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The Hepatoprotective Effects of Spirulina on Fatty Liver in Human Studies


Human (30 male)

Human (25 patients)

Human (23 patients, age 2-13)


High cholesterol, mild hypertension and hyperlipidemia

Obese subjects Obesity

Type-2 diabetes mellitus

Hyperlipidemic nephritic syndrome

Spirulina dose

4.2 g/day for 4 weeks

2.8 g/three times daily for 4 weeks 2 g/day for 2 months

1 g/day for 2 months


Hypocholesterolemic, hypoglycemic and hypotriacylglycer-olemic effects; reduced LDL Hypocholesterolemic effect; weight loss Hypoglycemic and hypolipidemic effects; reduced HbA-Ic Hypoglycemic and hypocholesterolemic effect

Reference Nayaka et al., 1988

Becker et al., 1986

Parikh et al., 2CC1

Samuels et al., 2CC2

process that follows a repeated or chronic insult of sufficient intensity to begin a "wound healing"-like reaction.134,135 The fibrotic process arises from excessive production of the extracellular matrix (ECM). Various cells and factors participate in fibrogenesis. HSC, Kupffer cells, and the recruited mononuclear cells are the main cells that are responsible for the process. In addition, transforming growth factor j1 (TGFj 1) is essential for the fibrotic diseases.136-138

Activated fibroblasts with myofibroblast characteristics are central in hepatic fibrosis.139 Both animal models and human studies indicate three subpopulations of myofibroblasts—in the portal area or fibrous septal, at interface between the liver cells and the stroma of the portal area, and the perisinusoidal HSC.135 The amplification of ECM production by activated HSC is the primary cause of hepatic fibrosis.140-142 Most HSC, formerly called Ito cells, lipocytes, perisinusoidal cells, and fat-storing cells are present in the Disse's space, which is between the hepatocytes and the sinusoidal endothelial cells; they are also present in the perivascular space around portal area.134,135,143 The major functions of quiescent HSC in normal liver include the secretion of cytokines, the production of ECM, the storage of vitamin A in lipid vacuoles, and the regulation of blood flow.144 HSC transforms from a quiescent phenotype to the activated state following a fibrogenic stimulus. The activation results in multitudinous changes in cellular morphology, metabolism, and gene expression. The changes in the morphology of activated HSC that are observed in animal models are also seen in tissue culture models. One of the most significant changes appears to be the transformation from quiescent HSC to myofibroblast-like cells. The modified myofibroblast-like HSC are characterized by enhanced expression of smooth muscle a-actin and desmin, proliferation, contractility, and migration, as well as altered ECM synthesis (a drastic increase in type I collagen level) and loss of retinol stores 134,135,145-148

The early progress of the activation of stellate cells is recognized as initiation, which develops later into perpetuation. Significant changes in phenotype and induction of early genes149 during the initiation of activation of stellate cells are responses to a series of profibrogenic stimuli, including an imbalanced redox state, altered early ECM composition and paracrine stimulation from injured hepatocytes, sinusoidal endothelial cells, and Kupffer cells. The initiation stage is also associated with an up-regulated kruppel-like transcription factor (Zf9)150 and adhesion molecule (ICAM-1).151 Injured sinusoidal endothelial cells activate HSC by producing variant fibronectins (EIIIA isoform).152 Furthermore, the activation of Kupffer cells and the up-regulated redox-sensitive transcription factors, such as activator protein-1 (AP-1) and nuclear factor kB (NF-kB), intensify this stage.153,154 In the perpetuation stage, accelerated fibrosis proceeds with amplified cellular events through enhanced paracrine and autocrine activities and continued ECM remodeling.135,140 Events in perpetuation of transdifferentiated HSC include proliferation, fibrogenesis, contractility, matrix disruption, and inflammation. Many of these responses are associated with RTK-mediated interactions between cytokines and corresponding up-regulated receptors.155

Expression of platelet-derived growth factor (PDGF) receptor appears to be important in HSC proliferation,156 in which the ERK/mitogenic-activated protein kinase (MAPK) pathway156 and the activation of phosphatidylinositol 3-kinase (PI 3K)156,157 are involved. Moreover, heightened activities of PDGF-regulated Na+/H+ and Na+/Ca2+ exchangers are reported in activated HSC158 and injured liver,159 sustaining extracellular calcium intake and altered pH for proliferation. Recent evidence reveals that proteinase-activated receptor agonists, thrombin, and MC tryptase also regulate HSC proliferation and collagen production.160

Another predominant mediator in fibrogenesis is the up-regulated transforming growth factor j1 (TGFj 1), which induces ECM genes like collagen and fibronectin.161,162 TGFj 1 knockout mice with acute liver injury that exhibit a marked reduction in collagen accumulation reveal the essential role of TGFj 1 in fibrosis.163 Most TGFj 1 in normal liver are from Kupffer cells, while some are from endothelial cells. However, in fibrotic rat liver, elevated autocrine TGFj 1 expression in HSC is evident.142,164 The injury-induced activity occurs not only at the transcriptional level but also through proteolysis by a urokinase-type plasminogen activator.165 Enhanced TGFj 1 results in the strengthened response of activated HSC to injury by interaction with receptors to produce type I collagen, which is low in normal liver ECM.165 The collagen type III content increases before that of collagen type I following liver injury.134 The accumulated collagen subsequently switches from type III to type I, becoming fibril-forming. The fibrosis further proceeds to sclerosis and cirrhosis when 60-70% of all of the collagen is type I.166,167 The deposition of excessive collagen in fibrotic liver impairs the exchange of nutrients and metabolites between paren-chymal cells and blood flow (capillarization). The altered fibrillar ECM interacts with integrins and RTKs on the plasma membrane, further activating HSC.

As the amount of fibril-forming collagen in ECM increases, the normal matrix is decomposed by several enzymes such as matrix metalloproteinase-2 (gelatinase A), 3 (stromelysin 1) and 9 (gelatinase B) (MMP-2, 3 and 9),168-172 and membrane-type MMPs.134 HSC expresses MMP-2, MMP-3 and the recently reported

MMP-9, disrupting basement-membrane collagen IV and subendothelial ECM. Furthermore, as fibrosis progresses, fully activated HSC releases TIMP1 and 2, inhibiting the activity of MMP, leading to advanced collagen accumulation and scar formation.169,173-175

Portal vein resistance, an important event in advanced fibrosis, results from the contractility of activated myofibroblast-like HSC, through the stimulation of autocrine-derived endothelin-1 (ET-1).176 This enzyme also stimulates the proliferation of quiescent HSC and inhibits the growth of activated HSC.177 Increased portal resistance compresses the fibrillar ECM, reducing the blood supply to hepatocytes through the bypassing effect of the connection of afferent portal veins and efferent hepatic veins.178 The retraction of fibrotic tissue results in hepatocyte ischemia and portal hypertension, which have been seen in typical sclerosis and cirrhosis. Accelerated fibrosis can be induced through the migration of activated HSC to the site of injury. This chemotaxis effect is mediated by PDGF and monocyte chemotactic protein-1 (MCP-1).179,180 The recruitment of leukocytes, which is critical to the perpetuation of HSC activation, together with up-regulated adhesion molecules and HSC-released autocrine cytokines and chemokines like colony-stimulating factor and MCP-1, amplify inflammation and accelerate fibrosis.181 The ROS generated by Kupffer cells and hepatocytes promotes inflammation.

The role of hepatic macrophages in hepatic fibrosis has recently been emphasized. The activation of macrophages have been proposed to exhibit two distinct mechanisms: (1) the classic macrophages are activated by TH 1 lymphokines, bacteria, and fungal cell wall components and (2) the alternatively activated macrophages are activated by TH 2 lymphokines, apoptotic cells, and corticosteroids.182,183 The classic macrophages release proinflammatory mediators and are involved in matrix decomposition,184 whereas the alternative activated macrophages produce anti-inflammatory cytokines such as IL-10 and TGF-^, and promote matrix accumulation upon incubation with myofibroblasts.185,186 These two characteristics of macrophages are evident in injured tissues.187 CD11b-DTR transgenic mice injured by carbon tetrachloride were selectively subject to depletion of macrophages during fibrosis and recovery,183 to clarify the duality of macrophages in liver injury and repair. The antifibrotic effect was significant during the depletion of macrophages during liver injury. On the contrary, a substantial matrix accumulated during depletion in the early recovery phase. This investigation demonstrates the opposite role of hepatic macrophages in liver inflammatory scaring. In addition, whether this difference in behavior resulted from the same or different subpopulations remains unclear. However, the evidence suggests that one subset undergoes phenotype switching during the progressing and recovery phases.188,189 Figure 6.4 shows a summary of recent studies on liver fibrosis.

Oxidative Stress and Liver Fibrosis

Liver fibrosis is usually associated with variations in the extent of oxidative stress.134 Oxygen-derived reactive species from hepatocyte lipid peroxidation trigger cultured HSC proliferation and collagen type I synthesis.190 Administering of antioxidants, such as vitamin E, carotenoids and flavonoids reduces fibrosis, often repairing


Hepatic stellate cells tgf ß

Kupffer cells/hepatic macrophages Kupffer cells/hepatic macrophages

Lipid perosidation

Initiation of activated stellate cells

Fibrillar ECM „_TGF/3j collagen synthesis

Initiation of activated stellate cells

Fibrillar ECM „_TGF/3j collagen synthesis

Hormal ECM degradation

Collagen synthesis

Hepatocyte death

Hormal ECM degradation

Collagen synthesis

Perpetuation of activated stellate cells Collagen synthesis

Perpetuation of activated stellate cells Collagen synthesis

Stellate cells reversion Apoptotic stellate cells I lTIMP-1

MMPs (MMP1/MMP13) In human In rodents

Stellate cells reversion Apoptotic stellate cells I lTIMP-1

MMPs (MMP1/MMP13) In human In rodents

Collagen degradation (fibrosis regression)

FIGURE 6.4 Summary of hepatic fibrosis and regression.

injury by scavenging free radicals. ROS mainly released from Kupffer cells and hepatocytes,191 initiate HSC activation and further cause fibrosis through the regulation of autocrine cytokines by redox-sensitive transcription factors like AP-1 and NF-kB. AP-1 is important to the transcription of liver fibrosis-related factors such as TGF^ 1, collagen type I and matrix metalloproteinases.192,193 NF-kB, mostly regulated at the post-translational level, assist in the transcription of inflammatory mediators (TNFa, IL-2, IL-6, IL-8), and adhesion molecules (intercellular, endothelial, and vascular cell adhesion molecules; ICAM, ECAM, and VCAM).134 Furthermore, NF-kB behaves differently from AP-1 in response to 4-hdroxy-2-nonenal (HNE),194,195 a lipid peroxidation-derived aldehyde. Significantly increased AP-1 nuclear binding rather than NF-kB is observed after Kupffer and stellate cells are treated with HNE. This fact is indicative of the fact that ROS and HNE trigger different mechanisms for enhancing fibrosis. ROS promotes inflammation, while HNE may contribute to the initiation of HSC activation.

Oxidative stress is also caused by alcohol, in a process that is catalyzed by cytochrome P450 2E1 (CYP2E1), an isoform of P450 in hepatocytes. This enzyme generates more ROS than cytochrome P450 by reducing O2 to O^-, and the subsequent converting of O2- to H2O2 through superoxide dismutases. The enhanced expression of CYP2E1 in HSC promotes the generation of ROS and collagen I.196 Antioxidant treatment suppresses the collagen I gene (COL1A2) expression. Furthermore, acetaldehyde, an alcohol metabolite, produced by alcohol metabolism through hepatic CYP2E1, activates collagen synthesis in activated HSC in a paracrine manner. The phagocytosis of alcohol-induced hepatocyte apoptotic bodies by Kupffer cells and HSC may lead to elevated expression of TGF^ 1 and activation of HSC. The activation of HSC may also result from the stimulation of ROS and TGF^ 1, which are released by Kupffer cells.197

The progress of fibrosis depends not only on the autocrine effects of cytokines and chemokines, but also on such reactive species as H2O2 and HNE, which can permeate the cell plasma menbrane.198

Spirulina and Potential Resolution of Liver Fibrosis

Liver fibrosis may resolve by the reversion of the normal matrix, the attenuation of inflammation or the reversion/apoptosis of HSCs.140 Whether HSC can revert to the quiescent state is unknown. However, the evidence indicates that HSC remains quiescent when cultured on a normal matrix.199 Moreover, IL-10, secreted as a negative feed back signal to down-regulate inflammation, increases interstitial collagenase activity, to reduce fibrosis through modifying the collagen structure.200,201 Recently, a-melanocyte-stimulating hormone (a-MSH) gene therapy has demonstrated that the reversion of carbon tetrachloride-induced liver fibrosis in mice by regulating collagen metabolism, including reducing the mRNA expression of liver TGF^ 1, collagen a1, and adhesion molecules; attenuating the activities of a-smooth muscle actin (a-SMA) and COX-2; increasing the activity of MMP, and deactivating TIMP.202 The induction of HSC apoptosis associated with attenuated TIMP-1 expression during the recovery phase of liver injury is another approach for reducing fibrosis.203 Furthermore, HSC apoptosis with elevated levels of Fas ligand, NF-kB, p53 and Bcl-2 during spontaneous activation, has been documented.204-206

Innate immunity, including Kupffer cells/macrophages, natural killer (NK) cells, and NKT cells, together with interferon-a and y (IFN-a, y ), has been indicated to regulate fibrosis progression and development.197 Macrophages and NK cells have been reported to kill activated HSC and attenuate fibrosis through matrix degradation during recovery.189,207 Besides, IFN-a and y inhibit fibrosis by means of blocking TGF£ 1 signaling and HSC activation.208

Spirulina has been reportedly associated with the attenuation of fibrosis by the induction of HSC apoptosis and the antioxidative activity, which is involved in the reduction of oxidative stress25 and a decrease in proinflammatory cytokine gene expression.209 In addition, Cpc, a pigment from blue-green algae including Spirulina, reduces the extent of Kupffer cell phargocytosis.209

Oxidative stress promotes the activation of HSC, whereas antioxidants may suppress this process.210,211 Antioxidants such as the natural phenolic compounds resveratrol and quercetin markedly inhibit HSC proliferation.211 Spirulina, which contains many antioxidants such as phycocyanins, carotenoids, selenium, and some phenolics,212 suppresses oxidative stress and the up-regulation of proinflammatory cytokine expression. It may further attenuate the progress of liver fibrosis. The administration of a suitable antioxidant supplement has been established to prevent significantly lipid peroxidation and fibrotic autocrine cytokine expression in rat liver, induced by CCU213

Natural phenolics modulate the activity of receptor tyrosine kinases and the expression of cell cycle protein cyclin D1, thereby modulating the functions of stellate cells.211 In addition, sulfhydryl antioxidants regulate stellate cells by exhibiting reducing activity.210 Natural phenolic compounds such as resveratrol and quercetin have been suggested to be potent inhibitors of the growth of stellate cells by perturbing the signal transduction pathway and the expression of the cell cycle protein.211 Furthermore, quercetin selectively inhibits growth and causes apoptosis in hepatic tumor cells rather than in normal cells.214 Spirulina extracts are likely to constitute compounds, such as phenolics or phycocyanin, that are potential anticancer or antifibrosis agents.

Pycocyanin, like Cpc, has been reported to reduce significantly carbon phagocytosis and carbon-induced O2 uptake on perfused rat liver by exploiting its antioxidant and anti-inflammatory capacities.209 Cpc also reduces 3,3',5-triiodothyronine (T3)-induced (thyroid calorigenesis) serum nitrite and TNF-a levels and hepatic nitric oxide synthase (NOS) activity. TNF-a, a profibrogenic factor that is released from activated macrophages, up-regulates in response to a net increase of ROS.215 The hep-atoprotective/antifibrosis effect of Cpc corresponds to the decline in the formation of reduced ROS and proinflammatory cytokine.

The apoptosis of key cells of fibrogenesis may also have the antifibrosis effect. Hepatic macrophages and stellate cells are central to fibrosis. Hepatic macrophages play different roles in fibrosis or antifibrosis during liver injury and the recovery stage.183 Hepatic macrophages have been suggested to secrete tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and other stimuli to provoke the apoptosis of activated stellate cells.216 Activated HSC also undergoes apoptosis during spontaneous activation that is associated with Bcl, NF-kB, and p53/p21 WAF1 system.206 Accordingly, a possible means of recovering from liver fibrosis is to trigger the apoptosis of activated HSC.140,217

Hepatic macrophage apoptosis at an early specific stage of fibrosis may help to attenuate the progress of fibrosis due to the reduced level of profibrotic cytokine, such as TGF^ 1. Cpc has been reported to induce macrophage apoptosis in a cultured LPS-stimulated RAW 264.7 macrophage cell line.28 Cpc not only selectively inhibits COX-2 activity, but also inhibits the growth and multiplication of RAW 264.7 macrophages in an arresting cell cycle at sub-G0/G1 phase. Moreover, Cpc apoptosis is independent of Bcl-2 and mediated by the release of cytochrome c.

Our earlier study demonstrated the apoptosis of rat-activated HSC and Hep G2, a human hepatocellular carcinoma cell line, by treating it with Spirulina aqueous extract.14 The algae extract-treated cells underwent pronounced morphological changes such as cell shrinkage, the formation of membrane blebs, and DNA fragmentation. The dose-dependent suppression of cell proliferation by treatment of both sets of cells with Spirulina aqueous extract suggests that Spirulina may have the potential to reduce liver fibrosis and probably liver tumors. The data herein are

Hepatic steatosis fibrosis

Quiescent hepatic stellate cells

î ALDH activity

Hypolipidemic and hypochlesterolemic effects

Activated stellate cells


Hypolipidemic and hypochlesterolemic effects


î Innate immunity


Oxidative stres/Lipid perosidation (ROS) (aldehyde)

FIGURE 6.5 Effects of Spirulina on liver fibrosis.

consistent with the results of clinical and laboratory studies,33 which have found that administering Spirulina is a valid treatment for chronic diffuse liver conditions because of its hepatoprotective properties. The results herein also show that aqueous Spirulina extract arrests the HSC cell cycle in the G2/M phase, suppressing proliferation and further inducing apoptosis, as verified by annexin-V analysis and the hypodiploid peak. Moreover, activation of innate immunity is an alternative approach to kill activated HSC. Spirulina may kill activated HSC through the enhanced innate immunity by means of activating NK cells and macrophages; increasing production of interferons.53,57 In conclusion, these findings are evidences of the potential antifibrotic action of Spirulina, which may partially explain the beneficial effects of Spirulina on liver diseases. The effects of Spirulina on liver fibrosis are illustrated in Figure 6.5.

Collective research results explore numerous effects of Spirulina, including antioxidative, anti-inflammatory, anticancer, antiviral, neuroprotective, hepatoprotective and immunoenhancing. Many of the effects are associated with the anti-oxidative effect, which appears to be involved in synergistic effects of a series of phytochemicals, such as selenium, carotenoids, phenolics, phycocyanins, and essential fatty acid GLA. The oxidative stress and subsequent induced inflammation substantially lead to liver damages such as hepatic steatosis, fibrosis, and carcinoma. Considerable results suggest that Spirulina is able to control hepatic steastosis through its antioxidative and anti-inflammatory effects, induction of PGE2 production by GLA, hypolipidemic and hypochlesterolemic effects, and activated ALDH activity. In addition, ROS and related oxidative intermediates have been recognized as the potential stimuli for the accumulation of connective tissue in the liver. Various types of cells are involved in liver fibrosis. Kupffer cells and HSC are mainly responsible for

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