Progerin, a truncated unprocessed lamin A protein, causes tissue aging and degeneration. In this study we explored the role of progerin in the pathogenesis of intervertebral disc degeneration (IDD). We also examined the effect of sulforaphane (SFN) on progerin accumulation and mitochondrial dysfunction in IDD.

Intervertebral disc degeneration (IDD) has been generally accepted as the major cause of low back pain (LBP), which causes an enormous socioeconomic burden. Previous studies demonstrated that the apoptosis of nucleus pulposus (NP) cells and the dyshomeostasis of extracellular matrix (ECM) contributed to the pathogenesis of IDD, and effective therapies were still lacking. Quercetin, a natural flavonoid possessing a specific effect of autophagy stimulation and SIRT1 activation, showed some protective effect on a series of degenerative diseases. Based on previous studies, we hypothesized that quercetin might have therapeutic effects on IDD by inhibiting the apoptosis of NP cells and dyshomeostasis of ECM via the SIRT1-autophagy pathway. In this study, we revealed that quercetin treatment inhibited the apoptosis of NP cells and ECM degeneration induced by oxidative stress. We also found that quercetin promoted the expression of SIRT1 and autophagy in NP cells in a dose-dependent manner. Autophagy inhibitor 3-methyladenine (3-MA) reversed the protective effect of quercetin on apoptosis and ECM degeneration. Moreover, SIRT1 enzymatic activity inhibitor EX-527, suppressed quercetin-induced autophagy and the protective effect on NP cells, indicating that quercetin protected NP cells against apoptosis and prevented ECM degeneration via SIRT1-autophagy pathway. In vivo, quercetin was also demonstrated to alleviate the progression of IDD in rats. Taken together, our results suggest that quercetin prevents IDD by promoting SIRT1-dependent autophagy, indicating one novel and effective therapeutic method for IDD.

Introduction: Bone tissue engineering has become one of the most effective methods to treat bone defects. Silk fibroin (SF) is a natural protein with no physiological activities, which has features such as good biocompatibility and easy processing and causes minimal inflammatory reactions in the body. Scaffolds prepared by electrospinning SF can be used in bone tissue regeneration and repair. Graphene oxide (GO) is rich in functional groups, has good biocompatibility, and promotes osteogenic differentiation of stem cells, while bone morphogenetic protein-2 (BMP-2) polypeptide has an advantage in promoting osteogenesis induction. In this study, we attempted to graft BMP-2 polypeptide onto GO and then bonded the functionalized GO onto SF electrospun scaffolds through electrostatic interactions. The main purpose of this study was to further improve the biocompatibility of SF electrospun scaffolds, which could promote the osteogenic differentiation of bone marrow mesenchymal stem cells and the repair of bone tissue defects. Materials and methods: The successful synthesis of GO and functionalized GO was confirmed by transmission electron microscope, X-ray photoelectron spectroscopy, and thermogravimetric analysis. Scanning electron microscopy, atomic force microscopy, mechanical test, and degradation experiment confirmed the preparation of SF electrospun scaffolds and the immobilization of GO on the fibers. In vitro experiment was used to verify the biocompatibility of the composite scaffolds, and in vivo experiment was used to prove the repairing ability of the composite scaffolds for bone defects. Results: We successfully fabricated the composite scaffolds, which enhanced biocompatibility, not only promoting cell adhesion and proliferation but also greatly enhancing in vitro osteogenic differentiation of bone marrow stromal cells using either an osteogenic or non-osteogenic medium. Furthermore, transplantation of the composite scaffolds significantly promoted in vivo bone formation in critical-sized calvarial bone defects. Conclusion: These findings suggested that the incorporation of BMP-2 polypeptide-functionalized GO into chitosan-coated SF electrospun scaffolds was a viable strategy for fabricating excellent scaffolds that enhance the regeneration of bone defects.

Mitochondrial dysfunction plays a major role in the development of intervertebral disc degeneration (IDD). Sirtuin 5 (SIRT5) participates in the maintenance of mitochondrial homeostasis through its desuccinylase activity. However, it is still unclear whether succinylation or SIRT5 is involved in the impairment of mitochondria and development of IDD induced by excessive mechanical stress. Our 4D label-free quantitative proteomic results showed decreased expression of the desuccinylase SIRT5 in rat nucleus pulposus (NP) tissues under mechanical loading. Overexpression of Sirt5 effectively alleviated, whereas knockdown of Sirt5 aggravated, the apoptosis and dysfunction of NP cells under mechanical stress, consistent with the more severe IDD phenotype of Sirt5 KO mice than wild-type mice that underwent lumbar spine instability (LSI) surgery. Moreover, immunoprecipitation-coupled mass spectrometry (IP-MS) results suggested that AIFM1 was a downstream target of SIRT5, which was verified by a Co-IP assay. We further demonstrated that reduced SIRT5 expression resulted in the increased succinylation of AIFM1, which in turn abolished the interaction between AIFM1 and CHCHD4 and thus led to the reduced electron transfer chain (ETC) complex subunits in NP cells. Reduced ETC complex subunits resulted in mitochondrial dysfunction and the subsequent occurrence of IDD under mechanical stress. Finally, we validated the efficacy of treatments targeting disrupted mitochondrial protein importation by upregulating SIRT5 expression or methylene blue (MB) administration in the compression-induced rat IDD model. In conclusion, our study provides new insights into the occurrence and development of IDD and offers promising therapeutic approaches for IDD.

Intervertebral disc (IVD) degeneration (IVDD) that greatly affected by regional biomechanical environment is a major cause of low back pain. Injectable hydrogels have been commonly studied for treatment of IVDD due to their capability of mimicking extracellular matrix structure to support cellular behavior and clinical prospects in minimally invasive treatment. However, most hydrogels suffer from complicated chemistry, potential uncertainty and toxicity from in-situ gelation, and mismatch with IVD mechanical environment that limit their therapeutic effects or clinical translation in IVDD or intervertebral disc defect repair. For IVD lesion repair, the study aims to develop a novel hydrogel with shear-thinning enabled injectability, high bio-safety, and mechanical properties adaptable to the IVD environment, using a simple chemistry and method. And therapeutic efficacy of the novel hydrogel in the treatment of IVDD or intervertebral disc defect will be revealed. A glycerol cross-linked PVA gel (GPG) was synthesized based on multiple H-bonds formation between glycerol molecules and PVA chains. The rheological and mechanical properties were tested. The swelling ratio was measured. The micro-architecture was observed through scanning and transmission electron microscopes. Nucleus pulposus (NP) cells were cultured in GPG-coated plates or silicone chambers treated under hydrostatic or dynamic loading in vitro, and examined for proliferation, vitality, apoptosis, expression of catabolic and anabolic markers. GPG was injected in needle puncture (IDD) or NP discectomy (NPD) models in vivo, and examined through magnetic resonance imaging, micro-computed tomography scanning and histological staining. GPG had a highly porous structure consisting of interconnected pores. Meanwhile, the GPG had NP-like viscoelastic property, and was able to withstand the cyclic deformation while exhibiting a prominent energy-dissipating capability. In vitro cell tests demonstrated that, the hydrogel significantly down-regulated the expression of catabolic markers, maintained the level of anabolic markers, preserved cell proliferation and vitality, reduced apoptotic rate of NP cells under pathologically hydrostatic and dynamic loading environments compared to cells cultured on untreated plate or silicone chamber. In vivo animal studies revealed that injection of GPG efficiently maintained NP structural integrity, IVD height and relative water content in IDD models, and stimulated the fibrous repair in NPD models. This study showed that GPG, with high injectability, NP-like viscoelastic characteristics, good energy-dissipating properties and swelling capacities, preserved NP cells vitality against pathological loading, and had therapeutic effects on IVD repair in IDD and NPD models. Effective clinical strategy for treatment of intervertebral disc degeneration (IVDD) is still lacking. This study demonstrates that injection of a hydrogel with nucleus pulposus-matched viscoelastic property could remarkably prevent the IVDD progress. Prepared with simple chemistry and procedure, the cell/drug-free GPG with high bio-safety and shear-thinning enabled injectability bears great translational potential for the clinical treatment of IVDD via a minimally invasive approach.