ABSTRACT
Intervertebral disc degeneration (IVDD) is a complex process involving many factors, among which excessive senescence of nucleus pulposus cells is considered to be the main factor. Our ...previous study found that metformin can inhibit senescence in nucleus pulposus cells; however, the mechanism of such an action was still largely unknown. In the current study, we found that metformin inactivates the cGAS-STING pathway during oxidative stress. Furthermore, knockdown of STING (also known as STING1) suppresses senescence, indicating that metformin might exert its effect through the cGAS-STING pathway. Damaged DNA is a major inducer of the activation of the cGAS-STING pathway. Mechanistically, our study showed that DNA damage was reduced during metformin treatment; however, suppression of autophagy by 3-methyladenine (3-MA) treatment compromised the effect of metformin on DNA damage. In vivo studies also showed that 3-MA might diminish the therapeutic effect of metformin on IVDD. Taken together, our results reveal that metformin may suppress senescence via inactivating the cGAS-STING pathway through autophagy, implying a new application for metformin in cGAS-STING pathway-related diseases.
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Osteoarthritis (OA) is a progressive joint disease characterized by the degradation and destruction of articular cartilage, which is involved with pathological microenvironmental ...alterations induced by damaged chondrocytes and inflammatory macrophages. However, the current therapies cannot effectively alleviate the progression of OA. Our previous studies have shown that the pathological process of OA progression is accompanied by DNA damage, and inhibition of STING, a key molecule in DNA damage, may become a potential method for the treatment of OA. Itaconate, a metabolite highly expressed in macrophages under inflammatory conditions, has shown a wide range of anti-inflammatory effects, but its effect on OA and its underlying mechanism has not yet been studied. In this study, we found that exogenous supplementation of itaconate can activate Nrf2, and accordingly inhibit the STING-dependent NF-κB pathway, thereby alleviating the inflammation, ECM degeneration and senescence of chondrocytes stimulated by IL-1β. In addition, itaconate can regulate the polarization of RAW264.7 macrophages, further reducing the apoptosis of chondrocytes. In vivo, intra-articular injection of itaconate reduces the degradation of cartilage and inflammation of synovial membrane in the mouse OA model. In conclusion, the present work suggests that exogenous supplementation of itaconate inhibits the inflammation, senescence and ECM degeneration of chondrocytes through the Nrf2/STING/NF-κB axis and regulates the polarization of synovial macrophages, thereby ameliorating the progression of OA, which supports that itaconate as a potential drug for the treatment of OA.
Intervertebral disc degeneration (IDD) is a complicated process that involves both cellular apoptosis and senescence. Metformin has been reported to stimulate autophagy, whereas autophagy is shown to ...protect against apoptosis and senescence. Therefore, we hypothesize that metformin may have therapeutic effect on IDD through autophagy stimulation. The effect of metformin on IDD was investigated both in vitro and in vivo. Our study showed that metformin attenuated cellular apoptosis and senescence induced by tert-butyl hydroperoxide in nucleus pulposus cells. Autophagy, as well as its upstream regulator AMPK, was activated by metformin in nucleus pulposus cells in a dose- and time-dependent manner. Inhibition of autophagy by 3-MA partially abolished the protective effect of metformin against nucleus pulposus cells' apoptosis and senescence, indicating that autophagy was involved in the protective effect of metformin on IDD. In addition, metformin was shown to promote the expression of anabolic genes such as Col2a1 and Acan expression while inhibiting the expression of catabolic genes such as Mmp3 and Adamts5 in nucleus pulposus cells. In vivo study illustrated that metformin treatment could ameliorate IDD in a puncture-induced rat model. Thus, our study showed that metformin could protect nucleus pulposus cells against apoptosis and senescence via autophagy stimulation and ameliorate disc degeneration in vivo, revealing its potential to be a therapeutic agent for IDD.
ABSTRACT
Diabetes mellitus may lead to intervertebral disc degeneration (IVDD). Matrix metalloproteinase‐13 (MMP‐13) is one of the major catabolic factors in extracellular matrix (ECM) metabolism of ...nucleus pulposus cells (NPCs) and contributes to diabetic IVDD. Bromodomain‐containing protein 4 (BRD4) is a member of the bromodomain and extraterminal protein family and is implicated in chronic inflammation. Here, we report that the expression of BRD4 and MMP‐13 was elevated in diabetic nucleus pulposus tissues as well as in advanced glycation end products (AGEs)‐treated NPCs; also, the regulatory effect of BRD4 on MMP‐13 was studied. We found that MMP‐13 was regulated by MAPK and NF‐κB signaling as well as autophagy in AGEs‐treated NPCs. Next, we explored the role of BRD4 in regulation of MAPK, NF‐κB signaling, and autophagy. The results showed that BRD4 is the upstream regulator of all of these 3 factors, and inhibition of BRD4 may suppress MAPK and NF‐κB signaling while activating autophagy in AGEs‐treated NPCs. Finally, we demonstrated that BRD4 inhibition may suppress MMP‐13 expression in diabetic NPCs in vitro as well as in vivo; meanwhile, it may preserve ECM in diabetic rats. Our study demonstrates that inhibition of BRD4 may suppress MAPK and NF‐κB signaling and activate autophagy to suppress MMP‐13 expression in diabetic IVDD, and diabetic IVDD may be compromised by BRD4 inhibitors.—Wang, J., Hu, J., Chen, X., Huang, C., Lin, J., Shao, Z., Gu, M., Wu, Y., Tian, N., Gao, W., Zhou, Y., Wang, X., Zhang, X. BRD4 inhibition regulates MAPK, NF‐κB signals, and autophagy to suppress MMP‐13 expression in diabetic intervertebral disc degeneration. FASEB J. 33, 11555–11566 (2019). www.fasebj.org
Excessive apoptosis and senescence of nucleus pulposus (NP) cells are major pathological changes in intervertebral disc degeneration (IVDD) development; previous studies demonstrated ...pharmacologically or genetically stimulation of autophagy may inhibit apoptosis and senescence in NP cells. Transcription factor EB (TFEB) is a master regulator of autophagic flux via initiating autophagy-related genes and lysosomal biogenesis. This study was performed to confirm whether TFEB was involved in IVDD development and its mechanism.
TFEB activity was detected in NP tissues in puncture-induced rat IVDD model by immunofluorescence as well as in tert-Butyl hydroperoxide (TBHP), the reactive oxygen species (ROS) donor to induce oxidative stress, treated NP cells by western blot. After TFEB overexpression in NP cells with lentivirus transfection, autophagic flux, apoptosis and senescence percentage were assessed. In in vivo study, the lentivirus-normal control (LV-NC) or lentivirus-TFEB (LV-TFEB) were injected into the center space of the NP tissue, after 4 or 8 weeks, Magnetic resonance imaging (MRI), X ray, Hematoxylin-Eosin (HE) and Safranin O staining were used to evaluate IVDD grades.
The nuclear localization of TFEB declined in degenerated rat NP tissue as well as in TBHP treated NP cells. Applying lentivirus to transfect NP cells, TFEB overexpression restored the TBHP-induced autophagic flux blockage and protected NP cells against apoptosis and senescence; these protections of TFEB are diminished by chloroquine-medicated autophagy inhibition. Furthermore, TFEB overexpression ameliorates the puncture-induced IVDD development in rats.
Experimental IVDD inhibited the TFEB activity. TFEB overexpression suppressed TBHP-induced apoptosis and senescence via autophagic flux stimulation in NP cell and alleviates puncture-induced IVDD development in vivo.
Low back pain, primarily caused by intervertebral disc degeneration (IVDD), lacks effective pharmacological treatments. Oxidative stress has been identified as a significant contributor to IVDD. This ...study aims to establish an in vitro model of IVDD induced by oxidative stress and identify potential therapeutic agents and their underlying mechanisms. By screening the natural product library, fisetin emerged as the most promising compound in suppressing cell death induced by oxidative stress in nucleus pulposus cells (NPCs). Furthermore, our investigation revealed that the cell death induced by oxidative stress was predominantly associated with ferroptosis, and fisetin demonstrated the ability to inhibit ferroptosis in NPCs. Mechanistic exploration suggested that the impact of fisetin on ferroptosis may be mediated through the Nrf2/HO-1 (Nuclear factor erythroid 2-related factor 2/heme oxygenase-1) axis. Notably, the in vivo study demonstrated that fisetin could alleviate IVDD in rats. These findings highlight fisetin as a potential therapeutic option for IVDD and implicate the involvement of the Nrf2/HO-1 pathway in its mechanism of action.
Intervertebral disc degeneration (IVDD) is a prevalent degenerative disease with significant adverse implications for patients' quality of life and socioeconomic status. Although the precise etiology ...of IVDD remains elusive, the senescence of nucleus pulposus cells is recognized as the primary pathogenic factor of IVDD; however, drugs that may targetedly inhibit senescence are still lacking. In the current study, we evaluated the small-molecule active drug 20-Deoxyingenol(20-DOI) for its effects on combating senescence and delaying the progression of IVDD. In vitro experiments revealed that the administration of 20-DOI displayed inhibitory effects on senescence and the senescence-related cGAS-STING pathway of nucleus pulposus cells. Additionally, it exhibited the ability to enhance lysosome activity and promote autophagy flux within nucleus pulposus cells. Subsequent investigations elucidated that the inhibitory impact of 20-DOI on nucleus pulposus cell senescence was mediated through the autophagy-lysosome pathway. This effect was diminished in the presence of transcription factor EB (TFEB) small hairpin RNA (shRNA), thereby confirming the regulatory role of 20-DOI on the autophagy-lysosome pathway and senescence through TFEB. In vivo experiments demonstrated that 20-DOI effectively impeded the progression ofIVDD in rats. These findings collectively illustrate that 20-DOI may facilitate the autophagy-lysosomal pathway by activating TFEB, thereby suppressing the senescence in nucleus pulposus cells, thus suggesting 20-DOI as a promising therapeutic approach for IVDD.
Blood–spinal cord barrier (BSCB) disruption following spinal cord injury (SCI) significantly compromises functional neuronal recovery. Autophagy is a potential therapeutic target when seeking to ...protect the BSCB. We explored the effects of lithium chloride (LiCl) on BSCB permeability and autophagy-induced SCI both in a rat model of SCI and in endothelial cells subjected to oxygen–glucose deprivation. We evaluated BSCB status using the Evans Blue dye extravasation test and measurement of tight junction (TJ) protein levels; we also assessed functional locomotor recovery. We detected autophagy-associated proteins in vivo and in vitro using both Western blotting and immunofluorescence staining. We found that, in a rat model of SCI, LiCl attenuated the elevation in BSCB permeability, improved locomotor recovery, and inhibited the degradation of TJ proteins including occludin and claudin-5. LiCl significantly induced the extent of autophagic flux after SCI by increasing LC3-II and ATG-5 levels, and abolishing p62 accumulation. In addition, a combination of LiCl and the autophagy inhibitor chloroquine not only partially eliminated the BSCB-protective effect of LiCl, but also exacerbated TJ protein degradation both in vivo and in vitro. Together, these findings suggest that LiCl treatment alleviates BSCB disruption and promotes locomotor recovery after SCI, partly by stimulating autophagic flux.
•Lithium chloride.•Blood–spinal cord barrier.•Spinal cord injury.•Autophagic flux.
The pathophysiology of spinal cord injury (SCI) involves primary injury and secondary injury. For the irreversibility of primary injury, therapies of SCI mainly focus on secondary injury, whereas ...inflammation is considered to be a major target for secondary injury; however the regulation of inflammation in SCI is unclear and targeted therapies are still lacking. In this study, we found that the expression of BRD4 was correlated with pro‐inflammatory cytokines after SCI in rats; in vitro study in microglia showed that BRD4 inhibition either by lentivirus or JQ1 may both suppress the MAPK and NF‐κB signalling pathways, which are the two major signalling pathways involved in inflammatory response in microglia. BRD4 inhibition by JQ1 not only blocked microglial M1 polarization, but also repressed the level of pro‐inflammatory cytokines in microglia in vitro and in vivo. Furthermore, BRD4 inhibition by JQ1 can improve functional recovery and structural disorder as well as reduce neuron loss in SCI rats. Overall, this study illustrates that microglial BRD4 level is increased after SCI and BRD4 inhibition is able to suppress M1 polarization and pro‐inflammatory cytokine production in microglia which ultimately promotes functional recovery after SCI.
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