Effect Of Dyslipidaemia On Intervertebral Disc Degeneration

Lumbar disc herniation(LDH)is a major cause of low back pain and sciatica,while intervertebral disc degeneration(IVDD)has been considered to be the pathological basis of it,however,the underlying pathophysiologic mechanism remains unclear.A synergistic effect involving many factors plays a role in the development of IVD degeneration,including abnormal mechanical loading,nutrition-related metabolic disorders,upregulation of matrix metalloproteinases(MMPs),and ADAMTS expression and activation of cytokines.Among them,nutrition-related metabolic disorders is one of the reasons for IVDD,which we can not be ignored.The intervertebral disc is a structure with avascular tissue whose primary nutritional supply is via blood capillary penetration of the vertebral bodies through to the endplate.Dyslipidaemia,as performance of lipid metabolic disorders,is a well-known risk factor for the development of atherosclerosis.Atherosclerosis may decrease blood supply to the vertebral body and reduce the amount of endplate nutrients,a process that eventually leads to intervertebral disc degenerative disease.we were prompted to investigate whether there was a relationship between IVD degeneration and abnormal serum lipid levels.We therefore designed a case-control study to determine the serum lipid levels in patients with lumbar disc herniation.Whereafter,we investigate the effect of dyslipidaemia in degenerated disc through in vivo experiments.Histomorphological changes were detected in both hyperlipidaemia rats and normal rats in order to identify the possible mechanisms by which dyslipidaemia aggravates the degeneration of discs.Moveover,we examined the changes of extracellular matrix and metabolic factors.Finally,observe the effects of palmitic acid(PA)on apoptosis of nucleus pulposus cells(NPCs)and its molecular mechanism,exploring the effect of lipid metabolism to nucleus pulposus cells under the cellular level.Part I The correlation between serum lipid levels and lumbar degenerative diseasesObjective:Abnormal serum lipid levels have been shown to be associated with the occurrence of atherosclerosis,but little is known about the relationships of them with the risk of developing intervertebral disc degeneration(IVDD)in Chinese population.The purpose of this study was to assess the relationship between serum lipid level and the risk of lumbar disc degeneration.Methods:We performed a case-control study to assess the relationship between serum lipid levels and lumbar disc degeneration.A total of 790 Chinese patients were recruited for this study at the time of hospitalization.We examined fasting serum lipid levels of total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C).396 patients(235 men and 161 women;mean age:41.07 years)underwent surgery for single-level lumbar disc herniation.A control group of 394 patients(225 men and 169 women;mean age:42.1 years)underwent surgery for wounded lower limbs during the same period.Patients in the control group were collected randomly from among patients who were age-and sex-matched patients with the case group.Results:Patients with lumbar disc herniation had significantly higher TC and LDL-C serum concentrations(P<0.001 for both)than controls.Percentage of High-TC,High-TG,High-LDL-C,borderline High-TC and borderline High-LDL-C were significantly higher in the disc herniation group(P=0.017,P=0.002,P=0.039,P=0.002 and P<0.001,respectively).Ratios of TC/HDL-C and LDL-C/HDL-C were significantly associated with disc herniation(P<0.001 for both).Logistic regression revealed that patients with higher serum LDL-C levels had a higher risk of disc herniation,in which odds ratio(OR)was 1.462 and confidence interval(CI)was 1.179-1.813.Moreover,patients with High-TG and borderline High-LDL-C had a higher probability of disc herniation(OR:2.974,Cl:1.488-5.945,statistical power:100%;OR:1.626,Cl:1.012-2.612,statistical power:61.4%,respectively).Hyperlipidaemia did not seem to be associated with the herniated segment of the lumbar intervertebral disc(p=0.374),however,the degenerative degree in hyperlipidemia group was more serious than control(P<0.001).Conclusions:The present study suggests that dyslipidaemia may be associated with a higher risk of developing lumbar disc herniation.Serum lipid levels could be a useful predictor for intervertebral disc degeneration in Chinese population.Part II Effect of hyperlipidaemia on intervertebral disc degeneration in rat modelsObjective:The purpose of this study is to investigate the relationship between hyperlipidaemia and IVDD in rat models and to identify the possible mechanism by which dyslipidaemia aggravates the degeneration of intervertebral discs.Methods:Hyperlipidaemia rats were induced,sixty male Wistar rats were randomly divided into two groups:normal chow diet control group(CON group,n=30)and high-fat diet group(HFD group,n=30).And then,the discs were harvested 4.6 and 8 months late,histomorphological analysis,immunohistochemistry staining for IL-1β,TNF-a,MMP-13,HIF-la,P65 and real-time PCR analysis for MMP-13,TIMP-1,BMP-2 were performed for all groups.Results:The degenerative histological score of disc in the HFD group was significantly higher than the CON group at 6 and 8 months.The decrease in disc height and increase in cartilage endplate thickness were observed at 8 month in the HFD group.Immunohistochemical analysis revealed remarkable reductions in aggrecan and collagen type II expressions,and significant increases in IL-1β,TNF-a,MMP-13,HIF-la and P65 expression in the HFD group at 6 and 8 month.RT-PCR showed that the mRNA levels of MMP-13 was higher in the HFD group at 6 and 8 month,the transcription of TIMP-land BMP-2 was higher in the HFD group at 6 month,while decreased in 8month.Conclusions:In conclusion,these results show that hyperlipidaemia may induce the progression of disc degeneration,and hyperlipidaemia may represent a risk factor for IVDD pathologyPart Ⅲ Effect of hyperlipidaemia to accelerate intervertebral discdegeneration in the injured rat caudal disc modelObjective:The purpose of this study is to investigate the histomorphological change,the expression of inflammatory cytokines and the metabolism of extracellular matrix in the injured rat caudal disc model with hyperlipidaemia.Methods:Hyperlipidaemia rats were induced,thirty male Wistar rats were randomly divided into two groups:normal chow diet control group(CON group,n=20)and high-fat diet group(HFD group,n=10)for 8 weeks.And then,a rat disc degeneration model was established,rats were divided into three experimental groups:the normal chow diet + sham surgery group(CON-Sham group,n = 10);the normal chow diet +needle puncture group(CON-NP group,n = 10);and the high-fat diet + needle puncture group(HFD-NP group,n = 10),all rats were continuely fed with normal chow diet or HFD 8 weeks.At the end of the experiment,histomorphological analysis,immunohistochemistry staining and real-time PCR were performed for all groups.Results:The degenerative histological score of disc in the HFD-NP group was significantly higher than the CON-NP group.Immunohistochemical analysis revealed remarkable reductions in aggrecan and collagen type II expressions,and significant increases in IL-1β,TNF-a,MMP-13,HIF-la and P65 expression in the HFD-NP group.RT-PCR showed that the mRNA levels of MMP-13 and TIMP-1 were higher in the HFD-NP group.Conclusions:Hyperlipidemia resulted in an exaggerated degenerative changes and altered expression and transcription of the degeneration-associated molecules in the rat disc tissue.These results raise the possibility that hyperlipidemia may accelerate the progression of disc degeneration,which may lead to new approaches against the IVD degeneration in the future.Part Ⅳ Palmitic acid induces apoptosis of nucleus pulposus cells and affects the metabolism of extracellular matrix in vitroObjective:The deleterious effects of palmitic acid(PA)on various cells have been well documented,however,the effects of PA on nucleus pulposus cells(NPCs)function have not been fully explored.The purpose of the present study was to observe the effects of palmitic acid(PA)on apoptosis of nucleus pulposus cells(NPCs)and its molecular mechanism,exploring the effect of lipid metabolism to nucleus pulposus cells under the cellular level.Methods:Rat NPCs were isolated,cultured in vitro,and placed in DMEM/F12 medium with 10%foetal bovine serum(FBS).After stimulated with PA at various concentrations and different times,the cell apoptosis was observed by flow cytometry;ECM metabolism-related genes,including aggrecan,collagen type II,MMP-13,TIMP-1 and BMP-2 were analyzed by RT-PCR;Western blot was used to detect the protein expression of aggrecan and collagen type Ⅱ.Then,NPCs cultured with high PA(800μmol/L)were pretreated with p38-MAPK inhibitor SB203580,the activation of p38-MAPK was measured by western blot,and the mRNA expression of aggrecan,collagen type Ⅱ,TNF-a and iNOS was analyzed by RT-PCR.Results:PA increased NPCs apoptosis in dose-and time-dependent manners;the expression of aggrecan,collagen type Ⅱ,MMP-13,TIMP-1 and BMP-2 was decreased with dose-and time-dependent effects,but the expression of MMP-13 was increased;PA also up-regulated the expression of TNF-a and iNOS,and actived the p38-MAPK signaling pathway,however these effects could be inhibited by p38-MAPK inhibitor SB203580.Conclusions:PA is able to exert a pro-inflammatory effect on NPCs via stimulating TNF-a and iNOS expression,partly through the p38-MAPK pathway,increasing the of apoptosis NPCs and imbalance of extracellular matrix metabolism(ECM).These findings might explain in part the role of PA to NPCs in the processes of intervertebral disc degeneration(IVDD).Therefore,these results suggest that aggressive PA control should be recommended to prevent or limit intervertebral disc degeneration.