Protective Effects of Yinchenhao Decoction on Cholesterol Gallstone in Mice Fed a Lithogenic Diet by Regulating LXR, CYP7A1, CYP7B1, and HMGCR Pathways

Abstract

The study attempted to elucidate whether lipid genes are closely associated with lipid metabolic abnormalities during the lithogenic time and how Yinchenhao Decoction (YCHD) works on the transcriptions of lipid genes against cholesterol gallstone model. C57BL/6J mice fed on lithogenic diet (LD) were used for model establishment and randomized into 5 groups. All groups received LD for different weeks with isometrically intragastric administration of YCHD or NS. Biochemical tests were measured and liver tissues were harvested for histological and genetic detection. It was found that all groups with increasing LD showed a following tendency of gallstone incidence, bile cholesterol, phospholipids, total bile acid, and cholesterol saturation index (CSI). Conversely, YCHD could significantly normalize the levels of gallstone incidence, bile lipids, and CSI (CSI<1). As lithogenic time progressed, ABCG5, ABCG8, PPAR-α, and ABCB4 were upregulated, and SREBP2, CYP7A1, and CYP7B1 were downregulated, while CYP7A1, CYP7B1, LXR, and HMGCR mRNA were increased 3-fold under the administration of YCHD. It was concluded that abnormal expressions of the mentioned genes may eventually progress to cholesterol gallstone. CYP7A1, CYP7B1, LXR, and HMGCR mRNA may be efficient targets of YCHD, which may be a preventive drug to reverse liver injury, normalize bile lipids, facilitate gallstone dissolution, and attenuate gallstone formation.

Figure 1

The gallbladder conditions and hepatic histological sections of different groups. (a)–(d) Analysis of gallstone evolution under 2× stereo microscope: (a) dark red liver, small and transparent gallbladder without gallstone; (b) cholestasis with greasy, fragile, light gray liver; (c) sludge or floccules in the bile; (d) massive, granulated, and white gallstones. (e)-(f) Analysis of cholesterol gallstone evolution with naked eye: (e) dense clumps of stones; (f) cholecystectasia. (g)-(i) HE staining of hepatic tissues under 400× microscope: (g) normal hepatocytes; (h) serious liver injury with fat droplets in loose cytoplasm extensively. Fatty vacuoles were filled with cytoplasm. Cells may exist with nuclear fragmentation, interstitial and double nuclear (arrow); (i) light liver injury with small vacuoles degeneration. The nucleus to cytoplasm ratio is slightly abnormal and some of the nucleus was loose and squeezed to the edge (arrow). HE: hematoxylin eosin.

Figure 2

Relative levels of biliary lipid profiles and CSI (n = 7). The mean of lipid profiles and CSI in 0W was set to 1 and all values were according to mean ± SD. “a” p < 0.001 versus 0W group; “b” p < 0.01 versus 4W group; “c” p < 0.001 versus NS group. W: weeks; NS: normal saline; YCHD: Yinchenhao Decoction; CSI: cholesterol saturation index; BC: bile cholesterol; PL: phospholipids; TBA: total bile acid.

Figure 3

Relative expressions of hepatic lipid genes on mice cholesterol gallstone. (a)–(c) Expressing lipid gene transcriptions in 0W, 4W, and 8W (n = 6-8); (d) Expressing lipid gene transcriptions in NS and YCHD (n = 7-9). GAPDH was as normalizing control gene and all values were according to mean ± SD. “∗” p < 0.05 versus 0W group; “#” p < 0.05 versus 4W group; “&” p < 0.05 versus NS group; “∗∗, ##, &&” p < 0.01; “∗∗∗, ###, &&&” p < 0.001. W: weeks; NS: normal saline; GAPDH: glyceraldehydes-3-phosphate dehydrogenase; YCHD: Yinchenhao Decoction.