Role of nuclear pregnane X receptor in Cu-induced lipid metabolism and xenobiotic responses in largemouth bass (Micropterus salmoides)

Abstract

The pregnane X receptor (PXR) is a master xenobiotic-sensing receptor in response to toxic byproducts, as well as a key regulator in intermediary lipid metabolism. Therefore, the present study was conducted to investigate the potential role of PXR in mediating the lipid dysregulation and xenobiotic responses under Cu-induced stress in largemouth bass (Micropterus salmoides). Four groups of largemouth bass (52.66 ± 0.03 g) were treated with control, Cu waterborne (9.44 μmol/L), Cu+RIF (Rifampicin, 100 mg/kg, PXR activator), and Cu+KET (Ketoconazole, 20 mg/kg, PXR inhibitor) for 48 h. Results showed that Cu exposure significantly elevated the plasma stress indicators and triggered antioxidant systems to counteract Cu-induced oxidative stress. Acute Cu exposure caused liver steatosis, as indicated by the significantly higher levels of plasma triglycerides (TG), lipid droplets, and mRNA levels of lipogenesis genes in the liver. Liver injuries were detected, as shown by hepatocyte vacuolization and severe apoptotic signals after Cu exposure. Importantly, Cu exposure significantly stimulated mRNA levels of PXR, suggesting the response of this regulator in the xenobiotic response. The pharmacological intervention of PXR by the agonist and antagonist significantly altered hepatic mRNA levels of PXR, implying that RIF and KET were effective agents of PXR in largemouth bass. Administration of RIF significantly exacerbated liver steatosis, and such alterations were dependent on the regulations on pparγ and cd36 rather than srebp1 signaling, which suggested that PXR-PPARγ might be another pathway for Cu-induced lipid deposition in fish. Whereas, KET administration showed reverse effects on lipid metabolism as indicated by the lower hepatic TG levels, suppressed mRNA levels of pparγ and cd36. Activation of PXR stimulated autophagy and inhibited apoptosis, leading to lower hepatic vacuolization; while inhibition of PXR showed higher apoptotic signals, inhibition of autophagic genes and stimulation of apoptotic genes. Taken together, PXR played a cytoprotective role in Cu-induced hepatotoxicity through regulations on autophagy and apoptosis. Overall, our data has demonstrated for the first time on the dual roles of PXR as a co-regulator in mediating xenobiotic responses and lipid metabolism in fish, which implying the potential of PXR as a therapy target for xenobiotics-induced lipid dysregulation and hepatotoxicity.

Keywords: Cu exposure; PXR; apoptosis; autophagy; lipid metabolism.

Figure 1

Plasma stress indicators in largemouth bass subjected to RIF or KET under Cu exposure (n = 6). Significant differences between CON and Cu groups, Cu and Cu+RIF groups, as well as Cu and Cu+KET groups were indicated by “#”, “*”, and “+” (P < 0.05).

Figure 2

Liver histology (H&E) and histochemistry (Oil Red O) observations of largemouth bass subjected to RIF or KET under Cu exposure (n = 6). (A, E) Control; (B, F) Cu exposure; (C, G) Cu+RIF; (D, H) Cu+KET; (I) relative areas of hepatocyte vacuolization; (J) relative areas of lipid droplets. Photomicrographs magnification (× 200) and scale bar (200 μm). H&E: hematoxylin and eosin staining; Oil Red O: Oil red O staining. Lipid droplets was red-colored and nuclei was blue-colored. Data represent means ± SEM and are normalized to percentage of field area. Significant differences between CON and Cu groups, Cu and Cu+RIF groups, as well as Cu and Cu+KET groups were indicated by “#”, “*”, and “+” (P < 0.05).

Figure 3

PXR responses and lipid metabolism related index in the liver of largemouth bass subjected to RIF or KET under Cu exposure (n = 6). (A) mRNA levels of PXR in the liver; (B) plasma triglycerides (TG); (C) hepatic mRNA levels of srebp1, acc and fas; (D) hepatic mRNA levels of pparγ, cd36 and scd-1; (E) hepatic mRNA levels of pparα and cpt1a. Significant differences between CON and Cu groups, Cu and Cu+RIF groups, as well as Cu and Cu+KET groups were indicated by “#”, “*”, and “+” (P < 0.05).

Figure 4

Enzyme activities and transcriptional levels of antioxidant systems in the liver of largemouth bass subjected to RIF or KET under Cu exposure (n = 6). (A) T-AOC: total antioxidant capacity; (B) SOD: superoxide dismutase; (C) CAT: catalase; (D) MDA: malondialdehyde; (E) hepatic mRNA levels of nrf2, keap1, sod, cat1. Significant differences between CON and Cu groups, Cu and Cu+RIF groups, as well as Cu and Cu+KET groups were indicated by “#”, “*”, and “+” (P < 0.05).

Figure 5

Hepatic mRNA levels of genes involved in autophagy in largemouth bass subjected to RIF or KET under Cu exposure (n = 6). Significant differences between CON and Cu groups, Cu and Cu+RIF groups, as well as Cu and Cu+KET groups were indicated by “#”, “*”, and “+” (P < 0.05).

Figure 6

Representative DAPI and TUNEL double staining of liver in largemouth bass subjected to RIF or KET under Cu exposure (n = 6). TUNEL-positive cells were stained and indicated by bright green fluorescence indicating apoptotic cells, and normal nuclei appear in blue. (A) TUNEL staining sections; (B) Apoptotic index: the number of apoptotic nuclei/The number of observed nuclei × 100%. Significant differences between CON and Cu groups, Cu and Cu+RIF groups, as well as Cu and Cu+KET groups were indicated by “#”, “*”, and “+” (P < 0.05).

Figure 7

Hepatic mRNA levels of genes involved in apoptosis in largemouth bass subjected to RIF or KET under Cu exposure (n = 6). Significant differences between CON and Cu groups, Cu and Cu+RIF groups, as well as Cu and Cu+KET groups were indicated by “#”, “*”, and “+”(P < 0.05).