cirrhosis

PharmaNutrition 5 (2017) 109–117

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Hepatoprotective effect of boldine in a bile duct ligated rat model of cholestasis/cirrhosis

MARK

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Reza Heidaria, Leila Moezia,b, , Behnam Asadia, Mohammad Mehdi Ommatia,c, Negar Azarpirad a

Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran c Department of Animal Sciences, School of Agriculture, Shiraz University, Shiraz, Iran d Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran b

A R T I C L E I N F O

A B S T R A C T

Chemical compounds studied in this article: Dichlorofluorescein Diacetate (PubChem CID: 101615877) Boldine (PubChem CID: 10154) 5,5′-Dithiobis-2-nitrobenzoic acid (PubChem CID: 6254) Thiobarbituric acid (PubChem CID: 2723628) Chloramine-T (PubChem CID: 3641960)

Liver fibrosis is a debilitating disease associated with chronic liver injury. Oxidative stress is known as a pivotal mechanism in the initiation and propagation of liver fibrosis. Boldine is a potent antioxidant molecule with several pharmacological effects. The current investigation aimed to evaluate the effect of boldine in bile duct ligated (BDL) rats as a model of cholestasis and cirrhosis. BDL animals received boldine (5, 10, and 20 mg/kg/ day, oral) for 14 days (Cholestatic rats) and 28 days (Cirrhotic rats). The serum biomarkers of liver injury were drastically increased in the BDL group. Moreover, the level of oxidative stress markers was significantly increased in BDL animals. Severe bridging fibrosis, tissue necrosis, and inflammation were also detected in BDL rats. It was found that boldine (5, 10, and 20 mg/kg/day, oral) restored the BDL-induced depletion of glutathione content and tissue antioxidant capacity. Moreover, histopathological changes and collagen deposition were markedly attenuated by the boldine treatment. The beneficial effects of boldine administration in cholestasis/cirrhosis might be associated with anti-fibrotic properties via antioxidant activities.

Keywords: Alkaloid Collagen deposition Hepatotoxicity Liver fibrosis Oxidative stress

1. Introduction Boldine (1, 10-dimethoxy-2, 9-dihydroxyaporphine; Fig. 1) is the major alkaloid in the bark and leaves of the boldo tree (Peumus boldus) [1,2]. This alkaloid is believed to be responsible for the majority of the health-promoting properties of the boldo extract [1,2]. A wide range of pharmacological properties is attributed to boldine [1,2]. Boldine has been shown to have cytoprotective, antitumor, anti-inflammatory, immunomodulatory, hepatoprotective, and antipyretic properties [1,3–6]. Moreover, this alkaloid is known as a potent antioxidant and radical scavenging molecule [1,7]. Several chronic diseases, such as viral hepatitis and alcoholism, might finally lead to liver fibrosis and hepatic failure [8]. Currently, the only effective available therapeutic option for liver fibrosis, cirrhosis, and liver failure is liver transplantation. However, many factors limit the impact of liver transplantation and mention the importance of effective hepatoprotective and antifibrotic therapies [9–11]. The mechanisms involved in the development of liver fibrosis have been established [12]. It has been found that free radicals and oxidative stress are key players in the initiation and propagation of liver injury

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Corresponding author. E-mail address: [email protected] (L. Moezi).

http://dx.doi.org/10.1016/j.phanu.2017.07.001 Received 3 March 2017; Received in revised form 15 July 2017; Accepted 21 July 2017 Available online 24 July 2017 2213-4344/ © 2017 Published by Elsevier B.V.

and fibrosis [13,14]. Hence, the administration of potent antioxidant molecules such as boldine could be a potential therapeutic approach in preventing liver injury. The antioxidant and radical scavenging properties of other aporphine structures also have been reported [1]. On the other hand, structure–activity relationship studies have provided data which indicate boldine as a potent antioxidant and radical scavenging molecule among aporphine-based alkaloids [1]. Bile duct ligation (BDL) is a very reproducible animal model of chronic liver injury, cholestasis, and cirrhosis [15,16]. This model induces a high yield of cirrhosis in animals obstructed for one month or longer [15]. The morphological changes in the liver of BDL animals are similar to human cholestasis/cirrhosis [15,16]. The antifibrotic and hepatoprotective effect of boldine in chronic liver injury has not been investigated so far. Hence, this study aimed to examine the potential protective effects of boldine in a rat model of cholestasis/cirrhosis induced by BDL. Changes in serum biomarkers of liver injury, parameters of oxidative stress in liver tissue, liver hydroxyproline content, and liver fibrotic changes were monitored to evaluate whether boldine has any hepatoprotective properties in BDL rats as an animal model of chronic liver injury.

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(Cholestasis model), and six animals were assessed 28 days after BDL operation (Cirrhosis model) [17,18]. Boldine doses were selected based on the previous investigations [19–21]. 2.5. Serum biochemistry A Mindray BS-200® auto analyzer and standard kits were used to measure serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT), and bilirubin [22]. 2.6. Liver histopathology For histopathological assessments, samples of liver were fixed in buffered formalin solution (0.4% sodium phosphate monobasic, NaH2PO4, 0.64% sodium phosphate dibasic, Na2HPO4, and 10% formaldehyde in distilled water). Paraffin-embedded sections of tissue (5 μm) were prepared and stained with hematoxylin and eosin (H & E) before light microscope viewing. Liver fibrotic changes was determined by Masson’s trichrome staining in BDL rats. The Ishak system which uses a six-point scale for fibrosis stage (0–6) was applied for scoring liver fibrosis in the current investigation [23,24]. Samples were analyzed by a pathologist in a blind fashion.

Fig. 1. Boldine chemical structure.

2. Material and methods 2.1. Chemicals N-chloro tosylamide (Chloramine-T), Trichloroacetic acid (TCA), Sodium acetate, Citric acid, n-Propanol, p-Dimethyl amino benzaldehyde, 5,5′-Dithionitrobenzoic acid (DTNB), Dithiothreitol (DTT), Sucrose, 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ), Thiobarbituric acid (TBA), Sodium citrate, Ethylenediamine tetra-acetic acid (EDTA), Phosphoric acid, 2‐amino‐2-hydroxymethyl-propane-1,3-diolHydrochloride (Tris-HCl), were obtained from Merck (Darmstadt, Germany). Boldine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Kits for evaluating biomarkers of liver injury, including ALT, LDH, AST, ALP, γ-glutamyl transpeptidase (γ-GT), and bilirubin, were obtained from Pars Azmun® (Tehran, Iran). All salts used for preparing buffer solutions were of analytical grade and obtained from Merck (Darmstadt, Germany).

2.7. Reactive oxygen species (ROS) formation Reactive oxygen species formation in liver was estimated by a previously described method [25,26]. Briefly, liver tissue (200 mg) was homogenized in 5 ml of ice-cooled Tris-HCl buffer (40 mM, pH = 7.4). Samples of the resulted tissue homogenate (100 μl) were mixed with Tris-HCl buffer (1 ml) and 2′, 7′‐dichlorofluorescein diacetate (Final concentration 10 μM). The mixture was incubated at 37 °C (30 min, in dark). Finally, the fluorescence intensity of the samples was assessed using a FLUOstar Omega® multifunctional microplate reader with λ excitation = 485 nm and λ emission = 525 nm [25,27].

2.2. Animals

2.8. Lipid peroxidation Male Sprague-Dawley rats (n = 60; 200–250 g weight) were obtained from the Center of Comparative and Experimental Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. Animals were housed in plastic cages over hardwood bedding. There was an environmental temperature of 23 ± 1 °C and a 12L: 12D photoschedule along with a 40% of relative humidity. The rats were allowed free access to a normal standard chow diet and tap water. Animals received humane care and all the experiments were performed in conformity with the guidelines for care and use of experimental animals approved by an ethic committee in Shiraz University of Medical Sciences, Shiraz, Iran (#94-01-36-10649).

The thiobarbituric acid reactive substances (TBARS) were measured as an index of lipid peroxidation in liver tissue [28]. The reaction mixture was consisted of 500 μl of tissue homogenate (10% w/v in KCl, 1.15% w/v), 1 ml of thiobarbituric acid (0.375%, w/v), and 3 ml of phosphoric acid (1% w/v, pH = 2). Samples were mixed well and heated in boiling water (100 °C) for 45 min. After the incubation period, the mixture was cooled, and then 2 ml of n-butanol was added. Samples were vigorously vortexed and centrifuged (10,000g for 10 min) [26]. Finally, the absorbance of developed color in n-butanol phase was measured at 532 nm using an Ultrospec 2000®UV spectrophotometer (Pharmacia Biotech, Uppsala, Sweden) [28].

2.3. Surgery 2.9. Hepatic glutathione content Animals were anesthetized (10 mg/kg of xylazine and 70 mg/kg of ketamine, i.p), a midline incision was made and the common bile duct was localized, doubly ligated, and cut between these two ligatures (Day = 0) [17]. The sham operation consisted of laparotomy and bile duct identification and manipulation without ligation.

Liver samples (200 mg) were homogenized in 8 ml of ice-cooled EDTA solution (40 mM). Then, 5 ml of the prepared homogenate were added to 4 ml of distilled water (4 °C) and 1 ml of trichloroacetic acid (50%; w/v). The mixture was vortexed and centrifuged (10,000g, 4 °C, 15 min). Then, 2 ml of the supernatant was mixed with 4 ml of Tris-HCl buffer (40 mM, pH = 8.9), and 100 μl of DTNB (0.01 M in methanol) [26]. The absorbance of the developed color was measured at 412 nm using an Ultrospec 2000®UV spectrophotometer (Pharmacia Biotech, Uppsala, Sweden) [28].

2.4. Experimental setup Animals (n = 60) were equally allotted into five groups containing 12 rats in each. Rats were treated as follows: 1) Sham-operated (Vehicle-treated); 2) BDL; 3) BDL + Boldine (5 mg/kg/day, oral, started from day 1 after BDL operation); 4) BDL + Boldine (10 mg/kg/ day, oral, started from day 1 after BDL operation); 5) BDL + Boldine (20 mg/kg/day, oral, started from day 1 after BDL operation). Six animals in each group were evaluated 14 days after BDL operation

2.10. Protein carbonylation in liver tissue Total protein carbonyl content of liver tissue was measured by a spectrophotometric assay after derivatizing the protein carbonyl 110

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adducts with 2,4-dinitrophenyl hydrazine (DNPH) [29]. Briefly, 500 mg of liver tissue was homogenized in a sodium phosphate buffer (0.1 M, pH = 7.4) containing Triton-X100 (0.1%; v/v). An aliquot of the liver homogenate (500 μl) was added to an equivalent volume (500 μl) of DNPH (0.1% w/v in 2 N HCl) and incubated for 1 h at room temperature in dark. This reaction was terminated and total cellular protein was precipitated by the addition of an equivalent volume of TCA (20%, w/ v). Cellular protein was rapidly pelleted by centrifugation (12,000g, 5 min) and the supernatant was discarded. Excess unincorporated DNPH was extracted three times using 1 ml of ethanol: ethyl acetate (1:1 v/v) solution each time. Following the extraction procedure, the recovered pellet was dissolved in 1 ml of Tris-HCl buffer (40 mM, containing 8 M guanidine‐HCl, pH = 7.2). The resulting solubilized hydrazones were measured at 380 nm by an Ultrospec2000® spectrophotometer (Pharmacia Biotech, Uppsala, Sweden) [30,31]. 2.11. Ferric reducing antioxidant power (FRAP) of liver tissue FRAP assay measures the formation of a blue colored Fe2+-tripyridyltriazine compound from the colorless oxidized Fe3+ form by the action of electron-donating antioxidants [32]. In the current study, the working FRAP reagent was prepared by mixing 10 vols of acetate buffer (300 mmol/L, pH = 3.6), with 1 vol of TPTZ (10 mmol/L in 40 mmol/L hydrochloric acid) and 1 vol of ferric chloride (20 mmol/L). All solutions were prepared freshly. Tissue was homogenized in an ice-cooled Tris-HCl buffer (250 mM Tris-HCl, 200 mM sucrose and 5 mM DTT, pH = 7.4). Then, 50 μl of tissue homogenate and 150 μl of deionized water was added to 1.5 ml of the FRAP reagent [33]. The reaction mixture was incubated at 37 °C for 5 min. Finally, the absorbance of developed color was measured at 595 nm by an Ultrospec 2000® UV spectrophotometer (Pharmacia Biotech, Uppsala, Sweden) [26,34]. 2.12. Liver hydroxyproline content Liver hydroxyproline content was assessed as an index of liver fibrosis in BDL rats. Briefly, 500 μl of liver tissue homogenate (20% w/v in phosphate buffered saline; PBS; pH = 7.4) was digested in 1 ml of hydrochloric acid (6 N) at 120 °C for 8 h. An aliquot of digested homogenate (25 μl) was added to 25 μl of citrate–acetate buffer (pH = 6). Then, 500 μl of chloramines-t-solution (56 mM) was added and the mixture was left at room temperature for 20 min. Then, 500 μl Ehrlich’s reagent (15 g of p-Dimethyl amino benzaldehyde in n-propanol/perchloric acid; 2:1 v/v) was added and the mixture was incubated at 65 °C for 15 min. After cooling, the intensity of developed color was measured spectrophotometrically at 550 nm using an Ultrospec 2000®UV spectrophotometer (Pharmacia Biotech, Uppsala, Sweden) [35].

Fig. 2. Growth pattern and liver/body weight ratio of different experimental groups. Bold: Boldine. BDL: Bile duct ligated. Data are given as Mean ± SD (n = 6). ***P < 0.001 between BDL vs. sham. a P < 0.001 between BDL vs boldine-treated. ns: not significant as compared with BDL group. Ψ Indicates not significant as compared with sham.

hepatocyte injury were also drastically increased in both cholestatic and cirrhotic animals (Figs. 3 and 4). Moreover, serum γ-GT and ALP level as markers of biliary tree injury were significantly increased in the BDL animals (Figs. 3 and 4). It was found that boldine administration significantly inhibited the hepatocyte damage, as was evident by the decreased serum level of liver injury biomarkers in the cholestatic/ cirrhotic animals (Figs. 3 and 4). Furthermore, boldine administration significantly decreased serum total bilirubin, γ-GT and ALP levels (Figs. 3 and 4). It was found that markers of oxidative stress were significantly elevated in both cholestatic and cirrhotic rats (Table 1). An increase in hepatic ROS formation, lipid peroxidation, and protein carbonylation was detected in BDL animals (Table 1). BDL also significantly distorted the hepatic GSH content and tissue antioxidant capacity (Table 1). Parameters of oxidative stress were significantly augmented by boldine administration (5, 10, and 20 mg/kg) to BDL animals (Table 1). BDL considerably increased the hydroxyproline content of liver in both cholestatic and cirrhotic rats compared with its level in the shamoperated group (Fig. 5). It was found that boldine administration (5, 10 and 20 mg/kg) significantly decreased the liver hydroxyproline content, in cholestatic and cirrhotic animals (Fig. 5).

2.13. Statistical methods Data are given as Mean ± SD. Comparison of data sets was performed by the one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons as the post hoc test. Values of P < 0.05 were considered statistically significant. 3. Results A significant decrease in the animals’ body weight was detected in cirrhotic rats (Fig. 2), but there were no statistically significant changes in body-weight of the cholestatic animals (Fig. 2). BDL operation induced a significant enlargement of the liver in both cholestatic and cirrhotic rats (Fig. 2). It was found that boldine treatment produced an increase in the animals’ weight (in the cirrhotic animals) and a decrease in BDL-induced hepatomegaly (Fig. 2). BDL was ensued by a significant elevation in serum total bilirubin (Figs. 3 and 4). The serum AST and ALT, and LDH as biomarkers of 111

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Fig. 3. Serum biochemical measurements in cholestatic rats (14 days after BDL operation). Data are given as Mean ± SD for six animals in each group. Bold: Boldine. BDL: Bile duct ligated. a Indicates significantly different as compared with sham-operated group (P < 0.001). Asterisks indicate significantly different as compared with BDL group (*P < 0.05, **P < 0.01, and ***P < 0.001). ns Not significant as compared with BDL group (P > 0.05).

and fibrogenesis were significantly inhibited in both cholestatic and cirrhotic rats which were treated with boldine (5, 10 and 20 mg/kg) (Table 2) (Figs. 6 and 7). No sign of tissue necrosis was detected in boldine-treated (10 and 20 mg/kg) BDL animals (Table 2) (Figs. 6 and 7). It is noteworthy that the effect of boldine on the parameters evaluated in the current investigation didn’t seem to be dose-dependent. However, several biochemical markers of liver injury were significantly

Liver histopathological changes in BDL rats revealed an extensive confluent and focal necrosis, inflammation and fibrotic changes of tissue, as assessed by Hematoxylin & Eosin and Masson’s trichrome staining (Figs. 6 and 7, Table 2). Boldine showed a hepatoprotective activity against the cholestatic/ cirrhotic condition, and liver fibrogenesis in accordance with the histopathological findings by H & E and Masson’s trichrome staining (Table 2) (Figs. 6 and 7). It was found that liver necrosis, inflammation, 112

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Fig. 4. Serum biochemical measurements in cirrhotic rats (28 days after BDL operation). Data are given as Mean ± SD for six animals in each group. Bold: Boldine. BDL: Bile duct ligated. a Indicates significantly different as compared with sham-operated group (P < 0.001). Asterisks indicate significantly different as compared with BDL group (*P < 0.05, **P < 0.01, and ***P < 0.001). ns Not significant as compared with BDL group (P > 0.05).

4. Discussion

different in higher doses of boldine in comparison with lower doses of this alkaloid (Figs. 3 and 4). Moreover, this alkaloid seems to have significantly higher efficacy against some parameters of oxidative stress in cholestatic or cirrhotic rats (Table 1).

Liver fibrosis represents a major health problem. Several chronic liver diseases such as viral hepatitis, and alcoholism, along with inborn

Table 1 Liver oxidative stress markers. Treatment

Cholestatic Rats Sham BDL BDL + Bold 5 mg/kg BDL + Bold 10 mg/kg BDL +Bold 20 mg/kg Cirrhotic Rats Sham BDL BDL + Bold 5 mg/kg BDL + Bold 10 mg/kg BDL +Bold 20 mg/kg

ROS formation (Fluorescent Intensity; FI)

Lipid peroxidation (nmol of TBARS /mg protein)

Glutathione (μmol of GSH /mg protein)

Protein carbonylation (OD at 370 nm)

Total antioxidant capacity (μM of VitC equivalent)

83889 ± 16040 177433 ± 17806a 151845 ± 8149 b

1.77 ± 0.06 3.15 ± 0.40a 2.14 ± 0.21 b

19.86 ± 3.64 7.9 ± 0.76a 8.10 ± 0.97 b

0.156 ± 0.02 0.354 ± 0.07 0.324 ± 0.05

3.60 ± 0.45 2.66 ± 0.03 2.82 ± 0.13

b

0.202 ± 0.02

b

3.20 ± 0.17

b

0.182 ± 0.02

b c

3.49 ± 0.36

b

166952 ± 8267

b

2.13 ± 0.19

b

9.19 ± 0.50

131253 ± 4373

b c

2.27 ± 0.18

b

12.45 ± 2.62

1.60 ± 0.52 5.02 ± 0.54a 2.54 ± 0.46 b

18.56 ± 2.87 5.87 ± 0.82a 8.58 ± 1.16

0.136 ± 0.14 0.390 ± 0.06 0.299 ± 0.09

95641 ± 10500 162720 ± 7875a 148697 ± 5568 b 154678 ± 7937

b

148484 ± 11803

b

b

b

b

3.77 ± 0.12 2.38 ± 0.15 2.95 ± 0.12

b

3.20 ± 0.55

b

9.84 ± 0.70

b

0.218 ± 0.03

b

3.28 ± 0.20

b

3.26 ± 0.49

b

9.55 ± 0.82

b

0.211 ± 0.02

b

3.20 ± 0.12

b

Data are given as Mean ± SD (n = 6). Bold: Boldine. BDL: Bile duct ligated. a Indicates significantly different as compared with sham group (P < 0.001). b Indicates significantly different as compared with BDL group (P < 0.01). c Indicates significantly different as compared with 5 mg/kg and 10 mg/kg boldine-treated groups (P < 0.05).

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Fig. 5. The hydroxyproline content of the liver tissue. Data are represented as Mean ± SD (n = 6). Bold: Boldine. BDL: Bile duct ligated. Asterisks indicate significantly different as compared with sham-operated group (*P < 0.05 and ***P < 0.001). a Indicates significantly different as compared with BDL group (P < 0.05). b Indicates significantly different as compared with BDL group (P < 0.01). ns No significant difference as compared with BDL group (P > 0.05).

Fig. 6. Liver photomicrographs showing histopathological changes in cholestatic rats (14 days after BDL operation). Upper row: H & E staining; lower row: Masson Trichrome staining for hepatic fibrotic changes (Scale bar, 1000 μm). A & F: Sham-operated, B & G: BDL, C & H: BDL + Boldine 5 mg/kg, D & I: BDL + Boldine 10 mg/kg, and E, J: BDL + Boldine 20 mg/kg. For histopathological grading and the level of tissue fibrotic changes refer to Table 2. Green arrow: Inflammation. White arrow: Necrosis; Blue arrow: Bile duct proliferation; Yellow arrow: Sinusoidal congestion; Black arrow: Tissue collagen deposition.

of the mechanisms of liver fibrosis, there is currently no permissive therapeutic agent for the treatment of this complication. Antioxidants are potential candidates against liver fibrosis and its associated complications [38–40]. In the current study, the histopathological changes in BDL animals revealed significant tissue fibrosis in both cholestatic and cirrhotic rats (Figs. 6 and 7; Table 2). Trichrome staining is widely used to dye abnormal collagen fibers and fibroplasia, and our results showed that boldine supplementation significantly reduced the positively stained areas (Figs. 6 and 7; Table 2), and decrease the liver hydroxyproline content as a “gold standard” for the degree of liver fibrosis (Figs. 6 and 7; Table 2). Boldine administration (5, 10, and 20 mg/kg) also mitigated other histopathological lesions in the liver of cholestatic/cirrhotic animals. This mention the significant anti-fibrotic properties of boldine as the main findings of our research. The antifibrotic properties of boldine could be applied through a variety of mechanisms such as antioxidant effects of this alkaloid. Boldine is known as one of the most potent antioxidants [1,2,6]. This alkaloid is rapidly absorbed after oral administration and

errors of metabolic enzymes, might lead to liver fibrosis. There is no promising therapeutic option against liver fibrosis. In the current investigation, we found that oral administration of boldine (5, 10, and 20 mg/kg) to cholestatic and cirrhotic rats significantly alleviated liver injury, as evident by the decrease in serum biomarkers of liver injury, tissue markers of oxidative stress, and hepatic histopathological lesions. Liver fibrogenesis is a dynamic response to chronic hepatocellular damage. The fibrosis process results from the massive accumulation of extracellular matrix. A complex biological procedure regulates liver fibrosis [12]. Hepatic stellate cells and macrophages (Kupffer cells) play a key role in the initiation and progression of liver fibrosis [36]. On the other hand, the participation of oxidative stress in the progression of liver fibrosis has been widely recognized [37]. The occurrence of oxidative stress was evident in our study as characterized by the increase in ROS formation, lipid peroxidation, and protein carbonylation, along with a decrease in liver antioxidant capacity and hepatic glutathione reservoirs. Although there have been significant advances in the understanding 114

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Fig. 7. Liver photomicrographs representing histopathological changes in cirrhotic rats (28 days after BDL operation). Liver tissue was stained with H & E (Upper row) and Masson’s trichrome (Lower row) (Scale bar, 1000 μm). Sham (A, F), BDL (B, G), BDL + Boldine 5 mg/kg (C, H), BDL + Boldine 10 mg/kg (D, I) and BDL + Boldine 20 mg/kg (E, J). For histopathological grading and the level of tissue fibrotic changes refer to Table 2. Green arrow: Inflammation; White arrow: Necrosis; Blue arrow: Bile duct proliferation; Yellow arrow: Sinusoidal congestion; Black arrow: hepatic collagen deposition.

Table 2 Liver histopathological changes in cholestatic rats. Treatment

Confluent Necrosis

Focal Necrosis

Portal Inflammation

Bile duct proliferation

Ishak stage of liver fibrosis

Cholestatic Rats Sham (vehicle-treated rats) Bile duct ligated (BDL) rats BDL + Boldine 5 mg/kg BDL + Boldine 10 mg/kg BDL + Boldine 20 mg/kg

– – – – –

– ++ + – –

– +++ + + +

– ++ – + +

Normal 4 3 2 2

Cirrhotic Rats Sham (vehicle-treated rats) Bile duct ligated (BDL) rats BDL + Boldine 5 mg/kg BDL + Boldine 10 mg/kg BDL + Boldine 20 mg/kg

– +++ ++ – –

– + + – –

– ++ + +++ +

– ++ – + +

Normal 6 3 3 3

The Ishak system (a six-point scale for fibrosis stage) was applied for scoring liver fibrosis in BDL rats (Figs. 5 and 6). BDL: Bile duct ligated.

fibrosis in the liver [14,37,39]. Oxidative stress is also involved in the activation of stellate and Kupffer cells as key players of the liver tissue fibrogenesis [37,45]. Accumulation of the cytotoxic bile acids during cholestasis is involved in the pathogeneses of severe oxidative stress in the liver [46]. These noxious chemicals affect a vast range of targets such as cellular mitochondria [47]. On the other hand, cytotoxic bile acids disrupt biomembranes because of their detergent properties [14,46]. In the current study, boldine administration (5, 10, and 20 mg/ kg) effectively mitigated the oxidative stress and associated complications such as lipid peroxidation and protein damage in the liver of cholestatic/cirrhotic animals. Hence, the antifibrotic properties of boldine might be, at least in part, associated with the antioxidant capacity of this alkaloid. In the current investigation, the differences between oxidative stress parameters were also not significantly different between cholestatic and cirrhotic animals (Table 1). We found that boldine effectively alleviated biomarkers of oxidative stress in both cholestatic and cirrhotic rats (Table 1) and we found no significant differences between the

preferentially concentrated in the liver [19]. It has been found that boldine acts as a very efficient scavenger of reactive oxygen species [41]. Boldine has been broadly shown to exert potent cytoprotective effects in models of oxidative stress-induced damage [1,6,20,41–43]. Boldine also effectively inhibits the oxidation of biomembrane lipids [41]. On the other hand, proteins are among the major targets of free radicals [44]. The protective effects of boldine in preserving protein structure and functionality also have been evident in previous studies [41]. In the current study, we found that boldine effectively decreased lipid peroxidation and protein carbonylation in the liver of cholestatic/ cirrhotic animals (Table 1). Hence, this alkaloid might protect the biomembranes and cell proteins in the liver and, finally, prevent hepatic injury in cholestatic/cirrhotic animals. The important role of oxidative stress in the initiation and propagation of liver fibrosis has been mentioned in previous investigations [14]. It has been found that oxidative stress-induced biomembrane disruption and reactive aldehydes end-product of lipid peroxidation (e.g reactive aldehydes) are important mediators of inflammation and 115

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beneficial in liver disorders involving oxidative stress. Future investigations on the effect of boldine on the other multiple mechanisms involved in liver fibrogenesis will enhance our understanding regarding the hepatoprotection provided by this alkaloid.

antioxidant properties of boldine in cholestatic and cirrhotic animals (Table 1). On the other hand, markers such as liver hydroxy proline content (Fig. 5) and liver histopathological changes (Figs. 6 and 7; Table 2) were significantly higher in cirrhotic animals in comparison with cholestatic ones. We found that confluent necrotic lesions were developed in cirrhotic animals in comparison with the cholestatic group (Figs. 6 and 7; Table 2). Moreover, the collagen deposition in liver tissue of cirrhotic animals was higher (Table 2 and Fig. 7). We found no significant differences between the hepatoprotective effects of boldine in these two experimental model and this alkaloid significantly alleviated the mentioned pathological changes in cholestasis and cirrhosis. This might mention the robust antioxidative properties of boldine which effectively alleviates oxidative stress and its deleterious consequences in different situations. As mentioned, oxidative stress is tightly interconnected to the fibrosis process [14]. Hence, boldine as a potent antioxidant molecule could prevent liver fibrosis in both cholestatic and cirrhotic animals. The effect of boldine on glutathione-S-transferase (GST) enzyme activity has been shown previously [48]. GST is responsible for glutathione conjugation and detoxification of a wide range of xenobiotics including cytotoxic bile acids [49]. Hence, boldine might also protect the liver in the BDL model by increasing the removal of potentially toxic substances such as hydrophobic bile acids. The effects of boldine on mitochondria have also been investigated [42,50]. Mitochondria have a central role in a variety of oxidative and toxic forms of cell injury, as well as apoptosis/necrosis induced by xenobiotics [51–53]. There is evidence of mitochondrial dysfunction in cholestatic liver injury [54,55]. Hydrophobic bile acids, which are accumulated during cholestasis, are mitochondrion-toxic agents [56,57]. Moreover, mitochondria play a role in the fibrogenesis process [37,58]. Hence, a part of the protective properties of boldine might be associated with its effects on hepatocytes mitochondria. The precise effect of boldine on hepatocytes mitochondria in different models of liver injury and fibrosis could be the subject of future investigations in this field. It has been reported that inflammatory cytokines play a major role in liver fibrosis [59]. It has been found that stellate and Kupffer cellsderived cytokines are involved in the activation of fibrogenesis process and deterioration of liver function [37,39,60]. On the other hand, the anti-inflammatory and immunomodulatory effects of boldine are also mentioned in several investigations [4,5,21]. Hence, the effects of boldine on inflammatory cells and cytokines might also play a role in its protective properties against liver fibrosis. Further investigations are needed to make clear the precise mechanism of boldine on inflammatory cytokines and stellate cells activation in different models of liver fibrosis. The anti-inflammatory and mitochondrial protection provided by boldine could originate from the antioxidant-independent mechanisms of boldine to protect cells. On the other hand, as hepatic stellate cells (HSCs) activation remains the most dominant pathway leading to hepatic fibrosis, an investigation into the effects of boldine on HSCs might shed some light on the precise protective effects of this alkaloid on liver fibrosis. In view of the previously established anti-oxidative and anti-inflammatory properties of boldine, this alkaloid might be a potential therapeutic option in the prevention of chronic liver injury (cholestasis/ cirrhosis) in humans. More investigations on the precise effect of boldine in preventing inflammation and tissue fibrogenesis might also provide evidence for its administration against a wide range of situations leading to tissue injury and fibrosis. There are numerous cellular, molecular and signaling pathways that are involved in the fibrogenesis process. Among these, cellular redox balance and oxidative stress seem to play an important role. Boldine might provide protection against liver cholestasis/cirrhosis by its potent antioxidant capacity and balance cellular redox environment. The results presented in this paper can suggest that, at least in part, the hepatoprotective and anti-fibrotic effects of boldine are mediated by decreasing oxidative stress in the liver. Hence, boldine might also be

Conflicts of interest The authors declare no conflicts of interest. Acknowledgments The technical facilities providing of Pharmaceutical Sciences Research Center of Shiraz University of Medical Sciences is gratefully acknowledged. This investigation was financially supported by the office of the Vice Chancellor of Research Affairs, Shiraz University of Medical Sciences (#94-01-36-10649). References [1] P. O’Brien, C. Carrasco-Pozo, Speisky H. Boldine, its antioxidant or health-promoting properties, Chem. Biol. Interact. 159 (2006) 1–17. [2] H. Speisky, B.K. Cassels, Boldo and boldine: an emerging case of natural drug development, Pharmacol. Res. 29 (1994) 1–12. [3] A. Lemberg, M.A. Fernández, Hepatic encephalopathy ammonia, glutamate, glutamine and oxidative stress, Ann. Hepatol. 8 (2009) 95–102. [4] N. Backhouse, C. Delporte, M. Givernau, B.K. Cassels, A. Valenzuela, H. Speisky, Anti-inflammatory and antipyretic effects of boldine, Agents Actions 42 (1994) 114–117. [5] M. Gotteland, I. Jimenez, O. Brunser, et al., Protective effect of boldine in experimental colitis, Planta Med. 63 (1997) 311–315. [6] R. Bannach, A. Valenzuela, B.K. Cassels, L.J. Núnez-Vergara, H. Speisky, Cytoprotective and antioxidant effects of boldine on tert-butyl hydroperoxide—induced damage to isolated hepatocytes, Cell Biol. Toxicol. 12 (1996) 89–100. [7] G. Schmeda-Hirschmann, J.A. Rodriguez, C. Theoduloz, S.L. Astudillo, G.E. Feresin, A. Tapia, Free-radical scavengers and antioxidants from peumus boldus mol. (Boldo), Free Radical Res. 37 (2003) 447–452. [8] A.E. Karimzadeh Toosi, Liver fibrosis: causes and methods of assessment, a review, Roman. J. Int. Med. 53 (2015) 304–314. [9] P. Wang, Y. Koyama, X. Liu, et al., Promising therapy candidates for liver fibrosis, Front. Physiol. 7 (2016). [10] D.Y. Kim, S.I. Chung, S.W. Ro, et al., Combined effects of an antioxidant and caspase inhibitor on the reversal of hepatic fibrosis in rats, Apoptosis 18 (2013) 1481–1491. [11] P. Wang, Y. Koyama, X. Liu, et al., Promising therapy candidates for liver fibrosis, Front. Physiol. 7 (2016). [12] S.L. Friedman, Mechanisms of hepatic fibrogenesis, Gastroenterology 134 (2008) 1655–1669. [13] V. Sánchez-Valle, C.N. Chavez-Tapia, M. Uribe, N. Méndez-Sánchez, Role of oxidative stress and molecular changes in liver fibrosis: a review, Curr. Med. Chem. 19 (2012) 4850–4860. [14] H. Tsukamoto, R. Rippe, O. Niemelä, M. Lin, Roles of oxidative stress in activation of Kupffer and Ito cells in liver fibrogenesis, J. Gastroenterol. Hepatol. 10 (1995) S50–S53. [15] J. Kountouras, B.H. Billing, P.J. Scheuer, Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat, Br. J. Exp. Pathol. 65 (1984) 305–311. [16] C.G. Tag, S. Sauer-Lehnen, S. Weiskirchen, et al., Bile duct ligation in mice: induction of inflammatory liver injury and fibrosis by obstructive cholestasis, J. Vis. Exp. 52 (2015) 524–538. [17] L. Moezi, R. Heidari, Z. Amirghofran, A.A. Nekooeian, A. Monabati, A.R. Dehpour, Enhanced anti-ulcer effect of pioglitazone on gastric ulcers in cirrhotic rats: the role of nitric oxide and IL-1b, Pharmacol. Rep. 65 (2013) 134–143. [18] L. Moezi, Z. Janahmadi, Z. Amirghofran, A.A. Nekooeian, A.R. Dehpour, The increased gastroprotective effect of pioglitazone in cholestatic rats: role of nitric oxide and tumour necrosis factor alpha, Int. J. Exp. Pathol. 95 (2014) 78–85. [19] I. Jiménez, H. Speisky, Biological disposition of boldine: in vitro and in vivo studies, Phytother. Res. 14 (2000) 254–260. [20] Y.S. Lau, X.Y. Tian, Y. Huang, D. Murugan, F.I. Achike, M.R. Mustafa, Boldine protects endothelial function in hyperglycemia-induced oxidative stress through an antioxidant mechanism, Biochem. Pharmacol. 85 (2013) 367–375. [21] M.C. Lanhers, M. Joyeux, R. Soulimani, et al., Hepatoprotective and anti-inflammatory effects of a traditional medicinal plant of Chile, Peumus boldus, Planta Med. 57 (1991) 110–115. [22] R. Heidari, H. Babaei, M.A. Eghbal, Amodiaquine-induced toxicity in isolated rat hepatocytes and the cytoprotective effects of taurine and/or N-acetyl cysteine, Res. Pharm. Sci. 9 (2014) 97–105. [23] Z.D. Goodman, Grading and staging systems for inflammation and fibrosis in chronic liver diseases, J. Hepatol. 47 (2007) 598–607. [24] E.M. Brunt, Grading and staging the histopathological lesions of chronic hepatitis: the Knodell histology activity index and beyond, Hepatology 31 (2000) 241–246. [25] R. Gupta, D.K. Dubey, G.M. Kannan, S.J.S. Flora, Concomitant administration of

116

PharmaNutrition 5 (2017) 109–117

R. Heidari et al.

[26]

[27]

[28]

[29] [30] [31]

[32]

[33]

[34]

[35]

[36] [37] [38] [39] [40] [41]

[42]

Moringa oleifera seed powder in the remediation of arsenic-induced oxidative stress in mouse, Cell Biol. Int. 31 (2007) 44–56. R. Heidari, N. Esmailie, N. Azarpira, A. Najibi, H. Niknahad, Effect of thiol-reducing agents and antioxidants on sulfasalazine-induced hepatic injury in normotermic recirculating isolated perfused rat liver, Toxicol. Res. 32 (2016) 133–140. D.J. Socci, K.B. Bjugstad, H.C. Jones, J.V. Pattisapu, G.W. Arendash, Evidence that oxidative stress is associated with the pathophysiology of inherited hydrocephalus in the H-Tx rat model, Exp. Neurol. 155 (1999) 109–117. R. Heidari, H. Niknahad, A. Jamshidzadeh, N. Azarpira, M. Bazyari, A. Najibi, Carbonyl traps as potential protective agents against methimazole-induced liver injury, J. Biochem. Mol. Toxicol. 29 (2014) 173–181. E. Shacter, Quantification and significance of protein oxidation in biological samples, Drug Metab. Rev. 32 (2000) 307–326. R. Mehta, L. Wong, P.J. O’Brien, Cytoprotective mechanisms of carbonyl scavenging drugs in isolated rat hepatocytes, Chem. Biol. Interact. 178 (2009) 317–323. S. Mostafalou, M.A. Eghbal, A. Nili-Ahmadabadi, M. Baeeri, M. Abdollahi, Biochemical evidence on the potential role of organophosphates in hepatic glucose metabolism toward insulin resistance through inflammatory signaling and free radical pathways, Toxicol. Ind. Health 28 (2012) 840–851. V. Katalinic, D. Modun, I. Music, M. Boban, Gender differences in antioxidant capacity of rat tissues determined by 2,2′-azinobis (3-ethylbenzothiazoline 6-sulfonate; ABTS) and ferric reducing antioxidant power (FRAP) assays, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 140 (2005) 47–52. R. Heidari, F. Jafari, F. Khodaei, B. Shirazi Yeganeh, H. Niknahad, The mechanism of valproic acid-induced fanconi syndrome involves mitochondrial dysfunction and oxidative stress in rat kidney, Nephrology (Carlton, Vic.) (2017), http://dx.doi.org/ 10.1111/nep.13012 (In-Press). M. Alía, C. Horcajo, L. Bravo, L. Goya, Effect of grape antioxidant dietary fiber on the total antioxidant capacity and the activity of liver antioxidant enzymes in rats, Nut. Res. 23 (2003) 1251–1267. N. Hamdy, E. El-Demerdash, New therapeutic aspect for carvedilol: antifibrotic effects of carvedilol in chronic carbon tetrachloride-induced liver damage, Toxicol. Appl. Pharmacol. 261 (2012) 292–299. R.K. Moreira, Hepatic stellate cells and liver fibrosis, Arch. Pathol. Lab. Med. 131 (2007) 1728–1734. G. Poli, Pathogenesis of liver fibrosis: role of oxidative stress, Mol. Asp. Med. 21 (2000) 49–98. G. Tahan, H. Akin, F. Aydogan, et al., Melatonin ameliorates liver fibrosis induced by bile-duct ligation in rats, Can. J. Surg. 53 (2010) 313. M. Parola, G. Robino, Oxidative stress-related molecules and liver fibrosis, J. Hepatol. 35 (2001) 297–306. C. Ara, H. Kirimlioglu, A.B. Karabulut, et al., Protective effect of resveratrol against oxidative stress in cholestasis, J. Surg. Res. 127 (2005) 112–117. H. Speisky, B.K. Cassels, E.A. Lissi, L.A. Videla, Antioxidant properties of the alkaloid boldine in systems undergoing lipid peroxidation and enzyme inactivation, Biochem. Pharmacol. 41 (1991) 1575–1581. Y.Y. Jang, J.H. Song, Y.K. Shin, E.S. Han, C.S. Lee, Protective effect of boldine on

[43]

[44] [45]

[46] [47]

[48]

[49] [50]

[51]

[52] [53]

[54]

[55] [56] [57]

[58] [59] [60]

117

oxidative mitochondrial damage in streptozotocin-induced diabetic rats, Pharmacol. Res. 42 (2000) 361–371. Y.S. Lau, W.C. Ling, D. Murugan, M.R. Mustafa, Boldine ameliorates vascular oxidative stress and endothelial dysfunction: therapeutic implication for hypertension and diabetes, J. Cardiovas. Pharmacol. 65 (2015) 522–531. E.R. Stadtman, R.L. Levine, Free radical-mediated oxidation of free amino acids and amino acid residues in proteins, Amino Acids 25 (2003) 207–218. S.W. Luckey, D.R. Petersen, Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats, Exp. Mol. Pathol. 71 (2001) 226–240. B.L. Copple, H. Jaeschke, C.D. Klaassen, Oxidative stress and the pathogenesis of cholestasis, Semin. Liver Dis. 30 (2010) 195–204. S. Krähenbühl, C. Talos, B.H. Lauterburg, J. Reichen, Reduced antioxidative capacity in liver mitochondria from bile duct ligated rats, Hepatology 22 (1995) 607–612. R. Kubinova, M. Machala, K. Minksova, J. Neca, V. Suchý, Chemoprotective activity of boldine: modulation of drug-metabolizing enzymes, Die Pharm. 56 (2001) 242–243. K. Mitamura, N. Hori, T. Iida, et al., Identification of S-acyl glutathione conjugates of bile acids in human bile by means of LC/ESI–MS, Steroids 76 (2011) 1609–1614. C.V. Klimaczewski, Saraiva RdA, D.H. Roos, et al., Antioxidant activity of Peumus boldus extract and alkaloid boldine against damage induced by Fe(II)–citrate in rat liver mitochondria in vitro, Ind. Crop Prod. 54 (2014) 240–247. R.J. Sokol, J.M. McKim Jr, M.C. Goff, et al., Vitamin E reduces oxidant injury to mitochondria and the hepatotoxicity of taurochenodeoxycholic acid in the rat, Gastroenterology 114 (1998) 164–174. D. Pessayre, A. Mansouri, D. Haouzi, B. Fromenty, Hepatotoxicity due to mitochondrial dysfunction, Cell Biol. Toxicol. 15 (1999) 367–373. H. Niknahad, R. Heidari, A.M. Alzuhairi, A. Najibi, Mitochondrial dysfunction as a mechanism for pioglitazone-induced injury toward HepG2 cell line, Pharm. Sci. 20 (2015) 169. N. Alptekin, G. Mehmetcik, M. Uysal, G. Aykac-Toker, Evidence for oxidative stress in the hepatic mitochondria of bile duct ligated rats, Pharmacol. Res. 36 (1997) 243–247. J. Sastre, G. Serviddio, J. Pereda, et al., Mitochondrial function in liver disease, Front. Biosci. 12 (2007) 1200–1209. C.M. Palmeira, A.P. Rolo, Mitochondrially-mediated toxicity of bile acids, Toxicology 203 (2004) 1–15. A.P. Rolo, P.J. Oliveira, A.J.M. Moreno, C.M. Palmeira, Bile acids affect liver mitochondrial bioenergetics: possible relevance for cholestasis therapy, Toxicol. Sci. 57 (2000) 177–185. E. Mormone, J. George, N. Nieto, Molecular pathogenesis of hepatic fibrosis and current therapeutic approaches, Chem. Biol. Interact. 193 (2011) 225–231. D.K. Stalnikowitz, A.B. Weissbrod, Liver fibrosis and inflammation: a review, Ann. Hepatol. 2 (2003) 159–163. H.L. Reeves, S.L. Friedman, Activation of hepatic stellate cells–a key issue in liver fibrosis, Front. Biosci. 7 (2002) 808–826.