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ObjectiveTo observe the effect of modified Da Chaihutang on cholesterol gallstone （CS） in mice due to damp-heat based on the farnesoid X receptor （FXR）/fibroblast growth factor 15 （FGF15）/fibroblast growth factor receptor 4 （FGFR4） pathway and explore the molecular biological mechanisms of CS differentiated into damp-heat syndrome from the perspective of correspondence between prescription and syndrome. MethodForty-eight six-week-old mice were randomly divided into the blank group， model group， modified Da Chaihutang （23.4 g·kg-1） group， and ursodeoxycholic acid （0.12 g·kg-1） group， with 12 mice in each group. The ones in the latter three groups were exposed to "internal dampness + external dampness + high-cholesterol diet" for 12 weeks for inducing CS due to damp-heat. Mice in the modified Da Chaihutang group and ursodeoxycholic acid group were gavaged with the corresponding drugs， while those in the model and blank groups with the same amount of normal saline for a total of four weeks. Before and after modeling， mice in each group were subjected to open field tests for determining their activities and mental states. Such general conditions as body mass， food intake， fur， and urine and stool of mice in each group were observed and recorded weekly for judging the damp-heat syndrome. After the intervention， the sampled liver and gallbladder tissues of mice in each group were stained with hematoxylin-eosin （HE） staining， and the serum γ-glutamyltransferase （GGT）， alkaline phosphatase （ALP）， and total bilirubin （TBIL） were determined. The total cholesterol （TC） and total bile acid （TBA） contents in bile were measured by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expression levels of FXR， FGF15， FGFR4， and cholesterol 7α-hydroxylase gene （CYP7A1） were assayed by real-time fluorescence quantitative polynucleotide chain reaction （Real-time PCR） and Western blot. ResultCompared with the blank group， the model group exhibited enlarged gallbladder， brown turbid bile with flocculent precipitation visible to the naked eye， obvious damp-heat syndrome， lipoid degeneration in the liver tissue， rough and thickened gallbladder wall， elevated ALP， GGT， and TBIL in serum （P<0.01） and TC in bile （P<0.01）， reduced TBA （P<0.01）， up-regulated FXR， FGF15， and FGFR4 mRNA and protein expression in ileum （P<0.05， P<0.01）， and down-regulated CYP7A1 mRNA and protein expression （P<0.01）. Compared with the model group， the two medication groups displayed improved bile turbidity， and the bile in the modified Da Chaihutang group became clearer. After intervention， the damp-heat syndrome of mice in the modified Da Chaihutang group was significantly alleviated. The liver and gallbladder lesions of mice in the two medication groups were significantly relieved， manifested as reduced serum ALP， GGT， and TBIL （P<0.01）. The reduction in ALP and TBIL of the modified Da Chaihutang group was more significant （P<0.01）. The TC contents in the bile of mice from the two medication groups were significantly lowered， whereas the TBA contents were elevated （P<0.01）， with more significant changes present in the modified Da Chaihutang group （P<0.01）. The mRNA and protein expression levels of FXR， FGF15， and FGFR4 in the modified Da Chaihutang group were down-regulated （P<0.05， P<0.01）， while the mRNA and protein expression levels of CYP7A1 rose （P<0.05）， except that the elevation in FGF15 and FGFR4 protein expression and reduction in CYP7A1 protein expression were not significant. The mRNA and protein expression levels of FXR， FGF15， and FGFR4 in the ursodeoxycholic acid group all decreased， among which the reduction in FXR was remarkable （P<0.05）， and the mRNA and protein expression levels of CYP7A1 were significantly up-regulated （P<0.05）. ConclusionModified Da Chaihutang significantly improves the stone， liver function， bile composition， abnormal cholesterol-bile acid metabolism， and damp-heat syndrome in the model mice of CS differentiated into damp-heat syndrome， which may be related to its regulation of key factors FXR， FGF15， FGFR4， and CYP7A1 mRNA and protein expression in the cholesterol-bile acid metabolism pathway.
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Background: Claudin protein family is an important component for construction of tight cell junction. Claudin-2 is a forming protein of paracellular channel and is highly expressed in hepatocytes and bile duct cells, which has selective permeability of water molecules and cations. Claudin-2 plays a vital role in the physiological regulation of digestive and urinary systems. Aims: To investigate the role and mechanism of claudin-2 in formation of cholesterol gallstone and bile secretion. Methods: Liver and gallbladder tissues from claudin-2 knockout (Cldn-2-/-) and wild type (Cldn-2+/+) mice were collected. Expressions of claudin family protein were determined by Western blotting. HE staining, immunofluorescence staining, electron microscope were used to analyze the effect of claudin-2 gene knockout on liver and gallbladder tissue structure. Regulatory roles of claudin-2 in bile component and flow were analyzed. Results: Claudin family proteins were highly expressed in liver and gallbladder tissue in Cldn-2+/+ mice. No significant difference in structure of liver and gallbladder tissue was found between Cldn-2-/- mice and Cldn-2+/+ mice. Compared with Cldn-2+/+ mice, bile flow rate was significantly decreased (P<0.05), concentration of bile was significantly increased (P<0.05), and contents of bile acid, cholesterol, bilirubin, phospholipid in liver and gallbladder bile were significantly increased in Cldn-2-/- mice (P<0.05). Conclusions: Claudin-2 regulates paracellular water flow which is required for proper regulation of bile composition and flow. Dysregulation of this process increases the susceptibility to cholesterol gallstone related diseases in mice.
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Background: Various clinical and biochemical parameters have been hypothesized to predict cholesterol gallstone formation. Hence, this study was structured to evaluate the degree of some of these suggested predictors among inhabitants of Port Harcourt in Nigeria.Methods: This was a retrospective study of the clinical and biochemical parameters of 42 cholesterol gallstones formers within a tertiary hospital in Nigeria. Records of age, gender, weight, height, calculated body mass index and plasma biochemical parameters (total cholesterol, total bilirubin, and total calcium) of cholesterol gallstone formers from 1st January 2008 to 31st December 2017 were abstracted from medical and laboratory records and analysed using SPSS version 20.Results: There were more females (70%) than males (30%) with a ratio of 2.3:1. The age ranged from 31-64 with mean 46.78±9.33. Obesity was observed among 40.5% of study population. Female gender (OR = 2.823; 95% CI = 2.446-3.200; p<0.001), obesity BMI status (OR = 1.534; 95% CI = 1.436 - 1.632; p = 0.012) and abnormal plasma cholesterol status (OR = 3.011; 95% CI = 2.916 - 3.106; p<0.001) were significant predictors of cholesterol gallstone formation. Abnormal plasma cholesterol status was the strongest of the predictors with AUC of 0.920 (p<0.001), seconded by female gender (AUC = 0.889; p<0.001) and obesity BMI status (AUC = 0.834; p<0.001).Conclusions: Abnormal plasma cholesterol status is the strongest independent predictor of cholesterol gallstone formation, seconded by female gender and high BMI status, among inhabitants of Port Harcourt in Nigeria.
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OBJECTIVE@#To investigate the distribution of pathogens and drug resistance in bile and the association between the pregnane X receptor (PXR) gene polymorphisms, traditional Chinese medicine (TCM) syndromes and the risk of cholesterol gallstone disease (CGD).@*METHODS@#A total of 392 samples were enrolled in this study from January 2014 to February 2015, among which 192 patients were with CGD, and 200 samples were healthy. Strains were isolated and susceptibility testing was the disk diffusion method susceptibility testing. The patients were divided into hepatochlic hygropyrexia, stagnation of liver-qi, and the accumulation of damp. The PXR gene polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism. The association between the PXR gene polymorphisms and the risk of CGD was examined by logistic regression analysis.@*RESULTS@#A total of 192 cases were detected in 230 of bile culture pathogens, including Gram-negative bacteria 133 (57.83%), Gram-positive bacteria 76 (33.04%), and fungi 21 (9.13%). The top five pathogens were Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Candida albicans, and Enterococcus feces, of which 110 cases was of single infection, 48 cases of mixed infection of two strains, eight cases of mixed infection of three bacteria. Among 59 Escherichia coli, the yield extended-spectrum beta-lactamases had 40 (67.80%). The hepatochlic hygropyrexia was the most TCM syndrome, followed by stagnation of liver-qi, and the accumulation of damp was least. Different pathogens and the rs6785049 genotypes distributed differently in cholelithiasis patients with different TCM syndromes (P < 0.05). In hepatochlic hygropyrexia patients the Gram-negative bacteria was most. There was significant differences between CGD group and control group in rs6785049 (P < 0.001). Comparison with wild-type portable GG, GA genotype increased the risk of the occurrence of gallstones (OR = 0.40, 95%CI: 0.16-0.79); likewise, carrying the GA+AA genotype also increased the risk (OR = 0.38, 95%CI: 0.19-0.81). There was no significant differences in rs2276707, rs3814055 site polymorphic loci alleles in CGD group and control group.@*CONCLUSIONS@#In the treatment of cholelithiasis, bile samples should be collected for bacterial culture and sensitivity test, and drugs should be strictly chosen based on the results. The rs6785049 polymorphisms in PXR gene may increase the risk of gallstones ontogeny, and gallstones can be early detected and prevented by detecting genotypes. rs6785049 polymorphisms in PXR gene may has relationship with TCM syndromes.
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OBJECTIVE@#To study the mechanism of insulin resistance in the cholesterol gallstone formation from insulin signal transduction pathway so as to reveal the possible mechanism and the effective role of Albiflorin Granule on preventing the cholesterol gallstones.@*METHODS@#Serum triglycerides (TG), free fatty acid (FFA), and total cholesterol (TC) from different groups were measured and liver cells InsR, PKB, IKK-β protein expression levels were detected by western blotting.@*RESULTS@#Albiflorin significantly decreased the cholesterol gallstone formation rate, increased glucose infusion rate in gallstone guinea pigs and improved insulin resistance. Compared with the normal group, insulin receptor and PKB protein expression in GS group were significantly reduced. IKK-β protein in the GS group increased significantly and Albiflorin could reduce IKK-β protein expression in guinea pig liver cells.@*CONCLUSIONS@#The model of insulin resistance in cholesterol gallstone guinea pig was successfully established, which plays an important role in the cholesterol gallstone formation. All aspects of insulin signaling pathway are involved in gallstone formation. Albiflorin can regulate various aspects of insulin signal transduction pathway to prevent the formation of gallbladder.
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Gallstone disease is highly prevalent in clinic,particularly in women and some specific ethnic groups.The formation of water-insoluble cholesterol crystals is due to a misbalance between the three major lipids present in the bile:cholesterol,bile salts,and phospholipids.Many proteins implicated in biliary lipid secretion in the liver are regulated by several transcription factors,including nuclear receptors LXR and FXR.Human and murine genetic,pathophysiological evidence is consistent with the relevance of these nuclear receptors in gallstone formation.In addition,there is emerging data that also suggests a role for estrogen receptor ESR1 in abnormal cholesterol metabolism leading to gallstone disease.A better comprehension of the role of nuclear receptor function in gallstone formation may help doctors to design new and more effective therapeutic strategies for this highly prevalent disease condition.
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Objective To observe the effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on cholesterol gallstones formation in C57BL/6 mice with diet-induced cholesterol gallstone,and then explore the potential mechanism.Methods Fifty C57BL/6 mice were randomly divided into 5 groups (10 mice in each group),referring to control group,experimental group,experimental plus DHA group,experimental plus EPA group,as well as experimental plus DHA and EPA group.The mice in control group were fed with regular diet,and the rest of the mice with lithogenic diet (LD).Subsequent to feeding the mice with separate diets for two weeks,EPA and/or DHA (70 mg · kg-1 · d-1) were orally administered for eight weeks,while the LD feeding was continued during this period.After a total of 10 weeks,the mice were dissected to observe the gallstone formation.The levels of serum lipids,total cholesterol (TC) and phospholipids (PL) in bile,and TC in the liver were tested,and the protein expression of HMGCR,SRBI,ABCG5/ABCG8,CYP7A1 and ABCB11genes in the liver of mice was measured.Results Compared with the experimental group,the experimental plus EPA group had significantly lower TC in liver (0.033 ±0.008 mmolo/g) and bile (1.807 ±0.381 mmolo/L),and lower relative protein expression levels of HMGCR (0.545±0.098),ABCG5 (0.418±0.089) and ABCG8 (0.501 ±0.151)in liver (P＜ 0.05).The contents of TC in liver and bile,and the protein expression of HMGCR,ABCG5andABCG8 in liver were 0.048 ± 0.006 mmol/g and 2.662 ± 0.339 mmolo/L,and 1.011 ± 0.213,1.037 ± 0.276 and 1.266 ±0.312,respectively.No significant differences were observed between experimental plus DHA group and experimental group (P ＞ 0.05).Conclusions EPA could prevent the cholesterol gallstone formation in mice by decreasing the expression of HMGCR and ABCG5/8 genes in liver,therefore reducing cholesterol synthesis and blocking cholesterol transport from liver to bile as well as diminishing cholesterol content in the bile.However,the inhibition effect of DHA on cholesterol gallstone formation was not obvious.
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OBJECTIVE:To compare clinical efficacy of Xiaoshi lidan capsules and Ursodeoxycholic acid capsules in the treat-ment of chronic cholesterol gallstone cholecystitis. METHODS:120 patients with chronic cholesterol gallstone cholecystitis in our hospital were selected and divided into observation group and control group according to random number table,with 60 cases in each group. Observation group was given Xiaoshi lidan capsules 1.2 g,po,tid(after the meal);control group was given Ursodeoxycholic acid capsules 250 mg,po,qd(after dinner). Both group received treatment for 6 months. Effective rate of litholysis were observed in 2 groups as well as abdominal pain score [PRI,VAS score,present pain intensity(PPI)],the thickness of gallbladder wall before and after treatment. Clinical efficacy and the occurrence of ADR were compared between 2 groups during treatment. RESULTS:3 pa-tients withdrew from the observation group and 1 patient withdrew from the control group. Before treatment,there was no statistical significance in PRI,VAS score,PPI and the thickness of gallbladder wall between 2 groups(P>0.05). After treatment,above index-es of 2 groups were decreased significantly,while PRI,VAS score and PPI in observation group was significantly lower than in con-trol group,with statistical significance (P0.05). There was no statistical signifi-cance in the incidence of ADR between 2 groups(P>0.05). CONCLUSIONS:Xiaoshi lidan capsules are similar to Ursodeoxycholic acid capsules in clinical efficacy for chronic cholesterol gallstone cholecystitis with good safety,and can be used as optional drug ex-cept for chronic cholesterol gallstone cholecystitis.
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Objective: To investigate the distribution of pathogens and drug resistance in bile and the association between the pregnane X receptor (PXR) gene polymorphisms, traditional Chinese medicine (TCM) syndromes and the risk of cholesterol gallstone disease (CGD). Methods: A total of 392 samples were enrolled in this study from January 2014 to February 2015, among which 192 patients were with CGD, and 200 samples were healthy. Strains were isolated and susceptibility testing was the disk diffusion method susceptibility testing. The patients were divided into hepatochlic hygropyrexia, stagnation of liver-qi, and the accumulation of damp. The PXR gene polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism. The association between the PXR gene polymorphisms and the risk of CGD was examined by logistic regression analysis. Results: A total of 192 cases were detected in 230 of bile culture pathogens, including Gram-negative bacteria 133 (57.83%), Gram-positive bacteria 76 (33.04%), and fungi 21 (9.13%). The top five pathogens were Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Candida albicans, and Enterococcus feces, of which 110 cases was of single infection, 48 cases of mixed infection of two strains, eight cases of mixed infection of three bacteria. Among 59 Escherichia coli, the yield extended-spectrum beta-lactamases had 40 (67.80%). The hepatochlic hygropyrexia was the most TCM syndrome, followed by stagnation of liver-qi, and the accumulation of damp was least. Different pathogens and the rs6785049 genotypes distributed differently in cholelithiasis patients with different TCM syndromes (P < 0.05). In hepatochlic hygropyrexia patients the Gram-negative bacteria was most. There was significant differences between CGD group and control group in rs6785049 (P < 0.001). Comparison with wild-type portable GG, GA genotype increased the risk of the occurrence of gallstones (OR = 0.40, 95%CI: 0.16-0.79); likewise, carrying the GA+AA genotype also increased the risk (OR = 0.38, 95%CI: 0.19-0.81). There was no significant differences in rs2276707, rs3814055 site polymorphic loci alleles in CGD group and control group. Conclusions: In the treatment of cholelithiasis, bile samples should be collected for bacterial culture and sensitivity test, and drugs should be strictly chosen based on the results. The rs6785049 polymorphisms in PXR gene may increase the risk of gallstones ontogeny, and gallstones can be early detected and prevented by detecting genotypes. rs6785049 polymorphisms in PXR gene may has relationship with TCM syndromes.
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Objective To study the mechanism of insulin resistance in the cholesterol gallstone formation from insulin signal transduction pathway so as to reveal the possible mechanism and the effective role of Albiflorin Granule on preventing the cholesterol gallstones. Methods Serum triglycerides (TG), free fatty acid (FFA), and total cholesterol (TC) from different groups were measured and liver cells InsR, PKB, IKK-β protein expression levels were detected by western blotting. Results Albiflorin significantly decreased the cholesterol gallstone formation rate, increased glucose infusion rate in gallstone guinea pigs and improved insulin resistance. Compared with the normal group, insulin receptor and PKB protein expression in GS group were significantly reduced. IKK-β protein in the GS group increased significantly and Albiflorin could reduce IKK-β protein expression in guinea pig liver cells. Conclusions The model of insulin resistance in cholesterol gallstone guinea pig was successfully established, which plays an important role in the cholesterol gallstone formation. All aspects of insulin signaling pathway are involved in gallstone formation. Albiflorin can regulate various aspects of insulin signal transduction pathway to prevent the formation of gallbladder.
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Objective To investigate the role and possible mechanism of caveolin-1 (CAV1) in the forming of cholesterol gallstone in mice fed with lithogenic diet.Methods Cholesterol gallstone susceptible C57BL/6 mice were study objects.The mice of control group (n=6) and experiment group (n=6) were fed with normal diet and lithogenic diet for four weeks respectively.The condition of cholesterol gallstone forming,changes of serum lipid and bile composition were measured,and the expressions of CAV1 and scavenger receptor classB member Ⅰ (SR-BⅠ) at mRNA and protein level in the liver and gallbladder were detected by realtime-polymerase chain reaction and Western blot,respectively.The t test was performed for mean comparsion between the two groups.Results The incidence rate of gallstone in experimental group was 100％ after fed with lithogenic diet for four weeks,the lipid level significantly increased,and the proportion of cholesterol in bile raised and bile salt decreased.Compared with those of control group,the expressions of CAV1 at mRNA and protein level in the liver and gallbladder tissues siginificantly decreased (in liver tissue,mRNA 0.53 ± 0.13 vs 1.00 ± 0.32,t =3.330,protein level 0.39 ± 0.07vs 0.92±0.06,t=10.280; in gallbladder tissue,mRNA 0.40±0.22 vs 1.00±0.22,t=3.823,protein level 1.04±0.07 vs 1.34 ± 0.04,t =6.367,all P＜0.01).There was no significant difference in the relative expression of SR-BⅠ at mRNA and protein level in the liver and gallbladder tissues between the mice of experiment group and control group.Conclusion The changes of CAV 1 expression at mRNA and protein level in liver and gallbladder tissues may affect lipids metabolism and cholesterol transportation in liver and gallbladder tissues of experiment mice,which might play an important role in the formation of cholesterol gallstone.
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Objective To investigate the role of osteopontin (OPN) in cholesterol gallstone formation.MethodsGallbladder bile was obtained from patients with cholelithiasis (n=36,the experimental group) and from donors of liver transplantation (n=19,the control group).OPN,calcium ion and lipid were analysed quantitively.The nucleating role of OPN in bile was evaluated using nucleating time (NT) approach.ResultsOPN inhibited cholesterol nucleation in a dose dependent manner.OPN (50 μg/ml and 100 μg/ml) prolonged NT by 48.90％ (91.51％) and 17.07％ (32.93％) in lithogenic and control bile,respectively.OPN (100 μg/ml) also inhibited the nucleating effect induced by calcium ion.Furthermore,a combination of OPN (50 μg/ml) and calcium prolonged NT by 75.78％ and 33.96％ in lithogenic and control bile,respectively.A combination of OPN (100 μg/ml) and calcium prolonged NT by 125.9％ and 62.26％ in the 2 groups.The contents of osteopontin and calcium were significantly lower in lithogenic bile than control bile (P＜0.05).On the other hand,the cholesterol saturation index and the contents of cholesterol,phospholipid and bile acid were significantly higher (P＜0.05).ConclusionsOPN inhibited cholesterol gallstone formation.It may be involved in the pathogenesis of cholelithiasis.
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Cholesterol gallstone disease is prevalent and its incidence is increasing in China. Supersaturation of biliary cholesterol is a prerequisite for gallstone formation.Recent studies show that disorders of hepatic-enteric metabolism of lipids play important roles in the pathogenesis of gallstone disease and these include： increased biliary cholesterol which originates from an increased uptake of plasma high density lipoprotein mediated by scavenger receptor B type 1,increased secretion of cholesterol into bile via hepatic canalicular cholesterol transporters, and increased intestinal cholesterol absorption in gallstone patients. These eventually lead to supersaturation of biliary cholesterol. Evidences also suggest that decreasing hepatic cholesterol loading, promoting biliary bile acids and phospholipids secretion, and/or inhibiting intestinal cholesterol absorption can moderate saturation of biliary cholesterol, and prevent gallstone formation.
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Objective To investigate the mRNA expression of liver receptor homolog 1 (LRH-1) gene in patients with cholesterol gallstone disease so that to elucidate the biomolecular pathogenesis of gallstone for- mation.Methods Twenty-seven patients with cholesterol gallstone (CGS) and 14 controls were included in this study.Biliary composition was assayed and mRNA expression of hepatic LRH 1 gene was determined by real time polymorphism chain reaction.Results In CGS patients,expression of LRH-1 was significantly higher than that in controls (14.18?9.37 vs 7.86?6.19,P＜0.05),and cholesterol of bile was oversaturated (1.17?0.27).Conclusion The formation of CGS may be related to increased expression of hepatic LRH-1 gene.
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Objective To clarify the significance of bacteria DNA in cholesterol gallstone. Methods Semi-quantitative PCR and comparative 16S rRNA analysis were used to detect bac teria DNA in gallbladder mucosa, bile and cholesterol gallstones. The influence of peri-operative use of antibiotics on the results of detection was a lso observed. Results (1) The positive rate of bacteria DNA in core and periphery part of gallstones, bile and gallbladder mucosa was 79%, 82%, 77% and 64% respectively, with no posi tive correlation existed among them. (2) The bacteria concentrations in core and periphery part of gallstones, bile and gallbladder mucosa were 3.19?2.09, 3.26 ?2.05, 3.23?2.14 and 3.28?2.70 cfu (log value) respectively. The bacteria con centration in core part of gallstone correlated with those of periphery ones ( r=0.822, P