ANTI-INFLAMMATORY AND ANALGESIC ACTIVITIES OF ETHANOL EXTRACT OF STEM-BARK OF HYMENODICTYON PACHYANTHA (UDELOSE) IN EXPERIMENTAL ANIMALS

ABSTRACT

This study was aimed at investigating the anti-inflammatory and analgesic activities of ethanol extract  of  Hymenodictyon  pachyantha  stem-bark as  well  as  the possible mechanisms  of anti- inflammatory action of the plant extract. The plant material was extracted using 3.5 litres of absolute ethanol. The anti-inflammatory activity of the ethanol extract was evaluated by determining  its  effect  on  egg  albumin-induced  rat  paw  oedema,  phospholipase  A2  activity, calcium chloride-induced platelet aggregatory response and membrane stabilization activity. The effect of the extract on acetic acid-induced writhing responses was also investigated. The percentage  yield  of  the  ethanol  extract  was  3.32%.  Phytochemical  analyses  of  the  extract revealed the presence of flavonoids (1359.268 ± 0.02 mg/100g), terpenoids (2154.695 ± 0.01 mg/100g), steroids (3.782 ± 0.05 mg/100g), saponins (0.405 ± 0.03 mg/100g), alkaloids (268.856

± 0.12 mg/100g), tannins (1375.930 ± 0.08 mg/100g) and  phenols (2900.169 ± 0.15 mg/100g). The acute toxicity test of the extract showed no toxicity up to 5000 mg/kg body weight. The stem-bark extract at 100, 200 and 400 mg/kg body weight significantly (p < 0.05) and dose- dependently inhibited egg albumin-induced rat paw oedema in both early and late stages of inflammation when compared with the untreated control, sustained over a period of 0.5 to 5 hrs. The standard anti-inflammatory drug (indomethacin, 10 mg/kg b. w.) followed a similar trend. The extract also significantly (p < 0.05) inhibited phospholipase A2  activity in a dose-related manner when compared to the control, with a range of 0.1 to 0.5 ml; inhibiting the enzyme activity by 78.92 to 95.59%. The extract also significantly (p < 0.05) and concentration- dependently inhibited platelet aggregatory response when compared to the control. The extract significantly (p < 0.05) inhibited hypotonicity-induced red blood cell membrane lysis in a concentration- dependent manner, similar to the standard drug indomethacin. Ethanol extract of Hymenodictyon pachyantha stem-bark (100, 200 and 400 mg/kg b. w.) significantly (p < 0.05) reduced the number of writhings induced by 0.6% acetic acid solution in a dose dependent manner counted over a period of 20 mins.  The results, therefore suggest that the mechanisms of the  anti-inflammatory  effect  may  be  due  to  the  stabilization  of  lysosomal  membrane,  by inhibiting phospholipase A2  and aggregation of platelets. Findings of this investigation provide empirical evidence for the use of Hymenodictyon pachyantha stem-bark extract in folkloric treatment of inflammatory disorders.

CHAPTER ONE

INTRODUCTION

The diverse natural products from medicinal plant continues to be an accepted form of treatment in the orient, and plant drugs based on traditional practice represent a huge portion of the pharmaceutical products in modern western countries (Dhami, 2013). Concerns have been raised that modern pharmaceutical practice too often involves costly drugs that produce unacceptable side effects. Experience shows that natural substances can apparently address several modern health concerns with fewer side effects. The acceptance that natural is better, fear or distrust of physicians, disappointment with allopathic care, and cultural or religion influences, increases interest in the use of natural bio-resources to manage chronic diseases and reduce issues of side effects and prices of pharmacological therapies. Medicinal plants with anti-inflammatory activity are considerably employed in the traditional treatment of several disorders of inflammation (Iwueke et al., 2006). Inflammation is a biological reaction to a disrupted tissue homeostasis (Medzhitov, 2008). Inflammation is a complex biological response of vascular tissues to harmful stimuli such as pathogens, damaged cells or irritants (Malaya et al., 2003). It can be acute or chronic (Ferrero-Miliani et al., 2007). Its purpose is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process, and to initiate tissue repair and restoration of functions. Inflammation is normally closely regulated by the body. When inflammation is too little, it could lead to progressive tissue destruction by the harmful stimulus (e.g. bacteria) and compromise the survival of the organism. On the other hand in chronic inflammation, the inflammatory response is out of proportion resulting in damage to the body. Inflammation is critically implicated in the development of many complex diseases and disorders including autoimmune diseases, metabolic syndromes, neurodegenerative diseases, cancers, and cardiovascular disease, heart attacks, Alzheimer’s disease and cancer (Coussens and Werb,

2002; Libby et al., 2002).

No matter the initiating stimulus, the classic inflammatory response is characterised by five clinical  signs:  calour  (warmth),  dolour  (pain),  rubour  (redness),  tumour  (swelling)  and functio laesa (loss of function) (Rigler, 1997). Cyclooxygenase (COX) is the key enzyme in the  synthesis  of  prostaglandins,  prostacyclins  and  thromboxanes  which  are  involved  in

inflammation, pain and platelet aggregation (Pilotto et al., 2010). The clinical symptoms such as fever, aches and pains associated with several diseases are directly or indirectly due to inflammatory disorders. Non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids are classically used to alleviate inflammation. Long term uses of NSAIDs causes side effects including gastric ulceration and renal toxicity (Payne, 2000; Ezekwesili et al., 2011) since they concurrently inhibit both isoforms of cyclooxygenase (COX). The development of NSAIDs which are selective COX inhibitors still have side effects as reports have connected these drugs with an increased risk of heart attack and stroke (Salmon, 2006; Nelson and Cox,

2008). There is therefore, the need for potent anti-inflammatory drugs with fewer side effects from natural sources as alternatives to these drugs. This has necessited the research into plants used in folk medicine to ameliorate inflammation. An example of such plant is Hymenodictyon pachyantha which is used in Nigeria folk medicine for the management of inflammatory disorders.

1.1 Description and uses of Hymenodictyon pachyantha

Hymenodictyon pachyantha is very distinct from the other Hymenodictyon species because of its recurved calyx lobes, which are longer than the mature corollas, and its elongate ovaries. Hymenodictyon is a genus of flowering plants in the family Rubiaceae comprising of about

30 species (David, 2008). The generic name is derived from two Greek words, hymen,

‘membrane’, and diktyon, ‘net’. It refers to the wing that surrounds each seed. Molecular phylogenetic studies have shown that Hymenodictyon is paraphyletic over the Madagascan genus Paracorynanthe (Ulrika and Birgitt, 2010). In Hymenodictyon and Paracorynanthe, the stipules bear large deciduous glands called colleters. The corolla tube is narrow at the base, gradually widening toward the apex. The fruit is a woody capsule. The species belonging to this genus are having oppositely arranged serrated leaves, small, clustered flowers and many seeded capsules. Stipules are linear to lanceolate or ovate 15 mm long, apex acuminate and pubescent.   Leaves   are   deciduous;   petioles   are   10-60   mm   long,   green-white-tinged, puberulous; secondary veins are seven to ten pairs per side. Fruit, a capsule, is ellipsoid, 2 to

2.5 cm long, growing on recurved, thick pedicels 5 to 12 mm long. Seeds are many, flat, winged all around the margin, about 1 cm long, including the wing. Bark is mostly furrowed and rough, 10-20 cm thick, exfoliating in irregularly shaped with soft scales. The wood of H. pachyantha is soft and has limited use, mostly for boxes. The wood is used as planks in

building houses and boats, making boxes, packing cases, pencils, toys model making and matches. In India, it is used as a cheaper grade of wood for making furniture, wrapper bobbins and wool boards. The bark obtained is useful for tanning purposes while leaves are useful for dyeing and as fodder for cattle while the remaining are only useful for timber wood (David, 2008).

1.1.1    Taxonomic classification of Hymenodictyon pachyantha

Kingdom:       Plantae Phylum:          Angiosperms Class:              Eudicots Order:             Gentianales Family:           Rubiaceae

Sub-family:    Cinchonoideae Genus:            Hymenodictyon Species:          pachyantha Source: (David, 2008)

1.1.2 Common names of Hymenodictyon pachyantha

Hymenodictyon pachyantha is commonly known in Igbo as Udeleose ( Enugu), (Jane and

Edwin, 2011) and óbadan (Farquhar) in Edo (Benin).

1.1.3 Geographical distribution

The genus comprises of trees and shrubs, distributed mostly in tropical and sub-tropical parts of Asia and Africa (Sylvain and Birgitta, 2006). H. pachyantha is mainly found in secondary forests at low altitudes, often about cliffs near the sea (Retief and Leistner, 2000). It is distributed in Nigeria (Benin, Enugu), Cameroon, and Ivory Coast.

1.2    Previous studies on Hymenodictyon pachyantha

Phytochemical studies carried out on species of the genus Hymenodictyon pachyantha  has shown considerable number of important phytoconstituents. The chemical constituents previously reported to be found in this plant were coumarins (Parichat et al., 2009) and anthraquinones.  The  stem  bark  contains  tannin,  toxic  alkaloid  hymenodictine,  a  bitter

substance, aesculin (Sylvain and Birgitta, 2006), an apioglucoside of scopoletin, hymexelsin (Prashant and Vijay, 2011). Anthraquinones, rubiadin and its methyl ether, lucidin, damnacanthal, nordamnacanthal, 2-benzylzanthopurpurin and anthragallol, (Gurung, 2002) have also been isolated from roots. Hymenodictyoline obtained from H. pachyantha is one of the few alkaloids which do not contain oxygen (Razafimandimbison and Brermer, 2002). Studies have also reported acetylenic fatty acids, a new triglyceride, and 11 known compounds,  among  them,  ursolic  acid,  oleaqnolic  acid,  uncarinic  acid  E,  b-sitosterol (Parichat et al., 2009). The roots of H. pachyantha also reported to contain anthragallol, 6- methylalizarin, soranjidiol, morindone and triterpenes (Sultana et al., 2015); oleanolic acid; uncarinic acid E (3β-hydroxy-27-(E) (Nareeboon et al., 2009).

Figure 1: Leaves and Stem-bark of Hymenodictyon pachyantha

1.3 Overview of inflammation

Inflammation is a pervasive phenomenon that operates during severe perturbations of homeostasis, such as infection, injury and exposure to contaminants (Ashley et al., 2012). Inflammation is the result of concerted participation of a large number of vasoactive, chemotactic and proliferative factors at different stages (Gobinda et al., 2011). Inflammatory response is  brought  about  or mediated  by  inflammatory  mediators  such  as  chemokines, cytokines, cell adhesion molecules, extracellular matrix proteins (Simon et al., 2000; Nanyak et al., 2013) which when in excess are deleterious (Liu and Hong, 2002). It is triggered by infectious agents such as, viruses, fungi, bacteria and protozoa. It is also due to trauma,

physical  and  chemical  agents,  tissue  necrosis  and  immune  reactions.  The  mechanisms involved in the inflammatory process are common to all, regardless of the triggering factor (Ferrero-Miliani et al., 2007).

Figure 2: The inflammatory response to injury

Source: (Marieb and Mitchell, 2007)

It involves a cascade of biochemical events comprising of the local vascular system and the immune system (Da Silveira e Sá et al., 2013). It also involves the production of factors that could cause damage to tissues when not properly regulated. Although inflammation is a defense mechanism, the complex events and mediators involved in inflammatory reaction can be induced, maintained and aggravated by many diseases (Anosike et al., 2012a), thus inflammation  is  critical  for  the  development  of  many  complex  diseases  and  disorders including autoimmune diseases, metabolic syndrome, neurodegenerative diseases, cancers, and cardiovascular diseases. Inflammation comes in two types: chronic inflammation, which can be defined as a dysregulated form of inflammation, and acute inflammation, which can be defined as a regulated form.

1.3.1 Inflammatory mediators

Inflammatory mediators are substances triggered by inflammatory stimuli (Bhadrapura and Sudharshan, 2016). They are derived from inflammatory cells, or released as plasma proteins (Vishal et al., 2014). They also mediate inflammatory response by either acting as pyrogens, thereby triggering off the synthesis and release of prostaglandin to the insult or by causing the lysis of cells, especially the mast cells in the case of injury of infections. Most mediators bind to  specific target  receptors on  the cells  to  elicit  their effects.  Exceptions  are lysosomal enzymes that have a direct enzymatic effect and reactive oxygen species (ROS) that have a direct toxic effect. Mediators can stimulate target cells to release secondary mediators. The local lipid mediators constitute a new genus of anti-inflammatory and pro-resolving endogenous compounds that have proven to be very potent in treating a number of inflammation-associated models of human disease.

1.3.1.1 Cell derived mediators

These are mediators derived from stimulated inflammatory cells. They may be preformed and stored in the granules of these cells or may be synthesized de novo and secreted as needed. Circulating platelets, basophils, PMNs, endothelial cells monocyte and macrophages, tissue mast  cells  and  the  injured  tissue  itself  are  all  potential  cellular  sources  of  vasoactive mediators. In general, these mediators are;

(i)  derived from metabolism of phospholipids and arachidonic acid (e.g., prostaglandins, thromboxanes, leukotrienes, lipoxins, platelet-activating factor PAF),

(ii) preformed and stored in cytoplasmic granules (e.g., histamine, serotonin, lysosomal hydrolases), or

(iii)derived from altered production of normal regulators of vascular function (e.g., nitric oxide and neurokinins).

1.3.1.1.1 Vasoactive amines

Vasoactive amines include histamine and serotonin. The latter is also known as 5- hydroxytryptamine   (5-HT).   Histamine   (β-Imidazolylethylamine)   is   a   vasodilator,   a constrictor of smooth muscle, and a potent stimulant of vascular permeability, respiratory mucus, and gastric acid secretion. It exerts its effects on a variety of cell types including

smooth muscle cells, neurons, glandular cells (endocrine and exocrine), blood cells, and cells of the immune system. In addition to its role in immediate hypersensitivity reactions, histamine can exert H2-receptor-mediated anti-inflammatory activity including inhibition of human neutrophil lysosomal enzyme release, inhibition of IgE-mediated histamine release from peripheral leukocytes, and activation of suppressor T-lymphocytes. Histamine is synthesized in the golgi apparatus of mast cells and basophils by decarboxylation of the amino acid histidine and is then stored in secretory granules in complexes with heparin, protein, or both. Histamine is  released during antigen reaction with mast cell bound antibody molecules and during the inflammatory response to skin injury (Trautmann et al., 2000). It is also released from basophils and platelets. It causes the contraction of endothelial cells of venules leading to increased vascular permeability. Its effect is rapidly inactivated by histaminase. Serotonin is released from the platelets along with histamine and acts similarly to histamine (Stone et al., 2010).

1.3.1.1.2 Prostaglandins

Phospholipase A2 (PLA2), in response to any disturbance of the cell membrane activates the hydrolysis of phospholipids from the lipid bilayer into free fatty acids such as, arachidonic acid. Arachidonic acid plays an important role in many metabolic pathways and is useful when produced in moderation (George et al., 2014). When produced in excess, it acts as a substrate for COX (or prostaglandin H2 synthase) to release prostanoids, comprising of prostaglandins  (PGs),  thromboxanes  (TXs)  and  prostacyclins  (Ricciotti  and  FitzGerald,

2011). Examples of PGs are prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2). Prostaglandin D2 is a major prostaglandin produced by mast cells. It is a bronchoconstrictor and also acts as a chemoattractant for leukocytes (Stone et al., 2010). Prostaglandin E2 which is more widely distributed causes pain, vasodilatation and increases vascular permeability. All cells are capable of synthesizing PGs, apart from non-nucleated erythrocytes.

1.3.1.1.3 Thromboxane A2 and prostacyclin

Thromboxanes  and  Prostacyclins  are  also  produced  through  the  action  of  COX  on arachidonic acid. Thromboxane A2 (TxA2) is a potent platelet-aggregating agent and a vasoconstrictor. It is unstable and is rapidly converted to inactive Thromboxane B2  (TXB2). Prostacyclin  or prostaglandin  I2   (PGI2) on  the other hand  is  a vasodilator and  a potent

inhibitor of platelet aggregation (Pilotto et al., 2010). It increases blood flow as well as blood vessel permeability by assisting in the release of NO from the endothelium (Vishal et al.,

2014).

1.3.1.1.4 Cytokines

These are soluble immune signalling proteins of low molecular weight that modulate the differentiation, proliferation and function of immune cells, and coordinate inflammatory responses (Kidd and Urban, 2001). They are secreted primarily by activated tissue macrophages, lymphocytes and endothelial cells. Their main effects are to induce the acute phase reaction and to activate vascular endothelium, leukocytes, platelets and fibroblasts, thus, initiating the cascade of vascular, cellular and humoural events which together comprise the inflammatory response (Tan et al., 1999). Several cytokines play essential roles in orchestrating the inflammatory process, especially interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-α) which are monokines, produced by monocytes and macrophages (Simopoulos, 2002). Lymphokines such as interferon‐gamma (IFN-γ) are produced by lymphocytes  and  they  play  an  important  role  in  the  first  line  of  defense  against  viral infections.

1.3.1.1.5 Leukotrienes

Leukotrienes (LTs), a family of lipid mediators, play a key role in the pathogenesis of inflammation.  LTs  are classified  into  two  classes:  LTB(4) and  cysteinyl  LTs  (CysLTs). LTB(4) is one of the most potent chemoattractant mediators of inflammation. It exerts its actions through a seven transmembrane-spaning through G protein receptors, LTB4 R-1 and LTB4 R-2 CysLTs (LTC(4), LTD(4), and LTE(4)). Leukotrienes (LTs) like prostanoids, are released from excess arachidonic acid due to the activity of 5-lipoxygenase (5-LOX). The lipoxygenase  (LOX)  pathway  is  a  parallel  inflammatory  pathway  to  the  COX  pathway (George et al., 2014). Leukotrienes are released from leukocytes and mast cells and are generally pro-inflammatory. Leukotriene B4 (LTB4) is a potent chemotactic agent for neutrophils. It also promotes their adhesion to vascular endothelial cells, their trans- endothelial migration and stimulates the synthesis of pro-inflammatory cytokines from macrophages   and   lymphocytes   (Dalgleish   and   O’Byrne,   2002;   Medzhitov,   2008). Leukotriene B4 also promotes degranulation and the generation of ROS. Cysteinyl-containing

leukotriene C4 (LTC4), leukotriene D4 (LTD4) and leukotriene E4 (LTE4) cause intense vasoconstriction, bronchospasm and increased vascular permeability in venules. Leukotriene (LT) C4 and its products, LTD4 and LTE4, make up the biologic mixture previously known as the slow-reacting substance of anaphylaxis.

1.3.1.1.6 Chemokines

Chemokines  such  as  IL-8,  macrophage  inflammatory  protein-1α,  macrophage chemoattractant protein-1, -2, and -3, and rantes function primarily as chemoattractants and activators (Kolaczkowska and Kubes, 2013). Chemokines are inducible molecules. They include, C-X-C (or α) chemokines and C-C (or β) chemokines. C-X-C chemokines are so called  because  they  have  an  intervening  amino  acid  between  the  first  two  of  the  four conserved cysteine residues at their amino terminal. C-C chemokines on the other hand, do not have an intervening amino acid between their first two amino-terminal cysteine residues (Tan et al., 1999). C-X-C chemokines are primarily neutrophils chemoattractants while C-C chemokines are primarily chemoattractants for monocytes and T-cells. Example of C-X-C chemokine is interleukin-8 (IL-8) while an example of C-C chemokine is monocyte chemoattractant protein-1 (MCP-1). Interleukin-8 and MCP-1 also induce degranulation and respiratory burst in neutrophils and monocytes respectively.

1.3.1.1.7 Nitric oxide

A free radical gas produced endogenously by a variety of mammalian cells including macrophages and endothelial cells, synthesized from arginine by nitric oxide synthase. Nitric oxide is one of the endothelium-dependent relaxing factors released by the vascular endothelium and mediates vasodilation. It also inhibits platelet aggregation, induces disaggregation of aggregated platelets, and inhibits platelet adhesion to the vascular endothelium. Nitric oxide activates cytosolic guanylate cyclase and thus elevates intracellular levels of cyclic GMP. Three major isoforms of NOS include two constitutively expressed forms, which are calcium-calmodulin dependent and are collectively known as constitutive nitric oxide synthase (cNOS). The third which is calcium-calmodulin independent, induced by cytokines and regulated in the gene by a variety of inflammatory mediators is inducible nitric oxide synthase (iNOS) (Kumar et al., 2013). Nitric oxide is toxic to bacteria and

directly inhibits viral replication. It also combines with ROS to yield peroxynitrate radicals which have potent antimicrobial activity (Chukwuka et al., 2011).

1.3.1.1.8 Platelet-activating factor

Platelet-activating factor (PAF) is an ether phospholipid which is released from most proinflammatory cells and platelets by the action of PLA2  (Sato et al., 2009). PAF- like molecules are also generated by activated vascular endothelium in a membrane-bound form. They are chemotactic for neutrophils, enhances the adhesion of platelets and leukocytes to endothelium, involved in vasoconstriction and bronchoconstriction and cause increased vascular permeability.

Figure 3: Generation of arachidonic acid metabolites and their roles in inflammation

Source: (Priyadarshini et al., 2013)

1.3.1.2 Plasma-derived mediators

Plasma proteins circulate in an inactive form waiting to undergo proteolytic cleavage to become active. Mediator-producing systems in plasma include: complement, clotting, fibrinolytic and kinin systems.

1.3.1.2.1 Complement system

Complement is part of the humoral immune system. Its role is to generate biologically active products from the pathways of complement activation which include: classical, lectin, alternative, properdin and thrombin pathways (Neher et al., 2011). Peptides generated by complement activation play a critical role in the elimination of invading pathogens. They include, C3b and C4b which opsonise pathogens for phagocytosis, the anaphylatoxins C3a and C5a which acts as chemoattractants for leukocytes and the membrane attack complex (MAC or C5b-9) involved in the direct lyses of pathogens (Alexander et al., 2008; Griffiths et al., 2009). The generation of anaphylatoxins C3a and C5a further induces degranulation of mast cells, basophils and eosinophils. They also induce the expression of adhesion molecules on endothelial cells and cause smooth-muscle contraction (Klos et al., 2009).

1.3.1.2.2 Clotting or coagulation system

The mechanism of coagulation involves activation, adhesion and aggregation of platelets, along with the deposition and maturation of fibrin. Fibrin activation involves two pathways, the intrinsic and  the extrinsic cascades.  Following  vascular injury, the extrinsic clotting cascade is triggered as activated endothelium expresses tissue factor (TF) on their luminal surfaces and expresses increased levels of plasminogen activator inhibitor-1 which inhibits fibrinolysis (Tan et al., 1999). In the presence of clot activating factors, prothrombin is converted to thrombin by prothrombinase. Thrombin in turn converts fibrinogen to fibrin, which is the major product involved in clot formation. The intrinsic cascade occurs with the engagement of Hageman factor (factor XII) which interacts with factors Va and VIIIa and becomes converted to its active form, factor XIIa. Factor XIIa in turn activates factor Xa which directly converts prothrombin to thrombin and the subsequent generation of fibrin

from fibrinogen (Ward, 2010). Fibrin is deposited on aggregated platelets to bring about thrombus formation.

1.3.1.2.3 Fibrinolytic system

The fibrinolytic system acts in opposition to the coagulation system, to counterbalance clotting. It is activated by urinary plasminogen activator (uPA) and tissue plasminogen activator (tPA) which converts plasminogen to plasmin. Plasmin directly interacts with fibrin to bring about the breakdown of fibrin clots as they are formed within the intravascular compartment (Ward, 2010).

1.3.1.2.4 Kinin System

Kinins are vasoactive peptides generated through the kinin-generating cascade. The most important product of this cascade is the plasma protein, bradykinin. Prekallikrein is converted to the protease, kallikrein by factor XIIa from the clotting cascade. Kallikrein interacts with high molecular weight kininogen (HMWK) to bring about the hydrolysis and release of bradykinin (Stankov, 2012). Bradykinin is a powerful vasopermeability agent. It also causes pain, vasodilatation and oedema, all contributing to inflammation.

1.3.2 Cells associated with inflammation

The cellular component involves leukocytes, which normally reside in blood and must move into the inflamed tissue via extravasation to aid in inflammation. Some act as phagocytes, ingesting bacteria, viruses, and cellular debris. Others release enzymatic granules that damage pathogenic invaders. Leukocytes also release inflammatory mediators that develop and maintain the inflammatory response. In general, chronic inflammation is mediated by agranulocytes such as monocytes and  lymphocytes whereas acute inflammation is mediated by granulocytes.

1.3.2.1 Agranulocytes

These leukocytes are characterised by the apparent absence of granules in their cytoplasm are referred to as agranulocytes. Among these categorization includes the macrophages, monocytes and lymphocytes.

1.3.2.1.1 Macrophages

Macrophages are a type of white blood cell that engulfs and digests cellular debris, foreign substances, microbes, cancer cells, and anything else that does not have the types of proteins specific to the surface of healthy body cells on its surface in a process called phagocytosis. They are found in essentially all tissues (Ovchinnikov, 2008) where they patrol for potential pathogens by amoeboid movement. They play a critical role in non-specific defense (innate immunity), and also help initiate specific defense mechanisms (adaptive immunity) by recruiting other immune cells such as lymphocytes. In humans, dysfunctional macrophages cause severe diseases such as chronic granulomatous disease that result in frequent infections. Beyond increasing inflammation and stimulating the immune system, macrophages also play an important anti-inflammatory role and can decrease immune reactions through the release of cytokines. Macrophages that encourage inflammation are called M1 macrophages, while those that decrease inflammation and encourage tissue repair are known as M2 macrophages (Mills, 2012). This difference is reflected in their metabolism, M1 macrophages have the unique ability to metabolize arginine to the killer molecule nitric oxide, whereas M2 macrophages have the unique ability to metabolize arginine to the repair molecule ornithine. Human   macrophages   are   produced   by   the   differentiation   of   monocytes   in   tissues. Macrophages are essential for wound healing. They replace polymorphonuclear  neutrophils as the predominant cells in the wound  two days after injury. The main role of macrophages is to  phagocytize  bacteria  and  damaged  tissue,  and  they  also  debride  damaged  tissue  by releasing proteases. Macrophages also secrete growth factors and other cytokines, especially during the third and fourth post-wounding days. These factors attract cells involved in the proliferation stage of healing to the area. Macrophages may also restrain the contraction phase (Newton et al., 2004). Macrophages are stimulated by the low oxygen content of their surroundings to produce factors that induce and speed angiogenesis (Greenhalgh, 1998) and they also stimulate cells that re-epithelialize the wound, create granulation tissue, and lay

down a new extracellular matrix (Stashak et al., 2004). By secreting these factors, macrophages contribute to pushing the wound healing process into the next phase.

1.3.2.1.2 Monocytes

Monocytes play multiple roles in immune function. Such roles include replenishing resident macrophages under normal states and in response to inflammation signals, monocytes can move quickly (approximately 8–12 h) to sites of infection in the tissues and differentiate into macrophages and dendritic cells to elicit an immune response. Half of them are stored in the spleen (Swirski et al., 2009). Monocytes are also capable of killing infected host cells via antibody-dependent cell-mediated cytotoxicity. Vacuolization may be present in a cell that has  recently  phagocytized  foreign  matter.  Microbial  fragments  that  remain  after  such digestion can serve as antigens. The fragments can be incorporated into MHC molecules and then trafficked to the cell surface of monocytes (and macrophages and dendritic cells). This process is called antigen presentation and it leads to activation of T lymphocytes, which then mount a specific immune response against the antigen. Antimicrobial activity of monocytes includes oxygen-dependent mechanisms such as the respiratory burst, which through a complex series of reactions forms highly reactive hydroxyl radicals that damage host and microbial membranes.

1.3.2.1.3 Lymphocytes

A lymphocyte is one of the subtypes of white blood cell in a vertebrate’s immune system. They consists of natural killer cells (NK cells) (which function in cell-mediated, cytotoxic innate immunity), T cells (for cell-mediated, cytotoxic adaptive immunity), and B cells (for humoral, antibody-driven adaptive immunity). They are the main type of cell found in lymph, which prompted the name lymphocyte. The three major types of lymphocyte are T cells, B cells and natural killer (NK) cells. Lymphocytes can be identified by their large nucleus.

1.3.2.1.3.1 T-cells and B-cells

T cells (thymus cells) and B cells (bone marrow- or bursa-derived cells) are involved in the acquired or antigen-specific immune response given that they are the only cells in the organism able to recognize and respond specifically to each antigenic epitope. The B Cells have the ability to transform into plasmocytes and are responsible for producing antibodies

(Abs). Thus, humoral immunity depends on the B Cells while cell immunity depends on the T Cells (Yang et al., 2010). B cells respond to pathogens by producing large quantities of antibodies which then neutralize foreign objects like bacteria and viruses. In response to pathogens some T cells, called T helper cells, produce cytokines that direct the immune response, while other T cells, called cytotoxic T cells, produce toxic granules that contain powerful enzymes which induce the death of pathogen-infected cells. Following activation, B cells and T cells leave a lasting legacy of the antigens they have encountered, in the form of memory cells. Throughout the lifetime of an animal, these memory cells will remember each specific pathogen encountered, and are able to mount a strong and rapid response if the pathogen is detected again.

1.3.2.1.3.2 Natural killer cells

Natural killer (NK) cells are part of the innate arm of the immune system. Their function is to eliminate aberrant cells, including virally infected and tumorigenic cells. For this purpose NK cells store cytotoxic proteins within secretory lysosomes, specialized exocytic organelles that are also known as lytic granules. NK cells are believed to be relatively short-lived, and at any one time there are likely more than 2 billion circulating in an adult (Blum and Pabst, 2007). NK cells distinguish infected cells and tumors from normal and uninfected cells by recognizing changes of a surface molecule called MHC (major histocompatibility complex) class  I.  NK  cells  are  activated  in  response  to  a  family  of  cytokines  called  interferons. Activated NK cells release cytotoxic (cell-killing) granules which then destroy the altered cells (Janeway et al., 2001). They were named natural killer cells because of the initial notion that they do not require prior activation in order to kill cells which are missing MHC class I.

1.3.2.2 Granulocytes

These WBCs are characterised by the presence of granules within their cytoplasm. Granulocytes comprise of mast cells, neutrophils, T-cells and B-cells, eosinophils and basophils.

1.3.2.2.1 Mast cells

Mast cells are found in mucosal and  epithelial tissues, surrounding blood cells, smooth muscle, and hair follicles throughout the body and in all vascularized tissues except for the central nervous system and the retina (da Silva et al., 2014). They are also seen in tissues surrounding digestive tract, conjunctiva, nose and mouth (Prussin and Metcalfe, 2003). Mast cells are located at the junction point of the host and external environment at places of entry of antigen such as gastrointestinal tract, skin, respiratory epithelium (Metcalfe and Boyce,

2006; Jamur et al., 2005). The cytoplasm of the mast cell contains 50–200 large granules that store  inflammatory   mediators,   including   histamine,   heparin,   a  variety   of  cytokines, chondroitin sulfate, and neutral proteases (da Silva et al., 2014). Mast cell is derived from the myeloid stem cell. It contains many granules rich in histamine and heparin. In order for mast cells to migrate to their target locations, the coordinated effects of integrins, adhesion molecules, chemokines, cytokines, and growth factors are necessary (Collington et al., 2011). They participate in wound healing, angiogenesis, defense against pathogens and blood-brain barrier function (da Silva et al., 2014; Polyzoidis et al., 2015). They are also known to play roles in allergy and anaphylaxis. Mast cells look very much like basophils but are different cells as they develop from different hematopoietic stem cells. Unlike basophils, which leave the bone marrow in the mature form, mast cells circulate in an immature form and only mature when they get to their target tissue site (Prussin and Metcalfe, 2003). Mast cells play important roles in the inflammatory process. They release histamine which dilates post- capillary venules, activates the endothelium, increases blood vessel permeability leading to local oedema and also depolarizes the nerve endings leading to pain or itching. In the skin, antigens, via IgE, activate mast cells in the deep layers of connective tissue. Mast cells release histamine as well as other vasoactive molecules, which cause urticaria (hives).

1.3.2.2.2 Neutrophils

Neutrophils are a type of phagocyte and are normally found in the bloodstream. During the acute phase of inflammation, particularly as a result of bacterial infection, environmental exposure (Jacobs et al., 2010) and some cancers (Waugh and Wilson, 2008; De Larco et al.,

2004), neutrophils are one of the first-responders of inflammatory cells to migrate towards the site of inflammation.  They migrate through the blood vessels, then through interstitial tissue, following chemical signals such as Interleukin-8 (IL-8), C5a, fMLP and Leukotriene B4 in a process called chemotaxis. They are the predominant cells in pus, accounting for its whitish/yellowish appearance. Neutrophils are recruited to the site of injury within minutes following trauma, and are the hallmark of acute inflammation (Cohen and Burns, 2002). However, due to some pathogens being indigestible, they can be less useful alone. With the eosinophil and the basophil, they form the class of polymorphonuclear cells.

1.3.2.2.3 Basophils

Basophils arise and mature in bone marrow (Voehringer, 2009). When activated, basophils degranulate   to   release   histamine,   proteoglycans   (e.g.   heparin   and   chondroitin),   and proteolytic enzymes (e.g. elastase and lysophospholipase). They also secrete lipid mediators like leukotrienes (LTD-4), and several cytokines (Nakanishi, 2010). Histamine and proteoglycans are pre-stored in the cell’s granules while the other secreted substances are newly generated. Each of these substances contributes to inflammation.

1.3.2.2.4 Eosinophils

Eosinophil granulocytes are white blood cells and one of the immune system components responsible for combating multicellular parasites and certain infections in vertebrates. Along with mast cells, they also control mechanisms associated with allergy and asthma. They are granulocytes that develop during hematopoiesis in the bone marrow before migrating into blood. These cells are eosinophilic or ‘acid-loving’ as shown by their affinity to coal tar dyes: Normally transparent, it is this affinity that causes them to appear brick-red after staining with eosin, a red dye, using the Romanowsky method. Eosinophils persist in the circulation for 8–12 h, and can survive in tissue for an additional 8–12 days in the absence of stimulation (Young et al., 2006).

1.3.2.3. Platelets

Platelets adhere to these sub-endothelial matrix proteins and become activated. Activated platelets lead to the formation of TXA2 which is a potent vasoconstrictor and stimulus of platelet  aggregation.  Thromboxanes  are  known  toinduce  blood  vessel  constriction  and platelet aggregation by increasing intracellular calcium ion (Ca2+), which promote fusion of dense and alpha platelet granules with the platelet membrane, thus, releasing their contents.

Adenosine  diphosphate  (ADP)  which  also  mediates  platelet  aggregation  is  released. Activated platelets also trigger the coagulation cascade leading to the formation of thrombin. Thrombin and other platelet agonists such as, ADP, PAF and adrenaline bind to specific receptors  on  platelet  membrane  and  stimulate  aggregation  in  which  more  platelets  are recruited and begin to adhere to each other. This forms haemostatic plug onto which fibrin may  be  deposited,  thus,  stabilizing  the  clot  formed.  The  degranulation  of  platelets  also releases P-selectin and histamine. Histamine increases vascular permeability while P-selectin facilitates leukocyte migration from blood to tissues (Zarbock et al., 2006).

1.3.3 Classification of inflammation

Inflammation could be classified as  either acute or chronic, depending on the type and duration of the antigen challenge and is mediated by some chemical substances such as histamine, serotonin, slow reacting substances of anaphylaxis (SRS-A), prostaglandins and some  plasma enzyme systems  such  as  the  complement  system,  the clotting  system,  the fibronolytic system and the kinin system.

1.3.3.1 Acute inflammation

In a classical acute inflammatory response, cellular events are temporally activated. Acute inflammation is a rapid response characterized by classical symptoms of redness, heat and oedema. It is defined as a series of tissue responses that occur within the first few hours following injury. It is initiated by resident tissue macrophages, mast cells and endothelial cells (Anosike et al., 2012b). Upon initial challenge, protein exudation increases and polymorphonuclear leukocytes (neutrophils) accumulate in inflamed tissue. Neutrophils are the most prominent cells in acute inflammation (Phillipson and Kubes, 2011; Sakic et al.,

2011). Neutrophil infiltration follows a rapid response from sentinel cells prestationed in the tissues at the time of injury, including macrophages and mast cells (Makriyannis and Nikas,

2011).   As primary defenders, neutrophils transmigrate into tissues in large numbers to neutralize pathogens and promote the clearance of cellular and other debris by phagocytosis. As  the lesion  matures,  neutrophils  accumulate  in the local  tissue  and  die via apoptosis (programmed cell death). The initial accumulation of neutrophils is followed by a second wave of cellular infiltration, of mononuclear phagocytes (monocytes). Differentiation of monocytes into macrophages promotes the removal of apoptotic neutrophils and debris by

nonphlogistic phagocytosis. Macrophages that have completed the elimination of apoptotic neutrophils are cleared from the inflamed tissue either by egression to the lymphatic system or by apoptosis, by a process termed efferocytosis. This temporal regulation of inflammation requires that this conserved cellular phenotype is highly regulated to clear the original insult (Savill et al., 2002). Outcomes of acute inflammation include, complete resolution, healing by connective tissue replacement, abscess formation or progression to chronic inflammation. Failure to resolve the inflammatory response, or continuous activation of the responses, become harmful to the tissue and consequently develop into the chronic lesion that we call inflammatory diseases. Diseases associated with uncontrolled acute inflammation are characterized by a lack of activation of resolution programs and by the inappropriate release and maintenance of high levels of toxic substances and pro-inflammatory mediators, which may result in damage to host tissues and prolong the inflammatory response (Bannenberg et al., 2005).

1.3.3.1.1 Mechanism of acute inflammation

Inflammation consists of a tightly regulated cascade that is orchestrated by cytokines. Recognition of antigen is the first step of the inflammatory cascade. This is achieved by innate immune system as it detects a broad range of molecular patterns called pathogen- associated  molecular  patterns  (PAMPs)  that  are  commonly  found  on  pathogens  but  are foreign  to  the  host,  (Janeway  and  Medzhitov,  2002).  Alarmins  or  damage-associated molecular patterns (DAMPs) which are endogenous molecules generated in response to a sterile injury, such as burn, hypoxia or chemical insult are also recognized by the innate immune system  (Bianchi,  2007;  Osterloh  et  al.,  2009).  Detecting  these signals  helps  in minimizing inadvertent targeting of host cells and tissues. These damage signals are recognized by tissue-resident cells such as macrophages and mast cells, through multi-ligand pattern-recognition receptors (PRRs) expressed on their surfaces (McGhan and Jaroszewski,

2011). Examples of these receptors are transmembrane toll-like receptors (TLRs) and NOD- like  receptors  (NLRs).  Transmembrane  TLRs  recognize  and  bind  to  PAMPs  while intracellular NLRs bind DAMPs.

Ligation of PRRs leads to the activation of signal transduction pathways that regulate diverse transcriptional and post-transcriptional processes. For example, TLRs couple to the adaptor

protein, myeloid differentiation primary response gene 88 (MyD88), resulting in activation of nuclear factor kappa β (NF-κβ) and mitogen-activated protein kinase (MAPK) pathways. These in turn control the activities of multiple signal dependent transcription factors that include members of the NF-κβ, activator protein-1 (AP-1) and interferon regulator factor (IRF) families (Glass et al., 2010). Nuclear factor kappa β is found in virtually all cell types and remains in an inactive state bound to an inhibitor protein known as, inhibitor of kappa β (Iκβ).   Upon   transduction   of   the   signal,   NF-κβ   is   released   from   Iκβ   through   the phosphorylation of Iκβ by inhibitor of kappa β kinase (IKK). Active NF-κβ translocates to the nucleus, where transcription is upregulated through binding to target inflammatory genes (Ashley et al., 2012).

Transcription and translation of genes lead to the third stage of the inflammatory cascade, which is the inducible expression of pro-inflammatory cytokines, such as interleukin-1-beta (IL- 1β), interleukin-6 (IL-6), TNF-α, IFN-γ and chemokines. Several pro-inflammatory proteins  such  ascyclooxygenase-2  (COX-2)  and  iNOS  are  also  expressed  (Russo-Marie,

2004). NOD-like receptors on the other hand, signal the inflammasome, which activates caspase-1 to convert cytokines into active forms (Martinon et al., 2009). In conjunction with chemokines, these cytokines facilitate the recruitment of effector cells, such as blood-borne neutrophils and monocytes, to the inflammatory site by chemotaxis. Neutrophils then eliminate pathogens usingdegranulation, NETs and phagocytosis. Macrophages and dendritic cells also participate in phagocytosis of antigen.

1.3.3.1.2 Responses in acute inflammation

Acute inflammation involves vascular and cellular responses. The vascular responses are vasodilatation and increased vascular permeability, while the cellular component involves the emigration of leukocytes from the vascular to the extravascular compartment.

1.3.3.1.2.1 Vasodilatation

This involves changes in the vascular caliber leading to increased blood flow to tissues. It is caused  by  arteriolar  dilation  which  sometimes  occurs  after  transient  vasoconstriction, resulting in heat and redness. It is also due to the opening of new capillaries. It occurs

primarily as a result of histamine and NO acting on the vascular smooth muscle (Ferrero- Miliani et al., 2007).

1.3.3.1.2.2 Increased vascular permeability

This involves the contraction of endothelial cells, leading to the escape of plasma protein-rich fluid known as exudates from the vascular compartment into the extravascular space. This process is called exudation. It leads to increased extravascular osmotic pressure leading to oedema (Stankov, 2012).

Figure 5: Overview of vascular changes in acute inflammation (Levison et al., 2008) This vascular leakage of exudates can be chemically mediated or injury induced. Chemical mediation involves the retraction of endothelial cells by mediators of acute inflammation such as histamine, bradykinins, LTs and PGs released by mast cells and  tissue resident macrophages. These mediators bind to their receptors on endothelial cells leading to vasodilatation, contraction of endothelial cells and widening of intercellular junctions (Da Silveira e Sá et al., 2013). Injury induced vascular leakage is an abnormal leakage caused by toxins and physical agents as they may cause necrosis of the vascular endothelium.

₦5,000.00 – DOWNLOAD FULL PROJECT DOWNLOAD FULL PROJECT Added to cart

---

---

---

---

Related Project Topics :