Reishi Mushroom Attenuates Hepatic Inflammation and Fibrosis Induced by Irradiation Enhanced Carbon Tetrachloride in Rat Model

This work was undertaken to establish a new experimental model of hepatic fibrosis by gamma irradiation and CCl4 and to study the hepatoprotective effect of Reishi Mushroom (RM) against hepatic fibrosis induced in that model. Our results revealed that oral co-administration of 110 mg/kg RM by gavage to fibrotic rats offered an obvious hepatic protection as assured by the significant decrement in ALT and AST, HP content, MDA and NO levels with elevation of the antioxidant enzymes activities. The levels of TGF-β, TNF-α, HO-1 and type-1 collagen and their m-RNA expression were markedly declined as compared with those of fibrotic rats. Microscopical examination revealed that the exposure of rats to radiation aggravated the effect of CCl4 causing extensive collagen deposition and marked pseudolobulation of the hepatic parenchyma indicative of bridging fibrosis. While, oral co-administration of RM obviously improved the state of steatosis and apparently suppressed hepatic fibrogenesis.


Introduction
Liver is the key organ of metabolism and excretion of many substances; hence it is often exposed to variety of xenobiotics and therapeutic agents.Hepatic fibrosis induced by chronic liver injuries as a result of hepatitis viruses, chronic alcohol intake, lipidperoxidative products and various drugs could end by cirrhosis [1].Chronic liver diseases are usually associated with inflammatory reaction that is considered a key contributor of hepatocellular damage with a resultant progression to liver fibrosis and may be to hepatocellular carcinoma [2].
Radiation-induced liver disease is mainly due to oxidative damage, leading to liver inflammation and fibrosis [3].
CCl 4 has been widely reported to induce acute and chronic tissue injuries.A single dose of CCl 4 can result in centrizonal necrosis and steatosis [4].While, prolonged administration of CCl 4 can cause hepatic fibrosis caused by its highly active metabolite trichloromethyl (CCl 3 ) that is produced by Cytochrome p450 during CCl 4 metabolism in hepatocytes [5].Those trichloromethyl radicals are known to trigger a cascade of events that result in hepatic inflammation and fibrosis [6] through activation of hepatic stellate cells (HSCs) [7].Marked changes in the extracellular matrix (ECM) take place as hepatic fibrogenesis progresses with more expression of type-1 collagen which is the most abundant ECM protein in hepatic fibrosis [8].The activation of HSCs is mediated by reactive oxygen species and various cytokines, including transforming growth factor (TGF)-β, tumor necrosis factor (TNF)-α [9] and interleukin-6 (IL-6), which are able to stimulate collagen synthesis [10].
Mushroom is widely used everywhere all over the world as an important source of nutrition and therapy [11].Reishi Mushroom (RM), also known as Ganoderma lucidum (GL) is a traditional oriental medicinal mushroom.Its role in the treatment of chronic hepatopathy with little or no side effects is widely known [12].Generally, many constituents in Mushrooms are known to have varieties of biological activities including antioxidant [13], antiinflammatory [14] and hepatoprotective properties [15].Furthermore, a protective action of water or ethanol extracts of G. lucidum against acute hepatitis in rats and mice have been recorded [16].In addition, among many constituents present in G. lucidum, triterpenoids and polysaccharides have proved to inhibit hepatitis and liver fibrosis [17] [18].
Aim of work: The main objective of the present study was to establish a fibrosis model using gamma irradiation and CCl 4 and to demonstrate the protective effect of RM aqueous solution against the induced hepatic fibrosis with focusing on the precise cellular and molecular mechanisms.

Animals
Fifty-five male albino rats with an average weight of 145 -160 gm were obtained from the animal house belonging to Research Institute of Ophthalmology, Giza, Egypt.Rats were housed in regular designed cages and maintained in good ventilation, at a temperature of 25˚C ± 5˚C, 60% humidity, with suitable illumination conditions (light/dark cycle) and were allowed standard pellet diet and fresh water ad libitum.Animals were left one week for acclimatization on lab environment before starting the onset of the experiment.Animal care and the protocol of animal treatment were approved by the Animal Care Committee of the National Centre for Radiation Research and Technology (NCRRT), Cairo, Egypt, and in accordance with the recommendations of the proper care and use of laboratory animals.

Irradiation Facilities
Irradiation was performed through the use of a Canadian Gamma Cell-40 ( 137 Cs) at the National Centre for Radiation Research and Technology (NCRRT), Cairo, Egypt.The dose rate was 0.675 Gy/minute.

Induction Models of Liver Fibrosis in Rats
Two models of hepatic fibrosis were established in the current study.The 1 st model was established using CCl 4 alone as; CCl 4 in olive oil (50% V/V) at a dose of 2 ml/Kg body weight was delivered subcutaneously three times/ week for six weeks.While in the 2 nd model, gamma irradiation was used to promote and enhance the CCl 4 induced hepatic fibrosis.Rats were exposed to six fractions (each of 2 Gy) of gamma irradiation once/week up to cumulative dose of 12 Gy concurrently with CCl 4 as in the 1 st model.

Experimental Design
Rats were randomly divided into five groups.Group (Gp) 1; control rats were s/c injected with the vehicle olive oil (0.2 ml).Gp2; rats in this group were treated with RM aqueous solution (1100 mg/kg) by gavage three times/ week.Gp3; represented the 1 st model of fibrosis; rats were subcutaneously injected with CCl 4 in olive oil (2 ml/kg)3 times/week for six weeks.Gp4; represented the 2 nd model of fibrosis; rats of this group were subjected to gamma irradiation and CCl 4 as described previously.Gp5; (RM + IRR + CCl 4 treated group) rats were treated with RM as in group 2 and were exposed to both IRR + CCl 4 as in group 4. The time interval between CCl 4 injection and RM administration was taken into account to be at least 5 hours to avoid the interference of the metabolic substances.Carful observation was carried out for all animals along the experimental period and body weights were recorded weekly.At the end of the experimental period, rats were sacrificed under gentle diethyl ether anesthesia prior to which blood samples were collected into heparinized test tubes for plasma separation for biochemical analysis.Through PM examination was carried out and livers were dissected out, washed with saline, dried on a filter paper and weighted.Each liver was divided into two parts; one was fixed in 10% buffered neutral formalin for histopathological examination while the second part was kept at −20˚C till used for biochemical analysis.

Biochemical Parameters Investigated in Blood Plasma
ALT and AST activities, total Protein (TP) and albumin (Alb) in plasma were determined using the available commercial kits purchased from Bio-Diagnostic Co., Cairo, Egypt.Plasma TGF-β and TNF-α were measured by ELISA kit immunoassay supplied by R & D Quantikine USA (Catalog Number: RTA00, MB100B) according to manufacturer's instructions.

Hydroxyproline (HP) Assay
HP; an indicator for hepatic collagen amount was colorimetriclly assayed in liver tissue homogenate as previously described [19].

Heme Oxygenase-1 (HO-1) Activity Assay
Liver tissues were homogenized with 2.5 volume Tris-HCl buffer (10 m mol/L, pH 7.6) containing 250 mmol /L sucrose and 0.4 mmol/L phenyl methylsulfonyl fluoride.The homogenates were centrifuged at 800 g for 10 minutes and then centrifuged at 13.500 g for 20 minutes to produce the mitochondrial pellet.The supernatant was withdrawn.The protein content in liver homogenate was determined [25].The activity of HO-1 in the supernatant was determined as previously described [26].Billirubin formed was determined by calculation of the difference in absorbance between 464 nm and 530 nm.The HO-1 activity was expressed as picomoles of billirubin per milligram of protein per hour using standard billirubin curve.

RNA Extraction and RT-PCR Analysis for TGF-β1, TNF-α, HO-1 and Type-1 Collagen
Total RNA was isolated from liver tissue homogenate using RN easy Purification Reagent (Qiagen, Valencia, Callifornia) according to the manufacturer's protocol.Extracted RNA was quantified by spectrophotometer at 260 nm.Reverse transcription was carried out on 5 µg RNA from each liver sample using MMuLV reverse transcriptase in a 50 µL reaction volume.Mixtures of the reverse transcription were used for amplification of fragments specific forTGF-β1, TNF-α, HO-1 and Type-1 collagen by PCR using the primer pairs listed in Table 1.The levels of expression of all transcripts were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the same tissue sample.The real time PCR was performed using the QuantiTect SYBR green PCR Kit (Qiagen, Germany) according to the manufacturer's instructions, by Applied Biosystems 7500 Instrument, USA.The PCR reaction mix was carried out in a total volume of 25 μL, containing 2 × QuantiTect SYBR green PCR master mix, 20 pmol/μL specific primer.Subsequently, cDNA was synthesized from purified RNA.The protocol consisted of 45 amplification cycles, each conducted as follows; 10 min at 95˚C (holding stage), 15 sec for denaturation at 95˚C, 30 sec for annealing at 60˚C and another 15 sec for elongation at 60˚C.RT-PCR was carried out as has been described previously [27].

Histopathological Studies
Formalin fixed liver specimens were routinely processed using conventional paraffin embedding technique.Paraffin blocks were serially sectioned at 4 -5 um thickness and stained with H & E [28].Azan stain was used to demonstrate collagen fibers and the progress of fibrosis [28].

Statistical Analysis
Data were analyzed using SPSS Version 20.0.Differences between experimental groups were analyzed using one-way analysis of variance.All differences were considered statistically significant at P < 0.05.

Clinical and Postmortem Examination
Rats of CCl 4 and IRR + CCl 4 treated groups appeared depressed with lusterless fur and decreased appetite.Livers of rats of CCl 4 were swollen with rounded borders, irregular surface and fatty appearance.While that of IRR + CCl 4 treated rats were severely distorted with fatty appearance.

The Effect of RM on Body Weight and Liver Weight Index of Fibrotic Rats
As shown in Table 2, a significant (P < 0.001) decrease in body weight gain coupled with significant increase (P < 0.001) in liver weight index was observed in both models of fibrosis generated in this study as compared to the control set.Moreover, more pronounced changes in body weight (P < 0.001) and in liver index (P < 0.01) were noticed in IRR + CCl 4 treated rats than in those treated with the sole CCl 4 alone.On contrary, RM co-administration to IRR + CCl 4 treated rats caused significant restoration (P < 0.001) in body weight gain and liver weight index near to the normal values (P > 0.05).

Effect of RM on ALT and AST Activities, TP, Alb, TNF-α and TGF-β Levels of Fibrotic Rats
It was observed that; RM treated rats did not show any significant (P > 0.05) changes of any of the investigated parameters in their blood plasma as well as in hepatic tissue compared to those of control rats.A significant (P < 0.001) elevation in ALT and AST activities, TNF-α and TGF-β with a significant (P < 0.001) reduction in plasma TP and Alb concentration was noticed in plasma of CCl 4 and IRR+CCl 4 treated groups when compared with controls (Figures 1-3).The highly significant (P < 0.05) changes were noticed in IRR + CCl 4 treated rats compared with those of the sole CCl 4 treatment.On contrary, the administration of RM to IRR + CCl 4 treated rats resulted in significant (P < 0.001) improvement of all of the above altered parameters compared to those of model 2 rats.

Effect of RM on Hydroxyproline Content in Liver Homogenate of Fibrotic Rats
Hydroxyproline content was significantly (P < 0.001) increased following CCl 4 treatment and showed higher significant (P < 0.01) increase following IRR + CCl 4 treatment than its level in model 1.By contrast, oral administration of RM with IRR + CCl 4 significantly restored the hepatic hydroxyproline content compared with its level in model 2 rats (Figure 4).

Effect of RM on Antioxidant Status in Hepatic Tissue of Fibrotic Rats
Data in (Figure 5 and Figure 6) revealed significant (P < 0.001) elevation in MDA and NO levels accompanied with significant (P < 0.001) decrease in GHS content and SOD and CAT activities in liver homogenates of CCl 4 as well as IRR + CCl 4 treated rats.Nevertheless, the changes in the antioxidant markers were higher in IRR + CCl 4 treated group (P < 0.01) compared to the corresponding values in CCl 4 alone treated rats.Oral administration of RM significantly restored the altered levels of the investigated antioxidants in hepatic tissue of Gp5 rats compared with Gp4.

Effect of RM on Heme Oxygenase-1 (HO-1) Activity in Hepatic Tissue of Fibrotic Rats
The activity of HO-1 in hepatic tissue was significantly increased in the two models (P < 0.001).A highly significant increase in HO-1 activity was recorded in the 2 nd model in comparison with the 1 st one.Concurrent administration of RM along with IRR + CCl 4 could significantly (P < 0.05) reduced the HO-1 activity as compared to IRR+ CCl 4 treated rats (Figure 6).

Effect of RM on the mRNA Levels of TGF-β, TNF-α, HO-1, and Type-1 Collagen in Hepatic Tissue of Fibrotic Rats
As shown in (Figure 7(A) and Figure 7(B)) the mRNA levels of TGF-β, TNF-α, HO-1 and type-1 collagen were significantly (P < 0.001) increased in the two rats' models as compared with control set, with a more conspicuous (P < 0.001) increase in their mRNA expression in the 2 nd model when compared to the 1 st model.However, treatment of fibrotic rats (IRR + CCl 4 ) with RM was able to significantly (P < 0.001) down regulate the mRNA expression of the previous parameters in hepatic tissue when compared to model 2.

Histopathological Examination
Liver of control as well as RM treated rats' revealed normal histological appearance.While examination of different liver sections of CCl 4 treated rats revealed massive destruction and alteration in the normal hepatic histology.Centrilobular congestion was evident with disorganization of the hepatic cords and massive fatty change that reached to marked fat steatosis.Necrosis of the hepatocytes as small groups of cells and as single cell necrosis (apoptosis) (Figure 8(A)) was clearly observed in most cases.The portal triads showed inflammatory cells infiltration and marked oval cell hyperplasia Figure 8(B)) that begins to insinuate among the hepatic parenchymal cells with an obvious abundant collagen fibers accumulation (Figure 8(C)).The hepatic normal architecture was unclear with expansion of fibrosis in portal area and peripherally.The expanded fibrosis formed slender fibrous septa that led to conspicuous bridging fibrosis (that demonstrated by Azan stain) (Figure 8(D) and Figure 8 (E)) at which the neighboring portal areas were bridged by fibrous septa with beginning of pseudolobulation formation of the hepatic parenchyma.That septa contained inflammatory cells, fat steatotic cells, hyperplastic oval cells and fibroblasts (Figure 8(F)).On the other hand, microscopical examination of livers' sections of IRR + CCl 4 treated rats revealed that the exposure of rats to irradiation aggravated the effect of CCl 4 in fibrosis induction.The arrangement of the hepatic plate was completely distorted with remarkable fat steatosis.The hepatocytes showed necrosis in fragments with appearance of remnants of degenerated and necrotic hepatocytes containing acidophilic hyaline globules (Figure 9(A)).
Extensive collagenous formation was observed that was even showed along the hepatic sinusoids and surrounded the necrotic hepatocytes (Figure 9(B) and Figure 9(C)).Bridging fibrosis was marked and the collagenous septa were much thicker than those of the sole CCl 4 treated group causing evident pseudolobuli formation.
It was noticed that RM co-administration to IRR + CCl 4 treated rats could undoubtedly improve the state of steatosis (Figure 9(D)) and increased number of apoptotic bodies as well as apparently suppressed hepatic fibrogenesis.The later was evidenced either by absence of fibrotic septa or in few cases by reducing the thickness of bridging fibrotic septa although collagen fibers accumulation and proliferation in the portal triad was still existed (Figure 9(E)).No obvious pseudolobulation (Figure 9(F)) was observed in most cases.

Results of Immunohistochemistry
Control and RM treated rats showed positive α-SMA staining around central vein and portal vein indicating normal expression of myofibroblasts.While, in CCl 4 and IRR + CCl 4 treated groups, α-SMA positive cells were markedly increased in the portal area, around the central vein and along the bridging fibrosis, exhibited the spread of collagen fibers from portal area (Figure 10(A)).Stronger pattern of positivity were observed in IRR + CCl 4 treated rats that even extended in the perisinasiodal spaces (Figure 10(B)).However, the expression of α-SMA positive cells was decreased in RM + IRR + CCl 4 treated rats and only observed in the portal area along the accumulated collagen fibers and around the central vein.

Discussion
In the current study, two models of hepatic fibrosis were generated experimentally in rats along six weeks.It was observed that the exposure to gamma irradiation in the 2 nd model could promote and enhance CCl 4 induced fibrosis more than the sole use of CCl 4 in the 1 st model.Moreover, the pathological extent of two models of fibrosis  and the protective effect of RM against the 2 nd model were further evaluated by biochemical and histopathological analysis in this study.
It is well known that: 1) CCl 4 is metabolized by cytochrome P450 in liver into the highly reactive trichloromethyl radicals (CCl 3 + ) and trichloromethylperoxy radicals (CCl 3 O 2 + ) resulting in initiation of cascade of lipid peroxidation, cell necrosis, steatosis, inflammation; 2) and this compound further promotes progression of hepatic [29] [30] which is largely mimics hepatic fibrosis in human diseases [31].Moreover, the exposure to ionizing radiation can lead to an increase in the generation of ROS leading to lipid peroxidation and oxidative stress [32].The involvement of ROS and lipid peroxidation in hepatic fibrosis has been reported [33].Accordingly, in the present work aggravation of fibrosis in the 2 nd model was expected due to excessive production of ROS and lipid peroxidation by gamma irradiation [32].
There was a significant decrease in body weight gain of animals of both fibrotic models, which could be contributed to anorexia.Similar reduction in body weight caused by IRR as well as CCl 4 has been well documented [34].Moreover, the increase in liver weight index in the two models follows the same pattern of previous researchers [35] who reported that the increase or decrease in either absolute or index of an organ weight after administration of a chemical or drug could be contributed to the toxic effect of that chemical.The co-treatment of RM to IRR + CCl 4 treated rats caused significant restoration in weight gain and significantly reduced the increase in liver weight index, which provided an evidence of the hepato-protective of RM.
The present study showed that the levels of ALT and AST, TP, Alb, TNF-α, TGF-β were significantly increased in the rats of the two models compared to control.The increased levels of hepatic function markers have been attributed to the liver injury and the release of these enzymes into the blood circulation after the administration of hepatotoxine; such as CCl 4 [36].In addition, the damaged effect of hepatocytes could be due to liberation of large amount of free radicals that induced peroxidative degeneration of membrane lipids and formation of peroxides which probably caused membrane damages [37] and thus cellular alteration.
Total protein and albumin are clinically useful markers of hepatic synthetic function [38].In agreement with other reports [39], our data indicated that induction of liver fibrosis in rats caused a significant diminution in total protein and albumin which was a further indication of liver damage.However, simultaneous administration of RM with IRR + CCl 4 contributes a hepatoprotective mechanism which stimulated protein synthesis and subsequently accelerates the process of regeneration and production of new hepatic cells [40].
Moreover, the observed increase in plasma levels of TNF-α and TGF-β in rats of the two models was accompanied with increased their hepatic mRNA expression.Accumulating evidence supports the concept that CCl 4 causes lipid peroxidation that leads to hepatocellular membrane damage and followed by the release of proinflammatory mediators, which are thought to potentiate CCl 4 -induced hepatic damage [41].Others demonstrated that irradiation induced chronic inflammatory mediators such as rapid activation of TGF-β [42] and TNF-α [43] resulted in fibrosis [44].RM administration to IRR + CCl 4 treated rats significantly reduced the elevated levels of the two cytokines in plasma associated with reduction in their mRNA expression in liver tissue.This result is in close relationship with early results which suggested that the suppression that suppression of inflammation and reduced TGF-β level have been proposed as a molecular mechanism involved in protection against hepatic fibrosis [45].
Furthermore, our study indicated high HP content in the two established fibrosis models indicated the progression of fibrosis which was assured by the observed up regulation of collagen expression especially in the 2 nd model.Hydroxyproline, is an amino acid and a characteristic product of collagen metabolism, its content indicated the total collagen present in liver.In consistent with our findings Fu et al. reported an elevated level of HP in response to CCl 4 induced fibrogenesis [46].This result could be due to activation of the pro-fibrogenic cytokine TGF-β and promotion of fibrosis via activation of HSCs [47] and expression of collagen.In addition, gamma irradiation was demonstrated to cause rapid activation of TGF-β [42].However, oral administration of RM could reduce HP concentration and consequently reduce collagen deposition and suppress the fibrogenic effect induced by CCl 4 alone or by combined IRR + CCl 4 treatments.
In the current investigation, a significant elevation in MDA and NO levels was recorded associated with significant reduction in GSH concentration, SOD and CAT activities in both fibrotic groups.This is an indication of fibrosis induced oxidative stress in liver.In agreement with our results, early reports demonstrated enhancement of lipid peroxidation after whole body irradiation [48] and CCl 4 treatment [49].The increase in MDA and NO levels could be attributed to high degree of oxidative stress and over production of free radicals and ROS which attack macromolecules such as lipids and proteins initiating lipid peroxidation, thereby, causing depletion of antioxidants.
However, administration of RM to model 2 rats significantly lowered MDA and NO levels, prevented the depletion of GSH and enhanced the activity of antioxidant enzymes as well.This result could be due to the strong free radical scavenger and antioxidant activity of RM which endorsed by the presence of active compounds that are responsible for the hepatoprotective activity as well as the reduction of the free radicals that induce oxidative damage to the liver [50].
Regarding, heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism that is induced by a variety of stimuli including oxidative stress and pro inflammatory cytokines.Our current investigation revealed significant elevation in HO-1 activity and its mRNA expression in hepatic tissue of fibrotic rats.Early reports recorded an elevation in HO activity and its mRNA expression in hepatic tissue caused by whole body gamma irradiation [51].Moreover, in a recent study, an abnormal activity of HO-1 after CCl 4 treatment has been evidenced [52].That elevation may be probably due to oxidative stress and inflammation induced by both CCl 4 and gamma irradiation.However, concurrent treatment of RM along with IRR + CCl 4 brought down the HO-1 activity and reduced its mRNA expression in hepatic tissue.The protective effect of RM could be due to its strong antioxidant activity.
Our histopathological results revealed marked hepatic tissue alterations as a result of CCl 4 and IRR + CCl 4 treatments.In addition, the induction of fibrosis models by using composite factors both of IRR and CCl 4 was succeeded as observed by more clear bridging fibrosis and pseudolobulation of livers of rats exposed to IRR + CCl 4 which encouraged the role of IRR in aggravation of hepatic fibrogenesis induced by CCl 4 .The later effect could be related to the production of large amount of free radicals by both CCl 4 and gamma irradiation which by their direct toxic effect could lead to lipid peroxidation and activation of an immune-inflammatory mechanisms that could result in functional and morphological alterations and even cell death [53] [54].
Our results are in agreement with those of [55] [56] who noted that CCl 4 administration to mice could induce portal fibrosis and bridging fibrosis as well as regenerating nodules.The observed bridging fibrosis and pseudo lobulation in IRR + CCl 4 treated rats were accompanied by increased positivity of α-SMA wherever the collagen fibers deposited.That immunopositivity of α-SMA indicates activation of HSCs which were responsible for the fibrosis occurrence in CCl 4 and IRR + CCl 4 treated rats.The later attribution is in agreement with that of [57] who mentioned that; at HSCs activation, the levels of α-SMA and desmin increase with decreased GFAP expression due to their transformation into myofibroblast-like cells.Moreover, it has been reported that hepatocytes which undergoing oxidative stress and release of ROS stimulate HSC activation.Prior studies have demonstrated that chronic hepatic fibrosis and inflammation accompanied with Kupffer cell accumulation, HSC activation and collagen deposition [58] [59].The observed amount of esinophilic hyaline globules of variable sizes in fibrotic lesions of IRR + CCl 4 treated rats was correlated with increased collagen deposition and the degree of liver fibrosis.Such bodies or globules could be seen in the hepatocytes in alcoholic and nonalcoholic liver disorders [60].Those bodies represent abnormal protein aggregates that could be reverted to normal state by molecular chaperones or degradated by proteasomes [61].However, if reparation or degradation processes fail, abnormal proteins became segregated in the cytoplasm as inclusion bodies.On the other hand, RM administration to IRR + CCl 4 treated rats' revealed great ameliorative action of RM on the deleterious effects of both CCl 4 and IRR as evidenced by clear absence of bridging fibrosis and pseudolobulation.Such hepato protective and fibrosis-inhibition effects of RM have been recorded [17] [18].This effect per se may be related to the antioxidant effect of RM' constituents and its scavenging activity of free radicals that reflected on saving the integrity of cellular membranes thus suppressing the inflammatory response and attenuated the fibrosis action.

Conclusion
In summary, this study provided evidence for experimental model of hepatic fibrosis using gamma irradiation and CCl 4 .The present study also suggests that RM aqueous solution showed a considerable hepatoprotective activity against IRR + CCl 4 induced hepatic fibrosis and injury in rats.This protective effect could be due to its membrane cellular protection, free radicals scavenging activity, enhancing the endogenous antioxidant system, suppressing the inflammatory responses and attenuation of fibrogenesis.The histopathological study confirmed the biochemical findings.

Figure 8 .
Figure 8. Livers of CCl 4 treated rats showing: (A) area of hepatocellular necrosis with multiple apoptotic bodies (arrow); (B) Portal triads with inflammatory cells infiltration and marked oval cell hyperplasia; (C) Abundant collagen fibers accumulation in the portal area and its extension peripherally; (D) Bridging fibrosis between the neighboring portal areas with apparent pseudolobulation formation; (E) Bridging fibrosis (Azan stain); (F) Fibrous septa contained inflammatory cells and fat steatotic cells with hyperplastic oval cells and fibroblasts.(H & E ×400, 100 and 200).

Figure 9 .
Figure 9. (A), (B), (C) Livers of IRR+ CCl 4 treated rats showing: (A): Complete distortion of the hepatic plate with necrosis of hepatocytes in fragments; (B) and (C): Extensive collagenous formation with its interpolation along the hepatic sinusoids and surrounded the necrotic hepatocytes; (D)-(F): Livers of RM + IRR + CCl 4 treated rats showing; (D): Improvement of the state of steatosis with marked apoptotic bodies (arrow); (E): Collagen fibers accumulation and proliferation only in the portal triad.(F): Inhibition of hepatic fibrogenesis with no obvious pseudolobulation.(H & E ×400, 100 and 200).

Figure 10 .
Figure 10.(A) Liver of CCl 4 treated rat showing positive α-SMA along the fibrous septa; (B) Liver of IRR + CCl 4 treated rat showing strong positivity of alpha-SMA in the portal areas and extending along the pseudolobulation; (C) Livers of RM + IRR + CCl 4 treated rats showing expression of α-SMA only in the portal area around portal vein.

Table 1 .
Primer sequences used for real time PCR.