Chronic Hepatotoxicity Of Oleanolic Acid In Mice

Objective: To study the hepatotoxic effects of oleanolic acid(OA) at different low doses for the long-term, to examine the relationship between the plasma concentrations of OA and liver toxicity and the expression of genes such as Nrf2 signaling pathways. Methods: Forty SPF male Kunming mice were randomly divided into 4 groups: normal control group(Corn oil), OA low dose group(45.7 mg/kg), middle dose group(91 mg/kg), and high dose group(137 mg/kg), 10 mice in each group. Mice were orally administered OA continuously for 90 days, one hour after the last administration, blood and livers were collected to determine the plasma ALT, AKP activities and total bilirubin(TBIL) contents; livers were subjected to HE staining, Masson staining, Sirius red staining to observe the effects of OA on liver morphology; the concentrations of OA in plasma and livers were determined by LC-MS; the effects of OA on the gene expression of the TGF-β/Smad pathway, Hedgehog pathway, bile acid synthesis pathway, and Nrf2 pathway were examined by RT-PCR; Western-blot was used to detect the expression of Nrf2 protein in the nucleus. Results: 1. Mice were given low doses of OA for 90 days, the body weights did not change significantly, and the liver weight index increased, the activities of ALT and AKP were increased, but the content of TBIL in the liver was unchanged. In normal control group, the structure of hepatic lobule was intact, hepatic cells appeared slightly enlarged in the OA low dose group, liver cells showed spotty necrosis in the middle dose group, and the necrosis area of liver cells were increased in the high does group by HE staining; Masson staining showed that in the normal group, the blue collagen fibers were not apparent, in OA low dose group, the area of blue collagen fibers increased around portal area, in the middle dose group, fibrosis was evident in portal area, forming fibrous septa, in OA high dose group, periportal fibroblasts proliferation, collagen formation, fibrous septa with disordered lobule structure were widespread; Sirius red staining showed the area of fibrosis was increased with OA does. 2. LC-MS was used to detect the concentration of OA in plasma and livers. Plasma OA increased with the increase of OA dosage, but OA concentration in the liver was basically the same and independent of OA dose. 3. Oral administration of OA to mice for long-time could significantly inhibit the expression of Smad7 mRNA in the TGF-β/Smad pathway(P<0.05); OA decreased the expression of Shh, Smo mRNA in the Hedeghog pathway(P<0.05); the expression of Cyp27a1 and the SHP mRNA were also decreased in the bile acid synthesis pathway(P<0.05); OA increased the expression of Nrf2, GCLC, NQO1, GST, HO-1, MRP2 and MRP3 mRNA at the low does(P<0.05), but not evident at the high dose. 4. Western-blot was used to detect the expression of Nrf2 in the nucleus of hepatic cell, the expression of Nrf2 was induced significantly in high group(P<0.05). Summary: Oral administration of OA at low dose for long-time induced liver fibrosis, and produced the elevation of plasma OA concentration, and associated with the expression of Nrf2.