TBK1-medicated DRP1 phosphorylation orchestrates mitochondrial dynamics and autophagy activation in osteoarthritis.

Mitochondrial dynamics, including mitochondrial fission and fusion, are critical for maintaining mitochondrial functions. Evidence shows that TANK-binding kinase 1 (TBK1) regulates mitochondrial fusion and fission and then mitophagy. Since a previous study demonstrates a strong correlation between mitophagy and osteoarthritis (OA), we herein investigated the potential role of TBK1 in OA process and mitochondrial functions. We demonstrated a strong correlation between TBK1 and OA, evidenced by significantly downregulated expression of TBK1 in cartilage tissue samples of OA patients and in the chondrocytes of aged mice, as well as TNF-α-stimulated phosphorylation of TBK1 in primary mouse chondrocytes. TBK1 overexpression significantly attenuated TNF-α-induced apoptosis and abnormal mitochondrial function in primary mouse chondrocytes. Furthermore, TBK1 overexpression induced remodeling of mitochondrial morphology by directly phosphorylating dynamin-related protein 1 (DRP1) at Ser637, abolishing the fission of DRP1 and preventing its fragmentation function. Moreover, TBK1 recruitment and DRP1 phosphorylation at Ser637 was necessary for engulfing damaged mitochondria by autophagosomal membranes during mitophagy. Moreover, we demonstrated that APMK/ULK1 signaling contributed to TBK1 activation. In OA mouse models established by surgical destabilization of the medial meniscus, intraarticular injection of lentivirus-TBK1 significantly ameliorated cartilage degradation via regulation of autophagy and alleviation of cell apoptosis. In conclusion, our results suggest that the TBK1/DRP1 pathway is involved in OA and pharmacological targeting of the TBK1-DRP1 cascade provides prospective therapeutic benefits for the treatment of OA.

Dual regulation of microglia and neurons by Astragaloside IV-mediated mTORC1 suppression promotes functional recovery after acute spinal cord injury.

Inflammation and neuronal apoptosis contribute to the progression of secondary injury after spinal cord injury (SCI) and are targets for SCI therapy; autophagy is reported to suppress apoptosis in neuronal cells and M2 polarization may attenuate inflammatory response in microglia, while both are negatively regulated by mTORC1 signalling. We hypothesize that mTORC1 suppression may have dual effects on inflammation and neuronal apoptosis and may be a feasible approach for SCI therapy. In this study, we evaluate a novel inhibitor of mTORC1 signalling, Astragaloside IV (AS-IV), in vitro and in vivo. Our results showed that AS-IV may suppress mTORC1 signalling both in neuronal cells and microglial cells in vitro and in vivo. AS-IV treatment may stimulate autophagy in neuronal cells and protect them against apoptosis through autophagy regulation; it may also promote M2 polarization in microglial cells and attenuate neuroinflammation. In vivo, rats were intraperitoneally injected with AS-IV (10 mg/kg/d) after SCI, behavioural and histological evaluations showed that AS-IV may promote functional recovery in rats after SCI. We propose that mTORC1 suppression may attenuate both microglial inflammatory response and neuronal apoptosis and promote functional recovery after SCI, while AS-IV may become a novel therapeutic medicine for SCI.

Prognostic Determinants Analysis and Nomogram for Bone Malignant Vascular Tumors: A Surveillance, Epidemiology and End Results (SEER) Analysis.

BACKGROUND The aim and objective of our investigations were to explore the prognostic value of various clinical and pathological factors of bone malignant vascular tumors and establish a nomogram for their outcome predictions. MATERIAL AND METHODS All data of primary bone malignant vascular tumors (MVTs) patients were randomly selected from the Surveillance, Epidemiology and End Results (SEER) database. However, selected patients were clinically diagnosed with various cancers during 1988-2015. The potential prognostic factors were analyzed using SPSS (Windows, version 22.0). All prognostic factors were combined to formulate a nomogram to predict the overall survival (OS). RESULTS A total of 266 selected patients were included in our study. In the univariate model, age (P.

BRD4 inhibition attenuates inflammatory response in microglia and facilitates recovery after spinal cord injury in rats.

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.

Genistein protects intervertebral discs from degeneration via Nrf2-mediated antioxidant defense system: An in vitro and in vivo study.

Oxidative stress has been reported to be closely associated with the development of intervertebral disc degeneration (IDD). IDD is one of the major causes of low back pain. Genistein (GES), one of the main isoflavones of soybean, has been shown to exert multiple biological functions on different diseases. Here, we tested the therapeutic potential of GES for IDD. In vitro experiments, we confirmed GES was nontoxic to rat nucleus pulposus cells (NPCs) within the concentration of 100 µM. Furthermore, GES was able to suppress apoptosis in tert-butyl hydroperoxide (TBHP)-treated NPCs. In the aspect of extracellular matrix (ECM), GES not only reduced metalloproteinase-13 (MMP-13) and a disintegrin-like and MMP thrombospondin type 1 motif 5 expression, but also increased aggrecan and type II collagen levels. Also, we found GES might rescue TBHP-induced NPCs degeneration by enhancing Nrf2-mediated antioxidant defense system. Silencing Nrf2 partly abolished the protective effects of GES on apoptosis and ECM disruption in TBHP-treated NPCs. Correspondingly, GES ameliorated IDD in a rat model by preserving morphology of degenerative intervertebral discs and promoting Nrf2 expression. To sum up, our study suggests that GES exerts protective effects in NPCs against degeneration and reveals the underlying mechanism of GES on Nrf2 activation in NPCs.

Itaconate attenuates osteoarthritis by inhibiting STING/NF-κB axis in chondrocytes and promoting M2 polarization in macrophages.

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-1ß. 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.

The therapeutic effect of TBK1 in intervertebral disc degeneration via coordinating selective autophagy and autophagic functions.

Introduction: While its innate immune function has been known, recent works of literature have focused on the role of Tank binding kinase 1 (TBK1) in regulating autophagy and it is unknown whether TBK1 protects against intervertebral disc degeneration (IVDD) through affecting autophagy. Objectives: Here, we aim to explore whether TBK1 is implicated in the pathogenesis of IVDD, and investigated the potential mechanism. Methods: Western blotting and immunohistochemistry were used to detect the TBK1 expression in human and rat NP tissue. After TBK1 overexpression in NP cells with lentivirus transfection, autophagic flux, apoptosis and senescence percentage were assessed. Si-RNA , a utophagy inhibitors and protein phosphatase inhibitors were applied to study the mechanism of autophagy regulation. In vivo study, we further evaluated the therapeutic action of lentivirus-TBK1(Lv-TBK1)injection in a rodent IVDD model. Results: The TBK1 level was reduced in rat and human NP tissue. TBK1 overexpression protected against apoptosis and premature senescence. These functions of TBK1 were abolished by chloroquine-medicated autophagy inhibition.P-TBK1, an activation form of TBK, is involved in selective autophagy through directly phosphorylating P62 at Ser 403, and the activation of TBK1 is also dependent on Parkin manner. TBK1 also activated NPCs autophagy to relieve puncture injury in vivo. Conclusion: We demonstrated that TBK1 overexpression attenuated senescence and apoptosis and promoted NPCs survival via upregulating autophagy. TBK1 represents a promising avenue for IVDD treatment.

Promoting Nrf2/Sirt3-Dependent Mitophagy Suppresses Apoptosis in Nucleus Pulposus Cells and Protects against Intervertebral Disc Degeneration.

One of the causes of intervertebral disc degeneration (IVDD) is nucleus pulposus cell (NPC) death, possibly apoptosis. In this study, we explored the role of the Nrf2/Sirt3 pathway and tert-butylhydroquinone (t-BHQ) in IVDD and elucidated the potential working mechanism. Reactive oxygen species (ROS) assay kits and malondialdehyde (MDA) assay kits were used to assess oxidative stress. Western blot and TUNEL staining were used to examine apoptosis. After siRNA against Nrf2 or lentivirus against Sirt3 was transfected into NPCs, the mechanism of the effect of the Nrf2/Sirt3 pathway on NPCs was assessed. The interaction between t-BHQ and its potential interacting protein NRF2 was further investigated through protein docking analysis. ChIP examined the binding affinity between Nrf2 and Sirt3 promoter. In vivo experiments, X-ray, hematoxylin-eosin (HE) staining, Safranin O staining, and immunohistochemistry were used to evaluate IVDD grades. The results demonstrated that activation of the Nrf2/Sirt3 pathway inhibited tert-butyl hydroperoxide- (TBHP-) induced apoptosis and mitochondrial dysfunction in vitro. In addition to apoptosis, upregulation of the Nrf2/Sirt3 pathway induced by t-BHQ restored TBHP-induced autophagic flux disturbances. However, its protective effect was reversed by chloroquine and Si-ATG5. Furthermore, t-BHQ ameliorated IVDD development in a rat model. In conclusion, our findings indicate that the Nrf2/Sirt3 pathway and its agonist represent a potential candidate for treating IVDD.

RNA-binding protein HuR suppresses senescence through Atg7 mediated autophagy activation in diabetic intervertebral disc degeneration.

OBJECTIVES: Diabetes is a risk factor for intervertebral disc degeneration (IVDD). Studies have demonstrated that diabetes may affect IVDD through transcriptional regulation; however, whether post-transcriptional regulation is involved in diabetic IVDD (DB-IVDD) is still unknown. This study was performed to illustrate the role of HuR, an RNA-binding protein, in DB-IVDD development and its mechanism. MATERIALS AND METHODS: The expression of HuR was evaluated in nucleus pulposus (NP) tissues from diabetic IVDD patients and in high glucose-treated NP cells. Senescence and autophagy were assessed in HuR over-expressing and downregulation NP cells. The mRNAs that were regulated by HuR were screened, and immunoprecipitation was applied to confirm the regulation of HuR on targeted mRNAs. RESULTS: The results showed that the expression of HuR was decreased in diabetic NP tissues and high glucose-treated NP cells. Downregulation of HuR may lead to increased senescence in high glucose-treated NP cells, while autophagy activation attenuates senescence in HuR deficient NP cells. Mechanistic study showed that HuR prompted Atg7 mRNA stability via binding to the AU-rich elements. Furthermore, overexpression of Atg7, but not HuR, may ameliorate DB-IVDD in rats in vivo. CONCLUSIONS: In conclusion, HuR may suppress senescence through autophagy activation via stabilizing Atg7 in diabetic NP cells; while Atg7, but not HuR, may serve as a potential therapeutic target for DB-IVDD.

Hydroxysafflor yellow A promotes multiterritory perforating flap survival: an experimental study.

The use of perforator flaps is a common surgical technique in wound repair. However, the area surrounding the multiterritory perforating flap often becomes necrotic due to ischemia. Hydroxysafflor yellow A (HSYA), a traditional Chinese medicine extracted from edible safflower, can be used medicinally to promote angiogenesis, inhibit apoptosis, and alleviate oxidative stress and other biological activities. Here, we investigated the effect of HSYA on perforator flap survival and its potential mechanism. Our results demonstrate that HSYA significantly improves the survival area of perforator flaps, increases blood supply, reduces tissue edema, and increases mean vascular density. HSYA treatment promotes angiogenesis and inhibits oxidative stress, apoptosis, and autophagy in perforator flaps, suggesting many potential mechanisms for flap survival.

Chemerin facilitates intervertebral disc degeneration via TLR4 and CMKLR1 and activation of NF-kB signaling pathway.

Now days, obesity is a major risk factor for intervertebral disc degeneration (IDD). However, adipokine, such as chemerin is a novel cytokine, which is secreted by adipose tissue, and are thought to be played major roles in various degenerative diseases. Obese individuals are known to have high concentration of serum chemerin. Our purpose was to study whether chemerin acts as a biochemical relationship between obesity, and IDD. In this study, we found that the expression level of chemerin was significantly increased in the human degenerated nucleus pulposus (NP) tissues, and had higher level in the obese people than the normal people. Chemerin significantly increased the inflammatory mediator level, contributing to ECM degradation in nucleus pulposus cells (NPCs). Furthermore, chemerin overexpression aggravates the puncture-induced IVDD progression in rats, while knockdown CMKLR1 reverses IVDD progression. Chemerin activates the NF-kB signaling pathway via its receptors CMKLR1, and TLR4 to release inflammatory mediators, which cause matrix degradation, and cell aging. These findings generally provide novel evidence supporting the causative role of obesity in IDD, which is essentially important to literally develop novel preventative or generally therapeutic treatment in the disc degenerative disorders.

Proanthocyanidins-Mediated Nrf2 Activation Ameliorates Glucocorticoid-Induced Oxidative Stress and Mitochondrial Dysfunction in Osteoblasts.

The widespread use of therapeutic glucocorticoids has increased the frequency of glucocorticoid-induced osteoporosis (GIOP). One of the potential pathological processes of GIOP is an increased level of oxidative stress and mitochondrial dysfunction, which eventually leads to osteoblast apoptosis. Proanthocyanidins (PAC) are plant-derived antioxidants that have therapeutic potential against GIOP. In our study, a low dose of PAC was nontoxic to healthy osteoblasts and restored osteogenic function in dexamethasone- (Dex-) treated osteoblasts by suppressing oxidative stress, mitochondrial dysfunction, and apoptosis. Mechanistically, PAC neutralized Dex-induced damage in the osteoblasts by activating the Nrf2 pathway, since silencing Nrf2 partly eliminated the protective effects of PAC. Furthermore, PAC injection restored bone mass and promoted the expression of Nrf2 in the distal femur of Dex-treated osteoporotic rats. In summary, PAC protect osteoblasts against Dex-induced oxidative stress and mitochondrial dysfunction via the Nrf2 pathway activation and may be a promising drug for treating GIOP.

Downregulating Akt/NF-κB signaling and its antioxidant activity with Loureirin A for alleviating the progression of osteoarthritis: In vitro and vivo studies.

Osteoarthritis(OA) is one of the most common diseases in orthopedics. It is characterized by degeneration of articular cartilage and chronic inflammation. In this study, we aim to elucidate the mechanism of Loureirin A's therapeutic effect in OA progression. In vitro, Loureirin A pretreatment can significantly inhibit production of NO, PGE2, COX-2, TNF-α, iNOS andIL-6 induced by IL-1ß in mouse articular chondrocytes. Moreover, Loureirin A suppressed the expression of matrix metalloproteinase-9(MMP-9), which leads to degradation of the extracellular matrix. The degradation of aggrecan and type II collagen protein in the extracellular matrix (ECM) stimulated by IL-1ß was reversed. For signal pathway research, Loureirin A dramatically inhibited the phosphorylation of AKT and subsequent NF-κB entering into the nucleus caused by IL-1ß in chondrocytes. Besides, a number of related indicators suggested that Loureirin A has a strong antioxidant activity in the treatment of osteoarthritis via increasing content of SOD2 and suppressing MDA and ROS. In addition, in vivo study demonstrated that Loureirin A could ameliorated the progression of OA in mice DMM model In conclusion, all results showed that Loureirin A may be a potential therapeutic candidate for the OA.

Stabilization of HIF-1α alleviates osteoarthritis via enhancing mitophagy.

Mitochondrial dysfunction leads to osteoarthritis (OA) and disc degeneration. Hypoxia inducible factor-1α (HIF-1α) mediated mitophagy has a protective role in several diseases. However, the underlying mechanism of HIF-1α mediated mitophagy in OA remains largely unknown. This current study was performed to determine the effect of HIF-1α mediated mitophagy on OA. Therefore, X-ray and tissue staining including HE staining, safranin O-fast green (S-O) and Alcian Blue were used to assess imageology and histomorphology differences of mouse knee joint. Transcriptional analysis was used to find the possible targets in osteoarthritis. Western blot analysis, RT-qPCR and immunofluorescence staining were used to detect the changes in gene and protein levels in the vitro experiment. The expression of HIF-1α was increased in human and mouse OA cartilage. HIF-1α knockdown by siRNA further impair the hypoxia-induced mitochondrial dysfunction; In contrast, HIF-1α mediated protective role was reinforced by prolylhydroxylase (PHD) inhibitor dimethyloxalylglycine (DMOG). In addition, HIF-1α stabilization could alleviate apoptosis and senescence via mitophagy in chondrocytes under hypoxia condition, which could also ameliorate surgery-induced cartilage degradation in mice OA model. In conclusion, HIF-1α mediated mitophagy could alleviate OA, which may serve as a promising strategy for OA treatment.

The homogeneous and heterogeneous risk factors for occurrence and prognosis in lung cancer patients with bone metastasis.

PURPOSE: To analyse the homogeneous and heterogeneous risk factors for occurrence and prognosis in lung cancer patients diagnosed with bone metastasis (BM) by using the Surveillance, Epidemiology, and End Results (SEER) database. PATIENTS AND METHODS: The medical records of lung cancer patients with or without bone metastasis were identified in the SEER database between 2010 and 2015. A multivariate logistic regression analysis was performed to identify risk factors, and a multivariate Cox regression was used to determine the prognostic effects of every variable on survival. RESULTS: In total, 34,585 eligible patients from the SEER database were included in the analysis. Male gender and metastasis to the liver were factors that were both positively associated with a risk for the development and prognosis of bone metastasis in patients with lung cancer. Younger age, poor tumour differentiation grade, higher N stage (N3), adenocarcinoma and metastasis to the brain were all positively correlated with a risk of occurrence of BM, but these factors were not correlated with an unfavourable prognosis. Age, race, marital status, tumour size and pathologic type were independent risk factors for the prognosis of bone metastasis. CONCLUSION: The morbidity of bone metastasis in lung cancer patients is dismal, with a rate of 25.9%. The findings of this study estimate the homogeneous and heterogeneous risk factors for the occurrence and prognosis of bone metastasis in lung cancer patients, which may provide clinical guidelines for physicians.