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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 32  |  Issue : 2  |  Page : 116-125

Saussurea lappa root extract accelerates the reversion of liver fibrosis induced by carbon tetrachloride in rats


1 Department of Anatomy and Embryology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
2 Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt

Date of Submission16-Sep-2015
Date of Acceptance29-Nov-2015
Date of Web Publication14-Apr-2016

Correspondence Address:
Hassan Reda Hassan Elsayed
MSc, Department of Anatomy and Embryology, Mansoura University, 60, El Gomhoria Street, 35516 Mansoura
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-208X.180324

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  Abstract 

Background and aim of work
Liver fibrosis is a major health problem associated with high morbidity and mortality, particularly in Egypt. It is mainly regulated by hepatic stellate cells, which acquire a fibrogenic character in response to oxidant stress and inflammatory cytokines. We tried to test the efficacy of Saussurea lappa (Sl) root extract on the reversion of the already established liver fibrosis as this extract was reported to have anti-inflammatory and antioxidant activities.
Materials and methods
A total of 24 albino rats were divided into four groups: negative control group, carbon tetrachloride (CCl 4 ) fibrosis model group, spontaneous resolution group, CCl 4 followed by Sl root extract group. In all rats, serum alanine transaminase, aspartate transaminase, liver malonaldehyde and liver reduced glutathione were measured. Histopathological assessment were carried out through haematoxylin and eosin (H&E), sirius red staining and α-smooth muscle actin immunohistochemical staining with evaluation of the fibrosis grade and percentage of the area occupied by collagen fibres.
Results
Administration of Sl root extract for 4 weeks, after 8 weeks of CCl 4 injection, caused a significant decrease in CCl 4 -induced rise in plasma levels of alanine transaminase and aspartate transaminase, liver malonaldehyde and the percentage of collagen area and an increase in liver glutathione with almost preserved liver architecture, less inflammatory infiltration, less collagen deposition, fewer thinner septa, less bridging fibrosis and less positive reaction for α-smooth muscle actin as compared with the spontaneous resolution group.
Conclusion
These data indicate that Sl root extract can accelerate matrix degradation and reversion from liver fibrosis induced by CCl 4 in rats. This might be through antioxidant, anti-inflammatory activities and through inhibition of the activated hepatic stellate cells or probably inducing their apoptosis.

Keywords: Carbon tetrachloride, costus, hepatoprotective, liver fibrosis, Saussurea lappa


How to cite this article:
Elsayed HR, Abd-Elmonem MM, Gabr OM, Badria FA. Saussurea lappa root extract accelerates the reversion of liver fibrosis induced by carbon tetrachloride in rats. Benha Med J 2015;32:116-25

How to cite this URL:
Elsayed HR, Abd-Elmonem MM, Gabr OM, Badria FA. Saussurea lappa root extract accelerates the reversion of liver fibrosis induced by carbon tetrachloride in rats. Benha Med J [serial online] 2015 [cited 2017 Nov 18];32:116-25. Available from: http://www.bmfj.eg.net/text.asp?2015/32/2/116/180324


  Introduction Top


Liver fibrosis is a major health burden, particularly in Egypt. Apart from the original cause of the injury, liver fibrosis results from a persistent wound-healing reaction to repetitive injury. However, liver fibrosis represents a dynamic process and has the potential to regress after removal of the cause of injury. But when the injury is chronic, cirrhosis develops and limits the potential for resolution [1].

One of the first events in hepatic fibrosis is Kupffer cell activation and recruitment of additional cells; neutrophils and platelets produce multiple important cytokines, transforming growth factor-β, platelet-derived growth factor, tumour necrosis factor-α (TNF-α), interleukin-1, interleukin-6 and reactive oxygen species. Under these circumstances, hepatic stellate cells (HSCs) present in the perisinusoidal space become activated and transdifferentiate into proliferative, contractile and fibrogenic myofibroblasts, with loss of its retinoid, and exhibit increased expression of α-smooth muscle actin (α-SMA). This is a significant step in fibrogenesis, responsible for the secretion of extracellular matrix collagen, fibronectin and elastin, which characterizes liver fibrosis [2].

Activated HSCs increase the expression of the potential inhibitors of metalloproteinases (TIMPs). Consequently, the equilibrium between the action of matrix-degrading metalloproteinases (MMPs) and their inhibitors is changed, favouring matrix accumulation into the fibrotic scars [3].

CCl 4 model is a unique model that mimics all important properties of human liver fibrosis, particularly in hepatitis C virus patients, including inflammation, portal hypertension, ascites, fibre formation and potentially fibrosis regression with easy comparability, excellent reproducibility and multiple previous publications [4].

CCl3 - , the toxic metabolite of CCl 4 , binds to lipoprotein and leads to peroxidation of lipids with increase in liver malonaldehyde (MDA), and release of reactive oxygen species and a decrease in liver glutathione (GSH). This is followed by a change in the physical and chemical properties of hepatocyte plasma, mitochondrial and lysosomal membrane, causing hepatocyte swelling, lysis increasing serum alanine transaminase (ALT) and aspartate transaminase (AST) [5].

Development of liver fibrosis is formed of three phases: acute injury, beginning of fibre formation and advanced fibrosis. The phase of acute CCl 4 -mediated liver fibrosis is characterized by Kupffer cells activation and initiation of an inflammatory response, promoting production of cytokines, chemokines, with attraction and activation of neutrophils, and lymphocytes contributing to liver necrosis [6].

Fibrosis can be seen after 2 weeks of CCl 4 application. Fibrosis markers are easily detectable at that time. Distinct bridging fibrosis may be seen after 4-6 weeks of constant administration. On continuing CCl 4 application, severe centrilobular necrosis is followed by distinct bridging fibrosis that may finally lead to cirrhosis and hepatocellular carcinoma [7].

After stoppage of the cause of liver fibrosis, spontaneous reversion and remodelling of extracellular matrix occur. Reversibility of liver fibrosis was achieved in different cases, and this suggests that reversibility is generic rather than disease-specific [8].

The explanation for this reversibility of liver fibrosis may be that the myofibroblasts, the principal scar-producing cells in the liver fibrosis, show rapid loss by apoptosis after stoppage of the cause of fibrosis [9]. In addition, during fibrosis resolution a rapid decrease in TIMP levels occurs, reversing the MMP-TIMP balance, resulting in increased matrix degrading activity [10]. In addition, the contact between activated myofibroblasts and collagen 1, which is the main collagen in hepatic fibrosis, promotes cell survival and fibrogenic activity, and thus the loss of that collagen causes apoptosis of myofibroblast [11]. In addition, emerging evidence implicates macrophages as central mediators not only of fibrogenesis but also of fibrosis resolution [12].

However, Issa et al. [10] reported that cross-linking of matrix, presence of elastin and collagen-1 fibres in the paucicellular mature scars make the fibrotic bands resistant to degradation and full resolution, even with the lengthening of the recovery times.

Saussurea lappa Clarke (Asteraceae) is well known for its wide therapeutic uses in India. Madhuri et al. [13] reviewed the traditional uses and different therapeutic activities of Saussurea lappa (Sl) root extract. This extract exhibits several therapeutic activities for a variety of diseases: antiarthritic [14], antiulcer [15] and antifungal activities [16].

Other researches on Sl root extract have shown antioxidant activity [17]. In addition, cynaropicrin isolated from this plant exhibited anti-inflammatory activity [18]. Compounds from Sl root, like santamarine and reynosin [19], costunolide and dehydrocostus lactone [20], and saussureamine [21], have shown inhibitory effect on expression of TNF-α and nitric oxide from activated macrophages. Moreover, the cytotoxic activity of this plant against cancer cells has been explained by different extracted compounds: costunolide [22] and dehydrocostus lactone [23]. Furthermore, calcium channel blocking constituents have been identified in Sl [24]. The hepatoprotective activity of Sl root extract against d-galactosamine and lipopolysaccharide-induced acute liver hepatitis in mice had been reported [25]. However, the number of studies of that plant on liver diseases is still very few.


  Materials and methods Top


Chemicals

Carbon tetrachloride solution was purchased from El-Gomhoria Company (Mansoura, Egypt).

Preparation of plant material

Dried roots of Sl were purchased from a local herbalist. Plant material was cleaned, ground to powder and extracted with 70% methanol in a container for 24 h. The solvent was then removed and fresh solvent was added to the plant material. The extraction method was repeated twice. It was filtered through filter paper and concentrated into thick semisolid viscous mass under reduced pressure on an evaporator, with a yield of 5% approximately. The extract was soluble in normal saline and distilled water as described by Yaeesh et al. [25].

Animals

A total of 24 adult female albino rats, with average body weight between 150-200 g, used in this study were obtained from the Mansoura experimental research center (MERC), Egypt. They were housed in stainless steel, meshed cages under control condition of temperature (23 ± 3°C) and relative humidity. The animals were permitted a free access to standard basal commercial diet and tap water ad libitum with a 12-h light-dark cycle throughout acclimatization and experimental periods. All rats were kept in the animal house, under specific pathogen-free conditions. All the experiments were carried out according to the rules and regulations laid down by the committee on animals' experimentation of Mansoura University.

Experimental design

The rats were randomly divided into four groups: group 1 (negative control group), in which six rats were fed a basal diet only; group 2 (fibrosis model group CCl 4 ), in which six rats received intraperitoneal injection of sterile CCl 4 in a dose of 1 ml/kg dissolved in corn oil at 1 : 1 ratio twice weekly for 8 weeks as previously described by Jang et al. [26], and then the rats were killed 3 days after the last CCl 4 injection; group 3 (spontaneous resolution group), in which six rats received intraperitoneal injection of CCl 4 for only 8 weeks, as described in group 2, after which the rats were allowed to survive for 4 weeks after stopping CCl 4 injection without treatment, and then were finally killed; group 4 (CCl 4 followed by Sl root extract), in which six rats received intraperitoneal injection of CCl 4 for only 8 weeks, as described in group 2 and 3, followed by the administration of Sl root extract for 4 weeks through a gastric tube in a dose of 400 mg/kg daily according to Sutar et al. [27].

Then, the rats were killed and subjected to laboratory, histopathological and immunohistochemical study.

Sampling protocol

Obtaining samples

Rats were killed by cervical dislocation. Blood samples, collected by direct cardiac puncture into sterilized and heparinized syringes, were used to estimate ALT and AST. The liver was cut out and a portion was fixed for histopathology staining with haematoxylin and eosin, sirius red stains and immunohistochemical staining for α-SMA, and was processed for microscopic examination. Another portion of liver was homogenized for quantitative analysis of MDA and GSH in tissue homogenate.

Estimation of serum alanine transaminase and aspartate transaminase

The serum samples were obtained by blood centrifugation for 10 min at 5000g at 4°C as previously described by Jia et al. [28] The levels of serum ALT and AST were assayed according to the routine biochemical analysis system using clinical test kits spectrophotometrically (Elitech, London, UK) according to Reitman and Frankel [29].

Assessment of lipid peroxidation and oxidative stress

Portions of livers were homogenized (10% weight/volume) in ice-cold 0.1 mol/l Tris-HCl buffer (pH = 7.4). The homogenate was centrifuged at 3000 rpm for 10 min at 4°C. Oxidative stress markers were detected in the resultant supernatant of liver homogenate as previously described by Jia et al. [28]. The appropriate kits (Biodiagnostic kits; Giza, Egypt) were used for the determination of reduced GSH and lipid peroxidation, which was measured by the formation of MDA.

Processing of the specimens for light microscopic examination

Immediately after sacrifice, the liver was removed and a part of it was immediately fixed in 10% phosphate buffered formalin and processed by routine histology procedures. The specimens were processed for paraffin sectioning by gradual dehydration using ascending graded concentrations of alcohol, cleared in xylene and embedded in soft then hard paraffin wax, cut using microtome at a thickness of 5-6 μm per piece, and mounted on the slide for staining according to Bisen [30]. The tissues were stained with H&E as described by Bancroft and Layton [31], and with sirius red as described by Chun and Inoue [32] and were processed for histopathological examination and immunohistochemical staining for α-SMA antibody as described by Yoshiji et al. [33].

Analysis of liver histopathology

We commented on the pathological alterations, fibrosis, inflammatory infiltration and nodule formation. Sirius red staining facilitated the accurate assessment of the degree of hepatic fibrosis. The grade of liver fibrosis in liver sections of all animals was estimated according to Ishak modified hepatic activity index score [34].

Image analysis of the area occupied by collagen fibres

The sirius red-stained sections of all groups were subjected to image analysis. Quantitative assessment of liver fibrosis was performed with morphometry on sections processed with sirius red, which specifically stains collagen.

The data were obtained using image analyzer computer system. The image analyzer consisted of a colored video camera, colored monitor and the hard disc of personal computer connected to the microscope. These measurements were done using an objective lens of magnification 4 - that is, of total magnification 40.

Several readings were obtained from the different slides of the six animals of each group and at least six random fields were measured in each slide, and then the fibrosis area was masked by a black binary color to be measured using program ImageJ (version 1.48, Wayne Rasband, National Institutes of Health, Bethesda, Maryland, USA).

Statistical analysis

Serum ALT and AST, liver MDA and GSH, and the percentage of area occupied by collagen tissue were all presented as mean and SD, and the mean of two groups were compared by Student's t-test. Statistical analysis was carried out using SPSS program (version 22, IBM, New York, USA). P-value of less than 0.05 was considered significant.


  Results Top


The liver sections of the rats receiving CCl 4 for 8 weeks (group 2), stained by haematoxylin and eosin, showed hydropic degeneration of hepatocytes, inflammatory cell infiltration and large thick fibrous septa and marked distortion of hepatic architecture [Figure 1]. The serum levels of ALT, AST, liver MDA, and the percentage of the area of collagen fibres, in the image analysis of sirius red stained sections, showed significant increase with significant decrease (P < 0.05) in liver GSH levels compared with the negative control group [Table 1],[Table 2] and [Table 3] and [Graph 1 [Additional file 1]],[Graph 2 [Additional file 2]],[Graph 3 [Additional file 3]],[Graph 4 [Additional file 4]] and [Graph 5 [Additional file 5]].
Figure 1: H and E-stained sections (×400). (a) Negative control group 1 showing flat, anastomosing plates of hepatocytes with normal portal tract; (b) CCl4-treated group 2 showing thick fi brous septa (arrows) radiating from the vessels with inflammatory infiltration along them and hydropic degeneration in periseptal hepatocytes (stars); (c) spontaneous resolution group 3 showing fibrous septa with moderate inflammatory infiltration (arrows); (d) Saussurea lappa-treated group showing almost preserved architecture, normal portal tract with mild inflammatory infiltration along thin short fibrous septa (arrows).

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Table 1: The percentage of the area occupied by collagen fibres and fibrosis grade in the different groups

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Table 2: Serum levels of alanine transaminase and aspartate transaminase in the different groups

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Table 3: Liver malonaldehyde and glutathione levels in the different groups

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Sirius red-stained liver sections of the rats, receiving CCl 4 for 8 weeks, group 2, showed pericentral and periportal collagen deposition with abundant septa seen radiating from portal tracts and central veins, marking bridging fibrosis, and pseudolobule formation [Figure 2] The fibrosis grade was evaluated according to Ishak score, to be grade 5.08 ± 0.51 [Table 1]. In addition to the positive α-SMA immunohistochemical reaction normally detected in the walls of portal veins, hepatic arteries and terminal hepatic venules in the negative control group, liver sections of the rats of the CCl 4 -treated group showed additional positive reaction in the perisinusoidal spaces at the leading edge of the fibrosis, and occupied the whole thickness of the fibrous tissue bands facing the liver parenchyma [Figure 3].
Figure 2: Sirius red-stained sections (×40). (a) Negative control group 1 showing only vessels stained in red; central veins and portal tracts (arrows), which are normal places for collagen deposits. (b) CCl4- treated group 2 showing excessive collagen deposition around vessels and thick bridging fibrous septa (arrows) radiating from the vessels and connecting them. (c) Spontaneous resolution group 3 showing thinner septa (arrows) with less bridging fibrosis as compared with group 2. (d) Saussurea lappa-treated group showing thinner shorter septa (arrows) with less bridging fibrosis as compared with group 3.

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Figure 3: ¦Á-Smooth muscle actin (¦Á-SMA) immunohistochemical staining (×400). (a) Negative control shows positive staining in walls of portal vein and hepatic artery (arrows), which are normal places for ¦Á-SMA. (b) CCl4-treated group 2 shows strong positive staining perivascular along fibrous septa and in perisinusoidal space (arrows). (c) Spontaneous resolution group 3 shows moderate positive staining (arrows) perivascular along fibrous septa and in perisinusoidal space. (d) Saussurea lappa-treated group shows less positive staining
(arrows) perivascular along fibrous septa and in perisinusoidal space as compared with group 3.


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The liver sections of the rats of the spontaneous resolution group 3 received CCl 4 for 8 weeks, were left for 4 weeks without treatment, and were then killed. They showed less distortion of structural integrity of hepatic tissue, diminished portal inflammation, smaller and thinner fibrous septa in haematoxylin and eosin-stained sections compared with the CCl 4 -treated group 2 [Figure 1] The levels of serum ALT, AST, liver MDA, and the percentage of the area of collagen fibres in the image analysis of the sirius red-stained sections showed significant decrease with significant increase (P < 0.05) in liver GSH levels in the spontaneous resolution group 3, compared with the CCl 4 -treated group 2 [Table 1],[Table 2] and [Table 3] and [Graph 1],Graph 2],[Graph 3],[Graph 4] and [Graph 5].

Sirius red-stained liver sections of rats of the spontaneous resolution group 3 showed decreased pericentral and periportal collagen deposition, fewer septa radiating from portal tracts and central veins, and fewer bridging fibrosis as compared with CCl 4 -treated group 2 [Figure 2]. The fibrosis grade in the spontaneous resolution group 3 was evaluated according to Ishak score, to be grade 3.83 ± 0.72 [Table 1]. As for α-SMA immunohistochemical study, liver sections of rats of the spontaneous group 3 showed less positive reaction in the perisinusoidal spaces and in the fibrous tissue bands as compared with the CCl 4 -treated group 2 [Figure 3].

The liver sections of the rats of group 4 received CCl 4 for 8 weeks, followed by administration of Sl root extract for 4 weeks and were then killed. They showed almost preserved structural integrity of hepatic tissue, diminished portal, smaller and thinner fibrous septa, and fewer bridging in haematoxylin and eosin-stained sections as compared with the spontaneous resolution group 3 [Figure 1]. The levels of serum ALT, AST, liver MDA, and the percentage of the area of collagen fibres in the image analysis of the sirius red-stained sections showed significant decrease with significant increase (P < 0.05) in liver GSH levels as compared with the spontaneous resolution group 3 [Table 1],[Table 2] and [Table 3] and [Graph 1],[Graph 2],[Graph 3],[Graph 4] and [Graph 5].

Sirius red-stained liver sections of rats of the Sl-treated group 4 showed decreased pericentral and periportal collagen deposition, fewer septa radiating from portal tracts and central veins, and fewer bridging fibrosis as compared with the spontaneous resolution group 3 [Figure 2]. The fibrosis grade in the Sl-treated group 4 was evaluated according to Ishak score, to be grade 2.08 ± 0.79 [Table 1]. As for α-SMA immunohistochemical study, liver sections of rats of the Sl-treated group 4 showed decreased positive reaction in the perisinusoidal spaces and in the fibrous tissue bands as compared with the spontaneous group 3 [Figure 3].


  Discussion Top


In the current study, we tried to test the efficacy of Sl root extract on the reversion of liver fibrosis induced by CCl 4 in rats.

Liver fibrosis is a universal health problem associated with significant high morbidity and mortality, particularly in Egypt [35]. Liver fibrosis is mainly regulated by HSCs, which undergo activation because of viral hepatitis, toxins, nonalcoholic steatohepatitis and autoimmune disorders [36].

We used Sl root extract as previous studies proved its anti-inflammatory activity [18], possible cytotoxic activity [22] and antioxidant activity [17]. Sl root extract was tested once as a hepatoprotective preparation but against acute hepatitis only [25]. In our study, we tried for the first time to test its efficacy against chronic hepatitis and liver fibrosis.

We used CCl 4 model as it is a unique model that mimics all important properties of human liver fibrosis, particularly of patients with hepatitis C, including inflammation, portal hypertension, ascites, fibre formation, and potentially fibrosis regression with easy comparability, excellent reproducibility, multiple previous publications, moderate burden on the animals, and shorter time to induce liver fibrosis, with less mutagenicity and less carcinogenicity compared with other hepatotoxins [4]. Rats were used as they mimic human in many aspects [37].

In the present study, a prepared solution of Sl root extract was given orally to rats through a gastric tube in a dose of 400 mg/kg/day, which represent the 1/5 of the median lethal dose (LD50) of Sl root extract according to Sutar et al. [27] who performed acute toxicity studies and found that a dose of 2000 g/kg of Sl root extract did not cause toxicity or mortality in rats.

In the present study, CCl 4 was dissolved in corn oil in a ratio of 1 : 1. Corn oil was recently proved to be inert with no hepatoprotective or hepatotoxic effects, and thus corn oil is better than olive oil as a solvent for CCl 4 , as the olive oil was proved to be hepatoprotective and may minimize the degree of liver fibrosis induced by CCl 4 [38]. Induction of liver fibrosis was successfully produced by intraperitoneal injection of sterile CCl 4 in a dose of 1 ml/kg twice weekly for 8 weeks as described by Jang et al. [26].

As Shi et al. [48] found earlier, the peak effect of acute CCl 4 toxicity and for maximal hepatocytes apoptosis was 72 h after last injection of CCl 4 , and thus the animals in the present study were killed and liver specimens were taken 72 h (3 days) after the final injection of CC1 4 for studying the fibrosis, and the procedure was repeated in all the other groups.

The liver sections of the rats treated with CCl 4 for 8 weeks, group 2, were stained by haematoxylin and eosin, and showed inflammatory cellular infiltration, parallel to the data reported by Heindryckx et al. [6]. Large thick fibrous septa and pseudolobule formation with multiple bridging were found and were similar to the results reported by Kisseleva et al. [7].

The serum levels of liver enzymes ALT and AST in CCl 4 -treated group 2 showed significant increase (P < 0.05) as compared with the negative control group fed on basal diet only. This is in parallel to the studies of Afzal et al. [39] and Mihailovic et al. [40] who reported a change in the physical and chemical properties of cellular membranes, thus affecting their fluidity and permeability for ion exchange, resulting in leakage of enzymes in blood.

MDA levels in the liver homogenate of CCl 4 -treated group 2 showed significant increase (P < 0.05) in comparison with the negative control group 1. This can be explained by the fact that a toxic metabolite trichloromethyl (CCl3 - ) radical is produced from CCl 4 by CYP2E1 in hepatocytes. CCl3 - binds to lipoprotein and leads to peroxidation of lipids of the endoplasmic reticulum causing increase in the hepatic MDA level [5],[41].

GSH levels in liver homogenate of CCl 4 -treated group 2 showed significant decrease (P < 0.05) in comparison with the negative control group 1, and this indicates that oxidative stress, mediated by CCl 4 -induced hepatocyte damage, led to the depletion of reduced GSH, which is in parallel to the results of the studies conducted by Abbas et al. [42] and Adeneye et al. [43].

Masson trichrome and sirius red-stained liver sections of CCl 4 -treated group 2 showed pericentral and periportal collagen deposition, with abundant septa seen radiating from portal tracts and central veins, marked bridging fibrosis and pseudolobule formation, and this is parallel to the results of the study by Kisseleva et al. [7]. This was confirmed by image analysis of sirius red-stained sections, which showed significant increase in the area of collagen fibres (P < 0.05) of the CCl 4 -treated group, as compared with the negative control group 1. The fibrosis grade in group 2 was evaluated according to Ishak score [34], to be grade 5.08 ± 0.51.

In addition to the positive α-SMA immunohistochemical reaction normally detected in the walls of portal veins, hepatic arteries and terminal hepatic venules of the rat livers of the negative control group 1, the liver sections of the rats of the CCl 4 -treated group 2 showed additional positive reaction in the perisinusoidal spaces at the leading edge of the fibrosis, and occupied the whole thickness of the fibrous tissue bands facing the liver parenchyma. These results are in agreement with those of the previous studies that reported that immunohistochemical staining for α-SMA revealed marked increase in the number of activated HSCs in the fibrous septa with phenotypic modulation of HSCs, and that α-SMA is a good immunohistochemical marker for the activated HSCs [44],[45].

Although the CCl 4 -treated female rats in our study showed significant difference in serum ALT and AST, liver MDA and GSH as compared with the negative control group, the difference was approximately two-fold to three-fold only. This can be explained by the fact that the fibrotic response of the female liver to CCl 4 treatment is significantly weaker than that of the male liver. It suggested that physiological levels of oestrogen have an antifibrogenic effect as it suppresses hepatic collagen content, reduces the areas of HSC positive for α-SMA and lowers the synthesis of hepatic type I collagen [46].

To test the reversibility of liver fibrosis and its capacity to undergo spontaneous resolution after stoppage of the cause of injury, the rats of group 3 received intraperitoneal injection of CCl 4 for 8 weeks, after which they were left for 4 weeks without treatment and then killed. The liver sections of rats of this group, group 3, showed less distortion of structural integrity of hepatic tissue confirmed by haematoxylin and eosin staining showing diminished inflammatory cellular infiltration, smaller and thinner fibrous septa and fewer bridging, as compared with the CCl 4 -treated group 2.

The levels of serum ALT, AST and liver MDA of the spontaneous resolution group 3 showed significant decrease (P < 0.05), with significant increase in GSH level of the liver homogenate, as compared with the CCl 4 - treated group 2.

Masson trichrome staining and sirius red-stained liver sections of rats of the spontaneous resolution group 3 showed fewer septa seen radiating from portal tracts and central veins, fewer bridging fibrosis and occasional pseudolobule formation as compared with CCl 4 -treated group 2. This was confirmed by image analysis of sirius red-stained liver sections showing significant decrease in the area of collagen fibres (P < 0.05) of the spontaneous resolution group 3, as compared with the CCl 4 -treated group 2. The fibrosis grade in group 3, according to Ishak score, decreased to be grade 3.83 ± 0.72.

As for α-SMA immunohistochemical study, liver sections of rats of the spontaneous resolution group 3 showed decreased positive reaction in the perisinusoidal spaces and in the fibrous tissue bands as compared with the CCl 4 -treated group 2.

The results of the spontaneous resolution group 3 proved the reversibility of liver fibrosis even without treatment and ensured the spontaneous resolution capacity of the liver tissue. These results are similar to the data reported by Iredale [8].

The explanation for this reversibility of liver fibrosis may be that during fibrosis resolution, a rapid decrease in TIMP levels occurs, reversing the MMP-TIMP balance and resulting in increased matrix degrading activity [10]. In addition, the contact between activated myofibroblasts and collagen 1, which is the principal collagen in hepatic fibrosis, promotes cell survival and fibrogenic activity, and thus loss of that collagen causes apoptosis of myofibroblast [11]. The myofibroblasts, the principal scar-producing cells in the liver fibrosis, show rapid loss by apoptosis after stoppage of the cause of fibrosis [9]. In addition, emerging evidence implicates macrophages as central mediators not only of fibrogenesis but also of fibrosis resolution [12].

However, in our study, the livers of rats of the spontaneous resolution group 3 did not completely resolve or regain normal histological and biochemical parameters. When compared with negative control group 1, the livers of the rats of the spontaneous resolution group 3 showed significant increase (P < 0.05) in the area of collagen, fibrosis grade, serum ALT, AST and liver MDA levels with significant decrease in liver GSH levels, which is parallel to the results of a study conducted by Issa et al. [10] who reported that cross-linking of matrix, presence of elastin and collagen-1 fibres in the paucicellular mature scars make the fibrotic bands resistant to degradation and full resolution, even with the lengthening of the recovery times.

To test the efficacy of Sl root extract against already established liver fibrosis, the rats of group 4 received intraperitoneal injections of CCl 4 for 8 weeks to induce liver fibrosis, were then treated with Sl root extract through gastric tube for 4 weeks in a dose of 400 mg/kg daily, as previously described by Sutar et al. [27] to be the 1/5 of (LD50), and were finally killed.

The liver sections of rats injected with CCl 4 for 8 weeks and then treated by Sl root extract for 4 weeks, group 4, showed less distortion of structural integrity of hepatic tissue, diminished inflammatory cellular infiltration, smaller and thinner fibrous septa as confirmed by haematoxylin and eosin staining, with significant decrease (P < 0.05) in serum ALT, AST and liver MDA with a significant increase in the liver GSH, as compared with the spontaneous resolution group 3.

Masson trichrome and sirius red-stained liver sections of rats injected with CCl 4 for 8 weeks and then treated by Sl root extract for 4 weeks, group 4, showed less collagen deposition around portal tracts and central veins, and fewer bridging fibrosis as compared with the spontaneous resolution group 3. This was confirmed by the image analysis of sirius red-stained liver sections showing significant decrease in the area of collagen fibres (P < 0.05) in the Sl-treated group 4, as compared with the spontaneous resolution group 3. The fibrosis grade in the Sl-treated group 4 was evaluated according to Ishak score to be grade 2.08 ± 0.79.

As for α-SMA immunohistochemical study, liver sections of animals of the Sl-treated group 4 showed markedly decreased reaction as compared with the spontaneous resolution group 3, and became localized only to the portal vein and hepatic arterial walls and few positive cells in the perisinusoidal spaces and along the fibrous tissue septa. This indicates that Sl root extract can accelerate the reversion from the already established liver fibrosis. This is partially parallel to the results of Yaeesh et al. [25] who reported the hepatoprotective activity of Sl root extract.

Yaeesh et al. [25] were the first and the only one who reported and published the hepatoprotective activity of Sl root extract. However, the present study differs from that of Yaeesh et al. [25] in that the present study evaluated the efficacy of Sl root extract against chronic liver toxicity induced by CCl 4 in rats, not only in prevention of liver fibrosis but also in treatment of the already established liver fibrosis, applying different techniques: haematoxylin and eosin, Masson trichrome, sirius red stains, immunohistochemical staining for α-SMA, measuring serum ALT, AST and liver MDA, GSH levels, but Yaeesh et al. [25] were evaluating the efficacy of Sl root extract against acute hepatitis induced by injection of d-galactosamine (D-GalN) and lipopolysaccharide in mice, using different dosage, applying routine biochemical and histological techniques.

The possible antifibrotic effect of Sl root in our results may be due to the following reasons: (a) anti-inflammatory activity, as cynaropicrin isolated from this plant has explained the ability of this plant to exhibit anti-inflammatory activities, as reported in a study by Cho et al. [18]; (b) compounds from Sl root, like santamarine and reynosin [19], costunolide, dehydrocostus lactone [20] and saussureamine [21], have shown inhibitory effect on the expression of TNF-α and nitric oxide from activated macrophages; (c) antioxidant activity of this plant, as demonstrated in a study by Pandey et al. [17]; (d) possible cytotoxic activity against stellate cells explained by other extracts, such as costunolide [22] and dehydrocostus lactone [23]; and (e) calcium channel blockers have been found to be hepatoprotective as they inhibit the contractile character of HSC against sinusoidal wall, thus inhibiting hypoxia and inhibiting new angiogenesis [47], and calcium channel blocking constituents have been identified in Sl [24].

However, the livers of the rats of group 4, injected with CCl 4 for 8 weeks and then treated with Sl root extract for 4 weeks, were still showing significant increase (P < 0.05) in the area of collagen and fibrosis grade, significant increase (P < 0.05) in serum ALT, AST and liver MDA levels and significant decrease (P < 0.05) in liver GSH levels compared with the negative control group 1. This proves the role of Sl in accelerating the reversion from liver fibrosis but not to the normal range. However, lengthening the therapeutic time and using adjuvant drugs with different strategies in treatment of liver fibrosis may give beneficial results.


  Conclusion Top


Sl root extract was shown to accelerate the resolution and reversibility of the already established liver fibrosis after stoppage of the cause of injury (CCl 4 ), proved by the significant decrease in serum ALT and AST, and significant decrease in the fibrosis grade and fibrosis area and rapid restoration of liver architecture, may be through its antioxidant effect, proved by the decrease in liver MDA and the increase in liver GSH, anti-inflammatory activity and its ability to inhibit the activated HSC.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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