| Objective:Low back pain is a common disease,which has seriously affected people’s life and work worldwide.According to clinical observations and substantial investigations,lumbar disc degeneration is recognized as one of the most important contributors of LBP.There is a positive correlation between the degree of IDD and LBP.However,the current treatments for this degenerative disease are mainly to alleviate symptoms instead of addressing its etiology and pathogenesis,and there are still few approaches for preventing or reversing disc degeneration.Therefore,it is imperative to find new genetic targets and noval therapeutic approaches in the treatment of intervertebral disc degeneration.In this study,we plan to collect intervertebral disc tissue samples clinically,constructed rat models with IDD and degenerative NP cell models,and use Western Blot,q RT-PCR,histological staining,MTT and sh RNA gene knockdown techniques and methods to deeply analyze the function and molecular mechanism that BBR regulates GDF-5 to alleviate intervertebral disc degeneration,which will provide new targets and ideas for the prevention and treatment of this degenerative disease.Methods:Firstly,19 intervertebral disc tissue samples were collected from patients during operation,and these tissues were graded according to the Pfirrmann system based on patients’ preoperative MRI images of the intervertebral discs,4 disc tissues of grade II were designated as normal group and 15 tissue of grade III-V were degenerated group.The protein expression of GDF-5,Col II and MMP-13 in normal and degenerative disc tissues was quantified by WB,and the expression of Col II and MMP-13 was analyzed qualitatively by IHC,and the correlation between GDF-5 and disc degeneration was analyzed in combination with the changes of degenerative disc specific indexes.Secondly,30 rats were divided into the blank group(Control),the intervertebral disc degeneration group(IDD),and the BBR gavage group(BBR),and the rats in the IDD and BBR groups were performed with acupuncture to induce intervertebral disc degeneration,and the rats in the BBR group were treated with berberine by gavage.The Control group was raised normally and the BBR group was treated with BBR dilution at 150 mg/ kg/day for 8 weeks by continuous gavage,while the IDD group was treated with the same dose of saline gavage for 8 weeks.The expression of Col II and Agg(main components of the ECM),and MMP-13 was detected by q RT-PCR.Histomorphological changes of intervertebral discs’ tissues were observed by staining with saffron O and HE,and the ECM metabolic changes of rats intervertebral discs in BBR group were analyzed to identify the influence of BBR on rats with IDD.Finally,to establish an IL-1β-induced degenerative NP cell model that was intervened with BBR.And to divide cells into blank group(Control/Mock),degenerative group(IL-1β),berberine intervention group(BBR),sh NC negative interference group(sh NC+IL-1β+BBR)and sh GDF-5 interference group(sh GDF-5+IL-1β+BBR).WB and q RT-PCR were performed to determine the alterations of Col II,Agg,MMP-13,and GDF-5 protein and gene expression for further verifying the effects of BBR on the ECM metabolism,and to demonstrate the function and molecular mechanism that BBR alleviates IDD by regulating GDF-5.Results:First part,for clinically collected intervertebral disc tissue samples,both WB and IHC results showed higher expression of Col II in disc tissues with Pfirrmann grade II and lower expression in degenerated disc tissues with grades III-V,but the opposite was true for matrix degrading enzyme MMP-13expression(p<0.05),indicating that the grading criteria for disc degeneration are basically reliable.Second part,the histological staining of the caudal intervertebral disc tissues of rats was performed at 4th and 8th weeks after modeling.Four-week HE staining showed that the intervertebral disc tissues of the control group were structurally intact,with neatly arranged fibers,high density and normal morphology of chondrocytes and NP cells;the intervertebral disc tissues of the IVDD group were disorganized,of which the fibers were not aligned,and with reduced density and abnormal morphology of chondrocytes and NP cells;while the disc tissues of the BBR group had recovered both in structure,density and morphology of chondrocytes and NP cells compared with those of the IVDD group.Eight-week stainning presented the disc tissues of rats in the control group were rich in chondrocytes and stroma with clear and dark extracellular matrix proteoglycan staining,clear boundary between NP and AF,and high density and normal morphology of nucleus pulposus cells.In the IVDD group,there were less chondrocyte and stromal content,the boundary between the nucleus pulposus and the fibrous ring was unclear,and the density of nucleus pulposus cells was reduced.In contrast,the material content of the extracellular matrix and the density of nucleus pulposus cells were restored in the BBR group.Meanwhile,the results of q RT-PCR indicated that the gene expression of Col II and Agg was decreased,and the expression of MMP-13 was significantly increased in the caudal disc tissues of rats in the modeling group at 4th and 8th weeks compared with the control group.While,the increased gene expression of Col II and Agg was observed in the BBR group,but the expression of MMP-13 has decreased compared with the modeling group(p<0.05),which fully verified the therapeutic effect of BBR on rats with IDD.Third part,the degenerative NP cell model induced by IL-1β was constructed.The optimal concentration of BBR delivery was 100μg/m L.q RT-PCR and WB results suggested a lower level of the gene and protein expression of Col II and Agg,the main components of NP cell extracellular matrix,and a higher level of MMP-13 after modeling.However,the synthesis of Col II and Agg has been increased after BBR intervention,while the expression of MMP-13 was decreased a lot.The expression of GDF-5 was significantly reduced in the extracellular matrix of the degenerative NP cells,and was increased after BBR intervention(p<0.05),which suggest that BBR may have an upregulatory effect on GDF-5.When it was intervened with sh GDF-5(knockdown of GDF-5 gene)and sh NC(negative control),there was a trend of decreased Col II and Agg synthesis and increased MMP-13 expression in the sh GDF-5+IL-1β+BBR and sh NC+IL-1β+BBR groups,and the trend was more pronounced in the sh GDF-5 intervention group.This indicated that sh GDF-5 may attenuate the effect of BBR in inhibiting MMP-13 synthesis and preventing degradation of the ECM to an extent.BBR could inhibit the IL-1β-induced ECM degradation of NP cell by targeting GDF-5 and up-regulating its expression.Conclusion:1.GDF-5 is widely expressed in human normal and degenerated intervertebral disc tissues,and is negatively related with the degree of IDD.2.BBR,a single active ingredient of the Coptis chinensis Franch with anti-inflammatory properties,has clear therapeutic effects in rats with intervertebral disc degeneration.By inhibiting the synthesis of MMP-13,it will increase the expression of Col II and Agg,which are the main components of the extracellular matrix,and recover the morphology and structure of the degenerated intervertebral disc.3.BBR inhibits matrix degrading enzyme MMP-13 by up-regulating GDF-5 expression,and promotes the synthesis of Col II and Agg,which facilitates the metabolism of the extracellular matrix,and eventually alleviates IDD.4.In summary,this study has elaborated the mechanism that BBR regulates GDF-5 to alleviate IDD,which will provide a theoretical basis for the study of strategies to promote disc degeneration repair. |
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