Role And Mechanism Of Interstitial Cells Of Cajal Impairment In The Pathogenesis Of Cholesterol Gallstone Formation

Background and aimsGallstone disease is one of the most prevalent and costly digestive diseases. Westernization of the Asian diet has resulted in an increased incidence of CG. Impaired gallbladder emptying can prolong residence of bile in the gallbladder and contribute to nucleation of cholesterol crystals from supersaturated bile. In addition, delayed gallbladder emptying is an independent risk factor for gallstone recurrence after successful treatment with gallbladder preserved, such as extracorporeal shockwave lithotripsy and cholangioscopic cholecystolithotomy. Interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal (GI) tract. Loss of ICC or abnormalities of their pacemaker activities are associated with many GI motility disorders. A tyrosine kinase receptor, c-Kit, is a marker of ICC which allowing easy recognition of ICC. For this reason, c-Kit labelling is widely used in morphologic and functional studies of ICC. ICC were recently discovered in the wall of the human gallbladder and in the bile ducts. These reports attracted increasing attention of investigators. In 2013, a study reported that the number of ICC in the gallbladder wall was significantly lower in patients with gallstones when compared with controls. This founding suggests a possibility that damage to ICC participates in gallstone formation. However, whether ICC impairment is involved in gallstone formation by inducing gallbladder hypomotility, the mechanisms underlying these effects and ICC injury have not been illustrated. Cholecystokinin (CCK) is a GI hormone which can induce significant contraction of gallbladder. Previous studies have demonstrated that the lack of CCK or CCK1 receptor (CCK1) impaired gallbladder motility, enhanced cholesterol crystallization and finally promoted gallstone formation. Increasing research reveals that ICC is an important target cell of CCK. CCK1 have been discovered on the rat gastric antral ICC and guinea pig gallbladder ICC; we previously reported that CCK could attenuate the pacemaker activities of ICC via CCK1. Thus, the first aim of the present study was to investigate the relationship between ICC loss and gallbladder hypomotility in cholesterol gallstone disease and to explore the role of CCK between them. Secondly, we established cholesterol gallstone animal models to determine the roles of ICC loss in gallbladder hypomotility and gallstone formation, as well as the effect of high cholesterol diet on ICC impairment. Finally, the effect of high cholesterol level on the growth of ICC was evaluated to investigate the possible mechanisms underlying ICC loss.Methods1. Research of correlation between gallbladder ICC impairment and gallbladder hypomotility carried out on clinical specimens Fresh cholecystectomy tissues and bile were obtained from patients who undergone cholecystectomy with gallstones (study group) or without gallstones (control group, patients who were elective ly treated for pancreatic head tumors or right hepatic tumor and had no pre-or intraoperative signs of cholelithiasis or jaundice).(1) Double-immunohistochemical staining were performed to examine co-localization of CCK1 and c-Kit.(2) Gallbladder muscle strips were made to record in vitro contractions in the presence of cholecystokinin-8 (CCK-8).(3) Immunohistochemistry of c-Kit labeling and double-immunofluorescent staining of c-Kit and mast cell tryptase (MCT) were performed to label c-Kit+ cells and ICC (c-Kit+MCT). C-Kit+ and ICC density were counted. The correlation between density of c-Kit+ cells/ICC and contractile ability of gallbladder muscle strips induced by CCK-8 were analyzed.(4) Double-immunofluorescent staining of c-Kit and CCK1 was also performed to observe the relationship between CCK1 expression and c-Kit expression.(5) Sections from each gallbladder were examined after routine staining with hematoxylin and eosin (H&E). The thickness of lamina propria & muscularis propria was measured. Protein xpression of c-Kit were examined by Western blot.(6) Cholesterol levels in bile were measured with a biochemical analyzer.2. Research of roles of ICC loss in the gallbladder hypomotility and gallstone formation carried out on animal models(1) Establishment of cholesterol gallstone guinea pig model. Fourty gunea pigs (4 weeks old) were randomly assigned into two groups:regular diet (RD) group and lithogenic diet (LD) group.(2) B-mode ultrasonography were employed to monitor the changes of bile composition and gallstone formation. After 6 weeks, the guinea pigs were sacrificed and the rates of gallstone formation were calculated.(3) The in vitro contration of gallbladder muscle strip was recorded.(4) c-Kit and CCK1 mRNA expression levels were measured by real-time PCR. The correlation of contractility and c-Kit mRNA level was analyzed.3. Research of mechanism underlying gallbladder ICC loss in cholesterol gallstone disease:the effect of high cholesterol level on the growth of cultured ICC(1) Cells were isolated from the murine gallbladder by enzymolysis. Single-cell suspensions were incubated with the following antibodies:c-Kit was labeled with PE-Cy (PC) 7-c-Kit antibody; mast cells and other leukocytes were identified using PC5-CD45 antibody;PC5-F4/80 and PC5-CDllb antibodies to used to label macrophages; PE-CD34 antibody was employed to identify ICC precursors. Cells were sorted on a BD FACSAria II instrument. Mature ICC were identified as c-Kit+CD45-F4/80-CD 11 b-CD34- and harvested.(2) Real-time PCR was used to assess the specificity of the ICC sorting and immunofluorescence was used to identify cultured ICC.(3) Water-soluble cholesterol (WSC) intervention:cells were randomly divided into 5 groups:control group and 5mM, lOnM,20mM,40mM WSC group. The cells were cultured for 72h. The expression of c-Kit mRNA was analyzed by real-time PCR.Results1. Research of correlation between gallbladder ICC impairment and gallbladder hypomotility carried out on clinical specimens(1) Ten cases were included in the control group and 30 cases in the study group. There were no significant differences in average age and gender composition between the two groups.(2) Co-localization of CCK1 and c-Kit was observed in gallbladder tissues of both groups, indicating that human gallbladder ICC probably express CCK1.(3) The maximal contractilities of gallbladder muscle strips in response to CCK were significantly decreased in the study group, as compared with the control group.(4) The c-Kit+ cell and ICC density were markedly reduced in the study group, as compared with the control group. There was a positive correlation between the gallbladder contractility and the number of ICC or c-Kit+cells.(5) The expression of CCK1 was decreased in parallel with reduced c-Kit expression in the study group.(6) The thickness of lamina propria & muscularis propria was increased in the study group when compared with the control group. Protein expression of c-Kit was significantly reduced in the study group.(7) Bile cholesterol levels were much higher in the study group than those in the control group.2. Research of roles of ICC loss in the gallbladder hypomotility and gallstone formation carried out on animal models(1) Both stones and sludge were not evident in the RD group. Gallstones were found in 60% guinea pigs in the LD group.(2) Compared with the control group, the maximal contractilities of gallbladder muscle strips from guinea pigs with gallstones or without gallstones in the LD group all apparently decreased in the presence of CCK-8.(3) The expression of c-Kit mRNA significantly decreased in the study group when compared with the control group.(4) There was a positive correlation between the gallbladder contractility and the expression level of c-Kit mRNA.3. Research of mechanism underlying gallbladder ICC loss in cholesterol gallstone disease:the effect of high cholesterol level on the growth of cultured ICC(1) Purification of ICC by Fluorescence-activated Cell Sorting. Mature ICC represented 10.32±2.21% of the total cells isolated from the murine gallbladder.(2) Culture and identification of ICC. ICC were attached to the culture dishes when cultured for 12h. Immunofluorescent staining of c-Kit or ANO1 (another marker of ICC) were used to identify ICC. When staining ICC with anti-c-Kit antibody, the membrane and cytoplasm of ICC were labeled. When staining ICC with anti-ANO1 antibody, the membrane, cytoplasm and nuclei were all labeled. To examine the purity of ICC sorting, we performed real-time PCR with both unsorted cells and sorted ICC. The results indicated a 102±3.18-fold (P＜0.0001) enrichment of c-Kit expression in sorted ICC relative to unsorted cells.(3) WSC intervention. Compared with the control group, the number of ICC significantly decreased when treated with lOmM,20mM or 40mM WSC for 72h, coinciding with decreased c-Kit mRNA levels (P＜0.05).Conclusions1. Research carried out on clinical specimens indicated that:ICC in the human gallbladder wall probably express CCK1; the biological effects of CCK-8 probably occur via CCK1 located on ICC; loss of ICC is correlated with gallbladder hypomotility in cholesterol gallstone disease, which is probably caused by decreased CCK1 expression; bile cholesterol levels were much higher in the cholesterol gallstone group, than those in the control group.2. Research carried out on cholesterol gallstone guinea pig model indicated that: long-term high level cholesterol diet may contribute to gallbladder hypomotility and cholesterol gallstone formation; in addition, gallbladder hypomotility occurred before gallstone formation.3. Research carried out on cultured ICC indicated that:high level cholesterol could markedly inhibite the growth of ICC. ICC loss in the gallbladder wall may be associated with high bile cholesterol level.Background and aimsIn the gastrointestinal (GI) tract, phasic contractions are caused by electrical activity termed slow waves, which are generated and propagated by interstitial cells of Cajal (ICC), The initiation of pacemaker activity in ICC is caused by rhythmic cytoplasmic Ca2+ oscillation. CCK is a bioactive peptide that regulates a variety of physiological functions, acting as both hormone and neurotransmitter in the GI tract. CCK1 were found in the rat gastropyloric ICC, suggesting a role for the ICC in the mediation of CCK effects. The aims of the present study were:(1) to study whether ICC in the murine gastric antrum express CCK1; (2) to investigate the effects of CCK on Ca2+ oscillation in the cultured murine gastric antral ICC and the underlying mechanisms.Methods1. Whole-mount preparations and sectioned murine gastric antral tissues as well as cultured ICC were double-immunostained with anti-c-Kit and anti-CCK1 antibodies.2. The effects of CCK on Ca2+ oscillation in the murine gastric antral ICC: (1) ICC were isolated from murine gastric antrum and cultured; (2) The responsiveness of [Ca2+]i to CCK in cultured ICC was measured by using Fluo-3/AM. Selective CCK1 antagonist lorglumide, intracellular Ca2+-ATPase inhibitor thapsigargin, type III inositol 1,4,5-triphosphate (InsP3) receptor blocker xestospongin C and L-type voltage-operated Ca2+ channel inhibitor nifedipine were employed to examine the mechanisms of [Ca2+]i elevation caused by CCK-8S;Results1. There was co-localization of c-Kit and CCK1 in the murine gastric antrum.2. The effects of CCK on Ca2+ oscillation in the murine gastric antral ICC:(1) Cultured ICC were identified by immunofluorescence stain;(2) CCK significantly increased the mean [Ca2+]i of ICC by (59.30 ± 4.85)%. Pretreatment with lorglumide, thapsigargin and xestospongin C completely abolished the CCK-induced effect. Addition of nifedipine also decreased CCK-induced effect.ConclusionsCCK probably increased [Ca2+]i in ICC via CCK1. This effect depends on the release of intracellular Ca2+ stores.