Haematococcus pluvialis Carotenoids Enrich Fractions Ameliorate Liver Fibrosis Induced by Thioacetamide in Rats: Modulation of Metalloproteinase and Its Inhibitor

Hepatic fibrosis is a consequence of chronic liver diseases. Metalloproteinase and its inhibitor have crucial roles in the resolution of liver fibrosis. The current relevant study is aimed to evaluate the therapeutic effect of Haematococcus pluvialis (H. pluvialis) extract, astaxanthin-rich fraction, astaxanthin ester-rich fraction, and β-carotene-rich fraction as well as their mechanisms of action in curing hepatic fibrosis induced by thioacetamide (TAA). Liver fibrosis was induced using TAA (intraperitoneal injection, two times a week for 6 weeks), in a rat model and H. pluvialis extract (200 mg/kg), and other fractions (30 mg/kg) were orally administered daily for 4 weeks after the last TAA injection. Based on HPLC analysis, H. pluvialis extract contains β-carotene (12.95 mg/g, extract) and free astaxanthin (10.85 mg/g, extract), while HPLC/ESI-MS analysis revealed that H. pluvialis extract contains 28 carotenoid compounds including three isomers of free astaxanthin, α or β-carotene, lutein, 14 astaxanthin mono-esters, 5 astaxanthin di-esters, and other carotenoids. H. pluvialis and its fractions reduced liver enzymes, nitric oxide, collagen 1, alpha-smooth muscle actin, and transforming growth factor-beta as well as elevated catalase antioxidant activity compared to the TAA group. Also, H. pluvialis extract and its fractions exceedingly controlled the balance between metalloproteinase and its inhibitor, activated Kupffer cells proliferation, and suppressed liver apoptosis, necrobiosis, and fibrosis. These findings conclude that H. pluvialis extract and its fractions have an antifibrotic effect against TAA-induced liver fibrosis by regulating the oxidative stress and proinflammatory mediators, suppressing multiple profibrogenic factors, and modulating the metalloproteinase and its inhibitor pathway, recommending H. pluvialis extract and its fractions for the development of new effective medicine for treating hepatic fibrosis disorders.


Introduction
Liver fibrosis is induced by a chronic hepatic insult and associated with liver dysfunction and life-threatening complications [1]. Chronic liver diseases affected millions of patients worldwide; 30% of patients are likely to develop fibrosis and cirrhosis. Also, this condition is evolved towards liver cancer and an elevation in the mortality rate [2,3]. In fibrosis and chronic liver diseases, there is an accumulation of fibril-lar extracellular matrix (ECM) [4] and stimulation of sinusoidal endothelial cells releasing cytokines and activating hepatic stellate cells (HSCs) [5]. Also, Kupffer cells, hepatic macrophages, play an important role in the resolution of liver fibrosis [6] via the production of a large spectrum of matrix metalloproteinase (MMPs) expression [7]. MMP9 is a gelatinase enzyme produced by Kupffer cells and is implicated in the pathological process of hepatic injury [8]. The presence of MMP9 suppressed transforming growth factor-beta (TGF-β1) activation and reduced ECM components accumulation as collagen 1 and α-Smooth muscle actin (α-SMA) during fibrogenesis in the liver [9]. The expression MMP9 was elevated during fibrosis resolution in Kupffer cells [10]. In the liver fibrogenesis process, the most critical cytokine involved is TGF-β [11]. TGF-β1 can also control MMPs and tissue inhibitors of matrix metalloproteinase (TIMPs) expression. MMPs carry out an important function in degrading ECM components, and TIMPs have the ability to adverse MMP function [12,13].
Carotenoids including β-carotene and astaxanthin showed abundant awareness lately due to their powerful antioxidant properties that trigger the protection of the organism against different oxidative stresses related disorders. Different carotenoids accumulate primarily in the liver, then they are transported by divers lipoproteins for releasing to the blood circulation from which it deposited in some organs and tissues including skin, kidneys, adrenal glands, adipose tissues, testes, and the prostate [14]. The accumulation of these carotenoids as antioxidants and their different metabolites in the liver can improve the hepatocyte metabolism and regulate the cellular oxidative conditions in certain liver complications [15] Astaxanthin is one of the most potent active carotenoids exhibiting an antioxidant effect 10 times greater than βcarotene and 100 times stronger than vitamin E, and it exists mostly in the form of fatty acid esters such as that produced by the microalgae H. pluvialis. Besides, astaxanthin and astaxanthin esters H. pluvialis microalgae synthesized β-carotene [16]. As a result of their potent competency to scavenge different reactive oxygen species and quench singlet oxygen, the antioxidant capability of different carotenoid individuals, including astaxanthin and β-carotene, is of prominent importance to human health [17] through their ameliorative ability against oxidative stress; the main promoter of many diseases including liver disorders [18,19].
The health advantages of supplements containing H. pluvialis extract enrich in astaxanthin and astaxanthin esters have been the hot topic of several in vitro and clinical studies because of its potent antiaging influence, anti-inflammatory, gastroprotective, immunoprotective, cardioprotective, nephroprotective, neuroprotective, antiasthmatic, antidiabetic, and anticancer properties [20][21][22][23]. Besides astaxanthin, β-carotene has anticancer activity especially against lung and prostate cancers [24]. β-Carotene also has anti-inflammatory potentials by suppressing different proinflammatory markers including redox-based nuclear factor-kappa-β (NF-κβ), NADPH oxidase protein, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) was reported [25,26]. Also, β-carotene from other algae Dunaliella salina (D. salina) has an improvement potential against liver fibrosis injures induced by thioacetamide (TAA) in rats [19]. In this study, we evaluated the therapeutic effect of H. pluvialis extract and its astaxanthin-rich fraction, astaxanthin esterrich fraction, and β-carotene-rich fraction and their mechanisms of action in curing hepatic fibrosis induced by TAA in rats via modulation of MMP and its inhibitor as a target to ameliorate liver fibrosis.

Materials and Methods
2.1. Cultivation of H. pluvialis. H. pluvialis was isolated from the freshwater community of River Nile water at Cairo, Egypt, in October 2011, and grown on BG11 media [27], which contains NaNO 3 , 1.5 g/l; K 2 HPO 4 , 0.04 g/l; MgSO 4 .7H 2 O, 0.075 g/l; CaCl 2 .7H 2 O, 0.036 g/l; citric acid, 0.006 g/l; Na 2 CO 3 , 0.02 g/l; Na 2 EDTA, 0.001 g/l; ferric ammonium citrate, 0.006 g/l; and 1 ml/l of trace-metal mix A5; A5 contains H 3  After growing for 10 days, the culture was transferred to a fully automated and computer-controlled vertical photobioreactor (Varicon Aqua Solutions Ltd, United Kingdom) with a capacity of 4000 L. Carbon dioxide was injected into the culture, by a gas control unit of the photobioreactor, as a carbon source. The culture was left to grow until the biomass reached 2-2.5 g/l, which is monitored by daily calculation of algal biomass weight (g/l). Algal biomass was harvested by centrifugation at 2000 rpm for 15 min using basket centrifuge. Samples were washed twice with water, dried in an oven at 50°C, ground into a homogenous powder, and stored for further chemical and biological investigation.

Preparations of Algal Extract and Enrich
Fractions. The fine powder of H. pluvialis (100 g) was soaked with 1000 ml of dichloromethane: methanol (3 : 1, V/V) in a 2000 ml conical flask and kept on an orbital shaker (Stuart, England) at 160 rpm at room temperature for 24 h. Then, the extract was centrifuged using a centrifuge (Sigma 3-18 ks Centrifuge, Germany) at 5000 rpm for 20 min at 25°C to separate cell debris from the supernatant. The extraction step was repeated twice, and the pooled supernatants were concentrated using a vacuum rotary evaporator (Heidolph Unimax 2010, Germany) at 40°C to dryness giving the crude extract. The crude extract was subjected to a silica gel column chromatography using silica gel, 60-120 μm (Sigma-Aldrich Co., USA), and hexane/ethyl acetate as mobile phase with increasing polarity (0, 10, 30, and100% ethyl acetate) then ethyl acetate: methanol (1 : 1) that afforded 10 fractions that collected in 50 ml per each fraction. The 10 fractions were subjected to TLC (20 × 20 cm aluminum sheets coated with silica gel 60 F254, Merck, Germany) to detect the presence of phytocompounds that were visualized by ultraviolet (UV) fluorescent colors at 254/366 nm UV lamps. Fractions were combined into 3 fractions (1-3) based on TLC results and concentrated to dryness using a rotary evaporator. β-Carotene enriches fraction (Fraction 1) and astaxanthin esters enrich fraction (Fraction 2) and astaxanthin enriches fraction (Fraction 3) were confirmed by HPLC. All the used solvents 2 BioMed Research International were Analar grade from Sigma-Aldrich Co., USA. All the extraction and column chromatography fractionation steps were performed in dim light.
The following solvents were used at a flow rate of 1.25 ml/min: (A) acetone and (B) methanol: H2O (9 : 1 v/v) containing 0.05% BHT. The separation of β-carotene and astaxanthin was achieved by a gradient between solvents A and B for 40 min as follows: B was run at 80 to 20% for 25 min, 20% for 10 min, and 20 to 80%for 5 min [28]. The peaks were integrated at 450 nm to quantify β-carotene and astaxanthin. β-Carotene (C4582-5MG) and all-transastaxanthin (SML0982-50MG) were purchased from Sigma-Aldrich Co., USA, and used as standard. β-Carotene and all-trans-astaxanthin were identified and quantified by comparing retention time and the peak area of the unknown peak with the β-carotene and all-trans-astaxanthin standards. All the used solvents were HPLC grade from Sigma-Aldrich Co., USA. At the end of the experiment, rats were anesthetized with pentobarbital sodium. Blood samples were withdrawn from the retro-orbital plexus of the rats under the same anesthesia; then, rats were sacrificed by decapitation [33] under the same anesthesia for the collection of liver samples. A 0.5 gm of the liver of each animal was rapidly dissected out, washed, and homogenized using phosphate-buffered saline (PBS, 50 mM potassium phosphate, pH 7.5) at 4°C to produce a 20% homogenate using a homogenizer (Heidolph, DIAX 900, Germany). The homogenates were centrifuged at 2000 × g for 20 min at 4°C then stored at -80°C till the time of analysis [34]. Another part of liver tissues was kept in 10% formalinsaline for histopathological examination.

Histological Examination.
For histopathologic assessment, the parts of the livers were fixed in 10% formalin solution then dehydrated in ascending grades of alcohol and embedded in paraffin. Four sections/group, at 4 μm thickness, were taken and stained with hematoxylin and eosin (H&E). The severity of histopathological alternation was semiquantitatively assessed based on liver histology evaluated by a blinded pathologist using a scoring system in which score 0 indicated no alternation; score 1, mild alternation; score 2, alternation activity; and score 3, severe alternation.

Statistical Analysis.
All the values are presented as means ± standard error of the means (SE). Data were evaluated by one-way analysis of variance followed by Fisher's LSD comparisons test. GraphPad Prism software, version 5 (Inc., San Diego, USA) was used to carry out these statistical tests. The difference was considered significant when P < 0:05.

Effects of H. pluvialis Crude Extract and Its Fractions
(Astaxanthin, Astaxanthin Ester, and β-Carotene) on Serum Hepatic Function Biomarkers. Injection of TAA, for 6 weeks, led to a significant elevation in ALT and AST activities as well as total bilirubin serum level with a decrease in albumin serum level compared to those in the control rats. Treatment with silymarin, for 4 weeks, resulted in a significant decrease in serum level of ALT, AST, and total bilirubin with an increase in albumin serum level as compared to the TAA group. Administration of H. pluvialis crude extract and its fractions (astaxanthin, astaxanthin ester, and β-carotene) for 4 weeks reduced serum levels of ALT, AST, and total bilirubin with an increase in albumin serum level as compared to the TAA group. Moreover, crude extract-treated rats showed a decrease in serum level of ALT and AST by 10% and 17%, respectively, as compared to the silymarin group, returning the serum liver biomarkers to their normal values (Table 3).

Effects of H. pluvialis Crude Extract and Its Fractions
(Astaxanthin, Astaxanthin Ester, and β-Carotene) on Oxidative Stress Biomarkers. Serum catalase activity was decreased, and NO level was elevated with TAA injection as compared to normal group values. On the other hand, treatment with silymarin, for 4 weeks, resulted in a significant increase in serum activity of catalase with a significant decrease in NO serum level, as compared to the TAA group. Treatment with H. pluvialis crude extract and its fractions (astaxanthin, astaxanthin ester, and β-carotene) for 4 weeks resulted in an elevation in catalase activity with a decrease in NO level, as compared to the TAA group. Treatment with crude extract showed an increase in catalase activity with a decline in NO level by 28% and 9%, respectively, as compared to the silymarin group, returning the serum levels of catalase and NO to their normal values (Table 4). 4 BioMed Research International      (Figure 7).

Effects of H. pluvialis Crude Extract and Its Fractions
(Astaxanthin, Astaxanthin Ester, and β-Carotene) on Proinflammatory Cytokine Biomarkers. Induction of liver fibrosis in rats using TAA for 6 weeks resulted in a significant elevation in hepatic contents of IL-6 and TNF-α, as compared to the normal group. Treatment with silymarin, for 4 weeks, reduced hepatic contents of IL-6 and TNF-α as compared to the TAA group. Moreover, treatment with H. pluvialis crude extract and its fractions (astaxanthin, astaxanthin ester, and β-carotene) for 4 weeks significantly decreased the elevated hepatic IL-6 and TNF-α as comparing to the TAA group. Treatment with crude extract showed a decrease in hepatic contents IL-6 and TNF-α by 39% and 31%, respectively, as compared to the silymarin group. Treatment with crude extract returned hepatic contents of IL-6 and TNF-α to their normal values (Figure 8). Figure 9 summarised the effects of H. pluvialis crude extract and its fractions (astaxanthin, astaxanthin ester, and β-carotene) on histopathological alteration. The liver section of the control normal rat showed no histopathological alteration, and the normal histological structure of the central vein and surrounding hepatocytes in the parenchyma was recorded (Figure 10(a)). The liver section of the TAA-treated group that showed fine fibroblastic cell proliferation was dividing the degenerated and necrobiotic changes hepatocytes into lobules (Figure 10(b)). Liver section of the silymarin-treated group there was few pigmented cells infiltration surrounding and adjacent to the dilated central vein (Figure 10(c)). The liver section of the crude extract showed diffuse Kupffer cell proliferation was detected in between the degenerated hepatocytes ( Figure 10(d)). The    (Figure 10(e)). The liver section of astaxanthin showed mild degeneration in the hepatocytes adjacent to the congested central vein. A focal hemorrhage was detected in the parenchyma adjacent to the portal area ( Figure 10(f)). The liver section of β-carotene showed dilatation in the central vein associated with focal hemorrhage in the parenchyma. Fine fibroblastic cell proliferation was dividing the degenerated and necrobiotic changed hepatocytes into lobules (Figure 10(g)).

Histopathological Findings.
The liver section of the portal area of the TAA-treated group showed fibrosis with inflammatory cell proliferation between the congested portal vein and hyperplastic bile ducts (Figure 11(a)). In the liver section of the silymarin-treated group, the portal area showed inflammatory cell infiltration (Figure 11(b)). The liver section of crude extract in the portal area showed few inflammatory cell infiltration (Figure 11(c)). Liver section of astaxanthin ester associated with inflammatory cells infiltration in the portal area (Figure 11(d)). The liver section of astaxanthin showed a focal hemorrhage detected in the parenchyma adjacent to the portal area ( Figure 11(e)). The liver section of β-carotene the portal area showed congestion in the portal vein and mild fibrosis with inflammatory cell infiltration in between (Figure 11(f)).

Discussion
The HPLC analysis of H. pluvialis carotenoid extract showed the presence of free astaxanthin and β-carotene based on the available standers; these results agree with [21]. Because the main crude extract and astaxanthin ester rich fraction showed the potent effects against liver fibrosis as compared  Data are presented as the mean ± S:E: of (n = 8) for each group and % of the TAA group. Statistical analysis was carried out by one-way analysis of variance followed by Fisher's LSD comparisons test. a Statistically significant from the control group. b Statistically significant from the TAA group. c Statistically significant from the silymarin group. d Statistically significant from crude extract group. e Statistically significant from astaxanthin ester group at P < 0:05.  The potent activity of crude extract and astaxanthin esterrich fraction of H. pluvialis against liver fibrosis in this study might be thanks to the association of astaxanthin with different fatty acids that increase the bioavailability of astaxanthin in different tissues accordingly increases its bioactivity [38].
Liver fibrosis is closely related to oxidative stress. As previously mentioned, TAA induced oxidative stress, hepatocellular fibrosis, and necrosis [32], overexpressed the iNOS gene, and produced apoptotic DNA fragmentation [39]. Antioxidants have a protective effect in animal models and clinical trials on liver fibrosis [40]. In the current study, the potent competency of different carotenoid individuals including astaxanthin and β-carotene is their ameliorative ability against oxidative stress in liver diseases. TAA increased ALT, AST, and total bilirubin serum levels with a reduction in albumin serum level as shown in a previous study [41], however, administration of H. pluvialis crude extract and its fractions (astaxanthin, astaxanthin ester, and β-carotene) effectively reduced liver function with an increase in albumin serum level. Only the administration of H. pluvialis crude extract returned the serum levels of ALT, AST, total bilirubin, and albumin to their normal levels. In these contexts, the H. pluvialis carotenoids alleviated doxorubicin-induced liver injury through decreasing liver function and regulating Keap1/Nrf2/HO-1 pathway [42]. Also, the liver is more vulnerable to TAA-induced liver oxidative damage evidenced by higher NO serum level and lower catalase activity, while all treatments especially crude extract have antioxidant effects as confirmed by the detection of oxidant and antioxidant indicators as NO serum level and catalase activity. The administration of H. pluvialis crude extract and its fractions (astaxanthin, astaxanthin ester, and β-carotene) effectively reduced the liver oxidative damage and inflammation induced by TAA administration in this study, especially the administration of H. pluvialis crude extract returning the levels of NO, CAT, IL-6, and TNF-α to its normal levels, this might be thanks to the potent antioxidant activity of the different carotenoids including astaxanthin and β-carotene identified in H. pluvialis crude extract by LC/ESI-MS. The administration of H. pluvialis   Figure 9: Effects of crude extract and its fractions (astaxanthin, astaxanthin ester, and β-carotene) on histopathological alteration. Data are presented as the mean ± S:E: of (n = 8) for each group. Statistical analysis was carried out by one-way analysis of variance followed by Fisher's LSD comparisons test. A Statistically significant from the control group. B Statistically significant from the TAA group. C Statistically significant from the silymarin group. D Statistically significant from the crude extract group. E Statistically significant from the astaxanthin ester group at P < 0:05. carotenoids astaxanthin enhanced the levels of SOD and CAT and decreased the lipid peroxidation against liver injury induced by doxorubicin in rats [42]. Also, β-carotene, from other algae D. Saline, exerted its antioxidant through inducing catalase and thioredoxin enzymes that attenuated STZinduced diabetic neuropathy [43] and elevated the levels of GSH with a decrease in MDA opposing hepatic fibrosis induced by TAA in rats [44]. β-Cryptoxanthin, one of the other carotenoids, found in our crude extract has an antioxidant effect against oxidative DNA damage and lipid peroxidation [45]. Moreover, it has anti-inflammatory activity modulating macrophage immune response [46]. This carotenoid suppressed proinflammatory cytokines secretion (TNF-α and IL-6) [46] reversing inflammation, steatosis, and fibrosis progression in NASH by repressed macrophage activation [47]. In patients with nonalcoholic fatty liver disease (NAFLD), β-cryptoxanthin intake elevated antioxidant and anti-inflammatory effects [15]. Lutein is one of the natural H. pluvialis carotenoids; its supplementation alleviated hepatic lipid accumulation, preventing NAFLD induced by a high-fat diet [48]. HSCs, collagen-producing cells, contribute to ECM deposition [49] and have a pivotal role in liver fibrosis [50]. Our results explored that TAA expressed TGF-β activating HSCs and increased liver contents of α-SMA and collagen 1, while H. pluvialis crude extract and its fractions (astaxanthin, astaxanthin ester, and β-carotene) suppressed the activation of HSCs, inhibited profibrotic gene expression

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BioMed Research International via preventing the release of TGF-β, and decreased liver contents of α-SMA and collagen 1. Especially, astaxanthin ester has a direct method of preventing and treating liver fibrosis through reducing the activation of HSCs and decreasing ECM components and deposition that evidenced in this study by decreased liver contents of collagen-1 and α-SMA. Further evidence has shown that astaxanthin (80 mg/kg), during liver fibrosis, inhibited the expression of TGF-β and activation of HSCs [51,52]. Also, β-carotene from D. salina treating liver fibrosis via decrement of α-SMA and COL-1 liver contents [19].
MMPs, in fibrosis, can degrade ECM protein components and promote activated HSC apoptosis [53]. This current study is the first to explore the effects of crude extract and its carotenoid enrich fractions on liver fibrosis induced by TAA via increasing MMP9 and decreasing its inhibitor TIMP1expression. Some molecules become stimulated as MMPs during liver fibrosis restoration, and other molecules become repressed as MMP inhibitor. Our results exhibited that crude extract and its carotenoid enrich fractions play a dual role in liver fibrosis as preserved the balance between MMP9/TIMP1 ameliorating liver fibrosis. These results were simultaneously confirmed in a histopathological study that showed amelioration of liver fibrosis and inflammatory cell proliferation in all treatment groups, especially crude extract. These results might be owing to the presence of the potent antioxidant carotenoids (astaxanthin and β-carotene) in the used extract of H. pluvialis; these results are in accord with other studies that confirmed the antifibrotic effect of astaxanthin in the CCL4 model of liver fibrosis [54]. The antifibrotic effect of β-carotene of D. salina through the inhibition of the TGF-β expression and increasing MMP9 was also reported previously [55].
Kupffer cell is a sensor for tissue integrity and homeostasis in the liver and functions as a primary defense line against invading microorganisms and maintaining immunological tolerance and providing an anti-inflammatory in the liver and in [56]. For instance, it secretes interleukin-10, the anti-inflammatory cytokine [57]. Crude extract and its carotenoid enrich fractions in this study, for the first time, increased the number of Kupffer cells, as shown in the current histopathological study, that expressed MMP9, downregulated inflammation, and mediated fibrosis restoration. These results provide a new prospect for treating liver fibrosis, apoptosis, and necrobiosis induced by TAA.

Conclusion
This study elucidated the therapeutic effect of H. pluvialis crude extract and its fractions (astaxanthin, astaxanthin ester, and β-carotene) in treating hepatic fibrosis via decreasing ECM components and its deposition, regulating oxidative stress and MMP9/TIMP1 as well as finally activating Kupffer cell. Also, this study showed the mechanisms responsible for the hepatic fibrosis process, increasing interest concerning the translation of H. pluvialis and its carotenoid enrich fractions into clinical applications in the future.

Data Availability
Data are available with the corresponding author.

Conflicts of Interest
The authors declare that they have no conflicts of interest.