Dehydroandrographolide enhances innate immunity of intestinal tract through up-regulation the expression of hBD-2.

BACKGROUND: Dehydroandrographolide (DA) is one of major active components in the well-known oriental herbal medicine Andrographis paniculata (Burm.f) Nees which belongs to the Acanthaceae family. DA is used for the treatment of infections in China. However, DA has not been found to significantly inhibit bacterial and viral growth directly. The current study investigates the effect of DA on the expression of human ß -defensin-2 (hBD-2) in human intestinal epithelial cells and the possible signaling pathways. METHODS: Human intestinal epithelial HCT-116 cells were incubated with 1-100 µM DA for 2-24 h. RT-PCR and Western blot were used to assess the expression of hBD-2. The specific inhibitors were used and the levels of phosphorylation of signaling molecules were detected for dissecting the signaling pathways leading to the induction of hBD-2. RESULTS: MTT assay showed there was no obvious cytotoxicity for HCT-116 cells by 1-100 µM DA treatment. RT-PCR and Western blot assays showed that DA (1-100 µM) could up-regulate the expression of hBD-2, and the effect lasted longer than 24 h. By using SB203580 and SB202190 (inhibitors of p38), the enhancement of hBD-2 expression were significantly attenuated. However, inhibitor of ERK and inhibitor of JNK could not block the effect of DA. Furthermore, Western blot found activation of p38 but not ERK and JNK in DA-treated HCT-116 cells. CONCLUSION: The results suggested that DA enhanced innate immunity of intestinal tract by up-regulating the expression of hBD-2 through the p38 MAPK pathways.

Protective effects of Carthamus tinctorius injection on isoprenaline-induced myocardial injury in rats.

CONTEXT: Carthamus tinctorius injection (CTI) is a traditional Chinese medicine (TCM) specifically used for the treatment of cerebral ischemia and myocardial ischemia. OBJECTIVE: This study evaluated the protective effects of CTI on isoprenaline-induced acute myocardial ischemia (AMI) in rats and explored the underlying mechanisms. MATERIALS AND METHODS: (i) Sprague-Dawley rats were randomly divided into 5 groups: control, myocardial ischemia model, and high-, low-dose of CTI groups (2.5 and 0.625 g/kg, respectively, i.p. for 5 days), and Xiang-Dan (20 g/kg) group (n = 10 in each group). AMI was induced by isoproterenol (5 mg/kg) by intraperitoneal injection. Assessment of electrocardiograms (ECG) was carried out. (ii) Another 40 rats were randomly divided into 5 groups, the concentration of IL-6 and TNF-α in serum were measured by radioimmunological assay; Bcl-2 and Bax protein expression were measured by immunohistochemistry. RESULTS: CTI (2.5 and 0.625 g/kg) significantly inhibited the typical ECG S-T segment elevation, reduced concentration of IL-6 and TNF-α in serum, suppressed overexpression of Bax protein and also inhibited the reduction of Bcl-2 expression and markedly depressed the Bax/Bcl-2 ratio. DISCUSSION AND CONCLUSION: These findings demonstrate that CTI is cardioprotective against AMI in rats and is likely to related to decrease inflammatory response mediated by TNF-α and IL-6, down-regulate protein level of Bax and up-regulate that of Bcl-2 in the heart tissue.

Calycosin stimulates proliferation of estrogen receptor-positive human breast cancer cells through downregulation of Bax gene expression and upregulation of Bcl-2 gene expression at low concentrations.

BACKGROUND: Calycosin is one of main components in the herb radix astragali and is considered a typical phytoestrogen. It has either estrogenic or antiestrogenic effects that mainly depend on estrogen levels in vivo. This study investigated the effects and mechanisms of calycosin on estrogen receptor (ER)-positive human breast cancer (MCF-7) cells in vitro. METHODS: ER-positive MCF-7 cells were treated with different concentrations of calycosin. Effects of calycosin on the proliferation of ER-positive MCF-7 cells were determined by the MTT assay. Apoptosis in these treated cells was examined by flow cytometry. The mRNA and protein levels of Bcl-2 and Bax in these treated cells were also determined by reverse-transcription polymerase chain reaction and immunohistochemical staining, respectively. RESULTS: Compared with the vehicle control, calycosin stimulated proliferation of ER-positive MCF-7 cells at low concentrations (2, 4, and 8 µmol/L). Furthermore, at these concentrations, calycosin decreased the percentage of early apoptosis in MCF-7 cells, downregulated mRNA and protein levels of Bax, and upregulated those of Bcl-2 at low concentrations. On the other hand, calycosin at higher concentrations (16 and 32 µmol/L) inhibited cell proliferation. CONCLUSION: At relatively low concentrations, calycosin has stimulatory effects on the proliferation of MCF-7 cells, with the estrogenic effect the mechanism.

[Effect of human beta defensin 2 on Staphylococcus aureus infection: in vivo study with transgenic mice].

OBJECTIVE: To investigate the effect of human beta defensin 2 (HBD-2) on Staphylococcus aureus infection. METHODS: A minigene of HBD-2 containing pCMV promoter, full length of HBD-2 cDNA, and BGH polyA tail was generated by PCR amplification and introduced into the fertilized oocytes of C57/ICR hybridized mouse by microinjection. After gestation of 3 - 4 weeks, immunohistochemistry was used to detect the expression of HBD-2 peptide in different tissues of the transgenic young mice. Staphylococcus aureus was cultured and injected intraperitoneally to wild type mice and transgenic mice to observe their surviving status. RESULTS: PCR showed that the HBD-2 fragment had been successfully integrated into the chromosome of the mice. A widespread expression of HBD-2 gene was found in many tissues of the transgenic mice: trachea, lung, intestine, esophagus, testis, spleen, skin, endothelium, brain, etc. Four of the 7 transgenic mice survived the Staphylococcus aureus infection, and 10 wild type mice all died within 24 hours. CONCLUSION: HBD-2 may play an important role ion the host defense against Staphylococcus aureus infection.

[Production of HMGN2 polyclonal antibody by immunization with recombinant GST-HMGN2 fusion protein and its application to analysis of HMGN2 distribution in human monocytes].

OBJECTIVE: To prepare high mobility group chromosomal protein N2 (HMGN2) polyclonal antibodies and determine the subcellular localization of HMGN2 in human monocytes. METHODS: The recombinant prokaryotic expression vector pGEX-1lambdaT-HMGN2 was constructed and E. coli-based product of GST-HMGN2 fusion protein was prepared and used to immunize rabbit for producing the anti-serum against HMGN2. The polyclonal antibodies were partially purified by caprylic acid and ammonium sulfate precipitation. The titter of specific polyclonal antibodies against HMGN2 was detected by ELISA. The immunocytochemical staining was performed to determine the distribution of HMGN2 in THP-1 cells. RESULTS: Gel electrophoresis of the enzyme-digested recombinant plasmid and the DNA sequencing confirmed that the recombinant prokaryotic expression vector pGEX-1lambdaT-HMGN2 was correctly constructed. After IPTG induction, the recombinant-transformed E. coli produced a bulk of GST-HMGN2 fusion protein. The polyclonal antibodies to HMGN2 was obtained from the serum of rabbit immunized with GST-HMGN2 fusion protein and its ELISA titer was 1:2000. The immunocytochemistry staining indicated that when stimulated with LPS, HMGN2 was present not only in THP-1 nucleus but also in the cytoplasm. The presence of HMGN2 was also detected in the culture supernatant. CONCLUSION: This result suggests that recombinant peptide fusion protein could be used to produce peptide antibody. HMGN2 could be present in the cytoplasm of monocytes and release to the extracellular environment when stimulated with lipopolysaccharide (LPS).