The Study On The The Effects Of Bushen Zhuangdu Recipe On The Expression Of Bcl-2 And Caspase-3 In Rat Models Of Lumbar Intervertebral Disc Degeneration

ObjectiveThe study is to investigate whether Bushen Zhuangdu recipe can slow LIDD and explore the mechanism of action,so as to provide theoretical and experimental basis for the prevention and treatment of LIDD by traditional Chinese medicine（TCM）by establishing the rat models of lumbar intervertebral disc degeneration（LIDD）,and observing the effects of Bushen Zhuangdu recipe onthe expression of Bcl-2 and caspase-3,which are both the regulatory proteins of mitochondrial pathway of apoptosis.Methods1.The experiment of establishing models:20 male SD rats weighing0.2-0.3kg,were randomly divided into control group and annulus puncture group,10 in each group.Sham operation group:after general anesthesia,cut the skin,subcutaneous tissue from the incision in the middle of the right side of the rats abdomen in turn and opened the abdominal cavity;Then along the intermuscular space of psoas opened the retroperitoneum and protected large blood vessels,so that the L5/6 disc was exposed and then we sutured layer by layer.Annulus puncture group:exposed the L5/6 disc with the same surgical approach,with 21G needle punctured the whole layer of the L5/6 disc annulus fibrosus.At postoperative 4^thh week,observed the degeneration of L5/6 disc through the lumbar spine X-ray,HE and immunohistochemical staining of typeⅠcollagen.2.TCM intervention experiment:100 male SD rats weighing 0.2-0.3kg,were randomly housed into five groups:sham operation group（group A）,model group（group B）,low dose of TCM group（group C）,medium dose of TCM group（group D）and high dose of TCM group（group E）,20 rats in each group.Group A:after exposure of the L4 to L6 disc,suture layer by layer.Other groups:Annulus puncture method was used to induce model of LIDD.The next day after modeling,the rats in groups C,D and E were given low dose,medium dose and high dose of Bushen Zhuangdu recipe decoction by gavage.While Group A and group B were given equal volume of normal saline by gavage.After 4 weeks,randomly select 10 rats from each group and execute them for L4 to L6intervertebral disc.The IDD of each group was observe by HE staining.The apoptosis of nucleus pulposus cells was detected by TUNEL method.And the expression of the Bcl-2 and caspase 3 protein in rat nucleus pulposus in degeneration was tested by Western blot.Results1.The experiment of establishing models:The 4^thweek after surgery,compared with sham operation group,the intervertebral disc relative height decreased significantly in annulus puncture group,the pathological scores of IDD rise obviously.And the average gray value of type I collagen staining decreased significantly（P<0.05）.2.TCM intervention experiment:Take the test results of HE staining,TUNEL method and western blot,we have found that,the pathological scores of IDD and lumbar nucleus pulposus cells apoptosis index of group B were significantly higher,and the expression of Bcl-2 decreased while the expression of activated caspase 3 increased significantly in intervertebral disc tissue,compared with those of group A,and the difference had statistical significance（P<0.05）.The pathological scores of IDD and lumbar nucleus pulposus cells apoptosis index of groups C,D and E were significantly lower,and the Bcl-2 expression in intervertebral disc tissue increased significantly while activated caspase3 expression decreased obviously（P<0.05）,compared with those of group B.The pathological scores of IDD and lumbar nucleus pulposus cells apoptosis index of group D and E were significantly lower than those in the C group,the Bcl-2 expression in intervertebral disc tissue increased significantly while activated caspase 3 expression decreased obviously,the difference was statistically significant（P<0.05）.And the comparison between groups D and E indicated that the difference in all of the above had no statistical significance（P>0.05）.Conclusion1.A reliable rats model of LIDD can be successfully established by the method of puncturing the whole layer of the annulus fibrosus.And the degree of degeneration can be controlled by choosing different types of needle,different puncture depth and duration,and different postoperative observation points,to meet the study needs of the different LIDD stages.2.The apoptosis of nucleus pulposus cell increases obviously in the degenerated lumbar disc tissue of rat,and the degree of IDD and the apoptosis of nucleus pulposus cell were positively correlated.Moreover,the degree of apoptosis are negatively correlated with the expression of Bcl-2,which is a main antiapoptotic protein in mitochondrial pathway of apoptosis while are positively correlated with the expression of caspase-3,which is a main apoptosis executive protein.Therefore,the study confirmed that the increasement of the apoptosis of nucleus pulposus cellsmediated by mitochondrial plays an important role in the process of LIDD.3.Bushen Zhuangdu recipe could slow the histological degeneration of intervertebral disc,reduce the apoptosis of nucleus pulposus cells,increase the expression of Bcl-2 and lower the expression of caspase-3,thus postponing the rat lumbar disc degeneration,and its mechanism of action is associated with reducing the apoptosis of nucleus pulposus cellsmediated by mitochondrial,and we have certain dose dependent effect.