| Background and objectivesThe incidence of cervical spondylosis,spinal stenosis,lowback pain and other degenerative disc diseases(DDD)is extremely high,and has a significantly increasing trend.Anatomical studies show that 83% to 95% of adult corpses exists intervertebral disc degeneration(IVDD).IVDD is mainly affected by age,genetic,mechanical,immune and so on,and eventually lead to changes in the internal biological properties of the disc,such as cell apoptosis,extracellular matrix changes.The role of cytokines such as interleukin(IL),tumor necrosis factor-?(TNF-?)and transforming growth factor-?(TGF-?)in IVDD has been extensively studied,while Smad3 and Smad7 act as the intracellular transmission medium of TGF-?,there are less study of its role and influence in IVDD.In this study,the animal DDD model established to detect the expression of Smad3 and Smad7 in degenerated intervertebral disc,observe the changes in the process of IVDD,analyze the relationship between it and IVDD,so as to provide new ideas and methods for the prevention and treatment of DDD.Materials and methods60 rats,meeting the criteria as adult,healthy,male,no specific pathogens,SPF grade SD were randomly divided into the experimental group and control group,each group was divided into three observation stages: one month,three months and five months,each stage have 10 of each group.The rats in the experimental group were treated with static and dynamic imbalance to establish cervical disc degeneration model,the muscles attached to the spinous process and the lamina were transected horizontally,then supraspinal ligament and interspinal ligaments between the neck 2 and 7 were removed,and were directly sutured;the control group was treated with sham operation,and only the skin was cut and sutured directly.The rats were sacrificed at 1 month,3 months and 5 months respectively.The cervical disc was removed;the degree of disc degeneration was observed by HE staining;the expression of Smad3 and Smad7 protein was detected by immunohistochemistry(IHC);RT-PCR was used to detect the expression of Smad3 and Smad7 m RNA.Data were analyzed by SPSS21.0.Results1.In the course of the experiment,two of the rats died in 1 month and one of the rats died in 2 month in the experimental group,one of the rats died in 1 month in the control group.2.The histological score of the intervertebral disc: The histological score of the intervertebral disc in the experimental group(1.67 ± 1.23,3.56 ± 1.13,4.44 ± 0.88)was higher than that in the control group(0.56 ± 0.73,0.78 ± 0.83,1.22 ± 0.97),the difference was statistically significant(p <0.05);the number of intervertebral disc cells in the experimental group was generally less than that in the control group,and were irregularly arranged,the fibers of annulus fibrosus appear irregularly arranged,the annulus fibrosus and nucleus pulposus are blurred,slab structure cracks,the spinal cord cells in nucleolus-like structure are lost,cartilage-like cells are increased,nucleus pulposus mucous degeneration occurred,fracture formation and so on,osteogenesis occurred in the intervertebral disc,the above situation has aggravated with time;in the control group,the annulus fibrosus and nucleus pulposus cells of intervertebral disc were relatively arranged in order,cracks? mucous degeneration and osteogenesis were relatively slight,the difference among the three phases is relatively small.3.The expression of Smad3 and Smad7 protein in the intervertebral discs of the rats: in three stages,the expression of Smad3 protein in the experimental group(22.90±2.72,33.52±4.99,46.48±10.42)was significantly higher than that in the control group(18.13±3.09,19.06±5.06,20.96±3.38),the difference was statistically significant(p <0.05);in three stages,the expression of Smad7 protein in the experimental group(18.61±7.52,42.68±15.50,24.46±9.20)was significantly higher than that in the control group(11.44±3.75,14.46±6.43,15.08±6.31),the difference was statistically significant(p <0.05);the expression of Smad3 protein in the experimental group was statistically significant at 1,3 and 5 months(p <0.05),the expression of Smad7 protein in the experimental group was statistically significant between 1 month and 3 month,3 month and 5 month(p<0.05);the expression of Smad7 protein of experimental group between 1 month and 5 month and that of Smad3 and Smad7 proteins of control group at each stage was not statistically significant(p > 0.05).4.The expression of Smad3 and Smad7 mRNA in the intervertebral discs of the rats: in three stages,the expression of Smad3 m RNA in the experimental group(0.441±0.111?0.532±0.153?0.794±0.090)was significantly higher than that in the control group(0.346±0.101?0.409±0.122?0.413±0.114),the difference was statistically significant(p <0.05);in three stages,the expression of Smad7 m RNA in the experimental group(0.351±0.124?0.610±0.206?0.430±0.165)was significantly higher than that in the control group(0.181±0.066?0.208±0.072?0.223±0.089),the difference was statistically significant(p <0.05);the expression of Smad3 m RNA in the experimental group was statistically significant between 1 month and 5 month,3 month and 5 month(p <0.05),the expression of Smad3 m RNA of experimental group between 1 month and 3 month and that of Smad3 m RNA of control group at each stage was not statistically significant(p > 0.05);the expression of Smad7 m RNA in the experimental group was statistically significant between 1 month and 3 month,3 month and 5 month(p <0.05);the expression of Smad7 m RNA of experimental group between 1 month and 5 month and that of Smad7 m RNA of control group at each stage was not statistically significant(p > 0.05).5.The correlation analysis between the histological score of IVDD and Smad3,Smad7 expression: the correlation coefficients between Smad3 protein,m RNA expression and histological score were respectively r=0.527,r=0.640,and both were positively correlated,the difference was statistically significant(p <0.05);the correlation coefficients between Smad7 protein,m RNA expression and histological score were respectively r=0.118?r=0.276,the difference was not statistically significant(p >0.05);the correlation coefficients between Smad3 protein and Smad7 protein was r=0.203,the difference was not statistically significant(p >0.05);the correlation coefficients between Smad3 m RNA and Smad7 m RNA was r=0.249,the difference was not statistically significant(p >0.05);Smad3 expression of the intervertebral disc was positively correlated with the intervertebral disc degeneration,the expression of Smad7 increased firstly and then decreased with the aggravation of IVDD and the increased expression of Smad3.Conclusions1.Smad3 was highly expressed in the degenerated intervertebral disc,and positively correlated with the degree of IVDD.2.Smad7 was highly expressed in the degenerated intervertebral disc,and with the trend of increasing firstly and then decreasing with the aggravation of IVDD.3.There is not a clear correlation between Smad3 and Smad7 expression in the process of IVDD,the expression of Smad7 increased firstly and then decreased with the expression of Smad3,and the expression of both was not only affected by the feedback of the other.4.Regulation of Smad3 and Smad7 expression in the intervertebral disc may provide a new method for the prevention and treatment of IVDD. |
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