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. 2019 Sep 4;9(1):12740.
doi: 10.1038/s41598-019-49095-z.

Combination treatment with n-3 polyunsaturated fatty acids and ursodeoxycholic acid dissolves cholesterol gallstones in mice

Affiliations

Combination treatment with n-3 polyunsaturated fatty acids and ursodeoxycholic acid dissolves cholesterol gallstones in mice

Sung Ill Jang et al. Sci Rep. .

Abstract

The increasing prevalence of cholesterol gallstone disease places an economic burden on the healthcare system. To identify novel therapeutics, we assessed the effects of n-3 polyunsaturated fatty acids (PUFA) in combination with UDCA in a mouse model of cholesterol gallstones. Gallstone dissolution, gallbladder wall thickness, mucin gene expression in the gallbladder, and levels of phospholipids, cholesterol, and bile acids in bile and serum were analysed. RNA was extracted from the liver for mRNA sequencing and gene expression profiling. Combination treatment resulted in greater gallstone dissolution compared with the control group, and PUFA and combination treatments reduced the thickness of the gallbladder wall. Expression levels of mucin genes were significantly lower in the UDCA, PUFA, and combination groups. Transcriptome analyses revealed that combination treatment modulated hepatic lipid metabolism. The PUFA and combination groups showed elevated bile phospholipid and bile acid levels and a lower cholesterol saturation index. Combination treatment with PUFA and UDCA dissolves cholesterol gallstones in mice by decreasing mucin production, increasing levels of phospholipids and bile acids in bile, and decreasing cholesterol saturation. Further studies of the therapeutic effects of combination PUFA and UDCA treatment in patients with cholesterol gallstones are warranted.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Body weight, liver weight, and gallbladder stone weight and size before and after treatments. Body weight, liver weight, and gallbladder stone weight and size were checked during the pre-treatment (after the feeding with lithogenic diet for 8 weeks) and post-treatment stages (after administration of the therapeutic agent with a regular diet for 12 weeks) in the four groups (control, ursodeoxycholic acid [UDCA], polyunsaturated fatty acids [PUFA], and combination group). The variables were evaluated in 15 mice in each group during the pre-treatment stage, and in 25 mice in each group during the post-treatment stage. No significant differences were observed among the groups in (A) body weight or (B) liver weight, and no systemic side effects were noted. (C) Gallstone weight decreased significantly in the combination group. (D) Gallstone size, as measured using a 6-point grading system, decreased significantly in the UDCA and combination groups. *P < 0.05 vs. control group.
Figure 2
Figure 2
Gross findings of the liver, gallbladder, and gallstones. The photographs demonstrate the gross findings of the liver, gallbladder, and gallstones. (A) In the pre-treatment stage (after feeding with lithogenic diet for 8 weeks), gallstone formation was confirmed and the number and size of the gallstones were measured in 15 mice in each of the four groups (BE). Gallstone number and size were compared between the control group (B) and three treatment groups ((C) UDCA group; (D) PUFA group; (E) combination group) during the post-treatment stage (after administering the therapeutic agent with the regular diet for 12 weeks). The number and size of the gallstones grossly reduced in all three treatment groups (UDCA, PUFA, and combination group) compared to the pre-treatment group.
Figure 3
Figure 3
Histological mucosal changes, thickness of the gallbladder wall, and expression levels of MUC genes in the gallbladder. The photographs demonstrate the microscopic features of the gallbladder. (A) Lithogenic diet (LD) induced mucosal hypertrophy of the gallbladder in the pre-treatment stage (after feeding the lithogenic diet for 8 weeks). (BE) Gallbladder wall thickening was compared between the control group and three treatment groups ((B) control group; (C) UDCA group; (D) PUFA group; and (E) combination group) in the post-treatment stage (after administration of the therapeutic agent with the regular diet for 12 weeks). The treatments reduced the thickness of the hypertrophied gallbladder wall. (F) The thickness of the gallbladder wall decreased significantly in the PUFA and combination groups. (G) Muc2, (H) Muc5ac, (I) Muc5b, and (J) Muc6 expression levels in the gallbladder decreased significantly in the UDCA, PUFA, and combination groups. *P < 0.05 vs. control group.
Figure 4
Figure 4
The CSI and levels of total cholesterol, phospholipids, and bile acids. (A) Total cholesterol level in the bile increased significantly in the PUFA group. (B,C) Both the PUFA and combination groups had significantly increased bile acid (B) and total phospholipid levels in bile (C). (D) As a result, the CSI decreased significantly in the PUFA and combination groups. (E) Total serum levels of cholesterol were similar among the groups. (F) Serum levels of phospholipids increased significantly in the combination group. *P < 0.05 vs. control group.
Figure 5
Figure 5
Heat maps of bile phospholipid, cholesterol, and bile acid classes. Heat map analysis of (A) phospholipid classes (e.g., phosphatidylcholine, lysophosphatidylcholine, lipoprotein (a), PS, sphingomyelin, dihydroceramide 1-phosphate, ceramide 1-phosphate), (B) cholesteryl ester and cholesterol, and (C) various bile acids. Lipid subclass profiles across the four types. Each colored four types on the map depends on a concentration value. Data in the heat map of measured by Euclidean distance using Ward clustering algorithm.
Figure 6
Figure 6
Cholesterol and bile acid production and transport, and expression levels of related genes. Hepatic expression levels of genes related to (A) canalicular efflux, (B) cholesterol synthesis, (C) bile acid synthesis and bile acid transporters, (D) lipogenesis, and (E) fatty acid transporters. Expression levels were normalized to that of β-actin. (F) Hepatic triglyceride levels. (G) Microscopic features of the liver. *P < 0.05 and **P < 0.01 vs. control group. Data are mean ± standard error of the mean (SEM).
Figure 7
Figure 7
Transcriptome analyses and protein–protein interaction networks. (A) DEGs in the treatment groups; 129 DEGs were common across all treatments. (B) Heatmap of log2-fold changes in DEGs. (C) GO enrichment analyses of the 129 common target genes; bars represent enrichment scores (−logP-value). (D) Physical and functional interactions among the 129 common target genes. (E) Number of degrees for the main hub genes in (A).

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