| Objective Firstly,a rat model of lumbar intervertebral disc degeneration(IVDD)was constructed.Then,tension was provided using a three-dimensional fixed-point balanced traction bed for IVDD rat.At the animal level,it was investigated whether tension could improve the level of autophagy in intervertebral disc cells via MAPK/ERK/m TOR signaling pathway,and improve the degeneration of rats.Secondly,human nucleus pulposus(NP)cells were induced by lipopolysaccharide(LPS)to simulate the degeneration model of intervertebral disc NP cells.Then,the Flexcell FX-6000 Tension System was used to load the tension on NP cells.To explore whether tension inhibits the degradation of extracellular matrix(ECM)by inducing autophagy and improves the degeneration of NP cells,the effect of tension on regulating autophagy of NP cells will be observed based on MAPK/ERK/m TOR signaling pathway.The current study might play an important role on the prevention and treatment of IVDD.Methods1.Animal experiments(1)Model construction: A rat model of lumbar IVDD was established by annulus fibrosus puncture method,and a sham operation group was also established.(2)Groups and intervention: The model rats were randomly divided into model group and tension group,and the rats in each group were given different intervention measures.Model group: daily routine feeding without any intervention;Tension group: a three-dimensional fixed-point balanced traction bed(patent number: 202021343721.X)was used on IVDD rats to provide tension;Sham operation group: the rats were only fixed on the three-dimensional fixed-point balanced traction bed without any intervention.(3)Detection indicators:(1)HE staining was used to observe the histopathological changes of IVDD rats.(2)The serum levels of inflammatory factors IL-6,TNF-α and IL-1βin IVDD rats were detected by ELISA.(3)The protein expressions of CollagenⅡ and COX-2 in IVDD rats was detected by Immunohistochemistry.(4)The protein expressions of MMP-2,MMP-3 and MMP-9 in IVDD rats was detected by Immunohistochemistry.(5)RT-q PCR and Western blot were used to detect the m RNA and protein expressions of autophagy-related factors Beclin-1,LC3 and p62 in IVDD rats.(6)Western blot was used to detect the expressions of MAPK/ERK/m TOR pathway-related proteins in IVDD rats.2.Cell experiments(1)LPS inducing NP cells: Human NP cells were induced and stimulated by LPS to simulate the pathological process of IVDD.(2)NP cells loaded with tension: Flexcell FX-6000 Tension System(Flexcell International,North Carolina,USA)was used to load the tension on NP cells.(3)Groups: There were four groups including NP cells group,NP cells + tension group,NP cells + LPS group,and NP cells + tension + LPS group.(4)Detection indicators:(1)Transmission Electron Microscopy(TEM)was used to observe the autophagosomes and autophagolysosomes of NP cells.(2)The NP cells were transfected with LC3 dual-fluorescence lentivirus,and then mmunofluorescence experiments were performed.The changes in autophagic flux in NP cells were observed using confocal laser microscopy.(3)RT-q PCR and Western blot were used to detect the m RNA and protein expressions of autophagy-related factors Beclin-1,LC3 and p62 in NP cells.(4)Western blot was used to detect the expressions of MAPK/ERK/m TOR pathway-related proteins in NP cells.(5)The protein expressions of Collagen Ⅱ and COX-2in NP cells was detected by Western blot.(6)The protein expressions of MMP-2,MMP-3and MMP-9 in IVDD rats was detected by Western blot.Results1.Animal experiments(1)Histopathological changes in IVDD rats: In model group,HE staining was uneven in rat intervertebral disc tissues.Moreover,NP was shrunken with a large number of chondrocyte-like necrosis,nuclear fragmentation or dissolution,and cytoplasmic necrosis,even with a large amount of hemorrhage and exudation.However,after giving tension treatment,the pathological morphology of in rat intervertebral disc tissues was significantly improved compared with model group.The results suggested that tension might have a protective effect on IVDD rats.(2)Detection the serum levels of IL-6,TNF-α and IL-1β in IVDD rats: After the rats were giving tension treatment,the levels of TNF-α and IL-1β in the serum of rats were significantly higher than those in model group(P < 0.05).The results suggested that tension could inhibit IVDD by improving the inflammatory response of IVDD rats.(3)Protein expressions of Collagen Ⅱ and COX-2 in IVDD rats: After the rats were giving tension treatment,the protein levels of Collagen Ⅱ in tension group were significantly higher than those in model group(P < 0.05).In addition,the protein levels of COX-2 were significantly lower than those in model group(P < 0.05).The results suggested that tension could improve IVDD by reducing the degradation of CollagenⅡand inhibiting the expression of inflammatory factor COX-2 in IVDD rats.(4)Protein expressions of MMP-2,MMP-3 and MMP-9 in IVDD rats: After the rats were giving tension treatment,the protein levels of MMP-2,MMP-3 and MMP-9 in tension group were significantly lower than those in model group(P < 0.05).The results suggested that tension could improve IVDD by inhibiting the expression of MMP in IVDD rats.(5)Expressions of autotroph-related factors Beclin-1,LC3 and p62 in IVDD rats: After the rats were giving tension treatment,the protein and m RNA levels of Beclin-1and LC3 in tension group were significantly higher than those in model group(P < 0.05),while the protein and m RNA levels of p62 in tension group were significantly lower than those in model group(P < 0.05).The results suggested that tension could improve IVDD by regulating autophagy of intervertebral disc cells(6)Expressions of MAPK/ERK/m TOR pathway-related proteins in IVDD rats: After the rats were giving tension treatment,the phosphorylation levels of MEK,ERK and m TOR in tension group were significantly lower than those in model group(P < 0.05).The results suggested that tension could improve IVDD by regulating autophagy of intervertebral disc cells via MAPK/ERK/m TOR signaling pathway.2.Cell experiments(1)Morphological changes of autophagosomes in NP cells: In NP cells + tension + LPS group,typical autophagolysosomes could be observed,which contained residual undigested organelles or cytoplasm,while the autophagosomes and autolysosomes of NP cells in other three groups were smaller in volume and less in numbers.The results indicated that tension had a promoting effect on autophagy of NP cells.(2)Changes of autophagic flow in NP cells: In NP cells + tension + LPS group,after red-green fluorescence was merged,both yellow spots(autophagosomes)and red spots(autophagolysosomes)were increased,and the fluorescence intensity of LC3-Ⅱ was enhanced,while there were fewer yellow and red spots in in other three groups,and the fluorescence intensity of LC3-Ⅱ was relatively weak.The results suggested that tension had a promoting effect on autophagy of NP cells and could promote the activation of autophagic flux.(3)Expressions of autophagy-related factors Beclin-1 and LC3 in NP cells: Compared with NP cells + LPS group,the m RNA/protein levels of Beclin-1 and LC3 were significantly increased(P < 0.05)in NP cells + tension + LPS group.The results suggested that tension could activate autophagy with the levels autophagy-related factors Beclin-1and LC3 increased.(4)Expressions of MAPK/ERK/m TOR pathway-related proteins in NP cells: in NP cells +LPS group,LPS increased the phosphorylation levels of MEK,ERK and m TOR compared with NP cells group(P < 0.05),while in NP cells + tension + LPS group,the phosphorylation levels of MEK,ERK and m TOR were decreased compared with NP cells+ LPS group(P < 0.05).The results suggested that tension could activate autophagy of NP cells via MAPK/ERK/m TOR signaling pathway.(5)Protein expressions of Collagen Ⅱ and COX-2 in NP cells: Compared with NP cells group,the protein expressions of CollagenⅡ in NP cells + LPS group was lower(P <0.05).After loading tension on NP cells,compared with NP cells + LPS group,the protein levels of Collagen Ⅱ in NP cells + tension + LPS group were significantly increased(P <0.05).These results indicated that LPS could induce inflammation degeneration of NP cells,while tension could reverse inflammation degeneration with reducing the degradation of CollagenⅡ in NP cells.In addition,compared with NP cells group,the protein levels of Collagen Ⅱ in NP cells + tension group was significantly increased(P < 0.05),suggesting that the tension might have a protective effect on NP cells without reducing the levels of CollagenⅡ.Compared with NP cells group,the protein expressions of COX-2 in NP cells + LPS group was increased(P < 0.05).After loading tension on NP cells,compared with NP cells+ LPS group,the protein levels of COX-2 in NP cells + tension + LPS group were significantly decreased(P < 0.05).These results indicated that LPS could induce inflammation degeneration of NP cells,while tension could reverse inflammation degeneration with increasing the levels COX-2 in NP cells.In addition,compared with NP cells group,the protein levels of Collagen Ⅱ in NP cells + tension group was significantly decreased(P < 0.05),suggesting that the tension might have a protective effect on NP cells without increasing the levels of CollagenⅡ.(6)Protein expressions of MMP-2,MMP-3 and MMP-9 in NP cells: Compared with NP cells group,the protein expressions of MMP-2,MMP-3 and MMP-9 in NP cells + LPS group was increased significantly(P < 0.05).After loading tension on NP cells,compared with NP cells + LPS group,the protein levels of MMP-2,MMP-3 and MMP-9 in NP cells + tension + LPS group were significantly decreased(P < 0.05).These results indicated that tension could improve the the degradation of ECM by reducing the protein levels of MMP.In addition,compared with NP cells group,the protein levels of MMP-2,MMP-3 and MMP-9 in NP cells + tension group were without significantly decreased(P >0.05),suggesting that tension might have protective effect on NP cells without increasing MMP protein levels.In conclusion,we could speculate that tension could inhibit the inflammatory degeneration of NP cells by reducing MMP activity.Conclusion(1)In IVDD rats,tension could increase the autophagy level of intervertebral disc,improve the inflammatory response,reduce the activity of MMP,and reduce the degradation of Collagen Ⅱ protein,via inhibiting MAPK/ERK/m TOR signaling pathway,thereby improving IVDD.(2)In LBS induced NP cells,tension could activate autophagy via MAPK/ERK/m TOR signaling pathway with inhibiting MMP activity,reducing CollagenⅡ protein degradation,reducing the expression of inflammatory factor COX-2,improving ECM degradation,and then delaying degradation of NP cells. |
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