This work was undertaken to establish a new experimental model of hepatic fibrosis by gamma irradiation and CCl 4 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 CCl 4 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.
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 [
Radiation-induced liver disease is mainly due to oxidative damage, leading to liver inflammation and fibrosis [
CCl4 has been widely reported to induce acute and chronic tissue injuries. A single dose of CCl4 can result in centrizonal necrosis and steatosis [
Mushroom is widely used everywhere all over the world as an important source of nutrition and therapy [
Aim of work: The main objective of the present study was to establish a fibrosis model using gamma irradiation and CCl4 and to demonstrate the protective effect of RM aqueous solution against the induced hepatic fibrosis with focusing on the precise cellular and molecular mechanisms.
CCl4 (C25630) was obtained from Sigma Chemical Co. (St. Louis, MO) USA. The kits used were purchased from Bio-diagnostic, Cairo, Egypt. Reishi Mushroom (RM) powder was provided by DXN marketing SD.BHD (283904-P), Malaysia.
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 was performed through the use of a Canadian Gamma Cell-40 (137Cs) at the National Centre for Radiation Research and Technology (NCRRT), Cairo, Egypt. The dose rate was 0.675 Gy/minute.
Two models of hepatic fibrosis were established in the current study. The 1st model was established using CCl4 alone as; CCl4 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 2nd model, gamma irradiation was used to promote and enhance the CCl4 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 CCl4 as in the 1st model.
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 1st model of fibrosis; rats were subcutaneously injected with CCl4 in olive oil (2 ml/kg)3 times/week for six weeks. Gp4; represented the 2nd model of fibrosis; rats of this group were subjected to gamma irradiation and CCl4 as described previously. Gp5; (RM + IRR + CCl4 treated group) rats were treated with RM as in group 2 and were exposed to both IRR + CCl4 as in group 4. The time interval between CCl4 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.
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.
HP; an indicator for hepatic collagen amount was colorimetriclly assayed in liver tissue homogenate as previously described [
Malondiladehyde (MDA); the end product of lipid peroxidation [
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 [
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
primer | sequence |
---|---|
TGF-β1 | Forward primer: 5'-AGGGCTACCATGCCACTTC-3' Reverse primer: 5'-GCGGCACGCAGCACGGTGAT-3' |
TNF-α | Forward primer: 5'-GAAAAGCAAGCAGCCAACCA-3' Reverse primer: 5'-CGGATCATGCTTTCTGTGCTC-3' |
HO-1 | Forward primer: 5'-TTTCAAAGGGTCAGGTGTC-3' Reverse primer: 5'-CCTTCTGCGGCAATCTTCTTC-3' |
Type-1 collagen | Forward primer: 5'-AATTGGAGCTGTTGGTAACGC-3' Reverse primer: 5'-CACCAGTAAGGCCGTTTGC-3' |
GADPH | Forward primer: 5'-CTCCCATTCTTCCACCTTTG-3' Reverse primer: 5'-CTTGCTCTCAGTATCCTTGC-3' |
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 [
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 [
Labeled streptoavidin-biotin method was used for immunohistochemical detection of α-SMA using a Histostatinplus bulk kit (Zymed Laboratories Ins., San Francisco, CA, USA). The primary antibody used was mononuclonal anti-smooth muscle actin, at a dilution of 1:800 (clone 1A4, Sigma Co., St. Lois, MO, USA).
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.
Rats of CCl4 and IRR + CCl4 treated groups appeared depressed with lusterless fur and decreased appetite. Livers of rats of CCl4 were swollen with rounded borders, irregular surface and fatty appearance. While that of IRR + CCl4 treated rats were severely distorted with fatty appearance.
As shown in
Treatments | Body Weight (g) | Liver Index (%) | |
---|---|---|---|
Initial | Final | ||
CT | 154.4 ± 2.4 | 268.4 ± 3.5 | 2.48 ± 0.19 |
RM | 154.8 ± 3.8 | 244.1 ± 6.8* | 2.78 ± 0.02 |
CCl4 | 145.6 ± 1.8 | 242.3 ± 4.7** | 3.9 ± 0.21*** |
IRR + CCl4 | 144.7 ± 1.3NS | 218.8 ± 5.7***,a | 4.76 ± 0.12***,# # |
RM + IRR + CCl4 | 146.3 ± 0.6NS | 239.8 ± 5.9**,b | 3.48 ± 0.11¥ |
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.
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.
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 (
Data in (
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 (
As shown in (
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.
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) (
hepatocytes containing acidophilic hyaline globules (
Extensive collagenous formation was observed that was even showed along the hepatic sinusoids and surrounded the necrotic hepatocytes (
It was noticed that RM co-administration to IRR + CCl4 treated rats could undoubtedly improve the state of steatosis (
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 (
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
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 [
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 [
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 [
Total protein and albumin are clinically useful markers of hepatic synthetic function [
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 [
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 [
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 [
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 [
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 [
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.
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