|Year : 2017 | Volume
| Issue : 2 | Page : 46-55
Nigella sativa seed reduced galectin-3 level and liver fibrosis in thioacetamide-induced liver injury in rats
Ahmed M Salem1, Karam A Mahdy2, Nahla S Hassan1, Gamila S.M El-Saeed2, Abdel Razik H Farrag3, Mahmoud A Abdel Monem2
1 Department of Biochemistry, Faculty of Science, Ain Shams University, Egypt
2 Department of Medical Biochemistry, National Research Centre, Cairo, Egypt
3 Department of Pathology, National Research Centre, Cairo, Egypt
|Date of Submission||05-Jun-2017|
|Date of Acceptance||24-Sep-2017|
|Date of Web Publication||29-Dec-2017|
Mahmoud A Abdel Monem
Department of Medical Biochemistry, National Research Centre, Cairo, 12622
Source of Support: None, Conflict of Interest: None
Background/aim Fibrosis represents the final common pathway of chronic tissue injury. Galectin-3 (Gal-3) is an important regulator of fibrosis that links chronic inflammation to fibrogenesis. We investigated the effect of Nigella sativa seed (black seed), a common hepatoprotective natural remedy, on Gal-3 level and progression of liver fibrosis in thioacetamide (TA)-induced liver injury in rats.
Materials and methods Forty male Wistar rats were used in this study and were divided into four groups of 10 rats each. Group I served as control, groups from II to IV were intoxicated by TA (200 mg/kg body weight); meanwhile group III was treated with silymarin (50 mg/kg body weight) and group IV was treated with black seed (50 mg/kg body weight). Gal-3, transforming growth factor β1 (TGF-β1), some antioxidant and oxidative stress biomarkers were determined in the liver tissue homogenate. Moreover, serum liver function parameters, total cholesterol, triacylglycerols, and plasma glucose were determined. Quantitative measurement of fibrotic areas was achieved using computerized image analysis system.
Results TA administration caused significant elevations in the levels of liver Gal-3, TGF-β1, malondialdehyde, nitric oxide and serum alanine transaminase and aspartate transaminase activities, total bilirubin, total cholesterol, triglycerides, and plasma glucose levels. Meanwhile, significant decreases were recorded in the level of liver total antioxidant capacity, catalase activity, and serum levels of total protein and albumin. Histopathological observation showed severe damage in the liver and presence of fibrotic areas. Treatment with silymarin and black seed resulted in decreasing levels of liver Gal-3 and TGF-β1 and marked improvement in liver functions, as well as reducing the fibrotic areas in the liver. Gal-3 exhibited positive correlation with TGF-β1, malondialdehyde, nitric oxide, alanine transaminase and aspartate transaminase, while it negatively correlated with total antioxidant capacity and catalase.
Conclusion Black seed reduced liver Gal-3 level and ameliorated fibrogenesis in liver due to TA administration.
Keywords: black seed, galectin-3, liver fibrosis, rats, thioacetamide
|How to cite this article:|
Salem AM, Mahdy KA, Hassan NS, El-Saeed GS, Farrag AH, Abdel Monem MA. Nigella sativa seed reduced galectin-3 level and liver fibrosis in thioacetamide-induced liver injury in rats. J Arab Soc Med Res 2017;12:46-55
|How to cite this URL:|
Salem AM, Mahdy KA, Hassan NS, El-Saeed GS, Farrag AH, Abdel Monem MA. Nigella sativa seed reduced galectin-3 level and liver fibrosis in thioacetamide-induced liver injury in rats. J Arab Soc Med Res [serial online] 2017 [cited 2019 Aug 18];12:46-55. Available from: http://www.new.asmr.eg.net/text.asp?2017/12/2/46/221887
| Introduction|| |
Many people around the world have been afflicted by some type of liver lesions including fatty liver, nonalcoholic steatohepatitis, hepatitis with its types (A, B, and C), cirrhosis and hepatocellular carcinoma . Fibrosis, and in certain, cirrhosis represent the final endpoint of chronic liver tissue injury, in which chronic inflammation leads to the formation of scar tissue, loss of tissue architecture, and organ failure . Liver fibrosis is one of the leading causes of morbidity and mortality worldwide .
Until now there is no effective and reliable drug for resolving fibrosis. However, there are many trials depending on approaches using several agents that block fibrogenic activation and extracellular matrix (ECM) production by myofibroblasts. This works well in culture and in some rodent models of liver fibrosis but carry a high risk of unwanted side effects in patients due to a lack of specificity for myofibroblasts ,.
Galectins are a category of animal lectins that can bind β-galactosides through their carbohydrate recognition domain, and each galectin prefers binding to an individual carbohydrate. They can be found inside cells in the cytoplasm and nucleus or in the ECM, and can also be found on the cell surface .
Among all the galectins, galectin-3 (Gal-3) was found to have important role in many biological functions such as, cell adhesion, cell signaling, cell proliferation, apoptosis and angiogenesis, immune reactions and fibrogenesis .
It is believed that the soluble part of Gal-3 is responsible for myofibroblast activation, leading to collagen secreting in the ECM and fibrogenesis. Also Gal-3 expression has been accompanied with the development of fibrosis in a variety of organs, such as the liver, kidney, gut, and heart ,. Gal-3 has a role in thioacetamide (TA) acute liver injury through the production of monocyte chemoattractant protein-1 and transforming growth factor-β1 (TGF-β1) . TGF-β1 is a cytokine that enhances fibrosis in many organs. It is mainly produced by hepatic stellate cells and induces the production of ECM components .
Nigella sativa seed (black seed) is a widely used natural food and medicinal plant throughout the world. It has been used in traditional medicine in Middle and Far East since ancient times . Black seed contains many active components such as thymoquinone (30–48%), p-cymene (7–15%), and carvacrol (6–12%). It is reported to have a wide spectrum of therapeutic properties such as antihypertensive, antidiabetic, immunomodulatory, anticancer, renalprotective and hepatoprotective, anti-inflammatory, and antioxidant properties ,,,. Many studies have shown the protective role of N. sativa against heavy metals, such as lead and cadmium, toxicity in liver tissues. Other studies reported the protective effect of N. sativa against hepatic lipid peroxidation introduced by toxic compounds, such as carbon tetrachloride .
TA is a hepatotoxicant that causes centrilobular necrosis in the liver. TA is widely used as a model compound for induction of acute and chronic liver injury and fibrosis, which closely resembles human micronodular cirrhosis ,.
Silymarin is among the drugs that are used in the treatment of hepatic dysfunction. Silymarin is a mixture of flavolignans and flavonoids with antioxidant properties, extracted from the plant Silybum marianum . It is used as a hepatoprotective agent against hepatic injury caused by many toxic substances such as CCl4, aflatoxin B1, and galactosamine .
This study was carried out to evaluate the effect of black seed on Gal-3 level in rats suffering from TA-induced hepatic injury. The study also investigated the correlation between Gal-3 level and other biochemical markers for liver injury. Additionally, this study compared the results with the standard drug, silymarin.
| Materials and methods|| |
Black seed and silymarin used
Black seed was purchased from the Ministry of Agriculture Selling Port, Giza, Egypt. The black seed was washed, dried, ground, and then suspended in water before use. Silymarin was obtained from the pharmacy as 10 sachets produced by SEDICO Pharmaceutical Co., 6 October City, Egypt. Each sachet contains 140 mg silymarin (calculated as silybin). It was freshly prepared and administered by dissolving the content of each sachet in water (50 ml).
Experimental animals and diet
A total of 40 male Wistar rats (90–130 g) were obtained from the National Research Centre, Animal House. The animals were maintained under standardized environmental conditions at 12 h light/dark cycle and at a constant temperature of 25±1°C. Animals were fed on basal diet , and water was supplied ad libitum. Rats were acclimated to laboratory conditions 1 week prior to the beginning of the experiment.
The animals were segregated into four groups, 10 rats per each, as follows. Group I (normal controls): rats received saline intraperitoneally (0.01 mg/kg body weight); group II (TA intoxicated group): rats injected intraperitoneally by 200 mg/kg body weight of TA two times per week for 7 weeks ; group III (silymarin and TA supplemented group): rats were treated with TA as in group II and received orally 50 mg/kg body weight of silymarin daily for 7 weeks; group IV (N. sativa and TA supplemented group): rats were treated with TA as in group II and received orally 50 mg/kg body weight of N. sativa daily for 7 weeks .
Collection of blood samples
At the end of the experiment, blood samples were collected after 16 h fasting using the orbital sinus technique of Sanford . Blood samples were left to clot in clean dry test tubes, and then centrifuged at 3000 rpm for 10 min. The clear supernatant serum was then separated and frozen at −20°C for the biochemical analysis. A small portion of blood was withdrawn into sodium fluoride coated tubes for determination of glucose level. After blood collection, the rats were killed by decapitation and the whole liver of each animal was rapidly dissected, thoroughly washed with isotonic saline and plotted. A small part of each liver was cut and weighed for preparing liver tissue homogenate; the rest of each liver was fixed in formaldehyde buffer (10%) for histological investigation.
Biochemical analyses methods
Liver Gal-3 and TGF-β1 level were determined by enzyme-linked immunosorbent assay technique, using the kit purchased from Sun Red Biotechnology (Shanghai, China), according to the method described by the manufacturer. Liver total antioxidant capacity (TAC), catalase, malondialdehyde (MDA), and nitric oxide (NO) were estimated using commercial kits purchased from Biodiagniostic, Egypt, based on the methods described by Koracevic et al. , Aebi , Satoh , and Montgomery and Dymock , respectively.
Serum alanine transaminase (ALT) and aspartate transaminase (AST) were estimated using kits of Vitro Scient (Hannover, Germany), based on the method described by Bergmeyer et al.  and Henry et al. , respectively. Serum total bilirubin was determined using the kit of Biodiagnostic (Dokki, Giza, Egypt), based on the method described by Walter and Gerade . Serum total protein, albumin, and plasma glucose were estimated using kits purchased from Spectrum (Hannover, Germany), based on the methods described by Cannon et al. , Doumas et al. , and Howanitz and Howanitz , respectively. Serum total cholesterol and triacylglycerols were estimated using kits of Vitro Scient, based on the methods described by Allain et al.  and Fossati and Prencipe , respectively.
Histopathological examinations and image analysis
The liver of rats of different groups were removed and fixed in 10% formal saline; 5 μm thick paraffin sections were stained with haematoxylin and eosin  and examined by light microscope. Formalin-fixed specimens were embedded in paraffin, sliced with a thickness of 4 μm and mounted on silanized glass slides. Van Gieson’s staining, described by Bancroft and Cook , was performed for quantitative measurement of fibrotic areas by using computerized image analysis (Leica Qwin 500) in Image Analyzer Unit, Pathology Department, National Research Centre. The image analyzer was first calibrated automatically to convert the measurement units (pixels) produced by the image analyzer program into actual micrometer units. Ten nonoverlapping fields were chosen in each specimen and the mean values were obtained.
The data obtained in the present work are represented as average mean±SE. Statistical analysis was evaluated using the Student t-test. P values of less than 0.05 were considered statistically significant .
| Results|| |
Biochemical analyses results
The results obtained in [Table 1] show liver Gal-3, TGF-β1, TAC, MDA, NO levels, and catalase activity. Liver Gal-3 and TGF-β1 levels exhibited significant increase (P<0.05) in groups II–IV when compared with normal controls (group I) and significantly decreased (P<0.05) in groups III and IV when compared with group II. Meanwhile, both the liver TAC level and catalase activity decreased significantly (P<0.05) in groups II and IV when compared with group I, while both of them increased significantly (P<0.05) in groups III and IV when compared with group II. Concerning liver MDA and NO levels, there was a significant increase (P<0.05) of their levels in groups II–IV when compared with group I; meanwhile, both of them decreased significantly (P<0.05) in groups III and IV when compared with group II.
|Table 1 Liver levels of galectin-3, transforming growth factor-β1, antioxidant, and oxidative stress parameters in the experimental groups|
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[Table 2] exhibits the results of serum liver function parameters, total cholesterol, triacylglycerols, and plasma glucose. It was found that each of serum ALT and AST activities and total bilirubin level increased significantly (P<0.05) in groups II–IV when compared with group I, while they decreased significantly (P<0.05) in groups III and IV when compared with group II. In comparison with group I, serum total protein and albumin levels of group II decreased significantly (P<0.05); however, both of them increased significantly (P<0.05) in group IV. Meanwhile both levels increased significantly (P<0.05) in comparison with group II. Regarding serum total cholesterol, triacylglycerols, and plasma glucose, their levels increased significantly (P<0.05) in group II when compared with group I. Moreover, the levels of the three parameters in groups III and IV decreased significantly (P<0.05) when compared with group II.
|Table 2 Serum levels of liver function parameters, total cholesterol, triacylglycerols, and plasma glucose in the experimental groups|
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Histopathological examinations and image analysis
Liver sections from normal control rats exhibit normal structure of hepatic lobules ([Figure 1]a). Examination of the liver sections from TA intoxicated rats showed nodular appearance of the liver lobule, bridging necrosis accompanied with mononuclear inflammatory cells, signs of vacuolar degeneration of hepatocytes with balloon cells, and focal proliferation of biliary ductules within the periportal tracts ([Figure 1]b).
|Figure 1 Micrographs of liver section from A): G (I) shows the normal architecture of the portal tract (arrow). At the wall of sinusoids, phagocytic irregular cells known as Von Kupffer cells are shown (arrowhead), B): G (II) shows nodular appearance of the liver lobule and bridging necrosis with accompanied mononuclear inflammatory cells (arrows). Also signs of vacuolar degeneration of hepatocytes with balloon cells (arrowhead) and focal proliferation of biliary ductules within the periportal tracts(green arrow) are visible, C): G (III) shows acute inflammatory infiltrate (arrow) within the periportal tract entering focally the liver lobule and vacuolar degeneration of single hepatocytes. Fibrotic septa are not seen, D): G (III) shows mild inflammation (arrow) but no fibrotic septa. Hepatocytes appear more or less like normal ones with small areas of hydropic degeneration, E): G (IV) shows mild inflammation (arrow) in portal and periportal tract without fibrotic septa. Hepatocytes appear more or less like normal ones, F): G (IV) shows hepatocytes appear more or less like normal ones (H&E, Scale bar: 20 μm).|
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Liver sections of rats given silymarin and TA showed acute inflammatory infiltrate within the periportal tracts entering focally the liver lobule and vacuolar degeneration of single hepatocytes. Fibrotic septa were seen ([Figure 1]c). In some rats from the same group, mild inflammation but no fibrotic septa were shown. Hepatocytes appeared more or less like normal ones with small areas of hydropic degeneration ([Figure 1]d).
The histopathological study of liver sections from N. sativa and TA supplemented group showed mild inflammation in the portal and the periportal tract without fibrotic septa. Hepatocytes appear more or less like normal ones ([Figure 1]e and [Figure 1]f).
Liver sections stained with Van Gieson’s stain from normal control rats show the normal hepatocytes architecture and mild fibrotic area within the periportal vein ([Figure 2]a).
|Figure 2 Micrographs of liver section from A): G (I) shows normal hepatocytes architecture and mild congestion within the periportal vein, B): G (II) shows intense fibrosis (arrows) dividing the liver lobule into the rounded contours or visible nodules, C): G (III) shows mild thin septa, D): G (III) shows red positive area that indicated moderate fibrosis within the periportal tract entering focally the liver lobule, E): G (IV) shows Occasional thin septa (arrows); may have portal expansion, F): G (IV) shows no septa or rare thin septa; may have portal expansion or mild sinusoidal fibrosis (Van Gieson stain, Scale bar: 20 μm).|
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Examination of the liver sections stained with Van Gieson’s stain from TA intoxicated rats indicated intense fibrosis that divide the liver lobules into rounded contours or visible nodules ([Figure 2]b). Image analysis showed significant increases in the areas of fibrosis in the group of rats treated with TA alone as compared with control. The mean of fibrotic areas found in the examined rat liver sections was 1247±335 μm2 ([Table 3]).
|Table 3 Fibrotic areas calculated from the examined rat liver sections of the different experimental groups|
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Using Van Gieson’s stain, liver sections of TA and silymarin group showed moderate thin septa ([Figure 2]c). In some sections, red positive fibrotic areas indicated mild fibrosis within the periportal tract entering the liver lobules ([Figure 2]d). In addition, the fibrotic areas calculated from the examined liver sections showed a significant decrease (P<0.05) in comparison with the TA group ([Table 3]).
On the other hand, the liver sections stained by Van Gieson’s stain in the N. sativa and TA group showed occasional thin septa; may have portal expansion ([Figure 2]e), and some liver sections showed no septa or rare thin septa; may have portal expansion or mild sinusoidal fibrosis ([Figure 2]f). In addition, the fibrotic areas calculated from the examined liver sections showed a significant decrease (P<0.05) in comparison with the TA group ([Table 3]).
Correlations between liver galectin-3 level and other markers
The data presented in [Table 4] exhibited that liver Gal-3 positively correlated with each of liver TGF-β1 (P<0.01), MDA (P <0.05), NO (P<0.05), and serum ALT and AST activities (P<0.01). Additionally, the results showed a negative correlation between liver Gal-3 and each of liver TAC (P<0.01) and catalase activity (P<0.01). The other markers exhibited insignificant correlation with liver Gal-3.
|Table 4 Pearson’s correlation between liver galectin-3 and each of liver antioxidant and oxidative stress parameters and serum transaminases|
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| Discussion|| |
Liver fibrosis, and in certain cirrhosis, have become major endpoints in patients with chronic liver diseases. Chronic liver disease reduces the regenerative capacity of liver tissues, which becomes insufficient; besides, apoptosis or necrosis of hepatic parenchymal cells becomes more favoured. This results in the deposition of ECM components and formation of scar . Excessive formation of scar tissues distorts the vascular architecture of the liver tissue and may lead to liver dysfunction . Therefore, an urgent need for an effective antifibrotic drug has appeared, a drug that enhances resolving fibrosis independent of the etiologic cause of fibrosis.
Until now there is no standard treatment or specific drug for liver fibrosis, but a reduction in liver injury events such as cessation of alcohol intake or successful viral hepatitis treatment can control fibrosis progression. Nevertheless, these actions are often insufficient to avoid eventual progression to cirrhosis in the vast majority of patients . Recently, Gal-3 was found related to fibrogenesis in different organs. It is believed that Gal-3 has a vital role in TGF-β1-mediated activation of myofibroblasts to secrete ECM components, which is a key step in fibrogenesis ,,.
Finding a suppressor of Gal-3 expression can help in the development of novel potential antifibrotic therapies. Natural products with known hepatoprotective and antifibrotic history are important candidates for finding a probable safe inhibitor for Gal-3 with minimum or no side effects. The present study aimed to investigate the effect of N. sativa seed (black seed) on Gal-3 level and the histological features of the liver in TA-induced liver injury of rats. We focused on the biochemical changes elicited in liver tissues including TGF-β1, TAC, catalase, MDA, NO, and serum liver function parameters, plasma glucose, and lipid profile, in addition to the histopathological changes in liver cells.
TA administration resulted in a significant elevation of liver Gal-3 and TGF-β1 levels in comparison with the control group. These results are in coincidence with Henderson et al. , MacKinnon et al. , and Li et al. , who reported Gal-3 elevation as a result of tissue injury in different body organs. This coincides with our histopathological results showing large fibrotic areas. Through activation of MAPK/MEK pathway, TA-induced oxidative stress can trigger activation of activator protein-1 (AP-1), leading to the upregulation of Gal-3 expression . TA-induced oxidative stress causes hepatocellular injury and necrosis. These events activate macrophages, Kupffer cells, and cholangiocytes; resulting in the secretion of TGF-β1 and other cytokines ,.
Treatment with silymarin and black seed antagonized TA toxic effect on liver Gal-3 and TGF-β1 levels, and the levels of the two parameters in both groups III and IV were close. Silymarin effect on Gal-3 may be due to its interference with and downregulation of each of ERK1/2 induced activation of c-Jun/AP-1 and transcription factor NF-κB, which are two transcriptional factors regulating Gal-3 expression . In addition to AP-1 and NF-κB, silymarin inhibits hypoxia-inducible factor 1. All these transcriptional factors regulate TGF-β1 gene expression and were reported to be inhibited by silymarin ,.
The inhibitory effect of black seed on both of Gal-3 and TGF-β1 level may be related to thymoquinone, the most abundant component of N. sativa seed. Thymoquinone has inhibitory effect on the activation of the transcriptional factor NF-κB which regulates Gal-3 and TGF-β1 gene expression ,,.
The oxidative stress induced by TA active sulfur metabolite (TASO2) resulted in significant depression of liver TAC level and catalase activity besides significant elevation in liver MDA and NO levels. These results are in accordance with Chen et al. , Song and Chen , and Nehar and Kumari . Chronic oxidative stress leads to high consumption rate of antioxidant enzymes and molecules that exceeded biosynthesis rate. TASO2 attacks cell components especially cell membrane lipids causing lipid peroxidation leading finally to the formation of MDA . Oxidative stress stimulates immune cells like mast cells to produce and secrete cytokines that activate nitric oxide synthase (iNOS) expression. iNOS is responsible for NO production in macrophages .
Both silymarin and black seed exhibited a protective effect against TA-induced oxidative damage, represented in significant higher liver TAC level, catalase activity, and significant lower liver MDA and NO levels in groups III and IV when compared with group II. These results are in accordance with Jia et al. , Freitag et al. , Ismail et al. , and Umar et al. . Silymarin contains silybin and several flavonoids with antioxidant properties. It inhibits lipid peroxidation caused by free radicals; thus decreases MDA production which results in preventing cell injury leading to a decreased level of NO production. Silymarin also inhibits iNOS expression ,. Meanwhile black seed contains many phenolic compounds, mostly thymoquinone, p-cymene, and carvacrol which have been reported to have antioxidant properties. These compounds act as a shelter protecting against lipid peroxidation and preventing high rate of cellular antioxidants consumption and depletion .
The rats intoxicated with TA showed significant increased serum ALT and AST activities and total bilirubin level in comparison with control rats. These results agree with Bludovska et al.  and Amin et al. . TASO2 attacks lipids of cell membranes, leading to damage in hepatocytes cell membranes resulting in the leakage of intracellular components outside the cell . TA induces impairment in the liver function of uptaking biliurubin from blood due to hepatocellular necrosis and bile duct damage .
Treatment with silymarin and black seed significantly decreased serum ALT and AST activities and total bilirubin level in comparison with group II. The results were similar in the two treated groups III and IV. These results are in accordance with Eminzade et al. , Chen et al. , Nehar and Kumari , Saricicek et al. , and Elkhateeb et al. . The antioxidant properties of both silymarin and black seed ameliorate TA-induced oxidative damage on hepatocytes and bile ducts and maintain cell membrane integrity ,.
TA administration resulted in a significant depression of serum total protein, albumin, and significant elevation of serum total cholesterol and triacylglycerol levels in comparison with the control group. These results are in accordance with Khalaf et al. , Mustafa et al. , and Abdalla et al. . TA intoxication results in severe damage to polyribosomes found on the endoplasmic reticulum of hepatocytes, leading to depression in protein synthesis . The long period of administration of TA leads to necrosis of a great number of hepatocytes and exhausts the regenerative ability of the liver tissues. As a result, great loss occurs in liver functions of protein synthesis and uptaking total cholesterol and triacylglycerols from the blood, along with impairments in protein and lipid metabolism ,,.
Significant increase was found in serum total protein, albumin and significant decrease of serum total cholesterol and triacylglycerol levels in the groups treated with silymarin and black seed when compared with group II. These results agree with Eminzade et al. , Essawy et al. , Cacciapuoti et al. , and Saricicek et al. . These results probably are due to the antioxidant activities of both silymarin and black seed, which shelter hepatocytes from excessive oxidative damage induced by TA administration ,. This preserved the functionality of a greater portion of liver than in case of group II. Moreover, black seed has cholerectic activity that functions through reducing cholesterol biosynthesis by hepatocytes or by decreasing cholesterol fractional absorption by the small intestine . It is worth mentioning that serum total protein level of group IV increased significantly in comparison with the control group in coincidence with Saricicek et al. . This may be due to the increased rate of protein synthesis induced by black seed during healing and regeneration of injured hepatocytes.
Fasting plasma glucose decreases than the normal range only in cases of hepatic failure due to severe damage as in the last stage of cirrhosis , and in the present study we did not reach that stage; so fasting blood glucose level remained in the normal range in all the experimental groups.
TA administration resulted in great damage to the rat liver represented by nodular appearance of the liver lobule, bridging necrosis accompanied with vacuolar degeneration of hepatocytes. These results are parallel with De David et al.  and coincide with Chen et al. . This also comes in line with our biochemical analyses results. TA is a hepatotoxin that causes centrilobular necrosis; chronic liver injury stimulates the replacement of parenchymal cells by resident ECM which forms fibrosis .
Treatment with silymarin and black seed resulted in an improvement in the liver architecture represented in reduced areas of fibrosis in comparison with group II, minor fibrotic septa and mild inflammation. These results are in agreement with Chen et al. , Essawy et al. , and Jaswal and Shukla  and confirm our biochemical analyses results.
The correlation we found between Gal-3 and TGF-β1 may be related to the direct effect of Gal-3 on the activation of TGF-β1expresion pathway. Gal-3 stimulates macrophages to secrete TNF-α, which induces activation of the transcriptional factor TN-κB ,, which in turn induces expression of TGF-β1. Regarding the correlation between liver Gal-3 and liver TAC, MDA, NO levels, catalase, and serum ALT and AST activities, this relation can be related to a common factor caused the elevation which is oxidative stress.
| Conclusion|| |
In conclusion, the present findings provide clear experimental evidence for the inhibitory effect of black seed on Gal-3 level in the liver of rats after TA-induced injury. This inhibitory effect may be related to the direct effect of black seed on the transcriptional factors NF-κB and AP-1 which are both responsible for Gal-3 expression. Besides, there is an indirect role of black seed through its antioxidant properties fighting against oxidative stress caused after TA administration. Gal-3 showed significant positive correlations with other biomarkers for liver disease. These results suggest Gal-3 to be a reliable and significant indicator for screening liver disease progression.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Kim MN, Kim BK, Han KH. Hepatocellular carcinoma in patients with chronic hepatitis C virus infection in the Asia-Pacific region. J Gastroenterol 2013; 48:681–688.
Trautwein C, Friedman SL, Schuppan D, Pinzani M. Hepatic fibrosis: concept to treatment. J Hepatol 2015; 62:15–24.
Kandeel A, Genedy M, El-Refai S, Funk AL, Fontanet A, Talaat M. The prevalence of hepatitis C virus infection in Egypt 2015: implications for future policy on prevention and treatment. Liver Int 2017; 37:45–53.
Domvri K, Zarogoulidis P, Porpodis K, Koffa M, Lambropoulou M, Kakolyris S. Gene Therapy in liver diseases: state of the art and future perspectives. Curr Gene Ther 2012; 12:463–483.
Duval F, Moreno-Cuevas JE, González-Garza MT, Rodríguez-Montalvo C, Cruz-Vega DE. Protective mechanisms of medicinal plants targeting hepatic stellate cell activation and extracellular matrix deposition in liver fibrosis. Chinese Med 2014; 9:27.
Bharat KV, Sirsiker M, Delphine SW. Galectin-3: a novel biomarker. Intl J Chem Pharm Res 2013; 2:081–094.
Hrynchyshyn N, Jourdain P, Desnos M, Diebold B, Funck F. Galectin-3: a new biomarker for the diagnosis, analysis and prognosis of acute and chronic heart failure. Arch Cardiovasc Dis 2013; 106:541–546.
Wijesundera KK, Juniantito V, Golbar HM, Fujisawa K, Tanaka M, Ichikawa C et al.
Expressions of Iba1 and galectin-3 (Gal-3) in thioacetamide (TAA)-induced acute rat liver lesions. Exp Toxicol Pathol 2013; 65:799–808.
Wijesundera KK, Izawa T, Murakami H, Tennakoon AH, Golbar HM, Kato-Ichikawa C. M1- and M2-macrophage polarization in thioacetamide (TAA)-induced rat liver lesions; a possible analysis for hepatopathology. Histol Histopathol 2014; 29:497–511.
De Oliveira SA, De Freitas Souza BS, SÁ Barreto EP, Kaneto CM, Neto HA, Azevedo CM et al.
Reduction of galectin-3 expression and liver fibrosis after cell therapy in a mouse model of cirrhosis. Cytotherapy 2012; 14:339–349.
Bourgou S, Pichette A, Marzouk B, Legault J. Antioxidant, anti-inflammatory, anticancer and antibacterial activities of extracts from Nigella sativa
L. (black cumin) plant parts. J Food Biochem 2012; 36:539–546.
Khaled AAS. Gastroprotective effects of Nigella Sativa
oil on the formation of stress gastritis in hypothyroidal rats. Int J Physiol Pathophysiol Pharmacol 2009; 1:143–149.
Assayed ME. Radioprotective effects of black seed (Nigella sativa
) oil against hemopoietic damage and immunosuppression in gamma-irradiated rats. Immunopharmacol Immunotoxicol 2010; 32:284–296.
Abdel-Zaher AO, Abdel-Rahman MS, Elwasei FM. Protective effect of Nigella sativa oil against tramadol-induced tolerance and dependence in mice: role of nitric oxide and oxidative stress. Neurotoxicology 2011; 32:725–733.
Abel-Salam BK. Immunomodulatory effects of black seeds and garlic on alloxan-induced diabetes in albino rat. Allergol Immunopathol (Madr) 2012; 40:336–340.
Woo CC, Kumar AP, Sethi G, Tan KH. Thymoquinone: potential cure forinflammatory disorders and cancer. Biochem Pharmacol 2012; 83:443–451.
Chilakapati J, Shankar K, Korrapati MC, Hill RA, Mehendale HM. Saturation toxicokinetics of thioacetamide: role in initiation of liver injury. Drug Metab Dispos 2005; 33:1877–1885.
Fujisawa K, Miyoshi T, Tonomura Y, Izawa T, Kuwamura M, Torii M, Yamate J. Relationship of heat shock protein 25 with reactive macrophages in thioacetamide induced rat liver injury. Exp Toxicol Pathol 2011; 63:599–605.
Loguercio C, Festi D. Silybin and the liver: from basic research to clinical practice. World J Gastroenterol 2011; 17:2288–2301.
Morales-González JA, Gayosso-Islas E, Sánchez-Moreno C, Valadez-Vega C, Morales-González A, Esquivel-Soto J. Protective effect of silymarin on liver damage by xenobiotics. In: Morales-Gonzalez JA, editor. Oxidative stress and chronic degenerative diseases-a role for antioxidants. XXX: In Tech; 2013. DOI: 10.5772/51502
Reeves BG, Nielson FH, Fahmy GC. Reported of the American institute of nutrition. Adhoc-Wrilling Committee on the reformulation of the AIN 1979. Ardent Diet J Nutr 1993; 123:1939–1951.
National Research council. Nutrient requirements of laboratory animals, general considerations for feeding and diet formulation. Washington: National Academy Press 1995. 3–50
Kadir FA, Othman F, Abdulla MA, Hussan F, Hassandarvish P. Effect of Tinospora crispa on thioacetamide-induced liver cirrhosis in rats. Indian J Pharmacol 2011; 13:64–68.
Khadr ME, Mahdy KA, El-Shamy KA, Morsy FA, El-Zayat SR, Abd-Allah AA. Antioxidant activity and hepatoprotective potential of black seed, honey and silymarin on experimental liver injuries induced by CCl4
. J Appl Sci 2007; 7:3909–3919.
Sanford HS. Method for obtaining venous blood from the orbital sinus of the rat or mouse. Science 1954; 119:100.
Koracevic D, Koracevic G, Djordjevic V, Andrejevic S, Cosic V. Method for the measurement of antioxidant activity in human fluids. J Clin Pathol 2001; 54:356–361.
Aebi H. Catalase in vitro. Methods Enzymol 1985; 105:121–126.
Satoh K. Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clinica Chimica Acta 1978; 90:37–43.
Montgomery HAC, Dymock J. The determination of nitrite in water. Analyst 1961; 86:414–416.
Bergmeyer HU, Scheibe P, Wahlefeld AW. Optimization of methods for aspartate aminotransferase and alanine aminotransferase. Clin Chem 1978; 24:58–73.
Henry RJ, Chiamori N, Golub OJ, Berkman S. Revised spectrophotometric methods for the determination of glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase, and lactic acid dehydrogenase. Am J Clin Pathol 1960; 34:381–398.
Walter M, Gerade H. A colorimetric method for determination bilirubin in serum and plasma. Micro Chem J 1970; 15:231–236.
Cannon DC, Henry RJ, Winkelman JW. Proteins. In Henry RJ et al.
editor. Clinical chemistry − principles and technics. 2nd ed. New York: Harper & Row; 1974. 422–431.
Doumas BT, Watson WA, Biggs HG. Albumin standards and measurement of serum-albumin with bromocresol green. Clin Chim Acta 1971; 3:87–92.
Howanitz PJ, Howanitz JH. Carbohydrates. In: Henry JB, editor. Clinical diagnosis and management by laboratory methods. Philadelphia: W.B. Saunders, 1984, 168–169.
Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem 1974; 20:470–475.
Fossati P, Prencipe L. Serum triglycerides determined colourimetrically with an enzyme that produces hydrogen peroxide. Clin Chem 1982; 28:2077–2080.
Drury RAB, Wallington EA. Carleton’s histological technique. 4th ed. Oxford, New York, USA: Oxford University Press; 1980. 129–140.
Bancroft JD, Cook HC. Manual of histological techniques[ISBN: 0443028702]. New York: Churchill Livingstone; 1984.
Armitage P, Berry G. Comparison of several groups. Statistical methods in medical research. 2nd ed. Oxford: Blackwell Scientific Publications; 1987. 186–213.
Schuppan D. Liver fibrosis: common mechanisms and antifibrotic therapies. Clin Res Hepatol Gastroenterol 2015; 39:51–59.
Henderson NC, Mackinnon AC, Rooney C, Sethi T. In Galectins and disease implications for targeted therapeutics. Klyosov A et al.
editor. ACS symposium series. Washington: American Chemical Society 2012. 377–390.
MacKinnon AC, Gibbons MA, Farnworth SL, Leffler H, Nilsson UJ, Delaine T et al.
Regulation of transforming growth factor-b1-driven lung fibrosis by galectin-3. Am J Respir Crit Care Med 2012; 185:537–546.
Li L-C., Li J, Gao J. Functions of galectin-3 and its role in fibrotic diseases. J Pharmacol Exp Ther 2014; 351:336–343.
Jiang JX, Chen X, Fukada H, Hsu DK, Liu F, Török NJ. In: The role of galectin-3 in stellate cell activation and liver fibrosis. In galectins and disease implications for targeted therapeutics. Klyosov A et al.
editor. ACS symposium series. Washington: American Chemical Society; 2012. 391–395.
Schnur J, Oláh J, Szepesi A, Nagy P, Thorgeirsson SS. Thioacetamide-induced hepatic fibrosis in transforming growth factor beta-1 transgenic mice. Eur J Gastroenterol Hepatol 2004; 16:127–133.
Pohlers D, Brenmoehl J, Löffler I, Müller CK, Leipner C, Schultze-Mosgau S. TGF-β and fibrosis in different organs − molecular pathway imprints. Biochim Biophys Acta 2009; 1792:746–756.
Hsieh YS, Chu SC, Yang SF, Chen PN, Liu YC, Lu KH. Silibinin suppresses human osteosarcoma MG-63 cell invasion by inhibiting the ERK-dependent c-Jun/AP-1 induction of MMP-2. Carcinogenesis 2007; 28:977–987.
Polyak SJ, Morishima C, Shuhart MC, Wang CC, Liu Y, Lee DY. Inhibition of T-cell inflammatory cytokines, hepatocyte NF-kappaB signaling, and HCV infection by standardized Silymarin. Gastroenterology 2007; 132:1925–1936.
Garcia-Maceira P, Mateo J. Silibinin inhibits hypoxia-inducible factor-1a and mTOR/p70S6K/4E-BP1signalling pathway in human cervical and hepatoma cancer cells: implications for anticancer therapy. Oncogene 2009; 28:313–324.
El Gazzar MA, El Mezayen R, Nicolls MR, Dreskin SC. Thymoquinone attenuates proinflammatory responses in lipopolysaccharide-activated mast cells by modulating NF-kappaB nuclear transactivation. Biochim Biophys Acta 2007; 1770:556–564.
Sethi G, Ahn KS, Aggarwal BB. Targeting nuclear factor-kb activation pathway by thymoquinone: role in suppression of antiapoptotic gene products and enhancement of apoptosis. Mol Cancer Res 2008; 6:1059–1070.
El-Baba C, Mahadevan V, Fahlbusch FB, Mohan SS, Rau TT, Gali-Muhtasib H, Schneider-Stock R. Thymoquinone-induced conformational changes of PAK1 interrupt prosurvival MEK-ERK signaling in colorectal cancer. Mol Cancer 2014; 13:201–214.
Chen T, Zamora R, Zuckerbraun B, Billiar TR. Role of nitric oxide in liver injury. Curr Mol Med 2003; 3:519–526.
Song Y, Chen M. Zinc supplementation attenuates thioacetamide-induced liver injury and hyperglycemia in mice. Biol Trace Elem Res 2004; 92:174–180.
Nehar S, Kumari M. Ameliorating effect of Nigella sativa
oil in thioacetamide-induced liver cirrhosis in Albino rats. Ind J Pharm Edu Res 2013; 47:135–139.
Stankova P, Kucera O, Lotkova H, Roušar T, Endlicher R, Cervinkova Z. The toxic effect of thioacetamide on rat liver in vitro. Toxicol in Vitro 2010; 24:2097–2103.
Jia R, Cao L, Du J, Xu P, Jeney G, Yin G. The protective effect of silymarin on the carbon tetrachloride (CCl4)-induced liver injury in common carp (Cyprinus carpio). In Vitro Cell Dev Biol-Animal 2013; 49:155–161.
Freitag AF, Cardia GFE, Bruno da Rocha A, Aguiar RP, Silva-Comar FM, Spironello RA. Hepatoprotective effect of silymarin (Silybum marianum) on hepatotoxicity induced by acetaminophen in spontaneously hypertensive rats. Evid Based Complementary Altern Med 2015; 2015:8.
Ismail M, Al-Naqeep G, Chan KW. Nigella sativa thymoquinone-rich fraction greatly improves plasma antioxidant capacity and expression of antioxidant genes in hypercholesterolemic rats. Free Radic Biol Med 2010; 48:664–672.
Umar S, Zargan J, Umar K, Ahmad S, Katiyar CK, Khan HA. Modulation of the oxidative stress and inflammatory cytokine response by thymoquinone in the collagen induced arthritis in Wistar rats. Chem Biol Interact 2012; 197:40–46.
Neha A, Jaggi AS, Singh N. Silymarin and its role in chronic diseases. Gupta SC et al.
editor. Drug discovery from mother nature, advances in experimental medicine and biology, 929. Switzerland: Springer International Publishing; 2012.
Bai T, Yang Y, Wu YL, Jiang S, Lee JJ, Lian LH, Nan JX. Thymoquinone alleviates thioacetamide-induced hepatic fibrosis and inflammation by activating LKB1-AMPK signaling pathway in mice. Int Immunopharmacol 2014; 19:351–357.
Bludovska M, Mistrova E, Kotyzová D, Chottová-Dvořáková M. The influence of chronic administration of thioacetamide on levels of ALT, AST and GLDH in serum and markers of oxidative stress in the liver and kidneys of female Wistar rats. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2013; 157:65–69.
Amin ZA, Bilgen M, Alshawsh MA, Ali HM, Hadi AHA, Abdulla MA. Protective role of Phyllanthus niruri extract against Thioacetamide-induced liver cirrhosis in rat model. Evid Based Complementary Altern Med 2012; 2012:9.
Eminzade S, Uras F, Izzettin FV. Silymarin protects liver against toxic effects of anti-tuberculosis drugs in experimental animals. Nutr Metab 2008; 5:18–25.
Chen I, Chen Y, Chou C, Chuang R, Sheend L, Chiub C. Hepatoprotection of silymarin against thioacetamide-induced chronic liver fibrosis. J Sci Food Agric 2012; 92:1441–1447.
Saricicek E, Tarakcioglu M, Saricicek V, Gulsen MT, Karakok M, Baltaci Y. Effect of Nigella sativa
on experimental liver fibrosis. Biomed Res 2014; 25:32–38.
Elkhateeb A, El Khishin I, Megahed O, Mazen F. Effect of Nigella sativa Linn oil on tramadol-induced hepato-and nephrotoxicity in adult male albino rats. Toxicol Rep 2015; 2:512–519.
Jaswal A, Shukla S. Therapeutic efficacy of Nigella sativa Linn. seed extract against CCl4 induced hepatic injury in wistar rats. Indian J Exp Biol 2015; 53:44–50.
Khalaf AA, Mekawy MM, Moawad MS, Ahmed AM. Comparative study on the protective effect of some antioxidants against CCl4
hepatotoxicity in rats. Egy J Nat Toxins 2009; 6:59–82.
Mustafa HN, El Awdan SA, Hegazy GA. Protective role of antioxidants on thioacetamide-induced acute hepatic encephalopathy: biochemical and ultra-structural study. Tissue Cell 2013; 45:350–362.
Abdalla O, Elboshy M, Risha E, Mohammed FH, Abu-Ghanema W. Hepatoprotective and antioxidant effects of pomegranate against thioacetamide toxicity in rats. Ann Veterinary Animal Sci 2016; 3:28–40.
Essawy AE, Abdel-Moneim AM, Khayyat LI, Elzergy AA. Nigella sativa seeds protect against hepatotoxicity and dyslipidemia induced by carbon tetrachloride in mice. J Appl Pharm Sci 2012; 2:21–25.
Cacciapuoti F, Scognamiglio A, Palumbo R, Forte R. Silymarin in non alcoholic fatty liver disease. World J Hepatol 2013; 5:109–113.
Buriro MA, Tayyab M. Effect of Nigella sativa
on lipid profile in albino rats. Gomal J Med Sci 2007; 5:28–31.
De David C, Rodrigues G, Bona S, Meurer L, GonzaLez-Gallego J, Tunon MJ, Marroni MP. Role of quercetin in preventing thioacetamide-induced liver injury in rats. Toxicol Pathol 2011; 39:949–957.
Granado-Serrano AB, Martın MA, Bravo L, Goya L, Ramos S. Quercetin attenuates TNF-induced inflammation in hepatic cells by inhibiting the NF-κB pathway. Nutr Cancer 2012; 64:588–598.
Mahdy KA. Impact of Galectin-3 deficiency on gene expression in the liver of mice 48-h post-treatment with olive oil or CCl4
. JASMR 2011; 6:81–90.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]