Protection of Flos Lonicerae against acetaminophen-induced liver injury and its mechanism

This study aims to observe the protective action of Flos Lonicerae (FL) aqueous extract against acetaminophen (AP)-induced liver injury and its mechanism. Results show that FL decreases AP-increased serum alanine/aspartate transaminases (ALT/AST) activity, as well as total bilirubin (TB) amount, in mice. Histological evaluation of the liver further confirms the protection of FL against AP-induced hepatotoxicity. TdT-mediated biotin-dUTP nick-end labeling (TUNEL) assay shows that FL reduces AP-increased apoptotic cells. Furthermore, AP-decreased liver glutamate-cysteine ligase (GCL) enzymatic activity and glutathione (GSH) amount are both reversed by FL because of the increased expression of the catalytic sub- unit of GCL (GCLC) protein. The amount of chlorogenic acid (CGA), caffeic acid, and luteolin, the main active compounds in FL, is detected by high-performance liquid chromatography (HPLC). In addition, cell viability assay demonstrates that polyphenols in FL, such as CGA, caffeic acid, as well as isochlorogenic acids A, B, and C, can reverse AP-induced cytotoxicity. In conclusion, FL can prevent AP-induced liver injury by inhibiting apoptosis. The cellular antioxidant enzyme GCL is also involved in such protection. Polyphenols may be the main active hepato-protective ingredients in FL.

1. Introduction

Flos Lonicerae (FL), derived from the dried flower buds of Lonicera japonica Thunb., is a commonly used Chinese herbal medicine also known as Jin-Yin-Hua in Chinese Pharmacopoeia. In Chinese medicine, FL is characterized by cold temperature characteristic and is generally used under excessive heat con- ditions, such as fevers, skin rashes, and sore throat. These excessive heat conditions are essentially inflammatory pro- cesses involving heat, redness, pain, and swelling, which are often attributed to external pathogenic factors, such as bacte- ria and viruses. The cold nature of FL has made it a widely used clinical treatment for acute fever, headache, pharyngodynia, upper respiratory infection, and epidemic febrile diseases, etc. (The State Pharmacopoeia Committee of China, 2010; Shang et al., 2011). FL is also often used as raw material for the pro- duction of various health care products, such as tea, wine, and cola, which are commonly sold in Asian markets. The tea made from FL provides relief from summer heat. FL is also the major ingredient in several nutritional and health prod- ucts, such as ImmunoPhase®, that support natural immune function and maintain immune system.
Acetaminophen (AP), also called paracetamol, is a widely used over-the-counter (OTC) analgesic and antipyretic drug. The side effects of AP are mild or non-existent in rec- ommended doses, but AP overdose may result in severe hepatotoxicity because of its reactive metabolite N-acetyl-p- benzoquinone imine (NAPQI) (James et al., 2003). Hepatotoxi- city from AP is the most common cause of acute liver failure in both the United States and the United Kingdom (Lee, 2004; Larson et al., 2005). N-acetylcysteine (NAC), the precursor of cellular glutathione (GSH) synthesis, is the sole clinical anti- dote for AP-induced hepatotoxicity (Kelly, 1998).

Numerous anti-cold drugs and traditional Chinese medicines in the market, such as Tylenol, Panadol, and Vitamin C Yinqiao tablets, contain AP, and most of them are indexed in the OTC directory in China. In several cases, patients use a variety of anti-cold drugs, which increases the possibility of AP overdose. FL is also a com- mon anti-cold medicine used in various compounds, such as Shuang–huang–lian mixture and Qing–kai–ling oral liquid (Liu et al., 2011; Zhang et al., 2006). In addition, at least three com- pounds contain both AP and FL, which are indexed in Chinese Pharmacopoeia and other local Pharmacopoeia. The main compounds in FL are phenolic acids, including chlorogenic acid (CGA), isochlorogenic acids, and caffeic acid (Yuan et al., 2012). Among these compounds, CGA is a biomarker used by the Chinese Pharmacopoeia for evaluating FL quality (The State Pharmacopoeia Committee of China, 2010). Meanwhile, several studies have reported on the protective activity of CGA and caffeic acid against ischemia/reperfusion, lipopolysac- charide, or chemical-induced liver injury (Yun et al., 2012; Xu et al., 2010; Janbaz et al., 2004). The present study inves- tigates the protective effects of FL aqueous extract against AP-induced liver injury and its preliminary mechanism.

AP and other reagents, unless otherwise indicated, were purchased from Sigma Chemical Co. (St. Louis, MO). Rabbit antibodies against the catalytic subunit of glutamate–cysteine ligase (GCLC) and the regulatory subunit of glutamate- cysteine ligase (GCLM) were from Santa Cruz (Santa Cruz, CA). Peroxidase-conjugated goat anti-Rabbit IgG (H + L) was purchased from Jackson ImmunoResearch (West Grove, PA).

2.2. Preparation of FL aqueous extract powder

Dried FL (100 g) was heated under reflux with 1000 mL of dis- tilled water for 4 h. The extracts were filtered, and the residue was re-extracted under the same conditions. The extracts were combined, concentrated to 100 mL under reduced pres- sure, and further lyophilized to fine powder for long-term storage.

2.3. Experimental animals

Specific pathogen-free male ICR mice (16–20 g body weight) were purchased from Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China). All animals were fed with a standard laboratory diet and given free access to tap water. The animals received humane care in compliance with the institutional animal care guidelines approved by the Experimental Animal Ethical Committee of Shanghai University of Traditional Chinese Medicine. The ani- mal room was maintained at a temperature of 22 ± 1 ◦C and 65 ± 5% humidity, with a 12 h light–dark cycle.

2.4. Treatment of animals

Mice were divided into seven groups with eight mice each: (1) vehicle control; (2) AP (300 mg/kg); (3) AP (300 mg/kg) + FL (87.5 mg/kg); (4) AP (300 mg/kg) + FL (175 mg/kg); (5) AP (300 mg/kg) + FL (350 mg/kg); (6) AP (300 mg/kg) + NAC (600 mg/kg); and (7) FL (350 mg/kg). FL powder was dis- solved in 0.5% CMC-Na. Mice were pre-administered orally with various doses of FL for seven consecutive days. After the administration of FL for 1 h on the last day, mice were given a single dose of AP (300 mg/kg, intragastrically). Mice were killed 4 h after AP intoxication, and blood and livers were collected.

2. Materials and methods

2.1. Materials, chemicals, and reagents

FL was purchased from Shanghai Kangqiao Herbal Pieces Co. Ltd. and identified as Lonicera japonica Thunb. The voucher sample was deposited in the Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine. CGA, caffeic acid, luteolin and other chemical compounds with 98% purity were purchased from Shanghai Hitsanns Co. Ltd. (Shanghai, China) or Internet Aladdin Reagent Database Inc. (Shanghai, China). TdT-mediated biotin-dUTP nick-end labeling (TUNEL) kit was purchased from Merck Calbiochem (Darmstadt, Germany). The kits for determining serum ALT/AST activity and TB amount were obtained from the Shanghai Rongsheng Biotech Corporation (Shanghai, China).

2.5. ALT, AST, and TB assay

Blood obtained from the mice was placed at room temper- ature for 60 min to clot. After centrifugation at 3000 × g for 15 min, the serum was collected in new tubes. Serum ALT and AST activity, as well as TB amount, were determined by their respective kits according to the manufacturer’s instructions.

2.6. Histological observation

Liver slices were fixed in 10% phosphate-buffered saline (PBS)- formalin and embedded in paraffin for further histological assessment of tissue damage. Samples were subsequently sectioned (5 µM), stained with hematoxylin and eosin, and examined under a light microscope (Olympus, Japan) to eval- uate liver damage.

2.7. TUNEL assay

Apoptotic hepatocytes were labeled in situ using a TUNEL apoptotic detection kit according to the manufacturer’s instructions. The procedure was described as follows: slides of paraffin-embedded liver slices were deparaffinized and rehy- drated, and the entire slide was covered with 100 µL proteinase K (20 µg/mL) for 15 min at room temperature. After washing in PBS, the slides were titrated with 3% H2O2 to inactivate the endogenous peroxidases, followed by the incubation with TdT enzyme at 37 ◦C for 90 min in the dark. The labeling reaction was then stopped and detected by blocking buffer with perox- idase streptavidin conjugate solution. Thereafter, the entire slide was incubated with 100 µL DAB solution at room tem- perature and counterstained by methyl green. Finally, a light microscope (Olympus, Japan) was used for the observation. A dark brown DAB signal indicates positive staining (apoptotic cells). The apoptotic hepatocytes were counted manually in at least three random fields per slide at a magnification of ×200.

2.8. Western-blot analysis

Liver tissues (approximately 50 mg) were homogenized in ice- cold lysis buffer containing 50 mM Tris (pH 7.5), 1 mM EDTA, 150 mM NaCl, 20 mM NaF, 0.5% NP-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 10 µg/mL pepstatin A. The homogenate was centrifuged at 3000 × g for 3 min, and then the supernatants were transferred to new tubes. Proteins were separated by SDS-PAGE and probed with the appropriate combination of primary and HRP-conjugated secondary antibodies.

2.9. Measurement of liver glutamate-cysteine ligase (GCL) activity

Livers were homogenized in ice-cold PBS (pH 7.0), and soni- cated at 4 ◦C twice. The homogenate was centrifuged at 4 ◦C and 10,000 × g for 15 min, and then supernatants were used for GCL activity assay. GCL activity was determined and ana- lyzed according to the procedures described in our previously published paper (Liang et al., 2011).

2.10. Measurement of liver glutathione (GSH) and oxidized GSH (GSSG) amounts

The amounts of liver reduced GSH and GSSG were measured as described in our previously published paper (Liang et al., 2011).

2.11. High-performance liquid chromatography (HPLC) analysis

Chromatography was performed on a Shiseido C18 column (4.6 mm × 250 mm, 5 µm) at temperature of 30 ◦C. The wave- length used to detect CGA, caffeic acid, and luteolin was set at 327 nm. The mobile phase consisted of a mixture of acetonitrile – 0.4% H3PO4 adjusted to pH 3.0 with sodium hydroxide (0.2 g/mL), and at 0–10 min (10:90, v/v), 10–20 min (10:90, v/v), 20–40 min (30:70, v/v). The mobile phase was prepared daily, filtered under vacuum through a 0.45 µm mem- brane filter, and degassed before use. The flow rate was set at 1.0 mL/min. FL powder was dissolved in methanol and filtered through a 0.22 µm membrane filter before loading into the HPLC system.

Fig. 1 – FL decreased AP-induced increase in serum ALT/AST activity and TB amount. (A) Serum ALT and AST activity. (B) Serum TB amount. Data were expressed as means ± SEM (n = 8). ***P < 0.001 compared to control; #P < 0.05, ##P < 0.01 compared to AP.

The CGA, caffeic acid and luteolin contents in FL were deter- mined using the external standard method and expressed as milligram of CGA per gram of FL. The peak area of CGA, caf- feic acid and luteolin in FL was calculated as the means of three parallel measurements for CGA, caffeic acid and luteloin quantification. The peak of CGA, caffeic acid and luteolin in the FL powder solvent was identified by comparing the retention time with that of the CGA, caffeic acid and luteolin references.

Fig. 2 – Histological observation of liver injury. After given AP for 4 h, the livers were removed, fixed, sectioned (5 µm) and processed for staining with hematoxylin and eosin. Typical images were chosen from each experimental group. (A) Vehicle control, (B) AP (300 mg/kg), (C) AP (300 mg/kg) + FL (87.5 mg/kg), (D) AP (300 mg/kg) + FL (175 mg/kg), (E) AP (300 mg/kg) + FL (350 mg/kg), (F) FL (350 mg/kg) and (G) AP (300 mg/kg) + NAC (600 mg/kg) (magnification ×100).

2.12. Cell viability assay

Human normal liver L-02 cells were seeded into 96-well plates at a density of 1 × 104 cells per well. Cells were pre- treated with various compounds (50 µM) for 15 min, and then incubated with AP (10 mM) for 48 h. After treatments,cells were incubated with 500 µg/mL 3-(4,5-dimethylthiazol- 2-yl) 2,5-diphenyltetrazolium bromide (MTT) for 4 h. The functional mitochondrial succinate dehydrogenases in sur- vival cells can convert MTT to formazan, which generates a blue color. Finally, formazan was dissolved in 10% SDS–5% isobutanol–0.01 M HCl. The plates were read at 570 nm with 630 nm as a reference, and cell viability was normalized as a percentage of control.

Fig. 3 – FL prevented AP-induced apoptotic cell death. (A) TUNEL assay. After given AP for 4 h, the livers were removed, fixed, sectioned (5 µm) and processed for TUNEL assay. (a) Control, (b) AP (300 mg/kg), (c) AP (300 mg/kg) + FL (350 mg/kg) and (d) FL (350 mg/kg). Typical images were chosen from each experimental group (magnification ×200). (B) The apoptotic hepatocytes were counted manually in at least three random fields every section. Data were expressed as means ± SEM (n = 4). ***P < 0.001 compared to control; ###P < 0.001 compared to AP.

2.13. Statistical analysis

Data were expressed as means ± standard error of the mean (SEM). The significance of differences between groups was evaluated by one-way ANOVA with LSD post hoc test; and P < 0.05 indicated statistically significant differences.

3. Results

3.1. FL decreased AP-induced the increase in ALT, AST activity and TB amount

As shown in Fig. 1A, FL (350 mg/kg) decreased the AP-induced elevation of ALT and AST enzymatic activity (P < 0.05, P < 0.01), two important biomarkers that reflect liver injury. NAC, the commonly used antidote for AP in clinic, also decreased AP-induced increase of ALT activity (P < 0.05). Meanwhile, Fig. 1B showed that FL (350 mg/kg) and NAC (600 mg/kg) both decreased the AP-induced increase in serum TB amount (P < 0.05, P < 0.01). However, FL (87.5 mg/kg and 175 mg/kg) had no effect on AP-induced the increase of ALT, AST and TB.

3.2. Liver histological evaluation of AP-induced liver injury

As shown in Fig. 2B, the mice treated with AP (300 mg/kg) demonstrated severe liver damage, as indicated by intrahep- atic hemorrhage, lymphocyte infiltration, and destruction of the liver structure. However, various FL doses ameliorated such liver injury (Figs. 2(C)–(E), of which FL at 350 mg/kg
showed the best effect (Fig. 2E). FL (350 mg/kg) itself demon- strated not much different from normal control mice (Fig. 2F). NAC (600 mg/kg) also obviously ameliorated AP-induced liver injury (Fig. 2G).

3.3. FL prevented AP-induced hepatic apoptosis

TUNEL assay is one of the main methods used to detect apo- ptosis. The results of TUNEL assay (Figs. 3A) showed that there was increased brown-stained apoptotic hepatocytes in AP- treated mice, whereas FL (350 mg/kg) decreased the increased apoptotic hepatocytes. Furthermore, the results of apoptotic cell counting (Fig. 3B) showed that AP increased the number of apoptotic hepatocytes (P < 0.001), whereas FL (350 mg/kg) obviously decreased such increase (P < 0.001).

3.4. FL reversed AP-induced the decrease in GCL enzymatic activity and GSH amount

As shown in Fig. 4A, AP decreased the enzymatic activity of GCL (P < 0.001), whereas FL (350 mg/kg) prevented such decrease (P < 0.01). GCL enzyme is composed of GCLC and GCLM subunits. Furthermore, Fig. 4B reveals that GCLC and GCLM protein expressions both decreased in AP-treated mice (P < 0.05, P < 0.001), whereas FL (350 mg/kg) prevented AP- decreased protein expression of GCLC, but not of GCLM (P < 0.05). Furthermore, results of liver GSH and GSSG amount assay (Fig. 4C) showed that although AP evidently decreased liver GSH amount (P < 0.001), it had no effect on GSSG amount. FL (350 mg/kg) reversed the AP-induced decrease in GSH level (P < 0.05).

3.5. The amount of CGA, caffeic acid and luteolin in FL was detected by HPLC

The amount of CGA, caffeic acid, luteolin in FL was analyzed by HPLC, and their chemical structure was shown in Fig. 5A. Fig. 5B showed the HPLC chromatogram of reference standard of CGA, caffeci acid, and luteolin. Fig. 5C showed the HPLC chromatogram of CGA, caffeic acid, and luteolin in FL. Through analysis, the amount of CGA, caffeic acid, and luteolin in FL was measured as 5.43%, 0.069%, 0.024%, respectively.

3.6. Effects of various compounds in FL on AP-induced hepatotoxicity

We observed the preventive action of CGA and other com- pounds, which are all distributed in FL, against AP-induced hepatotoxicity. Table 1 showed that CGA, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and caffeic acid all reversed AP-induced cytotoxicity in human normal liver L-02 cells (P < 0.01, P < 0.001). However, other compounds, including flavonoids such as hyperoside, luteolin, luteoloside, iridoids such as swertiamarin, and volatile oils such as geran- iol, and linalool, had no evident protection against AP-induced hepatotoxicity (Tables 1 and 2).

Fig. 5 – HPLC chromatogram of chlorogenic acid (CGA), caffeic acid and luteolin in FL. (A) Chemical structure of CGA, caffeic acid, and luteolin. (B) HPLC chromatogram of CGA (a), caffeic acid (b), and luteolin (c) in FL. The general HPLC condition was as described in Section 2.

4. Discussion

ALT, AST, and TB are commonly used as biomarkers and are generally increased in chemical toxin-induced liver injury; such toxins include carbon tetrachloride, AP, and α- naphthylisothiocyanate (Jayakumar et al., 2006; Ding et al., 2012; Zhao et al., 2011). The results of serum ALT/AST activity, and TB amount analysis demonstrated that FL can pre- vent AP-induced liver injury. Meanwhile, the protection of FL against AP-induced liver injury is further confirmed by the liver histological evaluation. As a traditional anti-cold drug, the anti-inflammatory and anti-bacterial activities of FL have already been reported in previous studies (Xiong et al., 2013; Park et al., 2005, 2012b). Recent studies show that FL extract can induce cell cycle arrest and apoptosis in hepatoma HepG2 cells (Park et al., 2012a) and prevent hydrogen peroxide- induced apoptosis in human neuroblastoma SH-SY5Y cells (Kwon et al., 2011), which may be due to the different cell line, dosage or stimuli. The present study demonstrates the protection of the aqueous extract of FL against AP-induced liver injury. Our results suggest that FL may have nutritional hepato-protective function and will be helpful for enhancing the capacity of the liver to prevent exogenous toxin-induced liver injury.

Apoptosis has been involved in various mechanisms of liver disease progression (Guicciardi and Gores, 2012). In situ detection of apoptosis by the TUNEL assay is a commonly used method to detect apoptotic cells (Loo, 2011). Previous studies have not drawn a conclusion about whether AP-induced liver injury is due to cell death or apoptosis, but several reports evidenced the involvement of apoptotic signals in regulat- ing AP-induced liver injury (Jaeschke and Bajt, 2006; Bantel and Schulze-Osthoff, 2012). Meanwhile, various published papers demonstrated that L-2-oxothiazolidine-4-carboxylate, fucoidan, and hesperidin can alleviate AP-induced liver injury partly via abrogating apoptosis (Choi et al., 2013; Hong et al., 2012; Ahmad et al., 2012). The results of our TUNEL staining assay demonstrated that FL prevented AP-induced apoptotic liver cell death.

GCL is the rate-limiting enzyme for the biosynthesis of cellular GSH, which is an important antioxidant involved in numerous cellular activities, including detoxification, antiox- idant defense, and maintenance of cellular redox status (Lu, 2009). The heterodimeric GCL is composed of GCLC and GCLM, each encoded by a unique gene, and generally the increased expression of GCLC or GCLM protein will contribute to GCL activity (Soltaninassab et al., 2000; Wild and Mulcahy, 2000; Franklin et al., 2009). Meanwhile, several studies have reported on the regulation of numerous stimuli on its activity (Soltaninassab et al., 2000; Wild and Mulcahy, 2000; Franklin et al., 2009). GCL is reported to play important roles in reg- ulating AP-induced liver injury, and the enzymatic activity and protein expression of GCL are both decreased after AP administration (Akai et al., 2007; Shinohara et al., 2010). Our results showed that FL prevented AP-induced decrease in GCL enzymatic activity, which may be due to the increased expression of the GCLC protein. Further results demonstrated that FL reversed the decrease of GSH level induced by AP, which suggests that FL may prevent AP-induced liver injury via enhancing GSH amount by increasing GCL enzymatic activity. Thus, our results demonstrate that GCL performs crucial func- tions in regulating the prevention of FL against AP-induced liver injury.

Previous studies have shown that the main compounds in FL are phenolic acids, flavonoids, iridoids, and saponins (Chen et al., 2007; Tang et al., 2008). The results of the protec- tion of various compounds against AP-induced hepatotoxicity showed that all effective compounds belong to phenolic acids, which have already been reported to prevent liver injury induced by various chemical agents such as carbon tetrachlo- ride, thioacetamide, and others (Mancini-Filho et al., 2009; Wu et al., 2007). Additionally, CGA and caffeic acid have already been reported to prevent AP-induced liver injury (Ji et al., 2013; Janbaz et al., 2004). Our results suggest that pheno- lic acids, including CGA and caffeic acid, may be the main hepato-protective compounds against AP-induced hepatotox- icity in FL. Phenolic acids are well-known antioxidants and are generally used as nutritional supplements to enhance the antioxidant capacity of body. Our results further evidenced the nutritional value of such phenolic acids as the hepato- protective ingredients.

Overall, the present study shows that FL ameliorates AP- induced liver injury by preventing apoptotic cell death and increasing the expression of cellular important antioxidant enzyme GCL. This research also demonstrates that CGA and other phenolic acids are the main hepato-protective com- pounds against AP-induced hepatotoxicity in FL. The present study is the first report about the protection of FL aganist AP-induced hepatotoxicity, which indicates that the frequent supplementation of FL and its ex229 related health care products may be beneficial for protection against AP-induced liver injury.