Anti-oxidant and Anti-inflammatory Effects of Ethanol Extract from Polygala sibirica L. var megalopha Fr. on Lipopolysaccharide-Stimulated RAW264.7 Cells.

OBJECTIVE: To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L. var megalopha Fr. (EEP) on RAW264.7 mouse macrophages. METHODS: RAW264.7 cells were pretreated with 0-200 µg/mL EEP or vehicle for 2 h prior to exposure to 1 µg/mL lipopolysaccharide (LPS) for 24 h. Nitric oxide (NO) and prostaglandin (PGE2) production were determined by Griess reagent and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin-1beta (IL-1ß), and IL-6 were determined using reverse transcription polymerase chain reaction (RT-PCR). Western blot assay was used to determine the protein expressions of iNOS, COX-2, phosphorylation of extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK), inhibitory subunit of nuclear factor Kappa B alpha (Iκ B-α) and p38. Immunofluorescence was used to observe the nuclear expression of nuclear factor-κ B p65 (NF-κ B p65). Additionally, the anti-oxidant potential of EEP was evaluated by reactive oxygen species (ROS) production and the activities of catalase (CAT) and superoxide dismutase (SOD). The 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), superoxide anion (O2-) radical and nitrite scavenging activity were also measured. RESULTS: The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g. With EEP treatment (100 and 150 µg/mL), there was a notable decrease in NO and PGE2 production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions (P<0.01 or P<0.05). Furthermore, with EEP treatment (150 µg/mL), there was a decrease in the mRNA expression levels of TNF-α, IL-1ß and IL-6, as well as in the phosphorylation of ERK, JNK and p38 mitogen-activated protein kinase (MAPK, P<0.01 or P<0.05), by blocking the nuclear translocation of NF-κ B p65 in LPS-stimulated cells. In addition, EEP (100 and 150 µg/mL) led to an increase in the anti-oxidant enzymes activity of SOD and CAT, with a concomitant decrease in ROS production (P<0.01 or P<0.05). EEP also indicated the DPPH, OH, O2- radical and nitrite scavenging activity. CONCLUSION: EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κ B pathway and protected against oxidative stress.

[Expression of ß-catenin in Skin Lesions of Patients with Scleroderma and Its Effect on Epithelial-Mesenchymal Transition of Human Epidermal Keratinocytes].

OBJECTIVE: To investigate the expression of ß-catenin in the skin lesions of patients with systemic scleroderma (SSc) and its effect on epithelial-mesenchymal transition (EMT) of human epidermal keratinocytes. METHODS: The expression of ß-catenin, Snail1 and E-cadherin in the skin lesions sample of 45 SSc patients and normal skin sample from 20 healthy adults was detected with SP immunohistochemistry. HaCaT, the human epidermal keratinocytes, were treated with different concentrations of Wnt10b (0 ng/mL (control), 2 ng/mL and 4 ng/mL) for 48 h. then detected the localization of ß-catenin in HaCaT cells by immunofluorescence assay, determined the mRNA levels of Snail1 and Snail2 in HaCaT cells by real-time fluorescent quantitative PCR, detected the proteins expression of ß-catenin, Vimentin, N-cadherin and E-cadherin in HaCaT cells by Western blot. RESULTS: The positive rates of ß-catenin, Snail1 and E-cadherin in skin lesions of SSc patients were 100%, 88.89% and 2.22% respectively, while in healthy adult skin, the corresponding positive rates were 0%, 10.00%, and 95.00%. The difference between the two groups was significant. Compared with control group, treatment with different concentrations of Wnt10b (2 ng/mL and 4 ng/mL) induced up-regulation of ß-catenin expression and promoted translocation of ß-catenin from cytoplasm to nucleus, increased the mRNA levels of Snail1 and Snail2 (P < 0.05), and up-regulated the proteins expression of Vimentin, N-cadherin, down-regulated the E-cadherin protein expression in HaCaT cells (P < 0.05). CONCLUSIONS: Abnormally activated Wnt/ß-catenin signaling pathway and abnormally expressed EMT-related proteins are observed in SSc lesions. Activation of Wnt/ß-catenin signaling pathway may promote EMT in HaCaT cells.

Synthesis and antitumor activity of conjugates of 5-Fluorouracil and emodin.

A series of conjugates of 5-Fluorouracil (5-FU) and emodin were synthesized by coupling trimethyl emodin with N(1), N(3) dialkylated 5-FU. The 5-FU moiety contained various substituents at the N(3)-position were linked to the 2-position of trimethyl emodin via a methylene linkage. Their cytotoxicity against three cancer cell lines and one noncancerous cell were studied. The results revealed that some of conjugates exhibited better or comparable in vitro antitumor activity to 5-FU and emodin and low toxicity in the normal cell. The structure-activity relationship study showed N(3)-aromatic substituent was important for their cytotoxic activity.

Downregulation of both gene expression and activity of Hsp27 improved maturation of mouse oocyte in vitro.

BACKGROUND: Heat shock protein 27 (Hsp27), a member of the small heat shock protein family, is an apoptosis regulator. Our previous proteomic study showed that Hsp27 mainly expressed in human oocyte, and that Hsp27 expression was downregulated in the ovaries derived from women with the polycystic ovary syndrome (PCOS), a well known endocrinal disorder with abnormal apoptotic activity and folliculogenesis. However, the exact effects of Hsp27 downregulation on oocyte development have not yet been clarified. METHODS: The expression of Hsp27 gene was downregulated in the mouse oocytes cultured in vitro using siRNA adenovirus infection, while the activity of Hsp27 was decreased by microinjection of polyclonal Hsp27 antibody into the cytoplasm of germinal vesicle (GV) oocytes. Oocyte maturation rate was evaluated by morphological observation. Early stage of apoptosis was determined using Annexin-V staining analysis and some critical apoptotic factors and cytokines were also monitored at both mRNA level by real time RT-PCR and protein expression level by immunofluorescence and western blot. RESULTS: Hsp27 expressed at high level in maturing oocytes. Infection with AdshHsp27, and microinjection of Hsp27 antibody into GV oocytes, resulted in the improved oocyte development and maturation. Germinal vesicle breakdown (GVBD) rates were significantly increased in two AdshHsp27-treated groups (88.7%, 86.0%) and Hsp27 antibody-injected group (77.0%) when compared with control (76.2% in AdGFP, 64.4% in IgG-injected), respectively. In addition, the rates of metaphase II (MII) development in two AdshHsp27-treated groups (73.8%, 76.4%) and Hsp27 antibody-injected group (67.3%) were higher than that in the controls (59.6% in AdGFP, 55.1% in IgG-injected). We also found that the rates of early stage of apoptosis in Hsp27 downregulated groups (46.5% and 45.6%) were higher than that in control group (34.1%) after 8 h of IVM. Similarly, downregulation of Hsp27 caused a significantly enhanced the expression of apoptotic factors (caspase 8, caspase 3) and cytokines (bmp 15 and gdf 9). CONCLUSIONS: Downregulation of Hsp27 improved the maturation of mouse oocytes, while increased early stage of apoptosis in oocytes by inducing the activation of extrinsic, caspase 8-mediated pathway.

Polymorphism in the first intron of follicle stimulating hormone beta gene in three Chinese pig breeds and two European pig breeds.

Follicle stimulating hormone (FSH) is a pituitary gonadotropin that plays a key role in the regulation of gonadal function in mammal. The gonadotropic hormones are composed of two subunits, the common alpha subunit and the hormone-specific beta subunit, which determine the binding to specific receptors and induction of biological response. The aim of this study was to investigate the polymorphism of the first intron of FSH-beta gene present in different pig breeds and its influence on the expression of FSH-beta gene. Genomic DNA was isolated from blood of three Chinese local pig breeds and two European pig breeds. The first intron of FSH-beta gene was amplified by polymerase chain reaction (PCR) and the PCR product was then determined by sequencing. The correlation between the expression level of FSH-beta gene and the sequence polymorphism was evaluated by RT-PCR using 29 heterozygous pig breeds. Three patterns of the PCR amplified fragments were observed in five different pig breeds. They are 500bp, 220bp, and 500/220bp in length representing three different genotypes with respect to the size of the first intron of FSH-beta gene. After sequencing the whole intron 1 of FSH-beta, we have found that the different size of the PCR amplified fragments was attributed to an insertion element, which was only found in the larger fragment. The insertion, specific in pig genome, showed high similarity to short interspersed nucleotide elements (SINE). Moreover, The Chinese pig breeds, Xiang, Nuogu and Kele pig, exhibited a statistically significant higher frequency of the allele with this SINE in FSH-beta gene than the European pig breeds did. To elucidate whether the SINE insertion affects the expression of FSH-beta gene, 29 heterozygous Chinese and European pig breeds were tested by RT-PCR. The results confirmed that the major transcript in the heterozygous pigs was from SINE- allele but not detected from SINE+ allele. These data suggested that the SINE insertion negatively regulated the expression of FSH-beta gene and the dominant character related to propagation was gained from the SINE- allele of FSH-beta locus in heterozygous breeds of pigs.

The immunogenicity of fusion protein linking the carboxyl terminus of the B subunit of Shiga toxin 2 to the B subunit of E. coli heat-labile enterotoxin.

To augment the immunogenicity of the subunit B of Shiga toxin (Stx2e B) produced by Escherichia coli and protect piglets from edema disease in china, a fusion gene was constructed consisting of Stx2e B genetically linked at the N-terminus of the B subunit of heat-labile enterotoxin (LTB) in a translational fusion. After being induced with IPTG, the expressed fusion protein of Stx2e B-LTB was about 8.8% of total proteins, approximately 13 microg/ml of the bacteria culture. The Stx2e B-LTB fusion protein was found to be nontoxic to Vero cells at the dose higher than 1 microg/ml and to mice less than 100 microg/ml. Antibody titer against the fusion protein Stx2e B-LTB was 1:76,800, much higher than that of the recombinant Stx2e B protein (1:12,800) alone. All of the mice immunized with the Stx2e B-LTB fusion protein survived when challenged with a lethal dose (LD) of Stx2e toxin. The results showed that the poor immunogenicity of Stx2e B was overcome by conjugating the stx2e B to ltB. The immunogenicity of the constructed fusion protein Stx2e B-LTB in the present study was highly qualified to protect animals against Shiga toxin produced from Shiga toxin-producing Escherichia coli (STEC). The fusion protein of Stx2e B-LTB could be a candidate for a vaccine against edema disease and post-weaning diarrhea simultaneously in piglets.