Effects of Andrographis paniculata on α–Naphthylisothiocyanate

(ANIT)-Induced Cholestatic Liver Injury in Rats

Tanaporn Khamphaya1, Pawinee Piyachaturawat1, 2and Jittima Weerachayaphorn2

ABSTRACT

Andrographis paniculata (Burm. f.) Nees (A. paniculata) or Fa Tha Lai Jon is a medicinal

plant, which has traditionally been used in medicinal system of many Asian countries. It has an anti-

inflammatory effect and protects liver injuries. The present study aimed to investigate the

hepatoprotective effect of A. paniculata against α-naphthylisothiocyanate (ANIT)-induced intrahepatic

cholestasis in adult male rats. The A. paniculata ethanol extract was orally administered to rats for 4

consecutive days. On day-2, rats were intraperitoneally injected with ANIT to induce intrahepatic

cholestasis. ANIT markedly increased serum activities of alanine aminotransferase (ALT) and alkaline

phosphatase (ALP), which were accompanied with a massive inflammation of epithelial cells on bile

duct at 48 hours after ANIT injection. Treatment with the A. paniculata extract substantially reduced

serum ALT and ALP with minimal bile duct damage in the ANIT treated rats. A. paniculata also

decreased the hepatic mRNA expression of TNF-α and MCP-1 in rats treated with ANIT. Taken

together, our results suggest that A. paniculata possesses the hepatoprotective potential against

cholestatic liver injury insulted by hepatotoxin such as ANIT.

Key words: Andrographis paniculata, andrographolide, cholestasis, ANIT, TNF-α, MCP-1

e-mail address: jittima.wee@mahidol.ac.th 1Toxicology Graduate Program, Faculty of Science, Mahidol University, Bangkok, 10400 2Department of Physiology, Faculty of Science, Mahidol University, Bangkok, 10400

 

INTRODUCTION

Andrographis paniculata (Burm. f.) Nees (A. paniculata) or Fa Tha Lai Jon belongs to

Acanthaceae family, which has traditionally been used for the treatment of common colds and upper

respiratory tract infections (Caceres et al., 1997). It has a broad range of pharmacological activities

such as anti-inflammatory (Yang et al., 2013) and hepatoprotective effects. Therapeutic efficacy of A.

paniculata extract is mainly attributed to andrographolide. Andrographolide has been reported to

have anti-hepatotoxic activity against ethanol- (Singha et al., 2007), carbon tetrachloride- (CCl4)

(Akowuah et al., 2009; Ye et al., 2011), concanavalin-A- (Shi et al., 2012) and acetaminophen-

induced liver injury (Shukla et al., 1992; Visen et al., 1993). However, the effect of A. paniculata on

cholestasis has not been investigated yet.

Cholestasis is a clinical disorder defined as an impairment of bile flow, and that leads to toxic

bile acid accumulation in hepatocytes (Tanaka et al., 2009). The accumulation of potentially toxic bile

acids results in hepatocellular damage followed by inflammation, fibrosis and liver cirrhosis (Jin et al.,

2013). Currently, only ursodeoxycholic acid (UDCA) is available for primary biliary cirrhosis (PBC)

treatment. However, the potential beneficial effects of UDCA are limited only in the early stage of PBC

(He et al., 2011). Therefore, the search for novel therapeutic compounds is still needed.

Alpha-naphthylisothiocyanate (ANIT) is a widely used chemical to induce intrahepatic

cholestasis in experimental animals (Xu et al., 2004). ANIT is conjugated with glutathione in

hepatocytes and transported into bile through the canalicular efflux transporter, Mrp2. ANIT

conjugated with glutathione falls apart when get into the bile. The rapid accumulation and high

concentration of ANIT in bile directly damages bile duct epithelial cells. In response to ANIT, bile duct

epithelial cells release pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and

monocyte chemoattractant protein-1 (MCP-1). An increase in inflammatory cytokines results in

migration and infiltration of neutrophils leading to lipid peroxidation and bile duct epithelial cell

damage. (Jean and Roth, 1995; Hill et al., 1999; Sullivan et al., 2010).

Since A. paniculata has a hepatoprotective property against liver injury induced by various

toxicants, it appears to be an attractive compound for therapy of cholestatic liver diseases. To

determine the protective effect of A. paniculata on the intrahepatic cholestasis, we used ANIT as the

model of intrahepatic cholestasis in rats. The results obtained from this study will provide a novel

therapeutic option of A. paniculata for intrahepatic cholestasis diseases.

MATERIALS AND METHODS

1. Plant extract

The A. paniculata ethanol extract was obtained from Assoc. Prof. Noppamas

Soonthomchareonnon, Faculty of Pharmacy, Mahidol University.

2. Animals and treatments

The experimental protocol was approved by the Animal Care and Use Committee, Faculty of

Science, Mahidol University. Four-week-old male Wistar rats weight between 170 and 200 grams

were obtained from the National Laboratory Animal Center, Bangkok, Thailand. Animals were housed

in Faculty of Science Animal facility, kept in controlled light dark cycle (12-h light:Dark) and

temperature (25°±2C), and provided with food and water ad libitum. Animals were acclimatized for at

least 5 days prior to the experiments.

Rats (4 animals per group) were received daily oral gavage with 1% carboxymethyl cellulose

(CMC) as a control group or A. paniculata at a dose of 200 mg/kg BW for 4 consecutive days as an

experimental group. On day 2, rats were intraperitoneally injected with ANIT at a dose of 75 mg/kg

BW. All animals were sacrificed at 48 hours after ANIT injection. Blood sample and the part of liver

were collected for further biochemical and histological studies.

3. Biochemical analysis

Blood sample was collected from posterior aorta and the serum was separated by

centrifuged at 3000 rpm for 10 minuets at 4 °C. Serum alanine aminotransferase (ALT) and alkaline

phosphatase (ALP) levels were measured using a commercially available clinical test kit (Human

Society for biochemical and diagnostic, Germany).

4. Liver histology

Liver tissues taken from the largest lobe were fixed in 10 % buffer formalin for 24 hours and

then embedded in paraffin. Five micron-thick sections were obtained and performed hematoxylin and

eosin (H&E) staining. All sections were examined and photographed under light microscope using

Nikon Eclipse E600 microscope-fitted with Nikon digital camera DXM1200.

5. TaqMan real-time PCR

Total RNA was extracted from rat liver tissues by Trizol reagent according to the

manufacturer’s instructions. Five micrograms of total RNA were converted to cDNA by the RNA to

cDNA EcoDryTM Premix (Oligo dT) kit (Clontech, CA, USA). TaqMan real-time PCR primers, TNF-α,

MCP-1 and Gapdh were obtained from Applied Biosystems (ABI), and the real-time PCR reaction was

performed on the ABI 7500 Sequence Detection System.

6. Statistical analysis

All data are expressed as the mean ± standard deviation (S.D). Statistical analyses were

performed using ANOVA with the Dunnett’s post-hoc test (Graphpad Prism, La Jolla, CA). Differences

with p < 0.05 were considered to be statistically significant.

 

RESULTS AND DISCUSSION

1. Effects of A. paniculata extract on ANIT-induced cholestasis liver injury in rats. As shown in Figure 1 A and B, after treatment with ANIT for 48 hours, serum ALT and ALP

levels were markedly increased, indicating liver cell membrane damages. Pretreatment of A.

paniculata extract significantly decreased serum ALT level and tended to decrease ALP level in ANIT

treated rats. These results are in line with earlier reports showing that A. paniculata extract and

andrographolide reduce increases in ALT and ALP levels in several liver injury models including

ethanol- (Singha et al., 2007), carbon tetrachloride- (Akowuah et al., 2009; Ye et al., 2011),

concanavalin-A- (Shi et al., 2012) and acetaminophen-induced liver injury (Shukla et al., 1992; Visen

et al., 1993).

Figure 1 Effect of A. paniculata on (A) ALT, (B) ALP and (C) histological liver section in ANIT-induced

cholestatic liver injury in rats. Rats were received A. paniculata at a dose of 200 mg/kg BW

for 4 consecutive days and followed by intraperitoneally injected with ANIT (75 mg/kg BW)

on day 2. All animals were sacrificed at 48 hours after ANIT injection. *p < 0.05 vs. control. #p < 0.05 vs. ANIT. Arrow indicates bile duct damage around portal area, PV = portal vein,

CV = central vein. (n=4, original magnification x10).

Histological evaluation provided evidence of the protective effect of A. paniculata on ANIT-

induced cholestasis. The H&E staining liver sections exhibited normal morphological structure in

control rats. The liver specimens of ANIT-induced cholestasis rats revealed degenerative changes of

bile duct. In contrast, pretreatment of A. paniculata to ANIT treated rats completely prevented

histological damages (Figure 1C). Taken together, these findings clearly showed that A. paniculata

protected liver injury in ANIT-induced cholestatic liver injury in rats.

2. Effects of A. paniculata on TNF-α and MCP-1 mRNA expressions in ANIT-induced cholestasis in rats.

Since ANIT results in inflammatory cytokine accumulation, we further investigated the effects

of A. paniculata on hepatic TNF-α and MCP-1 mRNA expressions. We found that TNF-α and MCP-1

were significantly induced in the ANIT treated rats. In contrast, the expression of TNF-α and MCP-1

was suppressed by A. paniculata extract, suggesting that A. paniculata extract could inhibit the

inflammatory cytokine release and that protect liver injury induced by ANIT. This result is in agreement

with previous studies reported that andrographolide, an active compound of A. paniculata extract, can

inhibit the generation of TNF-α and exert the anti-inflammatory activity in an allergic lung inflammation

model (Abu-Ghefreh et al., 2009).

Figure 2 Effects of A. paniculata on hepatic (A) TNF-α and (B) MCP-1 mRNA expressions in ANIT

induced cholestasis in rats. Rats were received A. paniculata for 4 consecutive days and

followed by intraperitoneally injected with ANIT on day 2. Total RNA was extracted from liver

tissues and converted to cDNA. TaqMan real-time PCR reaction was performed on the ABI

7500 Sequence Detection System. * p < 0.05 vs. control. (n=4)

 

CONCLUSION

In summary, our findings provided evidence that A. paniculata extract could ameliorate the

ANIT-induced cholestatic liver injury in rats.

ACKNOWLEDGEMENTS

This research project was supported by Science Achievement Scholarship of Thailand (SAST)

and the Center of Excellence on Environmental Health, Toxicology, Science & Technology

Postgraduate Education and Research Development Office (PERDO).

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