Genetic Inactivation of ZCCHC6 Suppresses Interleukin‐6 Expression and Reduces the Severity of Experimental Osteoarthritis in Mice.
ANIMAL experimentation; CARTILAGE; CARTILAGE cells; DACTINOMYCIN; DNA-binding proteins; GENE expression; GENETIC aspects; IN vivo studies; INTERLEUKINS; MICE; MICRORNA; OSTEOARTHRITIS; PREVENTION; SEQUENCE analysis; SEVERITY of illness index; SYNOVITIS; TRANSCRIPTION factors
Objective: Cytokine expression is tightly regulated posttranscriptionally, but high levels of interleukin‐6 (IL‐6) in patients with osteoarthritis (OA) indicate that regulatory mechanisms are disrupted in this disorder. The enzyme ZCCHC6 (zinc‐finger CCHC domain–containing protein 6; TUT‐7) has been implicated in posttranscriptional regulation of inflammatory cytokine expression, but its role in OA pathogenesis is unknown. The present study was undertaken to investigate whether ZCCHC6 directs the expression of IL‐6 and influences OA pathogenesis in vivo. Methods: Human and mouse chondrocytes were stimulated with recombinant IL‐1β. Expression of ZCCHC6 in human chondrocytes was knocked down using small interfering RNAs. IL‐6 transcript stability was determined by actinomycin D chase, and 3′‐uridylation of microRNAs was determined by deep sequencing. Zcchc6−/− mice were produced by gene targeting. OA was surgically induced in the knee joints of mice, and disease severity was scored using a semiquantitative grading system. Results: ZCCHC6 was markedly up‐regulated in damaged cartilage from human OA patients and from wild‐type mice with surgically induced OA. Overexpression of ZCCHC6 induced the expression of IL‐6, and its knockdown reduced IL‐6 transcript stability and IL‐1β–induced IL‐6 expression in chondrocytes. Reintroduction of Zcchc6 in Zcchc6−/− mouse chondrocytes rescued the IL‐1β–induced IL‐6 expression. Knockdown of ZCCHC6 reduced the population of micro‐RNA 26b (miR‐26b) with 3′‐uridylation by 60%. Zcchc6−/− mice with surgically induced OA produced low levels of IL‐6 and exhibited reduced cartilage damage and synovitis in the joints. Conclusion: These findings indicate that ZCCHC6 enhances IL‐6 expression in chondrocytes through transcript stabilization and by uridylating miR‐26b, which abrogates repression of IL‐6. Inhibition of IL‐6 expression and significantly reduced OA severity in Zcchc6−/− mice identify ZCCHC6 as a novel therapeutic target to inhibit disease pathogenesis. [ABSTRACT FROM AUTHOR]
Ansari Mohammad Y; Khan Nazir M; Ahmad Nashrah; Green Jonathan; Novak Kimberly; Haqqi Tariq M
Arthritis & Rheumatology
2019
2019-04
<a href="http://doi.org/10.1002/art.40751" target="_blank" rel="noreferrer noopener">10.1002/art.40751</a>
Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis.
Humans; *Apoptosis; *Apoptosis; *ERK1/2; *Nrf2; *Osteoarthritis; *Redox; Oxidative Stress; Osteoarthritis/*metabolism; NF-E2-Related Factor 2/genetics/*metabolism; Up-Regulation; MAP Kinase Signaling System; Chondrocytes/*physiology; Caspases/metabolism; Cells; Cultured; 70-kDa/metabolism; Ribosomal Protein S6 Kinases; Carboxylic Ester Hydrolases/metabolism; ets-Domain Protein Elk-1/metabolism; Interleukin-1beta/immunology; 90-kDa/metabolism
Nrf2, a redox regulated transcription factor, has recently been shown to play a role in cartilage integrity but the mechanism remains largely unknown. Osteoarthritis (OA) is a multifactorial disease in which focal degradation of cartilage occurs. Here, we studied whether Nrf2 exerts chondroprotective effects by suppressing the oxidative stress and apoptosis in IL-1beta stimulated human OA chondrocytes. Expression of Nrf2 and its target genes HO-1, NQO1 and SOD2 was significantly high in OA cartilage compared to normal cartilage and was also higher in damaged area compared to smooth area of OA cartilage of the same patient. Human chondrocytes treated with IL-1beta resulted in robust Nrf2/ARE reporter activity, which was inhibited by pretreatment with antioxidants indicating that Nrf2 activity was due to IL-1beta-induced ROS generation. Ectopic expression of Nrf2 significantly suppressed the IL-1beta-induced generation of ROS while Nrf2 knockdown significantly increased the basal as well as
Khan Nazir M; Ahmad Imran; Haqqi Tariq M
Free radical biology & medicine
2018
2018-02
<a href="http://doi.org/10.1016/j.freeradbiomed.2018.01.013" target="_blank" rel="noreferrer noopener">10.1016/j.freeradbiomed.2018.01.013</a>
Autophagy plays an essential role in bone homeostasis.
osteoclast; autophagosome; autophagy; osteoblast; osteocytes
Autophagy is very critical for multiple cellular processes. Autophagy plays a critical role in bone cell differentiation and function.
Jaber Fatima A; Khan Nazir M; Ansari Mohammad Y; Al-Adlaan Asaad A; Hussein Nazar J; Safadi Fayez F
Journal of cellular physiology
2019
2019-02
<a href="http://doi.org/10.1002/jcp.27071" target="_blank" rel="noreferrer noopener">10.1002/jcp.27071</a>
A wogonin-rich-fraction of Scutellaria baicalensis root extract exerts chondroprotective effects by suppressing IL-1beta-induced activation of AP-1 in human OA chondrocytes.
Cells; Chemical Fractionation/methods; Chondrocytes/*drug effects/metabolism; Cultured; Cyclooxygenase 2/genetics/metabolism; Flavanones/*pharmacology; Gene Expression Regulation/drug effects; Humans; Interleukin-1beta/pharmacology; Interleukin-6/genetics/metabolism; Osteoarthritis/pathology; Plant Extracts/*pharmacology; Plant Roots/*chemistry; Protective Agents/pharmacology; Reactive Oxygen Species/metabolism; Scutellaria baicalensis/*chemistry; Transcription Factor AP-1/genetics/*metabolism
Osteoarthritis (OA) is a common joint disorder with varying degrees of inflammation and sustained oxidative stress. The root extract of Scutellaria baicalensis (SBE) has been used for the treatment of inflammatory and other diseases. Here, we performed activity-guided HPLC-fractionation of SBE, identified the active ingredient(s) and investigated its chondroprotective potential. We found that the Wogonin containing fraction-4 (F4) was the most potent fraction based on its ability to inhibit ROS production and the suppression of catabolic markers including IL-6, COX-2, iNOS, MMP-3, MMP-9,
Khan Nazir M; Haseeb Abdul; Ansari Mohammad Y; Haqqi Tariq M
Scientific reports
2017
2017-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1038/srep43789" target="_blank" rel="noreferrer noopener">10.1038/srep43789</a>
Deep sequencing and analyses of miRNAs, isomiRs and miRNA induced silencing complex (miRISC)-associated miRNome in primary human chondrocytes.
MicroRNAs, a group of small, noncoding RNAs that post-transcriptionally regulate gene expression, play important roles in chondrocyte function and in the development of osteoarthritis. We characterized the dynamic repertoire of the chondrocyte miRNome and miRISC-associated miRNome by deep sequencing analysis of primary human chondrocytes. IL-1beta treatment showed a modest effect on the expression profile of miRNAs in normal and osteoarthritis (OA) chondrocytes. We found a number of miRNAs that showed a wide range of sequence modifications including nucleotide additions and deletions at 5' and 3' ends; and nucleotide substitutions. miR-27b-3p showed the highest expression and miR-140-3p showed the highest number of sequence variations. AGO2 RIP-Seq analysis revealed the differential recruitment of a subset of expressed miRNAs and isoforms of miRNAs (isomiRs) to the miRISC in response to IL-1beta, including miR-146a-5p, miR-155-5p and miR-27b-3p. Together, these results reveal a complex repertoire of miRNAs and isomiRs in primary human chondrocytes. Here, we also show the changes in miRNA composition of the miRISC in primary human chondrocytes in response to
Haseeb Abdul; Makki Mohammad Shahidul; Khan Nazir M; Ahmad Imran; Haqqi Tariq M
Scientific reports
2017
2017-11
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1038/s41598-017-15388-4" target="_blank" rel="noreferrer noopener">10.1038/s41598-017-15388-4</a>
Epigenetics in osteoarthritis: Potential of HDAC inhibitors as therapeutics.
*DNA methylation; *Epigenetics; *HDACs; *lncRNA; *miRNA; *Osteoarthritis; Animals; Epigenesis; Genetic; Histone Deacetylase Inhibitors/*therapeutic use; Humans; Osteoarthritis/*drug therapy/*genetics
Osteoarthritis (OA) is the most common joint disease and the leading cause of chronic disability in middle-aged and older populations worldwide. The development of disease modifying therapy for OA is in its infancy largely because the regulatory mechanisms for the molecular effectors of OA pathogenesis are poorly understood. Recent studies identified epigenetic events as a critical regulator of molecular players involved in the induction and development of OA. Epigenetic mechanisms include DNA methylation, non-coding RNA and histone modifications. The aim of this review is to briefly highlight the recent advances in the epigenetics of cartilage and potential of HDACs (Histone deacetylases) inhibitors in the therapeutic management of OA. We summarize the recent studies utilizing HDAC inhibitors as potential therapeutics for inhibiting disease progression and preventing the cartilage destruction in OA. HDACs control normal cartilage development and homeostasis and understanding the impact of HDACs inhibitors on the disease pathogenesis is of interest because of its importance in affecting overall cartilage health and homeostasis. These findings also shed new light on cartilage disease pathophysiology and provide substantial evidence that HDACs may be potential novel therapeutic targets in OA.
Khan Nazir M; Haqqi Tariq M
Pharmacological research
2018
2018-02
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.phrs.2017.08.007" target="_blank" rel="noreferrer noopener">10.1016/j.phrs.2017.08.007</a>
Inhibition of cartilage degradation and suppression of PGE2 and MMPs expression by pomegranate fruit extract in a model of posttraumatic osteoarthritis.
*Phytotherapy; *Punicaceae; ACLT; Animal; Animals; Anterior Cruciate Ligament/drug effects/metabolism/pathology; Apoptosis; Cartilage/cytology/*drug effects/metabolism/pathology; Chondrocytes/drug effects/metabolism/pathology; Collagen Type II/genetics/metabolism; Dinoprostone/*metabolism; Disease Models; Disease Progression; Female; Fruit; Interleukins/metabolism; Joints/cytology/*drug effects/metabolism/pathology; Male; Messenger/metabolism; Metalloproteases/genetics/*metabolism; Mitogen-Activated Protein Kinases/metabolism; MMPs; NF-kappa B/metabolism; Osteoarthritis; Osteoarthritis/*drug therapy/etiology/metabolism/pathology; PGE(2); Plant Extracts/pharmacology/therapeutic use; Pomegranate; Rabbit; Rabbits; RNA; Synovial Fluid/metabolism
OBJECTIVE: Osteoarthritis (OA) is characterized by cartilage degradation in the affected joints. Pomegranate fruit extract (PFE) inhibits cartilage degradation in vitro. The aim of this study was to determine whether oral consumption of PFE inhibits disease progression in rabbits with surgically induced OA. METHODS: OA was surgically induced in the tibiofemoral joints of adult New Zealand White rabbits. In one group, animals were fed PFE in water for 8 wk postsurgery. In the second group, animals were fed PFE for 2 wk before surgery and for 8 wk postsurgery. Histologic assessment and scoring of the cartilage was per Osteoarthritis Research Society International guidelines. Gene expression and matrix metalloproteinases (MMP) activity were determined using quantitative reverse transcriptase polymerase chain reaction and fluorometric assay, respectively. Interleukin (IL)-1 beta, MMP-13, IL-6, prostaglandin (PG)E2, and type II collagen (COL2A1) levels in synovial fluid/plasma/culture media were quantified using enzyme-linked immunosorbent assay. Expression of active caspase-3 and poly (ADP-ribose) polymerase p85 was determined by immunohistochemistry. Effect of PFE and inhibitors of MMP-13, mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB was studied in IL-1 beta-stimulated rabbit articular chondrocytes. RESULTS: Safranin-O-staining and chondrocyte cluster formation was significantly reduced in the anterior cruciate ligament transaction plus PFE fed groups. Expression of MMP-3, MMP-9, and MMP-13 mRNA was higher in the cartilage of rabbits given water alone but was significantly lower in the animals fed PFE. PFE-fed rabbits had lower IL-6,
Akhtar Nahid; Khan Nazir M; Ashruf Omer S; Haqqi Tariq M
Nutrition (Burbank, Los Angeles County, Calif.)
2017
2017-01
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.nut.2016.08.004" target="_blank" rel="noreferrer noopener">10.1016/j.nut.2016.08.004</a>
Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis.
*Apoptosis; *ERK1/2; *Nrf2; *Osteoarthritis; *Redox
Nrf2, a redox regulated transcription factor, has recently been shown to play a role in cartilage integrity but the mechanism remains largely unknown. Osteoarthritis (OA) is a multifactorial disease in which focal degradation of cartilage occurs. Here, we studied whether Nrf2 exerts chondroprotective effects by suppressing the oxidative stress and apoptosis in IL-1beta stimulated human OA chondrocytes. Expression of Nrf2 and its target genes HO-1, NQO1 and SOD2 was significantly high in OA cartilage compared to normal cartilage and was also higher in damaged area compared to smooth area of OA cartilage of the same patient. Human chondrocytes treated with IL-1beta resulted in robust Nrf2/ARE reporter activity, which was inhibited by pretreatment with antioxidants indicating that Nrf2 activity was due to IL-1beta-induced ROS generation. Ectopic expression of Nrf2 significantly suppressed the IL-1beta-induced generation of ROS while Nrf2 knockdown significantly increased the basal as well as
Khan Nazir M; Ahmad Imran; Haqqi Tariq M
Free radical biology & medicine
2018
2018-02
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.freeradbiomed.2018.01.013" target="_blank" rel="noreferrer noopener">10.1016/j.freeradbiomed.2018.01.013</a>
Wogonin, a plant derived small molecule, exerts potent anti-inflammatory and chondroprotective effects through the activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes.
*ERK1/2; *Nrf2; *Osteoarthritis; *Redox; *Wogonin; Anti-Inflammatory Agents/administration & dosage; Chondrocytes/drug effects/pathology; Flavanones/*administration & dosage; Gene Expression Regulation/drug effects; Humans; Inflammation/*drug therapy/metabolism/pathology; Kelch-Like ECH-Associated Protein 1/chemistry/*genetics/metabolism; MAP Kinase Signaling System/drug effects; Molecular Docking Simulation; NF-E2-Related Factor 2/antagonists & inhibitors/chemistry/*genetics/metabolism; Osteoarthritis/*drug therapy/metabolism/pathology; Protein Binding; Reactive Oxygen Species/metabolism; Signal Transduction/drug effects
Osteoarthritis (OA), characterized by progressive destruction of articular cartilage, is the most common form of human arthritis. Here, we evaluated the potential chondroprotective and anti-inflammatory effects of Wogonin, a naturally occurring flavonoid, in IL-1beta-stimulated human OA chondrocytes and cartilage explants. Wogonin completely suppressed the expression and production of inflammatory mediators including IL-6, COX-2, PGE2, iNOS and NO in
Khan Nazir M; Haseeb Abdul; Ansari Mohammad Y; Devarapalli Pratap; Haynie Sara; Haqqi Tariq M
Free radical biology & medicine
2017
2017-05
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.freeradbiomed.2017.02.041" target="_blank" rel="noreferrer noopener">10.1016/j.freeradbiomed.2017.02.041</a>
Dataset of effect of Wogonin, a natural flavonoid, on the viability and activation of NF-kappaB and MAPKs in IL-1beta-stimulated human OA chondrocytes.
MAPK; Mass-spectrometry; NF-kappaB; Nrf2; Osteoarthritis; Wogonin
This article contains data related to the article "Wogonin, a plant derived small molecule exerts potent anti-inflammatory and chondroprotective effects through activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes" (Khan et al. 2017) [1]. The data are related to effects of Wogonin on the viability and IL-1beta-stimulated activation of NF-kappaB and ERK1/2, JNK1/2 and p38 MAPKs in human OA chondrocytes. Gene expression data representing the chondrogenic phenotype and the efficiency of Nrf2 knockdown in monolayer culture of human OA chondrocytes were shown. Moreover, mass spectrometric calibration curve of Wogonin used to quantify the intracellular uptake were also presented. The data are presented in the form of figures and significance of these has been given in the research article (Khan et al. 2017) [1].
Khan Nazir M; Haseeb Abdul; Ansari Mohammad Y; Devarapalli Pratap; Haynie Sara; Haqqi Tariq M
Data in brief
2017
2017-06
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.dib.2017.03.054" target="_blank" rel="noreferrer noopener">10.1016/j.dib.2017.03.054</a>
Wogonin, a natural flavonoid, intercalates with genomic DNA and exhibits protective effects in IL-1beta stimulated osteoarthritis chondrocytes.
Apoptosis/drug effects; Binding Sites; Cells; Chondrocytes/cytology/*drug effects/metabolism; Chondroprotective effects; Cultured; Denaturation; DNA binding; DNA/chemistry/*metabolism; Flavanones/chemistry/metabolism/*pharmacology; Flavonoids/chemistry/pharmacology; Fluorescence Resonance Energy Transfer; Humans; Intercalating Agents/chemistry/metabolism/*pharmacology; Interleukin-1beta/*pharmacology; Molecular Docking Simulation; Nucleic Acid Conformation; Osteoarthritis; Osteoarthritis/metabolism/pathology; Protective Agents/chemistry/metabolism/*pharmacology; Reactive Oxygen Species/metabolism; Up-Regulation/drug effects; Wogonin
Wogonin has recently been shown to possess anti-inflammatory and chondroprotective properties and is of considerable interest due to its broad pharmacological activities. The present study highlights that Wogonin binds DNA and exerts chondroprotective effects in vitro. Wogonin showed strong binding with chondrocytes genomic DNA in vitro. The mode of binding of Wogonin to genomic-DNA was assessed by competing Wogonin with EtBr or DAPI, known DNA intercalator and a minor groove binder, respectively. EtBr fluorescence reduced significantly with increase in Wogonin concentration suggesting possible DNA intercalation of Wogonin. Further, in silico molecular docking of Wogonin on mammalian DNA also indicated possible intercalation of Wogonin with DNA. The denaturation and FRET studies revealed that Wogonin prevents denaturation of DNA strands and provide stability to genomic DNA against a variety of chemical denaturants. The cellular uptake study showed that Wogonin enters osteoarthritis chondrocytes and was mainly localized in the nucleus. Wogonin treatment to OA chondrocytes protects the fragmentation of genomic DNA in response to IL-1beta as evaluated by DNA ladder and TUNEL assay. Treatment of chondrocytes with Wogonin resulted in significant suppression of IL-1beta-mediated induction of ROS. Further, Wogonin exhibited protective potential through potent suppression of extrinsic and intrinsic apoptotic pathways and induction of anti-apoptotic proteins in
Khan Nazir M; Ahmad Imran; Ansari Mohammad Y; Haqqi Tariq M
Chemico-biological interactions
2017
2017-08
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.cbi.2017.06.025" target="_blank" rel="noreferrer noopener">10.1016/j.cbi.2017.06.025</a>
A standardized extract of Butea monosperma (Lam.) flowers suppresses the
*Butea; Aged; Autophagy; Autophagy/drug effects/physiology; Butea monosperma (Lam.); Chondrocytes/drug effects/*metabolism; Dose-Response Relationship; Drug; Flowers; Gene Expression; Humans; Interleukin-1beta/*pharmacology; Interleukin-6/*biosynthesis/genetics; Matrix Metalloproteinase 13/biosynthesis/genetics; Matrix Metalloproteinase 3/biosynthesis/genetics; Matrix Metalloproteinase 9/biosynthesis/genetics; Matrix Metalloproteinases/*biosynthesis/genetics; Middle Aged; mTOR; Nutraceuticals; Osteoarthritis; Osteoarthritis/*metabolism; Plant Extracts/isolation & purification/pharmacology
BACKGROUND/OBJECTIVE: Osteoarthritis (OA) is a leading cause of joint dysfunction, disability and poor quality of life in the affected population. The underlying mechanism of joint dysfunction involves increased oxidative stress, inflammation, high levels of cartilage extracellular matrix degrading proteases and decline in autophagy-a mechanism of cellular defense. There is no disease modifying therapies currently available for OA. Different parts of the Butea monosperma (Lam.) plant have widely been used in the traditional Indian Ayurvedic medicine system for the treatment of various human diseases including inflammatory conditions. Here we studied the chondroprotective effect of hydromethanolic extract of Butea monosperma (Lam.) flowers (BME) standardized to the concentration of Butein on human OA chondrocytes stimulated with IL-1beta. METHODS: The hydromethanolic extract of Butea monosperma (Lam.) (BME) was prepared with 70% methanol-water mixer using Soxhlet. Chondrocytes viability after BME treatment was measured by MTT assay. Gene expression levels were determined by quantitative polymerase chain reaction (qPCR) using TaqMan assays and immunoblotting with specific antibodies. Autophagy activation was determined by measuring the levels of microtubule associated protein 1 light chain 3-II (LC3-II) by immunoblotting and visualization of autophagosomes by transmission electron and confocal microscopy. RESULTS: BME was non-toxic to the OA chondrocytes at the doses employed and suppressed the IL-1beta induced expression of inerleukin-6 (IL-6) and matrix metalloprotease-3 (MMP-3), MMP-9 and MMP-13. BME enhanced autophagy in chondrocytes as determined by measuring the levels of
Ansari Mohammad Y; Khan Nazir M; Haqqi Tariq M
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
2017
2017-12
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.biopha.2017.09.140" target="_blank" rel="noreferrer noopener">10.1016/j.biopha.2017.09.140</a>
A Polyphenol-rich Pomegranate Fruit Extract Suppresses NF-kappaB and IL-6 Expression by Blocking the Activation of IKKbeta and NIK in Primary Human Chondrocytes.
chondrocytes; Chondrocytes – Drug Effects; Chondrocytes/drug effects; Fruit; Fruit/chemistry; Gene Expression Regulation/drug effects; Genes – Drug Effects; Humans; I-kappa B Kinase/genetics/*metabolism; IkappaB; IKKbeta; IL-1beta; Interleukin 1 – Metabolism; Interleukin-1beta/metabolism; Interleukin-6/genetics/*metabolism; Interleukins; Interleukins – Metabolism; NF-kappa B; NF-kappa B – Metabolism; NF-kappa B/genetics/*metabolism; NF-kappaB; NIK; osteoarthritis; Phosphorylation – Drug Effects; Phosphorylation/drug effects; Plant Extracts; Plant Extracts – Pharmacodynamics; Plant Extracts/chemistry/*pharmacology; Polyphenols – Pharmacodynamics; Polyphenols/pharmacology; pomegranate; Pomegranate; Protein-Serine-Threonine Kinases/genetics/*metabolism; Proteins – Metabolism; Punicaceae/*chemistry; Signal Transduction – Drug Effects; Signal Transduction/drug effects; Transcription Factor RelA/metabolism; Transferases; Transferases – Metabolism
Pomegranate fruit extract (PE) rich in polyphenols has been shown to exert chondroprotective effects, but the mechanism is not established. Here, we used an in vitro model of inflammation in osteoarthritis (OA) to investigate the potential of PE to suppress interleukin 1 beta (IL-1beta)-stimulated expression of inflammatory cytokine IL-6, generation of reactive oxygen species (ROS) levels, and investigated the mechanism of NF-kappaB inhibition by analyzing the activation of the kinases upstream of IkappaBalpha in primary human chondrocytes. Total and phosphorylated forms of kinases and expression of IL-6 were determined at protein and mRNA levels by western immunoblotting and Taqman assay, respectively. Dihydrorhodamine 123 staining estimated ROS generation. Pomegranate fruit extract inhibited the mRNA and protein expression of IL-6, generation of ROS, and inhibited the IL-1beta-mediated phosphorylation of inhibitor of nuclear factor kappa-B kinase subunit beta (IKKbeta), expression of IKKbeta mRNA, degradation of IkappaBalpha, and activation and nuclear translocation of
Haseeb Abdul; Khan Nazir M; Ashruf Omer S; Haqqi Tariq M
Phytotherapy research : PTR
2017
2017-05
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1002/ptr.5799" target="_blank" rel="noreferrer noopener">10.1002/ptr.5799</a>
Sucrose, But Not Glucose, Blocks IL1-beta-Induced Inflammatory Response in Human Chondrocytes by Inducing Autophagy via AKT/mTOR Pathway.
*AUTOPHAGY; *CHONDROCYTES; *OSTEOARTHRITIS; *SUCROSE; Adult; Aged; Autophagy/*drug effects; Chondrocytes/*metabolism/pathology; Female; Glucose/*pharmacology; Humans; Inflammation/chemically induced/metabolism/pathology; Interleukin-1beta/*pharmacology; Male; Middle Aged; Osteoarthritis/*metabolism/pathology; Proto-Oncogene Proteins c-akt/*metabolism; Signal Transduction/*drug effects; Sucrose/*pharmacology; TOR Serine-Threonine Kinases/*metabolism
Pathogenesis of osteoarthritis (OA) is multifactorial but interleukin-1beta (IL-1beta) is known to be an important mediator of cartilage degradation. Autophagy is an essential cellular homeostasis mechanism and has been proposed to protect against cartilage degradation and chondrocyte death under pathological conditions. We investigated the role of autophagy activated by sucrose, a natural disaccharide, in suppressing inflammatory mediator's expression and cell death under pathological conditions in human chondrocytes. Autophagy activation was investigated by Western blotting for LC3 and Beclin-1, immunofluorescence staining for LC3 puncta, and measuring autophagic flux. Activation of mTOR, AKT, and P70S6K was evaluated by Western blotting. Chondrocyte apoptosis was evaluated by propidium iodide (PI) staining using flowcytometry, expression of Bax by Western blotting, gene expression by TaqMan assays and caspase 3/7 activity was measured using a luminescence-based assay. We found that sucrose-induced active autophagy in OA chondrocytes in vitro was dependent on the activation of AKT/mTOR/P70S6K signaling pathways but was independent of reactive oxygen species (ROS) production. Sucrose activated autophagy blocked IL-1beta-induced apoptosis and mRNA expression of MMP-13, COX-2, and IL-6 in human OA chondrocytes. Glucose or fructose, the two metabolites of sucrose, failed to induce autophagy indicating that autophagy was specifically mediated by sucrose. In conclusion, sucrose attenuated IL-1beta induced apoptosis and the expression of catabolic mediators by inducing autophagy, and the autophagy in part was mediated through the activation of AKT/mTOR/P70S6K signaling pathway in human OA chondrocytes. J. Cell. Biochem. 118: 629-639, 2017. (c) 2016 Wiley Periodicals, Inc.
Khan Nazir M; Ansari Mohammad Y; Haqqi Tariq M
Journal of cellular biochemistry
2017
2017-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1002/jcb.25750" target="_blank" rel="noreferrer noopener">10.1002/jcb.25750</a>
Genetic inactivation of ZCCHC6 suppresses IL-6 expression and reduces the severity of experimental osteoarthritis in mice.
OBJECTIVE: Cytokine expression is tightly regulated post-transcriptionally but high levels of IL-6 in osteoarthritis (OA) indicate disruption of regulatory mechanisms. ZCCHC6 enzyme is implicated in post-transcriptional regulation of inflammatory cytokine expression but its role in OA pathogenesis is unknown. Here we studied whether ZCCHC6 directs the expression of IL-6 and influence OA pathogenesis in vivo. METHODS: Human and mouse chondrocytes were stimulated with recombinant IL-1beta. We knocked down the expression of ZCCHC6 in human chondrocytes by siRNAs. IL-6 transcript stability was determined by Actinomycin-D chase and 3'-uridylation of miRNAs was determined by deep sequencing. Zcchc6-/- mice were produced by gene targeting. OA was surgically induced in the knee joints of mice and the disease severity was scored using a semi-quantitative scoring system. RESULTS: ZCCHC6 was markedly upregulated in the damaged cartilage from human OA patients and from wild type mice with surgically-induced OA. Overexpression of ZCCHC6 induced the expression of IL-6 and its knockdown reduced the IL-6 transcript stability and IL-1beta-induced expression in chondrocytes. Reintroduction of Zcchc6 in Zcchc6-/- chondrocytes rescued the IL-1beta-induced
Ansari Mohammad Y; Khan Nazir M; Ahmad Nashrah; Green Jonathan; Novak Kimberly; Haqqi Tariq M
Arthritis & rheumatology (Hoboken, N.J.)
2018
2018-10
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1002/art.40751" target="_blank" rel="noreferrer noopener">10.1002/art.40751</a>