| Objective:1.Intervertebral disc degeneration and vertebral osteoporosis are the most common disorders of spinal degenerative disease in elderly people,and which cause a series of clinical manifestations,such as low back pain and neural compressive symptoms.It have been the hotspot and difficulty of research for many years.The purpose of this study is to find out the pathogenic factors that may affect the degeneration of intervertebral disc,and to explore the inherent relationship of disc degeneration with vertebral osteoporosis and osteochondral metabolism,aiming to define the role of osteoporosis and bone metabolism in the intervertebral disc degeneration.2.Animal models were further made to validate and observe wether existing the successive occurrence of vertebral osteoporosis,endplate lesions,and intervertebral disc degeneration in spinal degeneration for clarifying the correlation between osteoporosis and disc degeneration.At the same time,TGF?1-Smad2/3 signaling pathway was observed in vertebra and disc tissues to define whether it participates in vertebral-intervertebral disc metabolism and mediates abnormal bone and cartilage metabolism as well as tissue remodeling,thus,to discuss the molecular mechanism of vertebral osteoporosis and disc degeneration.3.Based on the guidance of classical theory of traditional Chinese medicine(TCM),The Bushen Herbs were used to intervene mouse model and to observe whether it can influence the TGF?1-Smad2/3 signaling and regulate the metabolic environment of osteochondral interface as well as improve bone and joint structure in spinal degenerative diseases.The results can provide the experimental basis for clinical prevention and treatment of vertebral osteoporosis and intervertebral disc degeneration.Methods:1.Clinical researchThe relationship between lumbar disc degeneration and vertebral osteoporosis and serum bone metabolism80 cases of middle-aged and elderly patients with chronic low back pain were retrospectively collected,and their lumbar MRI images,bone mineral density(BMD),bone metabolism indexs and case data were obtained through the medical record management system.The degree of degeneration of L1-L4 disc was graded by Pfirrmann grade form 1-5 according to the lumbar MRI T2 images,and the degree of osteoporosis was graded 1-4 according to the L1-4 vertebral body bone mineral density test results.The correlation of the severity of two groups was statistically analyzed.In addition,analysis of the serum levels of bone metabolism indexs between different disc degeneration grades is needed for clear the difference between the different groups.2.Animal experimental research2.1 The structural and metabolic changes of vertebra and intervertebral disc under ovariectomy and mechanical imbalance conditionsForty SPF female C57BL/6J mice in 12 weeks old were randomly divided into 4 groups: control group(CT),ovariectomized group(OVX),instability group(SW)and combined model group(OS),10 mice per group.The mice were weighed weekly to observe the change of body weight.After 12 weeks,the uterus was observed in morphology and then weighed.Micro-CT scans of L4/5 segments were performed to detect vertebral and endplate bone mass,microstructure parameters and intervertebral disc volume.Paraffin sections were made.HE and Safranine O-green staining were used to observe the morphological changes of vertebral body,endplate and intervertebral disc.Immunohistochemistry or immunofluorescence staining was used to detect the expression of type I collagen(Col1),type II collagen(Col2)and vascular endothelial growth factor(VEGF).2.2 The effect of Bushen Herbs,alendronate sodium and TGF?receptor inhibitor SB505124 on the structure of vertebral body and discSeventy-five 12 weeks-old SPF female C57BL/6J mice were randomly divided into five groups: normal control group(CT),compound model group(OS),TGF? inhibitor group(SB),alendronate group(ALN)and Bushen Herbs group(HB),fifteen mice in each group respectively.Twelve weeks after the prophylactic administration,the L4/5 segments were scanned with Micro-CT and the L4 vertebral bone quantity,microstructure parameters,endplates volume and porosity as well as L4/5 intervertebral disc volume were analyzed.Paraffin sections were made and the morphological changes were observed with Safranin O-fast green staining.2.3 The regulative effects of Bushen herbs on key messenger proteins of TGF?/smad signaling pathway and osteochondral metabolism of vertebral body and intervertebral discAfter micro-CT scan of the L4/5 segments,the paraffin sections were then made.Immunohistochemistry was used to detect the expression of Col1,Col2,MMP9,CD31,TGF-? and P-Smad2/3 in intervertebral disc.Western blot was used to detect the expression of Smad3,P-Smad2/3 and VEGF in the intervertebral disc,and the expression of TGF?1,P-Smad2/3,MMP9 and Osterix in the vertebral body.Results:1.The clinical researchThe mid-aged and elderly patients with chronic low back pain often have reduced vertebral bone mass or different degrees of vertebral osteoporosis.Lumbar disc degeneration were more common in the L4/5 segment and the severity was most range from moderate to severe.The degree of intervertebral disc degeneration was positively correlated with the degree of vertebral osteoporosis(r = 0.459,P<0.001).The incidence and degenerative level of intervertebral disc degeneration increased in patients with severe lumbar vertebral osteoporosis.At the same time,in patients with high average degenerative degree of disc,the index of bone metabolism(PINP,CTX-1)were significantly higher than those with mild degeneration and the difference was statistics significant(P<0.05).2.Animal experimental research2.1 The structural and metabolic changes of vertebra and intervertebral disc under ovariectomy and mechanical imbalance conditionsWhen compared with CT group and SW group,OVX induced a significant increase in body weight and uterine atrophy(P <0.05).OVX and SW can lead to the occurrence of disc degeneration and OS causes more serious disc degeneration.The results of Micro-CT showed that OVX resulted in osteoporosis of the vertebral body.The BMDtv,BV/TV,Tb.N and Tb.Th in OVX group and OS group were significantly lower than those in CT group(P <0.05).SW could affects the subchondral bone to some extent.There was no significant difference between SW group and CT group(P> 0.05),but the BMDtv had an upward trend.Besides,the BV/TV was significantly higher in OS group compared with OVX group(P<0.05),and have an increasing trend in BMDtv,BS/TV,and Tb.N while a decreasing trend in Tb.Sp and SMI.OVX can causes ossification of endplate while SW could leads osteosclerosis of subchondral bone.When compared with CT group,the volume of intervertebral disc in SW group and OS group was significantly lower than that in CT group(P <0.05),but there was no significant difference between OVX and CT group(P>0.05).Pathological staining results showed that OVX could leads to sparse trabecular bone and endplate ossification,which were consistent with the results of micro-CT scan.At the same time,the cells loss can be found in vertebral body and disc of OVX group and OS group.SW can lead the destruction,disorder and fusion of the annulus fibrosus as well as the mechanical adaptive changes of subchondral bone.Of note,OS could promotes collapse and fusion of intervertebral discs.Immunohistochemistry or fluorescence analysis showed that the expression of Col2 was lower in OS group,OVX group and SW group than in CT group.Col1 was not significantly different among groups but a slight higher in the OVX group and OS group.Futhermore,the expression of VEGF in discs of OVX group and OS group were significantly higher than that of CT group and SW group,especially in the ossification remodeling area of the endplate.2.2 The effect of Bushen Herbs,alendronate sodium and TGF?receptor inhibitor SB505124 on the structure of vertebral body and discMicro-CT results showed that severe osteoporosis and endplate leision occurred in OS group.When compared with CT group,the BMDtv of vertebral body were significantly decreased(P<0.05),and the bone microstructure parameters got worse(P<0.05)in OS group.Besides,the endplate volume and porosity significantly increased(P<0.05)in OS group,and simultaneously,the disc volume significantly reduced(P<0.05).However,the BMDtv of vertebral body in SB,ALN and HB groups had an upward trend,and the bone microstructure parameters improved(P<0.05)when compared with OS group.In addition,the ossification of the endplate was inhibited in some degrees,the porosity and volume of the endplate were decreased(P<0.05),while the intervertebral disc volume increased(P<0.05)after intervention of SB,ALN and HB.The pathological results showed that the disc structure of the OS group was disordered and severely degenerated when compared with CT group.However,SB,ALN and HB could delay disc degeneration to some extent,and improved the histopathology of disc degeneration when compared with OS group.2.3 The regulative effects of Bushen herbs on key messenger proteins of TGF?/smad signaling pathway and osteochondral metabolism of vertebral body and intervertebral discThe results of immunohistochemistry showed that the expressions of TGF?1,P-Smad2/3,Col1,MMP9 and CD31 in OS group were significantly higher than those in CT group,while Col2 was significantly decreased.When compared with OS group,TGF?1,P-Smad2/3,MMP9 and CD31 expression in SB group,ALN group and HB group were decreased to some extent.There were no significant change of Col2 expression in SB and ALN groups,but the Col1 expression decreased,when compared with OS group.Interestingly,HB group showed an significant increase in Col2 and Col1 expression compared to OS group.The western blot results showed that the expression of Smad3,P-Smad2/3 and VEGF in disc of OS group were significant higher than that of CT group.However,the expression of Smad3,P-Smad2/3 and VEGF in the SB group,ALN group and HB group were significant lower than those in OS group,and the decrease of P-Smad2/3 in SB group and HB group was particularly obvious,which indicate that HB could inhibit the activation of TGF?/smad signaling.The WB results of vertebral body showed that the expression of TGF?1,P-Smad2/3,MMP9 in OS group were significant higher than those in CT group,while the osterix was decreased.When compared with OS group,TGF?1 expression in SB group was significant increased and P-Smad2/3 was significant decreased.We assume that this may be related to the body compensatory feedback due to the TGF?/smad signaling pathway inhibition.At the same time,it can promote the expression of osterix,while MMP9 expression did not change significantly.The expression of TGF?1 and MMP9 in ALN group was significant decreased and along with the significant decreasing of P-smad2/3 expression when compared with OS group,which indicated that inhibition of osteoclasts could significantly inhibit the release of TGF?1 and futher inhibit the activation of TGF?/smad signaling.However,the osterix expression of ALN group did not change significantly which suggests that ALN maybe had no effect on osteogenesis.HB group showed TGF?1 decreased and coupled with significant decrease of P-Smad2/3 expression,indicating that HB could has inhibitory effect on TGF?/smad pathway,which was similar to SB.In addition,the expression of osterix increased and the expression of MMP9 did not change significantly in HB group,suggesting HB could also have osteogenic effects.Conclusion:1.There is a positive correlation between intervertebral disc degeneration and vertebral osteoporosis.Osteoporosis of vertebrae may promote the development of disc degeneration.In addition,the concentration of serum bone metabolites are associated with the degree of intervertebral disc degeneration,which may be used as predictors of degenerative disc disease to some extent.2.Both of ovariectomy and mechanical imbalance could lead to the degeneration of intervertebral disc in mice and more serious degeneration of intervertebral disc occurred in compound model,indicating that osteochondral metabolism and structural changes of vertebral body and endplate may play an important role in the degenerative process of intervertebral disc.The vicious circle of mechanics and metabolics may be responsible for the progressive intervertebral disc degeneration.At the same time,the increased porosity and angiogenesis due to endplate remodeling do not provide adequate nutrition for the intervertebral disc,but rather promote the degeneration of the intervertebral disc,which may be related to the slow loss of internal pressure of the nucleus pulposus and the disruption of the intervertebral disc microenvironment.3.Metabolic disorders or mechanical stimulations could cause abnormal osteochondral matrix conversion.The release of numerous TGF?1 because of matrix remodeling will activate P-smad2/3 and mediate the uncoupling of osteogenesis and osteoclast as well as tissue fibrosis,which may be an important link in the development of vertebral osteoporosis and intervertebral disc degeneration.Bushen herbs could inhibit TGF?-Smad2/3 signaling pathway in vertebrae and intervertebral discs and improve the cartilage metabolism and structure of vertebra and endplate,futher ease the disc degeneration.Its role in prevention and treatment of vertebral osteoporosis and intervertebral disc degeneration may be achieved by regulating of TGF?-Smad2/3 signaling pathway. |
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