Evaluation of methanolic extract of Phragmites karka on carbon tetrachloride-induced liver fibrosis in rat
Abstract
Phragmites karka has been reported for its anti-inflammatory and analgesic activities. Here, extracts of leaf and rhizome of the plant were individually investigated in CCl4-induced hepatofibrosis in male Wistar rats by administering CCl4 intraperitoneally biweekly for 6 weeks. Afterwards the animals were investigated for liver fibrosis at biochemical, molecular and histological levels, and it showed a profound increase (p<0.001) in elevation of serum levels of transaminases, gamma-glutamyl transpeptidase, mRNA expression of alpha smooth muscle actin, collagen and transforming growth factor beta (TGF-beta), and extracellular matrix deposition and perilobular necrosis. Both extracts markedly (p<0.001) decreased the elevated levels of these markers. Histopathological investigations also substantiated the above results by exhibiting a decreased in extracellular matrix deposition in post-treatment animals. In conclusion, both extracts had substantially modified the biochemical and molecular markers of liver fibrosis, in addition to histological improvement in architecture of liver.
Introduction
Hepatic fibrosis is a continuous wound healing response to repeated liver injuries which may be caused by viral hepatitis, alcohol abuse and other hepatotoxic agents (Friedrich-Rust et al., 2009; Rehman, 2014). It is initially a critical intermediary step in the progression to liver cirrhosis and latterly hepatocellular carcinoma (Zhou et al., 2014). In hepatic fibrosis, hepatic stellate cells are activated and form an excessive extracellular matrix that includes collagens and fibronectins (Tsukada et al., 2006). Accumulation of extracellular matrix proteins forms a dense fibrous scar, thus interrupting normal architecture of liver tissue. It compromises the flow of blood in the liver and causes an oxidative burden on the organ, thus paving the way for regeneration of parenchymal nodules and ultimately leading to cirrhosis (Ismail and Pinzani, 2009). Furthermore, continuous hepatic stellate cells activation leads to myofibroblast transition via the release of pro-fibrogenic cytokines including transforming growth factor (TGF-beta) (Bisselle et al., 1995; Desmouliere et al., 1993; Dooley et al., 2012), platelet-derived growth factor, and connective tissue growth factor (Tsukada et al., 2006). Therefore, it is one important reason that hepatic stellate cells and expressed cytokines e.g., TGF-beta (Inagaki et al., 2005; Tiggelman et al., 1995 ) is investigated as a therapeutic target for treating liver fibrosis and cirrhosis.
In search of ideal molecules to treat the hepatic injury and hepatofibrosis, scientists are also diverting their attention towards the natural remedies (Ahmed et al., 20116; Gonzalez et al 2016; Gu et al., 2013). Phragmites karka belongs to the family Pocaceae, which is wildly distributed in Punjab and Kashmir regions of Pakistan and used as a folk medicine for the treatment of rheumatic pains and other inflammatory conditions. It has been reported to contain chemical entities such as pentosans, lignins, flavonoids, steroids, terpenoids, tannins and alkaloids (Abideen et al., 2015; Sultan et al., 2017). Its leaf exhibits the highest polyphenol contents, which may attribute to its reported antioxidant activity (Abideen et al., 2015). Its rhizome and root are known for the antiemetic and diaphoretic action, and are also used for the treatment of diabetes (Oyen 2011). The leaf exhibits analgesic, antidepressant and antinociceptive activities (Sultan et al., 2017).
Keeping in view, its folklore use as a medicinal plant in various inflammatory ailments, this study was thus designed to explore its role in an animal model of hepatofibrosis.
Materials and Methods
Plant Material
The plant was collected from the Lahore region and identified by a taxonomist of the Department of Botany, Government College University, Lahore, Pakistan. The specimens of the leaf and rhizome of the plant were deposited in the herbarium and voucher number GC Herb. Bot. 2916 was issued.
Preparation of the Extract
The leaf and rhizome of P. karka were thoroughly washed with water and dried in shade before grinding. Dried powdered leaf (500 g) and rhizome (300 g) of P. karka were separately macerated in methanol (2 L) for 8 days at an ambient room temperature with occasional shaking and stirring. The macerate was removed and the cake was macerated again for two more times. The macerates were filtered through the muslin cloth followed by filtration through Whatman filter paper No. 8. The extract was concentrated in a rotary evaporator (company) at 50°C and 90 rpm. The viscous mass obtained was dried to semisolid consistency and stored at 4°C.
Animals
Male Wistar rats weighing 150-250 g were given standard laboratory food, water ad libitum and maintained at the natural day-night cycle. The animals were acclimatized to the lab conditions for one week before starting the experiment.
Chemicals
Methanol and CCl4 were purchased from the Sigma-Aldrich (UK). Corn oil was purchased from the local manufacturer. Serum aminotransferases and gamma-glutamyl transpeptidase were performed using the kit (LABTEST, TRON, France).
Induction of Fibrosis and Treatment Plans
Liver fibrosis was induced in rats using previous protocol (Nakamura et al., 1999) with minor modifications. Rats were intraperitoneally (IP) injected with CCl4 dissolved in corn oil biweekly for 6 weeks. In the first week 0.5 µL CCl4/gram of body weight (g); in the second week 0.75 µL CCl4/g and for the rest of 4 weeks 1 µL CCl4/g). The control animals were administered (IP) corn oil only. The investigational treatment was started from the 3rd week onwards with the leaf and rhizome extract at indicated doses.
Experimental Groups
Male Wistar rats were randomly assigned to 6 groups (n=5 in each group). Group 1 served as control group and administered (IP) with corn oil. Group 2 and 3 were daily administered with the leaf extract (500 mg/kg) and rhizome extract (500 mg/kg) respectively, via oral gavage tube. Group 4 was fibrosis positive group and the rats were administered CCl4 (as mentioned above). Group 5 and 6 served as treatment groups. They were daily administered with leaf extract (500 mg/kg) and rhizome extract (500 mg/kg) respectively, in addition to CCl4 (IP). All the doses were administered in absence of anesthesia.
Collection of Serum and Liver Samples
Rats were anatomized under anesthesia one day after the last dose was given. Blood was collected by means of cardiac puncture. The serum was collected by centrifugation at 4,000 rpm for 4-5 min. Tissue samples of liver were stored in 10% formaldehyde for fixation. Liver tissues were also preserved at -20°C for real time PCR.
Liver Function Tests
Serum aminotransferases and gamma-glutamyl transpeptidase levels were determined using the manufactures' protocol.
Histopathological Evaluation
Liver tissues were sliced (5 um thickness) using a rotary microtome and stained with hematoxylin and eosin (H & E) stain by adopting standard lab procedure.
mRNA Expression Analysis
Total RNA extraction, complementary DNA (cDNA) and real-time PCR were carried out using the kits from WizBio Solution (Korea). To determine the mRNA levels of TGB-beta, alpha-SMA and collagen following primers sequences were used:
18S: F5'-CGG CTA CCA CAT CCA AGG A 3', R5'-CCA ATT ACA GGG CCT CGA AA3'; alpha-SMA: F 5'-GCC AGT CGC CAT CAG GAA C3', R5'-CAC ACC AGA GCT GTG CTG TCT T3'; Collagen: F 5'-TGG TGA ACG TGG TGT ACA AGG T3', R 5'-CAG TAT CAC CCT TGG CAC CAT3'; TGFbeta: F 5'-GGG CTA CCA TGC CAA CTT CTG 3', R 5'-GAG GGC AAG GAC CTT GCT GTA3'.
Statistical Analysis
All data are expressed as the mean ± standard deviation (SD). Statistically significant differences between the groups were analyzed by one-way analysis of variance (ANOVA) followed by Bonferonni's descriptive test. Differences with p<0.05 were considered statistically significant.
Results
Effects on serum aminotransferase and gamma-glutamyl transpeptidase
Figure 1 shows the effect of P. karka extract on serum aminotransferase and gamma-glutamyl transpeptidase. Initially, the corn oil treated control group was compared with groups of animals treated with leaf extract or rhizome extract alone where the data showed no significant increase in serum levels of aminotransferase and gamma-glutamyl transpeptidase. CCl4-treated group showed a significant (p<0.001) increase in the serum levels of aminotransferase and gamma-glutamyl transpeptidase when compared with the control. Thereafter, CCl4-treated control group was compared with group which were treated with CCl4 and leaf/rhizome extract. The data clearly indicated a significant (p<0.001) decline in the serum levels of aminotransferase and gamma-glutamyl transpeptidase compared to CCl4-treated group.
Figure 1: ALT (A), AST (B) and GGT (C) values of different groups; Data is represented as Mean ± SD; "a" Shows significant difference Vs Control Group; "b" Shows significant difference Vs CCl4 Group
mRNA expression evaluation of liver fibrosis markers
Figure 2 shows the mRNA expression evaluation of alpha-SMA, TGF-beta and collagen. The data showed that there was a 1-fold increase in mRNA expression of alpha-SMA in CCl4-treated group as compared to the control group (Figure 2A). Treatment with leaf extract alone lowered the levels of alpha-SMA by half as compared to the control (vehicle treated) group, whereas rhizome extract did not show any effect on alpha-SMA mRNA expression. Interestingly, both extracts exhibited a significant (p<0.001) down-regulation of mRNA (compare CCl4-treated and leaf or rhizome extract group with the CCl4-treated group. Moreover, leaf extract seemed to be more effective in terms of mRNA expression of alpha-SMA when compared with rhizome extract-treated group.
Figure 2: mRNA expression values of alpha- Smooth muscle actin (A), transforming growth factor-beta (B) and collagen (C); Data is represented as Mean ± SD; “a†Shows significant difference Vs Control Group. "b" Shows Significant difference Vs CCl4 Group
Figure 2B shows mRNA expression of TGF-beta. The data shows that there was a 2-fold increase in mRNA expression of TGF-beta in CCl4 -treated group as compared to the control group. Leaf extract did not show any change in the mRNA levels of TGF-beta as compared to the control group. However, rhizome extract profoundly reduced the mRNA expression levels of TGF-beta compared to control. Both extracts of P. karka exhibited a significant (p<0.001) down-regulation of mRNA levels of TGF-beta (compare CCl4-treated and leaf or rhizome extract group with the CCl4-treated group.
There was more than 1-fold increase in mRNA expression of collagen in CCl4-treated group as compared to the control group (Figure 2C). Both extracts did not modify the mRNA expression levels of the collagen as compared to the control group. However, leaf and rhizome extracts managed to reduce the CCl4-induced mRNA expression levels of collagen significantly (p<0.001)(compare CCl4-treated and leaf or rhizome extract group with the CCl4-treated group).
Histopathological and gross view findings from the liver tissue
Figure 3 shows microscopic view of liver tissue from various experimental groups, Figure 3A, B and C show normal arrangement of hepatocytes, while liver of animal treated with CCl4 showed disarrangement of hepatocytes, fatty degeneration and massive number of inflammatory cells in the liver tissue (Figure 3D). In comparison liver of animal treated with leaf extract (Figure 3) and rhizome extract (Figure 3F) in CCl4 intoxicated rats show improved features and lesser degree of hepatocyte disarrangement.
Figure 3: Microscopic evaluation of H & E stained liver tissue. Representative of n=5 from each group. A: Corn oil treatment, B: Corn oil + leaf extract (500 mg/kg), C: Corn oil + rhizome extract (500 mg/kg) D; CCL4 treated, E: CCL4 + leaf extract 500 mg/kg, F: Corn oil+ rhizome extract 500 mg/kg
Figure 4 shows the gross overview of livers from different experimental groups respectively and complements the results found in Figure 3. Liver from the control animal (Figure 4A) had smooth shiny surface while treatment with CCl4 (as shown in Figure 4D) led the color changed to whitish with decreased amount of blood, nodular appearance and felt very hard to touch as compared to the liver of control groups (Figure 4A). Figure 4E and F show gross appearance of liver of animal treated with leaf and rhizome extract in CCl4 intoxicated rats which showed lesser degree of nodulation as compared to CCl4 intoxicated rat livers.
Figure 4: Gross appearance of livers. Excised livers from the treated animals after dissection and photographed. Representative of n=5 from each group. A: Corn oil treatment, B: Corn oil + leaf extract (500 mg/kg), C: Corn oil + rhizome extract (500 mg/kg) D: CCL4 treated, E: CCL4 + leaf extract 500 mg/kg, F: Corn oil+ rhizome extract 500 mg/kg
Discussion
The results suggest that P. karka leaf and rhizome extracts prevent the experimentally induced liver damage and fibrosis by repressing the mRNA expression levels of fibrosis related genes, improving liver architecture and serum markers of liver damage.
This study used chronic model of liver injury to induced liver fibrosis and cirrhosis (Liedtke et al., 2013). In line with previously reported study (Gou et al., 2013; Kew C, 2000), administration of CCl4 led to hepatocyte destruction and increased levels of ALT, AST, and GGT in the serum of rats, which were reversed by treatment with the leaf and rhizome extract. Also, in line with earlier observation carried out by (Mohammed et al., 2016), CCl4 treatment caused profound induction of the mRNA levels of TGF-beta, alpha-SMA and collagen genes which were repressed on treatment with both extracts. It suggests that the chemical constituent (s) of both extracts may likely have some regulatory role on the expression of mRNA of the studied genes. However, it is still to be determined whether antifibrotic effects are due to the direct inhibitory action on hepatic stellate cells activation or by antioxidant potentials possessed by the extracts (Nimse et al., 2015; Van acker et al., 1996); since oxidative stress play crucial role in hepatic injury and hepatic stellate cell proliferation. (Dunning et al., 2013)
Another possible mechanism of action can be related to the presence of steroidal constituents present in P. karka. Sultan et al., has reported the presence of steroidal moieties in P. karka (Sultan et al., 2017), which suggests its diversified effects on prostaglandin synthesis pathway. Prostaglandins and leukotrienes are implicated in pathophysiology of liver injury and are synthesised via acrachidonic acid pathway (Fireman et al., 2004; Nanji et al., 1993; Op den Winkel et al., 2013). A sophisticated balance between leukotrienes and prostaglandins is crucial for physiological performance (Moore and Peters-Golden, 2006). Since, the synthesis of prostaglandins and leukotrienes can be inhibited by steroidal and non-steroidal anti-inflammatory agents and, P. karka is known for its steroidal content and analgesic action. Therefore, the steroidal constituents may involve in antifibrotic actions observed in this study. However, further studies required to elucidate these mechanisms. In general, our data is in agreement with the existing studies which have also shown hepatoprotective and antifibrotic actions in the liver (Mohammed et al., 2016).
Conclusion
CCl4-induced biochemical (ALT, AST, GGT) and molecular (TGF-beta, alpha-SMA and collagen) markers of liver damage and fibrosis have been profoundly modified toward normalcy in the hepatofibrotic rats. In addition, the histopathological markers analysis have also supported the above biochemical and molecular studies.
Ethical Issue
The experiments were approved by the Animal Research Ethics Committee of The University of Lahore, Pakistan. Care and handling of animals followed the internationally accepted procedures according to the Institute for Laboratory Animal Research's Guide for the Care and Use of Laboratory Animals.
Acknowledgement
Acknowledgement is due to the faculty of pharmacy administration, University of Lahore for supporting this research, Mr. Shoaib, Mr. Javed Tahir and Dr. M.H. Qazi from Center for Research in Molecular Medicine, The University of Lahore. Authors also thankful to Dr. Azmat Rasheed from Department of Botany, Government College University, Lahore, Pakistan for plant identification.
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