| Background and objectivesAccording to statistics, 80%-90% of people have ever suffered from lower back pain in the world, and lumbar intervertebral disc degeneration and protrusion is the most common cause. However, the precise pathogenesis remains unclear, and the therapeutic effect of lower back pain is still poor. Lumbar intervertebral disc degeneration, a major cause for lower back pain and lumbar intervertebral disc protrusion, is characterized by cellular and matrix composition changes, in which matrix composition changes directly lead to the loss of the mechanic properties of intervertebral discs (IVDs). It has been shown that the degradation of IVD extracellular matrix (ECM) and apoptosis due to impairment of matrix-cell adhesion underlie IVD degeneration. In matrix composition disorders resulting from the imbalance between matrix synthesis and degradation, matrix metalloproteinases (MMPs) as matrix catabolic enzymes play important roles. Tissue Inhibitors of Metalloproteinase (TIMPs) act as tissue-specific inhibitors for endogenous MMPs. TIMPs have selective affinity to MMPs. TIMPs bind irreversibly and non-covalently to their corresponding MMPs in a proportion of 1:1 (gram-molecular weight) and inhibit the activity of MMPs. MMPs/TIMPs have been found to closely relate to human IVD degeneration.Generally, there are three common types of lumbar IVD protrusion which require operation, i.e., protrusion, extrusion and sequestration. The three types of degeneration and protrusion also reflect the degree of breakage of fibrous ring of IVD and the protrusion of vertebral pulp tissue. In the present study, therefore, normal, non-protrusion IVDs and the three types of IVD protrusion were investigated to explore the relationship between MMP-9 and TIMP-1 and the pathogenesis of various degrees of lumbar IVD degeneration and protrusion.Methods45 vertebral pulp samples were collected from surgeries for lumbar IVD protrusion conducted between March 2005 and April 2006 at the Department of Orthopaedics, Southwest Hospital of the Third Military Medical University. Based on the degree of IVD degeneration and protrusion, the samples were divided into the IVD protrusion group, the IVD extrusion group, and the IVD sequestration group (n=15). 15 vertebral pulp samples obtained from non-protrusion IVDs were used as the normal controls. MMP-9 and TIMP-1 mRNA expression was determined by RT-PCR, MMP-9 and TIMP-1 protein expression by Western blotting, and MMP-9 and TIMP-1 activity by gelatinase zymography. Images were processed using a computer, and the gray scale value of each band was measured. The ratios of gray scale area of the target genes to the internal standard were deemed as semiquantitative data, which were then statistically analyzed.Results1.RT-PCR results showed certain degrees of MMP-9 and TIMP-1 mRNA expression in normal vertebral pulp and vertebral pulp of IVDs with various degrees of degeneration and protrusion. Table 1 shows the means of contents relative toβ-actin. MMP-9 and TIMP-1 increased as the degree of IVD protrusion increased. MMP-9 mRNA expression was increased by 156% in the protrusion group, 188% in the extrusion group and 230% in the sequestration group, as compared to the normal group (all P<0.01). With increasing degrees of IVD degeneration and protrusion, TIMP-1 mRNA expression was increased less than MMP-9 mRNA expression (137%, 163%, and 192%, respectively) (all P<0.01). RT-PCR analysis indicated that the ratio of MMP-9 /TIMP-1 mRNA expression increased by 117%, 121%, and 123%, respectively (all P≥0.05).2. Western blotting results showed certain degrees of MMP-9 and TIMP-1 expression in normal vertebral pulp and vertebral pulp of IVDs with various degrees of degeneration and protrusion. MMP-9 protein absorbance value increased by 134% in the protrusion group, 145% in the extrusion group, and 158% in the sequestration group, as compared to the normal group (all P<0.01). TIMP-1 expression was increased less than MMP-9 expression (123%, 141%, and 152%, respectively) (all P<0.01). Western blotting indicated that the ratio of MMP-9 /TIMP-1 protein expression increased by 108%, 109% and 120%, respectively (all P≥0.05).3. Gelatinase zymography results showed certain degrees of MMP-9 and TIMP-1 expression in normal vertebral pulp and vertebral pulp of IVDs with various degrees of degeneration and protrusion. MMP-9 was expressed as a 92KD enzyme precursor or a 82KD enzyme, and TIMP-1 as a 28KD protein. The absorbance value of MMP-9 protein increased by 183% in the protrusion, 292% in the extrusion group, and 379% in the sequestration group, as compared to the normal group (all P<0.01). The absorbance value of TIMP-1 protein increased by 164%, 178%, and 217%, respectively (all P<0.01). Gelatinase zymography indicated that the MMP-9 /TIMP-1 ratio increased by 131%, 171%, and 218%, respectively (all P<0.01).Conclusions1. There was certain MMP-9 gene and protein expression in normal vertebral pulp and vertebral pulp of IVDs with various degrees of degeneration and protrusion. The expression degree and enzyme activity of MMP-9 increased with aggravating degeneration and protrusion of lumbar IVDs. MMP-9 was closely associated with lumbar IVD degeneration and protrusion.2. There was certain TIMP-1 gene and protein expression in normal vertebral pulp and vertebral pulp of IVDs with various degrees of degeneration and protrusion. The expression degree of TIMP-1 increased with aggravating degeneration and protrusion of lumbar IVDs. The enzyme activity of TIMP-1 increased in lumbar IVDs with degeneration and protrusion compared to the normal group, but there was no difference in the enzyme activity of TIMP-1 between the degeneration and protrusion groups.3. Gene and protein detection indicated that the MMP-9/TIMP-1 ratio increased in all degeneration and protrusion groups as compared to that in the normal group, but there was no significant difference in the ratio between the degeneration and protrusion groups. Enzyme activity detection showed that the MMP-9 /TIMP-1 ratio increased as lumbar IVD degeneration and protrusion aggravated, suggesting that the enzyme activity lost as TIMP-1 bound irreversibly and covalently to MMP-9. However, with aggravating IVD degeneration and protrusion, MMP-9 was expressed more highly than TIMP-1, resulting in increased MMP-9 /TIMP-1 ratios. The results suggested that inappropriate MMP-9 /TIMP-1 ratios were closely related to the degree of IVD degeneration and protrusion.Taken together, we detected the gene and protein expression as well as the enzyme activity of MMP-9 and TIMP-1, and analyzed the relationship between the MMP-9/TIMP-1 ratios and various degrees of lumbar IVD degeneration and protrusion. It was demonstrated that MMP-9 expression and the MMP-9/TIMP-1 ratios increased with aggravating lumbar IVD degeneration and protrusion. It is suggested that MMP-9 may cause IVD degeneration and protrusion through degradation of IVD matrix, and TIMP-1 and MMP-9 expression imbalance is closely associated with IVD degeneration and protrusion. |
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