| BackgroundIntervertebral disc degeneration(IDD)is one of the main causes of low back pain in clinic,which seriously affects the life and work of patients.Meanwhile,it causes great economic burden to families and society.The main clinical imaging manifestations of IDD are the reduction of disc height,the decrease of vertebral mobility and the deterioration of vertebral stability.The histopathological manifestations are the decrease of water content,the expression of Aggrecan and collagen type II in nucleus pulposus.These two kinds of extracellular matrix proteins play an extremely important role in maintaining the morphology and physiological function of disc.Studies have confirmed that the imbalance of synthesis and decomposition of extracellular matrix is an important pathological factor leading to IDD.Research shows that abnormal stress is one of the important factors leading to IDD.The appropriate range of mechanical stimulation can promote the synthesis and secretion of extracellular matrix,while abnormal mechanical loading can lead to the change of extracellular matrix.However,the mechanism of IDD caused by abnormal stress remains unclear.Therefore,it is important to study the effect and mechanism of abnormal stress on the IDD.Our previous clinical studies found that Bushen Huoxue Recipe(BSHXR)could effectively improve the clinical symptoms of low back pain.Animal experiment found that BSHXR can stimulate nucleus pulposus cells to synthesize and secrete Aggrecan and collagen Ⅱ,but the effects and mechanisms of BSHXR on IDD are still not clear.By reviewing the literature and adopting the bioinformatics analysis,we found that Wnt/beta-catenin signaling pathway plays an important role in mechanical factors inducing IDD and drug delaying IDD.Therefore,we proposed that "mechanical factors regulate nucleus pulposus cells by mediating Wnt/beta-catenin signaling pathway to induced IDD" and "BSHXR regulate nucleus pulposus cells by mediating Wnt/beta-catenin signaling pathway to delay IDD".Part One:Bioinformatics analysis of intervertebral disc degeneration based on high-throughput microarrayObjective:To explore the potential target and mechanism of abnormal mechanical factors inducing IDD to provide basis for experimental research by bioinformatics technology.Methods:Adaptting bioinformatics technology to excavate IDD-related gene chips in GEO database,and exploring the genes and pathways involved in IDD process by screening differentially expressed genes(DEGs),functional enrichment analysis and construction of protein interaction network.Predicting the target and pathway of IDD caused by abnormal stress provides a basis.Results:915 IDD-related DEGs were obtained by GE02R function.GO analysis showed that DEGs were mainly concentrated in inflammatory response,positive regulation of apoptotic process,internal apoptotic signaling pathway of oxidative stress response,positive regulation of apoptotic cell clearance,Wnt signaling pathway and other biological processes.KEGG signaling pathway analysis showed that abnormal mechanical factors could participate in somatic cell genesis through metabolic pathway.Path,PI3K-Akt signaling pathway,p53 signaling pathway,signaling pathway regulating stem cell pluripotency,MAPK signaling pathway,amino acid biosynthesis,Wnt signaling pathway,TGF-beta signaling pathway and other eight main signaling pathways play the role of inducing IDD.Conclusion:Abnormal mechanical stimulation has various pathways and targets for IDD.Wnt/beta-catenin signaling pathway plays an important role in IDD.In the future,we can explore the mechanism of abnormal mechanical factors inducing IDD based on Wnt/beta-catenin signaling pathway.Part Two:The mechanism and effect of pressure on intervertebral disc degenerationObjective:To observe the mechanism and effect of pressure on IDD.Methods:After the execution of New Zealand white rabbits,we took out the complete spinal motion segments and randomly divided into blank control group,low pressure group(0.5 Kg),medium pressure group(1 Kg),high pressure group(3 Kg),activator group(1 Kg),inhibitor group(1 Kg)according to random number table.After 14 days of culture in vitro,HE staining was used to evaluate the effect of pressure on the degree of IDD.Immunohistochemistry(DAB staining)and Western blot(WB)were used to detect the effects of pressure on the expression levels of Wnt-3 alpha,GSK-3 beta,beta-catenin,Aggrecan and Collagen II in the nucleus pulposus of intervertebral disc.RT-PCR was used to detect the effects of pressure on the expression of Wnt-3 alpha,GSK-3 beta,beta-catenin,Aggrecan and Collagen II.Results:The effect of pressure on IDD:HE staining showed that the number of nucleus pulposus cells in the middle pressure group was more than other group,and the shape of cells are relatively regular.With the prolongation of time,the number of spindle cells and fibrous tissue in nucleus pulposus were less than those in the control group.WB and RT-PCR results:Compared with the same group at different time points,the protein and mRNA levels of Wnt-3a,beta-catenin,Aggrecan and Collagen II in the control group decreased gradually,while the protein levels of Wnt-3 alpha,GSK-3 beta,beta-catenin,Aggrecan and Collagen II increased first and then decreased in the low,medium and high pressure groups,the differences were statistical significance(P<0.05),but the mRNA of it decreased gradually(P<0.05);there were no statistical difference among the different groups at first(P>0.05);after 1 day,there were significant difference among groups at the same time(P<0.05),and the protein and mRNA expression levels of Wnt-3 alpha,beta-catenin,Aggrecan,Collagen II in the middle pressure group were more than other group after 1 day.The mechanism of pressure on IDD:DAB staining of showed that Aggrecan of activator group increased significantly compared with the control group on the 3rd day,while the Aggrecan of inhibitor group decreased significantly compared with the control group on the 3rd day,and the mean optical density(MOD)difference between the three groups were statistically significant(P<0.05).WB and RT-PCR results:On the 3rd day,there was no significant difference between activator group and control group on Wnt-3 alpha(P>0.05).The protein expression level of GSK-3 beta in activator group was significantly lower than control group(P<0.05).The protein and mRNA expression level of beta-catenin,Aggrecan and Collagen Ⅱ was higher than control group(P<0.05).There was no significant difference between inhibitor group and control group in Wnt-3 alpha,GSK-3beta and beta-catenin(P<0.05).The protein and mRNA expression levels of Aggrecan and Collagen Ⅱ decreased significantly(P>0.05),and the differences were statistically significant(P<0.05).Conclusion:Stress can regulate nucleus pulposus cells by mediating Wnt/beta-catenin signaling pathway to influence the process of IDD;Pressure affects the process of IDD:Appropriate pressure(1 Kg)can delay the process of IDD by promoting the expression of Aggrecan,Collagen Ⅱ and other proteins within 14 days,while abnormal pressure(0.5 Kg,3 Kg)can accelerate the process of IDD within 14 days.Part Three:The mechanism and effect of BSHXR on IDDObjective:To observe the mechanism and effect of BSHXR on IDD.Methods:After the execution of New Zealand white rabbits,we took out the complete spinal motion segments and randomly divided into blank control group,low dose group,medium dose group,high dose group,activator group and inhibitor group according to random number table.After 14 days of culture in vitro,HE staining was used to evaluate the effect of BSHXR on the degree of IDD.Immunohistochemistry(DAB staining)and Western blot(WB)were used to detect the effects of BSHXR on the expression levels of Wnt-3 alpha,GSK-3 beta,beta-catenin,Aggrecan and Collagen II in the nucleus pulposus of intervertebral disc.RT-PCR was used to detect the effects of BSHXR on the expression of Wnt-3 alpha,GSK-3 beta,beta-catenin,Aggrecan and Collagen II.Results:The effect of BSHXR on IDD:HE staining showed that the number of nucleus pulposus cells in the medium dose group was more than other group.With the prolongation of time,the number of spindle cells gradually increased,the extracellular matrix gradually decreased,the scar and fibrous tissue in nucleus pulposus gradually increased.WB and RT-PCR results:Compared with the same group at different time points,the protein and mRNA levels of Wnt-3 alpha,GSK-3 beta,beta-catenin,Aggrecan and Collagen Ⅱ increased first and then decreased in all group,and the differences were statistical significance(P<0.05);there were no statistical difference among the different groups at first(P>0.05);there were significant difference among groups at the same time after 1 day(P<0.05),and the protein and mRNA expression levels of Wnt-3 alpha,beta-catenin,Aggrecan,Collagen Ⅱ in the medium dose group were more than other group after 1 day,but GSK-3 beta was lower.The mechanism of BSHXR on IDD:Toluidine blue staining showed that the number of nucleus pulposus cells in nucleus pulposus tissue of the activator group increased significantly compared with the control group on the 3rd day,and the cell morphology was more regular,the fibrous tissue and scar in nucleus pulposus were less than the control group;while the number of nucleus pulposus cells in the inhibitor group decreased significantly,the cell morphology was spindle-shaped,and the fibrous tissue and scar in nucleus pulposus were more obvious than the control group.DAB staining of showed that the Collagen Ⅱ of activator group increased significantly compared with the control group on the 3rd day,while the Collagen Ⅱ of inhibitor group decreased significantly compared with the control group on the 3rd day,and the MOD difference between the three groups were statistically significant(P<0.05).On the 3rd day,there was no significant difference in Wnt-3 alpha between activator group and control group(P<0.05);the protein and mRNA expression level of GSK-3 beta in activator group was significantly lower than control group(P<0.05);the protein and mRNA expression level of beta-catenin,Aggrecan and Collagen Ⅱ was higher than control group(P<0.05).On the 3rd day,there was no significant difference between inhibitor group and control group in Wnt-3 alpha,GSK-3 beta and beta-catenin(P<0.05),the protein and mRNA expression levels of Aggrecan and Collagen Ⅱ decreased significantly,and the differences were statistically significant(P<0.05).Conclusion:BSHXR can regulate nucleus pulposus cells by mediating Wnt/beta-catenin signaling pathway to delay the process of IDD;BSHXR can delay the degeneration process of intervertebral disc,and medium dose of BSHXR can get a better result than low dose and high dose of BSHXR in delaying effect within 14 days. |
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