Co-culture Of Adipose Derived Mesenchymal Stem Cells Under Periodic Mechanical Stress Promotes Proliferation And Autophagy Of Nucleus Pulposus Cells

Objective: The aim of this study was to investigate whether the severity of lumbar disc degeneration is associated with the expression of integrin alpha 1(ITGA1)in nucleus pulposus tissue and to investigate the effect of adipose tissue and nuclei cocultured mesenchymal stem cells under mechanical stress physiological conditions underlying medullary cells.nucleus pulposus cells and further study the mechanism.Methods: The nucleus pulposus tissues undergoing surgical treatment due to lumbar disc degeneration were collected,grouped according to preoperative MRI images,and immunohistochemical techniques and Western Blot were used to detect the expression of related proteins.Adipose-derived mesenchymal stem cells and nucleus pulposus cells were cultured in vitro,and the nucleus pulposus cells were cultured separately or cocultured under periodic mechanical stress and horizontal pressure under physiological conditions,and CCK-8,WB and EDU(5-ethynyl-2’-deoxyuridine,5-ethynyl-2’deoxyuridine)to detect cell proliferation and expression of extracellular matrix.The mechanism of promoting the proliferation of nucleus pulposus cells and the expression of extracellular matrix in co-culture under periodic mechanical stress by RNA interference technology and WB.Finally,transmission electron microscopy,WB,RNA interference technology,immunofluorescence and flow cytometry were used to detect the autophagy level of nucleus pulposus cells co-cultured with adipose-derived mesenchymal stem cells under periodic mechanical stress,and whether it could rescue nucleus pulposus cells in Apoptosis in an inflammatory environment.Results: It was found that the expressions of ITGA1 and Collagen II(COL2A1)in nucleus pulposus decreased with the severity of intervertebral disc degeneration.The proliferation,expression and phosphorylation of related proteins(COL2A1,Aggrecan,ITGA1,Src,GIT1,ERK1/2)were effectively promoted by coculture cells under horizontal pressure or single culture under cyclic mechanical stress.The effect of coculture under cyclic stress was more obvious than that of other groups.The difference was statistically significant(P<0.05).When RNA interference inhibited endogenous ITGA1,the proliferative ability and extracellular matrix protein content of nucleus pulposus cells co-cultured with mechanical stress were significantly reduced,Src,GIT1 and ERK1/2 were reduced,and the phosphorylated levels were also significantly reduced,the difference There is statistical significance(P<0.05).It was found that the ITGA1/Src/GIT1/ERK1/2 axis was used to regulate proliferation and differentiation of nucleus pulposus cells and extracellular matrix expression under cyclic mechanical stress.The pressurized coculture cells were incubated in 100μm tertbutylhydrogen peroxide(TBHP),and the endogenous ITGA1 and ERK1/2 were inhibited by RNA interference technology and inhibitors.Under the projection electron microscope,the number of autophagosomes and autophagosomes were significantly reduced,and the expression level of autophagorelated proteins was also significantly decreased.The difference was statistically significant(P<0.05).At the same time,the fluorescence spots and autophagy flow of LC3 B in nucleus pulposus cells were also significantly decreased under fluorescence microscopy,with statistical significance(P<0.05).After using flow cytometry and live staining,it was observed that the apoptosis of nucleus pulposus cells increased significantly after ITGA1 and ERK1/2were knocked out and autophagy was inhibited in the mechanical stress co-culture of nucleus pulpocytes placed in TBHP environment.There were also significant differences in apoptosis-associated protein expression(cleaved Caspase3,BAX,BCL2)(p<0.05).Matrix metalloproteinase 13(MMP13)immunofluorescence techniques were used to detect the matrix-degrading enzymes levels,and found that the mechanical stress can reduce nucleus pulposus cells co-culture MMP13 expressed.Conclusions: This study confirmed that co-culture of nucleus pulposus cells with adipose mesenchymal stem cells under moderate periodic mechanical stress can effectively promote the proliferation of nucleus pulposus cells,the expression of extracellular matrix and up-regulate the level of autophagy.save its apoptosis in inflammatory environment.At the same time,it is proved that co-culture under periodic mechanical stress can regulate the physiological function of nucleus pulposus cells by up-regulating the receptor ITGA1 on the surface of nucleus pulposus cells and activating the Src/GIT1/ERK1/2 axis.This study provides a reliable theoretical basis for stem cells in the treatment of intervertebral disc degeneration.