Dioscin Protects ANIT-Induced Intrahepatic Cholestasis Through Regulating Transporters, Apoptosis and Oxidative Stress

Abstract

Intrahepatic cholestasis, a clinical syndrome, is caused by excessive accumulation of bile acids in body and liver. Proper regulation of bile acids in liver cells is critical for liver injury. We previously reported the effects of dioscin against α-naphthylisothio- cyanate (ANIT)-induced cholestasis in rats. However, the pharmacological and mechanism data are limited. In our work, the animals of rats and mice, and Sandwich-cultured hepatocytes (SCHs) were caused by ANIT, and dioscin was used for the treatment. The results showed that dioscin markedly altered relative liver weights, restored ALT, AST, ALP, TBIL, GSH, GSH-Px, MDA, SOD levels, and rehabilitated ROS level and cell apoptosis. In mechanism study, dioscin not only significantly regulated the protein levels of Ntcp, OAT1, OCT1, Bsep and Mrp2 to accelerate bile acids excretion, but also regulated the expression levels of Bak, Bcl-xl, Bcl-2, Bax, Caspase 3 and Caspase 9 in vivo and in vitro to improve apoptosis. In addition, dioscin markedly inhibited PI3K/Akt pathway and up-regulated the levels of Nrf2, GCLc, GCLm, NQO1 and HO-1 against oxidative stress (OS) caused by bile acids. These results were further validated by inhibition of PI3K and Akt using the inhibitors of wortmannin and perifosine in SCHs. Our data showed that dioscin had good action against ANIT-caused intrahepatic cholestasis through regulating transporters, apoptosis and OS. This natural product can be considered as one active compound to treat intrahepatic cholestasis in the future.

Keywords: PI3K/Akt pathway; cholestasis; dioscin; oxidative stress; α-naphthylisothiocyanate.

FIGURE 1

Dioscin rehabilitates ANIT-induced cholestasis in vivo. (A) Effects of dioscin on ANIT-induced liver injury based on H&E staining (× 200 original magnification). (B) Effects of dioscin on the relative liver weight and the serum levels of ALT, AST, ALP and TBIL in rats and mice. Values are expressed as the mean ± SD (n = 10). ^##p < 0.01 compared with control groups. ^∗p < 0.05 and ^∗∗p < 0.01 compared with model groups.

FIGURE 2

Dioscin attenuates ANIT-induced oxidative stress. (A) Effects of dioscin on the ROS levels in SCHs by immunofluorescence assay (× 400 magnification). (B) Effects of dioscin on the levels of GSH, GSH-Px, MDA and SOD in rats and mice. Values are expressed as the mean ± SD (n = 10). ^##p < 0.01 compared with control groups. ^∗p < 0.05 and ^∗∗p < 0.01 compared with model groups.

FIGURE 3

Dioscin regulates ANIT-induced cholestasis by transporters. (A–C) Effects of dioscin on the levels of Ntcp based on immunofluorescence assay in SCHs (× 400 original magnification) and animals (× 200 original magnification). (D–F) Effects of dioscin on the protein levels of Ntcp, OAT1, OCT1, Bsep and Mrp2 in SCHs, rats and mice.

FIGURE 4

Dioscin attenuates ANIT-induced apoptosis. (A–C) Representative images of TUNEL-stained SCHs ( × 400 magnification), and the livers of rats and mice ( × 200 magnification). (D–F) Effects of dioscin on the protein levels of Bcl-2, Bcl-xl, Bax, Bak, Caspase 9 and Caspase 3 in SCHs, rats and mice.

FIGURE 5

Dioscin inhibits PI3K/Akt signaling pathway. (A–C) Effects of dioscin on PI3K levels based on immunofluorescence assay in SCHs ( × 400 original magnification) and animals ( × 200 original magnification). (D–F) Effects of dioscin on the protein levels of pPI3K, p-Akt, Nrf2, GCLm, GCLc, NQO1 and HO-1 in SCHs, rats and mice.

FIGURE 6

Dioscin inhibits PI3K/Akt-mediated oxidative stress. (A) Effects dioscin on p-PI3K levels based on immunofluorescence assay in SCHs ( × 400 original magnification) treated by wortmannin and perifosine. (B) Effects of dioscin on the protein levels of p-PI3K, p-Akt, Nrf2, GCLm, GCLc, NQO1 and HO-1 in SCHs treated by wortmannin and perifosine. (C) Effects of dioscin on the mRNA levels of Nrf2, GCLm, GCLc, NQO1 and HO-1 in SCHs treated by wortmannin or perifosine. Values are expressed as the mean ± SD (n = 5). ^∗,^∗∗p < 0.01 compared with model group; ^&p < 0.05 and ^&&p < 0.01 compared with ANIT + Dio 800 + wortmannin group.

FIGURE 7

The schematic diagram of dioscin against ANIT-induced intrahepatic cholestasis. Dioscin regulated the levels of Ntcp, OAT1, OCT1, Bsep and Mrp2 to accelerate bile acids excretion, and then adjusted the levels of Bcl-2, Bcl-xl, Bax, Bak, Caspase 9 and Caspase 3 to alleviate apoptosis. In addition, dioscin markedly inhibited PI3K/Akt pathway and up-regulated the levels of Nrf2, GCLc, GCLm, NQO1 and HO-1 against oxidative stress caused by bile acids. Dioscin exhibited protective effect against ANIT-induced intrahepatic cholestasis via altering transporters, apoptosis and oxidative stress.