| Purpose:1.Firstly,we quote the preclinical medicine of TCM to expound the relationship of“Du meridian-Kidney-Bone”,Secondly,we analysis the disease name,symptoms,pathogenesis and treatment to give the evident that lumber degenerative disc disease（LDDD）is closely related with“Du meridian-Kidney-Bone disorder”.In summary,“Du meridian-Kidney-Bone”made through connections into integration from the anatomy and physiology view,and the pathogenesis of this disease can be summarized as"Du meridian-Kidney-Bone disorders".2.β-catenin（ex3）Co12ER mice conforms part of lumber degenerative disc desease phynotype,the mice also got pain behavior,pain is the most common symptoms of the disease.It proved that Wnt/β-catenin signaling pathway played an important role in lumbar disc degeneration disease.3.To observe the effect of Banlong Wan（representative formula for tonifying kidney and Du meridian）toβ-catenin（ex3）Co12ERo12ER mice,verify that whether it can rescue part of the lumber degenerative disc disease like phenotype and the pain symptom.Observe the effect of Banlong Wan to Wnt/β-catenin signaling pathway and HPA axis fuction.4.Observe the effect of Banlong Wan drug serum on annulus fibrosus cell and nucleus pulposus cell,prove that Banlong Wan drug serum can regulate Wnt/β-catenin signaling pathway and its downstream target gene mmp13,Adamts4 and Adamts5.5.Evaluate the therapeutic effect of Banlong Wan to lumber degenerative disc disease with in vivo and in vitro experiments from the overall level and cell level,assess if Banlong Wan can regulate Wnt/β-catenin signaling pathway and HPA axis fuction,testify therapeutic effect with curative effect,to provide the experimental basis for TCM treatment of lumbar disc degenerative disease.Material and method:1.Theoretical research:Describe the relationship of“Du meridian-Kidney-Bone”through sorting out the preclinical medicine of TCM,“Du meridian-Kidney-Bone”made through connections into integration from the anatomy and physiology view.Anlysis he disease name,symptoms,pathogenesis and treatment to explain the pathogenesis of lumber degenerative disc disease can be summarized as"Du meridian-Kidney-Bone disorders".2.Experimental research:In vivo experimental:β-catenin（ex3）Co12ER mice were bred,and mice were genoytping.The Cre+mice were divided into 4 groups according to the genoytping results,and the Cre-mice were the normal control group,with totally 10 males and females randomly in each group.The mice were administered tamoxifen（1 mg/10 g body weight,IP,daily for 5 days）.The high,medium and low dose of Banlong Wan gavage to the experimental group,while the blank control group and the normal control group were given the same dose of distilled water for 24weeks.Animal behavioral test was performed after the administration of the drug.First,the hot plate test was performed on the mice.Three tests were performed for each mouse,and the average of the three tests was compared between different groups.After the hot plate experiment,cat walk test was performed,and each mouse was performed three times to compare the differences in gait analysis indicator such as Print Area,Swing Speed,average pressure（Intensity）and Swing.After the animal behavior test,chloral was inject hydrate intra-peritoneally to anesthesia the mice.After anesthesia,the blood was collected by extirpating eyeballs,then the mice were sacrified.Perform the enzyme-linked immunoassay detection,detecte mice serum adrenocorticotropic hormone releasing hormone（CRH）,adrenocorticotropic hormone（ACTH）,and corticosterone（CORT）.The spinal tissue of the mouse was taken and the spinal tissue was detected by micro-CT.Then the sections were stained,fixation,decalcification,paraffin embedding and section for 3um,the sections were used to observe the morphological changes of the mice lumber spine.2.2 In vitro experiments:Rat annulus fibrosus cells（AF）and nucleus pulposus（NP）cells were cultured to prepare rat serum containing drugs,including the blank control group and high,medium and low dose groups.Rat AF cells and NP cells are divided into five groups,blank control group,model group,the Banlong Wan low dose group,middle dose group and high dose group,5 groups of cells with 20%FBS nutrient solution culture cells in a 10 mm petri dish,after being attached except blank control group,other four groups were given Bio（GSK-3βinhibitor）,2 days later give 20%rat medicated serum to each group.Results:1.According to the genotyping results,we have bredβ-catenin（ex3）Co12ER mice successfully,mice can be used for later experiment.2.Micro-CT data showed that compared with the normal control group,the blank control group mice showed lumbar disc stenosis and a large number of osteophytes formation appeared under lumbar intervertebral disc.However,the osteophyte formation around the lumbar intervertebral disc decreased in Banlong Wan different dosage groups,especially in high-dose group.Observed Alcian blue/Hematoxylin&Orange G staining results,facet joint phenotype of the normal control group is normomorph,articular cartilage surface is smooth and complete,cartilage layer is clear,chondrocyte under the articular cartilage arranged orderly.Mice facet joint of blank control group were disorganized,part of the cartilage surface appear damage,cartilage layer dye and thinning,chondrocyte arranged and local crack produces,cavity appear in cartilage lacuna,a substantial reduction in collagen fiber.Histologic results demonstrated a severe loss of cartilage,a reduced number of growth plate chondrocytes.Subchondral bone showed hyperplasia and hypertrophy.3.The results of hot plate pain threshold showed that the incubation period of thermal reduction was significantly reduced in blank control group compare with normal control group mice.There was a certain degree of increase in the incubation period between different dose therapeutic groups and the blank control group,but there was no significant difference between the three different dosage groups.Gait analysis results show that compared with normal control group,blank control group mice print area decline,swing speed and intensity decrease,but swing increase.P<0.01,the result have statistical significance.Compared with the blank control group,all data expect print area have significant change in different dosage of the treatment group.The swing speeds,intensity increase,and swing decrease.P<0.01,the result have statistical significance,there are no obvious differences between three different dosage treatment group.4.Imunohistochemistry demonstrated thatβ-catenin was express in chondrcyte,compared with normal control group mice,positive cell accouont that expressβ-catenin increased significantly,but positive cell accouont that expressβ-catenin in the different dosage of the treatment group decreased compare with blank control group.The results of enzyme-linked immunoassay experimental showed that compared with normal control group mice,CRH,ACTH and CORT significant decrease in blank control group mice serum,and the content of CRH,ACTH and CORT increased in different dose groups,P<0.05,which was statistically significant,but there was no significant difference between the three different dosage treatment groups.5.RT-PCR results indicate that compared with the normal control group,the mRNA expression ofβ-catenin,mmp13,Adamts4 and Adamts5 in AF cells and NP cells significant increase in blank control group,P<0.01,data was statistically significant.Differente dose of treatment group can downregulateβ-catenin,mmp13,Adamts4 and Adamts5 mRNA expression in AF cells and NP cells,P<0.01,data was statistically significant,but here was no significant difference between three different dosages of treatment group.Western blot result indicate that compared with the normal control group,protein level ofβ-catenin rise apparently in blank control group,P<0.05,data was statistically significant.Banlong Wan drug serum can downregulateβ-catenin protein level.Conclusion:1.“Du meridian-Kidney-Bone”made through connections into integration from the anatomy and physiology view.According to the disease name,symptoms,pathogenesis and treatment,the pathogenesis of this disease can be summarized as"Du meridian-Kidney-Bone disorders".2.β-catenin（ex3）Co12ERo12ER mice conforms part of lumber degenerative disc desease phynotype,the mice also got pain behavior.It suggested that Wnt/β-catenin signaling pathway played an important role in lumbar disc degeneration disease.3.Theβ-catenin（ex3）Co12ER mice show lumber degenerative disc like phenotype,different dosages of Banlong Wan can partially rescue the phenotype,and release the pain.4.Banlong Wan medicine serum may downregulate Wnt/β-catenin signaling pathway and its downstream target gene mmp13、Adamts4和Adamts5,indicate that Banlong Wan medicine serum may protect cartilage by inhibite extracellular matrix collagen degradation.5.Banlong Wan may inprove the function of the renal function by downregulate Wnt/β-catenin signaling pathway.There is a certain therapeutic effect of Banlong wan on lumbar disc degeneration disease. |
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