Resveratrol Stimulate The Extracellular Matrix Synthesis Of Degenerative Endplate Chondrocytes Via Activating SIRT1/IGF-1R/AKT Pathway

Disc degeneration is a common cause of neck and low back pain. The disc is the largesthypoxic tissue of human body.Only the outer layer of annulus fibrosus exists vasculardistribution, the inner layer of annulus fibrosus and nucleus have no vascular distribution.nutrients that the nucleus pulpous cells required are supplied by the penetration function ofendplate cartilage from the vertebral body to the nucleus pulpous. With aging, the endplatechondrocytes aged,the apoptosis increased, synthesis of extracellular matrix decreased whiledecomposition accelerated, subchondral vertebral bone calcified, leading to the dispersion functionreduced. these alterations not only hindered nutrients supply of the nucleus pulposus, such as sugarand oxygen,but also hindered the discharge of metabolic wastes, such as lactic acid, thuspromoting disc degeneration.Although the exact molecular mechanism of disc degeneration is not fully understood, but thereis no doubt that the degeneration of the endplate cartilage cells, and finally the reduction ofpermeability plays an important role in the process of disc degeneration. Therefore, to prevent andreduce the degeneration of the endplate cartilage cells become a potential target to prevent discdegeneration.In2003,KT Howitz and Sinclair et al[1]firstly discovered and reported that resveratrol, itbelongs to the sirtuin activation complexes (sirtuin activating compounds, STACs), and is morepotent than a series of phenols as SIRT1activator. From then on,resveratrol,as a activator of SIRT1,is widely used in scientific research.Many scholars[2-6]have confirmed that SIRT1is a key molecule in the process of retardingaging and extending lifespan in mammalian, and plays an important regulatory role in cell cycle, metabolism, differentiation. Dvir-Ginzbergand et al[7]have confirmed that the high expression levelof SIRT1in human articular chondrocytes can promote the expression of cartilage extracellularmatrix genes, such as aggrecan, collagen2a (α1),2b (α1), collagen9(α1). Gagarina VG[8]have confirmed SIRT1could inhibit chondrocyte apoptosis in patients with osteoarthritis.Previous studies have shown[9-11]that in a variety of sources of articular chondrocyte, IGF-1can significantly promote proliferation,stimulate the synthesis of chondrocytes specific extracellularmatrix, such as type II collagen and proteoglycan, enhance alkaline phosphatase activity of bonecells.This study explored the expression of SIRT1by resveratrol of different concentrations and timein degeneration intervertebral disc endplate cartilage cells and provide reference for furtherresearch; then the effect of resveratrol on promoting the synthesis of extracellular matrix and itsrelationship with SIRT1was discussed by followed by SIRT1-specific siRNA and SIRT1bioactivityinhibitor nicotinamide; finally, the role of IGF-1R/AKT pathway in the resveratrol stimulation wasexplored through interference by IGF-1R/AKT pathway-specific inhibitors.1Concentration and time-dependent induction of SIRT1on human degenerative endplatechondrocytes by resveratrol.Objective To explore the effect of different concentrations and time of resveratrolintervention on the expression of SIRT1in human degenerative endplate chondrocytes. MethodsDegenerative disc endplate chondrocytes(DEC) were harvested from elder patient with lumbar dischernia, and then cultured. Passage2DEC were stimulated by resveratrol of differentconcentrations and Time respectively, and then expression levels of SIRT1and SIRT1mRNAwere detected by the end of stimulation. Results Compared with the expression levels in controlgroup, in order to achieve significant difference of SIRT1level, the least requirements of resveratrolwere12.5μmol/L by24hours,50μmol/L by12hours (P <0.05); in order to achieve significantdifference of SIRT1mRNA level, the least requirements of resveratrol were12.5μmol/L by48hours,50μmol/L by24hours (P <0.05).Resveratrol more than the above dose could inducesignificant difference when post-hoc analysis were conducted. Conclusion Resveratrolintervention could significantly upgrade the expression levels of SIRT1and SIRT1mRNA in human degenerative endplate chondrocytes in a concentration-and time-dependent manner.2Resveratrol stimulate extracellular matrix synthesis of degenerative endplate chondrocytesby activating SIRT1pathwayObjective To explore the effect and mechanism of resveratrol on extracellular matrixsynthesize of degenerative endplate chondrocytes. Method Degenerative disc endplatechondrocytes(DECs),derived from elder patient with lumbar disc hernia, were randomly dividedinto5groups, namely, control group, dimethyl sulfoxide (DMSO) control group, resveratrol (Res)group, resveratrol+nicotinamide(RES+Nam) group, resveratrol+siRNA (RES+siRNA) group.DECs were stimulated with corresponding reagent for48hours. Expression levels of SIRT1, typeⅡcollagen, aggrecan and SIRT1mRNA were detected. Results100μmol/L Resveratrolintervention could increase the expression levels of both SIRT1and SIRT1mRNA,. stimulating thesynthesis of extracellular matrix. Nicotinamide and siRNA intervention effectively suppress SIRT1enzymatic activity and SIRT1mRNA expression respectively, inhibiting the synthesis ofextracellular matrix. Conclusion Resveratrol intervention could stimulate the extracellular matrixsynthesize of degenerative endplate chondrocytes via SIRT1pathway.3Resveratrol stimulate the extracellular matrix synthesis of endplate chondrocytes viaIGF-1R/AKT pathway.Objective To explore the effect of IGF-1R/AKT pathway on extracellular matrix synthesis ofdisc endplate chondrocytes stimulated by resveratrol Method P2generation of endplatechondrocytes were randomly divided into5groups, and stimulated with corresponding reagent for48hours. Expression levels of IGF-1R, pIGF-1R, AKT, pAKT, COLAII and aggrecan were detectedby Western blotting technique. Results Resveratrol intervention could significantly increase thephosphorylation levels of both IGFR and AKT, and stimulate the synthesis of COLAII and aggrecan.AG1024and SH-5significantly inhibit the phosphorylation of both IGFR and AKT, and inhibit thesynthesis of COLAII and aggrecan. Conclusion Resveratrol could stimulate the extracellular matrixsynthesis of disc endplate chondrocytes via IGF-1R/AKT regulatory pathway.