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.

Effect of icariin on early βdefensin2 and T cell subsets in rats after tracheotomy / 中南大学学报(医学版)

OBJECTIVES@#To investigate the effect of icariin (ICA) on early β-defensin-2 and T cell subsets in rats after tracheotomy.@*METHODS@#A total of 54 SPF male Sprague-Dawley rats were randomly divided into a normal control group (group A), a model group (group B), and a model+ICA treatment group (group C), with 18 rats in each group. A tracheotomy intubation model of the B and C group was prepared. After 6 h of surgery, ICA intervention was given to group C. Groups A and B were given the same amount of normal saline. Lung tissue, alveolar lavage fluid and peripheral blood were taken at 24 h, 72 h and 168 h, respectively. The expression of rat β-defensin-2 mRNA in lung tissue was detected by RT-PCR. The content of β-defensin-2 in alveolar lavage fluid and peripheral blood serum was detected by ELISA. The content of peripheral blood T cell subsets (CD3, CD4, CD8) was detected by flow cytometry, and the ratio of CD4/CD8 was calculated.@*RESULTS@#After tracheotomy, the levels of β-defensin-2 mRNA and β-defensin-2 in lung tissue from the group B were increased significantly at 24 h, then they were decreased gradually, and decreased most significantly at 168 h (0.05). The level of CD3 T cells in peripheral blood was significantly lower than that in the group A (0.05). After ICA intervention in group C: lung tissue, alveolar lavage fluid, peripheral blood serum β-defensin-2 content, and peripheral blood CD3 and CD4 T cell levels were gradually increased, significantly higher than those in the group B (<0.05). CD8 T cell level was significantly lower than that in the group A at 24 h (<0.05), the CD4/CD8 ratio was significantly higher at 168 h than those in the group A or B (both <0.01).@*CONCLUSIONS@#ICA can improve the early lung immune function in rats with tracheotomy, which might be related to up-regulation of β-defensin-2 in lung tissue and alveolar lavage fluid, concomitant with increases in CD3 and CD4 T cells and CD4/CD8 ratio in peripheral blood while reduction in CD8 cells.

Role of human βdefensin 2 on preventing oxidized low-density lipoprotein induced monocyte foaming / 中华传染病杂志

Objective To clarify the role of human β-defensin2 ( hBD2) on preventing oxidized low-density lipoprotein (OX-LDL) induced human leukemic monocyte (THP-1) foaming.Methods The monocyte foaming model was established using THP-1 cell induced by OX-LDL and the model was identified by oil red staining.The hBD2 was overexpressed on THP-1 cells by using lentivirus system and the effect of hBD 2 overexpression on THP-1 cell foaming induced by OX-LDL was detected.The levels of inflammatory factors including tumor necrosis factor (TNF)-α, interleukin (IL)-1βand IL-6 in cell supernatant of each group were detected by enzyme-linked immunosorbent assay ( ELISA).Differences between the groups were compared by using the t test.Results The gene transfection efficiency of the cells was close to 100%at 72 h after infection. The hBD2 protein levels were 0.122 ±0.024 in the control group, 0.123 ±0.022 in Lv-control infection group and 0.981 ±0.183 in Lv-hBD2 infection group; and the level in control group was statistically higher than that in hBD-2 infection group (t＝-3.175, P＝0.007).The relative levels of hBD2 mRNA at 72 h after virus infection were 0.131 ±0.021 in control group, 0.128 ±0.022 in Lv-control group and 1.001 ±0.105 in Lv-hBD2 infection group; and the level in control group was statistically higher than that in hBD-2 infection group (t＝-7.213, P＝0.003).The results of oil red staining showed that OX-LDL inducing THP-1 cells for 72 h could significantly induce lipid accumulation in cells.Overexpression of hBD2 could effectively inhibit lipid accumulation in THP-1 cells induced by OX-LDL.The expression of hBD2 mRNA in THP-1 group was significantly higher than that in THP-1+OX-LDL group (t＝3.237, P＝0.004); and the difference was also significant when comparing THP-1+Lv-hBD2+OX-LDL group with THP-1+OX-LDL group (t＝-6.021, P＝0.003).The level of hBD2 protein in THP-1 group was significantly higher than that in THP-1+OX-LDL group (t＝0.314, P＝0.006); and the difference was also significant when comparing THP-1+Lv-hBD2+OX-LDL group with THP-1+OX-LDL group (t＝-4.061,P＝0.007).The levels of TNF-α, IL-1βand IL-6 in the supernatant of THP-1 cells induced by OX-LDL for 72 h were significantly increased compared with those in THP-1group (t＝-3.825,-2.017 and -3.551, respectively; P＝0.007, 0.004 and 0.005, respectively). The levels of TNF-α, IL-1βand IL-6 in THP-1+Lv-hBD2+OX-LDL group were significantly lower than those in THP-1+OX-LDL group ( t＝4.132, 3.681, and 2.991, respectively; P＝0.003, 0.002, and 0.007, respectively).Conclusions hBD2 can effectively inhibit THP-1 foaming induced by OX-LDL, which may be related to its inhibition of inflammatory response.

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.

Effect ofβ-defensin 2 gene silencing on the inflammatory response induced by Haemophilus influenzae type B combined vaccine immunization in rats / 中华传染病杂志

Objective To investigate the effect of β‐defensin 2 (rBD‐2 ) silencing on the inflammatory response induced by Haemophilus influenzae type B vaccine immunization in rats .Methods A total of 144 SPF Sprague Dawley (SD) rats were divided into four groups .Group 1 were immunized with Haemophilus influenzae type B vaccine .Group 2 were immunized with Haemophilus influenzae type B vaccine and administered with lentivirus Lv‐shRNA‐rBD‐2 by intratracheal instillation .Group 3 were administered with the lentivirus Lv‐shRNA‐rBD‐2 by intratracheal instillation .Group 4 were blank controls .Each group was set with 12 rats . The serum was collected at 12 ,24 and 72 hours after immunization ,and tumor necrosis factor (TNF)‐α,interleukine (IL)‐1β and IL‐10 were assessed by enzyme‐linked immuno sorbent assay (ELISA ) .Expression levels of rBD‐2 in lung tissues of rats were measured by western blotting . Results The recombinant virus was successfully produced by a lipofectmaine transfection method .At 12 ,24 ,and 72 h of immunization ,serum levels of TNF‐αof rats in group 1 were (82 .0 ± 8 .0) ,(155 .0 ± 18 .2) ,and (272 .0 ± 32 .5) pg/mL ,respectively ,which were all higher than those in group 4 ([55 .0 ± 6 .2] ,[52 .0 ± 5 .8] ,and [56 .0 ± 4 .8] pg/mL ,respectively ;t=16 .034 ,P=0 .043 ;t=12 .411 ,P=0 .035 ;t=10 .530 ,P=0 .018 ,respectively) .Serum levels of TNF‐αat 12 ,24 ,and 72 h of immunization in group 2 were (66 .0 ± 8 .2) ,(90 .0 ± 12 .6) ,and (108 .0 ± 13 .6) pg/mL ,respectively ,which were all significantly lower than group 1 (t=12 .115 ,P=0 .039 ;t=12 .830 , P=0 .033 ;t=15 .522 ,P=0 .012 ,respectively) .At each time point ,serum levels of IL‐1βand IL‐10 in group 1 were all significantly higher than those in group 4 (IL‐1β:t=18 .032 , P=0 .048 ;t=15 .824 , P=0 .039 ;t=13 .518 , P=0 .021 ,respectively ;IL‐10 :t=15 .410 , P=0 .045 ;t=14 .294 , P=0 .032 ;t=13 .375 ,P=0 .013 ,respectively) .Serum levels of IL‐1βand IL‐10 at each time points in group 2 were all significantly lower than those in group 1 (IL‐1β:t=19 .012 , P=0 .043;t=16 .991 , P=0 .034;t=14 .862 , P=0 .027 ,respectively ;IL‐10 :t=15 .134 , P=0 .048 ;t=15 .264 , P=0 .036 ;t=11 .408 , P=0 .024 ,respectively) .Seventy‐two hours after immunization of SD rats ,the relative content of rBD‐2 protein in lung tissue of rats significantly increased .Protein level in group 1 was significantly higher than that in group 4 (t=10 .582 ,P=0 .035) ,while protein level in group 2 was significantly lower than that in group 1 (t=13 .250 ,P=0 .027) .Conclusions rBD‐2 gene silencing can improve the inflammatory response induced by Haemophilus inf luenzae type B combined vaccine in rats.

A study on ß-defensin-2 and histatin-5 as a diagnostic marker of early childhood caries progression

BACKGROUND: Recently, a continuous growth of interest has been observed in antimicrobial peptides (AMPs) in the light of an alarming increase in resistance of bacteria and fungi against antibiotics. AMPs are used as biomarkers in diagnosis and monitoring of oral cavity pathologies. Therefore, the determination of specific protein profiles in children diagnosed with early childhood caries (ECC) might be a basis for effective screening tests and specialized examinations which may enable progression of disease. METHODS: The objective of the studies was to determine the role of histatin-5 and ß-defensing-2 as a diagnostic marker of early childhood caries progression. In this work, results of concentration determination of two salivary proteins (histatin-5 and ß-defensin-2) were presented. In addition, bacterial profiles from dental plaque in various stages of ECC and control were marked. The assessment of alteration in the concentration of these two proteins in a study group of children with various stages of ECC and a control group consisting of children with no symptoms was performed by enzyme-linked immunosorbent assays. RESULTS: The statistical analysis showed a significant increase in the concentration of histatin-5 and ß-defensin-2 in the study group compared to the control group and correlated with the progression of the disease. CONCLUSIONS: The confirmation of concentration changes in these proteins during the progression of dental caries may discover valuable disease progression biomarkers.

Effects of recombinant fusion protein interleukin-17F/His on expression of immune-related inflammatory factors in mouse model with Streptococcus pneumoniae infection / 中华实用儿科临床杂志

Objective To evaluate the effects of recombinant fusion protein interleukin-17F/His (IL-17F/His) on expression of immune-related inflammatory factors in murine models with Streptococcus pneumoniae (SP) infection.Methods BALB/c mice were randomly divided into SP infection group,fusion protein intervention group and the normal control group.Before intranasal infection with SP,the mice were treated with PBS or IL-17F/His respectively.The number of bacteria and leukocytes in bronchoalveolar lavage fluid (BALF) was counted,and the mRNA levels of β-defensin-2 (Defb2),macrophage inflammatory protein (MIP)-lα and MIP-2β in lung tissue were detected by real-time fluorescent quantitative PCR,the concentrations of MIP-1 α,MIP-2β,IFN-γ and IL-4 in BALF,supematants of spleen cell and mediastinal lymph node cell were assayed by enzyme linked immunosorbent assay.The expressions of IL-17F and Defb2 in lung tissues were observed with immunocytochemistry.Results 1.Compared with the SP infection group,in the fusion protein intervention group,the total number of WBC (209.00 ± 18.23) was higher,the number of neutrophil,macrophage and lymphocyte[(8.67 ±2.16) × 106/L,(193.50 ± 16.50) × 106/L,(6.83 ± 1.17) × 106/L] were higher in BALF(U/F =38.097,28.854,32.942,27.810,all P ＜ 0.05),while the number of bacteria [2.75 (1.15-3.09) × 103/L] was significantly lower(P =0.02).2.Compared with SP infection group,the expression levels of Defb2 mRNA and MIP-1α mRNA(31.64 ±4.97,5.81 ± 1.09) in lung tissues were higher in the fusion protein intervention group,while the expression level of MIP-2β mRNA (6.69 ± 2.05) was lower (t =4.889,2.306,3.536,all P ＜ 0.05).3.Compared with the SP infection group,the concentration of MIP-2β [(64.15 ± 13.41) ng/L] was significantly decreased and concentration of IL-4 [(92.28 ± 4.52) ng/L] was significantly increased in BALF in the fusion protein intervention group(H/F =159,289,40.767,all P ＜0.01).4.Compared with the SP infection group,the concentrations of MIP-2β and IL-4 [(255.02 ± 13.95) ng/L,(107.02 ± 7.53) ng/L] were both significantly increased in spleen cell culture supematants in the fusion protein intervention group,the concentrations of MIP-1 α and IFN-γ [(413.61 ± 24.23) ng/L,(98.88 ± 5.84) ng/L] were both significantly decreased (all P ＜ 0.05).5.Immunocytochemistry analysis revealed that the expressions of IL-17F and Defb2 were both higher in the fusion protein intervention group than those in the SP infection group; there was no expression in the normal control group.Conclusions Intranasal inoculation of recombinant fusion protein IL-17F/His can promote pulmonary neutrophil and macrophage recruitment and increase bacterium clearance,and enhance the host defense against SP infection through increa-sing the expression of defensins,regulating the levels of MIP,IL-4 and IFN-γ.

Expressions of human β defensin-2 in eutopic and ectopic endometrium of women with adenomyosis / 中国综合临床

Objective To investigate the expression of human β defensin-2(hβD-2)in the eutopic and ectopic endometrial tissue in patients with adenomyosis and in women with normal endometrium.Methods Twenty-five hysteromyoma patients with adenomyosis(AM) and 25 hysteromyoma patients without endometriosis (EMS) were selected and divided into three groups:AM ectopic endometrium group,AM eutopic endometrium group and control group( endometrial tissue in patients with hysteromyoma).The level of mRNA expressions of hβD-2,interleukin-1β ( IL-1β ),interleukin-6 ( IL-6 ) and tumor necrosis factor-α (TNF-α) was investigated quantitatively using Real Time PCR (RT-PCR) and the corresponding protein level was detected by immunohistochemistry.Results Comparing the expression of hβD-2,IL-1β,IL-6,TNF-α genes among the three groups,there was no significant difference between ectopic endometrium group and eutopic endometrium group and there was also no significant difference between eutopic endometrium group and the control group.( P ＞ 0.05 ).The expression of hβD-2 and IL-1β were 0.0320 (0.0095 ～ 0.0690 ) and 0.0427 ( 0.0038 ～ 0.0975 ) in the ectopic endometrium group,and they were 0.0034(0.0025 ～0.0424) and 0.0080(0.0040 ～0.0251 ) in the control group,respectively.They were both significantly higher in ectopic endometrium group than in the control group (P ＜ 0.05 ).In the ectopic endometrium group hβD-2 expression was positively correlated with the level of TNF-α ( r =0.857,P =0.014 ),and it had no correlation with both IL-1β and IL-6 ( r =0.750,P =0.052 ; r =0.464,P =0.464; respectively)Conclusion HβD-2 might not play an important role in the formation of adenomyosis.It may be related to no significant up-regulation of inflammatory factors in ectopic lesion tissue.

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.

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.

Anti-infection effect of human beta-defensin-2 gene therapy in a rat urinary tract infection model / 中华泌尿外科杂志

Objective To assess the human β-defensin-2 ( hBD2 ) gene therapeutic efficacy in a rat urinary tract infection (UTI) model via intravesical liposome-mediated gene transfer.Methods Fifty-six female Wistar rats (class SPF) weighting 1 80 -220 were randomly divided into an experiment group and a control group ( each n =28 ).The animals were administered either 250 μl recombinant pCAGG-hBD2 or control vector pCAGG intravesically.After 48 h,rats in both groups were infected via intravesical inoculation with 200 μl of the bacterial suspension of UTI89 ( 1 × 108 CFU/ml).The rats were sacrificed at 4,24,48,and 72 h post-inoculation.The bladders were aseptically removed and bisected.One half was fixed in neutral buffered formalin for histological analysis.The remaining half-bladders were homogenized and titered for surviving bacteria.The clean-catch urine sample from each rat was collected in sterile before they were killed for bacterial titers determined and WBC counted.Results Numbers of bacterial colony-forming unit in urine and bladders from hBD2 gene treated UTI rats were significantly lower than those from the control vector administered UTI rats at 24,36,and 72 h after infection ( P ＜ 0.05 ).The amount of WBC in urine was significantly less in the defensin group than in the control group.In addition,in vivo expression of hBD2 could reduce mucosa damage,interstitium edema and inflammatory cells infiltration in UTI animals.Conclusions The successful inhibition of UTI progression could be obtained with hBD2 gene therapy.Recombinant beta-defensin-2 could kill UTI89 in vivo and suppress the subsequent infection-induced inflammatory responses.

Study on the mechanism of human-β-defensin-2 expression in human vaginal epithelial cells induced by Lactobacillus cell wall extract / 中华微生物学和免疫学杂志

Objective To investigate the molecular and cell signal transduction mechanisms by Lactobacillus cell wall extract(LCWE)inducing human-β-defensin-2(hBD-2)expression in human vaginal epithelial cells.Methods The induction of hBD-2 in human vaginal epithelial cells(WZV-1)by LCWE was observed using real-time PCR and Western blot.After stimulating WZV-1.the activation of NF-κB and p38MAPK signaling pathways were determined by Western blot.The induction of hBD-2 in WZV-1 cells by LCWE was observed with signaling pathways inhibitors of NF-κB and p38MAPK using real-time PCR and Western blot.Results The results showed that LCWE significantly upregulated hBD-2 expression in the time and dose-dependent manner.The maximal stimulatory effect of LCWE on the expression of hBD-2mRNA in WZV-1 cells were observed at the concentration of 50μg/ml after treatment for 8 h.After stimulation by 50μg/ml LCWE,Western blot analysis demonstrated that the phosphorylation of p38MAPK increased at 0.5 h significantly,peaked at 1 h,moreover the concentration of NF-κB in nucleus increased at 0.5 h significantly(P<0.05),peaked at 2 h.Blocking with inhibitor of NF-κB and(or)p38MAPK pathways results in decreased levels of HBD-2 expression.Conclusion These findings suggest that p38MAPK and NF-κB pathways play the important roles in induction of hBD-2 expression by LCWE in human vagihal epithelial cells.

Expression and function of human β-defensin-2 in patients with peptic ulcer / 中国医师进修杂志

Objective To investigate the relationship between the pathophysiology of Helicobacter pylori induced antral gastritis and the expression of human β -defensin (HBD) -2 in peptic ulcer before and after Helicobacter pylori eradication.Methods Patients in peptic ulcer with Helicobacter pylori-posilive (23 cases) or Helicobacter pylori-negative (20 cases) were included.And 30 normal individuals were as controls.Biopsied gastric mucosa specimens from Helicobacter pylori-positive or Helicobacter pylori-negative individuals were done and normal controls were used.The specimens were examined for pathophysiology diagnosis and HBD-2 expression by immunohistochemistry before and after Helicobacter pylori eradication.Results Helicobacter pylori infection was correlated with the histological severities of both inflammation activity and atrophy in antral gastritis (r =0.574,0.640,P ＜ 0.01 ).The expression of HBD-2 was positively correlated with Helicobacter pylori infection in all the specimens (r =0.533,0.577,P ＜ 0.01 ).Immunohistochemistry using anti-HBD-2 antiserum revealed that HBD-2 expression and inflammation activity had significantly decreased in gastric specimens obtained after Helicobacter pylori eradication(P ＜ 0.01 ).The expression of HBD-2 had no variance in the case failing in Helicobacter pylori eradication (P ＞0.05).Conclusions Helicobacter pylori infection induces HBD-2 expression in the human gastric epithelium.HBD-2 inhibits the growth of Helicobacter pylori in vitro,which suggests that HBD-2 plays an antibacterial or inhibition role in Helicobacter pylori induced gastritis.In other words,the absence of HBD-2 should enhance the histological inflammation of Helicobacter pylori induced antral gastritis.

Granulocyte-Derived Cationic Peptide Enhances Homing and Engraftment of Bone Marrow Stem Cells after Transplantation / 한국실험동물학회지

Current strategies to accelerate hematopoietic reconstitution after transplantation include transplantation of greater numbers of hematopoietic stem/progenitor cells (HSPCs) or ex vivo expansion of harvested HSPCs before transplant. However, the number of cells available for transplantation is usually low, and strategies to expand HSPCs and maintain equivalent engraftment capability ex vivo are limited. We noted that activated granulocyte-derived cationic peptides positively primed responsiveness of HSPCs to a CXCL12 gradient. Accordingly, we noted that accelerated homing/engraftment of beta-defensin-2, a well-known antimicrobial cationic peptide, primed bone marrow nucleated cells (BMNCs) compared to normal BMNCs after transplantation into lethally irradiated recipients. We envision that small cationic peptides, which primarily possess antimicrobial functions and are harmless to mammalian cells, could be applied to prime HSPCs before transplantation. This novel approach would be particularly important in cord blood transplantation, where the number of HSPCs available for transplantation is usually limited.

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.