Expression characteristics of the cyp19a1b aromatase gene and its response to 17ß-estradiol treatment in largemouth bass (Micropterus salmoides).

To investigate the regulatory role of the cyp19a1b aromatase gene in the sexual differentiation of largemouth bass (Micropterus salmoides, LMB), we obtained the full-length cDNA sequence of cyp19a1b using rapid amplification of cDNA ends technique. Tissue expression characteristics and feedback with 17-ß-estradiol (E2) were determined using quantitative real-time PCR (qRT-PCR), while gonad development was assessed through histological section observations. The cDNA sequence of LMB cyp19a1b was found to be1950 base pairs (bp) in length, including a 5' untranslated region of 145 bp, a 3' untranslated region of 278 bp, and an open reading frame encoding a protein consisting of 1527 bp that encoded 508 amino acids. The qRT-PCR results indicated that cyp19a1b abundantly expressed in the brain, followed by the gonads, and its expression in the ovaries was significantly higher than that observed in the testes (P < 0.05). After feeding fish with E2 for 30 days, the expression of cyp19a1b in the pseudo-female gonads (XY-F) was significantly higher than that in males (XY-M) (P < 0.05), whereas expression did not differ significantly between XX-F and XY-F fish (P > 0.05). Although the expression of cyp19a1b in XY-F and XX-F fish was not significantly different after 60 days (P>0.05), both exhibited significantly higher levels than that of XY-M fish (P<0.05). Histological sections analysis showed the presence of oogonia in both XY-F and XX-F fish at 30 days, while spermatogonia were observed in XY-M fish. At 60 days, primary oocytes were abundantly observed in both XY-F and XX-F fish, while a few spermatogonia were visible in XY-M fish. At 90 days, the histological sections' results showed that a large number of oocytes were visible in XY-F and XX-F fish. Additionally, the gonads of XY-M fish contained numerous spermatocytes. These results suggest that cyp19a1b plays a pivotal role in the development of ovaries and nervous system development in LMB.

Timing of early gonadal differentiation and effects of estradiol-17ß treatments on the sex differentiation in Largemouth bass (Micropterus salmoides).

In this study, an efficient estradiol-17ß (E2)-induced feminization method was established based on the timing of early gonadal differentiation in Largemouth bass (Micropterus salmoides). Histological section results showed that from 20 days post-hatch (dph) to 30 dph, the germ cells gradually differentiated into oogonium and spermatic deferent, respectively. Moreover, female-biased genes Foxl2 and Cyp19a1a were up-regulated to the first peak at 20 dph, while the male-biased genes Dmrt1 were up-regulated to the first peak at 30 dph. These results indicated that the timing of early gonadal differentiation in Largemouth bass was between 20 and 30 dph. Therefore, 15 dph Largemouth bass with a body length of 15.10 ± 0.09 mm were chosen, and four E2-treated diets were set as 0 (E0, control), 50 mg/kg E2 (E50), 100 mg/kg E2 (E100), and 200 mg/kg E2 (E200). After feeding with E2-treated diets for 60 days, female ratios were 55%, 100%, 100%, and 100% in E0, E50, E100, and E200 groups, respectively. No intersex fish were observed in all the groups. However, 30% of females in the E200 group possessed thinner ovaries, with smaller ovary cavity structures and a decreased number of primary oocyte cells than those in other groups. Besides, the Largemouth bass in the E0 group grew more than those in E50, E100, and E200 groups during the E2 treatments period (P < 0.05). In conclusion, our study suggested that 50-100 mg/kg E2-treated diets could effectively induce the feminization of 15 dph Largemouth bass within 60 days duration time, which provided valuable information for the breeding of the all-male Largemouth bass population.

Fabp4 contributes toward regulating inflammatory gene expression and oxidative stress in Ctenopharyngodon idella.

Fatty acid-binding protein (Fabp)-4 is a member of the FABP family. Mammalian fabp4 has been demonstrated to involve in inflammation and immunity, whereas the related data of fish fabp4 remain limited. Therefore, we further investigated the effects of fabp4 on immunity in Ctenopharyngodon idella. The fabp4 sequence spanned 405 bp was cloned first, sharing high identity to fabp4 from other fish and mammals. Fabp4 expression was the highest in the adipose tissue, followed by the heart, muscle, and liver. In vivo, lipopolysaccharide (LPS) triggered the expression of fabp4, toll-like receptor (tlr)-22, interleukin (il)-1ß, and tumor necrosis factor (tnf)-α in the kidney and spleen. In vitro, exposing C. idella CIK cells to LPS decreased their viability, and the expression of fabp4 was also increased by LPS. However, BMS309403, an inhibitor of FABP4, mitigated these effects. Furthermore, treating the cells with LPS or fabp4 overexpression plasmids resulted in reactive oxygen species (ROS) generation and upregulation of inflammatory genes expression, including tlr22, type-I interferon (ifn-1), interferon regulatory factor (irf)-7, tnfα, il-1ß, and interferon-ß promoter stimulator 1. These effects were ameliorated by preincubation with BMS309403. Moreover, incubating the cells with glutathione reduced the production of ROS and the expression of inflammatory genes that were evoked by LPS and plasmid treatments. These results showed that fabp4 acts as a pro-inflammatory molecule via elevating ROS levels, providing a novel understanding of the molecular regulation of innate immunity in teleosts.

[Chinese herbal medicine Xiayuxue Decoction inhibits liver angiogenesis in rats with carbon tetrachloride-induced liver fibrosis].

OBJECTIVE: To evaluate the effects of Xiayuxue Decoction, a compound traditional Chinese medicine, on liver angiogenesis in rats with carbon tetrachloride (CCl(4))-induced liver fibrosis. METHODS: Liver cirrhosis was induced by intraperitoneal injection of 50% CCl(4)-olive oil solution at the dose of 1 mL/kg body weight, twice per week for 9 consecutive weeks. After 3- and 6-week injection, 6 rats in the normal group and 6 rats in the model group were randomly sacrificed for dynamic observation. The survival rats of model group were randomly divided into model group (n=15) and Xiayuxue Decoction group (n=11). Six normal rats were used as a normal control. Xiayuxue Decoction was administered orally starting from the 7th week for 3 weeks. At the end of the ninth week, animals were sacrificed and liver tissues were harvested to measure histological changes, activities of matrix metalloproteinase-2 (MMP-2) and MMP-9 and protein expressions of platelet endothelial cell adhesion molecule-1 (CD31), von Willebrand factor (vWF), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2 (VEGFR2), complement decay-accelerating factor (DAF) and α-smooth muscle actin (α-SMA) in the liver tissues. RESULTS: Compared with the normal group, liver injury, fatty degeneration and collagen deposition were evidently observed in the model group and protein expressions of CD31, vWF, VEGF, VEGFR2, DAF and α-SMA were gradually increased. In addition, the activities of MMP-2 and MMP-9 in liver tissues were enhanced in the model group (P<0.01). Compared with 9-week model group, liver injury, fatty degeneration and collagen deposition were markedly inhibited by Xiayuxue Decoction; protein expressions of CD31, vWF, VEGF, VEGFR2,α-SMA and DAF and activities of MMP-2 and MMP-9 in the liver tissues were decreased in the Xiayuxue Decoction group (P<0.01). CONCLUSION: The angiogenesis is evident and aggravating gradually during the progression of liver cirrhosis induced by CCl(4). Xiayuxue Decoction inhibits the angiogenesis by decreasing the activities of MMP-2 and MMP-9, inhibiting the activation of hepatic stellate cells, and damaging the new vessel integrality.

[Mechanism of hepatocytes transdifferentiation to bile duct epithelial cells and intervention of huangqi decoction].

OBJECTIVE: To investigate the mechanism of hepatocytes transdifferentiation to bile duct epithelial cells (BECs) and intervention of Huangqi decoction (HQD) on hepatic fibrosis formation in rats with secondary cholestasis. METHODS: Seventy-five SD male rats were made into cholestatic hepatic fibrosis model animals by bile duct ligation, and randomized into the control group (n = 50) and the HQD group (n = 15). Starting from one week after modeling, they were administered orally with saline and HQD respectively for four weeks. Besides, a sham-operated group was set up with 10 rats operated by choledochus segregating only and administered after then with saline. Rats were killed in batches at different time points, i.e. each five from the control group and sham-operated group at the end of the 1st week, five from the control group for each time at the end of the 2nd, 3rd and 4th week, and all the remaining rats at the end of the 5th week. Their liver tissues were taken for histological change examination, content of hydroxyproline (Hyp) determination; protein expression of BECs marker cytokeratin 7 (CK7) and the hepatocyte specific antigen HepPar detection by Western blot, and CK7-Hep Par co-localization by laser confocal microscopy. Then IPP software was used to analyze Sirius red stained positive areas of CK7 and Hep Par, as well as the average IOD of CK7/Hep Par co-localization. RESULTS: Hepatocytes in hepatic tissues (Hep Par positive cell) in the model rats decreased gradually along was time went by after modeling (Sham > M1w > M2w > M3w > M4w > M5w), which was in parallel with the increase of BECs (CK7 positive cells), degree of fibrosis, Hyp content and CK7 protein expression. Increasing of co-localized positive cells of CK7/Hep Par began at 1 week and reached the peak 3 weeks after modeling, then it decreased gradually. The Hep Par protein expression was negatively correlated with that of CK7; the Hep Par positive cell expression was negatively correlated with CK7 positive cell expression and collagen deposition; while the CK7 positive cell expression was positively correlated with the collagen deposition in the liver tissue. Compared with the model control group, the mortality, CK7/Hep Par co-localized positive cells, fibrosis degree, Hyp content and CK7 protein expression were lesser obviously (P < 0.01), while Hep Par positive cell and protein expressions were higher significantly in the HQD group. CONCLUSIONS: Hepatocytes transdifferentiation to BECs might be a key pathological element for secondary cholestatic hepatic fibrosis formation; the restraining action of HQD is possibly a major action mechanism of HQD for effectively intervening and treating secondary cholestasis hepatic fibrosis.

[Huangqi decoction inhibits cholangiocyte proliferation and transdifferentiation in cholestatic liver fibrosis induced by BDL in rats].

OBJECTIVE: To elucidate the antifibrotic mechanism of Huangqi decoction in rats with BDL-induced cholestatic liver fibrosis. METHODS: Liver fibrosis model was induced by ligating the common bile duct (BDL) in rats. Sham-operation was performed in control rats. The BDL rats were randomly divided into two groups: the BDL group and the Huangqi decoction group. Huanqi decoction was given intragastrically for 4 weeks. At the end of the fifth week after BDL, animals were sacrificed. RESULTS: Compared with the sham control group, mortality rate in BDL group was 33.3% and incidence rate of ascites was 90%, and hepatic function was abnormal in most of the rats in BDL group. The number of Hepatocytes was decreased and the number of cholangiocytes significantly increased in BDL group. In addition, Hyp content of liver tissue and protein expression of CK 7 and a-SMA were significantly increased. Immunostaining indicated that CK 7 and a-SMA were co-localized in BDL group. These changes were markedly suppressed by the Huangqi decoction. CONCLUSIONS: These observations suggest that Huangqi decoction can inhibit cholangiocyte proliferation and cholangiocyte transdifferentiation.