| Part I Establishment of a Rat Model of Lumbar Disc Herniation and Verification by Dynamic Pathological StudyObjectives:To establish a rat model of lumbar disc herniation according to a synthetical reference study and find the best experimental timing after comparison of pathological results at different time points of the model.Methods:According to the references to establish a rat model of lumbar disc herniation by transplantating autologous nucleus pulposus and compare pathological and serological results and observe animal conditions on different time points(7th,14th,21rt,28th day)after modeling.Results:1.Condition observations:The control group(sham group)and model group both showed obvious left hind leg limping and weakness on the 3rd day after modeling with neurological functional grading over level 3.From the 7th day on,control group was on a recovery trend and resumed almost normal 14 days later,while model group showed persisting symptoms until the 28th day after modeling and then an improving trend appeared.2.Pathological inspection:Sham group showed angiogenesis and inflammatory cell infiltration but no nerve injury on the 7th day after modeling while model group showed inflammatory cells infiltration and nerve compression both.From the 14th day on,sham group showed a significant improvement on inflammatory cell infiltration and resumed almost normal 21 days later,while model group showed persisting inflammatory cell infiltration and advanced to fibrosis 28 days later.3.TNF-? expression:Serum TNF-? in sham group decreased significantly over time.Compared with sham group,TNF-? of model group was significantly increased on each time points(7th,14th,21rt,28th day)after modeling,until the 28th days after modeling it was still significantly higher than the sham group.Conclusions:Taken by transplantation of autologous nucleus pulposus can effectively simulate the model of lumbar disc herniation with radiculitis pain.Compared with sham group,the animal's conditions,pathological results and serum TNF-? of model group were significantly different.As model group showed a clear recovery trend 21 days after modeling,the 21st day is the best appropriate experimental time point.Part ? Mechanisms of Longdan Yaobi Pill:A study on LDH model rats induced by nucleus pulposusObjectives:To confirm the anti-inflammatory and analgesic effects of Longdan Yaobi Pill through classic hot-plate test,abdominal constriction test,xylene-induced ear edema model and albumin-induced paw edema model.To study the mechanisms of Longdan Yaobi Pill by testing serum expressions of TNF-??IL-1???-NGF by enzyme linked immuno sorbent(ELISA)and to explore the expression of Fractalkine/CX3CL1 and TIMP-1 as well as neural signal transduction pathways and chemotactic factors by protein chip and the in situ hybrid technology in a herniated intervertebral disc model of rat induced by nucleus pulposus.Methods:1.To confirm the anti-inflammatory and analgesic effects of Longdan Yaobi Pill through classic hot-plate test,abdominal constriction test,xylene-induced ear edema model and albumin-induced paw edema model.2.The herniated intervertebral disc model of rat were successfully established according to results of part one and 72 rats were'randomly classified into the following 8 groups:blank group,sham group,model group,Longdan Yaobi Pill(high dose)group,Longdan Yaobi Pill(medium dose)group,Longdan Yaobi Pill(low dose)group,Yaobi Kang Pill(TCM control)group,diclofenac sodium(western medicine control)group.21 days after modeling all rats were executed and the serum expressions of TNF-? IL-1???-NGF were tested by enzyme linked immuno sorbent(ELISA).Ganglia,spinal nerves and intervertebral disc tissue were observed by protein chip and the in situ hybrid technology.Results:1.The writhing time induced by acetic acid and threshold of heat were remarkably improved by low,medium,and high dose of Longdan Yaobi Pill.The xylene-induced mice ear edema and albumin-induced rat paw edema were also significantly suppressed by it.2.The white blood cell counts were significantly reduced by all doses of Longdan Yaobi Pill but no effects on hemoglobin and platelets.Compared with blank group and sham group,the white blood cell counts and proportions of neutrophils of model group were significantly rised;compared with model group,the white blood cell counts of all treat groups were significantly decreased while no changes on hemoglobin and platelets.3.The expression of serum TNF-?,IL-1 ? were significantly lowered by Longdan Yaobi Pill but no differences on the expression of beta-NGF.Compared with the model group,all treat groups(Longdan Yaobi Pill low,medium and high dose groups,TCM and western medicine control group)has shown remarkable decrease of TNF-?,IL-1? but no significant changes in expression of ?--NGF.4.Compared with blank group and sham group,the expression of Fractline(both mRNA and protein)and MMP-8 of the model group were significantly rised while TIMP-1 were lowered.5.The expression of Fractline(both mRNA and protein)were significantly lowered by Longdan Yaobi Pill as well as a lot of inflammation related cytokines.6.The expression of TIMP-1 mRNA were raised by Longdan Yaobi Pill while MMP-8 and Fas Ligand were decreased.Conclusions:Longdan Yaobi Pill has shown obvious anti-inflammatory and analgesic effects.The herniated intervertral disc may induce the release of inflammatory-related cytokines thereby attracting the expression of Fractalkine.Through lowering Fractalkine expression then regulating CX3CR1-mediate adhesion and attraction of inmmunocytes may be the main mechanisms for Longdan Yaobi Pill.The expression of TIMP-1 were increased by Longdan Yaobi Pill while the expression of MMP-8 and Fas Ligand were decreased. |
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