| BackgroundDegeneration of intervertebral disc is the fundamental factor for the occurrence of degenerative diseases of spine.It is a common and frequently-occurring disease of clinical orthopaedic spine.It is the main cause of pain and numbness in neck,shoulder and waist and leg.The main structure of the intervertebral disc is composed of the middle nucleus pulposus,annulus fibrosus and the upper and lower end plates.Its main function is to buffer the external pressure on the spine and maintain normal intervertebral space.The vertebral endplate is connected with the upper and lower ends of the vertebral body.It is located at the upper and lower ends of the intervertebral disc.It is a transitional structure between the intervertebral disc and the adjacent upper and lower vertebral bodies.It can be divided into the upper endplate and the lower endplate.It is a thin fibrous structure composed mainly of cartilage and bone.The endplate of intervertebral disc is mainly composed of cartilage tissue,which mainly contains proteoglycan,collagen and water.It can maintain the shape and function of the endplate cartilage tissue.Relevant studies have found that mechanical stimulation is an important factor in promoting chondrocyte proliferation,differentiation,apoptosis and maintaining structural integrity.Previous studies have found that abnormal stress stimulation will change the expression of related genes,thus affecting the content of type II collagen and proteoglycan in chondrocyte and cartilage metabolism,and accelerating the occurrence of intervertebral disc degeneration.Previously,by using bioinformatics technology,we found that differentially expressed senes were mainly concentrated in inflammatory response,positive regulation of apoptotic process,intrinsic apoptotic signaling pathway of oxidative stress response,positive regulation of apoptotic cell clearance,Wnt signaling pathway involved in somatic cell genesis and other biological processes.Moreover,abnormal mechanical factors can induce intervertebral disc degeneration through metabolic pathway,PI3K-Akt sisnaling pathway.p53 signaling pathway,signal pathway regulating stem cell pluripotency.MAPK signaling pathway,amino acid biosynthesis.Wnt signaling pathway,TGF-?signaling pathway and so on.Therefore,we speculate that Wnt/?-catenin signaling pathway can promote intervertebral disc degeneration.It plays an important role in the course of disease.HE staining showed that the number of nucleus pulposus cells in the middle pressure group was relatively large and arranged regularly.With the prolongation of time,the expression levels of Wnt-3a,?-catenin.proteoglycan and collagen ? were relatively high under 1kg of pressure,which could significantly alleviate the process of intervertebral disc degeneration,and the abnormal load lower or higher than the pressure,and its related indicators.The rate of intervertebral disc degeneration was further accelerated.According to the basic theory of traditional Chinese medicine,the main etiology and pathogenesis of degenerative diseases of intervertebral disc are insufficiency of liver and kidney,stagnation of blood stasis and qi,so tonifying kidney and activating blood circulation is the main method of treating these diseases in clinic.Professor Zhu Liguo of our hospital,on the basis of Qing'e Formula(Taiping Huimin He Prescription),combined with many years of clinical experience,used the dialectical treatment method to replace walnut kernel and garlic with Achyranthes bidentata,Wellingxian,Papaya and Salvia miltiorrhiza four traditional Chinese medicines,thus forming the prescriptions:Eucommia ulmoides 15g,Psoralea 10g,Achyranthes bidata 10g,Salvia Miltiorrhiza 12g,Wellingxian 10g,Papaya 9g.The basic prescription of kidney activating blood circulation.The whole prescription invigorates the kidney and invigorates the spleen in order to cure the root causes,invigorate blood circulation and dredge collaterals in order to cure the symptoms.Previous clinical studies of our group have found that Bushen Huoxue Recipe has a good effect in the treatment of discogenic low back pain in clinic,and basic research has found that Bushen Huoxue Recipe can stimulate the synthesis of chondrocyte and secrete proteoglycan and collagen ? in the end plate of intervertebral disc,but its specific effect and mechanism on the degeneration of intervertebral disc are still unclear.The purpose of this study is to investigate the relationship between Bushen Huoxue Recipe(BSHX)and endplate chondrocyte and Wnt/?-catenin signaling pathway in vitro spinal segmental model,and to elucidate the characteristics and mechanism of BSHX in regulating endplate chondrocyte induction/delaying intervertebral disc degeneration at the cellular and molecular level.Objective:To observe the effect of Bushen Huoxue Recipe on the pressure degeneration model of spinal motion segment in rabbits in vitro,and to elucidate the effect characteristics and mechanism of Bushen Huoxue Recipe in regulating the endplate chondrocyte to delay the degeneration of intervertebral disc.Methods:Forty healthy New Zealand rabbits were randomly divided into model control group,traditional Chinese medicine group,traditional Chinese medicine inhibition group and traditional Chinese medicine excitation group with 10 rabbits in each group.The control group:model+culture medium+drug-free serum;Chinese medicine group:model+culture medium+drug-containing serum of Bushen Huoxue Prescription;Chinese medicine agonist group:model+culture medium+SB216763+drug-containing serum of Bushen Huoxue Prescription.Chinese medicine inhibitor group:model+culture medium+ICG001+serum containing Bushen Huoxue Prescription.The culture medium contained 20%fetal bovine serum.25ug/nil ascorbic acid and 50mg/mL gentamicin.The osmotic pressure of the cell culture medium was adjusted to 410 mOsm/kg by NaCl.Each group was cultured in a constant temperature incubator at 5%CO2 and 37 C.The solution was changed every other day.After 14 days of culture in vitro,the effects of Bushen Huoxue Recipe on the degeneration of intervertebral disc were evaluated by HE staining and pathological morphology;the effects of Bushen Huoxue Recipe on the contents of proteoglycan,collagen ? and vascular endothelial growth factor in the cartilage of intervertebral disc endplate were detected by imunohistochemistry;the effects of Bushen Huoxue Recipe on Wnt-3a.GSK-3? and?-catenin were detected by fluorescence quantitative polymerase chain reaction(RT-PCR).Western Blot was used to detect the effects of Bushen Huoxue Recipe on the expression of Wnt-3a.GSK-3,p-catenin and vascular endothelial growth factor in cartilage tissue of intervertebral disc endplate.Results:The effect of BSHXR on IDD:HE staining showed that compared with the control group,the number of chondrocyte in the traditional Chinese medicine group was relatively large,and with time prolonging,the extracellular matrix in both groups decreased gradually,but the decrease in the control group was greater than that in the traditional Chinese medicine group.The chondrocyte layer was gradually broken and loosely arranged,especially in the control group.RT-PCR results showed that Wnt-3a and GSK-3 ? in the control group and the traditional Chinese medicine group were more obvious at different time points in the same group.There was no significant difference between groups at the same time point(P>0.05),but there was no significant difference between groups at the first day(P>0.05)and the control group at the same time point(P<0.05).There was significant difference between the other groups after 1 day(P<0.05),and after 1 day,there was no significant difference between the traditional Chinese medicine group and the control group(P<0.05).The expression levels of Wnt-3a and ?-catenin were relatively low.but the expression levels of GSK-3 p and VEGF were relatively high.Western Blot results showed that the expression levels of Wnt-3a,GSK-3 ? and ?-catenin in the control group and the traditional Chinese medicine group increased at first and then decreased,and the expression levels of vascular endothelial growth factor decreased gradually,the difference was statistically significant(P<0.05);at the same time,the expression levels of Wnt-3a,GSK-3 ? and ?-catenin in the Compared with the same group,there was no significant difference among the groups at 0 days(P>0.05),but there was significant difference among the groups after 1 day(P<0.05).The expression levels of Wnt-3a and VEGF protein in the Chinese herbal medicine group were relatively high after 1 day,but the expression levels of GSK-3 ? and p-catenin protein were relatively low.The mechanism of BSHXR on IDD:The results of immunohistochemistry showed that compared with the same group at different time points,the contents of proteoglycan and vascular endothelial growth factor in the control group and the traditional Chinese medicine group decreased gradually,while the content of collagen? increased first and then decreased,with statistical significance(P<0.05);at the same time point,there was no significant difference among different groups at 0 days(P>0.05).and there was significant difference among groups after 1 day(P<0.05)The contents of histone polysaccharide,collagen ? and vascular endothelial growth factor were relatively high one day later,and the contents of protein polysaccharide,collagen ? and vascular endothelial growth factor increased slightly in the agonist group at 3 days.Compared with the traditional Chinese medicine group,there were significant differences between the agonist group and the inhibitor group(P<0.05).RT-PCR results showed that Wnt-3a gene expression in the agonist group and the inhibitor group decreased,and the inhibitor group table showed that Wnt-3a gene expression in the There was no significant difference between the two groups(P>0.05).The expression level of GSK-3 ? gene in the agonist group and the inhibitor group was lower than that in the agonist group.There was a significant difference between the agonist group and the traditional Chinese medicine group(P<0.05).The expression level of p-catenin gene in the agonist group and the inhibitor group was lower than that in the traditional Chinese medicine group(P>0.05).The expression level of p-catenin gene in the agonist group was lower than that in the There were signicant differences between the two groups(P<0.05).The expression levels of VEGF gene in the agonist group and the inhibitor group were lower than those in the inhibitor group.There were significant differences between the two groups and the traditional Chinese medicine group(P<0.05).Western Blot results showed that there was no significant difference between Wnt-3a protein expression agonist group and inhibitor group and traditional Chinese medicine group(P>0.05).There was significant difference in GSK-3 p expression agonist group(P<0.05),while there was significant difference in ?-catenin and VEGF protein expression inhibitor group(P<0.05).while there was no significant difference in agonist group(P>0.05).Conclusion:(1)The serum containing Bushen Huoxue Recipe could delay the degeneration of intervertebral disc in a short period of time,which provided a basis for the early prevention and treatment of the degeneration of intervertebral disc by Bushen Huoxue Decoction.:(2)Bushen Huoxue Recipe can regulate endplate chondrocyte to delay IDD process by mediating Wnt/p-catenin signaling pathway. |
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