Essay: Effect of maslinic acid on the immune function of liver (Research Proposal)

Introduction

Maslinic acid, also known as crategolic acid or (2α, 3β)-2, 3-dihydroxyolean-12-en-28-oic acid is a pentacyclic triterpene widely spread in the plant kingdom. We examine the potential of one group of compounds called triterpenes, derived from traditional medicine and diet for their ability to suppress inflammatory pathways. In the last decades, and in response to an increasing interest to identify new natural molecules with beneficial effects on health, maslinic acid has been isolated not only from various plants used in traditional herbal medicine but also from edible vegetables and fruits. Scientific studies have shown triterpenoids to be potential anti-inflammatory and anticancer agents many triterpenoids derived from botanical sources play an important role in reducing inflammation. In parallel, the biological activities of maslinic acid have been assessed in different experimental models, from tumor cell lines to animal models of several diseases, As more than 20,000 triterpenoids are available in nature and it is difficult to describe them all, Historically, maslinic acid was named “crategolic acid”, since it was first isolated from Crataegus oxyacantha L. it as a triterpenoid carboxylic acid with molecular formula C30H48O4, Maslinic acid is a pentacyclic triterpenic acid, rich in Crataegus pinnatifida Bunge, red Jujubes and Canarium album Raeusch, all of which play roles in immune stimulation and anti-inflammation mainly found in the leaves of the above mentioned species (Sanchezgonzalez, Lozanomena et al. 2013), although with some controversy isolated from olive husks a triterpenic acid with molecular formula and structure identical to those of crategolic acid, and named it maslinic acid (Caputo, Mangoni et al. 1974). Terpenes and their metabolites are widely distributed in various plant systems that depend on various biotic and abiotic environmental factors and their metabolites play a very important role in a plant’s defense mechanism. They protect the plants from both constitutive and induced self-protective responses against insects and environmental stress (Franceschi, Krokene et al. 2005, Keeling and Bohlmann 2006). Hence, triterpenoids provide a very good protection shield for plants, indicating their potential for use in the prevention of various cancers and inflammatory diseases in humans.

Triterpenoids, such as maslinic acid, are a group of secondary metabolites derived from the cyclization of squalene, oxidosqualene or bis-oxidosqualene these precursors (C30) are a substrate of several types of triterpene synthases, which catalyze their cyclization through intermediate cations to a wide variety of triterpenes. Depending on the number of rings, the latter are classified as mono-, bi-, tri-, tetra- or pentacyclic triterpene alcohols. Lupeol, α- and β-amyrin are examples of pentacyclic triterpene alcohols, which not only constitute secondary metabolites themselves but also might undergo oxidation reactions to yield other derivatives, such as betulinic, ursolic and maslinic acids (Xu, Fazio et al. 2004). More recently postulated the biosynthetic pathway that leads to the formation of maslinic acid in the fruits of Olea europaea L., one of the main natural sources of this triterpene. The authors suggest that in the developing olive both the sterols (primary metabolites) and the non-steroidal triterpenoids (secondary metabolites) share oxi do squalene as a common precursor. The enzyme β-amyrin synthase catalyzes its cyclization into β-amyrin, and further oxidation steps give rise to the triterpenic alcohol erythroid followed by the hydroxy pentacyclic triterpene acids oleanolic and maslinic (Stiti, Triki et al. 2007).

On the other hand, maslinic acid can also be found in plants used for traditional Asian medicine, for example, Eriobotrya japonica (Stiti, Triki et al. 2007), Campsis Grandiflora (Goncalves, Malheiro et al. 2012), Geum japonica (Sanchezgonzalez, Lozanomena et al. 2013), and Agastache rugosa, which are used to treat miscellaneous inflammatory diseases. The pharmacological effects of maslinic acid have been reported in various experimental models including their antitumor (Lu, Xi et al. 2009), anti-inflammatory (Banno, Akihisa et al. 2005) (Martin, Vazquez et al. 2006, Huang, Guan et al. 2011), cardioprotective (Shaik, Rasool et al. 2012), antiviral, antimalarial (Shin and Kang 2003), neuroprotective (Romero, Garcia et al. 2010), and antioxidant activities(Lin, Huang et al. 2011). Considering its wide distribution in the plant kingdom and biological activities, it is suggested that maslinic acid is a natural and safe molecule. Maslinic acid has been assessed for its toxicity effects in animal models fed with high doses of this triterpene and it did not produce any signs of morbidity and mortality. Several other pentacyclic triterpenes are under clinical trials or ready to be launched in the market.

The anti-inflammatory effects of pentacyclic triterpenoids are largely ascribed to their ability to inhibit molecular targets such as 5-lipoxygenase (LOX), inducible nitric oxide synthase (iNOS), cyclooxygenase (COX) 2, and nuclear factor-kappa B (NF-𝜅B) activities. NF-κB, a ubiquitous transcription factor, was discovered in 1986 as a nuclear factor that binds to the enhancer region of the κB chain of immunoglobulin in B cells. It is present in all cells, and in its resting stage, this factor resides in the cytoplasm as a heterotrimer consisting of p50, p65, and inhibitory subunit IκBα. NF-κB is activated by free radicals, inflammatory stimuli, cytokine activation, Recently, some studies have shown that MA has moderate anti-cancer activities has been revealed to exert therapeutic effects on a variety of solid tumors, including in bladder, prostate, colon, esophageal, colorectal, cervical and ovarian cancer (Reyeszurita, Pachonpena et al. 2011, Zhang, Ding et al. 2014). However, the molecular mechanisms underlying the antitumor functions of maslinic acid remain elusive. However, the mechanisms of MA action in inflammation and cancers are still not clear, and the synergistic effects of MA and TNF alpha in inhibiting tumor growth and proliferation have not been investigated. Maslinic acid is arising as a safe and novel natural pentacyclic triterpene which are used to treat diverse inflammatory diseases as well as protective effects against chronic inflammatory diseases in various in vivo and in vitro experimental models. Understanding the anti-inflammatory mechanism of maslinic acid is crucial for its development as a potential dietary nutraceutical. These previous studies implied that MA was a potent hepatic protective agent. So far, it remains unknown if this triterpene could protect the liver against alcohol-induced injury.

The study of (Huang, Guan et al. 2011) revealed that MA was able to alleviate inflammatory stress through inhibiting NF-κB pathway in lipopolysaccharide-treated rat cortical astrocytes. Thus, it is possible that MA could exert anti-inflammatory activity for the liver via regulating signaling pathways.

Inflammation is derived from the Latin word ‘inflammable or inflammation’, which means, “to set on fire.” Inflammation is a basic defense mechanism in which the body reacts against infections, Irritations, or other injuries. The four key features of inflammation are redness, heat, swelling, and pain. Inflammation stimulates the immune response at the site of injury or infection and is itself stimulated by increases in blood supply and vascular permeability, which allow more infiltration of plasma and leukocytes from the blood into injured tissues. This particular type of immune response is important because it helps the body to ward off pathogens and also to initiate the healing process in the damaged tissues. This reaction is classified as acute inflammation. In addition to these, pro-inflammatory cytokines and chronic infections can play an important role in the stimulation of IKK activity, which leads to constitutive NF-κB activation (Karin, Cao et al. 2002).Several proinflammatory factors like TNFα and Toll-like receptor ligands such as lipopolysaccharide (LPS) normally activate these pathways (Karin and Greten 2005).TNF is released mainly from macrophages and regulates immune cells. Its dysregulation and overproduction lead to cancer and other diseases.

TNF also plays a role in the activation of NF-κB by binding to a TNF receptor present on the cell surface that in turn triggers a pathway that leads to the activation of IKK (Aggarwal, Vijayalekshmi et al. 2009). Interleukins are a group of cytokines released in the body from numerous cells in response to various stimuli. While IL-1β plays an important role in the inflammatory response against infection by increasing the expression of endothelial adhesion factors, thus allowing infiltration of leukocytes at the site of infection, IL-6 is a proinflammatory cytokine released in response to trauma or tissue damage.

The liver is the human body largest digestive and metabolic organ, in addition to being a very important immune organ. The liver has a unique dual blood supply, with 20% of the blood coming from the hepatic artery and 80% from the portal vein, which is rich in bacterial products, environmental toxins, and food antigens. The liver is the site of convergence of antigens from the gastrointestinal tract and innate immune system. The liver’s anatomical location and functional characteristics make it one of the most vulnerable organs in the human body. Therefore, the liver should have a perfect defensive immune system to ensure its normal operation and unaltered physiological function of the innate immune system. The liver is the site for the production of cytokines, complement components, and acute reactive proteins. The defensive immune system of the liver comprises a very complex cell population. The liver comprises parenchymal and non-parenchymal cells, which account for 80 and 20% of the total liver cells, respectively. The non-parenchymal cells mainly include liver sinusoidal endothelial cells (LSECs), dendritic cells (DCs), hepatic stellate cells (HSCs), and Kupffer cells (KCs) (Mohar, Brempelis et al. 2015). These cells have different structural functions and sources of differentiation, and they regulate the local and systemic immune function.

The natural immune system of the liver regulates inflammation by balancing the production of pro-inflammatory and anti-inflammatory cytokines. As an important natural immune organ, the liver plays an important role in preventing tumor transformation and liver injury, defending against microbial invasion, and repair. The anti-inflammatory action is a common property of many triterpenoids. Since the anti-inflammatory effects of oleanolic acid were described for the first time in the 1960s, numerous studies have corroborated this activity. Several mechanisms have been proposed to mediate the anti-inflammatory activity induced by oleanolic acid and structurally related triterpenoids. Oleanolic acid is able to attenuate histamine release from mast cells and inhibit 5-lipoxygenase and human leukocyte elastase (Safayhi and Sailer 1997).

Kupffer cells are macrophages that reside in the hepatic sinus. It is the major natural macrophage population in the human body, accounting for 80-90% of the total number of natural macrophages and 20% of the liver non-parenchymal cells (Duarte, Coelho et al. 2015). They are mainly found in the portal vein, which is an important part of the cellular immunity machinery of the human body. Kupffer cells were first discovered in 1876 by Karl Wilhelm von Kupffer and hence are named after him (Haubrich 2004). Initially these cells were thought to arise from inside of the liver so as to form the lining for liver’s blood vessels. But continuous research on this topic, especially by Tadeusz Browicz revealed that Kupffer cells were originally macrophages which were born in bone marrow. This discovery was made in 1898 and due to this contribution by Browicz, Kupffer cells are also known as Browicz-Kupffer cells at times (Naito, Hasegawa et al. 1997). After that several new functions and evidence about these cells has been point out by the scientists. Some of them include its part in the recycling of dead blood cells and also helping out the liver response to waste products in the blood. Kupffer cells are large in size and irregularly shaped, with their perikarya protruding into the gap cavity or completely free in the sinus cavity. Their filopodia are attached to the superficial of the endothelial cells, inserted into the endothelial gap, or extended into the Disse space through the fenestrae, and are thereby interleaved with hepatocyte microvilli. The structure of KCs lays a foundation for the mutual coordination and influence of liver cells and other cell functions. Kupffer cells not only carry out non-specific phagocytosis and clearance of bacteria and foreign bodies such as antigenic substance in the bloodstream, but also play roles in specific immune response, anti-tumor immunity, detoxification of endotoxin, anti-infection, regulating microcirculation, and metabolism.

The kupffer cells surface has many functional receptors and proteins that are related to immune response, such as immunoglobulin G (IgG) Fc receptors (including CD64, CD32, and CDl6), complement receptor (including the complement receptor L, complement receptor 3, and complement receptor 4), mannose receptor, I region-associated antigen, and other surface molecules (such as CDl3, CDl5, and CD68). In vivo, Kupffer cells are generally in the sleeping state. Upon stimulation by pathogens or cytokines, they can be activated and have an enhanced function. They synthesize and secrete TNF-α, IL-l, IL-6, oxygen free radicals, peanut arachidonic acid, maslinic acid and a number of bioactive substances involved in the body inflammatory response (Heymann, Peusquens et al. 2015).

Kupffer cells play a critical role in the innate immune response; their localization in the hepatic sinusoid allows them to efficiently phagocytize pathogens arriving from the portal or arterial circulation. Kupffer cells also serve as the first line of defense against particulates and immunoreactive material passing from the gastrointestinal tract via the portal circulation and may be considered as a final component in gut barrier function. Kupffer cells thus play a major anti-inflammatory role by preventing the movement of these gut-derived immunoreactive substances from traveling past the hepatic sinusoid. Kupffer cells are also highly poised for clearance of particles, as well as dead and dying erythrocytes and cells in the hepatic parenchyma, from the systemic circulation. Kupffer cells thus comprise the major phagocytic activity of what was classically termed the reticular endothelial system and now more properly called the mononuclear phagocytic system (Thomson and Knolle 2010).

A change in the functional activity of Kupffer cells is associated with a variety of disease states. While Kupffer cells can be protective in a number of situations, including drug-induced liver injury (Ju, Reilly et al. 2002) and toxin-induced fibrosis (Ramachandran and Iredale 2012); dysregulation in the precise control of inflammatory responses in Kupffer cells can contribute to chronic inflammation in the liver, including alcoholic and nonalcoholic fatty liver diseases (NAFLDs/NASH) (Nagy 2003) (Chiang, Pritchard et al. 2011).

The complement complex is another system which may be influenced by triterpenoids. An inhibitory effect of oleanolic acid on complement-mediated inflammatory responses has been described by inhibiting the C3-convertase classical complement course (Lee, Park et al. 2004). Its ability to reduce inflammatory cell infiltration and reduce the quantifier of exudates in experimentally induced pleuritis after administration of oleanolic acid has been also described (Herrera, Rodriguezrodriguez et al. 2006).

Macrophages can act as mediators of inflammation by generating pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF) – α. Maslinic acid has been reported to decrease oxidative stress and the generation of IL-6 and TNF- α in lipopolysaccharide (LPS)stimulated murine peritoneal macrophages (Martin, Vazquez et al. 2006). Moreover, the modulation of these cytokine and chemokine secretions by pomace olive oil triterpenoids in human mononuclear cells has been recently described (Marquezmartin, La Puerta et al. 2006). In this study, oleanolic acid, erythrodiol and avail inhibited the production of IL-1 β and IL-6 to some degree, and erythroid was the most potent anti-inflammatory compound. Both oleanolic acid and uvaol showed a biphasic response in terms of TNF- α production, whereas maslinic acid only attenuated IL-6 production at high concentrations (Marquezmartin, La Puerta et al. 2006).

The beneficial effect of oleanolic acid on inflammation has been also associated with its specific and inhibitory action against isoforms of cytochrome P450 such as CYP1A2 (Kim, Lee et al. 2004).

Several investigations have shown the inhibitory activity of oleanolic acid and maslinic acid against human immunodeficiency virus replication (Herrera, Rodriguezrodriguez et al. 2006). This activity has been associated with the inhibition provided by both triterpenoids on serine proteases. This activity also suggests that oleanolic acid and maslinic acid might be a potential molecule for the synthesis of new antiretroviral drugs (Zhu, Shen et al. 2001). Other activities evoked by olive oil triterpenoids, especially by oleanolic acid, include antibacterial (Cunha, Silva et al. 2007) and anti-ulcer genic activities (Astudillo, Rodriguez et al. 2002).

1.2 Aim of study

Considering all the above facts present study will be designed to assess the anti-inflammatory effect of maslinic acid on immune function of kupffer cells in the liver.

Specific objectives

i. To assess Effect of maslinic acid on immune function of kupffer cells in the liver.

ii. Weather tlr4 pathway is involved in maslinic acid regulating the immune function of kupffer cells.

iii. Examine the physiological factors of the liver which effect by the maslinic acid diet.

iv.

Research Methodology

This section presents an overview of the methods to use in the study. The area covered including the research design, sample, and sampling techniques, data collection, and analysis.

Material & methods

Chemical purchased from local market

Animals

Five-week-old male Balb/cA mice were obtained from the National Laboratory Animal Center. Mice was housed on a 12-h light–12-h dark schedule and fed with water and mouse standard diet (PMI Nutrition International LLC, Brentwood, MO, USA). Use of the mice was reviewed and approved by the China Medical University animal care committee.

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