Hepatoprotective effects of blue honeysuckle on CCl4‐induced acute liver damaged mice

Abstract The objective of this study was to evaluate the hepatoprotective effects of blue honeysuckle (BH) on carbon tetrachloride (CCl4)‐induced acute hepatic damage in mice. The experiment used a total of 60 ICR mice, which were divided into six groups. Except for the intact control groups, all groups received a single intraperitoneal injection of CCl4 after a 7 day pre‐treatment period with distilled water, BH extracts, or silymarin. Twenty‐four hours after the CCl4 injection, the following observations, representative of classical oxidative stress‐mediated centrolobular necrotic acute liver injuries, were observed: decreased body weight; small nodule formation and enlargement on the gross inspections with related liver weight increase; elevation of serum AST and ALT, increases in hepatic lipid peroxidation and related depletion of endogenous antioxidants and antioxidative enzymes; centrolobular necrosis; increases in apoptotic markers, lipid peroxidation markers, and oxidative stress markers. However, liver damage was significantly inhibited by the pre‐treatment with BH extracts. The present study demonstrated that oral administration of BH extracts prior to exposure to CCl4 conferred favorable hepatoprotective effects. These results demonstrated that BHe possessed suitable properties for use as a potent hepatoprotective medicinal food.

Generally, when the liver is exposed to high levels of environmental toxins, metabolic dysfunction of the liver may occur, which ranges from the transient elevation of liver enzymes to life-threatening hepatic fibrosis, liver cirrhosis, and even hepatocellular carcinoma (Sun et al., 2011). Substantial evidence implicates oxidative stress and inflammation in the etiology of liver injury (Berasain et al., 2009). Similar effects are caused by CCl 4 , an industrial solvent known to induce liver injury and liver diseases, which is widely used in experimental hepatopathy (Yang et al., 2015;Zou, Qi, Ye, & Yao, 2016).
CCl 4 -induced toxicity depends on the dose and duration of exposure. At a low dose, transient effects occur, including the loss of Ca 2+ sequestration, impaired lipid homeostasis, and the release of several cytokines. Longer exposures alter fatty acid metabolism and induce fibrosis, cirrhosis, and cancer (Cui, Yang, Lu, Chen, & Zhao, 2014).
CCl 4 -induced hepatotoxicity is the result of reductive dehalogenation reactions catalyzed by the hepatic cytochrome P-450, which forms unstable trichloromethyl and trichloromethyl peroxyl radicals capable of binding to proteins or lipids and initiating lipid peroxidation and liver damage (Cheng et al., 2013). Oxidative stress has been accepted as one of the principal causes of CCl 4 -induced hepatic injury, which is mediated by the production of free radical derivatives of CCl 4 and is responsible for cell membrane damage and the subsequent release of the marker enzymes of hepatotoxicity (Boll, Weber, Becker, & Stampfl, 2001;Weber, Boll, & Stampfl, 2003).
Although the need for medicines to protect against liver damage has emerged, modern medicine still lacks reliable hepatoprotective drugs; therefore, numerous traditional herbal medicines have been studied for the evaluation of their hepatoprotective efficiency (Lu et al., 2016). Silymarin is a flavonoid found in the herb milk thistle, Silybum marianum. Milk thistle grows wild in a variety of settings, including roadsides. Silymarin is a powerful antioxidant that protects liver cells from toxins (Wellington & Jarvis, 2001). The antioxidant effects of silymarin on CCl 4 -induced liver damage have been well documented (Cordero-Pérez et al., 2013;Vargas-Mendoza et al., 2014); therefore, it was selected as a reference agent in this study.
In the present study, we aimed to screen the efficacy of three different types of BH extracts in a mouse model of CCl 4 -induced acute hepatic damage for their potential as potent hepatoprotective medicinal foods. The effects of the BH extracts were compared with those of silymarin (mixed flavonoids purified from milk thistle, Silybum marianum) (Jain, Lodhi, Jain, Nahata, & Singhai, 2011;Wang, Feng, Zhu, Zhao, & Suo, 2016).

| Preparation of BH extracts
Three different types of BH extract, BHw, BHj, and BHe, were prepared. BHw and BHj were prepared and supplied by Bioport Korea Inc. (Busan, Korea). BHw is the freeze-dried powder of the hot water extract of BH; briefly, water and dried BH were mixed in a 10:1 (w/w) ratio and then boiled at 90-95°C for 3 h under reflux.
In addition, silymarin was purchased in the form of a reddishyellow powder from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA) and used as a reference drug (Jain et al., 2011;Wang et al., 2016).
All three different types of BH extracts were dissolved in distilled water to 20 mg/ml, and orally administered once per day for 7 days, consecutively. The administration volume was 10 ml/kg (equivalent to 200 mg/kg), which was applied through gastric gavage using a zonde attached to a 1 ml syringe. Silymarin was suspended into distilled water at 10 mg/ml, and orally administered in a volume of 10 ml/kg (equivalent 100 mg/kg) once per day for 7 days, consecutively, in accordance with the reference recommendation (Jain et al., 2011;Wang et al., 2016). In the intact vehicle and CCl 4treated control mice, equal volumes of vehicle (distilled water) were orally administered instead of the test substances.

| Animals and husbandry
A total of sixty, healthy male ICR mice (6 weeks old upon receipt from OrientBio, Seungnam, Korea) were used after acclimatization period of 7 days. Four or five animals were allocated to a polycarbonate cage in a temperature-(20-25°C) and humidity-(30-35%) controlled room. The light-dark cycle was 12 h/12 h and the rats were given ad libitum access to feed (Cat. No. 38057;Purinafeed,Seungnam,Korea) and water were accessed.
The animals were divided into six groups based on their body weight prior to test substances administration: Intact control: Distilled water (DW) orally administered and olive oil intraperitoneally (IP) treated mice; CCl 4 control: DW orally administered and CCl 4 0.5 mg/kg IP treated mice; Silymarin: Silymarin 100 mg/kg orally administered and CCl 4 0.5 ml/kg IP treated mice; BHw: BHw 200 mg/ kg orally administered and CCl 4 0.5 ml/kg IP treated mice; BHj: BHj 200 mg/kg orally administered and CCl 4 0.5 ml/kg IP treated mice; and BHe: BHe 200 mg/kg orally administered and CCl 4 0.5 ml/kg IP treated mice (Figure 1).

| Changes in body weights
Changes in body weight were measured each day, from 1 day before the administration of the initial test, throughout all experimental periods, by using an automatic electronic balance (Precisa Instrument, Zürich, Switzerland). To reduce the individual differences, the body weight gains from the day of initial test substance administration to 24 h after CCl 4 injection were calculated as follows: Body weight gains (g) during 7 days of the whole experimental period = Body weight at 24 h after CCl 4 treatment -Body weight on the day of initial test substance administration.

| Measurements of liver weights
All animals were sacrificed 24 h after the CCl 4 injection, gross inspection was conducted under anesthesia induced with 2-3% isoflurane (Hana Pharm. Co. Ltd, Hwasung, Korea) in a mixture of 70% N 2 O and 28.5% O 2 using by using a rodent inhalation anesthesia apparatus (Surgivet, Waukesha, WI, USA) and rodent ventilator (Model 687, Harvard Apparatus, Cambridge, UK) and the weight of liver was measured (absolute wet-weights). To reduce the differences from individual body weights, the relative liver weights (as a percentage of body weights) were also calculated from the following formula: relative liver weights (% of body weight) = (absolute liver wet-weights/ body weight at sacrifice) × 100.

| Measurement of liver lipid peroxidation
The separated hepatic tissues were weighed and homogenized in icecold 0.01 M Tris-HCl buffer (pH 7.4) and centrifuged at 12,000 g for 15 min, as described by Kavutcu et al. (1996). Tissue homogenates were stored in an ultradeep freezer below −150°C until analysis. The concentration of liver lipid peroxidation was determined through the estimation of MDA by using the thiobarbituric acid test and a UV/ Vis spectrophotometer (Model OPTIZEN POP, Mecasys, Daejeon, Korea) to measure the absorbance of the solution at 525 nm, and determined as nM of MDA/mg protein (Jamall & Smith, 1985). The total protein content was measured by a previously described method (Lowry, Rosenbrough, Farr, & Randall, 1951) using bovine serum albumin (Invitrogen, Carlsbad, CA, USA) as internal standard.

| Measurement of hepatic antioxidant defense systems
The prepared hepatic homogenates were mixed with 0.1 ml 25% trichloroacetic acid (Merck, West Point, CA, USA) and centrifuged (1,627 g, 40 min, 4°C). The GSH content was spectrophotometrically determined through the measurement of absorbance at 412 nm by using 2-nitrobenzoic acid (Sigma-Aldrich, St. Louis, MO, USA) (Sedlak & Lindsay, 1968). The decomposition of H 2 O 2 in the presence of CAT was followed at 240 nm by using a spectrophotometer (Aebi, 1974). CAT activity was defined as the amount of enzyme required to decompose 1 nM of H 2 O 2 per minute at 25°C and pH 7.8. The results were expressed as U/mg protein. The measurement of SOD activity was performed in accordance with the method of Sun, Larry, and Ying (1988). SOD estimation was based on the generation of superoxide radicals produced by xanthine and xanthine oxidase, which react with nitrotetrazolium blue to produce formazan dye. SOD activity, which was related to the degree of inhibition of this reaction, was then spectrophotometrically measured at 560 nm and expressed as U/mg protein. One unit of SOD enzymatic activity is equal to the amount of enzyme that diminishes the initial absorbance of nitroblue tetrazolium by 50% over 1 min.

| Histopathology
The left lateral lobes of the liver were separated and fixed in 10% neutral buffered formalin (NBF), embedded in paraffin, sectioned (3-4 μm), and stained with hematoxylin and eosin (H&E) for general histopathological analysis (Ki et al., 2013;Lee et al., 2014). The histopathological profiles of each sample were generated by observation under a light microscope (Eclipse 80i, Nikon, Tokyo, Japan). To observe more detailed changes, hepatic damage was evaluated by the modified HAI (histological activity index) grading scores based on a previous well-established semiquantitative histopathological scoring system (Ishak et al., 1995), which includes assessment of confluent necrosis, focal lytic necrosis, apoptosis, and focal and portal inflammation (Table 1). In addition, the percentage of degenerative regions (%/mm 2 ) in the liver that exhibited centrolobular necrosis, congestion, and inflammatory cell infiltrations on hepatic lobules was computed by using a software-based automated image analyzer (iSolution FL ver 9.1, IMT i-solution Inc., Vancouver, Quebec, Canada). The numbers of hepatocytes that showed degenerative changes, including necrosis, acute cellular swelling (ballooning), and severe fatty acid changes, and inflammatory cells infiltrated were also calculated by using an automated image analyzer and expressed as cells/1000 hepatocytes and cells/mm 2 of hepatic parenchyma in accordance with previously described methods (Ki et al., 2013;Lee et al., 2014). The histopathologist was blinded to the group distribution when the analysis was performed.

| Statistical analyses
All numerical data were expressed as the mean ± SD of 10 mice.
Multiple comparison tests for different dose groups were conducted. Variance homogeneity was examined by using the Levene test (Levene, 1981). If the Levene test indicated no significant deviations from variance homogeneity, the obtain data were analyzed by one-way ANOVA followed by a least-significant differ-  (Ludbrook, 1997). Differences were considered significant at p < 0.05. Statistical analyses were computed by using SPSS for Windows (Release 14.0K, SPSS Inc., Chicago, IL, USA).
In addition, the percentage change between intact control mice and CCl 4 control mice was calculated to evaluate the severity of hepatic damages, including the induction of centrolobular necrosis, and the percentage changes compared with the CCl 4 control and BH-or silymarin-treated mice were also calculated to help elucidate the efficacy of the test substances. These calculations were performed by Equations (1) and (2), respectively, in accordance with our previously established method (Kang et al., 2014).

| Changes in the serum AST and ALT levels
Significant (p < 0.01) elevations of serum AST and ALT levels (intracellular enzymes indicative of hepatic damages) were observed in the CCl 4 control compared with the intact control, but significant (p < 0.01) decreases were induced by the treatment of all BH extracts at 200 mg/kg, and by silymarin 100 mg/kg, compared with the CCl 4 control. The changes were largest in BHe, followed by silymarin, BHj, and BHw ( Figure 5).

| Effects on activity of SOD, another endogenous antioxidative enzyme
A significant (p < 0.01) decrease in hepatic SOD activity was detected in the CCl 4 control compared with the intact control.
However, significant (p < 0.01) increases in SOD activities occurred in mice that received pre-treatment with all BH extracts at 200 mg/ kg and silymarin 100 mg/kg compared with that of the CCl 4 control.

| Histopathological inspection
Classic centrolobular necrosis (hepatocyte vacuolation and ballooning, deposition of lipid droplets in hepatocytes, and infiltration of inflammatory cells) was observed after a single IP treatment of CCl 4 0.5 ml/kg. However, this microscopic centrolobular necrosis was markedly inhibited by 7 days of continuous oral pre-treatment of silymarin 100 mg/kg and by all BH extracts at 200 mg/kg compared with the CCl 4 control; BHe was the most effective, followed by silymarin, BHj, and BHw. The histomorphometrical and semi-quantitative analysis indicated significant (p < 0.01) increases in the percentage area of degenerative regions, the numbers of degenerative hepatocytes, and the numbers of inflammatory cells infiltrated in hepatic parenchyma; therefore, a related increase of modified HAI grading scores was observed in the CCl 4 control compared with the intact control.
These changes were significantly (p < 0.01) ameliorated by treatment with all BH extracts at 200 mg/kg and by silymarin 100 mg/kg compared with the CCl 4 control; BHe exerted the strongest effect, followed by silymarin, BHj, and BHw (Table 5, Figure 6).    Figures 7 and 8).

| Immunohistochemical analysis
The mean number of cleaved caspase-3 immunoreactive hepatocytes in the CCl 4 control was increased to 35810.00% compared TA B L E 5 General histomorphometrical analysis of hepatic tissues from CCl

Items (Unit) Groups
General histomorphometry Histological Activity Index (Scores;

| D ISCUSS I ON
The cesses (Lu et al., 2016;Yang et al., 2015), the need for hepatoprotective medicines has gradually emerged. Modern medicine is still hampered by a lack of reliable hepatoprotective drugs; therefore, numerous traditional herbal medicines have been studied for their hepatoprotective efficiency (Lu et al., 2016). BH is a rich source of ascorbic acid and phenolic components, particularly anthocyanins, flavonoids, and low molecular weight phenolic acids, which exert multiple biological activities, including strong antioxidant activity (Chaovanalikit et al., 2004;Svarcova et al., 2007). BH extracts were shown to have the strongest antioxidant potential among 12 types of colored berries  and possess hepatoprotective effects (Palíková et al., 2009). However, as more detailed studies on these hepatoprotective effects are lacking, we aimed to evaluated the effect of BH extracts against CCl 4 -induced acute hepatic damage in mice (Ferreira et al., 2010;Wang et al., 2013); we examined three different extract types, BHw, BHj, and BHe, for their suitability as potent hepatoprotective medicinal foods.
The decreased body weight after the administration of CCl 4 is considered to result from the direct toxicity of CCl 4 and/or indirect toxicity related to the liver damage; hence, the change in body weight after CCl 4 treatment has been used as a valuable index in the efficacy test of CCl 4 -related organ damage (Pradeep, Mohan, Anand, & Karthikeyan, 2005;Yang, Li, Wang, & Wu, 2010). All mice in the intact control group in this study experienced normal increases in body weight within the range of normal age-matched mice of the same strain (Fox, Cohen, & Loew, 1984;Tajima, 1989).
In the current study, a significant decrease in body weight was have been used as serum markers to represent liver damage (Sodikoff, 1995); these enzymes are markedly elevated in CCl 4induced hepatic damage (Lu et al., 2016;Yang et al., 2015) and were elevated in the CCl 4 control in the present study. Therefore, the results of our study provided further evidence that all three BH extracts tested in this study exerted favorable hepatoprotective effects against CCl 4 -induced liver injuries; the strongest effect was induced by BHe, followed by BHj and BHw, as demonstrated by the marked and significant inhibitions on the CCl 4 -induced serum AST TA B L E 6 Immunohistochemistrical-histomorphometrical analysis of hepatic tissues from CCl 4 -treated mice

Positive cells by immunohistochemistry (cells/1000 hepatocytes)
Cleaved caspase-3 and ALT elevations in these groups compared with the CCl 4 control.
Better inhibition of the CCl 4 -induced elevation of serum AST and ALT were observed in mice treated with BHe 200 mg/kg compared with those treated with silymarin 100 mg/kg.
Considerable experimental and clinical evidence supports the prominent role of oxidative stress in the pathophysiological processes of liver injury related to CCl 4 exposure (Yang et al., 2015;Zou et al., 2016). Lipid peroxidation is an autocatalytic mechanism that leads to the oxidative destruction of cellular membranes (Subudhi, Das, Paital, Bhanja, & Chainy, 2008;Videla, 2000). Such destruction can lead to cell death and to the production of toxic and reactive aldehyde metabolites called free radicals, of which MDA is one of the most important (Messarah et al., 2010;Venditti & Di Meo, 2006). It is known that reactive oxygen species (ROS) lead to the oxidative damage of biological macromolecules, including lipids, proteins, and DNA (Das & Chainy, 2001;Messarah et al., 2010), and that oxidative stress also influences adipocytes, causing decreases in body fat mass and, subsequently, body weight decrease (Voldstedlund, Tranum-Jensen, Handberg, & Vinten, 1995). As MDA is a terminal product of lipid peroxidation, the content of MDA can be used to estimate the extent of lipid peroxidation (Messarah et al., 2010). Marked increases of liver MDA content have been observed in CCl 4 -treated animals (Yang et al., 2015;Zou et al., 2016); in this study, MDA was increased at 24 h after single IP treatment of CCl 4 0.5 ml/kg. GSH is a representative endogenous antioxidant, which prevents tissue damage by suppression of ROS levels; furthermore, at certain cellular concentrations, it is accepted to be a protective antioxidant factor in tissues (Odabasoglu et al., 2006). SOD is another antioxidant enzyme that contributes to the enzymatic defense mechanisms and the enzyme CAT is responsible for the conversion of H 2 O 2 to H 2 O (Cheeseman & Slater, 1993). Decreases in antioxidant enzyme activities, such as SOD and CAT, and decreases in the content of GSH may be indicative of the failure of cells to respond to the oxidative stress induced by CCl 4 (Yang et al., 2015;Zou et al., 2016). Trichloromethyl radicals also react with the sulfhydryl groups of GSH leading to its deactivation (Al-Sayed et al., 2014;Srivastava & Shivanandappa, 2010 As previously reported (Ki et al., 2013;Lee et al., 2014), vacuolation (the deposition of lipid droplets), the ballooning of hepatocytes, and inflammatory cell infiltration were detected after single IP injection of CCl 4 0.5 ml/kg, which are indications of classic centrolobular necrosis. Damaged hepatocytes were mainly located around the central veins and the fatty changed cells were marginally located.
CCl 4 treatment-related acute hepatic damages were confirmed by using HAI grading scores, based on the assessment of confluent necrosis, focal lytic necrosis, apoptosis, and focal and portal inflammation, which provides a well-established semi-quantitative histopathological scoring system in which higher grade scores indicate severe hepatitis (Ishak et al., 1995). PARP is a nuclear DNA-binding protein that functions in DNA base excision repair (Trucco, Oliver, de Murcia, & Menissier-de Murcia, 1998). PARP cleavage results in decrease enzymatic repair functions and contributes to the progression of apoptosis, although it is not strictly necessary for apoptosis to proceed (Smulson et al., 1998).
Caspase 3, a downstream effector caspase, is responsible for the cleavage of several critical nuclear targets in the apoptotic cascade, including the inhibitor of caspase-activated deoxynuclease, which results in nuclear fragmentation, and PARP, which results in a defective DNA repair function (Smyth, Berman, & Bursztajn, 2002). It has been reported that severe apoptosis of liver hepatocytes has been observed in CCl 4 -induced acute liver injury (Sun et al., 2011), and the inhibition of cleaved caspase-3 and PARP have been regarded as hepatoprotective indicators (Jiang et al., 2012;Talwar et al., 2013;Yu et al., 2014). In our study, increased cleaved caspase-3 and PARP immunoreactive hepatocytes were also demonstrated in the CCl 4 control, mainly in the centrolobular regions, compared with the intact control. Noticeably, all BH extracts significantly reduced the CCl 4 -induced increases in the number of cleaved caspase-3 and PARP immunolabeled hepatocytes; at 200 mg/kg, BHe exerted the strongest effects, followed by BHj and BHw. These immunohistochemical results on the hepatic cleaved caspase-3 and PARP immunostained cells provided direct evidence that the hepatoprotective effects on CCl 4 -induced acute hepatic damage exerted by all BH extracts tested in this study may occur through anti-apoptotic activity. BHe exerted the strongest effect, followed by BHj and BHw; in particularly, mice treated with BHe 200 mg/kg consistently demonstrated more favorable anti-apoptotic effects against CCl 4 treatment compared with those induced by silymarin.
NT is a product of tyrosine nitration mediated by reactive nitrogen species, such as the peroxynitrite anion and nitrogen dioxide. It is detectable in many pathological conditions, including CCl 4 -induced acute and acute hepatic damages, and is considered to be marker of NO-dependent, reactive nitrogen species-induced nitrative stress Pacher et al., 2007). 4-HNE is an α, β-unsaturated hydroxyalkenal produced by lipid peroxidation in cells; both these compounds are considered as possible causative agents of numerous diseases, including chronic inflammation, neurodegenerative diseases, adult respiratory distress syndrome, atherogenesis, diabetes, and different types of cancer Smathers et al., 2011). The metabolism of CCl 4 also initiates the peroxidation of polyunsaturated fatty acids that produce α, β-unsaturated aldehydes, including 4-HNE and malondialdehyde (Hartley, Kolaja, Reichard, & Petersen, 1999;Sigala et al., 2006).
In the present study, marked and significant increases of NT and 4-HNE immunostained hepatocytes were observed in the CCl 4 control compared with the intact control, but were significantly

| CON CLUS ION
Through the assessment of the key parameters of the hepatoprotective effects on CCl 4 -induced acute liver injury in mice, the present work demonstrated that the oral pre-administration of BHe, BHw, and BHj exerted favorable hepatoprotective effects through the activation of hepatic antioxidant defense systems; the strongest effects occurred in BHe, followed by BHw and BHj. In particular, BHe 200 mg/kg showed more favorable hepatoprotective effects compared with those of silymarin 100 mg/kg on CCl 4 -induced acute liver damage in mice in the current study. Therefore, BHe is a suitable candidate BH extract for the development of potent hepatoprotective medicinal foods.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.