IGF-1 and PDGF-bb suppress IL-1β-induced cartilage degradation through down-regulation of NF-κB signaling: involvement of Src/PI-3K/AKT pathway.

Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that plays a key role in the pathogenesis of osteoarthritis (OA). Growth factors (GFs) capable of antagonizing the catabolic actions of cytokines may have therapeutic potential in the treatment of OA. Herein, we investigated the potential synergistic effects of insulin-like growth factor (IGF-1) and platelet-derived growth factor (PDGF-bb) on different mechanisms participating in IL-1β-induced activation of nuclear transcription factor-κB (NF-κB) and apoptosis in chondrocytes. Primary chondrocytes were treated with IL-1β to induce dedifferentiation and co-treated with either IGF-1 or/and PDGF-bb and evaluated by immunoblotting and electron microscopy. Pretreatment of chondrocytes with IGF-1 or/and PDGF-bb suppressed IL-1β-induced NF-κB activation via inhibition of IκB-α kinase. Inhibition of IκB-α kinase by GFs led to the suppression of IκB-α phosphorylation and degradation, p65 nuclear translocation and NF-κB-regulated gene products involved in inflammation and cartilage degradation (COX-2, MMPs) and apoptosis (caspase-3). GFs or BMS-345541 (specific inhibitor of the IKK) reversed the IL-1β-induced down-regulation of collagen type II, cartilage specific proteoglycans, β1-integrin, Shc, activated MAPKinase, Sox-9 and up-regulation of active caspase-3. Furthermore, the inhibitory effects of IGF-1 or/and PDGF-bb on IL-1β-induced NF-κB activation were sensitive to inhibitors of Src (PP1), PI-3K (wortmannin) and Akt (SH-5), suggesting that the pathway consisting of non-receptor tyrosine kinase (Src), phosphatidylinositol 3-kinase and protein kinase B must be involved in IL-1β signaling. The results presented suggest that IGF-1 and PDGF-bb are potent inhibitors of IL-1β-mediated activation of NF-κB and apoptosis in chondrocytes, may be mediated in part through suppression of Src/PI-3K/AKT pathway, which may contribute to their anti-inflammatory effects

Mummy Prevents IL-1β-Induced Inflammatory Responses and Cartilage Matrix Degradation via Inhibition of NF-қB Subunits Gene Expression in Pellet Culture System

Purpose: In Persian traditional medicine, application of Mummy material has been advised since hundred years ago for treatment of different diseases as bone fracture, cutaneous wounds and joint inflammation. Regarding to the claim of indigenous people for application of this material in the treatment of joint inflammation, the present study was designed to evaluate whether Mummy can revoke the inflammatory responses in chondrocytes stimulated with interleukin 1-β (IL-1β). Methods: Isolated chondrocytes at the second passage were plated in 50 ml conical tubes at density of 1x106 for pellet culture or were plated in T75 culture flasks as monolayer. Cells in both groups were treated as control (receiving serum free culture medium), negative control (receiving IL-1β (10ng/ml for 24 hr)) and IL-1β pre-stimulated cells which treated with Mummy at concentrations of 500 and 1000µg/ml for 72hrs. After 72 hrs, to evaluate whether Mummy can revoke the inflammatory response in chondrocytes, cell in different groups were prepared for investigation of gene expression profile of collagen II, Cox-2, MMP-13, C-Rel and P65 using real-time RT-PCR. Results: Treatment of chondrocytes with IL-1β (10ng/ml) resulted in a significant increase in expression level of Cox-2, MMP-13, C-Rel and P65 in pellet culture system, while treatment of IL-1β-stimulated choncrocytes with Mummy at both concentrations of 500 and 1000µg/ml inhibited the expression level of above mentioned genes. Compared to the pellet culture, Mummy did not affect expression level of genes in monolayer condition. Conclusion: The obtained data from this investigation revealed that Mummy can be used as a potent factor for inhibiting the inflammatory responses induced by IL-1β in chondrocytes probably through inhibition of NF-қB subunits activation

Effect of Platelet-Rich Plasma on Differentiation of Osteoblasts and Osteoclasts in the Presence of Three-Dimensional Scaffold

Background: Osteoblasts’ activity is prerequisite for prevention from and treatment of apical periodontitis and a relatively high proportion of endodontically treated teeth will require retrograde treatment in future. Therefore, the aim of the present study was to evaluate the effect of platelet-rich plasma (PRP) on differentiation of stem cells into osteoblasts and osteoclasts. Methods: Mesenchymal stem cells were isolated from human fetal umbilical cord and cultured on two polycaprolacton/hydroxyapatite (PCL/HA) polymer scaffolds. In addition to differentiation agents, 10% PRP was added to PRP containing subgroups. After 10 days, osteoblast differentiation was assessed evaluating the osteocalcin and osterix gene levels where, in the osteoclast differentiation group the expression of tartarate-resistant acid phosphatase (TRAP) gene was evaluated. Results: Expression of TRAP gene did not reveal any significant differences between the study and control groups. There was a significant difference in osterix expression between the control and the PRP-treated groups (p < 0.01) as well as osteocalcin gene (p < 0.05). Conclusion: The results showed that PRP increased the osteoblastic differentiation, while it does not cause any significant increase in osteoclastic differentiation

Mummy Material Can Promote Differentiation of Adipose Derived Stem Cells into Osteoblast through Enhancement of Bone Specific Transcription Factors Expression

Purpose: Application of Mummy material for treatment of different diseases such as bone fracture, cutaneous wounds and joint inflammation has been advised since hundred years ago in Persian traditional medicine. Due to the claims of indigenous people and advice of traditional medicine for application of this material in healing of bone fractures, this study has been designed to evaluate whether Mummy material can promote the differentiation of mesenchymal stem cells into osteoblasts and enhance the expression of bone specific genes and proteins. Methods: Adipose derived stem cells (ASCs) at fourth cell passage were divided into control, osteogenesis group (received osteogenic medium), Mummy group (received Mummy at concentration of 500 µg/ml). ASCs in the fourth group were treated with both osteogenic medium and Mummy (500µg/ml). Cells in all groups were harvested on days 7, 14 and 21 days for further evaluation through Real time RT-PCR, Von kossa staining, Immunocytochemistry and flowcytometery. Results: Treatment of ASCs with Mummy at concentration of 500µg/ml promotes the expression level of Osteocalcin, RUNX-2 and β1-integrin genes in different time points but that of the Osterix did not changed. Furthermore the expression of Osteocalcin protein enhanced significantly in ASCs treated with Mummy detected by Immunocytochemistry and flowcytometery technique compared to the control groups. The results of this study also showed that treatment of ASCs with Mummy resulted in formation of mineral deposits which was evaluated by Von Kossa staining method. Conclusion: Obtained data from this study reveals that Mummy is a potent enhancer for differentiation of ASCs into osteoblasts in in vitro system, probably through increasing the level of bone specific genes and proteins

Effect of hydroxychloroquine on oxidative/nitrosative status and angiogenesis in endothelial cells under high glucose condition

Introduction: Under the diabetic condition, sustained production of oxidative/nitrosative stress results in irreversible vascular injuries. A great number of diabetic pathologies, such as inefficient or aberrant neo-angiogenesis emerge following chronic hyperglycemic condition. Lack of enough data exists regarding hydroxychloroquine (HCQ) contribution on angiogenesis during diabetes mellitus. Methods: To better address whether HCQ could blunt or exacerbate oxidative status and angiogenesis under high glucose condition (HCG), human umbilical vein endothelial cells (HUVECs) were exposed to 30 µM HCQ in combination with 30 mM glucose over a course of 72 hours. Viability was measured was evaluated by MTT assay. We used Griess method and TBARS assay to monitor changes in the levels of NO and MDA followed by flow cytometric analysis of ROS using DCFDA. To show the impact of HCQ on cell motility and in vitro angiogenic properties, we exploited routine scratch test and in vitro tubulogenesis, respectively. Results: Our data showed that HCQ diminished cell viability under 5 and 30 mM glucose contents. HCQ significantly decreased the total levels of nitric oxide (NO), malondialdehyde (MDA), and reactive oxygen species (ROS) in both sets of environments. Additionally, inhibitory effects were observed on cell migration after exposure to HCQ (P &lt; 0.001). Anti-angiogenic activity of HCQ was confirmed by the reduction of tube areas under a normal or surplus amount of glucose (P &lt; 0.001). Conclusion: In overall, results suggest that HCQ changes the oxidative/nitrosative status of HUVECs both in 5 and 30 mM conditions. HCQ is able to reduce migration and angiogenic activity of HUVECs irrespective of the glucose content

A Study of the Protective Effects of Vitamin E and Fennel Extract on Mitochondria Changes in Mice Ovary Due to Electromagnetic Field Exposure

Objective: Everyday use of different types of electrical instruments and appliances has caused a large number of people to constantly be under the influence of electromagnetic fields. Materials and Methods: For the purpose of this study, 40 female rats were randomly chosen from among 3 months old rats from the animals’ laboratory and they weighed 20 + 200 g. Then, they were randomly divided into 4 groups; control (n = 10), experiment 1 (Ex1) (n = 10), experiment 2 (Ex2) (n = 10), and experiment 3 (Ex3) (n = 10). During the experiment, all 4 groups were maintained in the same conditions and received the same feeding. The experiment groups 1, 2, and 3 were under the influence of a 50 Hz electromagnetic field (EMF) for 8 weeks. Subsequently, the second and third groups were kept away from the EMF effect for another 8 weeks. At the end of the study, after removal of the ovaries by glutaraldehyde, they were prepared for examination using an electron microscope. Group Ex2 rats were not sacrificed and were maintained in the normal laboratory environment for another 8 weeks away from the impacts of EMF. The rats were fed vitamin E(100 mg/kg) and fennel extract (1.5 g per body weight) every day orally and at the end of the second 8 weeks samples were taken. During the second 8 weeks, group Ex3 was kept in normal conditions without the use of vitamin E and fed fennel extract, and then, samples were taken. Samples were taken simultaneously from 10 rats of the control group and Ex1 group. Results: The results from the mitochondria in the ovary in the groups under the influence of electromagnetic waves indicated that this intracellular organ, compared to samples from the control group, was deformed and the majority of the organs were vacuolated. The mitochondrial vacuolization of the first to fourth groups were 1 ± 0.55, 9 ± 0.55, 6 ± 0.55, and 11 ± 0.55, respectively. Conclusion: Vitamin E and fennel extract can reduce the damaging effects of non-ionizing radiation with 50 Hz frequency on the ovarian follicles

An Ultrastructural Study of the Antioxidant Effects of Vitamin E and Fennel Extract on Zona Pellucida Cell Changes of Rat Ovaries under Non-Ionizing 50Hz Electromagnetic Fields

Objective: Everyday use of various electronic tools and appliances has caused a large number of people to constantly be under the influence of electromagnetic fields (EMFs). Materials and Methods: For the purpose of this study, 40 female rats were randomly selected from the animals’ laboratory. The rats chosen for the study were 3 months old and weighted 20 + 200 g. The animals were then randomly divided into 4 groups; Control (n = 10), Experiment 1 (n = 10), Experiment 2 (n = 10), and Experiment 3 (n = 10). During the experiment, all 4 groups were maintained in the same conditions and received the same feeding procedure. Test groups 1, 2, and 3 were under the influence of a 50 Hz EMF for 8 weeks. Subsequently, the second and third groups were kept away from the effects of EMF for another 8 weeks. At the end of the study, after removing the ovarian using glutaraldehyde, they were prepared for electron microscopy study. Ex2 group rats were not sacrificed, and were maintained for another 8 weeks in normal laboratory environment away from the impacts of EMF. The rats were fed vitamin E (100 mg/kg) and fennel extract (1.5 gr/kg/body weight) was added to their daily food. Samples were taken from this group at the end of the second 8 weeks. Samples from the Ex3 group were taken at the end of the second 8 weeks which were maintained in normal conditions without the use of vitamin E and fennel extract. The 10 rats from the control group were biopsied simultaneously with the Ex1 group sampling. Results: This study showed that in the groups that had been exposed to electromagnetic radiation, zona pellucida cells had lost their microvilli and mitochondrial crystal structure. In the groups that were exposed to vitamin E and fennel extract, these changes were reduced. Conclusion: The use of vitamin E in combination with fennel extract can reduce the damaging effects of non-ionizing radiation with 50 Hz frequency on the zona pellucida cells of rat ovaries

Study of Foeniculum vulgare (Fennel) Seed Extract Effects on Serum Level of Estrogen, Progesterone and Prolactin in Mouse

Objective: The Foeniculum vulgare (FVE) or fennel has a long history of use as both a food and medicine. The seed of this plant has been used to promote menstruation, alleviate the symptoms of female climacteric, and increase the number of ovarian follicles. The aim of this study is to evaluate the fennel extract effects on serum level of estrogen, progesterone and prolactin in female mice. Materials and Methods: A total of 28 virgin female albino mice were divided into four groups (n = 7). Groups 1 and 2 (experimental groups) were administered FVE at 100 and at a concentration of 100 and 200 mg/kg for 5 days, intraperitoneally. Group 3 (negative control) received ethanol and Group 4 (positive control) received normal saline. Animals were scarified at 6th day, sera were collected and the level of estrogen, progesterone, and prolactin hormones was analyzed using Elisa Kit. Results: Data analysis revealed that there is a significant difference in the mean level of serum estrogen, progesterone and prolactin between four different groups. P value in experimental groups compared with the control groups was (P < 0.0001). Conclusion: Fennel extract can increase the serum level of estrogen, progesterone, and prolactin in female mice; it can be introduced as a novel medicine for treatment of infertilit

The Role of Autophagy in Osteoclast Differentiation and Bone Resorption Function

Autophagy is an evolutionary conserved and highly regulated recycling process of cellular wastes. Having a housekeeping role, autophagy through the digestion of domestic cytosolic organelles, proteins, macromolecules, and pathogens, eliminates unnecessary materials and provides nutrients and energy for cell survival and maintenance. The critical role of autophagy and autophagy-related proteins in osteoclast differentiation, bone resorption, and maintenance of bone homeostasis has previously been reported. Increasing evidence reveals that autophagy dysregulation leads to alteration of osteoclast function and enhanced bone loss, which is associated with the onset and progression of osteoporosis. In this review, we briefly consolidate the current state-of-the-art technology regarding the role of autophagy in osteoclast function in both physiologic and pathologic conditions to have a more general view on this issue