGF2+PD group and IL-1 ? group,and the expression level in IL-1 ?+FGF2+PD group was higher than that in IL-1 ? group(P<0.05).The proliferation activity of cells in the three treatment groups increased with time at different time points(P<0.05).At the same time point,the proliferative activity of the three groups was compared,which indicated that:? at 0 h,the proliferative activity of the three groups had no difference(P>0.05);?at 12 h,the proliferative activity of IL-1 ?+FGF2 group and IL-1 ?+FGF2+PD group were higher than that of IL-1 ? group,but there was no difference between IL-1 ?+FGF2 group and IL-1 ?+FGF2+PD group(P>0.05);?The level of IL-1 ?+FGF2+PD group was significantly higher than that of IL-1 ?+FGF2 group and IL-1 ? group at 24,48 and 72 h,and the level of IL-1 ?+FGF2 group was higher than that of IL-1 ? group.Annexin V-FITC/PI double staining showed that the apoptosis rate of the three groups was not the same,and showed a continuous decrease(P<0.05).The comparison of apoptosis rate among three groups at the same time points indicated that:? there was no difference in the apoptosis rate of three groups at 0 and 12 h after culture(P>0.05);? at 24,48 and 72 h after culture,the level of IL-1 ?+FGF2+ PD group was lower than that of IL-1 ?+FGF2 group and IL-1 ? group,and the apoptosis rate of IL-1 ?+FGF2 group was lower than that of IL-1 ? group(P<0.05).ConclusionThis study elucidates the role of FGF2 in chondrocyte degeneration.The multiple roles of FGF2 in intervertebral disc homeostasis depend on the stage of degeneration and the type of disease process.In normal chondrocytes,FGF2 may act as a kind of synthetic metabolic medium,stimulate the expression of TIMP-4 and type ? collagen,decrease the content of MMP-13,induce cell proliferation and reduce apoptosis.FGF2 may be used as a catabolic mediator to stimulate the expression of MMP-13,inhibit the synthesis of proteoglycan,up regulate the expression of Caspase-3 and down regulate the expression of Bcl-2,induce apoptosis and aggravate intervertebral disc degeneration.The abnormal activation of FGFR1 in this pathological process may be the main factor to promote catabolism.Targeted blocking the expression of FGFR1 can effectively reduce the decomposition of ECM,which has positive significance in improving the treatment of intervertebral disc cartilage degeneration.Therefore,the combination of FGF2/FGFR1 antagonists is expected to be a potential new treatment to prevent disc cartilage degeneration and promote the regeneration and repair of endplate cartilage. |
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