Identification and functional characterization of the transcription factor NF-κB subunit p65 in common carp (Cyprinus carpio L.).

p65 is an important subunit of the transcription factor NF-κB in the regulation of immune response. In the present study, the p65 cDNA was identified from common carp (Cyprinus carpio L.) (named Ccp65). Phylogenetic analysis revealed that Ccp65 located in the same clade as piscine p65 and exhibited closest relationship to that of Ctenopharyngodon idella. Ccp65 was constitutively expressed in all the examined tissues. Aeromonas hydrophila and poly(I:C) can induce the expression of Ccp65 in the designated tissues and the Ccp65 expression was up-regulated in HKLs following LPS and poly(I:C) stimulation. In addition, the nuclear localization signal (NLS) and C-terminal domain are the important elements of Ccp65. Immunofluorescence assay revealed that the nuclear localization signal deletion mutation of Ccp65 (Ccp65ΔNLS) failed to translocate to the nucleus even though stimulation with poly(I:C) or LPS, and the C-terminal domain deletion mutation of Ccp65 (Ccp65ΔC) did not up-regulate the luciferase activity. Furthermore, Ccp65 can induce the expression of il-1ß and tnf-α. And LPS and poly(I:C) inducing the expression of il-1ß and tnf-α, is dependent on the Ccp65. Taken altogether, these findings lay the foundations for future research to investigate the mechanisms underlying fish p65.

ANTI-INFLAMMATORY ACTIVITY OF PLATYCODIN D ON ALCOHOL-INDUCED FATTY LIVER RATS VIA TLR4-MYD88-NF-κB SIGNAL PATH.

BACKGROUND: The current study was designed to evaluate the effect of Platycodin D (PD), triterpenoid saponins extracted from the roots of Platycodon grandiflorum (PG) on alcohol-induced fatty liver (AFL) and investigate the possible mechanism. METHODS AND MATERIALS: A rat model was set up by feeding ethanol and fish oil to experimental rats, which then were treated with PD of 10, 20, 30 mg/kg body weight/day for 4 weeks, respectively, whereafter, liver function enzymes, endotoxin of serum and liver lipid were assayed by biochemical methods, cytokines, histochemistry of hepatic tissue, the protein expression of CD14 and TLR4, the mRNA expression of MD-2, MyD 88 and TRAF-6 were assayed. RESULTS: Treatment with PD on AFL rats significantly decreased the levels of serum ALT, AST and TBIL, coefficient of liver index and the hepatic tissue contents of TG, additionally and dramatically decreased serum endotoxin levels, down-regulated MD-2 and CD14 levels, as well as the mRNA expression of TLR4, MyD88 and TRAF-6, accordingly suppressed NF-κB: p65 as well as endotoxin-mediated inflammatory factors such as TNF-α and IL-6. CONCLUSIONS: Treatment with PD effectively protects against AFL through anti-inflammatory and anti-endotoxic process, and the confirmed mechanism is that PD treatment ameliorate alcoholic-induced liver injury mainly via TLR4-MyD88-NF-K: B signal path in AFL rat. List of Abbreviations: AFL: alcoholic-induced fatty liver, CD14: cluster of differentiation 14, LPS: lipopolysaccharide, LBP: lipopolysaccharide-binding protein, TLR4: toll-like receptor 4, MD-2: molecule myeloid differential protein-2, MyD 88: myeloid differentiation primary response protein 88, TRAF-6: TNF-receptor associated factor-6, NF-κB: nuclear transcription factor kappa B, IL-6: interleukin-6, TNF-α: tumor necrosis factor-α, PG: Platycodon grandiflorum, PD: Platycodin D.

Molecular characterization of hepcidin gene in common carp (Cyprinus carpio L.) and its expression pattern responding to bacterial challenge.

Hepcidin is a cysteine-rich cationic antimicrobial peptide (AMP), which plays an important role in host innate immune system and iron regulation. A great many of hepcidin genes have been identified from vertebrates, including various fish species. Here we report the cloning and identification of a hepcidin cDNA from the liver of common carp (Cyprinus carpio L.). The full-length cDNA of the common carp hepcidin was 647 bp, which contained an ORF of 276 bp encoding a prepropeptide of 91 amino acid residues. The predicted preprohepcidin consisted of three domains: a signal peptide of 24 amino acids, a prodomain of 42 amino acids and a mature peptide of 25 amino acids, which containd eight cysteine residues at the identical conserved position. The genomic organization of common carp hepcidin was identified, which contained three exons and two introns, similarly to corresponding genes in mammals and other fish species. Sequence alignment and phylogenetic analysis showed that hepcidins were conserved in different species, and common carp hepcidin was type 1 hepcidin according to the phylogenetic tree, which had the highest identity with mud loach and zebrafish. Real-time PCR assay showed that the hepcidin gene was mostly expressed in liver, and expressed in all the other tested tissues of common carp in different levels. When challenged with pathogenic bacterium, Vibrio anguillarum, the expression level of common carp hepcidin was quickly up-regulated in liver, spleen, head kidney and hindgut, implying that hepcidin may be an important component of the innate immune system of common carp and involved in mucosal immune response against invading pathogens.

Anti-atherosclerotic activity of platycodin D derived from roots of Platycodon grandiflorum in human endothelial cells.

This study examined the effects of platycodin D (PD), a triterpene saponin from the the root of Platycodon grandiflorum A.DC on human umbilical vein endothelial cells (HUVECs) in vitro, which were pre-treated with PD (0.01, 0.15, 0.25 mg/mL), respectively, and treated with 50 mg/L oxidized low-density lipoprotein (oxLDL). The levels of nitric oxide (NO) and malonaldehyde (MAD) in the culture medium, vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) mRNA expression in endothelium cells and the adhesion of monocytes to endothelial cells were measured. The results showed that PD increased NO concentration and decreased MDA level induced by oxLDL in the medium of endothelial cells. Moreover, PD significantly inhibited the oxLDL-induced increase in monocyte adhesion to endothelial cells as well as decreasing mRNA expression levels of VCAM-1 and ICAM-1 on these cells. Based on these results, it is suggested that PD is a promising anti-atherosclerotic activity, which is at least in part the result of its increasing NO concentration, reducing the oxLDL-induced cell adhesion molecule expression in endothelial cells and the endothelial adhesion to monocytes.

Apoptosis of hepatoma cells SMMC-7721 induced by Ginkgo biloba seed polysaccharide.

AIM: To study the apoptosis of hepatoma cells SMMC-7721 induced by polysaccharide isolated from Ginkgo biloba seed. METHODS: Ginkgo biloba seed polysaccharide (GBSP) was isolated by ethanol fractionation of Ginkgo biloba seed and purified by Sephadex G-200 chromatography. The purity of GBSP was verified by reaction with iodine-potassium iodide and ninhydrin and confirmed by UV spectrophotometer, cellulose acetate membrane electrophoresis and Sepharose 4B gel filtration chromatography. The Scanning Electron Microscope (SEM) and Flow Cytometry (FCM) were used to examine the SMMC-7721 cells with and without GBSP treatment at 500 mg/ml for 36 h. RESULTS: GBSP product obtained was of high purity with the average molecular weight of 1.86 X 10(5). Quantitative analysis of SMMC-7721 cells in vitro with FCM showed that the percentages of G(2)-M cells without and with GBSP treatment were 17.01+/-1.28 % and 11.77+/-1.50% (P<0.05), the debris ratio of the cells were 0.46+/-0.12 % and 0.06+/-0.06 % (P<0.01), and the apoptosis ratio of cells was 3.84+/-0.55 % and 9.13+/-1.48 % (P<0.01) respectively. Following GBSP treatment, microvilli of SMMC-7721 cells appeared thinner and the number of spherical cells increased markedly. Most significantly, the apoptosis bodies were formed on and around the spherical cells treated with GBSP. CONCLUSION: GBSP could potentially induce the apoptosis of SMMC-7721 cells.