The Role Of MiR-503 In The Induction Of Extracellular Matrix Degradation Of Human Nucleus Pulposus By IL-1?

Objective:Intervertebral degenerative disc(IVDD)is the pathological basis of degenerative diseases of the spine and causes an important cause of low back pain.At present,the clinical treatment of IVDD is mainly to relieve clinical symptoms and does not prevent,delay or reverse the pathological process of IVDD.There is no effective prevention and treatment measures.IVDD is a tedious process.Its occurrence and recurrence rate are high and harmful.It is regulated by multiple factors and complex mechanisms.The specific mechanism for IVDD is still unclear.Studies have shown that apoptotic and reduced numbers of nucleus pulposus cells lead to reduced synthesis of extracellular matrix,disrupted synthesis and degradation of extracellular matrix dynamic balance,can lead to disc degeneration.In the process of spinal degeneration,inflammation is one of the major pathological changes.Non-coding microRNAs(miRNAs)are involved in the regulation of cell proliferation,differentiation,apoptosis,and inflammation.However,the role of MicroRNAs in the regulation of nucleus pulposus extracellular matrix degradation under inflammatory conditions is not fully understood.Therefore,this study aims to:culture nucleus pulposus cells in vitro and construct an inflammatory injury model of nucleus pulposus cells,study the role of miRNA-503 in the degradation of extracellular matrix of human nucleus pulposus induced by IL-1?,and provide a theoretical basis for preventing and treating intervertebral disc degeneration.And experimental basis.Methods:1.In the first part,(1)10 cases of intervertebral disc nucleus pulposus removed under intervertebral disc herniation due to disc herniation were collected[improved Pfirrmann's MRI score 2-3,within one year of history,the average age was 26 years old,(age The range is 22-35 years old];First,enzymatic digestion was used to extract nucleus pulposus cells from the freshly isolated nucleus pulposus and cultured in monolayer.The morphological structure and biological characteristics of the cells were observed,and toluidine blue staining and fluorescence quantification were performed.The cytokeratin 19 was detected by PCR to identify the nucleus pulposus cells;(2)The cultured nucleus pulposus cells were used to construct an inflammatory injury model of the nucleus pulposus cells in vitro.The IL-1? 0 ng/ml,IL-1? 1 ng/ml,IL-were used.1? 10ng/ml stimulated nucleus pulposus cells,and the effects of IL-1?,an inflammatory cytokine,on the nucleus pulposus cell viability were measured at 4h,8h,16h,24h,and 48h.(3)IL-1? Ong was used.The nucleus pulposus cells were stimulated with/ml and IL-1? 10 ng/ml,and the apoptosis of nucleus pulposus cells was detected by Hoechst 33258 at 4 h,8 h,16 h,24 h,and 48 h.The dual staining flow cells(FITC Annexin V/PI)were used.Flow cytometry and qRT-PCR analysis to detect changes in apoptosis rate in nucleus pulposus tissue,using fluorescence Needle(DCFH-DA)content in the nucleus pulposus detect ROS.2.In the second part,the expression of miR-503,MMP-9,ADAMTS-4,collagen ?,TIMP-3 and caspase3 was detected by real-time fluorescence quantitative PCR after stimulation of nucleus pulposus cells with IL-1?(10ng/ml,16h).In the third part,nucleus pulposus cells were transfected with miR-503mimic,miR-503antagomir,and their negative controls.After transfection,IL-1?(10ng/ml,16h)was added and flow cytometry was used to detect the apoptosis rate.Fluorescent quantitative PCR was used to detect the expression of MMP-9 and TIMP-3.Results:1.The first part of the experimental results:(1)Isolation and culture of nucleus pulposus cells,microscopic observation of the cell morphology is long spindle,polygonal,toluidine blue is a positive performance,keratin 19 was significantly higher in human nucleus pulposus cells;(2)The OD value in the IL-1?(10 ng/ml)group was significantly lower(P<0.01),indicating that the nucleus pulposus cells were significantly reduced in activity after induction of injury by IL-1?(10 ng/ml).Dependent on the gradient;(3)IL-1?(10ng/ml,16h)stimulated nucleus pulposus cells to show obvious apoptosis,cell nuclear fragmentation,nuclear condensation,apoptosis rate increased significantly(P<0.01);IL-1? The ROS content in the nucleus pulposus cells(10 ng/ml,16 h)was significantly higher than that in the control group(P<0.01),and ROS were abundantly produced in the nucleus pulposus cells.This indicated that the nucleus pulposus cell apoptosis was observed during IL-1?(10 ng/ml,16 h).The rate is most suitable for subsequent experiments.2.The second part of the experimental results:After IL-1?(10ng/ml,16h)acted on nucleus pulposus cells in vitro,the relative expression of MiR503,MMP9,ADAMTS4,Caspase3 mRNA was up-regulated compared with the control group(P<0.05);The relative expression of COL2A1 and TIMP3 mRNA was down-regulated compared with the control group(P<0.05).3.The third part of the experimental results:IL-1?+miR-503 mimics group than the IL-1?+miR-503 group MMP-9 mRNA expression was upregulated(P<0.05);IL-1? +miR-503 mimics group The expression of TIMP-3 mRNA in IL-1?+miR-503 group was down-regulated(P<0.05).Conclusion:1.Explored the most obvious apoptosis of nucleus pulposus cells induced by IL-1?at 10 ng/ml 16 h.2.High expression of miR-503 in human intervertebral disc degeneration induced by IL-1?.3.miR-503 regulates the degradation of extracellular matrix of human nucleus pulposus induced by IL-1?.