WT mice were purchased from Fudan University (Shanghai, China). OPN^−/− mice in congenic background were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed at 22±2°C and 60±10% relative humidity in a specific pathogen-free environment, with a 12:12 h light: dark cycle. WT and OPN−/− male mice between 8 and 10 weeks of age were fed a chow diet (CD) or LD (CD supplemented 15% fat, 2% cholesterol, and 0.5% cholic acid) for up to 8 weeks. Bile was collected, and crystal analysis was immediately performed. Blood was collected via right ventricle heart puncture. Feces was collected from individually housed mice for 72 h. The tissues were harvested and then snap-frozen in liquid nitrogen for protein and RNA isolation or fixed in 4% paraformaldehyde overnight for histological analysis. All animals received humane care and all experimental procedures were conducted in conformance with principles of the National Institutes of Health guide for the care and use of laboratory animal (NIH Publications No. 8023) and approved by the Animal Ethics Committee of Fudan University.

Gallstone were defined as macroscopically visible stones, whereas crystals were defined with polarizing light microscopy (Olympus, Tokyo, Japan). The fecal lipids and bile acids were extracted as previously described (8,22). Cholesterol, bile acids and triglyceride levels were measured with assay kits from KHB (Shanghai, China), and phospholipid content was measured using an assay kit from Wako (Osaka, Japan) according to the manufacturers' instructions. All the biochemical measures were assayed in triplicate 3 times. Then the cholesterol saturation index (CSI) was calculated according to Carey's critical tables (23).

Total RNAs were collected from the proximal segment of small intestine which was equally randomly divided into 5 segments or liver in each mouse with the RNAprep Pure Tissue kit (TianGen, Beijing, China) according to the manufacturer's instructions. Random primers (Takara, Shiga, Japan) were used for reverse transcription of total RNA to complementary DNA. Quantitative real-time PCR was performed using SYBR-Green I chemistry (TianGen) and the ABI 7900HT Fast Real-Time PCR System (Applied Biosystem, Shanghai, China). The gene-specific primer sequences are shown in Table I. Messenger RNA (mRNA) expression levels were calculated relative to the housekeeping gene GAPDH and further normalized to the expression levels of the respective controls following the basis of the relative expression method (24).

The entire proximal segment of small intestine that was randomly divided into 5 equal segments were lysed in ice-cold RIPA buffer supplemented with protease inhibitors. Protein concentration of the extracts was measured by the BCA Protein Assay kit (Thermo, Shanghai, China). The protein was analyzed by 8% SDS-PAGE and transferred to Poly (vinylidene fluoride) membranes. Anti-NPC1L1 antibody (Santa Cruz, Shanghai, China) was used at a 1:300 dilution. A 1:10,000 dilution of rabbit anti-goat immunoglobulin G-HRP (Santa Cruz) was used as a secondary antibody. After probing individual antibodies, the antigen-antibody complex was visualized by Enhanced Chemiluminescence Reagents Supersignal (TianGen, Beijing, China). The relative average protein level was determined by densitometry using Image J software (NIH, Maryland, and USA).

Tissues were fixed in 4% paraformaldehyde overnight and then transferred to 70% ethanol prior to routine processing and staining with hematoxylin and eosin. Formalin-fixed tissues were embedded in paraffin and cut into 3 mm sections. Sample slides were incubated with anti-NPC1L1 antibodies (Santa Cruz) with a 1:75 dilution, followed by 30 min incubation with an HRP-labeled polymer secondary antibody (Santa Cruz). Sections were viewed with a Nikon ECLIPSE E600 microscope (Nikon, Tokyo, Japan) using ×10 objective lenses, and images were acquired with a SPOT INSIGHT™ digital color camera, model 3.2.0 (Sterling, Heights, MI). Quantification of immunoreactivity was performed on digitally captured color images saved as TIFF files and analyzed using Image-Pro plus 6.0 software (Media Cybernetics, Florida, USA). The blind histopathological and immunohistochemistry analysis were conducted by a pathologist of Fudan University.

Data are demonstrated as mean ± standard deviation (SD) unless otherwise noted. The statistical significance of differences between the means of the experimental groups was evaluated with unpaired Student's t test (GraphPad, La Jolla, CA, USA). A difference was considered to be statistically significant at P<0.05.

Neither WT mice (0/6) nor OPN−/− mice (0/6) showed crystals or gallstones when fed a CD. When fed with LD for 8 weeks, all WT mice (6/6) developed gallstones, whereas the penetrance in OPN−/− mice was 16.7% (1/6). Compared to those of OPN−/− mice, the gallbladder bile of WT mice appeared turbid and full of precipitates and stones. Microscopic examination of the gallbladder bile revealed cholesterol crystals in WT mice, whereas those of OPN−/− mice were largely free of cholesterol precipitates (Fig. 1).

The histology examinations of gallbladder and ileum were similar in WT mice and OPN−/− mice (Fig. 2). And the body weight and gallbladder volume between two strains showed no difference (Table II). The cholesterol content of two genotypes mice were also similar when fed CD. After being fed with LD for 8 weeks, OPN−/− mice exhibited reduced in biliary cholesterol content and serum cholesterol profile compared to WT mice. But the fecal cholesterol content showed no statistically difference in two LD-fed strains (Table II). There was no difference in serum phospholipid, triglyceride and bile acids profiles between two LD-fed genotypes (Table III). And the fecal bile acids content showed no statistically difference in two strains. However, the LD-fed OPN−/− mice increased in biliary bile acids and biliary phospholipid content compared to LD-fed WT mice (Table III). This combined effect of biliary biochemical alterations led to a decreased CSI in LD-fed OPN−/− mice (Table II), providing a biochemical mechanism for the phenotype that protecting OPN−/− mice from cholesterol gallstone formation.

To understand the mechanisms responsible for the changes in biochemical contents, we profiled the expression of intestinal related genes. There was no significant difference in genes expression between the two genotypes on CD (Fig. 3A and B). When challenged with the LD for 8 weeks, OPN−/− mice showed no difference in the expression of apical sodium-dependent bile acid transporter (ASBT), ileal lipid binding protein (ILBP) and organic solute transporters α/β (OSTα/β), which are involved in intestinal reabsorption of bile acids (Fig. 3C). Among the intestinal cholesterol transporters, the expression of NPC1L1 had an almost 50% reduction in LD-fed OPN−/− mice, whereas the expression of ABCG5/8, ATP-binding cassette, sub-family A, member 1 (ABCA1), and ACAT2 was not affected (Fig. 3D). Then we detected the gene expression of liver X receptor α (LXRα) and peroxisome proliferator activated receptor δ (PPARδ), and found it was similar between two mice strains (Fig. 3D). Then we challenged the protein expression of intestinal NPC1L1 with immunohistochemistry, and found that the protein expression of NPC1L1 was similar in two CD-fed strains but decreased in LD-fed OPN−/− mice (Fig. 4). And the changed expression of NPC1L1 protein was also confirmed by western blot analysis (Fig. 5).

Because the homeostasis of cholesterol is regulated by both absorption in intestine and synthesis in liver, we also measured the expression of hepatic related genes. There were no significant differences in those genes between the two genotypes fed a CD (Fig. 6A). When challenged with the LD for 8 weeks the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9) tended to be decreased in OPN^−/− mice, but the change was not statistically significant. The expression of other hepatic related genes also showed no difference between two mice strains fed a LD (Fig. 6B).