Effects of ganmao shuangjie heji on the inflammatory damage in the lungs of mice infected by IV FM1

AIM: To establish the influenza infected mouse model and study the anti-inflammatory effect of ganmao shuangjie heji. METHODS: IV FM1 infected mice were used as the animal model. The changes of pathology and the cytokine TNF-α, IFN-γ and IL-10 in the lung were observed by HE staining and ELISA (double antibody sandwich enzyme linked immunosorbent assay) after ganmao shuangjie heji treatment. RESULTS: After infected by influenza virus, severe interstitial pneumonia was induced in the model group. Mild interstitial pneumonia was observed in ganmao shuangjie heji treated group. The protein expressions of cytokine TNF-α, IFN-γ and IL-10 were higher in model group than those in the control group. The protein expressions of TNF-α and IFN-γ in ganmao shuangjie heji treated group decreased and IL-10 expression increased significantly compared with model group. CONCLUSION: Ganmao shuangjie heji decreases the expressions of TNF-α and IFN-γ, and increases the expression of IL-10, thus, alleviates inflammatory injury. The clinical application of this medicine can shorten the course of disease.     

Study on the immunological function of sodium butyrate-induced immature human monocyte-derived dendritic cells

AIM: To investigate the immunological function of sodium butyrate-induced immature dendritic cells in vitro.METHODS: The human monocyte-derived dendritic cells were induced in the presence of human granulocyte macrophage-colony stimulating factor(GM-CSF) and interleukin-4 (IL-4), combined with sodium butyrate. The immunological function of sodium butyrate-induced dendritic cells was detected by the FCM, endocytic activity, T cells stimulatory proliferation capacity, and interleukin-12 (IL-12) production.RESULTS: Sodium butyrate could down-regulate the major histocompatibility complex(MHC) class II and costimulatory molecules of dendritic cells, increase the endocytic activity, induce a stage of T-cell anergey, and inhibit the T helper cell type 1-skewing factor IL-12 production. CONCLUSION: Sodium butyrate inhibits the maturation of dendritic cells and induces production of immature dendritic cells, which may help to explore the machenism of its epigenitic modification.     

The levels of 2′, 5′ oligoadenylate synthetase,interleukin 2 and interleukin 12 in hepatitis B virus infection

AIM: In order to study the effect of endogenous interferon system and Th1 response modes on hepatitis B virus infection, the 2′, 5′ oligoadenylate synthetase (2-5OAS), IL-2 and IL-12 were selected as the research parameters. METHODS: The activity of 2-5OAS in peripheral blood mononeuclear cells was determined by sensitive radioenzymatic assay. IL-2 and IL-12 were determined by ELISA. RESULTS: Compared to normal control, the 2-5OAS, IL-2 or IL-12 were not significantly changed (P>0.05) in the asymptomatic HBsAg carricer group. The 2-5OAS, IL-2 and IL-12 were significantly up-regulated (P<0.01) in the group of acute hepatitis, but in the groups of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, the 2-5OAS, IL-2, IL-12 were significantly down-regulated (P<0.05). Moreover, with the progression of patient′s conditions and with the complications of liver cirrhosis and hepatocellular carcinoma, the 2-5OAS, IL-2 and IL-12 decreased progressively, the 2-5OAS, IL-2, IL-12 were the lowest in guoups of liver cirrhosis and hepatocellular carcinoma (vs each groups of chronic hepatitis, P<0.05). CONCLUSION: The endogenous interferon system and Th1 response are significantly alterable in the different period of hepatitis B virus infection and among the different clinical types. The cellular immunity plays an important role in recovery from HBV infection.     

Mechanism of systemic inflammatory response syndrome induced by EC DNA

AIM: To investigate whether the bacterial DNA participates in SIRS and its possible mechanism. METHODS: Escherichia coli genomic DNA (EC DNA) was extracted and purified from Escherichia coli 25922 by alkaline lysis method. Mortality of mice challenged with EC DNA and the changes of TNF-α and IL-6 in rat serum were observed. ANA-1 cells were cultured in vitro, after the cells were stimulated by different concentrations of EC DNA and LPS, the level of TNF-α and IL-6 in supernatant were tested. Meanwhile,expression of TLR9 and TLR4 on cell surface was measured. Activation of NF-JB was also observed. RESULTS: The lethal effect of EC DNA on mice with an obvious dose-effect relationship was observed. The death happened within 24 hours. Calf thymus DNA and DNase I-treated EC DNA did not lead to mice to die. The changes of serum TNF-α and IL-6 in rats induced by EC DNA and LPS were similar, but TNF-α peak level of EC DNA group appeared 1 hour earlier than that of LPS group. In vitro, large amount of TNF-α and IL-6 were released from ANA-1 cells stimulated by EC DNA. High expression of TLR9 and TLR4 was observed on surfaces of THP-1 cells. In particularly, LPS induced strong activation of NFκB. The results suggested other pathway possibly took part in the signal transduction inducea by EC DNA. CONCLUSION: EC DNA has the abilities to lead to death of mice, andinduces serum TNF- αand IL- 6 level to increase in rats and ANA- 1 cells to release cytokines in vitro. High expression of TLR9 and TLR4, strong activation of NF- κB may be its importantmolecular mechanism, but other pathway probably exists to play an important role.     

团头鲂IL-12表达及功能的初步研究

白细胞介素-12（IL-12）是一种连接先天性和适应性免疫系统的重要细胞因子,在免疫反应和炎症反应中均发挥着重要作用。本实验以团头鲂为研究对象,克隆了团头鲂IL-12的4个亚基IL-12A、IL-12Ba、IL-12Bb和IL-12Bc的开放阅读框（ORF）序列,长度分别为588、993、939和837 bp。实时荧光定量PCR（RT-qPCR）结果表明,团头鲂IL-12各亚基在检测的10个不同的成鱼健康组织中都有表达,但是在不同组织中的表达模式不同;腹腔注射嗜水气单胞菌后,团头鲂IL-12各亚基在免疫相关组织中显著差异表达;脂多糖（LPS）刺激分离的团头鲂头肾淋巴细胞后,4个IL-12亚基均被快速诱导表达。进一步通过大肠杆菌原核表达系统和变性复性方法得到团头鲂IL-12Bb、IL-12Bc重组蛋白并刺激分离的头肾淋巴细胞,显著上调TNF-α、IL-1β、IFN-γ的表达,表明获得的重组蛋白具有生物学活性。最后对获得的蛋白进行抑菌活性检测,结果表明重组IL-12Bb、IL-12Bc单体蛋白对嗜水气单胞菌、大肠杆菌和金黄色葡萄球菌都有明显的抑制作用。综上,本研究结果表明,团头鲂IL-12在调节免疫应答的过程中发挥着重要作用。     

Gonadotropins I and II do not stimulate thein vitro secretion of 17α,20β-dihydroxy-4-pregnen-3-one 20-sulphate by rainbow trout gonads during final sexual maturation

Thein vitro secretion of 17,20-dihydroxy-4-pregnen-3-one 20-sulphate (17,20-P-sulphate) and the free steroid 17,20-dihydroxy-4-pregnen-3-one (17,20-P), by rainbow trout (Oncorhynchus mykiss) gonads, in response to gonadotropin (GTH) I and GTH II, were studied during the final stages of sexual maturation. Substantial amounts of 17,20-P-sulphate were produced, by both mature ovaries and testes, indicating considerable 20-hydroxysteroid sulphotransferase (20-HST) activity within these tissues. In the post-ovulatory ovary the level of 17,20-P-sulphate (36.6 ng ml^–1) greatly exceeded that of 17,20-P (8.59 ng ml^–1). The amount of 17,20-P-sulphate produced in incubations of both mature ovary and testes was unaffected by either GTH I or GTH II treatment at physiological concentrations up to 100 ng ml^–1. Similarly, incubations of maturing ovary and testes, treated with GTH I or GTH II, in the presence of added 17,20-P at 100 ng ml^–1 of medium, produced levels of 17,20-P-sulphate that were similar to those of the controls. In incubations of mature ovarian follicles at the stages of germinal vesicle breakdown and preovulation, both GTHs significantly stimulated secretion of 17,20-P, although GTH II was always more potent than GTH I. GTH II significantly elevated the levels of 17,20-P in testicular incubations from mature males more than 4-fold relative to GTH I and controls, which did not differ from one another.In conclusion, 20-HST, the enzyme responsible for the sulphate conjugation of 17,20-P, was found to be active in the ovaries and testes of rainbow troutin vitro. However, the levels of this enzyme do not appear to be regulated by either GTH I or GTH II.     

Estradiol-17β-induced calcium uptake and resorption in juvenile rainbow trout,Oncorhynchus mykiss

Juvenile rainbow trout, Oncorhynchus mykiss, were injected with estradiol-17β (E₂) in order to study the source of extra calcium needed during vitellogenesis. E₂-treatment increased the calcium uptake from the external medium as well as calcium mobilization from muscle and scale. Judged by the increase in plasma protein-bound calcium levels, the E₂-induced increase in calcium uptake is an apparent over-mobilization of calcium, i.e., the calcium uptake of the fish is in excess of what is found bound to plasma proteins. As the calcium excretion and calcium space (calculated from free plasma calcium levels) were unaffected, the excess calcium is suggested to be incorporated into internal calcium stores. This implies that the systems regulating vitellogenesis and calcium balance are integrated on the mechanistic or endocrine level, and that E₂ causes calcium mobilization of a magnitude geared to the needs of the sexually maturing female.     

Evidence that 17α,20β,21-trihydroxy-4-pregnen-3-one is a maturation-inducing steroid in spotted seatrout

Ovarian steroidogenesis during final oocyte maturation (FOM) in the spotted seatrout (Cynoscion nebulosus) was investigated by incubating ovarian fragments with tritiated pregnenolone, followed by chromatographic separation of the radioactive products. The major tritiated steroid produced during FOM comigrated with 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-dihydro-11-deoxycortisol, 20β-S) on HPLC and TLC. Only minor amounts of radioactive material coeluted with 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P), 11-deoxycorticosterone (DOC), estradiol-17β and testosterone standards in the HPLC system. Additional chromatography by TLC confirmed the presence of radioactive estradiol-17β and testosterone but not 17α,20β-P and DOC. All the ovarian steroids producedin vitro during FOM were assayed for their ability to induce germinal vesicle breakdown (GVBD) of spotted seatrout oocytes. Twenty grams of ovarian tissue were incubated with human chorionic gonadotropin and exogenous pregnenolone. The steroidal products were purified by HPLC and TLC. Most of the maturation-inducing activity was confined to steroidal material which comigrated in these systems with 20β-S. This material was active at a concentration of 1 ng steroid/ml medium in the GVBD assay. Smaller amounts of material which coeluted with 11-deoxycortisol, DOC, 17α,20β-P and several minor unidentified fractions induced GVBD at concentrations of 10 ng steroid(s)/ml. The structure-activity relationships of authentic steroids in inducing GVBD of spotted seatrout oocytes was investigated. Hydroxylation at the 17α, 20β or 21 positions increased potency to induce GVBD. Steroids with multiple hydroxyl groups at the 17α and 20β positions (17α, 20β-P) and at the 17α, 20β, and 21 positions (20β-S) had maximum biological activity in the GVBD bioassay. The results suggest that 20β-S is a major maturation-inducing steroid in spotted seatrout.