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. Author manuscript; available in PMC: 2024 Jan 23.
Published in final edited form as: Eur J Pharmacol. 2016 Jul 27;789:254–264. doi: 10.1016/j.ejphar.2016.07.032

Effects of andrographolide on intrahepatic cholestasis induced by alpha-naphthylisothiocyanate in rats

Tanaporn Khamphaya a, Piyachat Chansela b, Pawinee Piyachaturawat c, Apichart Suksamrarn d, Michael H Nathanson e, Jittima Weerachayaphorn c,e,*
PMCID: PMC10804355  NIHMSID: NIHMS1955759  PMID: 27475677

Abstract

Cholestasis is a cardinal manifestation of liver diseases but effective therapeutic approaches are limited. Therefore, alternative therapy for treating and preventing cholestatic liver diseases is necessary. Andrographolide, a promising anticancer drug derived from the medicinal plant Andrographis paniculata, has diverse pharmacological properties and multi-spectrum therapeutic applications. However, it is unknown whether andrographolide has a hepatoprotective effect on intrahepatic cholestasis. The aims of this study were to investigate the protective effect and possible mechanisms of andrographolide in a rat model of acute intrahepatic cholestasis induced by alpha-naphthylisothiocyanate (ANIT). Andrographolide was administered intragastrically for four consecutive days, with a single intraperitoneal injection of ANIT on the second day. Liver injury was evaluated biochemically and histologically together with hepatic gene and protein expression analysis. Rats pretreated with andrographolide prior to ANIT injection demonstrated lower levels of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, as well as bilirubin and bile acids as compared to rats treated with ANIT alone. Andrographolide also decreased the incidence and extent of periductular fibrosis and bile duct proliferation. Analysis of protein expression in livers from andrographolide-treated cholestatic rats revealed markedly decreased expression of alpha-smooth muscle actin and nuclear factor kappa-B (NF-κB). In conclusion, andrographolide has a potent protective property against ANIT-induced cholestatic liver injury. The mechanisms that underlie this protective effect are mediated through down-regulation of NF-κB expression and inhibition of hepatic stellate cell activation. These findings suggest that andrographolide could be a promising therapeutic option in prevention and slowing down the progression of cholestatic liver diseases.

Keywords: Cholestasis, Andrographolide, Alpha-naphthylisothiocyanate, Bile duct, Hepatic stellate cells, Bile acid transporters

1. Introduction

Cholestasis is associated with impaired hepatocellular secretion of bile, resulting in intrahepatic accumulation of toxic biliary constituents, such as bile acids and bilirubin, with progressive liver injury (Trauner et al., 1998; Wagner et al., 2009). Prolonged cholestasis leads to progressive liver fibrosis and ultimately to end-stage liver disease, which is the main indication for liver transplantation (Carey and Lindor, 2012). Nearly one in five liver transplants in the United States is for chronic cholestatic conditions (Carbone and Neuberger, 2011). Currently, ursodeoxycholic acid (UDCA) is the only FDA-approved drug and only therapeutic choice for patients with cholestatic liver diseases (Paumgartner and Beuers, 2002; Trauner and Graziadei, 1999). However, not all cholestatic patients respond well to UDCA and the risk for disease progression still remains (Lindor et al., 2009). Therefore, there is no uniformly effective treatment for cholestasis, and additional therapeutic or preventive approaches for patients with cholestatic liver diseases are desirable.

The pathogenesis of cholestatic liver injury involves inflammation. During cholestasis, hepatocyte injury leads to an inflammatory response and the release of pro-inflammatory mediators, resulting in stimulation of extracellular matrix (ECM) synthesis by activation of quiescent hepatic stellate cells (HSC) and portal fibroblasts (Bataller and Brenner, 2005). The excessive synthesis of ECM proteins subsequently leads to the formation of scar tissue and development of liver fibrosis. Suppression of both inflammatory process and activation of HSC would be promising therapeutic targets to prevent liver fibrosis in cholestatic liver diseases. Thus, small molecules or natural compounds that possess anti-inflammatory activity may provide effective therapeutic approaches that help prevent the progression of cholestatic liver injury and improve liver function.

Andrographolide, a labdane diterpenoid that is the major active compound isolated from medicinal plant Andrographis paniculata (Burm.f.) Nees, has a broad range of therapeutic applications including anti-inflammatory (National Center for Biotechnology Information. PubChem Compound Database), immunostimulatory (Kumar et al., 2004), antiviral (Lee et al., 2014) and cardioprotective (Woo et al., 2008). In the last several years, increasing attention has been paid to andrographolide because of its diverse therapeutic properties, including its potential antineoplastic properties in breast cancer (Chao et al., 2013) and colon cancer (Chao et al., 2010; Jada et al., 2008). The pharmacological value of andrographolide’s liver protective property has been increasingly recognized as well. Andrographolide can protect against hepatocellular damage caused by carbon tetrachloride (Ye et al., 2011), acetaminophen (Roy et al., 2013), concanavalin-A (Burgos et al., 2005) and hexachlorocyclohexane (Trivedi et al., 2007). However, little is known about its exact mechanism of action. In addition, whether andrographolide could protect against intrahepatic cholestasis has not been investigated yet. Combining data from multiple experiments regarding potential therapeutic value of andrographolide, particularly in the liver, we hypothesized that andrographolide would be an effective pharmacological treatment to prevent cholestatic liver injury and fibrosis. We tested this hypothesis by evaluating the effect of andrographolide at preventing cholestatic liver injury by ANIT, which is an experimental animal model mimicking intrahepatic cholestasis in human, and further determined how andrographolide could protect against cholestatic liver injury.

2. Materials and methods

2.1. Reagents

Chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Andrographolide was provided by Dr. Apichart Suksamrarn, Ramkhamhaeng University, Thailand. Monoclonal antibody to alpha-smooth muscle actin (α-SMA) was obtained from Sigma-Aldrich. Multidrug resistance protein 2 (ABCC2) and multidrug resistance protein 3 (ABCC3) antibodies were purchased from ALEXIS Biochemicals (San Diego, CA, USA) and Abcam (Cambridge, MA, USA), respectively. Polyclonal antibodies against bile salt export pump (ABCB11) and multidrug resistance protein 4 (ABCC4) were purchased from Kamiya Biomedical (Seattle, WA, USA) and Everest Biotech (Oxfordshire, UK), respectively. NF-κB (p50), NF-κB (p65) and proliferating cell nuclear antigen (PCNA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

2.2. Ethics statement

The study protocol (animal protocol no. MUSC54-027-273) was approved by the Institutional Animal Care and Use Committee of the Faculty of Science, Mahidol University. All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health.

2.3. Animals and experimental protocols

Adult male Wistar rats (6–7 weeks old) weighing 170–200 g were obtained from the National Laboratory Animal Center, Nakhon Pathom, Thailand. All rats were housed in the animal facility under controlled temperature (22 ± 2 °C) with a 12-h dark-light cycle, and were provided with food and water ad libitum. Animals were acclimatized for 5 days prior to the experiments. Rats were randomized and pretreated orally with andrographolide at a dose of 50 and 100 mg/kg body weight that is known to be the pharmacological dose and in the safe dose range or the solvent control (1% carboxymethylcellulose, CMC) for 2 days prior to intraperitoneal injection of ANIT (75 mg/kg body weight in olive oil) to induce intrahepatic cholestasis for 48 h. The 48-h time point was chosen because serum levels of ALT and ALP reached peak at 48 h after ANIT injection (Table 1). Total duration for andrographolide treatment was 4 days. Four experimental groups (n=6–10 animals per group) were assigned as follows: 1) CMC – olive oil (Control); 2) CMC – ANIT; 3) andrographolide (50 mg/kg body weight in CMC) – ANIT, and 4) andrographolide (100 mg/kg body weight in CMC) – ANIT. At 48 h after ANIT administration, animals were fasted overnight prior to being killed. Serum and liver samples were collected. Serum liver enzyme measurements for clinical chemistry parameters indicative of liver damage were made immediately after processing of blood samples. Liver tissues were snap frozen in liquid N2 and stored in a −80 °C freezer. A part of the liver from each rat was fixed in 10% neutral buffered formaldehyde for histological study.

Table 1.

Changes in serum levels of ALT and ALP in rats given a single intraperitoneal injection of ANIT at a dose of 75 mg/kg body weight at different time points.

Time (h) ALT (U/L) ALP (U/L)
0 (Basal level) 26.37 ± 2.36 348.30 ± 8.75
24 116.32 ± 52.76 968.86 ± 12.01a
48 1309.40 ± 425.30a 1451.91 ± 108.30a
72 572.14 ± 207.10a 1254.44 ± 165.60a
96 167.74 ± 176.50 978.28 ± 149.90a
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a

P < 0.05 versus 0 h (basal level).

2.4. Hepatocellular function

The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), total bilirubin (TBIL) and direct bilirubin (DBIL) were measured as indicators of hepatic and bile duct injury using commercial kits from Human Gesellschaft für Biochemica and Diagnostica mbH, Wiesbaden, Germany. Hepatic bile acids were measured as previously described (Mennone et al., 2006). Bile acid concentrations in the serum and liver were determined using a commercial total bile acid kit (Diazyme Laboratories, Poway, CA, USA).

2.5. Liver histology and immunohistochemical analysis

Formalin-fixed liver was embedded in paraffin, sectioned and stained with hematoxylin and eosin. Immunohistochemical analysis of α-SMA and PCNA was performed on paraffin embedded rat liver sections. The liver tissues were deparaffinized in xylene and rehydrated through a graded alcohol series. Antigen retrieval using 10 mM sodium citrate (pH 6.0), in the microwave oven for 20 min (750 W) was done prior to overnight incubation with anti α-SMA or anti-PCNA antibodies. Tissue sections were followed by incubation with horseradish peroxidase (HRP)-linked anti-mouse IgG for 1 h at room temperature. Finally, tissue sections were developed for 60 s using the NovaRED staining kit (Vector Laboratories, Burlingame, CA, USA), and counterstained with hematoxylin. Tissue slides were examined and imaged using a Nikon Eclipse E600 microscope-fitted with Nikon digital camera DXM1200 (Nikon Inc., Melville, NY). All images were captured using the same setting, and images were processed with Adobe Photoshop CS6 (Mountain View, CA, USA).

2.6. Immunoblotting analysis

The rat liver was homogenized using the TissueLyser LT (QIAGEN, Valencia, CA, USA) in RIPA lysis buffer (Amresco, OH, USA) containing Halt protease inhibitor cocktail (Thermo Scientific, IL, USA). The protein concentration of each sample was determined using a BCA protein assay kit (Pierce, IL, USA). Western blot analysis was performed. The proteins separated by SDS-PAGE were blotted onto nitrocellulose membranes. Blots were probed with the primary antibody at 4 °C overnight and followed by incubation with secondary antibody conjugated to horseradish peroxidase (Abcam) for 1 h. The immunoreactive protein was detected with the SuperSignal West Pico Chemiluminescent Substrate kit (Pierce) and subjected to photographic films. Signals on the immunoblot were quantified using the ImageJ software developed at the National Institutes of Health (NIH, Bethesda, USA). The β-actin was used as internal control and was used as a reference to normalize data.

2.7. RNA isolation and quantitative real-time polymerase chain reaction

Total RNA was extracted from frozen livers using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and was purified using the RNA clean-up kit (Qiagen). 5 μg of RNA was reverse transcribed into cDNA with the use of the RT-PCR kit-RNA to cDNA EcoDry Premix (Clontech Laboratories, Mountain View, CA, USA). The abundance of transcripts was assessed by real-time PCR on an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster city, CA, USA) for TaqMan analysis. All primers were purchased from Applied Biosystems. The expression of target genes was normalized to Gapdh and quantification of relative expression was determined by the Pfaffl’s method (Pfaffl, 2001).

2.8. Statistical analysis

Data are presented as means ± S.E.M. Prism 6 software (GraphPad, La Jolla, CA, USA) was used for data analysis. Statistical analyses were performed using two-tailed unpaired Student’s t-test or One-way ANOVA when three or more groups were compared and followed by Dunnett’s multiple comparison test. Differences were considered statistical significant at P < 0.05.

3. Results

3.1. Effects of andrographolide on ANIT-induced cholestatic liver injury

Andrographolide treatment had no effect on body weight and liver weight in normal rats, and liver histology was also normal in rats receiving andrographolide. Thus, andrographolide alone had no discernable effect on normal rat livers. In contrast, as expected, ANIT exposure led to profound cholestasis and hepatocellular damage in rats (Figs. 1 and 2). Treatment of rats with ANIT resulted in significantly increased serum levels of ALT and AST, indicative of liver damage (Fig. 1A and B). Serum levels of ALP and GGT, as markers of cholestasis, were more than 2-fold increase in ANIT-treated rats (Fig. 1C and D). Serum bilirubin, serum bile acid and hepatic bile acid levels were also markedly elevated following ANIT treatment (Fig. 1E–H), which are in line with previous studies (Ohta et al., 1999; Tanaka et al., 2009). However, andrographolide treatment at a dose of 100 mg/kg body weight significantly improved the biochemical markers of liver injury ALT, AST, ALP, and GGT in ANIT-treated rats (Fig. 1A–D). A significant reduction in the level of bile acid in the serum and the liver was also observed in andrographolide/ANIT-treated rats (Fig. 1G and H). Taken together, this finding indicates that andrographolide at a dose of 100 mg/kg body weight had substantial, profound protection against ANIT-induced liver damage.

Fig. 1.

Fig. 1.

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Andrographolide improves biochemical markers of liver injury and cholestasis in ANIT-treated rats. Serum levels of (A) ALT, (B) AST, (C) ALP, (D) GGT, (E) total bilirubin, (F) direct bilirubin and (G) bile acid were measured in each group to evaluate the protective effect of andrographolide after ANIT treatment. (H) Hepatic bile acid concentrations, livers from andrographolide-pretreated ANIT-treated rats exhibited decreased hepatic bile acid concentrations. Data represent average ± S.E.M., n=6–10 animals. *P < 0.05. AP, andrographolide.

Fig. 2.

Fig. 2.

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Andrographolide protects against ANIT-induced liver damage. Representative photomicrographs of livers stained with hematoxylin and eosin from control and ANIT-treated rats pretreated with CMC or andrographolide (at a dose of 50 mg/kg body weight and 100 mg/kg body weight). Andrographolide/ANIT treated rats showed less severity of periductular fibrosis and less bile duct proliferation. Magnification, 20 × , Scale bars, 100 μm. AP, andrographolide.

3.2. Andrographolide suppresses hepatic stellate cell activation and attenuates liver damage

Consistent with the results of liver enzymes, histological analysis of liver sections from ANIT-treated rats exhibited large areas of cholangitis with marked bile duct proliferation (Fig. 2). In contrast, liver sections from the andrographolide/ANIT-treated rats showed smaller areas of cholangitis, less inflammatory cell infiltration, and less bile duct proliferation than in ANIT-treated rats (Fig. 2), further supporting that andrographolide treatment (at a dose of 100 mg/kg body weight) markedly ameliorated the cholestatic changes. In addition, the hematoxylin and eosin stained liver sections of ANIT-treated rats exhibited more periductal fibrosis of large bile ducts and were accompanied by a marked increase in the number of α-SMA-positive cells and increased hepatic α-SMA protein expression (Fig. 3A and B). In comparison, less α-SMA-positive cells and less periductal fibrosis were seen throughout in andrographolide (100 mg/kg body weight)/ANIT-treated rats (Fig. 3A), which were paralleled by a substantial reduction in α-SMA protein expression (Fig. 3B). Taken together, these findings indicate that andrographolide suppresses hepatic stellate cell activation and attenuates liver damage in the ANIT model of intrahepatic cholestasis.

Fig. 3.

Fig. 3.

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Andrographolide reduces periductular fibrosis and activation of hepatic stellate cells. (A) Representative immunohistochemical staining of α-SMA was visualized with NovaRED. Sections stained were from control and ANIT-treated rat livers pretreated with CMC or andrographolide (100 mg/kg body weight). α-SMA-positive cells are red; Magnification, 20 × , Scale bars, 100 μm. Andrographolide/ANIT-treated rats exhibited less periductular fibrosis and were accompanied by a marked decrease in the number of α-SMA-positive cells. (B) Immunoblot analysis of α-SMA normalized to β-actin in control and ANIT-treated rat livers pretreated with and without andrographolide (100 mg/kg body weight). Data are expressed in arbitrary units ± S.E.M. *P < 0.05. AP, andrographolide. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

3.3. Effect of andrographolide on bile duct proliferation

Cholestasis disorders are often associated with bile duct proliferation. In animal models of cholestatic liver injury including bile duct ligation (BDL) and administration of ANIT, cholangiocytes proliferate in response to cholestatic injury (Alpini et al., 2001; Sackett et al., 2009). In line with a previous report, proliferation of the bile ducts was seen in the portal areas after ANIT injection (Fig. 2). In contrast, andrographolide pretreatment (100 mg/kg body weight) significantly reduced the magnitude of bile duct cell proliferation to nearly that of control rats (Fig. 2). Furthermore, the degree of reduction of increased serum liver enzyme activities by andrographolide correlated well with less bile duct proliferation (Figs. 1 and 2). To quantify this, we therefore analyzed the expression of PCNA, a known marker of cell proliferation, in andrographolide (100 mg/kg body weight)/ANIT-treated rats. Analysis of PCNA immunohistochemical liver staining from rats receiving ANIT alone showed that ANIT injection substantially increased PCNA expression (Fig. 4A). Similarly, a more profound induction in hepatic PCNA protein expression was also seen in ANIT-treated rats than in the vehicle-treated rats (Fig. 4B). ANIT treatment increased PCNA protein expression approximately 3-fold compared to control rats. In contrast, andrographolide pretreatment (100 mg/kg body weight) resulted in a significant decrease in the number of PCNA-positive cells observed in the portal areas of andrographolide/ANIT-treated rats (Fig. 4A). This reduction in PCNA expression after andrographolide pretreatment was confirmed by Western blotting (Fig. 4B). These findings indicate that andrographolide reduces ductular proliferation induced by ANIT, thus potentially preventing damage of bile ducts that can contribute to cholestasis.

Fig. 4.

Fig. 4.

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Effect of andrographolide on bile duct proliferation. (A) Immunohistochemical staining of PCNA was visualized with NovaRED. PCNA-positive cells are red. Sections stained were from control and ANIT-treated rat livers pretreated with CMC or andrographolide (100 mg/kg body weight). After ANIT treatment, andrographolide reduced the magnitude of bile duct cell proliferation to nearly that of control rats. Magnification, 20 × , Scale bars, 100 μm. (B) Western blot analysis of hepatic PCNA expression demonstrated andrographolide treatment (100 mg/kg body weight) diminished the protein expression of PCNA after ANIT treatment. Band intensities were quantitated and expressed as fold-change relative to control rats receiving CMC alone. Data are expressed in arbitrary units ± S.E.M. *P < 0.05. AP, andrographolide. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

3.4. Andrographolide suppresses the expression of NF-κB and Cyclin D1

There is increasing evidence that NF-κB is important for the development of liver injury, including inflammation and proliferation (Sethi et al., 2008). We further investigated whether the protective effect of andrographolide (100 mg/kg body weight) is associated with inhibition of the NF-κB pathway. The protein expression of NF-κB p50 and p65 were markedly induced by ANIT injection (Fig. 5A and B). In contrast, andrographolide treatment significantly reduced the expression of NF-κB p50 and p65 compared to ANIT-treated rats (Fig. 5A and B). Because inhibition of NF-κB activation leads to down-regulation of gene products involved in proliferation, most notably Cyclin D1 (Hinz et al., 1999), the expression of Cyclin D1 was examined. Protein expression of Cyclin D1 was substantially increased in ANIT-treated rats, whereas andrographolide markedly decreased Cyclin D1 protein expression in ANIT-treated rats (Fig. 5C). This finding is consistent with the idea that andrographolide may reduce proliferation of bile duct cells in response to cholestatic liver injury through suppression of NF-κB-regulated activation of Cyclin D1.

Fig. 5.

Fig. 5.

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Andrographolide inhibits the NF-κB signaling. Immunoblot analysis of (A) NF-κB p50, (B) NF-κB p65 and (C) Cyclin D1 in control and ANIT-treated rat livers pretreated with CMC or andrographolide (100 mg/kg body weight). Band intensities were quantitated and expressed as fold-change relative to control rats receiving CMC alone. Values are normalized to β-actin. Data are expressed in arbitrary units ± S.E.M. *P < 0.05. AP, andrographolide.

3.5. Effects of andrographolide on hepatocellular transporter expression

Cholestasis causes intrahepatic accumulation of toxic bile acids (Allen et al., 2011). Down-regulation of hepatic basolateral influx transporters represents one defense mechanism by which hepatocytes shut off bile acid and bilirubin influx during cholestasis. Up-regulation of hepatic basolateral efflux transporters provides an alternative mechanism for eliminating bile acids in an attempt to prevent hepatocellular accumulation of toxic bile acids (Trauner and Halilbasic, 2011). To determine if induction of alternative efflux routes contributes to the observed favorable effects of andrographolide (100 mg/kg body weight), we examined protein expression of alternative basolateral efflux transporters in andrographolide-fed ANIT rats. ANIT administration significantly increased ABCC3 and ABCC4 protein expressions approximately 2.5-fold and 4-fold, respectively. However, much less pronounced induction of both ABCC3 and ABCC4 was observed in andrographolide-fed ANIT rats (Fig. 6A and B). In addition, we examined protein expression of the key canalicular transporters implicated in the pathogenesis of intrahepatic cholestasis. Protein expression of the bile salt export pump was unchanged by ANIT administration (Fig. 6C), while ABCC2 expression was significantly decreased by ANIT (Fig. 6D), consistent with what is observed in a previous report (Tanaka et al., 2009). However, andrographolide had no significant effects on protein expression of either ABCC2 or ABCB11. These data are consistent with the decrease in liver injury after andrographolide administration to ANIT-treated rats. Since these rats showed less accumulation of toxic products in the liver and less liver injury, there was less need for the adaptive up-regulation of alternative basolateral transporters. Thus, the protective effect of andrographolide in acute intrahepatic cholestasis is also reflected in the adaptive response of the liver to ANIT-induced injury.

Fig. 6.

Fig. 6.

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Effect of andrographolide on protein expression of hepatic bile acid transporters. Immunoblot analysis showed the expression of the basolateral membrane transporters (A) ABCC3 and (B) ABCC4, and the canalicular membrane transporters (C) ABCB11 and (D) ABCC2 in control and ANIT-treated rats pretreated with CMC or andrographolide (100 mg/kg body weight). Band intensities were quantitated and expressed as fold-change relative to control rats receiving CMC alone. Values are normalized to β-actin. Data are expressed in arbitrary units ± S.E.M. *P < 0.05. AP, andrographolide.

4. Discussion

Cholestasis leads to liver fibrosis and cirrhosis, which eventually results in liver failure. To date, effective pharmacologic agents and therapeutic approaches are limited. In this study, we have demonstrated that andrographolide prevents development of experimental cholestatic liver injury induced by ANIT. We have provided at least two potential mechanisms of action for andrographolide: suppression of hepatic stellate cell activation and modulation of NF-κB activation.

ANIT has long been employed to induce cholestatic liver injury (Goldfarb et al., 1962). ANIT is conjugated to glutathione in the hepatocytes and is transported across the canalicular membrane by ABCC2, resulting in damage to the cholangiocytes lining the bile ducts and the accumulation of bile acids in the liver (Kossor et al., 1995). Adaptive responses to cholestasis to compensate for the loss of biliary excretory function and the accumulation of potentially toxic biliary constituents have been reported in ANIT-induced cholestatic liver injury (Yang et al., 2012). Basolateral bile acid influx transporters, the sodium/bile acid and sulphated solute cotransporter 1 and the solute carrier organic anion transporter, are down-regulated to reduce the uptake of bile acids and other organic anions in cholestasis. Basolateral bile acid efflux transporters, ABCC3, ABCC4 and organic solute transporters subunit-α/β are up-regulated in cholestatic liver injury (Paumgartner, 2006). In the present study, profound compensatory changes in the expression of basolateral bile acid transporters, particularly ABCC3 and ABCC4, were decreased in andrographolide/ANIT-treated rats compared to ANIT-treated rats (Fig. 6). Having less up-regulation of ABCC3 and ABCC4 could imply that andrographolide/ANIT-treated rats would have less severe cholestatic liver injury. This idea was consistent with the remarkable improvement observed in serum liver enzyme markers and liver histology (Figs. 1 and 2).

How does andrographolide protect against ANIT-induced cholestatic liver injury? A widely appreciated pharmacological property of andrographolide is anti-inflammation, and a link between inflammation and cholestasis has been established (O’Brien et al., 2013). Miyake and coworkers reported that bile acids are capable of inducing Kupffer cells to release proinflammatory cytokines. In this study, we report that andrographolide exerts an anti-inflammatory effect through inhibition of the NF-κB pathway (Fig. 5). This impression was supported by the following findings: i) andrographolide treatment inhibited the protein expression of NF-κB p50 and NF-κB p65 and ii) as the activation of hepatic stellate cells is associated with NF-κB activation (Baghdasaryan et al., 2010; Kim et al., 2012), we found that andrographolide inhibited the activation of hepatic stellate cells as seen by a reduction in protein expression of α-SMA (Fig. 3); iii) we also found that andrographolide suppressed NF-κB transcriptional activation in HepG2 cells transfected with a NF-κB luciferase reporter together with NF-κB p65 (Supplementary Fig S1). In addition, the critical requirement of NF-κB in the expression of genes involved in proliferation, typified by Cyclin D1, has been reported (Sethi et al., 2008). The inhibition of NF-κB activation leads to down-regulation of the Cyclin D1 gene (Hinz et al., 1999). We found the protein level of Cyclin D1 was decreased in andrographolide/ANIT-treated rats in conjunction with the suppression of NF-κB activation (Fig. 5 and Supplementary Fig S1), thus further supporting the idea that andrographolide exerted an anti-inflammatory and anti-proliferative effect through inhibition of the NF-κB pathway.

In addition, the hydrophobic bile acid, chenodeoxycholate, has been shown to induce the mRNA expression of interleukin-1 and tumor necrosis factor-α (Tnf-α) (Miyake et al., 2000). In our study, Tnf-α mRNA expression also was found to be induced in ANIT-treated rat livers, and this induction trended toward a decrease in andrographolide/ANIT-treated rat livers (data not shown). A reduction in Tnf-α expression by andrographolide also correlates with reduction in serum markers of hepatic damage and with severity of liver histological damage (Figs. 1 and 2) consistent with the idea that andrographolide alleviates cholestatic liver injury through an anti-inflammatory mechanism.

One of the potential targets for the medical therapy of cholestasis is stimulation of the conversion of hydrophobic bile acids and other toxic compounds to more hydrophilic, less toxic metabolites (Paumgartner, 2006). Therapeutic concepts targeting nuclear receptor in cholestasis are also established. In particular, the pregnane X receptor (Pxr), a classical xenobiotic receptor, has been reported to have anticholestatic properties in cholestatic animals (Kliewer and Willson, 2002). Staudinger and coworkers revealed that activation of Pxr protects against severe liver damage induced by lithocholic acid, and Pxr agonists may be useful in the treatment of human cholestatic liver disease (Staudinger et al., 2001). Studies in humans showed that the Pxr ligand rifampicin induces genes involved in bile acid and bilirubin metabolism (Marschall et al., 2005), and indeed rifampicin has been used in the treatment of jaundice and pruritus associated with intrahepatic cholestasis (Wagner et al., 2010). Our findings also showed that treatment with andrographolide gave rise to induction of Pxr mRNA expression and led to increased mRNA expression of Cyp3a2 (also known as Cyp3a11) (Supplementary Fig S2), thereby promoting hepatic bile acid and bilirubin detoxification and elimination. Furthermore, Cyp7a1 is the rate-limiting enzyme in bile acid synthesis (Chiang, 2009). Andrographolide repressed Cyp7a1 mRNA expression in ANIT-treated rats (Supplementary Fig S2), suggesting that it reduced bile acid synthesis and this finding is in line with hepatic bile acid levels (Fig. 1H). Taken together, induction of Pxr and Cyp3a2 expression and repression of Cyp7a1 expression (Supplementary Fig S2) would prevent the accumulation of bile acids to toxic levels and render the bile composition to be less toxic for the injured epithelium. Our data are consistent with the idea that the anti-cholestatic effect of andrographolide may be mediated through Pxr. Because andrographolide, like rifampicin, is a Pxr agonist, our findings may have implications for the development of new therapeutic agents for the treatment of human cholestatic liver disease.

Because UDCA is the only agent with demonstrated efficacy in patients with various cholestatic disorders (Paumgartner and Beuers, 2002; Trauner and Graziadei, 1999), it is useful to compare the effects of andrographolide (100 mg/kg body weight) with those reported in a recent study using UDCA (100 mg/kg body weight) and ANIT (65 mg/kg body weight) for 48 h in rats (Chen et al., 2016). Based on these two reports, andrographolide is similar or superior to UDCA in terms of effects on serum transaminases, bilirubin and bile acids. Also, of note is that there may be no dose-dependent relationship of andrographolide in contrast to several other Chinese herbal substances used in this model (Chen et al., 2016). In particular, we found minimal effects of 50 mg/kg body weight andrographolide and others have found that 100 mg/kg body weight and 200 mg/kg body weight andrographolide both exhibit maximal effects (Lee et al., 2010). This is in contrast to a more typical dose-response seen in other Chinese herbals (Chen et al., 2016).

Notably, it is important to emphasize that our study examines an acute model of cholestasis, plus treatment with the therapeutic agent. Cholestasis is often a chronic condition though. Thus, the effects of andrographolide during chronic rather than acute cholestasis would be informative. Published evidence suggests that chronic ANIT does not induce a very different pattern from acute administration, perhaps because of an adaptive response (Cai et al., 2014). On the other hand, the effects of andrographolide during bile duct ligation, a recognized model of chronic cholestasis, does provide evidence for its efficacy in that setting (Lee et al., 2010), in which andrographolide attenuates both liver injury and fibrosis (Lee et al., 2010). Further studies will be needed to delineate the usefulness of andrographolide in acute versus chronic cholestasis and as a preventative versus therapeutic agent.

5. Conclusion

In conclusion, this is the first report providing in vivo evidence of hepatoprotection by andrographolide in an animal model of intrahepatic cholestatic liver disease. These data suggest that andrographolide may be an effective alternative agent and support the potential utility of andrographolide for the prevention liver fibrosis and intrahepatic cholestasis. Our findings together with the results of earlier studies indicate that andrographolide may be desirable not only for anti-tumorigenesis but also for the prevention of cholestatic liver disease. Future work may focus on confirmation of andrographolide’s effect in other cholestatic models, and may culminate in clinical trials with andrographolide for patients with intrahepatic cholestatic liver diseases.

Supplementary Material

Supplementary Figure S1
Supplementary Figure S2
Supplementary Materials and Methods

Acknowledgments

This study was supported by the Thailand Research Fund (TRF) and Mahidol University [Grant TRG-5680099] (J.W.), by Faculty of Science, Mahidol University, Thailand (J.W.), by the Science Achievement Scholarship of Thailand (T.K.), and in part by Grants from the National Institutes of Diabetes and Digestive and Kidney Diseases [Grant P01-DK57751], [Grant R01-DK45710], [Grant R01-DK61747] (M.H.N.), [Grant P30-DK34989] (Yale Liver Center). Authors thanks Dr. Carol J. Soroka for critical reading of the manuscript.

Footnotes

Conflict of interest

The authors declare that there is no duality of interest associated with this manuscript.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2016.07.032.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure S1
NIHMS1955759-supplement-Supplementary_Figure_S1.tif (6.2MB, tif)
Supplementary Figure S2
NIHMS1955759-supplement-Supplementary_Figure_S2.tif (11.7MB, tif)
Supplementary Materials and Methods
NIHMS1955759-supplement-Supplementary_Materials_and_Methods.pdf (80.8KB, pdf)

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