the normal values (P > 0.05).
Table 2. The effect of Reishi Mushroom (RM) on body weight and liver weight index in fibrotic rats.
Each value represents the mean ± SE of 5 variables/group. Significant difference versus corresponding CT group at ***P < 0.001, **P < 0.01, *P < 0.05. Significance change of CCl4 from IRR + CCL4 at #P < 0.05, ##P < 0.01. Significant difference of Gp5 (RM + IRR + CCl4) from IRR + CCl4 at ¥P < 0.001.
3.3. 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 CCl4 and IRR+CCl4 treated groups when compared with controls (Figures 1-3). The highly significant (P < 0.05) changes were noticed in IRR + CCl4 treated rats compared with those of the sole CCl4 treatment. On contrary, the administration of RM to IRR + CCl4 treated rats resulted in significant (P < 0.001) improvement of all of the above altered parameters compared to those of model 2 rats.
3.4. Effect of RM on Hydroxyproline Content in Liver Homogenate of Fibrotic Rats
Hydroxyproline content was significantly (P < 0.001) increased following CCl4 treatment and showed higher significant (P < 0.01) increase following IRR + CCl4 treatment than its level in model 1. By contrast, oral administration of RM with IRR + CCl4 significantly restored the hepatic hydroxyproline content compared with its level in model 2 rats (Figure 4).
3.5. 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 CCl4 as well as IRR + CCl4 treated rats. Nevertheless, the changes in the antioxidant markers were higher in IRR + CCl4 treated group (P < 0.01) compared to the corresponding values in CCl4 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.
3.6. 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 2nd model in comparison with the 1st one. Concurrent administration of RM along with IRR + CCl4 could significantly (P < 0.05) reduced the HO-1 activity as compared to IRR+ CCl4 treated rats (Figure 6).
3.7. 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 2nd model when compared to the 1st model.
Figure 1. Effect of RM on ALT and AST activities in fibrogenic rats. Each value represents the mean ± SE. Significant difference versus corresponding control group at ***P < 0.001. Significant difference of IRR + CCL4 versus corresponding CCL4 group at #P <0.05, ##P <0.001. Significant difference of RM + IRR + CCL4 versus corresponding IRR + CCL4 group at ¥P < 0.001.
Figure 2. Effect of RM on total protein (TP) and albumin (Alb) in fibrogenic rats. Each value represents the mean ± SE. N = 5/group. Significant difference versus corresponding control group at ***P < 0.001. Significant difference of RM + IRR + CCl4 versus corresponding IRR + CCL4 group at ¥P < 0.001.
Figure 3. Effect of RM on levels of TNF-α and TGF-β in plasma of fibrogenic rats. Each value represents the mean ± SEM. N = 5/group. Significant difference versus corresponding control group at ***P < 0.001, **P < 0.01. Significant difference versus corresponding CCl4 at ##P < 0.001, #P < 0.01. Significant difference of RM + IRR + CCl4 versus corresponding IRR + CCl4 group at ¥P < 0.001.
Figure 4. Effect of RM on hydroxyproline (HP) content in liver homogenate of fibrogenic rats. Each value represents the mean ± SEM. N = 5/group. Significant difference versus corresponding control group at ***P < 0.001, **P < 0.01. Significant difference versus corresponding CCl4 at #P < 0.01. Significant difference of RM + IRR + CCl4 versus corresponding IRR + CCl4 group at ¥P < 0.001.
Figure 5. Effect of RM on MDA and NO in liver homogenate of fibrogenic rats. Each value represents the mean ± SEM. N = 5/group. Significant difference versus corresponding control group at ***P < 0.001, **P < 0.01.Significant difference versus corresponding CCl4 at #P < 0.01. Significant difference of RM + IRR + CCl4 versus corresponding IRR + CCl4 group at ¥P < 0.001.
Figure 6. Effect of RM on GSH content, SOD and CAT activities and HO-1in liver homogenates of fibrogenic rats. Each value represents the mean ± SEM. Significant difference versus corresponding control group at ***P < 0.001, **P < 0.01. Significant difference of IRR + CCl4 versus corresponding CCl4 group at #P < 0.05. Significant difference of RM + IRR + CCL4 versus corresponding IRR + CCl4 group at ¥P < 0.05, ¥¥P < 0.01 ¥¥¥P < 0.001.
Figure 7. Effect of RM on (A): mRNA levels of TNF-α, TGF-β and collagen type-1; (B): HO-1 of fibrotic rats as analyzed by RT-PCR in liver homogenates. Each value represents the mean ± SEM. Significant difference versus corresponding control group at ***P < 0.001, **P < 0.01. Significant difference of CCl4 versus corresponding IRR + CCl4 group at #P < 0. 01, ##P < 0.001. Significant difference of RM + IRR + CCL4 versus corresponding IRR + CCl4 group at ¥P < 0.001.
However, treatment of fibrotic rats (IRR + CCl4) 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.
3.8. Histopathological Examination
Liver of control as well as RM treated rats’ revealed normal histological appearance. While examination of different liver sections of CCl4 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 + CCl4 treated rats revealed that the exposure of rats to irradiation aggravated the effect of CCl4 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
Figure 8. Livers of CCl4 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).
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 CCl4 treated group causing evident pseudolobuli formation.
It was noticed that RM co-administration to IRR + CCl4 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.
3.9. 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 CCl4 and IRR + CCl4 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 + CCl4 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 + CCl4 treated rats and only observed in the portal area along the accumulated collagen fibers and around the central vein.
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 2nd model could promote and enhance CCl4 induced fibrosis more than the sole use of CCl4 in the 1st model. Moreover, the pathological extent of two models of fibrosis
Figure 9. (A), (B), (C) Livers of IRR+ CCl4 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 + CCl4 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. (A) Liver of CCl4 treated rat showing positive α-SMA along the fibrous septa; (B) Liver of IRR + CCl4 treated rat showing strong positivity of alpha-SMA in the portal areas and extending along the pseudolobulation; (C) Livers of RM + IRR + CCl4 treated rats showing expression of α-SMA only in the portal area around portal vein.
and the protective effect of RM against the 2nd model were further evaluated by biochemical and histopathological analysis in this study.
It is well known that: 1) CCl4 is metabolized by cytochrome P450 in liver into the highly reactive trichloromethyl radicals (CCl3+) and trichloromethylperoxy radicals (CCl3O2+) resulting in initiation of cascade of lipid peroxidation, cell necrosis, steatosis, inflammation; 2) and this compound further promotes progression of hepatic   which is largely mimics hepatic fibrosis in human diseases  . Moreover, the exposure to ionizing radiation can lead to an increase in the generation of ROS leading to lipid peroxidation and oxidative stress  . The involvement of ROS and lipid peroxidation in hepatic fibrosis has been reported  . Accordingly, in the present work aggravation of fibrosis in the 2nd model was expected due to excessive production of ROS and lipid peroxidation by gamma irradiation  .
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 CCl4 has been well documented  . Moreover, the increase in liver weight index in the two models follows the same pattern of previous researchers  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 + CCl4 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 CCl4  . 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  and thus cellular alteration.
Total protein and albumin are clinically useful markers of hepatic synthetic function  . In agreement with other reports  , 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 + CCl4 contributes a hepatoprotective mechanism which stimulated protein synthesis and subsequently accelerates the process of regeneration and production of new hepatic cells  .
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 CCl4 causes lipid peroxidation that leads to hepatocellular membrane damage and followed by the release of pro- inflammatory mediators, which are thought to potentiate CCl4-induced hepatic damage  . Others demonstrated that irradiation induced chronic inflammatory mediators such as rapid activation of TGF-β  and TNF-α  resulted in fibrosis  . RM administration to IRR + CCl4 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  .
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 2nd 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 CCl4 induced fibrogenesis  . This result could be due to activation of the pro-fibrogenic cytokine TGF-β and promotion of fibrosis via activation of HSCs  and expression of collagen. In addition, gamma irradiation was demonstrated to cause rapid activation of TGF-β  . However, oral administration of RM could reduce HP concentration and consequently reduce collagen deposition and suppress the fibrogenic effect induced by CCl4 alone or by combined IRR + CCl4 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  and CCl4 treatment  . 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  .
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  . Moreover, in a recent study, an abnormal activity of HO-1 after CCl4 treatment has been evidenced  . That elevation may be probably due to oxidative stress and inflammation induced by both CCl4 and gamma irradiation. However, concurrent treatment of RM along with IRR + CCl4 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 CCl4 and IRR + CCl4 treatments. In addition, the induction of fibrosis models by using composite factors both of IRR and CCl4 was succeeded as observed by more clear bridging fibrosis and pseudolobulation of livers of rats exposed to IRR + CCl4 which encouraged the role of IRR in aggravation of hepatic fibrogenesis induced by CCl4. The later effect could be related to the production of large amount of free radicals by both CCl4 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   .
Our results are in agreement with those of   who noted that CCl4 administration to mice could induce portal fibrosis and bridging fibrosis as well as regenerating nodules. The observed bridging fibrosis and pseudo lobulation in IRR + CCl4 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 CCl4 and IRR + CCl4 treated rats. The later attribution is in agreement with that of  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   . The observed amount of esinophilic hyaline globules of variable sizes in fibrotic lesions of IRR + CCl4 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  . Those bodies represent abnormal protein aggregates that could be reverted to normal state by molecular chaperones or degradated by proteasomes  . 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 + CCl4 treated rats’ revealed great ameliorative action of RM on the deleterious effects of both CCl4 and IRR as evidenced by clear absence of bridging fibrosis and pseudolobulation. Such hepato protective and fibrosis-inhibition effects of RM have been recorded   . 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.
In summary, this study provided evidence for experimental model of hepatic fibrosis using gamma irradiation and CCl4. The present study also suggests that RM aqueous solution showed a considerable hepatoprotective activity against IRR + CCl4 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.
Cite this paper
Omama E. ElShawi,Sahar S. AbdEl-Rahman,Marwa Abd ElHameed, (2015) Reishi Mushroom Attenuates Hepatic Inflammation and Fibrosis Induced by Irradiation Enhanced Carbon Tetrachloride in Rat Model. Journal of Biosciences and Medicines,03,24-38. doi: 10.4236/jbm.2015.310004