Engineering of Microparticle Encapsulated Antioxidant to Mitigate Oxidative Stress in vitro and in vivo

Chronic low back pain (cLBP) is one of the leading causes of years lived with disability in the United States. Current treatments for cLBP have variable results across the patient population, and many patients struggle to find consistent relief. Most current treatments focus on the symptom of pain, not the root cause. In painful degenerated discs, oxidative stress and inflammation function in a vicious cycle and perpetuate degeneration, damage, and pain. Therefore, oxidative stress and inflammation are important targets in addressing the source of pain. This work characterizes an antioxidant, BuOE, encapsulated in a chondroitin sulfate microparticle as a novel treatment for cLBP. This work encompasses the evaluation of the drug’s abilities in a cellular model of oxidative stress, the fabrication and characterization of BuOE encapsulated in a carrier vehicle, and effect of the encapsulated treatment on a rodent model of cLBP. BuOE demonstrated the ability to reduce ROS in a stressed cellular environment. The encapsulated BuOE functioned as an antioxidant for 80 days and exhibited controlled release, was not cytotoxic, and was able to be engulfed by native disc cells. Interestingly, the treatment demonstrated mixed results in behavioral assays, exhibiting possible pain-like behavior trends in non-evoked assays, but not in evoked assays. These results demonstrate the ability of encapsulated BuOE to reduce ROS and the potential of the therapeutic to improve function in cLBP patients. Further work will be conducted to evaluate the mechanism of the treatment and the ability of encapsulated BuOE to reduce pain. Advisor: Rebecca A. Wach

Tea

This book addresses in a succinct way some of the state-of-the-art studies on the chemistry and pharmacology of teas. It starts with some of the reasons why tea is called the elixir of life, and looks at the world consumption of tea and its role in many western and eastern cultures. The book proceeds with a systematic study that establishes the predominant compositions of different types of tea. The effects of tea constituents on health are discussed, and a final chapter discusses some of the potential applications of tea in the food industry

Reactive Oxygen Species

The term “reactive oxygen species” (ROS) refers to a group of reactive molecules and free radicals produced by molecular oxygen. In recent decades, there has been great interest in the role of ROS in various diseases. From basic science research to clinical trials, biomedical scientists have made rapid progress toward a better understanding of ROS-metabolizing systems and their role in health and diseases. This book includes sixteen chapters that address topics such as the history of ROS, its role in autoimmunity, neurodegeneration, and aging, and recent advances in various antioxidants and their therapeutic potential

The effect of concurrent and non-concurrent (delayed) administration of mepivacaine hydrochloride and triamcinolone acetonide on inflamed equine osteochondral and synovial explants in co-culture

During equine lameness investigation, intra-articular administration of a local anesthetic followed by a corticosteroid is common for diagnosis and treatment of osteoarthritis (OA). Anecdotally, veterinarians are advised to separate diagnosis and treatment to allow recovery of articular tissues from the inflammatory effect of the local anesthetic; however, no scientific evidence supports this need for rest. 5 geldings and 1 mare (aged 3-18 years) were euthanized for reasons unrelated to the study. From each horse, 48 synovial explants and 12 osteochondral explants were harvested from the stifles and transferred to a 12-well plate (2 wells per group, 2 synovial explants and 1 osteochondral explant per well). Explants were stimulated with 10 μg/ml recombinant equine interleukin-1β (IL-1β) and 10 μg/ml tumour necrosis factor-α (TNF-α). They were treated as 6 groups: unstimulated control, stimulated control, triamcinolone acetonide (TA, 10-6 M), mepivacaine hydrochloride (MH, 4.4 mg/ml), MH + TA (concurrent) and MH + TA (delayed). The delayed group was treated with MH and, six days later, treated with TA. Media were replenished and analysed every 3 days for 9 days total. ELISA analyses for markers of tissue cytotoxicity (lactate dehydrogenase, LDH), inflammatory eicosanoids (prostaglandin E2, PGE2), and markers of matrix degradation (matrix metalloproteinase 13, MMP-13 and dimethylmethylene blue assay, DMMB, for glycosaminoglycan, GAG) were performed. Data were analysed with 2-way ANOVA or mixed-effects models with Tukey’s multiple comparisons. There were no differences in LDH, PGE2, MMP-13 and GAG between delayed and concurrent groups at any time point; no differences were found in culture medium levels of cytotoxicity, inflammation or matrix degradation between explants treated concurrently with MH and TA and those that had a 6-day delay between MH and TA treatments. In this model, no benefit of a 6-day delay between local anaesthetic and corticosteroid treatments was found

Complexation of syringic and ρ-coumaric acids with zinc(ii) to improve their antidiabetic efficacy

Thesis (Doctor of Health Sciences: Biomedical Technology)--Central University of TechnologyBackground: The concept of Zn(II) complexation has long been explored as a possible scientific approach to developing potential therapeutic agents for managing impaired glucose tolerance in diabetes. This has been partly due to the safety profile of zinc mineral and its functional role in insulin preservation, secretion and function. Many types of ligands have been explored as suitable ligands to develop potent antidiabetic Zn(II) complexes. However, due to the critical role of oxidative stress in the development and progression of diabetes and vascular complications, the use of polyphenols as alternative ligands for antidiabetic Zn(II) complexes has been explored by scientists. It is perceived that the antioxidant attributes of natural polyphenols may afford a Zn(II) complex an antioxidant perspective in combating diabetic oxidative stress and reducing the risk of diabetic complications. Data from some studies that have complexed zinc with dietary flavonoids support this perception to an appreciable extent. However, natural phenolic acids, which are known dietary antioxidant have been scarcely explored as antioxidant ligands for developing antidiabetic Zn(II) complexes. Therefore, in the present study, p-coumaric acid and syringic acid were used as alternative ligands to develop Zn(II) complexes, due to their promising antioxidant attributes and some other diabetes-related pharmacological attributes. Methodology: p-Coumaric acid and syringic acid were separately complexed with ZnSO4 in a 1:2 mole ratio, respectively. The complexes were characterized by spectroscopically using FT-IR, proton NMR and high-resolution mass spectroscopy. The cytotoxic effect of the complexes was evaluated in Chang liver cells and L-6 myotubes. The antioxidant and antihyperglycaemic potential of the complex and precursors were evaluated with different experimental models. Molecular docking with target proteins linked to diabetes was performed. The antioxidant and antihyperglycaemic potential of the complexes, relative to their respective precursors were determined using in vitro, cellular and ex vivo experimental models. The complexes and their respective phenolic acids were comparatively docked against protein targets linked to diabetes. The Zn(II) complex of syringic acid was subjected to in vivo antidiabetic and antioxidant study in diabetic rats. Diabetes was induced in SD rats using 10% fructose and 40 mg/kg bw streptozotocin and thereafter subjected to a 4-week treatment with the complex and its precursors (Syringic acid and zinc sulphate) for 4 weeks at predetermined doses. Then, the effect of the treatments on diabetes and oxidative stress related parameters was measured. Results: Spectroscopic analysis showed the complexes were formed as a double hydrate Zn(II)- biphenolate product [Zn(II)-bicoumarate.2H2O and Zn(II)-bisyringate.2H2O complexes], which each had a moiety of Zn(II) and 2 moieties of their respective phenolic acids. The in vitro radical scavenging, α-glucosidase inhibitory, antiglycation and anti-lipid peroxidative activities of the complexes were several folds stronger than their respective phenolic acids. In Chang liver cells and rat liver tissues, the complexes inhibited lipid peroxidation and GSH depletion, which was notably stronger than their respective phenolic acids and comparable to ascorbic acid. Zn(II) and the phenolic acids synergistically modulated glucose uptake in L-6 myotubes and rat muscle tissue, which may be majorly influenced by to the observed complexation-mediated increase in muscle zinc uptake. Tissue glucose uptake activity of the complexes was accompanied by increased muscle hexokinase activity, suggesting increased glucose utilization. Moreover, treatment increased muscle phospho-Akt/pan-Akt ratio, while the complexes had stronger molecular docking interaction with insulin signalling protein targets (GLUT-4, Akt/PKB, insulin receptor tyrosine kinase and IRS-1) than their respective phenolic acid precursors. The Zn(II)- bisyringate.2H2O complex was more potent than the Zn(II)-bicoumarate.2H2O, thus was subjected to in vivo antidiabetic and antioxidant study in diabetic rats. The complex improved diabetic polyphagia, polydipsia and weight loss. Complexing Zn(II) with syringic acid improved their antihyperglycaemic action by ∼28 – 36% and ∼37 - 40%, respectively, suggesting a complexationmediated synergism. This may be attributed to the observed modulatory action of the complex on insulin secretion and sensitivity, tissue glycogen production, muscle hexokinase activity and Akt phosphorylation, thus improving glucose tolerance in diabetic rats, relative to its precursors. Concomitantly, the complex reduced systemic and tissue lipid peroxidation and increased antioxidant enzymes activity in the diabetic rats, while outperforming its precursors. In some cases, the antidiabetic action of the complex was comparable to metformin. The molecular properties of the complexes (i.e., the Zn(II) and biphenolate moieties) appears to be influential in the improved bioactivities of the complexes relative to their respective precursors, suggesting a complexation mediated synergistic potential. Conclusion: Complexing Zn(II) with these phenolic acids (p-coumaric and syringic acid) may be an underexplored therapeutic approach to improving the effectiveness of therapies for diabetes and oxidative stress management

Osteoarthritis

Osteoarthritis is one of the most debilitating diseases affecting millions of people worldwide. However, there is no FDA approved disease modifying drug specifically for OA. Surgery remains an effective last resort to restore the function of the joints. As the aging populations increase worldwide, the number of OA patients increases dramatically in recent years and is expected to increase in many years to come. This is a book that summarizes recent advance in OA diagnosis, treatment, and surgery. It includes wide ranging topics from the cutting edge gene therapy to alternative medicine. Such multifaceted approaches are necessary to develop novel and effective therapy to cure OA in the future. In this book, different surgical methods are described to restore the function of the joints. In addition, various treatment options are presented, mainly to reduce the pain and enhance the life quality of the OA patients

Neuroprotective pathways in the retina

A major cause of blindness in the Western world is degeneration of photoreceptors as a result of point mutations in genes coding for either phototransduction-related proteins or other proteins important for retinal function. Despite the diversity of mutated genes and proteins involved in this heterogeneous group of progressive retinal dystrophies with homologous phenotypes, the final event leading to blindness is apoptosis of photoreceptors. This has led to intensive studies of the effects of neuroprotective agents on the survival of photoreceptors in animal models of retinitis pigmentosa. One such effective molecule discovered to date to exert substantial rescue of retinal photoreceptors is glial cell line-derived neurotrophic factor (GDNF). However, the molecular mechanism of action underlying GDNF-mediated neuroprotection remains unresolved. This dissertation and the herein described studies were carried out with the goal of elucidating neuroprotective mechanisms using the porcine retina as a model. This species was selected due to its morphological and anatomical similarities to human retina. In order to clarify possible cellular mechanisms involved in neuroprotection, the initial studies involved analysis of GDNF action in porcine retina. It soon became evident that the GDNF-receptive cell in retina was not the photoreceptor itself but rather retinal Mueller glial cells (RMG), which are the major retinal glial cells. Thus, primary RMG cell cultures prepared from porcine retina were established and characterised to analyse this cell type without extraneous effects from the retinal environment. Proteomic profiling revealed profound changes in expression of RMG-specific marker proteins as an effect of in vitro conditions. Thus, the in vitro experiments for studying GDNF-induced signalling were performed with primary RMG cultures in an early state (two weeks in vitro) in order to study cells resembling the in vivo phenotype. GDNF was found to induce the ERK, SAPK and PKB/AKT pathways, as well as upregulating basic fibroblast growth factor (bFGF). Application of bFGF to primary porcine photoreceptors in vitro promoted a concentration-dependent rescue. Therefore a model of RMG-mediated indirect survival promoting mechanism induced by GDNF could be proposed. The finding that RMG are mediators of photoreceptor survival prompted further screenings for RMG-specific, secreted molecules promoting photoreceptor survival. A large-scale primary photoreceptor survival assay (96well format) was developed, in which RMG-conditioned medium (RMG-CM) was tested for survival activity. Conditioned medium was observed as having specific photoreceptor survival-promoting activity stemming from previously unidentified protein/s. Reducing the complexity of RMG-CM by anionic chromatography revealed that the activity does not bind to anionic resins. Mass spectrometric identifications of the mono-Q flow-through identified 23 different proteins from the active fraction, among them three potential new candidates for neuroprotective activity in the context of photoreceptor survival: connective tissue growth factor (CTGF), insulin-like growth factor binding protein 5 (IGFBP5) and insulin-like growth factor binding protein 7 (IGFBP7). Expression cloning and re-testing of these candidates for their ability to promote photoreceptor survival revealed that CTGF and IGFBP5 were effective in protecting photoreceptors when applied in combination with the RMG-conditioned media. Taken together, these results indicate that such survival-promoting activity is multi-factorial. RMG are likely to support photoreceptors by either cell to cell-mediated paracrine signalling or by secreting factors into the intercellular space between retina and retinal pigment epithelium, which consists of a complex matrix of proteins and polysaccharides. This matrix, designated as interphotoreceptor matrix (IPM), directly borders three cell types: photoreceptors, RMG and the retinal pigment epithelium and predisposes the IPM to function as repository of neuroprotective molecules possibly secreted from adjacent cells to protect and support photoreceptors. In order to identify such novel neuroprotective substances, the composition of IPM was investigated in this thesis by comparative proteomics. Over 140 different proteins were identified, the majority of which had never been previously detected in the IPM. Among these, 13 candidates were found, which in other tissue systems have been already reported to have a functional role in neuroprotection

Food Additive

A food additive is defined as a substance not normally consumed as a food in itself and not normally used as a characteristic ingredient of food whether or not it has nutritive value. Food additives are natural or manufactured substances, which are added to food to restore colors lost during processing. They provide sweetness, prevent deterioration during storage and guard against food poisoning (preservatives). This book provides a review of traditional and non-traditional food preservation approaches and ingredients used as food additives. It also provides detailed knowledge for the evaluation of the agro-industrial wastes based on their great potential for the production of industrially relevant food additives. Furthermore the assessment of potential reproductive and developmental toxicity perspectives of some newly synthesized food additives on market has been covered. Finally, the identification of the areas relevant for future research has been pointed out indicating that there is more and more information needed to explore the possibility of the implementation of some other materials to be used as food additives

Oxidative Stress in Diabetic Retinopathy

The combination of an increasing prevalence of diabetes and the aging of populations enables the appearance of a greater number of associated complications such as diabetic retinopathy. Diabetic retinopathy is the leading cause of preventable vision loss in working-age adults. The objective of this Special Issue is to highlight the existing evidence regarding the relationship between oxidative stress and low-grade chronic inflammation induced by hyperglycemia with the development and progression of diabetic retinopathy, with an emphasis on the importance of early diagnosis and the use of antioxidant and anti-inflammatory approaches to prevent or delay the harmful effects of diabetes on retinal tissue