Effects of rhein on bile acid homeostasis in rats

Rhein, the active ingredient of rhubarb, a medicinal and edible plant, is widely used in clinical practice. In this work, we investigated the alterations of 14 bile acids and hepatic transporters after rats were administered rhein for 5 consecutive weeks. There was no obvious injury to the liver and kidney, and there were no significant changes in biochemical indicators. However, 1,000 mg/kg rhein increased the liver total bile acid (TBA) levels, especially taurine-conjugated bile acids (t-CBAs), inhibited the expression of Farnesoid X receptor (FXR) and (bile salt export pump) BSEP mRNA, and upregulated the expression of (cholesterol 7α-hydroxylase) CYP7A1 mRNA. Rhein close to the clinical dose reduced the amounts of TBAs, especially unconjugated bile acids (UCBAs), and elevated the expression of FXR and multidrug resistance-associated protein 3 (Mrp3) mRNA. These results denote that rhein is not toxic and is safe to use at a reasonable dose and timing.


Introduction
Bile acids play an important role in regulating the metabolism balance of lipids in vivo (1). They are converted from cholesterol in liver by a series of enzymes, and they maintain a dynamic balance through the uptake and efflux of hepatocellular transporter as well as the enterohepatic circulation (2). help reduce blood lipids and lose weight (13). In addition to being used as a laxative in Europe, rhubarb is frequently used in food as a vegetable or in the production of desserts, jams and fruits (14).
Moreover, the cultivated area of rhubarb in some Nordic countries is approximately 60 hectares.

Meanwhile, the rhubarb cultivation area in the United
States and Canada is approximately 7 times the cultivated area of Europe (15). Rhubarb is widely popular in North America as an ingredient in pie (16).
Rhubarb has the effects of promoting dampness and relieving jaundice (17), anti-inflammatory activity, kidney protection, preventing and treating high blood lipids and cholestasis, ameliorating fibrosis and hepatic encephalopathy, and promoting blood circulation and hemostasis (18). Anthraquinones are the active constituents of rhubarb, and they include rhein, emodin, chrysophanol, emodin methyl ether, and aloe-emodin (19); meanwhile, studies have indicated that rhein shows a higher bioavailability and is more easily absorbed and exposed than other anthraquinones (20,21). However, the effects of repeated intake of rhein on liver function and bile acid metabolism are rarely reported. Therefore, the present study was designed to investigate the influences of different doses of rhein on liver and bile acid metabolism and provide information for the reasonable use of rhein.

Physical effects of rhein
The body weights of rats in the 1,000 mg/kg group were notably reduced after treatment with rhein for 5 consecutive weeks compared with the control group (p＜0.01, Fig. S1). In addition, the urine volume of rats in the 10 mg/kg and 30 mg/kg groups were remarkably increased compared with the control (data not shown). The relative liver weights were not different from the blank control. No diarrhea was found during the study period, and there were no other abnormal signs in the rats during the administration period.

Multivariate regression analysis of bile acids in serum and livers
The LC/MS chromatograms of 14 serum and liver bile acids, including t-CBAs  Table 1. Through a clustering analysis of rat serum and liver bile acid data, we can intuitively determine any differences between the rhein-treated or non-treated groups. The results of the principal component analysis showed that both serum and liver bile acid data were remarkably separated from the control group after rhein administration (Data not shown). An orthogonal partial least-squares discrimination analysis (OPLS-DA) revealed a good segregation between the treatment groups and the control (Fig. 2a, d). Although the 10 mg/kg and 30 mg/kg groups were not completely separated, they were still divided from the control. The 1,000 mg/kg rhein group was not only better distinguished from the control but also separated from the 10 mg/kg and 30 mg/kg groups (Fig. 2). These results indicate that the treatment of rats with rhein markedly altered the composition and levels of serum and liver bile acids.
The analysis of the animal latent variable 1 (LV1) scores for both serum (Fig. 2b) and liver (Fig. 2e) showed that the bile acid levels in the rhein groups   The green points represented the control, while the blue, red and yellow points represented the 10 mg/kg, 30 mg/kg and 1,000 mg/kg rhein group, respectively, as shown on the plots. The t1 scores in the serum (b) and liver (e) are shown, respectively, according to the OPLS-DA score plots. The VIP plots of OPLS-DA highlighted the discriminatory species in serum (c) and liver (f).

Serum bile acids profiles after treatment with rhein
The amounts of 14 individual bile acids in serum were determined after the rats were treated with or without rhein (Table 1), and the contents of g-CBAs, t-CBAs, UCBAs and TBAs (sum of the 14 bile acids) were classified and analyzed (Fig. 3a, b). 115.5%↑ 、 TCA 108.7%↑) were decreased and increased, respectively, compared with the control.
The results demonstrate that different doses of rhein displayed a diverse influence on bile acid homeostasis, and 10 mg/kg and 30 mg/kg rhein may be beneficial to its therapeutic effect of reducing bile acids. Table 1 Concentrations of bile acids in serum and liver after rats were treated with rhein for 5 weeks. Data are presented as the means ± SD concentrations in serum measured using UPLC-MS/MS on 5 rats. *p < 0.05, **p < 0.01, ***p＜0.001, compared with the control group of the same bile acid.

Liver bile acid profiles after treatment with rhein
The LC/MS chromatograms of 14 liver bile acids are shown in Fig. 1 and CDCA in liver ( Table 1). As a result, the overall levels of UCBAs in the liver were significantly decreased versus in the control (p < 0.05 or p < 0.01).
The TBAs levels in the liver, mainly t-CBAs.

Impact of rhein on hepatic bile acid transport and gene expression
To investigate the mechanism of abnormal alterations of bile acids induced by rhein, we analyzed the gene expression of liver bile acid receptor farnesoid X receptor (FXR) and the transporters associated with bile acid synthesis, transport and excretion by quantitative real-time PCR.
It can be seen from Fig. 4

Serum biochemical parameters analysis
The serum biochemical indexes (Table 2) involved in liver function were investigated. The results show that there were no marked differences in serum ALT, AST and CHO levels after continuous intragastric administration of rhein for 5 weeks. The level of serum ALP in the 30 mg/kg group was lower than that in the control, but the change was not dose-dependent. The dose of 1,000 mg/kg rhein reduced the serum TBIL and γ-GT levels (p < 0. 05).
However, the reduction of the above indicators did not have toxicological significance. The results imply that rhein has no significant toxicological effect on liver function and kidney function (data not shown).

Histopathological examination of liver
Histopathological examination of the liver showed inflammatory infiltration and fat droplets at 1,000 mg/kg rhein. Furthermore, mild ductular proliferation was found in 1 of the 5 rats after it was given 1,000 mg/kg rhein for 5 continuous weeks (Fig.   5b). However, histological abnormalities were not observed in the control, 10 mg/kg and 30 mg/kg groups.

Statistical analyses
The data are shown as the mean (M) ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA with SPSS software (version 16.0). Bile acid data were also used to perform the OPLS-DA with SIMCA-P (version 12.0). Data were compared with the control group, and P values less than 0.05 were considered significant.

Discussion
According to previous reports, rhein exerts Previous studies have reported that partial UCBAs such as CA, DCA, CDCA and β-MCA display cytotoxicity due to high hydrophobicity (31,32).

Taurine-conjugated bile acids (TCA, T-α-MCA) and
glycine-conjugated bile acids, such as GCA, are less toxic (33). Therefore, the effect of rhein on bile acids is generally beneficial to the reduction of liver toxicity, and this advantage becomes evident when the level of rhein is close to clinical use (10 mg/kg and 30 mg/kg).
To elucidate the mechanism of the changes of leading to reduced liver bile acids, especially the UCBAs (Fig. 3c) that are relatively high toxic. This is beneficial to the hepatocytes. No significant abnormalities or hepatoxicity were found in either the 10 or 30 mg/kg groups. Therefore, rhein is safe to use at a reasonable dosage and timing.

Chemicals and reagents
Rhein (Fig.6) was purchased from Beijing Saibaicao Technology Co., Ltd., and its purity was more than 98% according to high-performance liquid chromatography. The chemical structure of rhein is shown in Figure S1. All reference bile acids

Conditions of chromatography and mass spectrometry
The mobile phase comprised 0.01% formic acid in water (A) and 0.01% formic acid in acetonitrile  Supplementary Table S1.
The total chromatographic operation was divided into several periods. The ion dwell time and transition for the bile acids were set reasonably. The mass spectrometer was operated with the source and desolvation temperatures set at 120 °C and 350 °C, respectively. The curtain gas was 40 psi; the ion spray voltage was -4500 V; the probe temperature was 600 °C; the collision gas was medium; and ion source gas 1 and ion source gas 2 were all 40 psi.  Table S4 and Table S5). This process was repeated 3 times over 3 days to evaluate the interday accuracy and precision. The extraction recoveries of 14 bile acids are shown in Supplementary Table S3.

Individual bile acid analysis
The  Table S6). Quantification was conducted using the ΔΔCT method. The quantity of messenger RNA was normalized with the internal standard GAPDH.

Statistical analysis
The results are shown as the mean ± SD. Data were analyzed using SPSS 16.0 software, and differences between groups were analyzed by Student's t test. Bile acid data were also determined by OPLS-DA using SIMCA-P 12.0 software. The data of the rhein treatment groups were compared with the control. Values significantly different from the control are indicated as **p <0.01 and *p <0.05.

Conclusion
In conclusion, 1,000 mg/kg rhein notably Consequently, we believe that rhein is safe to use at a reasonable dosage and timing. However, However, the high dose of rhein increased the TBAs levels, which may result in the accumulation of bile acids in the liver; thus, the T-α-MCA and TCA levels should be monitored when taking rhein in high doses over a long period.

Conflicts of Interest:
The authors declare no conflict of interest.