Emodin Rescues Intrahepatic Cholestasis via Stimulating FXR/BSEP Pathway in Promoting the Canalicular Export of Accumulated Bile

Abstract

Aim: Bile salt export pump (BSEP) have been confirmed to play an important role for bile acid canalicular export in the treatment of cholestasis. In this study, we investigated the stimulatory effect of emodin on BSEP signaling pathway in cholestasis.

Methods: Cell and animal experiments were given different concentrations of emodin. The BSEP upstream molecule farnesoid X receptor was down-regulated by small interfering RNA (siRNA) technology or guggulsterones and up-regulated by lentivirus or GW4064. Real-time PCR and Western blotting was employed to detect the mRNA and protein levels of BSEP in LO2 cell, rat primary hepatocytes and liver tissue. Immunohistochemistry (IHC) was used to examine the expression of BSEP in liver tissues. Rat liver function and pathological changes of liver tissue were performed by biochemical test and hematoxylin and eosin (HE) staining.

Results: Emodin could increase the mRNA and protein expression of BSEP and FXR. When down-regulating farnesoid X receptor expression with the siRNA or inhibitor guggulsterones, and up-regulating farnesoid X receptor expression with the lentivirus or agonist GW4064, emodin could increase the mRNA level of BSEP and FXR and the protein level of BSEP, FXR1, and FXR2. Emodin also had a notable effect on rat primary hepatocytes experiment, rat pathological manifestation, BSEP, FXR1, and FXR2 positive staining in liver tissues and the test of liver function.

Conclusion: Emodin has a protective effect and a rescue activity on cholestasis via stimulating FXR/BSEP pathways in promoting the canalicular export of accumulated bile.

Keywords: bile salt export pump; cholestasis; downregulation; emodin; signaling pathway; upregulation.

FIGURE 1

Effect of emodin on the expression of FXR and BSEP in normal LO2 cells. The mRNA levels of FXR (A) and BSEP (B) were detected by RT-PCR. The protein levels of FXR1, FXR2, and BSEP were measured by Western blot (C–F). Values are the means ± SD (n = 3, ^∗P < 0.05 compared to the control group; ^∗∗P < 0.01 compared to the control group, as determined by Student’s t-test).

FIGURE 2

Effect of emodin on the expression of FXR and BSEP in LO2 cell after guggulsterone stimulation. The mRNA levels of FXR (A) and BSEP (B) were detected by RT-PCR. The protein levels of FXR1, FXR2, and BSEP were measured by Western blot (C–F). Values are the means ± SD (n = 3, ^∗P < 0.05 compared to the guggulsterone group; ^∗∗P < 0.01 compared to the guggulsterone group; ^#P < 0.01 compared to the control group, as determined by Student’s t-test).

FIGURE 3

Effect of emodin on the expression of FXR and BSEP in LO2 cell after GW4064 stimulation. The mRNA levels of FXR (A) and BSEP (B) were detected by RT-PCR. The protein levels of FXR1, FXR2, and BSEP were measured by Western blot (C–F). Values are the means ± SD (n = 3, ^∗P < 0.05 compared to the GW4064 group; ^∗∗P < 0.01 compared to the GW4064 group; ^#P < 0.01 compared to the control group, as determined by Student’s t-test).

FIGURE 4

Effect of emodin on the expression of FXR and BSEP after FXR was knocked down by siRNA. The mRNA levels of FXR and BSEP were detected by RT-PCR (A,B). The protein levels of FXR1, FXR2, and BSEP were measured by Western blot (C). Values are the means ± SD (n = 3, ^#P < 0.01 compared to the control group, ^∗∗P < 0.01 compared to the FXR-siRNA group; P < 0.05 compared to the FXR-siRNA group; ^∗p < 0.05 compared to the UDCA group; p < 0.05 compared to the DXM group, as determined by Student’s t-test).

FIGURE 5

The expression of GFP was observed with a fluorescence microscope after lentivirus was introduced into LO2 cells for 48 h (A) and 72 h (B). The mRNA levels of FXR and BSEP were detected by RT-PCR (C). (n = 3, ^#P < 0.01 compared to the control group, ^∗∗P < 0.01 compared to the lentivirus group; ^∗P < 0.05 compared to the lentivirus group; p < 0.05 compared to the UDCA group, as determined by Student’s t-test).

FIGURE 6

The protein levels of FXR1, FXR2, and BSEP were detected by Western blotting (A,B). Values are the means ± SD (n = 3, ^#P < 0.01 compared to the control group, ^∗∗P < 0.01 compared to the lentivirus group; ^∗P < 0.05 compared to the lentivirus group; p < 0.05 compared to the UDCA group, as determined by Student’s t-test).

FIGURE 7

Effect of emodin on the SHP-BSEP signaling pathway in ANIT-treated rats. The mRNA levels of FXR and BSEP were measured by real-time PCR (A). The protein levels of FXR1, FXR2, and BSEP were detected by Western blotting (B,C). Data were expressed as the mean ± SD. n = 3. (^#P < 0.01 compared to the control group, ^∗∗P < 0.01 compared to the model group; p < 0.05 compared to the UDCA group; ^Δp < 0.05 compared to the DXM group, as determined by Student’s t-test).

FIGURE 8

Effects of emodin on molecules of BSEP pathway in rat primary hepatocytes. Immunofluorescence was used to detect CK-18 protein in hepatocytes, and the red fluorescence molecule Dylight488 in primary hepatocytes was observed under a fluorescence microscope (A–C). Cytotoxicity of emodin on primary hepatocytes was determined by CCK8 assay (D). The mRNA levels of FXR and BSEP were detected by qRT-PCR (E). The protein levels of FXR1, FXR2, and BSEP were detected by Western blotting (F,G). Data are shown as the mean ± SD. n = 5. (^∗P < 0.05 compared to the control group; ^∗∗P < 0.01 compared to the control group).

FIGURE 9

(1) Effect of emodin on pathological manifestation of hepatic tissue by HE staining at 400× magnification (A: emodin 20 mg/kg group; B: emodin 40 mg/kg group; C: emodin 80 mg/kg group; D: UDCA group; E: DXM group; F: model group; G: control group). (2) Effect of emodin on BSEP expression was examined with IHC at 400× magnification (A: emodin 20 mg/kg group; B: emodin 40 mg/kg group; C: emodin 80 mg/kg group; D: UDCA group; E: DEX group; F: model group; G: control group. Values are the means ± SD. n = 3, ^##P < 0.01 compared to the control group, ^∗∗P < 0.01 compared to the model group; p < 0.01 compared to the UDCA group; ^ΔΔp < 0.01 compared to the DXM group, as determined by Student’s t-test). (3) Effect of emodin on FXR1 expression was examined with IHC at 400× magnification (A: emodin 20 mg/kg group; B: emodin 40 mg/kg group; C: emodin 80 mg/kg group; D: UDCA group; E: DXM group; F: model group; G: control group. Values are the means ± SD. n = 3, ^##P < 0.01 compared to the control group, ^∗∗P < 0.01 compared to the model group; p < 0.01 compared to the UDCA group; ^ΔΔp < 0.01 compared to the DXM group, as determined by Student’s t-test). (4) Effect of emodin on FXR2 expression was examined with IHC at 400× magnification (A: emodin 20 mg/kg group; B: emodin 40 mg/kg group; C: emodin 80 mg/kg group; D: UDCA group; E: DEX group; F: model group; G: control group. Values are the means ± SD. n = 3, ^##P < 0.01 compared to the control group, ^∗∗P < 0.01 compared to the model group; p < 0.01 compared to the UDCA group; ^ΔΔp < 0.01 compared to the DXM group, as determined by Student’s t-test).