Erythropoietin alleviates astrocyte pyroptosis by targeting the miR-325-3p/Gsdmd axis in rat spinal cord injury.

BACKGROUND: Neuroinflammation plays an important role in spinal cord injury (SCI), and an increasing number of studies have focused on the role of astrocytes in neuroinflammation. Pyroptosis is an inflammation-related form of programmed cell death, and neuroinflammation induced by astrocytes in the form of pyroptosis has been widely reported in many central nervous system diseases. Recent studies have found that erythropoietin has significant anti-inflammatory and neuroprotective effects in SCI; however, it has not been reported whether erythropoietin can reduce neuroinflammation by inhibiting neural cell pyroptosis in SCI. METHODS: A GEO dataset (GSE153720) was used to analyse the expression of pyroptosis-related genes in sham astrocytes and astrocytes 7 days, 1 month and 3 months after SCI. TargetScan and miRDB databases were used to predict the miRNA that could bind to the 3'UTR of rat Gsdmd. Primary rat spinal astrocytes were used for in vitro experiments, and the modified version of Allen's method was used to establish the rat SCI model. Western blotting, quantitative real-time polymerase chain reaction, flow cytometry, immunofluorescence, lactate dehydrogenase release assay and propidium iodide staining were used to detect the pyroptosis phenotype. A dual luciferase reporter gene assay was used to verify that miR-325-3p can bind to the 3'UTR of Gsdmd. RESULTS: We found that pyroptosis-related genes mediated by the canonical NLRP3 inflammasome were highly expressed in astrocytes in an SCI animal model by bioinformatic analysis. We also observed that erythropoietin could reduce astrocyte pyroptosis in vivo and in vitro. In addition, we predicted miRNAs that regulate Gsdmd, the pyroptosis executor, and verified that erythropoietin inhibits astrocyte pyroptosis in SCI through the miR-325-3p/Gsdmd axis. CONCLUSIONS: We demonstrated that erythropoietin can inhibit astrocyte pyroptosis through the miR-325-3p/Gsdmd axis. This study is expected to provide a new mechanism for erythropoietin in the treatment of SCI and a more reliable theoretical basis for clinical research.

Identification of canonical pyroptosis-related genes, associated regulation axis, and related traditional Chinese medicine in spinal cord injury.

Neuroinflammation plays an important role in spinal cord injury (SCI), and pyroptosis is inflammatory-related programmed cell death. Although neuroinflammation induced by pyroptosis has been reported in SCI, there is a lack of systematic research on SCI pyroptosis and its regulation mechanism. The purpose of this study was to systematically analyze the expression of pyroptosis-related genes (PRGs) in different SCI models and associated regulation axis by bioinformatics methods. We downloaded raw counts data of seven high-throughput sequencings and two microarray datasets from the GEO database, classified by species (rat and mouse) and SCI modes (moderate contusive model, aneurysm clip impact-compression model, and hemisection model), including mRNAs, miRNAs, lncRNAs, and circRNAs, basically covering the acute, subacute and chronic stages of SCI. We performed differential analysis by R (DEseq2) or GEO2R and found that the AIM2/NLRC4/NLRP3 inflammasome-related genes, GSDMD, IL1B, and IL18, were highly expressed in SCI. Based on the canonical NLRP3 inflammasome-mediated pyroptosis-related genes (NLRP3/PRGs), we constructed transcription factors (TFs)-NLRP3/PRGs, miRNAs- Nlrp3/PRGs and lncRNAs/circRNAs/mRNAs-miRNA- Nlrp3/PRGs (ceRNA) networks. In addition, we also predicted Traditional Chinese medicine (TCM) and small, drug-like molecules with NLRP3/PRGs as potential targets. Finally, 39 up-regulated TFs were identified, which may regulate at least two of NLRP3/PRGs. A total of 7 down-regulated miRNAs were identified which could regulate Nlrp3/PRGs. ceRNA networks were constructed including 23 lncRNAs, 3 cicrRNAs, 6 mRNAs, and 44 miRNAs. A total of 24 herbs were identified which may with two NLRP3/PRGs as potential targets. It is expected to provide new ideas and therapeutic targets for the treatment of SCI.

Erythropoietin inhibits ferroptosis and ameliorates neurological function after spinal cord injury.

Ferroptosis is one of the critical pathological events in spinal cord injury. Erythropoietin has been reported to improve the recovery of spinal cord injury. However, whether ferroptosis is involved in the neuroprotective effects of erythropoietin on spinal cord injury has not been examined. In this study, we established rat models of spinal cord injury by modified Allen's method and intraperitoneally administered 1000 and 5000 IU/kg erythropoietin once a week for 2 successive weeks. Both low and high doses of erythropoietin promoted recovery of hindlimb function, and the high dose of erythropoietin led to better outcome. High dose of erythropoietin exhibited a stronger suppressive effect on ferroptosis relative to the low dose of erythropoietin. The effects of erythropoietin on inhibiting ferroptosis-related protein expression and restoring mitochondrial morphology were similar to those of Fer-1 (a ferroptosis suppressor), and the effects of erythropoietin were largely diminished by RSL3 (ferroptosis activator). In vitro experiments showed that erythropoietin inhibited RSL3-induced ferroptosis in PC12 cells and increased the expression of xCT and Gpx4. This suggests that xCT and Gpx4 are involved in the neuroprotective effects of erythropoietin on spinal cord injury. Our findings reveal the underlying anti-ferroptosis role of erythropoietin and provide a potential therapeutic strategy for treating spinal cord injury.

TNF-α up-regulates Nanog by activating NF-κB pathway to induce primary rat spinal cord astrocytes dedifferentiation.

AIMS: Astrocytes re-acquire stem cell potential upon inflammation, thereby becoming a promising source of cells for regenerative medicine. Nanog is an essential transcription factor to maintain the characteristics of stem cells. We aimed to investigate the role of Nanog in astrocyte dedifferentiation. MAIN METHODS: TNF-α was used to induce the dedifferentiation of primary rat spinal cord astrocytes. The expression of immature markers CD44 and Musashi-1 was detected by qRT-PCR and immunofluorescence. The Nanog gene is knocked down by small interference RNA. Nanog expression was measured by qRT-PCR and western blotting. BAY 11-7082 was used to suppress NF-κB signals in astrocytes. NF-κB signaling was evaluated by Western blotting. KEY FINDINGS: Our results showed that TNF-α promoted the re-expression of CD44 and Musashi-1 in astrocytes. Dedifferentiated astrocytes could be induced to differentiate into oligodendrocyte lineage cells indicating that the astrocytes had pluripotency. In addition, TNF-α treatment activated NF-κB signaling pathway and up-regulated Nanog. Knockdown of Nanog reversed the increase of CD44 and Musashi-1 induced by TNF-α without affecting the activation of NF-κB signaling. Importantly, blocking NF-κB signaling by BAY 11-7082 inhibited the expression of immature markers suggesting that TNF-α induces dedifferentiation of astrocytes through the NF-κB signaling pathway. BAY 11-7082 could also inhibit the expression of Nanog, which indicated that Nanog was regulated by NF-κB signaling pathway. SIGNIFICANCE: These findings indicate that activation of the NF-κB signaling pathway through TNF-α leads to astrocytes dedifferentiation via Nanog. These results expand our understanding of the mechanism of astrocytes dedifferentiation.

The CRD of Frizzled 7 exhibits chondroprotective effects in osteoarthritis via inhibition of the canonical Wnt3a/ß-catenin signaling pathway.

Osteoarthritis (OA) is a chronic inflammatory joint disease without effective drugs. Frizzled 7 (FzD7) binds its ligand Wnt3a through an extracellular cysteine-rich domain (CRD) to transduce the canonical Wnt/ß-catenin signaling pathway, which has been strongly implicated in OA pathogenesis. Effects of recombinant protein of FzD7 CRD on Wnt/ß-catenin signaling and chondral destruction was evaluated in this study. Firstly, increased protein levels of FzD7, Wnt3a and ß-catenin were detected in human OA cartilage implying that the canonical Wnt/ß-catenin signaling mediated by Wnt3a and FzD7 executes an essential role in OA. Then we showed that FzD7 CRD antagonized the Wnt3a/ß-catenin signaling pathway in a dose-dependent manner by binding Wnt3a. In addition, FzD7 CRD increased the expression of glycosaminoglycans (GAGs), Collagen II, aggrecan and reduced the expression of matrix metalloproteinase (MMP)-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) in Wnt3a-stimulated human chondrocytes. Furthermore, a single intra-articular injection of the FzD7 CRD was efficacious in destabilization of the medial meniscus (DMM) mouse OA model, significantly improving Osteoarthritis Research Society International (OARSI) histology scores compared to mice treated with PBS. The results indicate that the FzD7 CRD exhibits chondroprotective effects by binding Wnt3a to suppress the Wnt3a/ß-catenin signaling. Targeting the FzD7 CRD may be a novel therapy for the treatment of OA.

Knockdown of SGK1 alleviates the IL-1ß-induced chondrocyte anabolic and catabolic imbalance by activating FoxO1-mediated autophagy in human chondrocytes.

Osteoarthritis (OA) is a common joint disease characterized by the progressive degeneration of articular cartilage with no effective treatment methods available. Cartilage degeneration is closely related to an anabolic and catabolic imbalance in chondrocytes, and accumulating evidence has revealed that autophagy is a crucial protective mechanism that maintains the balance of anabolic and catabolic activities. Therefore, studies aiming to identify additional genes that regulate autophagy as a promising therapeutic strategy for OA are needed. In this study, we analyzed the GSE113825 datasets from Gene Expression Omnibus and validated that serum- and glucocorticoid-regulated kinase 1 (SGK1) was upregulated in OA cartilage. Based on the results from loss-of-function studies, SGK1 silencing promoted the deposition of glycosaminoglycans in interleukin 1 beta (IL-1ß)-treated chondrocytes, and significantly alleviated IL-1ß-induced downregulation of Collagen II and Aggrecan, as well as the upregulation of a disintegrin and metalloproteinase with thrombospondin motifs 5 and matrix metalloproteinase-13. Furthermore, SGK1 knockdown reversed the IL-1ß-induced chondrocyte anabolic and catabolic imbalance by activating autophagy. Moreover, SGK1 directly bound to forkhead box protein O1 (FoxO1) and increased its phosphorylation, which in turn resulted in its translocation from the nucleus. The decreased FoxO1 levels led to a decrease in LC3-I/LC3-II conversion and Beclin-1 levels, subsequently inhibiting autophagosome formation and increasing P62 levels, thus indicating a downregulation of autophagy. Taken together, we identified a critical role of SGK1 in the IL-1ß-induced chondrocyte anabolic and catabolic imbalance, which may represent a potential novel therapeutic target for OA.

Parallel comparison of fibroblast-like synoviocytes from the surgically removed hyperplastic synovial tissues of rheumatoid arthritis and osteoarthritis patients.

BACKGROUND: Fibroblast-like synoviocytes (FLS) are essential cellular components in inflammatory joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). Despite the growing use of FLS isolated from OA and RA patients, a detailed functional and parallel comparison of FLS from these two types of arthritis has not been performed. METHODS: In the present study, FLS were isolated from surgically removed synovial tissues from twenty-two patients with OA and RA to evaluate their basic cellular functions. RESULTS: Pure populations of FLS were isolated by a sorting strategy based on stringent marker expression (CD45-CD31-CD146-CD235a-CD90+PDPN+). OA FLS and RA FLS at the same passage (P2-P4) exhibited uniform fibroblast morphology. OA FLS and RA FLS expressed a similar profile of cell surface antigens, including the fibroblast markers VCAM1 and ICAM1. RA FLS showed a more sensitive inflammatory status than OA FLS with regard to proliferation, migration, apoptosis, inflammatory gene expression and pro-inflammatory cytokine secretion. In addition, the responses of OA FLS and RA FLS to both the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) and the anti-inflammatory drug methotrexate (MTX) were also evaluated here. CONCLUSION: The parallel comparison of OA FLS and RA FLS lays a foundation in preparation for when FLS are considered a potential therapeutic anti-inflammatory target for OA and RA.

Dopamine delays articular cartilage degradation in osteoarthritis by negative regulation of the NF-κB and JAK2/STAT3 signaling pathways.

BACKGROUND: The progressive loss of cartilage matrix and the breakdown of articular cartilage induced by inflammation play an essential role in osteoarthritis (OA) pathogenesis. Dopamine (DA) is a critical neurotransmitter that is not only involved in controlling exercise, emotion, cognition and neuroendocrine activity but also has anti-inflammatory effects. This study aimed to investigate the effects of DA on OA in vitro and in vivo. METHODS: OA progression was evaluated in a mouse model with surgically induced destabilization of the medial meniscus. Cartilage degradation and OA were analyzed using Safranin O/Fast Green staining. Additionally, qRT-PCR and Western blotting were applied to detect catabolic and anabolic factors involved in cartilage degeneration and underlying mechanisms in OA chondrocytes treated with Interleukin-1ß. RESULTS: In vitro, DA treatment inhibited the production of inducible nitric oxide synthase, cyclooxygenase-2, matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13, while increasing type II collagen and glycosaminoglycan content. Mechanistically, DA reversed IL-1ß-treated nuclear factor-kappa B activation and JAK2/STAT3 phosphorylation. Furthermore, DA suppressed the degradation of cartilage matrix and reduced Osteoarthritis Research Society International scores in the surgically induced OA models. CONCLUSION: DA may be a novel therapeutic agent for OA treatment.

Angiopoietin-like 2 upregulation promotes human chondrocyte injury via NF-κB and p38/MAPK signaling pathway.

Several cellular and molecular processes participate in the pathologic changes of osteoarthritis (OA). However, the core molecular regulators of these processes are unclear, and no effective treatment for OA disease has been developed so far. ANGPTL2 is well known for its tissue remolding and pro-inflammation properties. However, the role of ANGPTL2 in osteoarthritis (OA) still remains unclear. To explore the expression level of ANGPTL2 in human OA cartilage and investigate the function of ANGPTL2 in human chondrocytes injury, qRT-PCR, western blot and immunohistochemistry were employed to investigate the expression of ANGPTL2 between human OA and normal cartilage samples. Next, human primary chondrocytes were treated with IL-1ß to mimic OA progress in vitro, and the expression of ANGPTL2 were tested by qRT-PCR and western blot. Furthermore, the effect of ANGPTL2 in the expression of pro-inflammation cytokines (IL-1ß, IL-6), proteolytic enzymes (MMP-1, MMP-13) and component of the cartilage matrix (COL2A1 and aggrecan) in human primary chondrocyte were explored by gain-of-function and loss-of-function methods. Finally, the nuclear factor kappa B (NF-κB) and p38/MAPK signaling pathways were also tested by western blot analysis. In this study, firstly, the expression level of ANGPTL2 was elevated both in human OA cartilage samples and IL-1ß stimulated human chondrocytes. Secondly, ANGPTL2 upregulation promotes extracellular matrix (ECM) degradation and inflammation mediator production in human chondrocytes. Finally, ANGPTL2 activated the NF-κB and p38/MAPK signaling pathways via integrin α5ß1. This study, for the first time, highlights that ANGPTL2 secreted by human chondrocytes plays a negative role in the pathogenesis of osteoarthritis, and it may be a potential therapeutic target in OA.

ACY-1215 exhibits anti-inflammatory and chondroprotective effects in human osteoarthritis chondrocytes via inhibition of STAT3 and NF-κB signaling pathways.

Cartilage degeneration is a basic pathological feature of osteoarthritis (OA), and there is growing evidence that it is associated with inflammation. ACY-1215, a selective HDAC6 inhibitor, has been reported to have anti-inflammatory effects. Here, we investigated the anti-inflammatory and chondroprotective effects of ACY-1215 in IL-1ß-stimulated human primary chondrocytes and C28/I2 cells. The results suggested that ACY-1215 can markedly suppress the expression of inflammatory factors, including IL-1ß and IL-6 in human primary chondrocytes and C28/I2 cells. Furthermore, ACY-1215 exerts potent chondroprotection through the amelioration of cartilage degradation by inhibiting the expression of matrix-degrading proteases, including MMP-1 and MMP-13 in chondrocytes. These effects may be related to ACY-1215 induced down-regulation of NF-κB and STAT3 pathways in OA chondrocytes. Taken together, our results show that ACY-1215 may be a potential and promising therapeutic drug for the management of OA.