Firm evidence for the detoxification of senecionine-induced hepatotoxicity via N-glucuronidation in UGT1A4-humanized transgenic mice.

Uridine diphosphate glucuronosyltransferase (UGT)1A4 is responsible for N-glucuronidation of tertiary amines but is a pseudogene in commonly used rodent models in toxicity and safety assessment. As a continuation of our investigation into the toxicity and safety assessment of pyrrolizidine alkaloid (PA)-containing herbs, we generated a UGT1A4-humanized (hUGT1A4) transgenic mouse model to systematically study the toxicity, metabolism network, and toxicokinetic characteristics of senecionine (a representative toxic PA) and compared with that in the wide-type controls in parallel. As results, senecionine-induced toxicity was significantly decreased as approved by mortality, pathology, and biochemistry assays in hUGT1A4 mice and cultured primary hepatocytes. More importantly N-glucuronidation adduct was exclusively identified in all the hUGT1A4 mice, liver microsomes, and cultured primary hepatocytes, yet absent in the wide-type controls. The variation in toxicokinetic characters was also observed between hUGT1A4 mice and the wide-type controls with a notably inhibition of the toxification metabolites, i.e., pyrrole-protein adducts, in hUGT1A4 mice. Conclusively, UGT1A4 plays an important role in detoxification of senecionine and the hUGT1A4 mouse model is promising for the pre-clinical evaluation of the efficacy and toxicity of tertiary amine agents in drug development and safety assessment.

Bear bile powder attenuates senecionine-induced hepatic sinusoidal obstruction syndrome in mice.

Hepatic sinusoidal obstruction syndrome (HSOS) via exposure to pyrrolizidine alkaloids (PAs) is with high mortality and there is no effective treatment in clinics. Bear bile powder (BBP) is a famous traditional animal drug for curing a variety of hepatobiliary diseases such as cholestasis, inflammation, and fibrosis. Here, we aim to evaluate the protective effect of BBP against HSOS induced by senecionine, a highly hepatotoxic PA compound. Our results showed that BBP treatment protected mice from senecionine-induced HSOS dose-dependently, which was evident by improved liver histology including reduced infiltration of inflammatory cells and collagen positive cells, alleviated intrahepatic hemorrhage and hepatic sinusoidal endothelial cells, as well as decreased conventional serum liver function indicators. In addition, BBP treatment lowered matrix metalloproteinase 9 and pyrrole-protein adducts, two well-known markers positively associated with the severity of PA-induced HSOS. Further investigation showed that BBP treatment prevents the development of liver fibrosis by decreasing transforming growth factor beta and downstream fibrotic molecules. BBP treatment also alleviated senecionine-induced liver inflammation and lowered the pro-inflammatory cytokines, in which tauroursodeoxycholic acid played an important role. What's more, BBP treatment also decreased the accumulation of hydrophobic bile acids, such as cholic acid, taurocholic acid, glycocholic acid, as well. We concluded that BBP attenuates senecionine-induced HSOS in mice by repairing the bile acids homeostasis, preventing liver fibrosis, and alleviating liver inflammation. Our present study helps to pave the way to therapeutic approaches of the treatment of PA-induced liver injury in clinics.

mRNA Expression of Ovine Angiopoietin-like Protein 4 Gene in Adipose Tissues.

Angiopoietin-like protein 4 (ANGPTL4) is involved in a variety of functions, including lipoprotein metabolism and angiogenesis. To reveal the role of ANGPTL4 in fat metabolism of sheep, ovine ANGPTL4 mRNA expression was analyzed in seven adipose tissues from two breeds with distinct tail types. Forty-eight animals with the gender ratio of 1:1 for both Guangling Large Tailed (GLT) and Small Tailed Han (STH) sheep were slaughtered at 2, 4, 6, 8, 10, and 12 months of age, respectively. Adipose tissues were collected from greater and lesser omental, subcutaneous, retroperitoneal, perirenal, mesenteric, and tail fats. Ontogenetic mRNA expression of ANGPTL4 in these adipose tissues from GTL and STH was studied by quantitative real time polymerase chain reaction. The results showed that ANGPTL4 mRNA expressed in all adipose tissues studied with the highest in subcutaneous and the lowest in mesenteric fat depots. Months of age, tissue and breed are the main factors that significantly influence the mRNA expression. These results provide new insights into ovine ANGPTL4 gene expression and clues for its function mechanism.

Ontogenetic Expression of Lpin2 and Lpin3 Genes and Their Associations with Traits in Two Breeds of Chinese Fat-tailed Sheep.

Lipins play dual function in lipid metabolism by serving as phosphatidate phosphatase and transcriptional co-regulators of gene expression. Mammalian lipin proteins consist of lipin1, lipin2, and lipin3 and are encoded by their respective genes Lpin1, Lpin2, and Lpin3. To date, most studies are concerned with Lpin1, only a few have addressed Lpin2 and Lpin3. Ontogenetic expression of Lpin2 and Lpin3 and their associations with traits would help to explore their molecular and physiological functions in sheep. In this study, 48 animals with an equal number of males and females each for both breeds of fat-tailed sheep such as Guangling Large Tailed (GLT) and Small Tailed Han (STH) were chosen to evaluate the ontogenetic expression of Lpin2 and Lpin3 from eight different tissues and months of age by quantitative real-time polymerase chain reaction (PCR). Associations between gene expression and slaughter and tail traits were also analyzed. The results showed that Lpin2 mRNA was highly expressed in perirenal and tail fats, and was also substantially expressed in liver, kidney, reproductive organs (testis and ovary), with the lowest levels in small intestine and femoral biceps. Lpin3 mRNA was prominently expressed in liver and small intestine, and was also expressed at high levels in kidney, perirenal and tail fats as well as reproductive organs (testis and ovary), with the lowest level in femoral biceps. Global expression of Lpin2 and Lpin3 in GLT both were significantly higher than those in STH. Spatiotemporal expression showed that the highest levels of Lpin2 expression occurred at 10 months of age in two breeds of sheep, with the lowest expression at 2 months of age in STH and at 8 months of age in GLT. The greatest levels of Lpin3 expression occurred at 4 months of age in STH and at 10 months of age in GLT, with the lowest expression at 12 months of age in STH and at 8 months of age in GLT. Breed and age significantly influenced the tissue expression patterns of Lpin2 and Lpin3, respectively, and sex significantly influenced the spatiotemporal expression patterns of Lpin3. Meanwhile, Lpin2 and Lpin3 mRNA expression both showed significant correlations with slaughter and tail traits, and the associations appear to be related with the ontogenetic expression as well as the potential functions of lipin2 and lipin3 in sheep.