Effect And Mechanism Of Eupatilin On Nucleus Pulposus Cells And Intervertebral Disc Degeneration In Rats

Objective:To observe the effect of Eupatilin(EP)on nucleus pulposus(NP)cells and intervertebral disc degeneration(IDD)in rats.To explore the possible therapeutic effect and regulatory mechanism of Eupatilin on the degradation of rat NP extracellular matrix and the senescence of NP cells induced by inflammatory stimulation,and to provide theoretical basis and new thinking of diagnosis and treatment for the treatment of IDD disease in clinical rehabilitation.Methods:(1)Based on the CCK-8 experiment,the effect of Eupatilin on the proliferation of NP cells in vitro was determined to determine the maximum non-toxic concentration of NP cells.(2)In vitro,tumor necrosis factor-α(TNF-α)induced NP cell inflammation was used to establish the model of cellular inflammation.(3)The effects of Eupatilin on TNF-α induced activation of inflammatory signaling pathway in NP cells,which led to imbalance of NP extracellular matrix synthesis and metabolism,and senescence of NP cells were examined by high-density cell culture,Alcian blue staining and senescence β-Galactosidases staining kit.(4)RT-q PCR,Western blot and immunofluorescence techniques were used to find out the molecular signaling mechanism of Eupatilin in improving the extracellular matrix degradation and senescence of NP cells induced by TNF-α.(5)The IDD model was established by puncturing the intervertebral disc of the caudal vertebrae of rats with 20-caliber sterile needle.(6)In vivo,the efficacy of Eupatilin in the treatment of rat IDD model was evaluated by X-ray,MRI,Safranin Fast Green staining,H&E staining and immunofluorescence staining.Results:(1)The maximum non-toxic concentration of Eupatilin in the intervention of NP cells was 25 μM.(2)TNF-α,Compared with the control group,had significantly up-regulated the expression of MMPs,P21,P53,TNF-α and IL-1β,and down-regulated the expression of SOX9 and Col2a1.(3)Eupatilin treated group,compared with TNF-α induced group,were down-regulated in a dose-dependent manner the expression of MMPs,P21,P53,TNF-αand IL-1β,while the expression of SOX9 and COL2A1 was up-regulated.(4)TNF-α induced NP cells had significantly activate the expression of NF-κB and MAPK pathway related proteins,and the intervention of Eupatilin in NP cells can significantly inhibit the up-regulation of this pathway protein.(5)In vivo experimental data showed that the Eupatilin intervention group significantly reduced the damage of intervertebral disc structure compared with PBS group.Conclusion:(1)Eupatilin reduces TNF-α Induced degradation of NP extracellular matrix and senescence of NP cells.(2)In mechanism,Eupatilin inhibits the activation of MAPK and NF-κB inflammatory signaling pathways,thereby improving extracellular matrix degradation and cellular senescence induced by TNF-α-induced signaling activation.(3)The establishment of rat IDD model by puncture showed that the treatment of Eupatilin can improve the progress of IDD and has potential therapeutic effects.