Changes in the interstitial cells of Cajal in the gallbladder of guinea pigs fed a lithogenic diet

Abstract

Cholesterol cholelithiasis is a common disease and gallbladder hypomotility may underlie its pathogenesis. Interstitial cells of Cajal (ICCs) in the gallbladder serve vital roles in regulating gallbladder motility. The aim of the present study was to investigate changes in gallbladder ICCs during the development of cholesterol cholelithiasis. A total of 40 male guinea pigs were randomly assigned to four groups and fed a standard diet (SD) or lithogenic diet (LD) for 2 or 8 weeks. The LD significantly increased the total cholesterol levels in the serum and bile, as well as the serum levels of high-density lipoprotein-cholesterol and low-density lipoprotein-cholesterol after 2 and 8 weeks. The LD also significantly increased and decreased the number of gallbladder ICCs at 2 and 8 weeks, respectively, by regulating the stem cell factor/C-kit pathway. Moreover, the ultrastructure of gallbladder ICCs was significantly altered after 8 weeks, and the protein expression levels of connexin 43 in the gallbladder were differentially altered after 2 and 8 weeks. Finally, cholecystokinin receptor type A (CCK1R) expression in the gallbladder was assessed. In gallbladder ICCs, its expression was significantly increased and decreased after 2 and 8 weeks, respectively. In conclusion, these results demonstrate that the density, ultrastructure and CCK1R expression levels of gallbladder ICCs are differentially altered at various stages of cholesterol cholelithiasis progression, indicating that gallbladder ICCs may be considered a potential therapeutic target for treatment of cholesterol cholelithiasis.

Keywords: cholecystokinin-1 receptor; cholesterol cholelithiasis; gallbladder; interstitial cells of Cajal; lithogenic diet.

Figure 1

LD significantly increases T-CHOL levels in the serum and bile and the serum levels of HDL-C and LDL-C. Serum levels of T-CHOL, HDL-C and LDL-C were measured after (A) 2 and (B) 8 weeks in guinea pigs fed with SD or LD. The bile levels of T-CHOL were measured after (C) 2 and (D) 8 weeks in guinea pigs fed with SD or LD. ^**P<0.01 and ^***P<0.001 vs. SD/2 weeks or SD/8 weeks. SD, standard diet; LD, lithogenic diet; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; T-CHOL, total cholesterol.

Figure 2

LD alters the number of ICCs and protein expression levels of C-kit and SCF in the gallbladders of guinea pigs. (A) Immunofluorescence staining of ICCs in the gallbladders of guinea pigs fed a SD or LD. ICCs were labelled using a primary antibody targeting C-kit (green), and nuclei were labelled using DAPI (blue). White arrowheads indicate C-kit-positive gallbladder ICCs, which are shown in the inset. Magnification, x200. (B) The mean number of ICCs in a field of view of guinea pig gallbladder. (C and D) Western blotting of C-kit and SCF protein expression levels in guinea pig gallbladders of the SD and LD groups. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. SD/2 weeks or SD/8 weeks. SD, standard diet; LD, lithogenic diet; ICCs, interstitial cells of Cajal; SCF, stem cell factor.

Figure 3

LD alters the ultrastructure of gallbladder ICCs in SD-fed guinea pigs after 8 weeks and the protein expression levels of Cx43 in guinea pig gallbladders. (A) Ultrastructure of gallbladder ICCs in the guinea pigs fed with SD or LD was visualized using a transmission electron microscope. The white arrowhead indicated enlarged mitochondria and the white triangle indicates the vacuole in the cytoplasm. Magnification, x10,000. Scale bar, 2 µm. (B and C) Western blotting of Cx43 expression in guinea pig gallbladders fed with SD or LD. ^*P<0.05, ^**P<0.01 vs. SD/2 weeks or SD/8 weeks. SD, standard diet; LD, lithogenic diet; Cx43, connexin 43; ICCs, interstitial cells of Cajal.

Figure 4

CCK1R expression in gallbladder ICCs is altered in guinea pigs fed a LD. (A) Serum CCK8 levels were measured after 2 and 8 weeks in guinea pigs fed a SD or LD. (B and C) Western blotting for CCK1R protein expression in guinea pig gallbladders fed a SD or LD. (D) Immunofluorescence labelling of CCK1R (red) expression in C-kit (green)-positive gallbladder ICCs of guinea pigs fed a SD or LD. Magnification, x400. Cell nuclei were labelled using DAPI (blue). White arrowheads indicate C-kit-positive gallbladder ICCs. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. SD/2 weeks or SD/8 weeks. SD, standard diet; LD, lithogenic diet; ICCs, interstitial cells of Cajal; CCK1R, cholecystokinin receptor type A.