Plantamajoside Ameliorates Inflammatory Response of Chondrocytes via Regulating NF-κB/NLRP3 Inflammasome Pathway

The damage of articular cartilage in osteoarthritis involves the oxidative stress and inflammation. The aim of the present study was to explore the role of plantamajoside (PM) in chondrocytes and elucidate the underlying mechanism. The cell viability following treatment with PM or lipopolysac-charide (LPS) was assessed by cell counting kit-8 (CCK-8). Enzyme-Linked Immunosorbent Assay (ELISA) was supplied to determine the levels of pro-inflammatory cytokines. Moreover, the oxidative stress-related markers were evaluated via assay kits. TUNEL assay was employed to stain the apoptotic cells. The components of nuclear factor-κB (NF-κB) pathway and NLRP3 inflammasome were estimated by western blot analysis. LPS-insulted cell viability of ATDC5 was restored by PM. PM alleviated the inflammatory response and oxidative stress of ATDC5 cells induced by LPS. Furthermore, it was found that the apoptotic cells were reduced following PM treatment. The protein levels of NF-κB, IκB kinase β (IKKβ) and NLRP3 inflammasome were decreased by PM. These results suggested that PM protected the ATDC5 cells from LPS stimulation, alleviated the inflammatory response may through regulating the NF-κB and NLRP3 inflammasome.

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ALK7 Inhibition Protects Osteoblast Cells Against High Glucose-induced ROS Production via Nrf2/HO-1 Signaling Pathway

Current Molecular Medicine ◽

10.2174/1566524021666210614144337 ◽

2021 ◽

Vol 21 ◽

Author(s):

Zhen Zhao ◽

Yu Lu ◽

Huan Wang ◽

Xiang Gu ◽

Luting Zhu ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Viability ◽

Signaling Pathway ◽

High Glucose ◽

Collagen I ◽

Cell Counting ◽

Enzyme Linked Immunosorbent Assay ◽

Heme Oxygenase 1 ◽

Ros Production ◽

Alizarin Red

Background: Some studies demonstrated that under high-glucose (HG) condition, osteoblasts develop oxidative stress, which will impair their normal functions. The effects of activin receptor-like kinase 7 (ALK7) silencing on HG-induced osteoblasts remained unclear. Objective: The aim of this study was to explore the effect of ALK7 on HG-induced osteoblasts. Methods: MC3T3-E1 cells were treated with different concentrations of HG (0, 50, 100, 200 and 300mg/dL), and the cell viability was detected using cell counting kit-8 (CCK-8). HG-treated MC3T3-E1 cells were transfected with siALK7 or ALK7 overexpression plasmid or siNrf2, and then the viability and apoptosis were detected by CCK-8 and flow cytometry. The levels of reactive oxygen species (ROS), collagen I and calcification nodule were determined by oxidative stress kits, Enzyme-linked immunosorbent assay and Alizarin red staining. The expressions of NF-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and osteoblast-associated genes were determined by quantitative real-time PCR (qRT-PCR) and Western blot. Results: Cell viability was reduced with HG treatment. Silencing ALK7 inhibited the effect of HG on increasing cell apoptosis and ROS production, reduced cell viability, mineralized nodules, and downregulated collagen I and osteoblast-associated genes expression in MC3T3-E1 cells. ALK7 silencing activated the Nrf2/HO-1 signaling pathway by affecting expressions of HO-1 and Nrf2. ALK7 overexpression had the opposite effects. In addition, siNrf2 partially reversed the effects of ALK7 silencing on HG-induced MC3T3-E1 cells. Conclusion: ALK7 silencing protected osteoblasts under HG condition possibly through activating the Nrf2/HO-1 pathway.

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Endoplasmic Reticulum Stress Is Involved in Baicalin Protection on Chondrocytes From Patients With Osteoarthritis

Dose-Response ◽

10.1177/1559325818810636 ◽

2018 ◽

Vol 16 (4) ◽

pp. 155932581881063 ◽

Cited By ~ 1

Author(s):

Jiangang Cao ◽

Yu Zhang ◽

Tianyi Wang ◽

Bo Li

Keyword(s):

Oxidative Stress ◽

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Cell Viability ◽

Er Stress ◽

Messenger Rna ◽

Cell Counting ◽

Protective Effects ◽

Gene Expressions ◽

Protein Levels

Osteoarthritis (OA) affects elderly population worldwide and endoplasmic reticulum (ER) stress is known to be positively correlated with OA development. Previous reports prove the cytoprotective effects of baicalin on chondrocytes, whereas the mechanisms are hardly reported. Hence, we aimed to investigate the links between OA, ER stress, and baicalin. Chondrocytes from patients with OA were subjected to H2O2 treatment with or without baicalin pretreatment, and cell viability was assessed via Cell Counting Kit-8. Messenger RNA (mRNA) amounts of apoptosis-related genes (Bax, Bcl-2, and Caspase-3), extracellular matrix (ECM)-related genes (Collange I, Collange II, Aggrecan, and Sox9) and ER stress hallmarks (binding immunoglobulin protein [BiP] C/EBP homologous protein [CHOP]) were evaluated via quantitative real-time PCR. Bax, Bcl-2, BiP, and CHOP protein levels were analyzed via Western blot. Baicalin suppressed the changes in cell viability and apoptosis-related gene expressions caused by H2O2. Reactive oxygen species and glutathione/oxidized glutathione assay showed that H2O2 enhanced oxidative stress. Baicalin suppressed H2O2-induced downregulation of mRNA expression of ECM-related genes. Moreover, baicalin reduced H2O2-stimulated increase in oxidative stress and the expression of ER stress hallmarks. Endoplasmic reticulum stress inducer abolished the protective activities, whereas ER stress inhibitor did not exhibit extra protective effects. Baicalin pretreatment protected patient-derived chondrocytes from H2O2 through ER stress inhibition.

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Di-2-ethylhexyl phthalate (DEHP) induces apoptosis of mouse HT22 hippocampal neuronal cells via oxidative stress

Toxicology and Industrial Health ◽

10.1177/0748233720947205 ◽

2020 ◽

Vol 36 (11) ◽

pp. 844-851

Author(s):

Wei Tu ◽

Weifeng Li ◽

Xingen Zhu ◽

Linlin Xu

Keyword(s):

Oxidative Stress ◽

Cell Viability ◽

Neuronal Cell ◽

Underlying Mechanism ◽

Treated Group ◽

Dependent Manner ◽

Ht22 Cells ◽

Protein Levels ◽

Ethylhexyl Phthalate

Di-2-ethylhexyl phthalate (DEHP) has been widely used as a plasticizer in industry and can affect memory; however, the underlying mechanism remains unclear. In the present study, mouse HT22 cells, an immortalized hippocampal neuronal cell line, was utilized as an in vitro model. We showed that DEHP dramatically inhibited cell viability and increased lactate dehydrogenase (LDH) release from the cells in a dose-dependent manner, suggesting that DEHP could cause cytotoxicity of mouse HT22 cells. The protein levels of cleaved Caspase-8, cleaved Caspase-3, and Bax markedly increased in the DEHP-treated cells, whereas there was a significant decrease in the Bcl-2 protein level, implying that DEHP could induce apoptosis of mouse HT22 cells. DEHP exposure significantly increased the content of malondialdehyde, whereas it markedly decreased the level of glutathione and the activities of glutathione peroxidase and superoxide dismutase, suggesting that DEHP induced oxidative stress of the cells. Compared with the DEHP-treated group, the inhibition of cell viability and the release of LDH were rescued in the N-acetyl-l-cysteine plus DEHP group. Furthermore, inhibition of oxidative stress could rescue the induction of apoptosis by DEHP. Collectively, our results indicated that DEHP could induce apoptosis of mouse HT22 cells via oxidative stress.

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Zoledronic acid promotes osteoclasts ferroptosis by inhibiting FBXO9-mediated p53 ubiquitination and degradation

PeerJ ◽

10.7717/peerj.12510 ◽

2021 ◽

Vol 9 ◽

pp. e12510

Author(s):

Xingzhou Qu ◽

Zhaoqi Sun ◽

Yang Wang ◽

Hui Shan Ong

Keyword(s):

Gene Expression ◽

Zoledronic Acid ◽

Cell Viability ◽

Cell Model ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Underlying Mechanism ◽

Cell Counting ◽

Venn Diagram

Bisphosphonates (BPs)-related osteonecrosis of jaw (BRONJ) is a severe complication of the long-term administration of BPs. The development of BRONJ is associated with the cell death of osteoclasts, but the underlying mechanism remains unclear. In the current study, the role of Zoledronic acid (ZA), a kind of bisphosphonates, in suppressing the growth of osteoclasts was investigated and its underlying mechanism was explored. The role of ZA in regulating osteoclasts function was evaluated in the RANKL-induced cell model. Cell viability was assessed by cell counting kit-8 (CCK-8) assay and fluorescein diacetate (FDA)-staining. We confirmed that ZA treatment suppressed cell viability of osteoclasts. Furthermore, ZA treatment led to osteoclasts death by facilitating osteoclasts ferroptosis, as evidenced by increased Fe2+, ROS, and malonyldialdehyde (MDA) level, and decreased glutathione peroxidase 4 (GPX4) and glutathione (GSH) level. Next, the gene expression profiles of alendronate- and risedronate-treated osteoclasts were obtained from Gene Expression Omnibus (GEO) dataset, and 18 differentially expressed genes were identified using venn diagram analysis. Among these 18 genes, the expression of F-box protein 9 (FBXO9) was inhibited by ZA treatment. Knockdown of FBXO9 resulted in osteoclasts ferroptosis. More important, FBXO9 overexpression repressed the effect of ZA on regulating osteoclasts ferroptosis. Mechanistically, FBXO9 interacted with p53 and decreased the protein stability of p53. Collectively, our study showed that ZA induced osteoclast cells ferroptosis by triggering FBXO9-mediated p53 ubiquitination and degradation.

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Cullin3 Aggravates The Inflammatory Response of PDLSCs Via Regulation of Shh Signaling and Nrf2

10.21203/rs.3.rs-368145/v1 ◽

2021 ◽

Author(s):

Wanhong Chen ◽

Jiangling Su ◽

Shixiong Cai ◽

Chun Shi

Keyword(s):

Cell Viability ◽

Cell Apoptosis ◽

Cell Counting ◽

Enzyme Linked Immunosorbent Assay ◽

Tunel Staining ◽

Western Blots ◽

Alizarin Red ◽

Shh Signaling ◽

Protein Levels ◽

Alkaline Phosphatase Staining

Abstract Objective: Sonic Hedgehog (Shh) was found to be correlated with inflammation degree of patients with periodontitis. Cullin3 is an important ubiquitin ligase for controlling Shh signaling. In this study, we exerted ourselves to clarify the roles of Shh and Cullin3 in P. gingivalis-LPS (Pg-LPS)-treated periodontal ligament stem cells (PDLSCs). Methods: Cell viability was detected using cell counting kit-8 (CCK-8). The inflammatory cytokines of PDLSCs were estimated by enzyme-linked immunosorbent assay (ELISA). The protein levels of Shh, Gli1 and NF-E2-related factor2 (Nrf2) were determined via western blots. Alkaline phosphatase staining and Alizarin red staining were performed to evaluate the differentiation and mineralization capabilities of PDLSCs. The apoptotic cells were screened by TUNEL staining. Results: Pg-LPS inhibited cell viability and triggered inflammation of PDLSCs. Overexpression of Cullin3 impeded the differentiation and mineralization capabilities of PDLSCs. Moreover, Cullin3 overexpression aggravated inflammation and cell apoptosis induced by Pg-LPS. Of note, while the protein levels of Shh, Gli1 and Nrf2 were elevated in PDLSCs treated with Pg-LPS, overexpression of Cullin3 decreased the expressions of them. Conclusion: Shh/Gli1 and Nrf2 were involved in the inflammation and cell apoptosis of PDLSCs, which was dominated by Cullin3.

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Brucella-Induced Downregulation of lncRNA Gm28309 Triggers Macrophages Inflammatory Response Through the miR-3068-5p/NF-κB Pathway

Frontiers in Immunology ◽

10.3389/fimmu.2020.581517 ◽

2020 ◽

Vol 11 ◽

Author(s):

Xingmei Deng ◽

Jia Guo ◽

Zhihua Sun ◽

Laizhen Liu ◽

Tianyi Zhao ◽

...

Keyword(s):

Inflammatory Response ◽

Immune Responses ◽

Nlrp3 Inflammasome ◽

Inflammatory Responses ◽

Underlying Mechanism ◽

Bioinformatic Analysis ◽

Luciferase Reporter ◽

Non Coding Rnas ◽

First Time

ObjectivesThe underlying mechanism of the inflammatory response against Brucellosis caused by Brucella remains poorly understood. This study aimed to determine the role of long non-coding RNAs (lncRNAs) in regulating of inflammatory and anti-Brucella responses.Materials and methodsMicroarray analysis was performed to detect differentially expressed lncRNAs in THP-1 cells infected with an S2308 Brucella strain. The candidate lncRNAs were screened using bioinformatic analysis and siRNAs; bioinformatic prediction and luciferase reporter assay were also conducted, while inflammatory responses was assessed using RT‐qPCR, western blot, immunofluorescence, ELISA, HE, and immunohistochemistry.ResultsThe lncRNA Gm28309 was identified to be involved in regulating inflammation induced by Brucella. Gm28309, localized in the cytoplasm, was down-expressed in RAW264.7 cells infected with S2308. Overexpression of Gm28309 or inhibition of miR-3068-5p repressed p65 phosphorylation and reduced NLRP3 inflammasome and IL-1β and IL-18 secretion. Mechanistically, Gm28309 acted as a ceRNA of miR-3068-5p to activate NF-κB pathway by targeting κB-Ras2, an inhibitor of NF-κB signaling. Moreover, the number of intracellular Brucella was higher when Gm28309 was overexpressed or when miR-3068-5p or p65 was inhibited. However, these effects were reversed by the miR-3068-5p mimic.ConclusionsOur study demonstrates, for the first time, that LncRNAs are involved in regulating immune responses during Brucella infection, and Gm28309, an lncRNA, plays a crucial role in activating NF-κB/NLRP3 inflammasome signaling pathway.

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Co-treatment with disulfiram and glycyrrhizic acid suppresses the inflammatory response of chondrocytes

Journal of Orthopaedic Surgery and Research ◽

10.1186/s13018-021-02262-3 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Chao Li ◽

Li Li ◽

Tian Lan

Keyword(s):

Cell Proliferation ◽

Inflammatory Response ◽

Glycyrrhizic Acid ◽

Cell Model ◽

High Mobility ◽

Cell Counting ◽

Inflammatory Factors ◽

Enzyme Linked Immunosorbent Assay ◽

High Concentration ◽

Protein Levels

Abstract Background Osteoarthritis (OA) is a kind of systemic musculoskeletal disorder and a most important factor for causing disability and physical painfulness. Nevertheless, due to the fact that OA can be triggered by multiple etiological factors, this disease is hard to be cured. Therefore, it is of great necessity for us to find novel targets or drugs for OA treatment. Materials and methods The chondrocytes were treated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP) to induce pyroptosis in OA. The cell proliferation was detected by Cell Counting Kit-8 assay (CCK-8 assay). Enzyme-linked immunosorbent assay (ELISA) was used for the detection of pyroptosis-related inflammatory factors. Then, the antagonists for gasdermin D (GSDMD) (disulfiram) and high mobility group box 1 (HMGB1) (glycyrrhizic acid) were used to treat the cell model to observe the effects of disulfiram and glycyrrhizic acid on the proliferation of chondrocytes in OA. The protein levels of pyroptosis-related inflammatory factors were measured by western blot, and the levels of aldehyde dehydrogenase (ALDH) and reactive oxygen species (ROS) were measured by corresponding commercial kits. Results After chondrocytes were induced by LPS and ATP, the cell proliferation was decreased and the expressions of pyroptosis-related inflammatory factors were increased. Disulfiram and glycyrrhizic acid treatment led to enhanced cell proliferation and increased expressions of pyroptosis-related inflammatory factors, while disulfiram showed better alleviative effects on the inflammation in chondrocytes in OA. However, co-treatment with disulfiram at a high concentration and glycyrrhizic acid did not result in higher proliferation of chondrocytes and alleviated inflammation, but led to oxidative stress. Conclusion In conclusion, co-treatment with disulfiram and glycyrrhizic acid at a standard concentration suppresses the inflammatory response of chondrocytes, which may provide guidance for the use of the drugs in the treatment of OA.

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Sirt-1 Regulates Physiological Process and Exerts Protective Effects against Oxidative Stress

BioMed Research International ◽

10.1155/2021/5542545 ◽

2021 ◽

Vol 2021 ◽

pp. 1-12

Author(s):

Lei Liu ◽

Guangyuan Xia ◽

Peifan Li ◽

Yiming Wang ◽

Qian Zhao

Keyword(s):

Oxidative Stress ◽

Transcriptional Activity ◽

Cell Counting ◽

Luciferase Reporter ◽

Therapeutic Drug ◽

Mrna Levels ◽

P53 Expression ◽

Protein Levels ◽

Gene Assay

Background. Recent studies suggest a correlation between the reduced Sirt-1 expression with Alzheimer’s diseases (AD) and depression, respectively, suggesting a possible pathogenic role of the altered Sirt-1 expression in neuronal degenerative diseases, such as AD and depression. However, the molecular mechanisms underlying how Sirt-1 reduction impairs neuronal functions remain unknown. Methods. We used the SK-N-SH neuroblastoma cells to study the role of Sirt-1 expression on physiological roles in neuronal cells. Gain of Sirt-1 was achieved by transiently transfecting Sirt-1 expression plasmid. Sirt-1-specific shRNA was used to elucidate the role of Sirt-1 loss of function. CCK-8 (Cell Counting Kit-8) assay and flow cytometry were used to evaluate cell proliferation. Semiquantitative western blotting was used to detect relative protein levels. A further luciferase reporter gene assay was employed to examine the effect of Sirt-1 expression on the transcriptional activity of p53. RT-qPCR was used to determine the mRNA levels of p21, Bax, and Bcl-2, which were the downstream target genes of p53. Results. Sirt-1 suppressed the p53 downstream gene p21 transcription, while shRNA-mediated Sirt-1 knockdown resulted in a significant increase in p21 expression, implying a possibility that Sirt-1 promotes neuron proliferation through suppressing p53 transcriptional activity. The mRNA and protein levels of p53 were not affected by the altered Sirt-1 expression, suggesting that Sirt-1 regulates the transcriptional regulatory activity of p53 rather than p53 expression. Indeed, we further confirmed that Sirt-1 appeared to inhibit p53 transcriptional activity by attenuating its acetylation and resulted in a decrease of p53’s binding to the p21 promoter. Overexpressed Sirt-1 scavenged reactive oxygen species (ROS) production in SK-N-SH with H2O2. Knockdown of Sirt-1 presented opposite effect; the addition of EX527 (Sirt-1 inhibitor) increased ROS accumulation. Conclusions. Oxidative stress induces Sirt-1 in neuron cells, and Sirt-1 promotes proliferation in SK-N-SH cells, which protects them from oxidative stress-induced cell death, potentially via suppressing the transcriptional activity of p53. These results provide a molecular explanation underlying how the reduced Sirt-1 potentially causes the AD and depression-related diseases, supporting the idea that Sirt-1 can possibly be used as a diagnostic biomarker and/or therapeutic drug target for the AD and depression-related diseases.

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METTL3 Attenuates LPS-Induced Inflammatory Response in Macrophages via NF-κB Signaling Pathway

Mediators of Inflammation ◽

10.1155/2019/3120391 ◽

2019 ◽

Vol 2019 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Jinghua Wang ◽

Shushan Yan ◽

Hongying Lu ◽

Shufeng Wang ◽

Donghua Xu

Keyword(s):

Inflammatory Response ◽

Critical Role ◽

Underlying Mechanism ◽

Enzyme Linked Immunosorbent Assay ◽

Macrophage Cell Line ◽

Macrophage Cell ◽

Chain Reaction ◽

Key Enzyme ◽

Polymerase Chain

Methyltransferase-like 3 (METTL3), an RNA N6-methyladenosine (m6A) methyltransferase, is essential for the m6A mRNA modification. As a key enzyme of m6A methylation modification, METTL3 has been implicated in immune and inflammation regulation. However, little is known of the role and underlying mechanism of METTL3 in rheumatoid arthritis (RA). The aim of the present study is to elucidate the function and potential mechanism of METTL3 in RA pathogenesis. We used quantitative real-time polymerase chain reaction to detect the expression of METTL3 in RA patients and controls as well as the macrophage cell line. CCK-8 was used for cell proliferation assay. Enzyme-linked immunosorbent assay (ELISA) was adopted to estimate the generation of IL-6 and TNF-α in macrophages. Western blot and immunofluorescence were applied to evaluate the activation of NF-κB in macrophages. The expression of METTL3 was significantly elevated in patients with RA. It was positively associated with CRP and ESR, two common markers for RA disease activity. Besides, LPS could enhance the expression and biological activity of METTL3 in macrophages, while overexpression of METTL3 significantly attenuated the inflammatory response induced by LPS in macrophages. Moreover, the effect of METTL3 on LPS-induced inflammation in macrophages was dependent on NF-κB. This study firstly demonstrates the critical role of METTL3 in RA, which provides novel insights into recognizing the pathogenesis of RA and a promising biomarker for RA.

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MicroRNA-191-5p diminished sepsis-induced acute kidney injury through targeting oxidative stress responsive 1 in rat models

Bioscience Reports ◽

10.1042/bsr20190548 ◽

2019 ◽

Vol 39 (8) ◽

Cited By ~ 4

Author(s):

Yi Qin ◽

Guizhen Wang ◽

Zhiyong Peng

Keyword(s):

Oxidative Stress ◽

Acute Kidney Injury ◽

Caspase 3 ◽

Kidney Injury ◽

B Cell Lymphoma ◽

Cell Counting ◽

Health Concern ◽

Enzyme Linked Immunosorbent Assay ◽

Renal Cells ◽

Protein Levels

Abstract There is no effective treatment for septic acute kidney injury (AKI), which is considered a major public health concern in today’s world. Here, we studied the functions of miR-191-5p in septic AKI. MiR-191-5p mimic or mimic control was injected into rats from caudal vein before cecal ligation and puncture (CLP) surgery. Part of kidney tissues was stained by Hematoxylin and Eosin (H&E) for histological examination. The levels of serum cytokines were evaluated using enzyme-linked immunosorbent assay (ELISA). For cell transfection, renal cells were isolated from the kidneys of CLP rat model injected with mimic control and miR-191-5p mimic. With TargetScan prediction, serine/threonine-protein kinase OSR1 was identified as a target of miR-191-5p. Oxidative stress responsive 1 (OXSR1) overexpression vector was transfected into renal cells. Cell viability and apoptosis rate were determined by Cell Counting Kit-8 (CCK-8) and flow cytometry, respectively. We additionally measured the phosphorylation levels of p38 and p65. We found that the injection of miR-191-5p mimic could observably inhibit renal injury scores, and inhibit inflammatory cytokine productions and apoptotic protein levels in septic rats. After being transfected with OXSR1, the apoptosis rates and expressions of B-cell lymphoma-2 (Bcl-2), down-regulated Bax and Cleaved caspase-3 (C caspase-3) indicated overexpressed OXSR1 contributed to cell apoptosis. The up-regulated protein levels of p-p38 and p-p65 may suggest the involvement of p38 MAPK/NF-κB signaling pathway in the functions of OXSR1. Our results showed that the protective effects of miR-191-5p on kidney tissues of septic rats may rely on the repression of OXSR1.

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