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Protective effect of geraniin against carbon tetrachloride induced acute hepatotoxicity in Swiss albino mice
Biochemical and Biophysical Research Communications xxx (2017) 1e6
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Protective effect of geraniin against carbon tetrachloride induced acute hepatotoxicity in Swiss albino mice Hoda Aayadi a, Smriti P.K. Mittal a, Anjali Deshpande b, Makarand Gore b, Saroj S. Ghaskadbi a, * a b
Department of Zoology, Savitribai Phule Pune University, Pune 411007, India AJ Organica Pvt. Ltd., Pune 411057, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 March 2017 Accepted 4 April 2017 Available online xxx
Geraniin is a hydrolysable tannin, widely present in many plant species, specifically used in traditional medicines. It has been shown to exhibit strong antioxidant activity in vitro. This study was performed to investigate hepatoprotective activity of geraniin against carbon tetrachloride (CCl4) induced damage in Swiss albino mice. Mice were treated with 30 and 60 mg/kg geraniin for 10 days followed by CCl4 administration for 24 h. Increase in Serum biochemical marker enzymes and histological deteriorative changes of liver tissue after CCl4 administration were attenuated by geraniin. Geraniin significantly reduced CCl4 induced lipid peroxidation, increase in amount of glutathione, glutathione reductase and Heme oxygenase-1 (HO-1). On the other hand it inhibited significant reduction in catalase activity and expression caused by CCl4 administration. Pre-treatment with geraniin reduced phosphorylation of translation initiation factor eIF2a, at serine 51, caused by CCl4 exposure and reduced elevated expression of its upstream kinase, Heme-regulated Inhibitor (HRI). These results clearly demonstrate hepatoprotective activity of geraniin against CCl4-induced acute hepatotoxicity via its free radical scavenging and antioxidant activities. © 2017 Published by Elsevier Inc.
Keywords: Geraniin CCl4 Heme oxygenase-1 eIF2a HRI
1. Introduction Reactive oxygen species (ROS) are molecules, ions or free radicals derived from molecular oxygen and are able to highly react with biological molecules such as lipids, proteins and DNA, resulting in their damage. Cellular ROS are produced either endogenously as byproducts of aerobic respiration or due to exogenous sources such as xenobiotic compounds. Excessive production of ROS, overwhelming cellular antioxidant defense system, generates oxidative stress (OS). OS is known to play a critical role in
Abbreviations: CCl4, carbon tetrachloride; HO-1, Heme oxygenase-1; GR, glutathione reductase; CAT, catalase; eIF2a, eukaryotic initiation factor 2 alpha subunit; HRI, heme-regulated inhibitor; ROS, reactive oxygen species; OS, oxidative stress; SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum glutamic-pyruvic transaminase; LDH, lactate dehydrogenase; SOD, superoxide dismutase; GPx, glutathione peroxidise; GSH, reduced glutathione; MDA, Malondialdehydes; PMSF, phenylmethylsulfonyl fluoride; GST, glutathione-s-transferase; PKR, protein kinase RNA-activated; PERK, protein kinase R (PKR)-like endoplasmic reticulum kinase; GCN2, general control non-derepressible-2. * Corresponding author. E-mail address: [email protected] (S.S. Ghaskadbi).
initiation and progression of several liver diseases such as hepatitis, liver cancer, cirrhosis and fibrosis. Carbon tetrachloride (CCl4), on its metabolism, produces trichloromethyl (CCl3∙) and trichloromethylperoxyl (CCl3O2∙) radicals and exerts OS that causes acute liver damage. These radicals initiate lipid peroxidation and contribute to liver injury [1,2]. This makes CCl4 a commonly used hepatotoxin in experiments performed using rodents to determine hepatoprotective effect of any compound or a drug. Cells activate their defense mechanism to prevent damaging effects of ROS, by means of endogenous antioxidant molecules and enzymes. In addition to first line antioxidant enzymes SOD, Catalase and GPx, HO-1, which is highly induced in OS, has drawn lot of attention recently by virtue of its hepatoprotective roles [3]. Another instant cellular response to OS is inhibition of protein synthesis, mainly through increased phosphorylation of the a subunit of the translation initiation factor eIF2a at S51. This allows cells to either recover from stress or be eradicated if the damage is unrepairable. To support cellular antioxidant action, many times several dietary exogenous antioxidants such as vitamins, flavonoids and polyphenols are used [4,5]. Many studies have suggested that plant
http://dx.doi.org/10.1016/j.bbrc.2017.04.013 0006-291X/© 2017 Published by Elsevier Inc.
Please cite this article in press as: H. Aayadi, et al., Protective effect of geraniin against carbon tetrachloride induced acute hepatotoxicity in Swiss albino mice, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.013
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H. Aayadi et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
polyphenols exhibit strong antioxidant activity [6e8]. Geraniin is a hydrolysable tannin with a unique dehydrohexahydroxydiphenoyl moiety and is widely distributed among dicotyledonous plants. There have been many biological studies on geraniin, either isolated from plant extract or as a pure compound. It has been shown that it exhibits several biological activities such as anticancer, antiviral, antihypertensive, antihyperglycaemic and antioxidant activity [7e16]. In previous studies in our lab, geraniin has been demonstrated to scavenge free radicals [9] and confer protection against oxidative damage to biomolecules. It has also been shown to protect HepG2 cells, against H2O2 induced cytotoxicity by upregulating Nrf2-mediated antioxidant enzyme expression via PI3K/Akt and ERK1/2 pathway [17]. Thus the studies done in vitro demonstrated that geraniin exerted its antioxidant activity in cells by upregulating antioxidant enzymes through triggering specific signaling pathways. In earlier reports, hepatoprotective activity of geraniin has been demonstrated by estimating serum hepatotoxic marker enzymes in rats [18,19]. In present study, ability of geraniin to protect mouse liver from CCl4 induced damage is investigated by its effect on liver histology, activity and expression of antioxidant defence machinery and phosphorylation of important initiation factor of protein synthesis, eIF2a.
oxaloacetic transaminase (SGOT), serum glutamic-pyruvic transaminase (SGPT), Billirubin, Lactate dehydrogenase (LDH) and Total Protein [20,21]. 100 mg liver tissue was homogenized in 1 ml lysis buffer (50 mM Tris-Cl (pH-7.8), 5 mM EDTA, 0.1% Triton X-100), containing protease inhibitor cocktail and 1 mM PMSF. The homogenate was centrifuged at 10000 rpm for 10 min at 4 C and supernatant was processed for estimation of protein [22], catalase (CAT) [23], glutathione reductase (GR) [24], reduced glutathione (GSH) [25] and Malondialdehydes (MDA) [26] using standard protocols. 2.5. Histopathological studies A part of liver tissue was fixed in 10% formalin for 24 h, embedded in paraffin, sliced in to 5 mm sections using microtome, stained with hematoxylin-eosin dye and observed under light microscope for histopathological examination [27]. 2.6. RNA isolation and qRT-PCR
2. Materials and methods
RNA was extracted from liver tissue using Trizol reagent [28]. Isolated RNA was quantitated, cDNA was synthesized and RealTime PCR amplification (Applied Biosystems, USA) was carried out using respective forward and reverse primers (Supplementary Table 1).
2.1. Chemicals
2.7. Western blot analysis
Chemicals were purchased from Sigma, USA and Sisco Research Laboratories, India. Analytical grade CCl4 was procured from Fisher Scientific, USA. Kits for estimation of biochemical parameters were of Pathozyme Diagnostics, India and cDNA synthesis kit was of Thermo scientific, USA. Antibodies were purchased from Cell Signaling Technology, USA and Abcam, UK. ECL detection kit was of Abcam, UK.
Forty microgram (40 mg) protein from each sample was resolved in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinyl difluoride membrane (PVDF) over night. Membranes were blocked with bovine serum albumin, incubated with respective primary antibodies, followed by incubation with peroxidase-conjugated secondary antibody. Signals were detected using Optiblot ECL detection kit (Abcam, UK) and documented using Gel Doc system (Bio-Rad, USA) [29].
2.2. Animals Adult swiss albino mice (20e30 g) of either sex were procured from National Toxicology Center, Pune, India. Mice were maintained in groups of six animals per cage, as per standard laboratory conditions. All experimental protocols were reviewed and approved by Institute Animal Ethics Committee (Registration no. 538/02/c/CPCSEA).
2.8. Statistical analysis All experimental values were expressed as the mean ± SE of at least 4 independent experiments. Statistical calculations of the data were performed by one-way analysis of variance (ANOVA), followed by post-hoc test using SPSS 19.0 software and a probability of P < 0.05 was considered as statistically significant.
2.3. Experimental design 3. Results Animals were divided in to six groups (n ¼ 6, 3 animals/group) and fed with food and water ad libitum. Group I: Untreated control animals. Group II: mice were administered with CCl4 dissolved in olive oil (1 ml/kg body weight, 10% v/v), intra-peritoneally, 24 h prior to sacrifice. Group III: mice received silymarin (100 mg/kg body weight), a known hepatoprotectant, orally, for 10 days followed by CCl4 administration. Group IV and V: mice received 30 and 60 mg/kg body weight geraniin respectively, orally, for 10 days followed by CCl4 administration. Group VI: mice received 60 mg/kg body weight geraniin alone, orally, for 10 days. All animals were sacrificed by cervical dislocation on 11th day, blood samples were immediately collected and liver tissue was dissected out for histopathological and biochemical studies. 2.4. Evaluation of serum and hepatic biochemical markers Serum was separated from blood samples and was used for estimation of biochemical parameters such as serum glutamic
3.1. Geraniin exhibits hepatoprotective effect against CCl4-induced liver damage A significant increase in SGOT, SGPT, Billirubin and Lactate dehydrogenase was observed after CCl4 administration in mice indicating severe hepatic damage whereas these were reduced significantly in mice treated with geraniin. This reduction was almost similar to that found in case of mice treated with silymarin, a known hepatoprotectant (Table 1). To further confirm these results, histopathological study was performed on liver sections of mice from different groups. In the liver section of CCl4-intoxicated mice degenerative changes in hepatocytes, centrilobular necrosis, focal cellular swelling, granular and vacuolar changes in cytoplasm of hepatocytes around central vein and presence of sinusoidal congestions and haemorrhages were observed. These pathological changes were reduced significantly in the liver of mice treated with 60 mg/kg BW geraniin and silymarin. In mice treated with geraniin
Please cite this article in press as: H. Aayadi, et al., Protective effect of geraniin against carbon tetrachloride induced acute hepatotoxicity in Swiss albino mice, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.013
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Table 1 Geraniin exhibits hepatoprotective effect against CCl4-induced liver damage.
SGPT (IU/l) SGOT (IU/l) Billirubin (mg/dl) LDH (IU/l) Total Protein (mg/dl)
Control
CCl4
Silymarin þ CCl4
Geraiin30 þ CCl4
Geraniin60 þ CCl4
Geraniin60
52.75 ± 5.18 86.25 ± 1.65 0.19 ± 0.02 656.75 ± 10.61 7.47 ± 0.43
111.62 ± 3.96* 133 ± 4.26* 0.40 ± 0.03* 1189.50 ± 23.07* 9.35 ± 0.13*
81.50 ± 3.12** 104.50 ± 6.14** 0.26 ± 0.02** 645.75 ± 32.09** 8.87 ± 0.32
71.50 ± 3.37** 91.25 ± 1.49** 0.24 ± 0.01** 624 ± 33.52** 8.42 ± 0.15**
54.25 ± 1.93** 88.75 ± 1.49** 0.24 ± 0.00** 618.75 ± 32.05** 8.17 ± 0.26**
66.52 ± 5.83 86 ± 2.34 0.17 ± 0.01 622.75 ± 32.68 8.15 ± 0.08
Note: Geraniin (30 or 60 mg/kg body weight) or Silymarin (100 mg/kg body weight) was orally administered to mice for 10 days followed by CCl4 intoxication on 11th day. Data shown represents the mean values of 6 mice per group ±SE.*p < 0.05 vs. control. **p < 0.05 vs. CCl4.
alone no histopathological changes were observed compared to control mice (Fig. 1AeF). 3.2. Geraniin normalizes the altered antioxidant enzyme activity and expression caused by CCl4
inhibited. (Fig. 2E). Similarly, a significant increase was observed in hepatic malondiadehyde, lipid peroxidation product in CCl4 treated mice and this was effectively reduced in mice treated with geraniin (Fig. 2F). 3.4. Geraniin reduces CCl4-induced HO-1 induction
Activity of catalase and its gene expression was significantly reduced in liver from CCl4 intoxicated mice whereas this reduction was not seen in the liver tissue from mice treated with geraniin and silymarin (Fig. 2A and B). In case of glutathione reductase in liver from CCl4 intoxicated mice both enzyme activity and gene expression of GR was significantly increased compared to untreated, control mice. Treatment with geraniin inhibited this increase significantly. The effect seen in mice treated with 60 mg/kg BW geraniin was even better than silymarin, a known hepatoprotectant (Fig. 2C and D). 3.3. Geraniin restores an elevated level of hepatic glutathione and inhibits CCl4-induced lipid peroxidation Hepatic glutathione was estimated as one of the important antioxidant molecules. Glutathione, an important redox buffer of the cell was found to be significantly increased in CCl4 intoxicated mice whereas in mice treated with geraniin, this was significantly
Expression of HO-1, was found to be drastically increased in the liver from CCl4 intoxicated mice while this increase was not found in a concentration dependent manner in mice treated with geraniin (Fig. 3). 3.5. Geraniin inhibits phosphorylation of eIF2-alpha by reducing HRI expression in CCl4 intoxicated mice In stressed condition, eukaryotic initiation factor 2 (eIF2), a necessary factor for initiation of translation in eukaryotes, is phosphorylated at its alpha subunit, as a target of several upstream kinases (GCN2, PERK, PKR and HRI) and this results in inhibition of initiation of translation process and thereby protein synthesis. Marked increase in phosphorylated eIF2a in liver from CCl4 treated mice was observed (Fig. 4A), whereas in mice treated with geraniin this phosphorylation was significantly inhibited. Since there are four upstream kinases involved in eIf2a
Fig. 1. Liver damage is significantly reduced in mice pre-treated with geraniin. Liver sections from mice belonging to different groups. (A) Control, (B) CCl4 intoxicated, (C) Silymarin (100 mg/kg body weight) þ CCl4, (D) Geraniin (30 mg/kg body weight) þ CCl4, (E) Geraniin (60 mg/kg body weight) þ CCl4, (F) Geraniin (60 mg/kg body weight). CV: Central vein. HC: Hepatic cell. S: Sinusoid. N: Necrosis.
Please cite this article in press as: H. Aayadi, et al., Protective effect of geraniin against carbon tetrachloride induced acute hepatotoxicity in Swiss albino mice, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.013
Fig. 2. Geraniin normalizes both antioxidant activity and expression, restores elevated level of hepatic glutathione and inhibits lipid peroxidation in mice exposed to CCl4. (A) Catalase activity and (B) its expression. (C) GR activity and (D) its expression. (E) GSH was determined in terms of nmol GSH/mg protein and (F) Lipid peroxidation was estimated in terms of nmol MDA/mg protein, in liver tissue from mice belonging to different groups. Data shown represents the mean values of 6 mice per group ± SE.*p < 0.05 vs. control. **p < 0.05 vs. CCl4. Sily: Silymarin, Ger: Geraniin.
phosphorylation, expression of all these four kinases was checked by qRT-PCR. HRI alone was found to be significantly upregulated in CCl4 intoxicated mice and this was prevented in mice treated with geraniin (Fig. 4C). 4. Discussion
Fig. 3. HO-1 protein expression in liver tissue of mice from different groups. (A) HO-1 protein expression by western blot and (B) its quantitation by densitometric analysis. Data shown represents the mean values of 6 mice per group ± SE.*p < 0.05 vs. control. **p < 0.05 vs. Sily: Silymarin, Ger: Geraniin.
Geraniin has been shown to exhibit different biological activities, among which its antioxidant activity is due to significant HO-1 induction through upregulation of Nrf-2 pathway [17,30,31]. In this study, geraniin was used to check its hepatoprotective effect against CCl4-induced liver injury in mice. CCl4, is a commonly used hepatotoxin, on metabolism generates free radicals, leading to OS which induces hepatic injury, measured in terms of cellular leakage of hepatic enzymes [32e35]. In the present study, SGOT, SGPT and LDH, hepatic marker enzymes were found to be elevated in CCl4 intoxicated mice, while in those treated with geraniin this was significantly reduced, indicating maintenance of structural integrity of hepatocytes and stabilization of their membrane activity (Table 1). This was additionally supported by histological findings. Centrilobular necrosis and sinusoidal congestion of liver
Please cite this article in press as: H. Aayadi, et al., Protective effect of geraniin against carbon tetrachloride induced acute hepatotoxicity in Swiss albino mice, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.013
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Fig. 4. Geraniin inhibits CCl4 induced phosphorylation of eIF2a and expression of responsible kinase, HRI. (A) Western blot analysis of p-eIF2a and (B) its quantitation by densitometry, (C) qRT-PCR of HRI gene. Data shown represents the mean values of 6 mice per group ± SE.*p < 0.05 vs. control. **p < 0.05 vs. CCl4. Sily: Silymarin, Ger: Geraniin.
tissue in mice intoxicated with CCl4 was not seen in those pretreated with geraniin (Fig. 1). Significant elevation in MDA, a product of lipid peroxidation, observed in CCl4 intoxicated mice [36,37] was inhibited in mice pretreated with geraniin. GSH is an important antioxidant in liver, and its homeostasis is associated with various toxin-induced liver injuries [38,39]. It can be directly used as antioxidant molecule or in enzymatic reactions catalyzed by GST, GPx and is continuously regenerated by GR. In the present study GSH was found to be elevated in mice treated with CCl4 similar to earlier report [40]. This could be due to both reduction in utilization of this compound by enzyme such as GST [41] or elevation in GR activity [42]. We indeed observed increase in GR activity and its expression in CCl4 intoxicated mice, while that of GPx remained unaltered. For catalase, both activity and expression was found to be reduced significantly in CCl4 intoxicated mice, probably due to peroxy radical generated by CCl4 metabolism [43]. Pretreatment with geraniin significantly inhibited increase in GSH and GR and restored catalase in CCl4 intoxicated mice indicating amelioration of OS by geraniin. HO-1, the rate-limiting enzyme in heme catabolism, is known to be induced in OS and confer protection against oxidative tissue injuries [44]. This induction is reported to be mediated through a rapid increase in microsomal free heme concentration, presumably derived from hepatic cytochrome P450, in rats administered intraperitoneally with CCl4. This is further confirmed by inhibiting HO-1 in CCl 4 intoxicated rats which led to significant increase in liver injury [45]. In the present study, as expected, HO-1 was significantly increased in CCl4 treated mice which was not seen in those pretreated with geraniin, implying significant reduction in OS. Under OS, products of peroxidation, MDA and 4- hydroxynonenal, are known to inhibit protein synthesis and this inhibition is through phosphorylation of a subunit of the translation initiation factor eIF2a at S5 [46]. Phosphorylation of eIF2a inhibits exchange of GDP for GTP catalyzed by eIF2B and prevents formation of ternary complex leading to inhibition of protein synthesis [46]. Phosphorylation of eIF2a occurs by a family of kinases namely HRI, PKR, PERK and GCN2 [47]. In CCl4 intoxicated mice, significant amount to eIF2a was found to be phosphorylated through HRI (Fig. 4A) which was reduced significantly in mice treated with geraniin, indicating its protective effect.
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Protective effect of geraniin against carbon tetrachloride induced acute hepatotoxicity in Swiss albino mice Hoda Aayadi a, Smriti P.K. Mittal a, Anjali Deshpande b, Makarand Gore b, Saroj S. Ghaskadbi a, * a b
Department of Zoology, Savitribai Phule Pune University, Pune 411007, India AJ Organica Pvt. Ltd., Pune 411057, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 March 2017 Accepted 4 April 2017 Available online xxx
Geraniin is a hydrolysable tannin, widely present in many plant species, specifically used in traditional medicines. It has been shown to exhibit strong antioxidant activity in vitro. This study was performed to investigate hepatoprotective activity of geraniin against carbon tetrachloride (CCl4) induced damage in Swiss albino mice. Mice were treated with 30 and 60 mg/kg geraniin for 10 days followed by CCl4 administration for 24 h. Increase in Serum biochemical marker enzymes and histological deteriorative changes of liver tissue after CCl4 administration were attenuated by geraniin. Geraniin significantly reduced CCl4 induced lipid peroxidation, increase in amount of glutathione, glutathione reductase and Heme oxygenase-1 (HO-1). On the other hand it inhibited significant reduction in catalase activity and expression caused by CCl4 administration. Pre-treatment with geraniin reduced phosphorylation of translation initiation factor eIF2a, at serine 51, caused by CCl4 exposure and reduced elevated expression of its upstream kinase, Heme-regulated Inhibitor (HRI). These results clearly demonstrate hepatoprotective activity of geraniin against CCl4-induced acute hepatotoxicity via its free radical scavenging and antioxidant activities. © 2017 Published by Elsevier Inc.
Keywords: Geraniin CCl4 Heme oxygenase-1 eIF2a HRI
1. Introduction Reactive oxygen species (ROS) are molecules, ions or free radicals derived from molecular oxygen and are able to highly react with biological molecules such as lipids, proteins and DNA, resulting in their damage. Cellular ROS are produced either endogenously as byproducts of aerobic respiration or due to exogenous sources such as xenobiotic compounds. Excessive production of ROS, overwhelming cellular antioxidant defense system, generates oxidative stress (OS). OS is known to play a critical role in
Abbreviations: CCl4, carbon tetrachloride; HO-1, Heme oxygenase-1; GR, glutathione reductase; CAT, catalase; eIF2a, eukaryotic initiation factor 2 alpha subunit; HRI, heme-regulated inhibitor; ROS, reactive oxygen species; OS, oxidative stress; SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum glutamic-pyruvic transaminase; LDH, lactate dehydrogenase; SOD, superoxide dismutase; GPx, glutathione peroxidise; GSH, reduced glutathione; MDA, Malondialdehydes; PMSF, phenylmethylsulfonyl fluoride; GST, glutathione-s-transferase; PKR, protein kinase RNA-activated; PERK, protein kinase R (PKR)-like endoplasmic reticulum kinase; GCN2, general control non-derepressible-2. * Corresponding author. E-mail address: [email protected] (S.S. Ghaskadbi).
initiation and progression of several liver diseases such as hepatitis, liver cancer, cirrhosis and fibrosis. Carbon tetrachloride (CCl4), on its metabolism, produces trichloromethyl (CCl3∙) and trichloromethylperoxyl (CCl3O2∙) radicals and exerts OS that causes acute liver damage. These radicals initiate lipid peroxidation and contribute to liver injury [1,2]. This makes CCl4 a commonly used hepatotoxin in experiments performed using rodents to determine hepatoprotective effect of any compound or a drug. Cells activate their defense mechanism to prevent damaging effects of ROS, by means of endogenous antioxidant molecules and enzymes. In addition to first line antioxidant enzymes SOD, Catalase and GPx, HO-1, which is highly induced in OS, has drawn lot of attention recently by virtue of its hepatoprotective roles [3]. Another instant cellular response to OS is inhibition of protein synthesis, mainly through increased phosphorylation of the a subunit of the translation initiation factor eIF2a at S51. This allows cells to either recover from stress or be eradicated if the damage is unrepairable. To support cellular antioxidant action, many times several dietary exogenous antioxidants such as vitamins, flavonoids and polyphenols are used [4,5]. Many studies have suggested that plant
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Please cite this article in press as: H. Aayadi, et al., Protective effect of geraniin against carbon tetrachloride induced acute hepatotoxicity in Swiss albino mice, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.013
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polyphenols exhibit strong antioxidant activity [6e8]. Geraniin is a hydrolysable tannin with a unique dehydrohexahydroxydiphenoyl moiety and is widely distributed among dicotyledonous plants. There have been many biological studies on geraniin, either isolated from plant extract or as a pure compound. It has been shown that it exhibits several biological activities such as anticancer, antiviral, antihypertensive, antihyperglycaemic and antioxidant activity [7e16]. In previous studies in our lab, geraniin has been demonstrated to scavenge free radicals [9] and confer protection against oxidative damage to biomolecules. It has also been shown to protect HepG2 cells, against H2O2 induced cytotoxicity by upregulating Nrf2-mediated antioxidant enzyme expression via PI3K/Akt and ERK1/2 pathway [17]. Thus the studies done in vitro demonstrated that geraniin exerted its antioxidant activity in cells by upregulating antioxidant enzymes through triggering specific signaling pathways. In earlier reports, hepatoprotective activity of geraniin has been demonstrated by estimating serum hepatotoxic marker enzymes in rats [18,19]. In present study, ability of geraniin to protect mouse liver from CCl4 induced damage is investigated by its effect on liver histology, activity and expression of antioxidant defence machinery and phosphorylation of important initiation factor of protein synthesis, eIF2a.
oxaloacetic transaminase (SGOT), serum glutamic-pyruvic transaminase (SGPT), Billirubin, Lactate dehydrogenase (LDH) and Total Protein [20,21]. 100 mg liver tissue was homogenized in 1 ml lysis buffer (50 mM Tris-Cl (pH-7.8), 5 mM EDTA, 0.1% Triton X-100), containing protease inhibitor cocktail and 1 mM PMSF. The homogenate was centrifuged at 10000 rpm for 10 min at 4 C and supernatant was processed for estimation of protein [22], catalase (CAT) [23], glutathione reductase (GR) [24], reduced glutathione (GSH) [25] and Malondialdehydes (MDA) [26] using standard protocols. 2.5. Histopathological studies A part of liver tissue was fixed in 10% formalin for 24 h, embedded in paraffin, sliced in to 5 mm sections using microtome, stained with hematoxylin-eosin dye and observed under light microscope for histopathological examination [27]. 2.6. RNA isolation and qRT-PCR
2. Materials and methods
RNA was extracted from liver tissue using Trizol reagent [28]. Isolated RNA was quantitated, cDNA was synthesized and RealTime PCR amplification (Applied Biosystems, USA) was carried out using respective forward and reverse primers (Supplementary Table 1).
2.1. Chemicals
2.7. Western blot analysis
Chemicals were purchased from Sigma, USA and Sisco Research Laboratories, India. Analytical grade CCl4 was procured from Fisher Scientific, USA. Kits for estimation of biochemical parameters were of Pathozyme Diagnostics, India and cDNA synthesis kit was of Thermo scientific, USA. Antibodies were purchased from Cell Signaling Technology, USA and Abcam, UK. ECL detection kit was of Abcam, UK.
Forty microgram (40 mg) protein from each sample was resolved in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinyl difluoride membrane (PVDF) over night. Membranes were blocked with bovine serum albumin, incubated with respective primary antibodies, followed by incubation with peroxidase-conjugated secondary antibody. Signals were detected using Optiblot ECL detection kit (Abcam, UK) and documented using Gel Doc system (Bio-Rad, USA) [29].
2.2. Animals Adult swiss albino mice (20e30 g) of either sex were procured from National Toxicology Center, Pune, India. Mice were maintained in groups of six animals per cage, as per standard laboratory conditions. All experimental protocols were reviewed and approved by Institute Animal Ethics Committee (Registration no. 538/02/c/CPCSEA).
2.8. Statistical analysis All experimental values were expressed as the mean ± SE of at least 4 independent experiments. Statistical calculations of the data were performed by one-way analysis of variance (ANOVA), followed by post-hoc test using SPSS 19.0 software and a probability of P < 0.05 was considered as statistically significant.
2.3. Experimental design 3. Results Animals were divided in to six groups (n ¼ 6, 3 animals/group) and fed with food and water ad libitum. Group I: Untreated control animals. Group II: mice were administered with CCl4 dissolved in olive oil (1 ml/kg body weight, 10% v/v), intra-peritoneally, 24 h prior to sacrifice. Group III: mice received silymarin (100 mg/kg body weight), a known hepatoprotectant, orally, for 10 days followed by CCl4 administration. Group IV and V: mice received 30 and 60 mg/kg body weight geraniin respectively, orally, for 10 days followed by CCl4 administration. Group VI: mice received 60 mg/kg body weight geraniin alone, orally, for 10 days. All animals were sacrificed by cervical dislocation on 11th day, blood samples were immediately collected and liver tissue was dissected out for histopathological and biochemical studies. 2.4. Evaluation of serum and hepatic biochemical markers Serum was separated from blood samples and was used for estimation of biochemical parameters such as serum glutamic
3.1. Geraniin exhibits hepatoprotective effect against CCl4-induced liver damage A significant increase in SGOT, SGPT, Billirubin and Lactate dehydrogenase was observed after CCl4 administration in mice indicating severe hepatic damage whereas these were reduced significantly in mice treated with geraniin. This reduction was almost similar to that found in case of mice treated with silymarin, a known hepatoprotectant (Table 1). To further confirm these results, histopathological study was performed on liver sections of mice from different groups. In the liver section of CCl4-intoxicated mice degenerative changes in hepatocytes, centrilobular necrosis, focal cellular swelling, granular and vacuolar changes in cytoplasm of hepatocytes around central vein and presence of sinusoidal congestions and haemorrhages were observed. These pathological changes were reduced significantly in the liver of mice treated with 60 mg/kg BW geraniin and silymarin. In mice treated with geraniin
Please cite this article in press as: H. Aayadi, et al., Protective effect of geraniin against carbon tetrachloride induced acute hepatotoxicity in Swiss albino mice, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.013
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Table 1 Geraniin exhibits hepatoprotective effect against CCl4-induced liver damage.
SGPT (IU/l) SGOT (IU/l) Billirubin (mg/dl) LDH (IU/l) Total Protein (mg/dl)
Control
CCl4
Silymarin þ CCl4
Geraiin30 þ CCl4
Geraniin60 þ CCl4
Geraniin60
52.75 ± 5.18 86.25 ± 1.65 0.19 ± 0.02 656.75 ± 10.61 7.47 ± 0.43
111.62 ± 3.96* 133 ± 4.26* 0.40 ± 0.03* 1189.50 ± 23.07* 9.35 ± 0.13*
81.50 ± 3.12** 104.50 ± 6.14** 0.26 ± 0.02** 645.75 ± 32.09** 8.87 ± 0.32
71.50 ± 3.37** 91.25 ± 1.49** 0.24 ± 0.01** 624 ± 33.52** 8.42 ± 0.15**
54.25 ± 1.93** 88.75 ± 1.49** 0.24 ± 0.00** 618.75 ± 32.05** 8.17 ± 0.26**
66.52 ± 5.83 86 ± 2.34 0.17 ± 0.01 622.75 ± 32.68 8.15 ± 0.08
Note: Geraniin (30 or 60 mg/kg body weight) or Silymarin (100 mg/kg body weight) was orally administered to mice for 10 days followed by CCl4 intoxication on 11th day. Data shown represents the mean values of 6 mice per group ±SE.*p < 0.05 vs. control. **p < 0.05 vs. CCl4.
alone no histopathological changes were observed compared to control mice (Fig. 1AeF). 3.2. Geraniin normalizes the altered antioxidant enzyme activity and expression caused by CCl4
inhibited. (Fig. 2E). Similarly, a significant increase was observed in hepatic malondiadehyde, lipid peroxidation product in CCl4 treated mice and this was effectively reduced in mice treated with geraniin (Fig. 2F). 3.4. Geraniin reduces CCl4-induced HO-1 induction
Activity of catalase and its gene expression was significantly reduced in liver from CCl4 intoxicated mice whereas this reduction was not seen in the liver tissue from mice treated with geraniin and silymarin (Fig. 2A and B). In case of glutathione reductase in liver from CCl4 intoxicated mice both enzyme activity and gene expression of GR was significantly increased compared to untreated, control mice. Treatment with geraniin inhibited this increase significantly. The effect seen in mice treated with 60 mg/kg BW geraniin was even better than silymarin, a known hepatoprotectant (Fig. 2C and D). 3.3. Geraniin restores an elevated level of hepatic glutathione and inhibits CCl4-induced lipid peroxidation Hepatic glutathione was estimated as one of the important antioxidant molecules. Glutathione, an important redox buffer of the cell was found to be significantly increased in CCl4 intoxicated mice whereas in mice treated with geraniin, this was significantly
Expression of HO-1, was found to be drastically increased in the liver from CCl4 intoxicated mice while this increase was not found in a concentration dependent manner in mice treated with geraniin (Fig. 3). 3.5. Geraniin inhibits phosphorylation of eIF2-alpha by reducing HRI expression in CCl4 intoxicated mice In stressed condition, eukaryotic initiation factor 2 (eIF2), a necessary factor for initiation of translation in eukaryotes, is phosphorylated at its alpha subunit, as a target of several upstream kinases (GCN2, PERK, PKR and HRI) and this results in inhibition of initiation of translation process and thereby protein synthesis. Marked increase in phosphorylated eIF2a in liver from CCl4 treated mice was observed (Fig. 4A), whereas in mice treated with geraniin this phosphorylation was significantly inhibited. Since there are four upstream kinases involved in eIf2a
Fig. 1. Liver damage is significantly reduced in mice pre-treated with geraniin. Liver sections from mice belonging to different groups. (A) Control, (B) CCl4 intoxicated, (C) Silymarin (100 mg/kg body weight) þ CCl4, (D) Geraniin (30 mg/kg body weight) þ CCl4, (E) Geraniin (60 mg/kg body weight) þ CCl4, (F) Geraniin (60 mg/kg body weight). CV: Central vein. HC: Hepatic cell. S: Sinusoid. N: Necrosis.
Please cite this article in press as: H. Aayadi, et al., Protective effect of geraniin against carbon tetrachloride induced acute hepatotoxicity in Swiss albino mice, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.013
Fig. 2. Geraniin normalizes both antioxidant activity and expression, restores elevated level of hepatic glutathione and inhibits lipid peroxidation in mice exposed to CCl4. (A) Catalase activity and (B) its expression. (C) GR activity and (D) its expression. (E) GSH was determined in terms of nmol GSH/mg protein and (F) Lipid peroxidation was estimated in terms of nmol MDA/mg protein, in liver tissue from mice belonging to different groups. Data shown represents the mean values of 6 mice per group ± SE.*p < 0.05 vs. control. **p < 0.05 vs. CCl4. Sily: Silymarin, Ger: Geraniin.
phosphorylation, expression of all these four kinases was checked by qRT-PCR. HRI alone was found to be significantly upregulated in CCl4 intoxicated mice and this was prevented in mice treated with geraniin (Fig. 4C). 4. Discussion
Fig. 3. HO-1 protein expression in liver tissue of mice from different groups. (A) HO-1 protein expression by western blot and (B) its quantitation by densitometric analysis. Data shown represents the mean values of 6 mice per group ± SE.*p < 0.05 vs. control. **p < 0.05 vs. Sily: Silymarin, Ger: Geraniin.
Geraniin has been shown to exhibit different biological activities, among which its antioxidant activity is due to significant HO-1 induction through upregulation of Nrf-2 pathway [17,30,31]. In this study, geraniin was used to check its hepatoprotective effect against CCl4-induced liver injury in mice. CCl4, is a commonly used hepatotoxin, on metabolism generates free radicals, leading to OS which induces hepatic injury, measured in terms of cellular leakage of hepatic enzymes [32e35]. In the present study, SGOT, SGPT and LDH, hepatic marker enzymes were found to be elevated in CCl4 intoxicated mice, while in those treated with geraniin this was significantly reduced, indicating maintenance of structural integrity of hepatocytes and stabilization of their membrane activity (Table 1). This was additionally supported by histological findings. Centrilobular necrosis and sinusoidal congestion of liver
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Fig. 4. Geraniin inhibits CCl4 induced phosphorylation of eIF2a and expression of responsible kinase, HRI. (A) Western blot analysis of p-eIF2a and (B) its quantitation by densitometry, (C) qRT-PCR of HRI gene. Data shown represents the mean values of 6 mice per group ± SE.*p < 0.05 vs. control. **p < 0.05 vs. CCl4. Sily: Silymarin, Ger: Geraniin.
tissue in mice intoxicated with CCl4 was not seen in those pretreated with geraniin (Fig. 1). Significant elevation in MDA, a product of lipid peroxidation, observed in CCl4 intoxicated mice [36,37] was inhibited in mice pretreated with geraniin. GSH is an important antioxidant in liver, and its homeostasis is associated with various toxin-induced liver injuries [38,39]. It can be directly used as antioxidant molecule or in enzymatic reactions catalyzed by GST, GPx and is continuously regenerated by GR. In the present study GSH was found to be elevated in mice treated with CCl4 similar to earlier report [40]. This could be due to both reduction in utilization of this compound by enzyme such as GST [41] or elevation in GR activity [42]. We indeed observed increase in GR activity and its expression in CCl4 intoxicated mice, while that of GPx remained unaltered. For catalase, both activity and expression was found to be reduced significantly in CCl4 intoxicated mice, probably due to peroxy radical generated by CCl4 metabolism [43]. Pretreatment with geraniin significantly inhibited increase in GSH and GR and restored catalase in CCl4 intoxicated mice indicating amelioration of OS by geraniin. HO-1, the rate-limiting enzyme in heme catabolism, is known to be induced in OS and confer protection against oxidative tissue injuries [44]. This induction is reported to be mediated through a rapid increase in microsomal free heme concentration, presumably derived from hepatic cytochrome P450, in rats administered intraperitoneally with CCl4. This is further confirmed by inhibiting HO-1 in CCl 4 intoxicated rats which led to significant increase in liver injury [45]. In the present study, as expected, HO-1 was significantly increased in CCl4 treated mice which was not seen in those pretreated with geraniin, implying significant reduction in OS. Under OS, products of peroxidation, MDA and 4- hydroxynonenal, are known to inhibit protein synthesis and this inhibition is through phosphorylation of a subunit of the translation initiation factor eIF2a at S5 [46]. Phosphorylation of eIF2a inhibits exchange of GDP for GTP catalyzed by eIF2B and prevents formation of ternary complex leading to inhibition of protein synthesis [46]. Phosphorylation of eIF2a occurs by a family of kinases namely HRI, PKR, PERK and GCN2 [47]. In CCl4 intoxicated mice, significant amount to eIF2a was found to be phosphorylated through HRI (Fig. 4A) which was reduced significantly in mice treated with geraniin, indicating its protective effect.
Thus, all these data indicate that pretreatment with geraniin significantly reduced CCl4 induced hepatic injury by reducing OS. Conflict of interest Authors declare that they have no conflict of interest. Acknowledgements Authors acknowledge the financial support received from University Grant Commission-Center for Advance Studies, Government of India (UGC-CAS), Department of Science and TechnologyPromotion of University Research and Scientific Excellence (DSTPURSE), Government of India, India. HA was recipient of fellowship from Indian Council for Cultural Relations (ICCR). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2017.04.013. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.04.013. References [1] W.J. Brattin Jr., E.A. Glende, R.O. Recknagel, Pathological mechanisms in carbon tetrachloride hepatotoxicity, J. Free Radicals Biol. Med. 1 (1985) 27e38. [2] R.O. Recknagel Jr., E.A. Glende, J.A. Dolak, R.L. Waller, Mechanisms of carbon tetrachloride toxicity, Pharmacol. Ther. 43 (1989) 139e154. [3] Y. Aniya, T. Koyama, C. Miyagi, M. Miyahira, C. Inomata, S. Kinoshita, T. Ichiba, Free radical scavenging and hepatoprotective actions of the medicinal herb, Crassocephalum crepidioides from the Okinawa Islands, Biol. Pharm. Bull. 28 (2005) 19e23. [4] U. Latha, M.G. Rajesh, M.S. Latha, Hepatoprotective effect of an ayurvedic medicine, Indian Drug 36 (1999) 470e473. [5] S.K. Mitra, S.J. Seshadri, M.V. Venkataranganna, S. Gopumadhavan, U. Venkatesh, D.N. Udupa, K. Sarma, Effect of HD-03-a herbal formulation in galactosamine-induced hepatopathy in rats, Ind. J. Physiol. Pharm. 44 (2000) 82e86. [6] J.N. Dhuley, S.R. Naik, Protective effect of Rhinax, a herbal formulation against CCl4-induced liver injury and survival in rats, J. Ethnopharm 56 (1997) 159e164.
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Please cite this article in press as: H. Aayadi, et al., Protective effect of geraniin against carbon tetrachloride induced acute hepatotoxicity in Swiss albino mice, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.013