Selective interference of mTORC1/RAPTOR protects against inflammation-induced apoptosis, senescence, and matrix catabolism possibly through Akt and autophagy induction in human disc cells.

IntroductionNutrient deprivation is a likely contributor to intervertebral disc (IVD) degeneration. Silent mating type information regulator 2 homolog 1 (SIRT1) protects cells against limited nutrition by modulation of apoptosis and autophagy. However, little evidence exists regarding the extent to which SIRT1 affects IVD cells. Therefore, we conducted an in vitro study using human IVD nucleus pulposus (NP) cells.MethodsThirty-two IVD specimens were obtained from patients who underwent surgical intervention and were categorized based on Pfirrmann IVD degeneration grades. Cells were isolated from the NP and cultured in the presence of recombinant human SIRT1 (rhSIRT1) under different serum conditions, including 10 % (v/v) fetal bovine serum (FBS) as normal nutrition (N) and 1 % (v/v) FBS as low nutrition (LN). 3-Methyladenine (3-MA) was used to inhibit autophagy. Autophagic activity was assessed by measuring the absorbance of monodansylcadaverine and immunostaining and Western blotting for light chain 3 and p62/SQSTM1. Apoptosis and pathway analyses were performed by flow cytometry and Western blotting.ResultsCells cultured under LN conditions decreased in number and exhibited enhanced autophagy compared with the N condition. Medium supplementation with rhSIRT1 inhibited this decrease in cell number and induced an additional increase in autophagic activity (P < 0.05), whereas the combined use of rhSIRT1 and 3-MA resulted in drastic decreases in cell number and autophagy (P < 0.05). The incidence of apoptotic cell death increased under the LN condition, which was decreased by rhSIRT1 (P < 0.05) but increased further by a combination of rhSIRT1 and 3-MA (P < 0.05). Under LN conditions, NP cells showed a decrease in antiapoptotic Bcl-2 and an increase in proapoptotic Bax, cleaved caspase 3, and cleaved caspase 9, indicating apoptosis induction via the mitochondrial pathway. These changes were suppressed by rhSIRT1 but elevated further by rhSIRT1 with 3-MA, suggesting an effect of rhSIRT1-induced autophagy on apoptosis inhibition. Furthermore, the observed autophagy and apoptosis were more remarkable in cells from IVDs of Pfirrmann grade IV than in those from IVDs of Pfirrmann grade II.ConclusionsSIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human IVD NP cells, suggesting that rhSIRT1 may be a potent treatment agent for human degenerative IVD disease.

Surgical treatment for spinal metastases is associated with significant improvement in health state value. In orthopaedic surgery, an ICUR less than $50,000/QALY gained is considered acceptable cost-effectiveness. Our results indicate that surgical treatment could be cost-effective.

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BACKGROUND CONTEXT Intervertebral disc (IVD) degeneration, a major cause of low back pain, is considered to be induced by daily mechanical loading. Mechanical stress is widely known to affect cell survival and extracellular matrix metabolism in many cell types. Although the involvement of integrin α5β1 transmembrane mechanoreceptor in IVD degeneration has been reported, the precise function of integrin α5β1 remains obscure. PURPOSE To reflect IVD tissue response to mechanical stress using a dynamic loading organ culture system and elucidate the functional impact of integrin α5β1 on the pathomechanism of IVD degeneration. STUDY DESIGN An ex vivo study using a dynamic loading organ culture system. METHODS Ninety-six rat IVD explants were examined. Intervertebral discs were subjected to 1.3 MPa, 1.0 Hz dynamic compressive load in the presence or absence of an Arg-Gly-Asp (RGD) peptide with affinity to the fibronectin binding-site of integrin α5β1. Cell viability and histomorphology were assessed. The localization of integrin α5β1 in the IVD was assessed by immunohistochemistry. Gene expression levels of IVD cells were evaluated using real-time reverse transcription-polymerase chain reaction. RESULTS In the nucleus pulposus (NP), cell density and viability were reduced by dynamic compressive load. Histologic degenerative alterations, mainly seen in the NP, were the morphologic changes of NP cells. In both NP and annulus fibrosus (AF), immunohistochemistry revealed localization of integrin α5β1 and that the messenger-RNA expression of integrin α5β1 was increased by dynamic load. Dynamic load induced a catabolic effect, the stimulation of matrix metalloproteinase-3 and -13 gene expressions by NP and AF cells. The RGD peptide partially blocked the histologic alterations and the catabolic effect. CONCLUSIONS The dynamic loading organ culture system simulated cellular responses to mechanical loading of the IVD. Our results suggest that IVD cells recognize the mechanical stress through RGD integrins, particularly the α5β1 subtype that is highly expressed in NP and AF cells. Further experiments using this system will provide information about pathomechanisms of IVD degeneration through the mechanotransduction pathways.