inhibition of asics reduces rat hepatic stellate cells activity and liver fibrosis: an in vitro and...

Cell Biology International ISSN 1065-6995doi: 10.1002/cbin.10287

RESEARCH ARTICLE

Inhibition of ASICs reduces rat hepatic stellate cells activity and liverfibrosis: An in vitro and in vivo studyChun-xiao Pany, Fan-rong Wu*y, Xiao-yu Wang, Jie Tang, Wen-fan Gao, Jin-fang Ge and Fei-hu Chen*

School of Pharmacy, Anhui Medical University, Hefei, Anhui, China

Abstract

Hepatic fibrosis is a chronic inflammation-associated disease, which is involved in the infiltration of inflammatory cells andreleasing of proinflammatory cytokines. In the pathological process, protons are released by damaged cells and acidosis isconsidered to play a critical role in cell injury. Although the underlyingmechanism (s) remain ill-defined, ASICs (acid-sensingion channels) are assumed to be involved in this process. The diuretic, amiloride, is neuroprotective in models of cerebralischemia, a property attributable to the inhibition of central ASICs by the drug. However, the effect of inhibition of ASICs byamiloride in the liver fibrotic process remains unclear. We found that amiloride (25, 50, or 100mM) could restrain acid-induced HSCs at pH6 in vitro. In vivo experiments showed that amiloride could significantly alleviate liver injury, decreasinglevels of profibrogenic cytokines, collagen deposition, and reducing pathological tissue damage. In summary, amilorideinhibits hepatic fibrosis in vivo and in vitro, which is probably associated with the downregulation of ASICs.

Keywords: activation; amiloride; ASICs; hepatic stellate cells; liver fibrosis

Introduction

Liver fibrosis, recognized as a common type of liver diseasewith significant morbidity, is characterized by the excessiveaccumulation of extracellular matrix (ECM) caused by bothincreased synthesis and deposition of connective tissue(Zhang et al., 2013). Activation of hepatic stellate cells(HSCs) is important in fibrotic progression (Safadi andFriedman, 2002), which involves the transdifferentiationfrom a quiescent state to myofibroblast-like cells with theappearance of a-smooth muscle actin (a-SMA) and loss ofcellular vitamin A storage (Mormone et al., 2011)

Acid-sensing ion channels (ASICs) are cationic channelswhich belong to the degenerin/epithelial Naþ channel(DEG/ENaC) superfamily (Waldmann et al., 1997). Todate, six additional subunits of ASICs (1b1, 1b2, 2a, 2b, 3,and 4) have been identified (Lingueglia, 2007; Huet al., 2012). A variety of functions have been attributed tothe ASICs, such as mediation of the migraine with tumors,ischemic injury, inflammation, and so on (Rong et al., 2012).Given that the common features of the diseases mentioned

above are local tissue acidification and lower pH value therole of ASICs in the pathogenesis of acid-associated diseaseshas drawn much attention (Chu and Xiong, 2013).

Protons are among the firstmediators released by damagedcells and extracellular pH may drop to as low as 5.4 in severeinflammation (Steen et al., 1992; Qiu et al., 2012), includingliver fibrosis. ASICs are probably involved in this process.Activation or sensitization of Ca2þ-permeable ASICs isresponsible for acidosis-mediated ischemic brain injurycaused by Ca2þ influx in neurons (Xiong et al., 2006). Ca2þ

influx in HSCs by both Ca2þ-dependent and Ca2þ-indepen-dent pathways is necessary to raise HSCs specific contractionand activation (Iizuka et al., 2011).However, the role ofASICsin activation of HSCs remains undefined.

We have examined the effects of amiloride on extracellu-lar acid-induced cell activation and the therapeutic effects onCCl4-induced of liver fibrosis in rats. The results suggest thattreatment with amiloride can protect rat HSCs fromextracellular acid induced activation in vitro. We alsoexplore the protective effect of amiloride on hepatic fibrosisin liver tissues in vivo.

�Corresponding author: e-mail: [email protected]; [email protected] authors contributed equally to this work.

1003Cell Biol Int 38 (2014) 1003–1012 © 2014 International Federation for Cell Biology

Materials and methods

Cells and cell culture

HSC-T6 cells were obtained from XiangYa Central Experi-ment Laboratory. Cells were maintained in DMEM mediumsupplemented with 10% FBS (GIBCO, CA, USA), 100 IU/mLpenicillin, and 100 IU/mL streptomycin. They were culturedat 37�C in a humidified air atmosphere with 5% CO2.

Cell treatments

HSCs were treated with final concentrations of amiloride of25, 50, and 100mM.After 30min, the cells were treated at pH6 with for 3 h in medium with MK801 (10mM), nimodipine(5mM), v-conotoxin MVIIC (3mM), and 1mM thapsigar-gin, which were to eliminate the effects of glutamatereceptors, voltage-gated Ca2þ channels, and Ca2þ release,respectively, from intracellular stores. After drug treatment,the cells continued in normal media for about 4 h.

Animals and treatments

Male Sprague-Dawley rats (5 weeks) weighing 180� 10 gwere kept at the Experimental Animal Center of AnhuiMedical University (Anhui, China). Animal care andexperimental procedures were carried out in accordancewith the Ethical Regulations for the Care and Use ofLaboratory Animals of Anhui Medical University, whichconform to the Guidelines for Laboratory Animals of theNational Research Council of the USA (1996). All rats werehoused under controlled conditions at 25� 2�C, relativehumidity of 60� 10%, room air changes 12–18 times/h, anda 12-h light/dark cycle. Food and water were available adlibitum. Following acclimatization for 1 week after arrival,90 rats were randomly divided into 6 groups: a normalcontrol group, animals received 2mL/kg corn oil intra-gastrically twice a week for 12 weeks and matchingequivalent normal saline daily from week 5 to week 12;and CCl4-treated model group, animals received 2mL/kgCCl4 (mixed 1:1 in olive oil) intragastrically twice a week for12 weeks and matching equivalent normal saline every dayfrom week 5 to week 12; colchicine-treated group (positivecontrol group), animals received 2mL/kg intragastric CCl4twice a week for 12 weeks, and colchicine (1mg/kg) dailyfrom weeks 5 to 12. The high-, medium-, and low-dosage ofamiloride-treated group, animals received 2mL/kg CCl4intragastrically twice a week for 12 weeks and amiloride (2.5,5, and 10mg/kg) daily from weeks 5 to 12. At the end of12 weeks, the animals were killed after ketamine hydrochlo-ride (30mg/kg b.w., i.v.) injection. Serum samples werecollected into heparinized tubes (50U/mL). Liver sampleswere dissected and washed immediately with ice-cold salineto remove excessive blood. The liver samples were divided

into two parts, one being immediately stored at �80�C forfuture analysis, and another excised and fixed in 10%formalin solution for histopathological examination.

Analysis of oxidative stress markers

The activity of serum alanine aminotransferase (ALT) andaspartate aminotransferase (AST) was determined usingcommercial kits (Jiancheng Institute of Biotechnology,Nanjing, China).

Assay of serum type III precollagen (PC-IIINP), type IVcollagen (col-IV), hyaluronic acid (HA), laminin (LN),interleukin-1 (IL-1), and interleukin-6 (IL-6)

Serum levels of hyaluronic acid (HA), type IV collagen (Col-IV), type III pro-collagen (PC-III), laminin (LN), IL-1, andIL-6 were determined by radioimmunoassay using com-mercially available kits (Beijing Furui BioengineeringResearch company, Beijing, China).

Pathological examination

The fixed liver tissue was processed by routine histologyprocedures to give 5mm sections mounted on slides, whichwere stained with H&E for histology. Other sections werestained with Masson’s trichrome method to identifyinterstitial collagen by its blue color to scoe fibrosis.

Immunohistochemistry

Liver sections were deparaffinized in xylene and rehydratedwith graded alcohols. The sectionswere placed in amicrowaveoven (300W) in citrate buffer for 10min at 100�C, andincubated in absolute methanol containing 3% hydrogenperoxide for 10min at room temperature. The sections weresequentially pre-incubated with 10% normal rabbit serum for10min at room temperature before incubation with theprimary antibody (Wuhan Boster Bioengineering ResearchInstitute). Immunoreactivity was visualized using the Strep-tavidin/Peroxidase (SP) method (Zhongshan Golden bridgeBiotechnology Co., LTD, Beijing, China) and diaminobenzi-dine (DAB) as the chromogen. The nuclei were lightlycounterstained with hematoxylin. The primary antibodieswere replaced with phosphate-buffered saline (PBS) contain-ing 0.1% bovine serum albumin as a negative control. Imageswere taken with a Nikon video microscope.

Reverse transcription-polymerase chain reaction (RT-PCR) analysis

Total RNA was prepared using Trizol reagent (Invitrogen).Five microgram total RNA was reverse transcribed intocDNA using RevertAid First Strand cDNA Synthesis Kit

ASICs and liver fibrosis C.-x. Pan et al.

1004 Cell Biol Int 38 (2014) 1003–1012 © 2014 International Federation for Cell Biology

(Fermentas, Vilnius, Lithuania). The primers were designedand synthesized by Shanghai Biology Engineering Corpora-tion according to the serial number fromGenbank (Table 1).b-actin was used as an internal control for all samples. PCRproducts were electrophoresed on a 1.2% agarose gel andvisualized with ethidium bromide, with the density of eachband being analyzed on a Bio-RadMultiAnalyst System. Themeasurements were repeated at least three timesindependently.

Western-blot analysis

Cells were washed twice with ice-cold PBS and lysed inbuffer for 20–30min on ice. Protein concentration wasmeasured by the Bradford assay. Equal amounts of proteinlysates (30mg) were separated on 12% SDS polyacrylamidegels (SDS-PAGE) and electrophoretically transferred ontopolyvinylidene fluoride membranes. The blots were probedwith the appropriate primary antibodies overnight at 4�C,using (Santa Cruz Biotechnology. Inc) followed by theappropriate horseradish peroxidase (HRP)-conjugated rab-bit anti-mouse or goat anti-rabbit IgG and visualized by ECLdetection kit (Pierce Chemical, Rockford, IL). Autoradio-graphs were scanned using a Image-Pro Plus Imaginganalysis software (Media Cybernetics, USA). Experimentswere performed three times with reproducible results.

Statistics

Data are expressed asmean� SD. Statistical analyses used SPSS16.0 software. Comparison among the different treatment

groups used analysis of variance (ANOVA) and unpairedStudent’s t test. P< 0.05 was considered significantly.

Results

Effect of amiloride on ASIC1a and epithelial Naþ channel(ENaC) in acid-induced HSC-T6

We verified that the mRNA and protein expression levels ofASIC1a were decreased in the amiloride groups comparedwith the acid-induced group, but there were no significantchanges of ENaC (a-,b-, andgENaC) in amiloride (Figure 1).

Amiloride attenuates acid-induced cell activation

The expression of a-SMA and TGF-b1 in HSCs wasquantified. a-SMA was higher in the model group than inthe normal control group. Amiloride significantly decreaseda-SMA expression (Figure 2A,B). Compared with thenormal group, the expression of TGF-b1 in the modelgroup increased, whereas expression of TGF-b1 wasdecreased, in the amiloride groups (Figure 2C, D).

ASICs and matrix metabolism and collagen production

The level of collagen I markedly increased in acid or CCl4induced liver fibrosis, but amiloride significantly reduce itcompared to the model group (Figure 3). The balancebetween MMP-13 and TIMP-1 had some effect on theproduction of collagen I. In the model group, MMP-13, amain component as the degradation of collagen content wasdecreased, whereas it was elevated in the treated group.

Table 1 Primers of liver fibrosis related gene amplified by PCR.

Serial number Primer Primer sequences Product length (bp) Tm (�C)

NM 024154.2 ASIC1a F:50-TTCTTCGACTCCTACAGCATCA-30 366 54R:50-CTGCTCGATGGTCTCATAGTTG-30

NM 031548.2 a-ENaC F:50-TACCCTTCCAAGTATACACAGC-30 406 53R:50-CAGAAGGAGACTCCGAATTAGT-30

NM012648.1 b-ENaC F:50-TGCAGGCCCAATGCCGAGGT-30 242 49.7R:50-GGGCTCTGTGCCCTGGCTCT-30

NM 017046.1 g-ENaC F:50-GGATCCTGAGAGAGAATCATGC-3’ 363 54R:50-GTGTCCAGCTATGCCCTTTAAC-30

NM 133530.1 MMP-13 F:50-GGTCCCAAACGAACTTAACTTACA-30 445 55.7R:50-CCTTGAACGTCATCATCAGGAAGC-30

NM 053819.1 TIMP-1 F:50-CCTTATACCAGCCGTTATAAGATCAAGAT-30 347 55R:50-GTCCACAAACAGTGAGTGTCACTC-30

NM 053304.1 Col-1 F:50-TACAGCACGCTTGTGGATG-30 256 51R:50-TTGAGTTTGGGTTGTTGGTC-30

NM 031004.2 a-SMA F:50-TGGCCACTGCTTCCTCTTCTT-30 422 60R:50-GGGGCCAGCTTCGTCATACTCCT-30

NM 021578.2 TGF-b1 F:50-CTGCTGCCGCTTCTGCTCCCACT-30 435 52R:50-ACGGGACAGCAATGGGGGTTCTG-30

NM 031144.2 b-actin F:50-TGGAATCCTGTGGCATCCATGAAAC-30 345 51R:50-ACGCAGCTCAGTAACAGTCCG-30

C.-x. Pan et al. ASICs and liver fibrosis


However, TIMP-1, an inhibitor of MMP-13, produced theopposite effect (Figure 3).

Animal vitality, and index of liver and spleen

Irritability, aggression, and weight loss of rats were seenpredominantly in the model group. Rats in the colchicinegroup, low and medium dose amiloride groups weighed morethan those in the model control group. Both liver index andspleen index in the low,medium, and high dosage groups were

significantly reduced compared with those in model group.Thus, hepatic intumescence had been alleviated by (Table 2).

Serum ALT, AST, type III precollagen, type IV collagen,hyaluronic acid, laminin, IL-1, and IL-6

ALT and AST activity in the model group was significantlyhigher, while those in the amiloride-treated group weresignificantly lower, as also in the colchicine-treated group(Figure 4A).

Figure 1 Effect of amiloride on the level of ASIC1a and epithelial Na+ channel (ENaC) in acid-induced HSC-T6. HSCs were treated with variousfinal concentrations of amiloride (25, 50, 100mM). After 30min, the cells were treatedwith extracellular solutions (pH 6.0) for 3 h; the control groupwasincubated in pH 7.4 extracellular solution without amiloride. The level of mRNA and protein expression was determined by RT-PCR and Western-blot,respectively. mRNA and protein expression was presented as the ratio of target gene band intensity to corresponding b-actin band intensity. The resultsshowed that the level of ASIC1a was reduced by the treatment of amiloride but there were not significant changes of ENaC (a-, b-, and g-ENaC).##P< 0.01 vs. control group. *P< 0.05, **P< 0.01 vs. model group.



Serum levels of PC III, col-IV, hyaluronic acid, laminin, IL-1,and IL-6 significantly increased in the model group comparedto the control. Treatment with amiloride and colchicineeffectivelydecreased the levels of the sixmarkers (Figure 4B,C).

Histopathological findings

The control showed normal lobular architecture with centralveins and radiating hepatic cords. In the CCl4-treated group,severe changes occurred, including fat degeneration, balloon-

ing, necrosis, collagen deposition, and infiltration ofinflammatory cells in liver, indicative of successful establish-ment of liver fibrosis. The groups treated with colchicine andamiloride had reduced pathological damage (Figure 5A, B).

Expression of ASIC1a, TGF-b1, col-I, and a-SMA in liver

We examined the protein expressions of ASIC1a, TGF-b1,col-I, and a-SMA in rat liver by immunohistochemicalassay. The livers of control mice were negative to ASIC1a.

Figure 2 Effect of amiloride ona-SMA, TGF-b1 inHSCs. The cells were cultured and treated as previously described. ThemRNA and protein levers ofa-SMA and TGF-b1 from HSCs were determined by RT-PCR and Western blotting. Compared with control group, acid solution exposure significantlyincrease the activity ofa-SMAand TGF-b1 inHSCs. However, treatmentwith amiloride could lessen the activity ofa-SMA and TGF-b1 comparedwith thepH6.0 solution group. ##P< 0.01 vs. control group. *P< 0.05, **P< 0.01 vs. pH 6.0 group.



Figure 3 Effect of amiloride on Type I collagen, MMP13, and TIMP1 in HSCs. The results showed that ASICs play a role in matrix metabolism andcollagen production in vitro and in vivo. ##P< 0.01 vs. control group. *P< 0.05, **P< 0.01 vs. model group.

Table 2 Effects of amiloride on body weight, liver index and spleen index of experimental animals (means� SD).

Body weight (g)

Group Doses (mg/kg) n Initial Final Liver index (%) Spleen index (%)

Normal – 13 180.9� 10.1 433.7� 40.9 1.68� 1.69 0.17� 0.26Model – 9 178.4� 12.8 275.3� 42.4## 4.23� 1.62# 0.69� 1.00#

Colchicine 1.0 11 185.9� 17.3 404.8� 35.7** 1.93� 3.2** 0.36� 0.22*Amiloride 2.5 10 177.6� 15.9 347.5� 62.2* 2.35� 1.81* 0.31� 0.24*

5.0 11 180.1� 16.7 341.4� 43.1* 2.49� 2.31* 0.34� 0.56*10 12 179.4� 16.9 328.3� 27.4 2.82� 2.01* 0.26� 0.06**

#P< 0.05, ##P< 0.01 as compared with normal control group. *P< 0.05, **P< 0.01 as compared with model group. Liver (spleen) index was liver(spleen) weight/body weight.



ASIC1a immunopositivity was diffusely found in hepato-cytes across necrotic areas in the CCl4 group,. In contrast,only sporadic ASIC1a immunopositivity was found in thelivers of mice treated with colchicine and amiloride(Figure 6A). Immunohistochemical assay showed thatTGF-b1, col-I, and a-SMA-stained areas were markedlyincreased in rats with hepatic fibrosis model groupcompared to control group. High, medium, and low dosesof amiloride significantly reduced the percentage of fibroticparenchyma areas (Figure 6B–D) compared to the modelgroup.

Discussion

The major finding is that amiloride inhibits acid-inducedHSC activation in vitro, as well as its antifibrotic activity invivo. Inhibition of ASICs could inhibit acid inducedprofibrogenic actions of activated rat HSCs, including cellactivation, collagen deposition and the imbalance of MMPs/TIMPs. Inhibition of ASICs with amiloride inhibits CCl4induced profibrogenic actions, including the level of ALTand AST, liver and spleen index, de novo synthesis of ECMcomponents and inflammatory cytokines.

Amiloride is an efficacious potassium-sparing diureticthat possesses natriuretic and anti-kaliuretic properties. Anacidic pH increased the activity of human ENaC by reducingNaþ self-inhibition (Collier et al., 2012). However, in thestudy, the level of ASIC1a was reduced by amiloride, but notsignificantly ENaC (a-, b-, and gENaC) in HSCs. ASICs arevoltage-independent, depolarizing cationic channels mainlypermeable to Naþ that are expressed in the mammaliannervous system and other tissues of cells (Wang andXu, 2011). Homomeric ASIC1a has also been reported toconduct Ca2þ ions. [Ca2þ]i is important in mediating HSCsactivation and stimulating contractility of HSCs (Pinget al., 2012). Our studies show that when ASICs wereblocked, a-SMA, a marker of transdifferentiation, wassignificantly reduced in vivo and in vitro.

TGF-b1 is themost potent fibrogenic cytokine in the liver.In HSCs, TGF-b1 favors the transition to myofibroblast-likecells, stimulates the synthesis of ECM proteins, and inhibitstheir degradation (Liu et al., 2011). Stimulation of activatedHSCs by TGF-b1may be the key fibrogenic response in liverfibrosis, and any strategy aimed at disrupting TGF-b1expression cold be a key approach in prevention andtreatment of liver fibrosis. We have indicated that pre-incubation with amiloride against ASICs reduces both theprotein and mRNA levels of TGF-b1 in vivo and in vitro.

During hepatic fibrogenesis, collagen deposition (primar-ily collagen types I and III) accounts for 50% of the totalproteins in the fibrotic liver (Li et al., 2013). Therefore, wefound the level of col-1 was effectively reduced by amirolidein vivo and in vitro. Homeostasis of ECM is precisely

Figure 4 (A) Effects of amiloride on liver function in CCl4-treatedfibrotic mice. Mice were injected with 10% CCl4 in olive oil (2mL/kg)twice a week for 12 weeks and administered amiloride (2.5–10.0mg/kg)orally everyday from week 5 to week 12. After 12-week treatment, theactivities of ALT and AST were assayed by using chemistry kits. (B, C)Effects of amiloride on serum type III pre-collagen, type IV collagen,hyaluronic acid, laminin, IL-1, and IL-6 levels. #P< 0.05, ##P< 0.01 vs.normal group. *P< 0.05, **P< 0.01 vs. model group.



maintained by MMPs and their inhibitors (TIMPs) innormal liver (Ding and Zhuo, 2013), whereas the balance isdysregulated in chronically damaged liver, which leads toECM deposition (Magness et al., 2004; Rabani et al., 2010).Our results show that MMP-13, a main component as thedegradation of collagen content, was markedly decreased,but elevated in the treated group. However, as for TIMP-1,the inhibitor of MMP13, the opposite was true.

In vivo, increased AST and ALT, biological markers ofhepatocellular damage, also confirmed the hepatic fibro-genesis in rats. Administration of amiloride, concurrent withCCl4 prevents the development of hepatic fibrosis in rats.Serum levels of Col-IV, PC III, HA, and LN are importantindices reflecting the degree of liver fibrosis (Yanget al., 2013). We found the contents of Col-IV, PC III,

HA, and LN in the serum were significantly higher in modelgroup than in the normal group, whereas they were allsignificantly decreased by amiloride, indicating that ami-loride can prevent hepatic fibrosis following chronic liverinjury, thus delaying the development of cirrhosis. Besidesthe direct mechanism on fibrogenesis, the protective effect ofamiloride on hepatic injury also contributes to its anti-fibrotic activity. In addition to the direct effect onfibrogenesis, the protective effect of amiloride also contrib-utes to its anti-fibrotic activity. IL-6 and IL-1 can induce liverinflammation and promote fibrosis (Shi et al., 2013), butboth were markedly decreased by amiloride treatment.

Amiloride can also block other cation channels andexchangers, such as T-type Ca2þ channels and Naþ/Hþ

antiporters, as well as other member proteins belonging to

Figure 5 Representative photomicrographs of liver sections stained with hematoxylin–eosin stain (A) and Masson’s trichrome (B) (originalmagnification, �200). a: control group; b: model group; c: colchicine (1.0mg/kg); d–f: amiloride (2.5, 5.0, 10.0mg/kg).



Figure 6 The effect of amiloride on expressions of ASIC1a (A), TGF-b1 (B), Col-1 (C) and a-SMA (D) in CCl4-induced liver fibrosis byimmunohistochemistry (magnification 200�). The results showed that TGF-b1, col-I and a-SMA-stained areas were markedly increased in rats withhepatic fibrosis model group compared to control group. Both high, medium, and low dose amiloride treatment significantly reduced the percentage offibrotic parenchyma areas compared to hepatic fibrosismodel group. a: Negative group; b: normal group; c, model group; d: colchicine (1.0mg/kg); e–g:amiloride (2.5, 5.0, 10.0mg/kg).



the DEG/ENaC superfamily. In particular, one possibility isthat the concentration of the drug used may modulate theactivities of epithelial sodium channels, which are alsoexpressed in HSCs. Therefore, further studies using selectiveinhibitors for each channel, knockout mice, and genesilencing should help determine the specific pathophysio-logical roles of ASICs.

In conclusion, amiloride can reduce liver inflammationand fibrosis in vitro and in vivo, a protective effect that maybe due to the inhibition of ASICs.

Acknowledgements

This project was supported by the National ScienceFoundations of China (No. 81100301, 81271949).

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Received 22 November 2013; accepted 20 March 2014.Final version published online 28 April 2014.



inhibition of asics reduces rat hepatic stellate cells activity and liver fibrosis: an in vitro and...

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