Clinical Efficacy of Maxingshigantang Enema in Treating Infants with Pneumonia and Asthma and Intervention Mechanism of Lung-intestine Microenvironment / 中国实验方剂学杂志

ObjectiveTo observe the clinical efficacy of Maxingshigantang enema in the treatment of infant viral pneumonia by comparing related indicators， and comprehensively evaluate the effect of traditional Chinese medicine （TCM） enema on the intestinal microenvironment. MethodSixty infants with viral pneumonia were selected and randomly divided into 3 groups. The dosage of enema drugs in high- （0.117 g·mL-1） and low-concentration （0.07 g·mL-1） TCM enema groups was same （3.5 g per time）， and the control group received normal saline enema， once a day for 7 days. Finally， the curative effect， total symptom score， salivary secretory immunoglobulin A （sIgA）， human beta defensin 2 （hBD2） and fecal calprotectin （CALP） of each group were statistically analyzed by SPSS 21.0， and the clinical efficacy of TCM enema in treating children with pneumonia and asthma was comprehensively evaluated. ResultThe curative effect of high-concentration TCM enema group （total effective rate 100%， χ2=7.059） was equivalent to that of low-concentration TCM enema group （total effective rate 95%， χ2=4.329）， higher than that of control group （total effective rate 70%） （P<0.017）. After treatment， compared with control group and low-concentration TCM enema group， high-concentration TCM enema group had higher total symptom score of children （P<0.05， P<0.01）. The proportion of coccobacillus was reduced in three groups， with high- and low-concentration TCM enema groups lower than control group （P<0.05）. The salivary sIgA concentration was increased in three groups （P<0.05）， with high-concentration TCM enema group higher than the other groups （P<0.01）. The hBD2 concentration was decreased in three groups， with high- and low-concentration TCM enema groups lower than control group （P<0.05）. The three groups reduced the fecal CALP concentration， and high-concentration TCM enema group had the highest reduction， followed by low-concentration TCM enema group （P<0.01）. ConclusionTCM enema outweighs western medicine in improving clinical symptoms， intestinal flora， and mucosal immune function， and reducing inflammation in children， and the high-concentration TCM enema group has better curative effect. Therefore， with easiness to operate， high compliance， and significant therapeutic effect， TCM enema is worthy of clinical promotion.

Expressions of intestinal alpha-defensin-5, beta-defensin-2 in acute liver failure / 中华实用儿科临床杂志

Objective To investigate the dynamic changes in intestinal alpha-defensin-5 (RD-5),beta-defensin-2 (BD-2) mRNA after acute liver failure(ALF),and to explore their role in ALF.Methods A total of 60 C57BL5 mice were divided into 4 groups by means of random number table method:normal control group,ALF group,E.coli via gavage group and ALF + E.coli via gavage group.Intraperitoneal injection of D-galactosamine (500 mg/kg) and lipopolysaccharide(10 μg/kg) to make the model,in addition,ALF mice were fed with E.coli,and the observation time was 6 hours,12 hours,and 24 hours after modeling,and each time point had 6 specimens.Real-time PCR was used to test the RD-5 mRNA and BD-2 mRNA levels in the ileum tissue.Results The levels of RD-5 and BD-2 showed dynamic change in the experiment of ALF.Compared with the levels of RD-5 and BD-2(11.25 ±0.74,23.86 ±0.39) of the normal control group,the levels of RD-5 and BD-2 in ALF group and E.coli via gavage group increased at 6 hours after modeling(14.19 ±0.39,26.79 ± 0.36 and 12.57 ± 0.68,26.45 ± 0.85),and the differences were significant(all P＜0.05);at 12 hours after modeling,the RD-5 and BD-2 reached to the maximum concentration(15.76 ±0.33,29.10 ± 0.61 and 12.90 ± 0.96,27.42 ± 0.71),and the differences were statistically signi-ficant (all P ＜ 0.05).The degree of elevation of BD-2 was higher than RD-5.Later,they gradually declined.Conclusions RD-5 and BD-2 may play an important role in the pathogenesis of intestinal endotoxemia in experimental ALF.

Construction of rat beta defensin-2 eukaryotic expression vector and expression in the transfected rat corneal epithelial cell / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To construct a recombinant eukaryotic expression vector of rat beta defensin-2(rBD-2), transfect it into the rat corneal epithelial cells with lipofection, determine the expression of target gene in the transfected cells, and discuss the potentiality of recombinant plasmid expressed in corneal epithelial cells, hoping to provide an experimental foundation for further study on the antimicrobial activity of rBD-2 <i>in vitro </i>and <i>in vivo</i> and to assess the probability of defensins as a new application for infectious corneal diseases in the future. <p>METHODS: The synthetic rBD-2 DNA fragment was inserted between the <i>Xho</i>I and <i>BamHI</i> restriction enzyme cutting sites of eukaryotic expression vector pIRES2-ZsGreen1 to construct the recombinant plasmid pIRES2-ZsGreen1-rBD-2, then transformed it into <i>E.coli DH5α</i>, positive clones were screened by kanamycin and identified with restriction endonucleases and sequencing analysis. Transfection into the rat corneal epithelial cells was performed by lipofection. Then the experiment was divided into three groups: rat corneal epithelial cell was transfected with the recombinant plasmid pIRES2- ZsGreen1-rBD-2, rat corneal epithelial cell was transfected with the empty plasmid pIRES2-ZsGreen1 and the non-transfected group. The inverted fluorescence microscope was used to observe the transfection process. At last, the level of rBD-2 mRNA expressed in the transfected cells and the control groups are compared by the real-time fluoresence relative quantitative PCR. <p>RESULTS: The recombinant eukaryotic expression vector of pIRES2-ZsGreen1-rBD-2 was successfully constructed. The level of rBD-2 mRNA in transfected cells was significantly higher than that in control groups through the real-time fluorescence relative quantitative PCR. <p>CONCLUSION: The recombinant eukaryotic expression vector pIRES2-ZsGreen1-rBD-2 could be transfected into rat corneal epithelial cells, and exogenous rBD-2 gene could be transcripted into mRNA in the transfected cells.

Expression of mouse beta defensin 2 in Escherichia coli and its broad-spectrum antimicrobial activity

Mature mouse beta defensin 2 (mBD2) is a small cationic peptide with antimicrobial activity. Here we established a prokaryotic expression vector containing the cDNA of mature mBD2 fused with thioredoxin (TrxA), pET32a-mBD2. The vector was transformed into Escherichia Coli (E. coli) Rosseta-gami (2) for expression fusion protein. Under the optimization of fermentation parameters: induce with 0.6 mM isopropylthiogalactoside (IPTG) at 34ºC in 2×YT medium and harvest at 6 h postinduction, fusion protein TrxA-mBD2 was high expressed in the soluble fraction (>95%). After cleaved fusion protein by enterokinase, soluble mature mBD2 was achieved 6 mg/L with a volumetric productivity. Purified recombinant mBD2 demonstrated clear broad-spectrum antimicrobial activity for fungi, bacteria and virus. The MIC of antibacterial activity of against Staphylococcus aureus was 50 µg/ml. The MIC of against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) was 12.5µg/ml and 25µg/ml, respectively. Also, the antimicrobial activity of mBD2 was effected by NaCl concentration. Additionally, mBD2 showed antiviral activity against influenza A virus (IAV), the protective rate for Madin-Darby canine kidney cells (MDCK) was 93.86% at the mBD2 concentration of 100 µg/ml. These works might provide a foundation for the following research on the mBD2 as therapeutic agent for medical microbes.

Expression of mouse beta defensin 2 in Escherichia coli and its broad-spectrum antimicrobial activity

Mature mouse beta defensin 2 (mBD2) is a small cationic peptide with antimicrobial activity. Here we established a prokaryotic expression vector containing the cDNA of mature mBD2 fused with thioredoxin (TrxA), pET32a-mBD2. The vector was transformed into Escherichia Coli (E. coli) Rosseta-gami (2) for expression fusion protein. Under the optimization of fermentation parameters: induce with 0.6 mM isopropylthiogalactoside (IPTG) at 34ºC in 2×YT medium and harvest at 6 h postinduction, fusion protein TrxA-mBD2 was high expressed in the soluble fraction (>95 percent). After cleaved fusion protein by enterokinase, soluble mature mBD2 was achieved 6 mg/L with a volumetric productivity. Purified recombinant mBD2 demonstrated clear broad-spectrum antimicrobial activity for fungi, bacteria and virus. The MIC of antibacterial activity of against Staphylococcus aureus was 50 µg/ml. The MIC of against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) was 12.5µg/ml and 25µg/ml, respectively. Also, the antimicrobial activity of mBD2 was effected by NaCl concentration. Additionally, mBD2 showed antiviral activity against influenza A virus (IAV), the protective rate for Madin-Darby canine kidney cells (MDCK) was 93.86 percent at the mBD2 concentration of 100 µg/ml. These works might provide a foundation for the following research on the mBD2 as therapeutic agent for medical microbes.

Effect of polymorphisms of Crohn disease related NOD2 gene and human beta-defensin 2 on expres-sion of human beta-defensin 2 / 中华消化内镜杂志

Objective To explore the effects of polymorphisms of Crohn's disease related NOD2 gene and human beta-defensin 2 (hBD-2) on transcription of hBD-2 gene and its mechanism. Methods HEK293T cells were transfected with hBD-2 gene and NOD2 eukaryotic expression plasmid, and were then stimulated with LPS, TNF-α, or BAY 11-7082 (antagonist of NF-κB), respectively. Transcriptional activity of hBD-2 was detected afterwards. Results LPS could suppress transcription of hBD-2 (P=0. 020), which was increased by TNF-α in a dose-dependent manner (P =0. 004). In the presence of LPS, there was sig-nificant difference in transcriptional activity of hBD-2 between wild-NOD2 transfected group and mutated NOD2 (P268S) transfected group (P=0. 008), but there was no significant difference between wild hBD-2 transfected group and mutated hBD-2 transfected group (P=0. 053). With the stimulation of TNF-α (5 ng/ml), there was a significant difference between mutated hBD-2 transfected group and wild hBD-2 transfected group (P=0. 006), but no significant difference between wild-NOD2 transfected and mutated NOD2 transfected group was defected (P = 0. 064). Pretreatment with BAY 11-7082 before TNF-α (5 ng/ml) significantly inhibited the transcriptional activity of hBD-2 (P < 0. 001). Conclusion The poly-morphism of NOD2 affects the innate expression of hBD-2, the polymorphism of site in hBD-2 promoter (-233) may lead to significant decline of the inducible expression of hBD-2, and NF-κB might be a key pathway that NOD2 protein mediates the expression of defensin.

TNF-alpha-induced Increase of Human beta-defensin-2 Expression in HaCaT Cell Lines / 대한피부과학회지

BACKGROUND: Several kinds of epithelial cells and focal lymph nodes are known to be involved in the skin's immune reaction. Especially, internal antimicrobial peptide play an important role in protecting microbial agents. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide which is produced by epithelial cells after stimulation with microorganisms or inflammatory mediators. hBD-2 participates in the increase of the cell-mediated immune reaction. It also affects the proliferation and differentiation of epithelial cells and fibroblasts, resulting in enhancement of wound healing. However, little is known as to whether the TNF-alpha induces the expression of hBD-2 in HaCaT cells through the NF-kappaB or MAPKs pathways. OBJECTIVE: Research was undertaken to investigate the roles of NF-kappaB and MAPKs transcription factors in the molecular pathway of TNF-alpha-induced hBD-2 expression in HaCaT cell lines. METHODS: The expression of hBD-2 in TNF-alpha-treated HaCaT cells was analyzed by immunofluorescence staining and reverse transcription polymerase chain reaction (RT-PCR). The expression of NF-kappaB was analyzed by Western blot analysis and electrophoretic mobility shift assay (EMSA). RESULTS: Strong positive hBD-2 immunofluorescence staining in TNF-alpha-treated HaCaT cells was observed. According to RT-PCR analysis, the expression of hBD-2 increased TNF-alpha-treated HaCaT cells by dose-dependent and time-dependent manners. In addition, according to Western blot analysis and EMSA, NF-kappaB was also activated in TNF-alpha-treated HaCaT cells. Interestingly, the expression of hBD-2 in TNF-alpha-treated HaCaT cells was attenuated in the presence of NF-kappaB inhibitors, PDTC or MG132. Furthermore, MAPKs inhibitors, especially SB (p38 inhibitor), partially attenuated the TNF-alpha-induced hBD-2 expession, but not PD (ERK inhibitor) and SP (JNK inhibitor). CONCLUSION: These results collectively suggest that hBD-2 is up-regulated in TNF-alpha-treated HaCaT cells through activation of NF-kappaB and p38 MAPKs pathway. Our data regarding the up-regulation of hBD-2 may help us to understand the antimicrobial mechanism in normal skin or in skin diseases.

Immunohistochemical Study of Human beta Defensin-2 Expression in Superficial Mycosis / 대한피부과학회지

BACKGROUND: Normal human skin is resistant to infection with various kinds of microorganisms by producing anti-microbial chemicals. Human beta defensin-2 (hBD-2) is an anti-microbial peptide that has recently been shown to be expressed in various epithelial cells and inflammatory diseases. However, the expression of hBD-2 in fungus-infected skin is not well-known. OBJECTIVE: This study was performed to investigate the expression pattern of hBD-2 in superficial mycosis. METHODS: Using the immunohistochemical method with formalin-fixed, paraffin-embedded sections, we checked the expression levels and localization of hBD-2 in lesional skin samples of tinea capitis (5 patients), tinea corporis (6 patients), candidiasis (3 patients), Malassezia folliculitis (2 patients), and psoriasis (3 patients) as positive control, and normal skin samples from 6 healthy subjects as negative control. RESULTS: The expression of hBD-2 was not observed in normal skin, but moderate to strong expression of hBD-2 was observed in the epidermis, and the papillary dermal infiltrating cells of psoriasis. In tinea capitis, strong hBD-2 expression was found in the upper spinous layer of epidermis and follicular epidermis, and perifollicular inflammatory cells. In tinea corporis and candidiasis, mild to strong expression of hBD-2 was found in the horny or spinous layer of epidermis and infiltrating inflammatory cells. Strong hBD-2 expression was found in the follicular epidermis and perifollicular inflammatory cells of Malassezia folliculitis. CONCLUSION: These results suggest that hBD-2 plays an important role in cutaneous innate immune defense against fungal infection.

Expression of Human beta-defensin 2 mRNA by Lipopolysaccharide in Human Corneal Epithelial Cells

Recently the transcriptional up-regulation of human beta-defensin 2 (HBD-2) by lipopolysaccharide (LPS) was found to be associated with NF-kappaB binding site. Although the general mechanisms of NF-kappaB activation by LPS stimulation are well understood, less is known about the signal transduction pathway leading to LPS-induced NF-kappaB activation in human corneal epithelial (HCE) cells. The aim of this study was to investigate the intracellular signals involved in LPS-induced HBD-2 mRNA expression in HCE cells. Pretreatments of inhibitors for NF-kappaB, protein tyrosine kinase, p38 mitogen activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) attenuated the LPS-induced NF-kappaB DNA binding activity and HBD-2 mRNA expression. Furthermore, pretreatments with inhibitors for protein kinase C (PKC), phosphatidylcholine-phospholipase C, phosphatidylinositol-phospholipase C, or phosphatidate phosphohydrolase prevented LPS-induced HBD-2 mRNA expression and HBD-2 prmoter-driven luciferase activity. However, the increased expression of HBD-2 mRNA and the increased DNA binding activity of NF-kappaB induced by LPS were not changed by the blockage of extracellular signal-regulated kinase (ERK) and of addition of antioxidants. Forskolin, a protein kinase A (PKA) agonist did not induce HBD-2 mRNA expression. These data demonstrate that LPS-induced HBD-2 mRNA expression via NF-kappaB is, at least in part, dependent on PKC, p38 MAPK, JNK, and protein tyrosine kinase status, but appears to be independent on PKA, ERK and ROS in HCE cells. Taken together, there may be more than one signaling pathways that lead to LPS-induced up-regulation of HBD-2 mRNA expression in HCE cells.

Expression of Human beta Defensin-2 in HaCaT Cell by Lipoteichoic Acid and Peptidoglycan / 대한피부과학회지

BACKGROUND: Defensin, a major family of antimicrobial peptides, is small cationic, cysteine rich peptides with wide range of antimicrobial activity against Gram negative and Gram positive bacteria, fungi, yeast, and virus. Expression of human defensin-2 is upregulated by bacteria, virus, fungus and pro-inflammatory cytokines. However, this peptide was found to be only bacteriostatic, but not bactericidal, against the Gram positive bacteria. OBJECTIVE: To evaluate human defensin-2 (hBD-2) expression after exposure of human skin keratinocytes to the cell wall component of Gram positive bacteria such as lipoteichoic acid (LTA) and peptidoglycan(PEN), and to compare quantitatively the amount of expression with that after their exposure to the cell wall component of Gram negative bacteria. METHODS: Expression of hBD-2 was measured by reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry(IHC). RESULTS: 1. In RT-PCR results, the amount of hBD-2 expression after exposure to LPS was larger than those of PEN and LTA at 6 and 12 hours (p=0.02). At 24 hours, hBD-2 expression showed a peak in PEN stimulated group (p=0.09). 2. In Western blot analysis, hBD-2 expressions, when treated with PEN and LTA, were stronger than that treated with LPS at 6 and 12 hours. 3. In IHC, hBD-2 was stained much stronger in LPS stimulated group than PEN or LTA stimulated groups at 12 hours. CONCLUSION: Our study demonstrated that exposure of human skin keratinocytes to the cell wall components of Gram positive bacteria such as LTA and PEN triggered production of hBD-2 in addition to the cell wall component of Gram negative bacteria such as LPS, however, the amounts of expression were relatively stronger in LPS treated group.

The Tissue Microarray Technology and Its Preliminary Application in Evaluation of the Expression and Clinical Significance of hBD in the Vocal Cord Polyp / 听力学及言语疾病杂志

0.05).?-defensin 2 was detected in the specimens of vocal cord polyp,but very little in the subjects of other two groups.Its expression level was significantly higher in the vocal cord polyp than that of the other two groups(P