| Objective: To investigate the mechanism of that whether the high mobility group protein 1(HMGB1)induces the osteogenic differentiation of human ligamentum flavum cells by activating the ERK signaling pathway.Methods: 1.Collect 3 cases of spine surgery diagnosed as thoracic spine ligamentum flavum ossification patients to collect the ossified ligamentum flavum and normal ligamentum flavum tissues.The relative and absolute quantification of TMT(Tandem Mass Tag)with isotope labeling combined with liquid chromatography-tandem mass spectrometry/mass spectrometry(LC-MS/MS)technology was used to analyze differential proteins,And perform GO function annotation analysis,use the correlation between inflammatory proteins or factors and the pathogenesis of TOLF,and screen the proteins of interest.2.Collect 8 specimens of ligamentum flavum from patients undergoing posterior fenestration of nucleus pulposus.Collagenase and tissue block method were used to separate,culture,and pass ligamentum flavum cells.Immunofluorescence staining was used to identify ligamentum flavum cells.3.(1)Experimental grouping: control group,different concentrations of HMGB1group(25 ng/mL,50 ng/mL,100 ng/mL),HMGB1(50 ng/mL)+U0126 group(ERK inhibitor),TPA group(ERK activator)(2)The results show that HMGB1 has an effect on the cytotoxicity of the ligamentum flavum by CCK-8 method detected.(3)Alkaline phosphatase(ALP)staining and quantification,alizarin red staining to detect ALP activity and mineralized nodule formation in ligamentum flavum cells.(4)Western Blot,RT-QPCR,to detect the expression of OSX and OCN in ligamentum flavum cells.(5)50ng/mL HMGB1 and U0126 and 50ng/mL HMGB1 were used to treat ligamentum flavum cells,and Western Blot was used to detect the expression of ERK1/2(p-ERK1/2)in the cells.Results: 1.A total of 586 differentially expressed proteins were identified through 5TMT combined with LC-MS/MS technology,of which 532 protein expression was up-regulated and 54 protein expression was down-regulated.According to GO function annotation analysis,23 inflammatory-related proteins are identified that are closely related to the pathogenesis of TOLF.Among them,calcium binding protein A10(S100A10),folate receptor β(FR-β),calcium binding protein A12(S100A12),and high mobility group protein B1(HMGB1)are closely related to osteogenic differentiation and calcification.2.The ligamentum flavum cells were successfully isolated and cultured by the method of collagenase and tissue block,and they were positive by immunofluorescence staining.3.(1)The 24 h and 48 h cytotoxicity test results of ligamentum flavum cells showed that HMGB1 had no toxic effect on ligamentum flavum cells.Compared with the control group,the 25 ng/mL,50 ng/mL,and 100 ng/mL(HMGB1)groups increased the ALP synthesis and activity of ligamentum flavum cells in a concentration-dependent manner,and the difference was statistically significant(P <0.001).4.Compared with the control group,the 25 ng/mL,50 ng/ml,and 100 ng/ml(HMGB1)groups showed the formation of orange-red concentric circular mineralized nodules,while the control group did not see the formation of mineralized nodules.5.RT-QPCR results showed that compared with the control group,the 25 ng/mL,50ng/mL,and 100 ng/mL(HMGB1)groups increased the expression of OSX and OCN m RNA in a concentration-dependent manner,the difference was statistically significant(P<0.001).Compared with the control group,the TPA group significantly increased the expression of OSX and OCN m RNA,and the difference was statistically significant(P<0.01).Compared with the 50 ng/mL HMGB1 group,U0126+50 ng/mL HMGB1 down-regulated the expression of OSX and OCNm RNA,and the difference was statistically significant(P<0.001).6.Western Blot results showed that compared with the control group,25 ng/mL,50ng/mL,100 ng/mL(HMGB1)up-regulated the expression of OSX and OCN proteins in a concentration-dependent mannerand the difference was statistically significant(P<0.001).Compared with the control group,the TPA group significantly increased the expression of OSX and OCN proteins,and the difference was statistically significant(P<0.001).Compared with 50 ng/mL HMGB1,U0126+50 ng/mL HMGB1down-regulated the expression of OSX and OCN proteins,and the difference was statistically significant(P < 0.05).Compared with the 50 ng/mL HMGB1 group,U0126+50 ng/mLHMGB1 down-regulated the protein expression of phosphorylated ERK1/2,and the difference was statistically significant(P<0.001).Conclusion: 1.Through the analysis of TMT combined with LC-MS/MS technology,S100A10,FOLR2,S100A12,HMGB1,etc.are related to osteogenic differentiation,bone formation and vascular calcification.2.HMGB1 can increase the ALP activity of human ligamentum flavum cells and promote the formation of mineralized nodules;HMGB1 may increase the expression of OSX and OCN genes by activating the ERK1/2 signaling pathway,thereby inducing the osteogenic differentiation of human ligamentum flavum cells. |
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