C-Kit expression in the gallbladder of guinea pig with chronic calculous cholecystitis and the effect of Artemisia capillaris Thunb on interstitial cells of Cajal

Headnote

ARTICLE INFO

Article type:

Original article

Article history:

Received: Mar 28, 2015

Accepted: Apr 28, 2016

Keywords:

Artemisia capillaries Thunb

C-Kit

Gallstone

Interstitial cells of Cajal

ABSTRACT

Objective(s): To study the c-Kit expression in the gallbladder of cholesterol lithogenic guinea pig model and the effect of Artemisia capillaris Thunb on interstitial cells of Cajal (ICCs).

Materials and Methods: A total of 45 guinea pigs were randomly assigned into three groups: the control group (guinea pigs fed a standard diet, normal group); the model group (guinea pigs fed a cholesterol gallstone-inducing diet); and the Chinese medicine group (guinea pigs fed the cholesterol gallstone-inducing diet and treated with A. capillaris through intragastric administration, therapy group). Each group had 15 guinea pigs. The gallbladders of the guinea pigs were harvested after 8 weeks. C-Kit expression was detected using an immunohistochemistry staining, real-time PCR, and Western blot analyses. The effect of A. capillaris on ICCs was evaluated by muscle strip contraction experiments.

Results: C-Kit expression significantly decreased in the gallbladder of model group, but increased in the Chinese medicine group. The Contractility of guinea pig gallbladder muscle strip significantly improved in the Chinese medicine group.

Conclusion: Our results indicated that A. capillaris improves gallbladder impairment by upregulating c-Kit expression, and it also can improve the contractile response of in vitro guinea pig gallbladder muscle strips.

Introduction

Gallbladder is an important compo nent of biliary tract and plays roles in inspissating, sto ring, and discharging bile thro ugh its rhythmic contraction and relaxation. Cholecystitis and chol elithiasis are mainly caused by insufficient dyn amic of gallbladder that might mak e pathological changes ( 1-2). T hus, explo ring the moto r function and mec hanism of gallbladder to preven t biliary diseases will be an important area for further study.

Gallbladder wall is similar to gastroi ntestinal wall in struc ture. Moreover, gallbladder contraction mainly depends on smooth muscle; it h as been demo nstrated that intersti tial cells of Cajal (ICCs) are widely distributed in the gastrointesti nal tract of mammals and play an important role in regulating gastrointesti nal motility due to close relation to neuron and smooth muscle cells (3). Studies have shown that biliary trac ts of hum an and guinea pigs contai n ICCs (4-5). The resting muscular tension of gallblad der smooth muscle is significantly reduced after remov al of gallbladder ICCs with m ethylene blue (6). Meanwhile, the expression of c-kit significantly decreas ed i n the gallbladder of guinea pigs on a high cholesterol diet (7).

We spec ulated that gallbladder ICCs possibly serve the same fu nction as in the gastrointestin al tract through occurrenc e of spontaneous rhythmic activity of smooth m uscle contraction i n gallbladder motility disorders (8-10). The expression of pro tooncogene c-Kit in ICCs has been studi ed before (11). C-Ki t is a recepto r ty rosine kinase type III which is specifically expressed in ICCs, thus, we could i dentify ICCs by detecting the expression of c-Kit. For the past few years, many stu dies fou nd ICCs in different animals by detecting the expression of c -Kit (12-15).

Arte misia capillaris Thunb is from the chapter of Yang Ming disease in Zhang Zho ngjin g's "Treatise on Febrile Diseases", this medicatio n is composed of A. capillaris, Fructus gardeniae, and Rheum pa lmatum L; which hav e functions of clearing away heat, removing dam pness by diuresis, and eliminating jaundice (16). T he A. capilla ries and F. gardeniae of this medicine can remov e dam p-heat, soothe the liver, and relieve bladder; Rheum palma tum L can decreas e blood stasis r. A. capillaris can usually treat yang jau ndice, and has shown significant efficacy in treating patients wi thout jaundice but having hepatoc hlic hygro pyrexia( 16). A. capillaris, F. gardenia, and Rheum pa lmatu m L and the combi ned drug (A. capilla ris) can significantly enhance the function of gallbladder and improve bile stasis (18); howev er, the exact m echanism remains unclear. 6,7dimetho xycoumarin is the main extrac t and transition com ponent of A. capillaris. Emodin is the active ingredient in Rheum pa lma tum L. while geniposide is the active i ngredient of Fructus gardenia (17-18). In o ur experiment, we firstly m ade guinea pigs model of c hronic calculous cholecystitis, and then detected the expression of c -Kit in gallbladder, meanwhile, we tes ted the expression of c-Kit after the treatment wi th A. capillaris in guinea pigs model group.

Materials and Methods

Animals

A total of 45 common guinea pigs were provided by the Laborato ry Animal Centre of D alian Medical University. Their weights ranged from 2 00-250 g. This study w as carried out in strict acco rdance with the recomm endations in the Guid e for th e Care and Use of L abo ratory Animals of the National Institutes of Health. The animal use pro tocol has been review ed an d app roved by the Institutio nal A nimal Care and Us e Committee (IACUC) of First Affiliated Hospital, Dalian Medical University.

The control (normal) group was supplied with normal feed by the Laboratory Animal Centre of Dalian Medical University. The model group and Chinese group was given common feed supplemented with 2% cholesterol, 5% sucrose, and 5% cod liver oil to induce cholesterol gallstone formation.

Groups

The 45 guin ea pigs w ere randomly assigned into three groups with 15 guinea pigs each. T he guinea pigs in th e control (no rmal) group were f ed a standard diet, the model gro up was fed the cholesterol gallstone-i nducing di et, and the Chinese Medicine (therapy) group was fed the chol esterol gallstone-inducing di et and treated wi th A. capilla ris via intragastric administration. All guinea pigs were fed for 8 weeks.

Tissue selection

After 8 w eeks, we an aesthetised the guinea pigs via an intraperi toneally injection of chloral hydrate, a nd harvested their gallbladders, which were immediately stored i n liquid nitrogen after washing. The guinea pigs were all euthanized.

Immunohistochemistry analysis

The guinea pig gallbladders were embedded in optimum cutting temperature, prepared for frozen sections using a freezing microtome (LEICA CM 1850), and fixed on glass slides treated with 3-Aminopropyltriethoxysilane (APES). After drying, the sections were fixed for 10 min in acetone at 4 °C. After washing 3 times with phosphate-buffered saline (PBS), slides were incubated with 10% normal goat serum for 30 min to block nonspecific background staining, and then incubated overnight with primary rabbit anti - mouse c-Kit antibodies (1:50, sc-168, Santa Cruz company) in a humid chamber at 4 °C. Following 3 times washing with PBS, the sections were incubated for 1 hr with species-specific secondary antibodies (sp9001, Beijing ZSGB-Bio Company) at room temperature, washed 3 times with PBS, stained with DAB, and washed with water. The sections were then observed under an inverted microscope (Nikon ECLIPSE Ti-U) and photographed. The results were analyzed to calculate the mean optical density (OD) using the Image Pro Plus software.

Western blot analysis

Total protein w as extracted and qu an tified via the Bradford method. After calculati ng pro tein concentration, the lysates were s eparated by gel electropho resis (for 4 hr), before blotting onto membranes. The nonspecific sites o n the membranes were blocked with 5% non-f at milk. The blots were incubated overnight wi th primary rabbit anti-mouse antibodies c-Kit (1:200, sc-168, Santa Cruz Com pany) and β-actin (1:1000, bs-0061R, Beijing Bosin) at 4 °C, washed 3 times with TBST, and i ncubated for 2 hr with goat anti-rabbit secondary antibodi es (1:5000, ZB-2301, ZSGB-Bio). After washing, the mem branes were s tained with an ECL kit (Thermo, USA), and the gel images were acqui red by UVP Bio Spec trum. Semiquantitative analysis was performed via UV Pgel densitometry using Quantity One software.

Real- time PCR

RT-PCR w as perform ed using the followin g primer sets via Gene B ank: C-Kit (sense) 5'CCAATTATTCCCTCATCGA-3', C-Ki t (antisense) 5'-GGGTTCAT CTTTAGCCAC-3', GAPDH (sense) 5'-ACCACAGTCCATGCCATCAC-3', GAPDH (antisense) 5'-T CCACCACCCTGTTGCTGTA -3'. Total RNA was extracted from the gallbladders using PCR kit for reverse cDNA. The PCR condi tions were as follows: 40 cycles of denaturation at 94 °C for 30 sec, annealing at 53 ° C for 3 0 sec, and extension at 72 °C for 1 min. Final extension was performed at 72 °C for 5 min. Amplification was visualized using UVP Bio Spectrum via 1.5% agarose gel electrophoresis. We used Qu antity One software to detect th e OD of all strips and relative OD (OD c-Kit/OD β-actin).

Prepar ation of in vitro muscle strip tissues and contraction experiments

Each guinea pig was intraperitoneally injec ted with 4-5 ml of 10% c hloral hydrate anesthesia. Su rgical instruments in the gallblad der were taken off from neck. Mes angial and blood vessels were removed and was hed 2-3 times in PBS solution. Along th e longitudin al axis of the gallblad der, the gallbladder smooth muscle strips with 8 mm len gth and 3 mm width w ere prepared. The muco usal and serosal layers were removed, and each gallbladder muscle strip w as divided into two. Th e prepared strips were attac hed to the muscle tension transduc er hook, the muscle perfusion bath tem perature was set to 37 °C, and the strips were infused into the freshly prepared Krebs solution. Then, 95% O2 and 5% CO2 gas mixtu re was supplied, and the ini tial tension of 1.0 g was given to the hook as preload. After appro ximately 15-20 min, smooth muscle strips appeared to hav e more regular amplitude of s pontaneous stabilized co ntractions. Successively, cholecystokinin octapepti de ( CCK-8) was added to the base tank Krebs to s timulate the smooth m uscle stri ps, which began to shrink significantly. Each gro up was given 1×1 0-5, 1×10-6, and 1 ×1 0-7 concentration gradients of CCK- 8. Based on records, after the additio n of CCK -8, gallbladder contracted frequ ency and contractions began to increase significantly after shrinkage. The degree of change in each contraction tension at the same stimuli and concentratio n in the three groups of smooth muscle strips were compared. CCK -8 was added 1 min prior to tension of the gallbladd er smooth muscle strips as a control value. After each addition of CCK-8, numerical values were used as the effect values, an d results w ere expressed as mean± SD. Comparison betw een the two grou ps were analyzed by t-test metho d; and P<0.05 was considered statistically significant.

Statistical analysis

Statistical analysis were performed using SPSS 16.0 software and all results w ere presented as mean±SD. Statistic ally significant differenc es between grou ps were determined using a Stud en t's t-test. Differenc es with P<0.05 were considered statistically significant.

Results

Immunohistochemistry analysis

The ICCs were dy ed brown and i rregularly distribu ted in the muscular layer of th e gallbladder, which pres ented shuttle or satellite shapes, and formed a network. Few positive-stai ning ICCs were observed in the model group and w hich were lighter in colour (Fi gure 1B) compared with the normal group (Figure 1A) and the therapy group (Figure 1C). The m ean OD ratios of the normal group, the model gro up, and the therapy gro up were 0. 1135±0. 0118, 0.0652±0. 0110, and 0.0871±0.0119, respectively (Figure 1D). This finding indicates that the c- Kitpositive ICCs in the mo del grou p w ere significantly fewer than th e norm al an d the therapy grou ps (P<0.05).

RT-PCR

The mRNA expression of c-Kit and GAPDH was examined using PCR (Figures 2A and 2B). The m ean OD ratios (C-Ki t/GAPDH) of the control group, the model group, and the therapy group were 0.3314±0.0110, 0.1779±0.0134, and 0. 2300±0.0117, respectively (Figure 2C). This data indicate th at the c-Kit mRNA expression in the mo del group was significantly lower than those i n the co ntrol and the therapy groups (P<0.05).

Western blot analysis

The protein expression of c-Kit and β-ac tin was examined using Wes tern blot analysis (Figures 3A and 3B). The mean OD ratios (C-Kit/β-ac tin) of the control group, the model group, and the therapy were 1.6056±0.0157, 0.2219±0.0154, and 1.5346±0. 124, respectively. This data indicate that the c -Kit protei n expression in the model group was significantly lower than those i n the control and the therapy groups (P<0.05; Figure 3C).

The respons es of gallbl adder muscle strips to different concentrations of CCK -8

Gallbladd er muscle strips were u nder 1.0 g of initial tension, wi thin 10-20 min; a gradual emergence of spontaneous voluntary contrac tion of the gallbladder was observed. The amplitude w as changed significan tly after the additio n of CCK-8 concentratio n gradients of 1×10-5, 1×10-6, and 1×10-7 mol/l in the normal, model, and therapy gro ups, respectively. After the addition of 3 conc entration gradients of CCK-8, gallbladder co ntractions accordin g to descending concentrations of CCK were as follows: in the normal gro up, 1.731±0. 312 g, 1.548±0.281 g, and 1.246±0.250 g (Figure 4A); in model group, 1.420±0.288 g, 1.268±0.270 g, and 1.029±0.233 g ( Figu re 4B); in the therapy grou p, 1.578±0.266 g, 1.326±0.237 g, and 1. 207±0.211 g (Figure 4C).

Discussion

Chronic calculous cholecystitis is a commo n disease. It h as been demo nstrated that disorders or conditions charac terized by altered gallbladd er motility predispose patients to gallstone form ation (19). Moreover, gallbladder em ptying and refilling, and even bile updating were all inhibited in patients wi th g a l lb la d der gallstone (20). Constriction rate (21) and co nstriction ac tivities of muscle strips (22) in gallbladder of chol esterol gallstone were both much lower than those in healthy gallblad der. Therefore, weak gallbladder motility is a main factor that lead to gallstone formation and cause secon dary histopatholo gic alteratio ns of cholecystitis and cholelithiasis (1-2).

ICCs widely distributed in the gastrointestinal tract of mammals and closely related wi th neuron and smooth muscle cells, play an important role in regulating gas trointestinal motility. Furthermore, regulatory effect of ICCs networks for gastrointesti nal motility will be a hot topic in the gastrointestinal dynamic abnormality field.

ICCs are classified into several different su btypes (1). ICC-M Y located between the circul ar and longitudinal m uscle layers, serve as pacemaker c ells and generate slow waves (2). ICC-IM are densely distributed th rou ghout th e circular and longitudinal cardi ac muscle layers. ICC-IM of the stomach and colon show a simple elongated spindle shape to transmit neural signal (3). ICC-DMP/I CC -SEP are located between the inner thin and outer thick sublayers of the circul ar muscle (4) ICC-SM are distributed under the mucous membrane (23).

ICCs located between gastrointestinal nerve and smooth muscle cells form a special network, called nerve-ICC-smooth muscle network. A gap junction that is consisted of junction proteins has been found between intestinal smooth muscle and ICC-DMP (24).

More and mo re studies have shown that gastroeso phageal reflux diseas e, gastrointestinal inflammatory diseases ( ulcerative colitis and Croh n's disease), and diabetic gastroparesis involve impaired morphological features o r func tions of ICCs and even impaired nerve-ICC-smooth m uscle network. A t present study, we consid ered that ICCs and nerve-I CC -smooth muscle, called enteric-nervo us system (ENS), were mediators to control gastrointestinal smooth muscle and were involved in gastrointesti nal neuro transmitter signal transduction. Thus, ENS will play very important role in gastrointestinal motility disorders diseases.

We studi ed whether mech anisms of gallbladder and gastrointestinal contraction function w ere the same, and ICCs could medi ate gallbladder contrac ted activities or not.

Previous studies h ave demo nstrated the following results:

Spontaneous myoel ectrical activity was obviously reco rded in gallbladders an d no significant differenc es of myoelectrical ac tivities were s een between smooth muscles of gallbladder and gastrointestinal smooth muscle (6).

Resting muscular tension of gallbladd er smooth muscle reduc ed significantly af ter remov al of gallbladder ICCs with methylene blue (6).

Both gallbladder ICCs and smooth muscles exhibi t spontaneous rhythmic Ca2+ flashes, however, the gap junc tion uncouples greatly reducedCa2+ flashes in gallbladder smoo th muscle, but they persisted in gallbladder ICCs (9).

Expression of c-kit was significantly decreased in the gallbladders of guinea pigs of high cholesterol diet (7).

Cholecystokinin (CCK)-A receptors (CCK-AR) on the gallbladder ICCs indicated CCK induced gallbladder m u s cle strip contractions through the ICCs or CCK-AR (10).

All above implied that gallbladder ICCs might mediate gallbladder spontaneous rhythmic activity and smooth muscle contraction. However, few studies researched how ICCs could regulate gallbladder motility. Some studies found that due to gallbladder wall chronic inflammation, gallbladder motility was decreased, even presented cholestasis (25-26); furthermore, Ca2+ concentration was obviously reduced in gallbladder smooth muscles based on laboratory tests. They demonstra-ted that Chinese medicine could be used to improve cholestasis and enhance gallbladder contraction function in cholesterol calculus model. Another study also found that the expression of c-kit was significantly decreased in the gallbladders of guinea pigs of high cholesterol diet (27), but it's possible mechanisms are not clear.

A. capillaris soup is a traditional Chinese medicine used for liver and gallbladd er diseases. It consist of A. capillaries, F. gardeniae, and Rhubarb, which co uld promote gallbladder contraction (16, 28).

Our study showed that the protein and mRN A expression of c-Kit in normal group was much higher than those in c holesterol gallstone model group, but significantly lower than those in therapy gro up that guinea pigs were treated wi th A. capillaris. This data indicated that ICCs were decreased o r impaired, this leads to depressed gallbladd er con traction in model gro up. Thus, we consid ered that ICCs could mediate regulatory effect of gallbladder con traction activities. Moreov er, A. capillaris enhanced gallbladder co ntractio n and inhibited gallbladd er ICCs to be impaired by high cholesterol diet. A. capillaris consists of three different ingredi ents, so, the respo nsible ingredient or ways that affect contraction func tion of ICCs and gallbladder should be further studied.

Conclusion

Our results indicate that A. capillaris improves gallbladder impairment by up-regulating c-Kit expression, and it also can improve the contractile response of in vitro guinea pig gallbladder muscle strips.

Acknowledgement

The results described in this paper w ere part of student th esis. This work was suppo rted by the National Natural Science Fo undation of China (81273922).

Sidebar

*Please cite this article as:

Feng H, Wang F, Wan g Ch. C-Kit expression in the gallbladder of guinea pi g with chronic calculous cholecystitis and effect of Artemisia capillaris Thunb on interstitial cells of Cajal. Iran J Basic Med Sci 2016; 19:720-725.

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