| In developed countries,low back pain(LBP)not only has an extremely high incidence,but is also one of several diseases with a high disability rate.About 84%of patients worldwide suffer from the physical and psychological problems caused by it all year round.Intervertebral disc degeneration(IVD degeneration,IDD)has been proven to be the predominant cause of low back pain and a variety of spinal diseases.Unfortunately,the exact mechanism of intervertebral disc degeneration is still unclear,and treatments are limited to anti-inflammatory or surgical treatments.While these treatments relieve the patient’s pain,they do not completely cure the related diseases.In contrast,they may even accelerate the degeneration of adjacent intervertebral discs.The Hedgehog signaling(Hh)pathway is highly conserved and closely related to the occurrence of most diseases,but its relationship with intervertebral disc degeneration is unclear.The genome sequence and intervertebral disc structure of mice are highly similar to those of humans.Moreover,there are various transgenic mouse tools available,making mouse one of the most attractive models for studying IVD degeneration.Studies have shown that the Hh pathway can regulate the development of intervertebral discs by influencing the formation of the notochord sheath during the embryonic period of mice.Preliminary studies in our laboratory have also found that inhibiting the Hh pathway in growing mice will cause some changes in the structure of the intervertebral disc,but it is still unclear whether these changes will eventually lead to the degeneration of the intervertebral disc.In addition,GDC,an inhibitor of the Hh pathway,has been clinically regarded as a very promising drug for the treatment of medulloblastoma caused by abnormal activation of the Hh pathway.However,it is still unclear whether application of this drug to adolescents will cause permanent spinal and intervertebral disc degeneration is still unclear.In this paper,we employed two different approaches,including systematically inhibiting with GDC(a small molecule)and IVDspecific ablation of Smo and/or Sufu with Agc1-CreERT2,to investigate the effect of inhibiting the Hh pathway on the homeostasis and degeneration of the intervertebral disc important role and underlying mechanisms of the Hh pathway in the maintenance of intervertebral disc in postemal growing mice and potentially provide some insights into molecular cause of intervertebral disc degeneration.Moreover,our study will provide a new theoretical basis for the prevention and treatment of intervertebral disc degeneration.Chapter 1:The effect of systemic inhibition of the Hh signaling pathway by a small molecule on intervertebral disc degeneration in postnatal growing mice[Experimental methods]We first used the drug GDC to systemically inhibit the Hh pathway in growing mice to observe the changes in the mouse spine and intervertebral discs to simulate the effect of GDC on the development of the spine in children with brain tumors.We divided 2-week-old C5 7BL/6 mice into a control group(Vehicle group)and an experimental group(GDC group).The experimental group mice were given with 50 mg/kg GDC via oral gavage twice a day for five consecutive days.And the control group was administered with solvent in the same manner,and the IVD samples were collected for analyses when the mice reached 4 months of age.①Examine the morphological changes of mouse spine and intervertebral discs by MRI,H&E staining and Safranin O&Fast Green staining;②Examine the degeneration situation of mouse intervertebral discs by immunohistochemistry and immunofluorescence staining methods.[Experimental results]①The results of MRI,H&E staining and Safranin O&Fast Green staining showed that GDC-treated mice have significantly shorter vertebral bodies,and IVD in these mice exhibited pathological changes similar to the intervertebral disc degeneration.② The immunofluorescence results further confirmed that systemically inhibiting the Hh pathway by GDC in the growing mice caused IVD degeneration-like phenotypes,including loss of vertebral body growth plate,thinning of cartilage endplate.fibrous annulus cracks and disorganzied expression of extracellular matrix,and appearance of chondrocyte-like cells in the nucler pulposus(NP)region.[Conclusion]Systemic inhibition of the Hh pathway in growing mice with drugs will lead to a degenerative phenotype of the intervertebral disc,which proves the important role of the Hh pathway in maintaining the stable growth of the mouse intervertebral disc and preventing its degeneration.Chapter 2:Specific knockout of Smo in intervertebral disc cells of growing mice leads to disc degeneration[Experimental methods]Having established that systemic inhibition of the Hh signaling pathway in growing mice leads to intervertebral disc degeneration,we intend to further determine whether Hh plays a cell-autonomous role in the IVD.To this end,we use Agc1-CreERT2 to specifically knock out Smo,the key component of the Hh pathway,in the IVD cells.Specifically,we injected 2-week-old Agc1-CreERT2;Smoflox/flox mice with TM daily for 5 consecutive days to induce ablation of Smo,and IVD samples were collected for observation and analysis during a specific period.①Extract RNA from IVDs from control and Smo-ablated mice,and then perform qPCR analyses to determine the knockout efficiency of Smo and other related genes;Using Glil-LacZ reporter mice to monitor Hh activity in control and Smo-ablated mice to clarify the effect of Smo knockout on the Hh pathway ②The effect of Smo knockout on mouse spine and intervertebral disc tissue morphology was observed by Micro-CT analyses,H&E and Safranin O&Fast Green staining;③The proliferation of intervertebral disc cells was detected by BrdU staining;TUNEL method to detect the apoptosis of intervertebral disc cells;④The expression of extracellular matrix of intervertebral discs was detected by immunofluorescence and immunohistochemical staining.[Experimental results]①The qPCR results showed that the expression of Smo in the intervertebral discs of Agc1-CreERT2;Smoflox/flox mice decreased by about 80%compared with the control group;the downstream expressions of Glil,Gli2,and Gli3 also showed a downward trend.In particular,the down-regulation of Gli2 the most obvious with statistical significance.Moreover,Ptch1 and Ihh also showed significant downward trend,but Dhh and Shh were not detected because of low content;LacZ staining results showed that Smo-ablated mice exhibited significantly reduced LacZ signaling and thus Hh activity,in the inner annulus fibrosus,growth plate and cartilage endplate,indicating that the Hh pathway is effectively inhibited in these regions;②Micro-CT results showed that the vertebrae development of Smo-ablated mice was stagnant and osteophytes initiated at 1-month-old,and further expanded with age;H&E staining and Safranin O&Fast Green staining showed that:At 1 month of age,the structure of Smo-ablated mice IVD was still largely normal,but appeared shorter growth piate,thicker annulus fibrosus,and enlarger nucleus pulposus.At 4 months of age,Smo-ablated mice showed the disappearance of growth plates,the calcification of cartilage endplates,and the appearance of fibrous annulus.At 16 months of age,Smo-ablated mice showed a acceleration of nucleus pulposus cells transforming to chondrocyte-like cells.These all aggravate the degeneration of the intervertebral disc;③BrdU and TUNEL staining were performed on 1-month-old mice.The results showed that the decreased proliferation and increased apoptosis of growth plate cells in Smo knockout mice were the main reasons for vertebral growth arrest;④The results of immunohistochemistry and immunofluorescence further confirmed that the specific inhibition of the Hh pathway in the IVD cells of the growing mouse will cause the cartilage endplate to be prematurely calcified,the fibrous annulus extracellular matrix composition changes,the transformation of nucleus pulposus cells into chondrocytelike cells,the chondrocyte-like cells apoptosis increase and other pathological changes of intervertebral disc degeneration.[Conclusion]Conditional knockout of Smo in the intervertebral disc cells of growing mice inhibits the Hh pathway,and accelerate the degeneration of the endplate,annulus fibrosus and nucleus pulposus of IVD in mice.Therefore,the Hh pathway in mouse intervertebral disc cells plays an important role in maintaining the IVD homeostasis and preventing its degeneration.Chapter 3:Knockout of Sufu in intervertebral disc cells of growing mice alleviates disc abnormalities caused by Smo ablation[Experimental methods]Sufu is an inhibitor of the Hh pathway and exerts its function by regulating downstream Gli transcription factors.In this chapter,we intend to investigate the role of Sufu in the regulation of IVD development and homeostasis by the Hh pathway by observing the alleviating effect of Sufu deletion on the disc abnormalities caused by Smo ablation.We injected 2-week-old Agcl-CreERT2;Smoflox/flox;Sufuflox/flox(DKOAgcl)with TM daily for 5 consecutive days to simultaneously ablate Smo and Sufu in IVD cells,and collected IVD samples at 2 months of ages to conduct follow-up observation and analysis.①Observe the changes in the morphology of mouse vertebrae and intervertebral discs when Smo and Sufu were both removed by Safranin O&Fast Green staining;②The expression of extracellular matrix was detected by immunofluorescence,immunohistochemical staining and ALP staining;③The proliferation of intervertebral disc cells was detected by Ki67 immunohistochemical staining;④Change of annulus fibrosus and nucleus pulposus were detected by immunohistochemistry and immunofluorescence staining.[Experimental results]①The results of Micro-CT,H&E staining and Safranin O&Fast Green staining showed that the Hh pathway was specifically inhibited in the intervertebral discs of growing mice while knocking out Sufu to relieve the shortening of the spine,and the complete structure returned to normal but showed too long growth plate;②Col10a1 and ALP staining results show that knocking down Smo and Sufu at the same time can restore the normal differentiation ability of the growth plate;③Ki67 immunohistochemistry results show that knocking down Smo and Sufu at the same time can restore the proliferation ability of growth plate cells;④Acan and Col10a1 staining results show that knocking down Smo and Sufu at the same time can restore the normal expression of extracellular matrix in the annulus fibrosus;Krt19 immunohistochemistry shows that the nucleus pulposus of heterozygous mice is enlarged,but in DKOAgcl mice nucleus pulposus returned to the level of control mice.[Conclusion]Ablation of Sufu can rescue the phenotypes of short spine and intervertebral disc degeneration in Smo-ablated mice,indicating that Hh pathway in the intervertebral discs of growing mice maintains intervertebral disc homeostasis by inhibiting Sufu. |
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