| BackgroundHypertrophy of the ligamentum flavum(HLF)is an important factor for lumbar spinal stenosis(LSS).In severe cases,it can compress nerve roots and cauda equina,causing lumbar pain and intermittent claudication.Although some studies have suggested that the ligamentum flavum(LF)hypertrophy may be related to various inflammatory factors,growth factors,aging and mechanical stress,the key molecules that cause the ligamentum flavum hypertrophy(LFH)and their regulatory mechanisms remain unknown.In addition,due to the lack of an effective animal model of ligamentum flavum hypertrophy,there are currently no in vivo studies on the molecular mechanism of ligamentum flavum hypertrophy.ObjectiveThe purpose of this study is:1.To find out the key regulatory proteins of the ligamentum flavum hypertrophy,and explore its mechanism of regulating the formation of extracellular matrix(ECM).2.To establish a new mouse model of ligamentum flavum hypertrophy,and verify the molecular mechanism in vivo.Methods1.The normal LF and HLF tissues were collected and the fibrosis of the two groups was detected by histological staining.Two groups of samples were analyzed by isobaric tags for relative and absolute quantitation(iTRAQ)technology,and the key protein:cytokine receptor like factor 1(CRLF1)was found by integrating the data of transcriptome and proteome.Immunohistochemistry and qPCR were used to verify the results.2.Human normal LF cells were isolated and cultured in vitro.TGF-?1 and mature recombinant human CRLF1(rhCRLF1)was used to treat the LF cells.The mRNA,protein expression of fibrosis related markers and the activation of SMAD3 and ERK1/2 signalling pathway were detected by immunofluorescence,Western blot and RT-qPCR.ERK1/2 pathway inhibitor was added to observe the biological effects of CRLF1 and TGF-?1.The effect of SMAD3 pathway inhibitor on TGF-?1 induced CRLF1 mRNA expression was observed.The expression of CRLF1 was inhibited by siRNA technology,and the effects of TGF-?1,IL-1? and mechanical stress-induced fibrosis were observed.3.To establish a model of HLF in bipedal standing mice.The stress on LF was analyzed by finite element analysis.Histologic staining was used to observe whether the pathological changes were the same as those of human hypertrophic ligamentum flavum.4.To construct the adeno-associated virus vector of CRLF1 overexpression and inhibition,and to verify the role of CRLF1 in the development of LF hypertrophy in vivo.Results1.Orderly elastic fibers in the HLF tissue were significantly reduced,irregular collagen fibers,the collagen fibers and myofibroblasts and the expression of p-ERK1/2 were significantly increased;CRLF1 was highly expressed in HLF.The expression of CRLF1 and a-SMA was positively correlated.2.TGF-?1 regulates the synthesis of fibrosis related markers at the transcriptional level,while CRLF1 mainly promotes the synthesis of fibrosis related markers at the post transcriptional level;The ERK1/2 signalling pathway was activated by CRLF1 and TGF-?1.The fibrogenic effects of CRLF1 and TGF-?1 were inhibited when the ERK1/2 pathway was blocked.TGF-?1 can promote the synthesis of CRLF1 mRNA,but the increase of CLCF1 mRNA was not obvious.CRLF1 mRNA was significantly down-regulated when SMAD3 pathway was blocked.Inhibition of CRLF1 can reduce fibrosis caused by TGF-?,IL-1? and mechanical stress.3.The stress of the ligamentum flavum in bipedal standing mice increased significantly,and the model of ligamentum flavum hypertrophy in bipedal standing mice could effectively simulate the pathological changes of human LFH.4.Compated to the control group,the number of CRLF1 positive cells in bipedal standing mice increased.CRLF1 over expression can lead the formation of HLF in vivo,and inhibition of CRLF1 reduced the formation of HLF caused by bipedal standing.ConclusionCRLF1 plays a key role in the process of multiple stimulation induced fibrosis.Inhibition of CRLF1 can reduce the formation of ligamentum flavum hypertrophy. |
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