HtrA1 Promotes The Expression Of Matrix Metalloproteinases In Nucleus Pulposus Cells And Its Mechanism

Objective Intervertebral disc degeneration(IDD)is an important cause of neck and back pain.The pathogenesis and mechanism of IDD is very complex,and its influencing factors are interrelated.High temperature requirement A1(Htr A1),belongs to serine protease(Htr A)family,There is increasing evidence that Htr A1 plays important role in bone,joint,and muscle disorders acts as a secreted protease.Matrix metalloproteinase(MMPs)is one of the indispensable proteolytic enzymes in the degradation of extracellular matrix,its biological role is involved in the metabolism of ECM,at present,more than 28 kinds of MMPs have been found,and the main research work in the IVD process are MMP1,MMP3,MMP7,MMP9,MMP13.MMP1,MMP3,MMP7,MMP9 and MMP13 play a more important role in the process of IVD.However,the intrinsic mechanism of Htr A1 and MMPs in IDD is not clear.Therefore,the purpose of this study was to detect the expression of Htr A1 and MMPs in the nucleus pulposus of intervertebral disc,and to analyze the correlation between Htr A1 and the expression of MMPs in the nucleus pulposus.Furthermore,explore the possible mechanism of Htr A1 effect on MMPs in human intervertebral disc nucleus pulposus cells.Methods:(1)clinical analysis: 32 patients with intervertebral disc degeneration were diagnosed as intervertebral disc degeneration in Affiliated Hospital of Jiangsu University from January 2015 to April 2016,there were 12 males and 6females with an average age of years 56.3(range,45~62 years).Separation of nucleus pulposus,The m RNA expression of Htr A1,MMP-1,MMP-3,MMP-7,MMP-9 and MMP-13 in nucleus pulposus was detected by RT-q PCR;The protein content of Htr A1,MMP-1,MMP-3,MMP-7,MMP-9,MMP-13 in nucleus pulposus was detected by Western-blot;The relationships between m RNA expression and MMP-1,MMP-3,MMP-7,MMP-9,MMP-13 and Htr A1 was anysised by Linear regression analysis.(2)r Htr A1 stimulated HNPCs and inhibitors intervention experiments:1)Expression of Htr A1 and MMPs after r Htr A1 stimulation in HNPCs:Stimulation of HNPCs with r Htr A1,the m RNA expression level of Htr A1,MMP-1,MMP-3 and MMMP-13 in HNPCs was detected by RT-q PCR;The protein content of Htr A1,MMP-1,MMP-3,MMP-13 in HNPCs was detected by Western-blot;The protein content of Htr A1,MMP-1,MMP-3,MMP-13 in the supernatant HNPCs was detected by ELISA.2)Inhibitor intervention: Stimulation of HNPCs with r Htr A1 and intervention with inhibitors of ERK1/2 and ROCK,the m RNA expression level of Htr A1,MMP-1,MMP-3 and MMMP-13 in HNPCs was detected by RT-q PCR;The protein content of Htr A1,MMP-1,MMP-3,MMP-13 in HNPCs was detected by Western-blot;The protein content of Htr A1,MMP-1,MMP-3,MMP-13 in the supernatant HNPCs was detected by ELISA.Reasults:(1)The expression of Htr A1 in patients with intervertebral disc degeneration of nucleus pulposus tissues compared with controls was significantly increased,and the expression of MMP-1,MMP-3 and MMP-13 have also increased;at the same time,the results of correlation analysis showed that Htr A1 was positively correlated with MMP-1,MMP-3,MMP-13.(2)RHtr A1 stimulated HNPCs,the m RNA and protein expression of MMP-1,MMP-3,MMP-13,were up-regulated,and r Htr A1 can cause the phosphorylation of ERK1/2 and ROCK.(3)After the intervention of ERK1/2 and ROCK inhibitors,the m RNA and protein expression of MMP-1,MMP-3,MMP-13 were down regulated,the levels of MMP-1,MMP-3 and MMP-13 in cell supernatant were also down regulated.