Effects of Zinc Adaptation on Histological Morphology, Antioxidant Responses, and Expression of Immune-Related Genes of Grass Carp (Ctenopharyngodon idella).

The study was conducted to evaluate the effect of zinc adaptation on histological morphology and antioxidant and immune responses of grass carp(Ctenopharyngodon idella). A total of 180 young grass carp (20.0 ± 2.0 g) was equally distributed into 9 groups, and triplicate groups were subjected to 0 µg/L Zn2+ (control group), 200 µg/L Zn2+, and 300 µg/L Zn2+ solution for 42 days, respectively. The results indicated that the liver and gill have obvious pathological changes after long-term adaptation to zinc except the intestine; the zinc adaptation can positively influence intestinal morphology. The activities of GPX (glutathione peroxidase activity), SOD (superoxide dismutase), and CAT (Catalase) were significantly increased in zinc treatment groups (P < 0.05). The genes expression levels of CuZnSOD (copper zinc superoxide dismutase), CAT, Hsp70 (heat shock protein-70), IL-1b (interleukin-1-b), and TGF-ß1 (transforming growth factor-ß1) were upregulated in the gill and intestine of grass carp following waterborne adaptation to zinc solution for 42 days (P < 0.05). In conclusion, zinc adaptation has different effects on organs of grass carp and may reduce the inflammatory response of the body's gills and intestines by improving the body's antioxidant and anti-stress defense capabilities.

Two Δ6-desaturase-like genes in common carp (Cyprinus carpio var. Jian): structure characterization, mRNA expression, temperature and nutritional regulation.

Δ6-Desaturase is the rate-limiting enzyme involved in highly unsaturated fatty acid (HUFA) biosynthesis. There is very little information on the evolution and functional characterization of Δ6Fad-a and Δ6Fad-b in common carp (Cyprinus carpio var. Jian). In the present study, the genomic sequences and structures of two putative Δ6-desaturase-like genes in common carp genome were obtained. We investigated the mRNA expression patterns of Δ6Fad-a and Δ6Fad-b in tissue, hatching carp embryos, larvae by temperature shock and juveniles under nutritional regulation. Our results showed that the two Δ6Fad genes had identical coding exon structures, being comprised of 12 coding exons, and with introns of distinct size and sequence composition. They were not allelic variants of a single gene. Both Δ6Fad genes were highly expressed in liver, intestine (pyloric caeca) and brain. The Δ6Fad-a and Δ6Fad-b mRNAs showed an increase in expression from newly hatched to 25 days after hatching. The expression levels of Δ6Fad-a were obviously regulated by temperature, whereas Δ6Fad-b was not affected by temperature. The regulation of Δ6Fad-a and Δ6Fad-b in response to dietary fatty acid composition was determined in liver, brain and intestine (pyloric caeca) of common carp fed with diets: diet1with fish oil (FO) rich in n-3 HUFA, diet2 with corn oil (CO, 18:2n-6) and diet3 with linseed oil (LO, 18:3n-3). The differential expression of Δ6Fad-a and Δ6Fad-b genes in liver, brain and intestine in common carps was fed with different oil sources, respectively. Further work is in progress to determine the mechanism of differential expression of the Δ6Fad-a and Δ6Fad-b genes in different tissues and the roles of transcription factors in regulating HUFA synthesis.

Influence of dietary fatty acids on muscle fatty acid composition and expression levels of Δ6 desaturase-like and Elovl5-like elongase in common carp (Cyprinus carpio var. Jian).

The effects of dietary fatty acids on muscle fatty acid composition and liver expression levels of Δ6 desaturase-like and Elovl5-like elongase were investigated in common carp (Cyprinus carpio var. Jian). Two Δ6 desaturase-like cDNAs (Fad6-a and Fad6-b) and two Elovl5-like elongase (Elovl5-a and Elovl5-b) cDNAs were cloned. Juvenile carp were fed three semi-purified diets (D1-3) for 6 weeks with different lipid sources: D1, fish oil with high highly unsaturated fatty acids (HUFAs); D2, corn oil with high linoleic acid (LA), but no HUFAs; and D3, linseed oil with high α-linolenic acid (LNA), but no HUFAs. Comparing muscle fatty acids among fish fed D1-3, the content of LA and arachidonic acid (AA) in common carp fed D2 and the content of LNA, EPA and DHA in common carp fed D3 were higher than initial levels (P<0.05), respectively. The liver transcript levels of Fad6-a and Elovl5-a in fish fed D2 and D3 were higher than initial levels (P<0.05), but Fad6-b and Elovl5-b levels were seldom affected by the diets. The dietary fatty acids affect the muscle fatty acid composition and the liver Fad6-a and Elovl5-a gene expression levels in common carp, and further studies should be undertaken.

[Inhibitory effects of Scutellaria barbate extracts on diethylnitrosamine-induced hepatocarcinoma in rats].

OBJECTIVE: To investigate the inhibitory effects of Scutellaria barbate extracts on diethylnitrosamine-induced hepatocarcinoma in rats. METHODS: Hepatocarcinoma model rats were induced by diethylnitrosamine (DEN). Sixty SD male rats were randomly divided into 4 groups: normal control group, hepatocarcinoma model group, ESB of high dose group and ESB of low dose group. All rats were killed in the 18th week, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), alkaline phosphatase (ALP), gamma-glutamyltransferase (gamma-GT) and alpha-L-fucosidase (AFU) in serum were measured by biochemical examinations; Hematoxy and eosin (HE) methods were used to examine the changes of liver pathology. RESULTS: The levels of ALT, AST, TBIL, ALP, gamma-GT, AFU in hepatocarcinoma model group and ESB groups were higher than that of control group (P < 0.05). ESB could relieve hepatic injures. The levels of liver function indexes in ESB groups were lower than that of model group. Histological examination demonstrated that the number of liver cancer nodus in ESB groups were lower than that of model group. Furthermore, ESB could attenuate the grade of cancer cell differentiation. CONCLUSION: ESB could inhibit experimental hepatocarcinoma and relieve hepatic injures in rats.

Matrine induces apoptosis in gastric carcinoma cells via alteration of Fas/FasL and activation of caspase-3.

AIM OF THE STUDY: Matrine, an alkaloid purified from the chinese herb Sophora flavescens Ait, is well known to possess activities including anti-inflammation, anti-fibrotic and anticancer. In this study, the mechanism of matrine inducing the apoptosis of gastric carcinoma cells was investigated. MATERIALS AND METHODS: Proliferation of SGC-7901 cells was examined by MTT assay. Cellular morphology was observed under transmission electron microscope. Flow cytometry (FCM) was used to observe the apoptosis of SGC-7901 cells by staining with annexinV-FITC/PI. The expression levels of Fas/FasL in SGC-7901 cells were monitored by FCM analysis using an indirect immunofluorescence method. Activity of caspase-3 enzyme was measured by spectrofluorometry. RESULTS: MTT assay showed that matrine inhibited SGC-7901 cells proliferation in a dose-dependent and time-dependent manner. Apoptosis induction was demonstrated by morphological changes under electron microscope and FCM analysis. Fluorescence intensity levels of Fas and FasL were found to be equally up-regulated after matrine treatment, which were both correlated with apoptosis rate. The activity of caspase-3 enzyme increased in matrine groups, positively correlated with apoptosis rate. CONCLUSIONS: Matrine could inhibit cell proliferation and induce apoptosis of SGC-7901 cells in vitro. The apoptosis induction appears to proceed by up-regulating Fas/FasL expression and activating caspase-3 enzyme.

Scutellaria barbate extract induces apoptosis of hepatoma H22 cells via the mitochondrial pathway involving caspase-3.

AIM: To study the growth inhibitory and apoptotic effects of Scutellaria barbata D.Don (S. barbata) and to determine the underlying mechanism of its antitumor activity in mouse liver cancer cell line H22. METHODS: Proliferation of H22 cells was examined by MTT assay. Cellular morphology of PC-2 cells was observed under fluorescence microscope and transmission electron microscope (EM). Mitochondrial transmembrane potential was determined under laser scanning confocal microscope (LSCM) with rhodamine 123 staining. Flow cytometry was performed to analyze the cell cycle of H22 cells with propidium iodide staining. Protein level of cytochrome C and caspase-3 was measured by semi-quantitive RT-PCR and Western blot analysis. Activity of caspase-3 enzyme was measured by spectrofluorometry. RESULTS: MTT assay showed that extracts from S. barbata (ESB) could inhibit the proliferation of H22 cells in a time-dependent manner. Among the various phases of cell cycle, the percentage of cells in S phase was significantly decreased, while the percentage of cells in G(1) phase was increased. Flow cytometry assay also showed that ESB had a positive effect on apoptosis. Typical apoptotic morphologies such as condensation and fragmentation of nuclei and blebbing membrane of apoptotic cells could be observed under transmission electron microscope and fluorescence microscope. To further investige the molecular mechanism behind ESB-induced apoptosis, ESB-treated cells rapidly lost their mitochondrial transmembrane potential, released mitochondrial cytochrome C into cytosol, and induced caspase-3 activity in a dose-dependent manner. CONCLUSION: ESB can effectively inhibit the proliferation and induce apoptosis of H22 cells involving loss of mitochondrial transmembrane potential, release of cytochrome C, and activation of caspase-3.

[Anti-proliferative and apoptosis-inducing activity of Scutellaria barbate containing serum on mouse's hepatoma H22 cells].

OBJECTIVE: To investigate the effects of Scutellaria barbate extract (ESB) on suppressing proliferation and inducing apoptosis of mouse hepatoma H22 cells. METHODS: H22 cells cultured in vitro were divided into 5 groups: blank control group, ESB in high, medium, low dose groups and 5-Fu group. H22 cells were cultured in media with serum containing different concentrations of ESB and blank serum. The proliferation of H22 cells was determined by microculture tetrazolium (MTT) assay. Fluorescence microscopy was utilized to observe the apoptosis of H22 cells by staining with Hoechst 33258. The cell cycle and apoptosis were analyzed by flow cytometry (FCM). RESULTS: The inhibition of serum containing ESB on the proliferation of H22 cells in vitro was observerd in a dose and time dependent manner. The typical morphological changes of apoptosis were observed after incubation with ESB-containing serum in high dose for 48 hours. Among the various phases of cell cycle, the percentage of cells in S phase decreased significantly, while the percentage of cells in G1 phase increased. Drug-containing serum showed positive effect on cell apoptosis. The apoptosis rate of blank control group, ESB in low, medium, high dose groups and 5-Fu group were 0.51%, 1.07%, 3.15%, 7.83%, 11.26%, respectively. CONCLUSION: ESB containing serum can inhibit proliferation and induce apoptosis of H22 cells in vitro.

[Effects of Scutellaria Barbata drug-containing serum on apoptosis and mitochondrial transmembrane potential of hepatoma H22 cells].

OBJECTIVE: To investigate the effects of serum containing Scutellaria Barbata extract (ESB) on apoptosis rate and mitochondrial transmembrane potential (MTP) of liver cancer cell line H22 from mice in vitro. METHODS: H22 cells were cultured in vitro and divided into 5 groups: blank control group, low-dose ESB group, medium-dose ESB group, high-dose ESB group and fluorouracil (5-Fu) group. Methyl thiazolyl tetrazolium assay was utilized to determine the proliferation rates of H22 cells. Cellular morphology was observed under a transmission electron microscope (EM). The rhodamine 123 was used as a fluorescence probe to label the H22 cells, and the fluorescence intensities were observed with a laser scanning confocal microscope. The fluorescence intensity of H22 cells indicated the MTP of H22 cells. RESULTS: The inhibition of serum containing ESB on the proliferation of H22 cells in vitro was observed in a time-dependent manner. The typical morphological changes of apoptosis were observed after incubation with ESB-containing serum in high dose for 48h. The apoptosis rates of blank control group, 5-Fu group, low-dose ESB group, medium-dose ESB group and high-dose ESB group were (0.51+/-0.32)%, (11.26+/-2.97)%, (1.07+/-0.46)%, (3.15+/-1.12)%, (7.83+/-2.25)% respectively. ESB could reduce the MTP of H22 cells from mice as compared with the untreated group. The MTPs of the blank control group, 5-Fu group, and low-, medium- and high-dose ESB groups were (245.45+/-67.37), (127.42+/-41.35), (213.68+/-65.52), (186.34+/-56.37) and (142.65+/-39.44) respectively, which were negatively correlated with the apoptosis rates. CONCLUSION: ESB-containing serum effectively induces apoptosis, which may be related to the decrease of MTP in H22 cells.