Taxifolin Disc Degeneration By Inhibiting Nucleus Pulposus Pyroptosis

Objective: Intervertebral disc degeneration(IVDD)is the pathological basis of a series of degenerative spinal diseases,which can cause a variety of symptoms and signs and seriously affect the quality of life of patients.Currently,clinical treatment for IVDD is limited.As a natural flavonoid,taxifolin(TAX)has anti-inflammatory,antioxidant and microcirculation improvement activities.The objective of this study was to explore the potential mechanism by which TAX alleviates intervertebral disc degeneration.Methods: In vitro experiments,we first used CCK-8 to verify the cytotoxicity of TAX to nucleus pulposus cells at different concentrations(10,20,50,100,200 μM).Then,we divided the experiment into three groups: the control group,LPS group and LPS+TAX group.After different treatments,flow cytometry and Western blotting(WB)were used to detect the pyroptosis of nucleus pulposus cells in each group.Second,to further explore the possible mechanism,we divided the experiment into four groups.The expression levels of p-p65/p65 and p IΚBα/IΚBα in the NF-κB pathway were investigated by WB after different treatments in the control group,LPS group,LPS+BAY 11-7082 group and LPS+TAX group to reveal the effect of TAX on the NF-κB pathway.In the in vivo experiment,24 male SD rats(weighing approximately 200 g)were randomly divided into3 groups: the sham puncture group(skin incision only),puncture degradation group(intramuscular injection of normal saline after puncture),and puncture +TAX group(intramuscular injection of TAX after puncture).MRI examinations were performed on the tail vertebrae of rats in each group 8 weeks after the puncture.After examination,the rats were executed and the corresponding intervertebral disc tissues were taken.WB was used to detect the expression levels of focal death-related proteins(NLRP3,GSDMD,IL-1β,caspase-1)in intervertebral disc tissue and to evaluate the causation of focal death and the degree of degeneration of intervertebral disc cells.Results: In vitro experiments: 1.CCK-8 results showed no significant cytotoxic effect on nucleus pulposus cells at the preset concentration range.2.Flow cytometry results showed that LPS could increase the death of nucleus pulposus cells,while the number of dead cells was significantly reduced after the addition of TAX,indicating that TAX could alleviate the apoptosis of nucleus pulposus cells.3.The expression of pyroptosis-related proteins in nucleus pulposus cells assayed by WB showed that LPS significantly increased the expression levels of NLRP3,GSDMD,IL-1β and caspase-1 in nucleus pulposus cells,while the expression levels of pyroptosis-related proteins decreased in the group treated with TAX.The results showed that TAX could relieve the pyroptosis of nucleus pulposus cells.4.The results of the detection of NF-κB pathway-related protein expression by WB indicated that the relative expression levels of p-p65/p65 and pIκBα/IκBα in the LPS-induced group were significantly higher than those in the blank control group.The relative expression levels of p-p65/p65 and p-IκBα/IκBα in the BAY11-7082 inhibitor group and TAX treatment group were significantly decreased.The results indicated that TAX could inhibit the activation of the NF-κB pathway.In vivo experiments: 1.MRI results of rat caudal vertebrae showed significantly lower T2-weighted nucleus pulposus signal intensity,misaligned disc structures and reduced height in the degenerated group compared with the sham puncture group.The improvement in the treatment group indicated that the injected drug could improve the disc degeneration.2.The expression of pyroptosis protein in intervertebral disc tissue detected by WB showed that intraintervertebral disc injection of TAX could relieve the pyroptosis of intervertebral disc cells.Conclusion:By inhibiting the NF-κB pathway,TAX can relieve nucleus pulposus cell pyroptosis and improve intervertebral disc degeneration.