Effect and related mechanism of Yinchenhao decoction on mice with lithogenic diet-induced cholelithiasis

Abstract

The aim of the present study was to investigate the effects and the underlying mechanisms of Yinchenhao Decoction (YCHD), a traditional Chinese medicine formulation, on C57BL/6 mice with lithogenic diet (LD)-induced cholelithiasis. The condition of cholelithiasis was evaluated using a six-level criteria. Levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) in the serum and liver tissue were measured using enzyme colorimetry. Concentrations of TC, phospholipids (PL) and total bile acids (TBA) in the bile were measured to calculate the cholesterol saturation index. Liver histopathology was microscopically observed and mRNA expression levels of ABCG5, ABCG8, SRBI, ABCB4, ABCB11 and NPC1L1 involved in cholesterol metabolism were measured using reverse transcription-quantitative PCR. The results showed that feeding mice the LD induced cholelithiasis, along with abnormal serum biochemical indices and imbalances in biliary cholesterol homeostasis. Increased ALT and ALP levels in the serum and ALT, ALP, TC and LDL-C levels in the serum and liver indicated the existence of hepatocyte injury, which were consistent with the pathological changes. YCHD treatment ameliorated the serum and hepatic biochemical abnormalities and adjusted the biliary imbalance. In addition, elevated expression of ATP-binding cassette subfamily G member 5/8, scavenger receptor class B type I and Niemann-Pick C1 Like 1 in the liver and small intestine were observed at the onset of cholelithiasis but were reversed by YCHD. Taken together, results from the present study suggest that YCHD ameliorated LD-induced cholelithiasis mice, which may be caused by improvements in biliary cholesterol supersaturation and regulation of cholesterol metabolism.

Keywords: Yinchenhao decoction; biliary cholesterol supersaturation; cholelithiasis; cholesterol metabolism.

Figure 1

Scheme of the present study. Oral, oral administration; qd, quaque die; YCHD, Yinchenhao Decoction; TUDCA, tauroursodeoxycholic acid.

Figure 2

Conditions of mice with cholelithiasis in the five different groups. (A) Representative images of gallbladders with each category of cholelithiasis, as graded using the six-level judgment criteria: Grade 0, gallbladder is filled with clear bile; grade I: A few fine crystals are found; grade II, ~10 fine crystals are found; grade III, fine crystals occupy ~50% of the gallbladder; grade IV: Crystals occupy >50% of the gallbladder; grade V: Round gallstones are found. The grade of cholelithiasis in mice after (B) treatment for 4 weeks (C) and for 8 weeks. ^**P<0.01. N, normal; M, model; L, low dose; H, high dose; YCHD, Yinchenhao Decoction; TUDCA, tauroursodeoxycholic acid.

Figure 3

Biliary lipid composition and cholesterol saturation index in mice from the five treatment groups. Measurements of cholesterol, phospholipids and total bile acid after treatment for (A) 4 weeks and (B) 8 weeks. ^*P<0.05, ^**P<0.01 vs. N; ^#P<0.05, ^##P<0.01 vs. M; ^&P<0.05, ^&&P<0.01 vs. YCHD-L. N, normal; M, model; L, low dose; H, high dose; YCHD, Yinchenhao Decoction; TUDCA, tauroursodeoxycholic acid; TC, total cholesterol; PL, phospholipid; TBA, total bile acid.

Figure 4

Total cholesterol levels in liver tissues isolated from mice from the five treatment groups. Measurements of cholesterol after treatment (A) for 4 weeks and (B) 8 weeks. ^**P<0.01 vs. N. ^##P<0.01 vs. M; ^&&P<0.01 vs. YCHD-L. N, normal; M, model; L, low dose; H, high dose; YCHD, Yinchenhao Decoction; TUDCA, tauroursodeoxycholic acid; TC, total cholesterol.

Figure 5

Hematoxylin and eosin staining of liver sections from mice from the five treatment groups. Representative histopathological images of mice from the five groups after treatment for (A) 4 weeks and (B) 8 weeks. Magnification, x100. N, normal; M, model; L, low dose; H, high dose; YCHD, Yinchenhao Decoction; TUDCA, tauroursodeoxycholic acid.

Figure 6

Expression of key components of the cholesterol metabolism pathway in the liver and small intestine tissues of mice from the five treatment groups. Measurements of ABCG5, ABCG8, NPC1L1, SRBI, ABCB11 and ABCB4 mRNA expression in liver and small intestine tissues of mice from the five treatment groups after treatment for (A) 4 weeks and (B) 8 weeks. ^**P<0.01 vs. N; ^#P<0.05, ^##P<0.01 vs. M; ^&&P<0.01 vs. YCHD-L. N, normal; M, model; L, low dose; H, high dose; YCHD, Yinchenhao Decoction; TUDCA, tauroursodeoxycholic acid; ABCG, ATP-binding cassette subfamily G; SRBI, scavenger receptor class B type I; ABCB, ATP-binding cassette subfamily B; NPC1L1, Niemann-Pick C1 Like 1.