Advanced Glycation End Products Induce PeroxisomeProliferator-Activated Receptor c Down-Regulation-Related Inflammatory Signals in Human Chondrocytesvia Toll-Like Receptor-4 and Receptor for AdvancedGlycation End Products

Accumulation of advanced glycation end products (AGEs) in joints is important in the development of cartilage destruction and damage in age-related osteoarthritis (OA). The aim of this study was to investigate the roles of peroxisome proliferator-activated receptor γ (PPARγ), toll-like receptor 4 (TLR4), and receptor for AGEs (RAGE) in AGEs-induced inflammatory signalings in human OA chondrocytes. Human articular chondrocytes were isolated and cultured. The productions of metalloproteinase-13 and interleukin-6 were quantified using the specific ELISA kits. The expressions of related signaling proteins were determined by Western blotting. Our results showed that AGEs enhanced the productions of interleukin-6 and metalloproteinase-13 and the expressions of cyclooxygenase-2 and high-mobility group protein B1 and resulted in the reduction of collagen II expression in human OA chondrocytes. AGEs could also activate nuclear factor (NF)-κB activation. Stimulation of human OA chondrocytes with AGEs significantly induced the up-regulation of TLR4 and RAGE expressions and the down-regulation of PPARγ expression in a time- and concentration-dependent manner. Neutralizing antibodies of TLR4 and RAGE effectively reversed the AGEs-induced inflammatory signalings and PPARγ down-regulation. PPARγ agonist pioglitazone could also reverse the AGEs-increased inflammatory signalings. Specific inhibitors for p38 mitogen-activated protein kinases, c-Jun N-terminal kinase and NF-κB suppressed AGEs-induced PPARγ down-regulation and reduction of collagen II expression. Taken together, these findings suggest that AGEs induce PPARγ down-regulation-mediated inflammatory signalings and reduction of collagen II expression in human OA chondrocytes via TLR4 and RAGE, which may play a crucial role in the development of osteoarthritis pathogenesis induced by AGEs accumulation

Detection of subtle neurological alterations by the Catwalk XT gait analysis system

BACKGROUND: A new version of the CatWalk XT system was evaluated as a tool for detecting very subtle alteration in gait based on higher speed sample rate; the system could also demonstrate minor changes in neurological function. In this study, we evaluated the neurological outcome of sciatic nerve injury intervened by local injection of hyaluronic acid. Using the CatWalk XT system, we looked for differences between treated and untreated groups and differences within the same group as a function of time so as to assess the power of the Catwalk XT system for detecting subtle neurological change. METHODS: Peripheral nerve injury was induced in 36 Sprague–Dawley rats by crushing the left sciatic nerve using a vessel clamp. The animals were randomized into one of two groups: Group I: crush injury as the control; Group II: crush injury and local application with hyaluronic acid. These animals were subjected to neurobehavior assessment, histomorphology evaluation, and electrophysiology study periodically. These data were retrieved for statistical analysis. RESULTS: The density of neurofilament and S-100 over the distal end of crushed nerve showed significant differences either in inter-group comparison at various time points or intra-group comparison from 7 to 28 days. Neuronal structure architecture, axon counts, intensity of myelination, electrophysiology, and collagen deposition demonstrate significant differences between the two groups. There was significant difference of SFI and angle of ankle in inter- group analysis from 7 to 28 days, but there were no significant differences in SFI and angle of ankle at time points of 7 and 14 days. In the Cat Walk XT analysis, the intensity, print area, stance duration, and swing duration all showed detectable differences at 7, 14, 21, and 28 days, whereas there were no significant difference at 7 and 14 days with CatWalk 7 testing. In addition, there were no significant differences of step sequence or regularity index between the two versions. CONCLUSION: Hyaluronic acid augmented nerve regeneration as early as 7 days after crush injury. This subtle neurological alteration could be detected through the CatWalk XT gait analysis but not the SFI, angle of ankle, or CatWalk 7 methods

Honokiol Induces Calpain-Mediated Glucose-Regulated Protein-94 Cleavage and Apoptosis in Human Gastric Cancer Cells and Reduces Tumor Growth

Background. Honokiol, a small molecular weight natural product, has been shown to possess potent anti-neoplastic and anti-angiogenic properties. Its molecular mechanisms and the ability of anti-gastric cancer remain unknown. It has been shown that the anti-apoptotic function of the glucose-regulated proteins (GRPs) predicts that their induction in neoplastic cells can lead to cancer progression and drug resistance. We explored the effects of honokiol on the regulation of GRPs and apoptosis in human gastric cancer cells and tumor growth. Methodology and Principal Findings. Treatment of various human gastric cancer cells with honokiol led to the induction of GRP94 cleavage, but did not affect GRP78. Silencing of GRP94 by small interfering RNA (siRNA) could induce cell apoptosis. Treatment of cells with honokiol or chemotherapeutics agent etoposide enhanced the increase in apoptosis and GRP94 degradation. The calpain activity and calpain-II (m-calpain) protein (but not calpain-I (mu-calpain)) level could also be increased by honokiol. Honokiol-induced GRP94 down-regulation and apoptosis in gastric cancer cells could be reversed by siRNA targeting calpain-II and calpain inhibitors. Furthermore, the results of immunofluorescence staining and immunoprecipitation revealed a specific interaction of GRP94 with calpain-II in cells following honokiol treatment. We next observed that tumor GRP94 over-expression and tumor growth in BALB/c nude mice, which were inoculated with human gastric cancer cells MKN45, are markedly decreased by honokiol treatment. Conclusions and Significance. These results provide the first evidence that honokiol-induced calpain-II-mediated GRP94 cleavage causes human gastric cancer cell apoptosis. We further suggest that honokiol may be a possible therapeutic agent to improve clinical outcome of gastric cancer

Inhibition of Nadph Oxidase-Related Oxidative Stress-Triggered Signaling by Honokiol Suppresses High Glucose-Induced Human Endothelial Cell Apoptosis

Angiopathy is a major complication of diabetes. Abnormally high blood glucose is a crucial risk factor for endothelial cell damage. Nuclear factor-kappaB (NF-kappaB) has been demonstrated as a mediated signaling in hyperglycemia or oxidative stress-triggered apoptosis of endothelial cells. Here we explored the efficacy of honokiol, a small molecular weight natural product, on NADPH oxidase-related oxidative stress-mediated NF- kappaB-regulated signaling and apoptosis in human umbilical vein endothelial cells (HUVECs) under hyperglycemic conditions. The methods of morphological Hoechst staining and annexin V/propidium iodide staining were used to detect apoptosis. Submicromolar concentrations of honokiol suppressed the increases of NADPH oxidase activity, Rac-1 phosphorylation, p22(phox) protein expression, and reactive oxygen species production in high glucose (HG)-stimulated HUVECs. The degradation of IkappaBalpha and increase of NF-kappaB activity were inhibited by honokiol in HG-treated HUVECs. Moreover, honokiol (0.125-1 microM) also suppressed HG-induced cyclooxygenase (COX)-2 upregulation and prostaglandin E(2) production in HUVECs. Honokiol could reduce increased caspase-3 activity and the subsequent apoptosis and cell death triggered by HG. These results imply that inhibition of NADPH oxidase-related oxidative stress by honokiol suppresses the HG-induced NF-kappaB-regulated COX-2 upregulation, apoptosis, and cell death in HUVECs, which has the potential to be developed as a therapeutic agent to prevent hyperglycemia-induced endothelial damage

Studies on the cellular signaling pathways in hyperglycemia-induced cell proliferation and apoptosis

糖尿病為一常見的內分泌代謝疾病，目前已經成為公共衛生問題的一部份。在世界各地隨著肥胖人數日驅增加，糖尿病的人數也隨之顯著提高。而糖尿病對身體造成最大的問題是對實質性組織器官的傷害；尤其是心血管系統以及腎臟的影響。也由於在世界各地糖尿病的病人愈來愈多，其導致的複雜變化以及合併病發症更值得我們去重視。 糖尿病病人體內血糖過高的訊息是不可逆的並且最後導致組織器官的功能障礙或損傷。由過去的文獻報導以及臨床病人報告中已知：糖尿病的病理過程可引起血管內皮細胞功能缺失，造成的血管功能障礙、加速冠狀動脈硬化並對預後不佳。然而進一步地導致血管內皮細胞凋亡的機制至今仍不十分清楚。探討此一異常訊息是如何造成人類血管內皮細胞凋亡？其機制為何？進而如何避免糖尿病在人類心血管系統功能障礙及合併症的產生，以及病程的惡化是十分重要的課題。於本論文第一部分的研究主要為探討高血糖的狀態下誘導人類血管內皮細胞凋亡之機制，以及PI3K所調控之環氧化蛋白酶表現在其中的角色。本實驗發現暴露於高血糖的環境中皆可測得早期血管內皮細胞凋亡訊號。在細胞凋亡的形態學上以Hoechst染色觀察細胞形態以及以Annexin V/Propidium Iodide偵測早期細胞凋亡訊號。高血糖狀態也會誘導環氧化蛋白酶的表現增加，同時也誘導環氧化蛋白酶的下游產物PGE2生成，進而促使凋亡酶-3 (caspase-3)活性增強而導致細胞走向凋亡。出乎意外地，給予PI3K抑制劑皆可有效地抑制高血糖狀態之環氧化蛋白酶蛋白表現、環氧化蛋白酶的下游產物PGE2生成、凋亡酶-3活性和細胞凋亡。高血糖誘導PI3K活化也依序促使Akt的磷酸化。更進一步地，高血糖引發氧化自由基的生成和NFDiabetes has become a public health crisis. With the incidence of obesity rising in the world, the number of diabetics will grow considerably. Of greatest concern is the impact this trend will have on damage to the kidneys and cardiovascular disease. The incidence of diabetes is increasing worldwide, with subsequent increase in the incidence of diabetic complication. The hyperglycemic signal exchange occurs ubiquitously and irreversibly in patients with diabetes mellitus, and its consequences are especially relevant to organ dysfunctions. In type-2 diabetes, a greater proportion of patients have overt nephropathy at shortly and vascular complications after diagnosis of diabetes. In this thesis, the studies are divided into two parts for description. Diabetes has been demonstrated to accelerate vascular dysfunction, coronary atherosclerosis, and the prognosis were worse following cardiac events. Therefore, the part I will investigate the regulation of abnormal signalings that promote human umbilical vein endothelial cells apoptosis under high glucose condition and relevant in understanding the intracellular signaling associated with vascular disease and preventing the development of vascular complication in diabetics, principally the evolution of this practice from its beginning until cell death. Diabetes mellitus causes endothelial dysfunction. The precise molecular mechanisms by which hyperglycemia causes apoptosis in endothelial cells are not yet well understood. The aim of this study was to explore the role of cyclooxygenases-2 (COX-2) and the possible involvement of phosphoinositide 3-kinase (PI3K) signaling in high glucose (HG)-induced apoptosis in human umbilical vein endothelial cells (HUVECs). For detection of apoptosis, the morphological Hoechst staining and Annexin V/Propidium Iodide staining were used. Glucose up-regulated COX-2 protein expression, which was associated with the induction of prostaglandin E2 (PGE2), caspase-3 activity and apoptosis. Unexpectedly, we found that PI3K inhibitors could suppress COX-2 expression, PGE2 production, caspase-3 activity, and the subsequent apoptosis under HG condition. Glucose-induced activation of PI3K resulted in the down stream effector Akt phosphorylation. PI3K inhibitors effectively attenuated the intracellular reactive oxygen species (ROS) generation and NF-TABLE OF CONTENTS 中文摘要 1 Abstract 5 List of abbreviations 9 PART I CHAPTER 1 Introduction 1.1 Diabetes "Basics" 11 1.2 Classification 11 1.3 Signs and complications 14 1.4 Endothelium cellS in cardiovascular system 16 1.5 The role of transcription factors in human umbilical vein endothelial cells pathophysiology of diabetes 17 1.6 The role of cyclooxygenase-2 (COX-2) in human umbilical vein endothelial cells pathophysiology of diabetes 18 1.7 The functions of glomerular mesangial cell 20 1.8 The role of glomerular mesangial cell in pathophysiology of diabetes 20 1.9 The role of COX-2 in glomerular mesangial cell 22 1.10 The role of NF- B in glomerular mesangial cell 24 1.11 The role of PI3K in glomerular mesangial cell 25 1.12 Purpose and rationales of present research 27 CHAPTER 2 Materials and Methods 28 2.1 Primary culture HUVECs 29 2.2 Apoptosis and Caspase-3/cpp32 activity 29 2.3 Phosphoinositide 3-kinase (PI3K) activity 30 2.4 Immunoblotting 30 2.5 Electrophoretic mobility shift assay (EMSA) 31 2.6 Reactive oxygen species production 31 2.7 PGE2 production 32 2.8 Mesangial cell culture 32 2.9 Cell proliferation assay MTS Assay 33 [3H]Thymidine incorporation 33 2.10 RT-PCR for COX-2 expression 34 2.11 Preparation of nuclear extracts 35 2.12 Transient transfection with dominant-negative vectors-DN-P85 and DN-Akt 35 2.13 Immunofluorescence staining for NF- B 36 2.14 Statistical analyses 36 CHAPTER 3 Results in endothelial cells 38 3.1 COX-2 protein expression and involvement in high glucose (HG)-induced apoptosis 38 3.2 PI3K activity and involvement in HG-induced COX-2 protein expression and apoptosis 38 3.3 Effects of PI3K inhibitors on HG-induced NF

High Glucose Induces Human Endothelial Cell Apoptosis through a Phosphoinositide 3-Kinase-Regulated Cyclooxygenase-2 Pathway

Objectives-: Diabetes mellitus causes endothelial dysfunction. The precise molecular mechanisms by which hyperglycemia causes apoptosis in endothelial cells are not yet well understood. The aim of this study was to explore the role of cyclooxygenase-2 (COX-2) and the possible involvement of phosphoinositide 3-kinase (PI3K) signaling in high glucose (HG)-induced apoptosis in human umbilical vein endothelial cells (HUVECs). Methods and Results-: For detection of apoptosis, the morphological Hoechst staining and Annexin V/propidium iodide staining were used. Glucose upregulated COX-2 protein expression, which was associated with the induction of prostaglandin (PG) E2 (PGE2), caspase- 3 activity, and apoptosis. Unexpectedly, we found that PI3K inhibitors could suppress COX- 2 expression, PGE2 production , caspase-3 activity, and the subsequent apoptosis under HG condition. Glucose-induced activation of PI3K resulted in the downstream effector Akt phosphorylation. PI3K inhibitors effectively attenuated the intracellular reactive oxygen species (ROS) generation and nuclear factor [kappa]B (NF-[ kappa]B) activation. Blocking the PI3K and Akt activities with the dominant-negative vectors greatly diminished the HG -triggered NF-[kappa]B activation and COX-2 expression and apoptosis. Conclusions-: These results suggest that HG, via PI3K/Akt signaling, induces NF-[kappa]B-related upregulation of COX-2, which in turn triggers the caspase-3 activity that facilitates HUVEC apoptosis. Also, HG may cause ROS generation in HUVECs through a PI3K/Akt-dependent pathway

Effects of honokiol on the expressions of GRP94 and GRP78 in human gastric cancer cell lines.

<p>(A) Western blot analyses for GRP94 and GRP78 in MKN45 cells treated with honokiol for 8 h in a dose-response manner. (B) Western blot analyses for GRP94 and GRP78 in MKN45 cells treated with honokiol (a, 20µM; b, 40 µM) in a time-response manner. (C) Comparison of GRP94 protein expression among human gastric cancer cell lines (SCM-1, AGS, N87, and MKN45), tumor isolated from MKN45 cells-inoculated mice (T), normal mouse gastric epithelium tissue (N), and human umbilical vein endothelial cells (HUVEC). All results shown are representative of at least four independent experiments.</p

Effects of honokiol on calpain-I and II protein levels and interaction of calpain and GRP94.

<p>Cells were treated with honokiol (20–60 µM) for various time courses as indicated. (A) Calpain-I and II protein levels were detected by Western blot analysis in honokiol-treated SCM-1 cells. (B) Primary antibodies for calpain-II and GRP94 were applied to the cells (MKN45 and SCM-1) followed by secondary antibodies coupled with FITC-conjugated or TRITC-conjugated, respectively. Co-localization of two labeled antigens was detected as a single image when the images from both channels were overlaid. (C) Interaction of calpain and GRP94 were detected in N87, AGS, MKN45 and SCM-1 cells. Immunoprecipitated proteins were collected and subjected to SDS-PAGE and immunoblotting with anti-calpain-II or anti-GRP94 antibodies. Results shown are representative of at least four independent experiments.</p

Honokiol induces calpain-mediated GRP94 degradation and apoptosis.

<p>Western blotting for determination of GRP94 degradation and calpain-I and II expression and activation of caspase-7 and caspase-12 in SCM-1 cells 24 h after honokiol (40 µM) treatment in the presence or absence of calpain inhibitors (ALLN and ALLM, 25 and 50 µM) was detected. In some experiments, SCM-1 cells were transfected with calpain-II-siRNA or control-siRNA. Results shown are representative of at least four independent experiments.</p