Ursodeoxycholic acid's impact on fat metabolism gene levels in mice with cholesterol gallstones

It is believed that the prevalence of gallstone disease (GSD) ranges from 10 to 30%, which has been observed in multiple studies [1, 2]. Due to the improvement in living standards and irrational eating habits [3], GSD has become one of the most widespread gastroenterological conditions, imposing a considerably socioeconomic burden on health care [4]. In addition, GSD is correlated with Mirizzi syndrome and insulin resistance [5, 6] and can extend into the liver, gallbladder and gastrointestinal tract. More than 90% of gallstones consist mainly of cholesterol and are formed within the gallbladder [7]. It is generally thought that the key pathological abnormalities are associated with an imbalance of gallbladder bile with cholesterol supersaturation relative to bile salts. In addition, phospholipids and gallbladder hypomotility are considered as prerequisites to gallstone disease [8, 9].

In 1882, Dr. Langenbuch performed the first cholecystectomy, which has been considered a significant routine surgery to eradicate gallstones. With the development of surgical technology, a laparoscopic cholecystectomy has become one of the most commonly performed surgical procedures worldwide [10]. However, cholecystectomy is invasive and has a risk of postoperative complications, and not all patients with symptomatic gallstones are candidates for surgery [11]. UDCA, a natural dihydroxy bile acid, promotes gallstone dissolution and has been attributed to have several other beneficial effects, which significantly affect the treatment of cholelithiasis [12]. Currently, UDCA is recommended as a pharmacological therapy to prevent gallstone formation primarily since it is noninvasive and has fewer complications [13]. In addition, increasing studies support the notion that UDCA may enhance gallbladder muscle contractility and gastrointestinal motility to empty the gallbladder, stomach and intestine [14, 15]. In recent decades, there have been numerous investigations emphasizing the association of lipid-related genes with GSD, although little is known about how UDCA influences nuclear receptors during the process of GSD [16].

Nuclear receptors include FXR, LXR and PPAR-α, which are hepatic lipid regulatory transcription factors of the candidate genes for gallstones [17, 18]. It is plausible that nuclear receptors are associated with the molecular mechanism of gallstone dissolution during UDCA intervention [7]. CYP7A1 is a rate-limiting enzyme of the classic pathway of bile acid biosynthesis and plays a significant role in the alternative pathway to catalyze cholesterol into chenodeoxycholic acid (CDCA) or cholic acid (CA). FXR inhibits bile acid synthesis through downregulating CYP7A1, whereas LXR may upregulate the transcription of CYP7A1 mRNA. On the other hand, LXR regulates ABCG5 and ABCG8 to facilitate cholesterol efflux [19–21]. Moreover, adenosine triphosphate-binding cassette subfamily B member 4 (ABCB4) and ABCB11 are determinants of phospholipids (PL) and total bile acid (TBA) secretion, both of which act as flippases and efflux transporters to mediate movement of PL and TBA from hepatocytes to the canalicular membrane. TBA is reabsorbed at the terminal ileum into the portal vein to complete the enterohepatic circulation of bile acid.

This study aimed to investigate the effect of an LD and an abnormal lipid metabolism on the development of gallstones in the liver and gallbladder, histological changes in the liver, alteration in bile composition and the relative mRNA expression of genes relating to the regulation of lipid and bile acid metabolism in a mouse model of gallstone disease over a period of 8 weeks. In addition, UDCA was used in this LD-induced gallstone disease mouse model and compared with control models to evaluate a potential mechanism of action.

In this study, gallstone-susceptible C57BL/J mice successfully expressed a 100% gallstone prevalence after feeding on an LD for 8 weeks. The results of this study are consistent with those of Liu [23]. Due to its abundance of fat and cholesterol, a lithogenic diet induced cholesterol gallstones in the mice by progressively influencing the imbalance of lipid metabolism with higher TC and PL, thus giving rise to a higher bile CSI and cholesterol crystallization until gallstone formation and acceleration of the deterioration of the liver into fatty liver occurred. The gallstone prevalence and liver deterioration were gradually increased with the extension of feeding time. Of note, mice that had been administered UDCA showed a lower gallstone prevalence of 30%. These results reinforce the findings from previous studies that UDCA may exhibit a tremendously potent activity in increasing the bile acid pool size and thus reduce the cholesterol saturation index and cholesterol gallstone formation.

ABCG5/8 gene products, predominantly localized on the canalicular membrane of hepatocytes, are heterodimerized to function as a sterol transporter to facilitate cholesterol secretion into the bile canaliculus. The ABCG5/8 genes are transcriptionally regulated under the activation of LXR, and the overexpression of the ABCG5/8 genes is attributed to GSD [24–26]. Biliary cholesterol secretion is primarily maintained by ABCG5/8, while recent investigation insists that almost 30% of the cholesterol output is transferred via an ABCG5/8-independent pathway [27]. Lithogenic food could induce the excess secretion of cholesterol and oxysterol congeners, thus adversely affecting the expression of ABCG5/8.

The expression of ABCG5 mRNA in the 4 + LD and 8 + LD groups showed a significantly higher tendency with respect to the 0 + LD group. Although the expression of LXR increased gradually with the extension of LD feeding time, the difference was not significant. For ABCG8, there was a significantly increased level in the 4 + LD group and a decrease in the 8 + LD group. Conversely, UDCA may have a potential ability to enhance the transcription of ABCG8 and to decrease LXR, compared to the model group. Bile acids are thought to regulate their own homeostasis via their sensor, the FXR, which could be directly activated by UDCA [28]. LXR are “cholesterol sensors” which, in response to excess cholesterol, stimulate its transport to the liver and biliary excretion [17]. The possible mechanism for this is that when UDCA enters the bile, it changes the bile salt concentration and increases cholesterol solubility. On one hand, UDCA can dissolve cholesterol stones/crystals in bile, and on the other hand, it can also stimulate ABCG8 to transport cholesterol forward into the bile. Therefore, the expression of ABCG8 is increased. LXR, as a cholesterol receptor, was passively downregulated to maintain cholesterol homeostasis when cholesterol transport increased. In addition, studies have shown that the ABCG5/8-independent pathway plays a critical role in regulating hepatic cholesterol secretion and in determining the susceptibility to cholesterol gallstones, working independently of the ABCG5/8 pathway, and that the hepatic LXR does not have an effect on the ABCG5/8-independent pathway for regulating biliary cholesterol secretion [29]. This is different from the classical mechanism of the LXR-ABCG5/8 pathway in regulating cholesterol metabolism. Because the concentration of bile salt is limited, when the concentration of bile salt reaches a constant level, it can affect the solubility of cholesterol, leading to cholesterol crystallization/stone formation. It may also be that UDCA is not effective for every patient or that stones recur after a period of time. Of course, many studies are needed to verify this in a follow-up study.

It is known that there are two pathways for the elimination of redundant cholesterol, where one is via the LXR-ABCG5/8 pathway to bile and the other is via bile acid synthesis to convert cholesterol into bile acid. One possible reason for the results obtained is that in response to persistent hypersecretion of cholesterol or oxycholesterol, the feed-forward loop may become active and the LXR mRNA may be motivated to decrease the cholesterol level in hepatocytes [30]. An increase in cholesterol can lead to an increase in oxysterols, which activates LXR to induce CYP7A1 and ABCG5/8 expression. The activation of LXR significantly influences ABCG5/8, thus increasing the transcription of ABCG5/8. The possible reason is that ABCG8 may contribute to the peak of the lithogenic effect after 4 weeks of lithogenic food. When the mice were fed with LD for 4 weeks, the expression of ABCG8 increased significantly, which was associated with the activation of this classical pathway. However, it is important to mention that LXR only induces CYP7A1 in rats and mice and not in hamsters, rabbits or humans [31].

It is known that excess cholesterol may participate in bile acid synthesis, which is coordinated by CYP7A1 and CYP27A1 [32]. These are two critical enzymes in the classic and alternative pathways to regulate cholesterol trafficking into CDCA or CA, which are concomitant with their downstream target genes to sustain cholesterol homeostasis. ABCB11 is a bile salt export pump that facilitates the efflux of bile acid to the canalicular membrane, thereby performing enterohepatic circulation of bile acid. Additionally, it was widely believed that the upregulation of LXR may exert a beneficial effect on the elevation of CYP7A1 expression, while FXR as a bile acid receptor may suppress CYP7A1 [33, 34]; however, in this experiment, no significant difference was observed in the FXR mRNA. It is thus controversial whether or not PPAR-α may downregulate CYP7A1 and CYP27A1 mRNA in fatty acid catabolism [35–37]. In the present study, the results suggested that the upregulation of PPAR-α may be implicated in the downregulation of CYP7A1. This is consistent with Xie’s research results [38]. In the LD-fed L-Fabp−/− mice, in the absence of L-Fabp (whose expression is generally regulated in a proximal to distal gradient), alterations in intestinal BA flux induce FXR expression, which results in transcriptional induction of downstream targets, including fibroblast growth factor (FGF) 15 [39]. Increased intestinal FGF15 expression would in turn be anticipated to suppress hepatic CYP7A1 expression [40, 41]. Thereafter, there was a remarkable finding that CYP7A1 and CYP7B1 mRNA showed downregulation at 4 weeks and, conversely, upregulation at 8 weeks, while PPAR-α may show a sustainable increase from 0 to 8 weeks. A different discrepancy in CYP7A1 may be attributed to the common control of LXR, FXR and PPAR-α, while CYP27A1 may be affected by the higher expression of PPAR-α. A prolonged lithogenic diet may produce serious injury to hepatocytes or the canalicular membrane, thus leading to the decrease in the CYP7A1, CYP7B1 or CYP27A1 translocators. Recently, studies have shown that UDCA may be an FXR antagonist [42, 43]. In liver, the majority of UDCA is conjugated with glycine and taurine to produce glycoursodeoxycholic acid (GUDCA) and tauroursodeoxycholic acid (TUDCA), which are transported into the gut and then reabsorbed into the ileum epithelial cells. This study revealed that GUDCA and TUDCA were bona fide FXR antagonists. Oral administration of GUDCA suppressed FXR signaling in the gut but did not affect FXR signaling in the liver. Because UDCA was found not to be a direct FXR antagonist, the metabolic benefits of UDCA may be due in part to GUDCA [44].

ABCB11 mRNA expression showed a trend of an increase first, followed by a decrease, among the 0 + LD, 4 + LD, and 8 + LD groups. This may be related to the influence of other factors on the expression of ABCB11, such as steroid receptor coactivator-2 (SRC-2), liver kinase B1 (LKB1), liver receptor homolog 1 (Lrh1), NF-E2-related factor-2 (Nrf2), and others [45–47]. Moreover, the expression of ABCB11 mRNA in the LD + UDCA group was significantly higher than that in the RD + NS and LD + NS groups. This indicated that under UDCA intervention, the expression of ABCB11 mRNA was upregulated, which was consistent with the results of Hu et al. The ABCB11 gene encodes a protein called the bile salt export pump, which transports bile salts from the hepatocyte into bile. The overexpression of ABCB11 significantly promoted biliary bile salt secretion and increased the circulating bile salt pool size and bile salt-dependent bile flow rate [48]. After that, cholesterol solubility can be increased, thus lowering the CSI and reducing cholesterol stone formation. The increased expression of ABCB11 could increase the rate of transport of bile salts within the hepatocyte, thereby decreasing their steady-state intracellular concentration and thus leading to enhanced signaling [49]. As a natural ligand of liver FXR, bile acid can play a role as a signaling molecule regulating liver metabolism [50]. Further studies are needed to confirm the possibility that lipid changes in bile may affect the expression of some genes.

To date, it has been discovered that the CYP27A1, LXR, PPAR-α, ABCG8 and ABCB11 genes involved in lipid metabolism predispose an individual to gallstone formation [51, 52]. However, their interactions with UDCA have not been fully ascertained yet.

With regard to the mouse groups on an LD for 0, 4, and 8 weeks, it should be emphasized that these alterations in the lipid-related genes may accelerate cholesterol accumulation and bile supersaturation while modulating bile lipids and the CSI to exert significant effects on gallstone formation. UDCA may achieve a cholagogic effect by decreasing the levels of TC, PL and CSI to normalize lipid composition. Thus, increasing the expression levels of ABCG8, ABCB11, CYP7A1 and CYP27A1 or decreasing the levels of LXR and PPAR-α can stimulate bile acid secretion and efficiently facilitate gallstone dissolution to reverse damage to the hepatocytes. UDCA may play a role in regulating the expression levels of some lipid-related genes directly or indirectly to ameliorate the development of gallstones. Therefore, UDCA can be used as the drug of choice for cholesterol gallstones if patients are unwilling to undergo surgery.

Not applicable.

YFC and SWW conceived and designed the study. KM, DHL and QYG performed the experiments. NF, KM and YQZ wrote the paper. NF and YH reviewed and edited the manuscript. The author(s) read and approved the final manuscript.

This study was funded by Natural Science Foundation of Tianjin Bureau of traditional Chinese Medicine13040, Key project of Tianjin science and technology support plan14ZCZDSY00021, Foundation of Tianjin Clinical Medical Research Center of Acute Abdomen with integrated Chinese and Western medicine15ZXLCSY00030, Project of the national science and technology program of Ministry of human resources and social security, Project of Tianjin thousands of people plan.

These funding sources are non-commercial.

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

All procedures conformed to the National Institutes of Health guidelines and were approved by the Animal Care and Use Committee in Tianjin Nankai Hospital.

Not applicable.ik

The authors declare that they have no competing interests.

Ning Fan, Email: fanningtcm@126.com.

Ke Meng, Email: mengke1104@163.com.

Yuqing Zhang, Email: zyq-088@163.com.

Yong Hu, Email: 575728745@qq.com.

Donghua Li, Email: nklidonghua@163.com.

Qiaoying Gao, Email: 843262515@qq.com.

Jianhua Wang, Email: wangjianhuaBCZY@163.com.

Yanning Li, Email: liyanningBCZY@163.com.

Shangwei Wu, Email: nkwushangwei@163.com.

Yunfeng Cui, Email: yfcuink@hotmail.com.