Mechanisms for a decrease in mortality of LPS-challenged mice by rhynchophylline

AIM: To observe effect of rhynchophylline （Rhy） on mortality and organ injury in endotoxemic mice and further investigate the mechanisms of its actions. METHODS: Male mice were randomly assigned into control, LPS, Rhy +LPS and Rhy group, and injected subcutaneously with normal saline (0.05 mL/10 g), or rhynchophylline once a day for 3 d, 1 h after subcutaneously treatment on day 3, LPS (20 mg/kg) or normal saline was injected intraperitoneally. Survival rate was recorded every 12 h for 6 d. In another experiment, 12 h after LPS injection, the left lung and intestine tissue sections were prepared for histological analysis and the right lung were used to determine the ratio of wet to dry lung tissue weight (W/D),the serum was collected to detect the concentrations of alanine aminotransferase（ALT）, aspartate aminotransferase (AST ), bloodureanitrogen (BUN) and creatinine (Cr). In addition, the concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β(IL-1β) and interleukin-10 (IL-10) in serum at 2 h after LPS challenge were detected by enzyme-linked immunosorbent assay. The concentration of NO in serum at 8 h was detected by enzymic method. The effect of Rhy on survival rate of mice subjected to cecal ligation and puncture (CLP) was also observed. RESULTS: Mortality of mice challenged with LPS alone was higher significantly than that in control at 24 h after LPS challenge, pretreated with Rhy at a dose of 8 or 16 mg/kg increased markedly the survival rate of LPS-challenged mice. However, Rhy at a dose of 8 mg/kg significantly increased mortality of mice subjected to CLP. In the histological analysis, severe inflammation was observed both in the lung and intestine tissues in the LPS group. LPS elevated lung W/D, the levels of ALT, AST, BUN, Cr, TNF-α, IL-1β, IL-10 and NO in serum. Pretreatment with Rhy had no obvious improvement in the lung and intestine tissue injury, and no significant depression in the lung W/D and the serum levels of ALT, AST, BUN, Cr, IL-1β, IL-10 and NO, but decreased the level of TNF-α in serum significantly in LPS -treated mice. CONCLUSION: Pretreatment with Rhy reduces the mortality in endotoxemic mice, but not decrease the mortality of mice challenged with CLP, at least in part, through inhibiting the synthesis and secretion of TNF-α.     

花茶对被动吸烟小鼠耐缺氧能力的影响

30只小鼠随机分为3组（n=10）,分别为对照组（CK）、被动吸烟级（B）、吸烟用茶组（C）;B、C组每天2次被动吸烟,每次1 h（6支/h）,C组每天饮用ST茶汤（30%）,第31 d进行密闭耐氧试验,测定3组小鼠的缺氧耐受时间,然后取血测定血红蛋白、肝糖原含量,并剖取胸腺和脾脏,计算其指数。结果表明,C组小鼠的缺氧存活时间显著长于B组,肝糖原含量、胸腺指数显著高于B组（p〈0.05）,血红蛋白含量和脾指数与B组差异不显著（p〉0.05）。说明ST能显著延长PS小鼠的耐缺氧时间,缓解PS对小鼠免疫器官的伤害。     

β-Glucan successfully stimulated the immune system in different jawed vertebrate species

Several reports have shown the positive effects of β-glucans on the immune. Howeverthese studies have a broad experimental design including β-glucans compounds. Consequently, a study using the same β-glucan molecule, administration route and experimental design is needed to compare the effects of β-glucan across vertebrate species. For this end, during 28 days we fed four different vertebrate species: mice, dogs, piglets and chicks, with two β-glucan molecules (BG01 and BG02). We measured the serum interleukin 2 as an indicator of innate immune response, the neutrophils and monocytes phagocytosis index as a cellular response and antibody formation as an adaptive response. The results clearly showed that the different β-glucan molecules exhibited biologically differently behaviors, but both molecules stimulate the immune system in a similar pattern in these four species. This finding suggests that vertebrates shared similar mechanisms/patterns in recognizing the β-glucans and confirms the benefits of β-glucans across different vertebrate species.     

Effects of Foxp3 transfection on splenocyte functions in asthma mice

AIM: To study the expression and the effects of Foxp3 on the immunologic functions by transfecting the Foxp3 eukaryotic expression plasmid into the splenocytes of the asthma mice. METHODS: The mice were sensitized and challenged by ovalbumin to make asthma model. The splenocytes were harvested and cultured. The Foxp3 expression vector pcDNA3.1(-)-Foxp3 was transfected into the splenocytes with electroporation. The splenocytes transfected with empty vector and control splenocytes (non-transfected) were also set up. The expression of Foxp3 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. The proportion of CD4⁺CD25⁺ Treg cells/CD4⁺ cells was measured by flow cytometry. Proliferation of the splenocytes was analyzed with MTT assay. ELISA was used to determine the levels of interleukin 4 (IL-4) and interferon γ (IFN-γ) in the supernatant of the splenocytes. RESULTS: The expression of Foxp3 at mRNA and protein levels in transfection group was significantly higher than that in empty vector group and control group. The proportion of CD4⁺CD25⁺Treg cells/CD4⁺ cells in transfection group was higher than that in empty vector group and control group. The proliferation of transfected cells was markedly inhibited compared with empty vector group and control group. The levels of IL-4 and IFN-γ were significantly lower in transfection group than those in empty vector group and control group. CONCLUSION: The transfected Foxp3 gene overexpresses in the splenocytes of asthma mice. Foxp3 increases the number of CD4⁺CD25⁺ T cells and inhibits the proliferation and production of Th1/Th2 cytokines in splenocytes.     

Primary culture,identification and in vitro angiogenesis of mouse pulmonary microvascular endothelial cells

AIM: To establish a simple and reliable method for primary culture of mouse pulmonary microvascular endothelial cells (PMVECs), and to investigate the ability of angiogenesis of the cells in vitro. METHODS: PMVECs from C57BL/6 new born mice were cultured with peripheral pulmonary tissue pasted method. The cells were observed under inverted phase-contrast microscope and identified by immunocytochemical and immunofluorescent methods. PMVECs were seeded at the density of 20 000 cells per well in 96-well plate on top of growth factor-reduced matrigel and incubated for 24 h at 37 ℃ in 5% CO₂. The plates were examined every 2 h for tube formation under an inverted microscope. RESULTS: The primary PMVECs showed fusiform shape or polygonal, regular cobblestone morphology after fusion to monolayer, and expressed factor Ⅷ -related antigen (ⅧF-Ag). Under fluorescence microscope, those cells demonstrated positive staining for uptaking acetylated low-density lipoprotein (DiI-Ac-LDL) and binding Bandeiraea simpli-cifolia lectin I (FITC-BSI). The purity of PMVECs was above 90%. PMVECs formed capillary-like structures within 4 h to 6 h after plated on matrigel and fully developed a "honeycomb" appearance at 10~12 h, and then the capillary tubes began to break apart. CONCLUSION: The tissue block pasted method for primary cultivation of PMVECs is easy to handle, with high yield and purity, and will further facilitate related studies of well-established tube-formation assay in vitro.     

大鼠小鼠妊娠子宫植物性神经变化——HRP法研究

应用HRP顺向追踪法,研究了大鼠和小鼠子宫植物性神经的分布及妊娠后的变化。HRP自子宫阔韧带引入。结果发现,HRP标记神经分布于子宫壁各层,在内膜和环行肌层最多,其直径3～9μm(平均:大鼠4.81μm,小鼠4.60μm)。光镜下对子宫切片整个横断面计数发现,妊娠组(妇娠14天)与非妊娠组相比,子宫各段HRP标记神经数量显著下降(P<0.01),神经纤维直径也有减少的趋势。表明妊娠子宫植物性神经数量减少。     

Inhibitory effects of Angelicae sinensis and sodium ferulate on acute inflammatory liver injury and expression of ICAM-1 and E-selectin in mice

AIM: To explore the effects of Angelicae sinensis preparation and sodium ferulate on inflammatory liver injury induced by lipopolysaccharide (LPS) and their molecule mechanism. METHODS: ICR mice were divided into five groups: Angelicae sinensis group, sodium ferulate group, dexamethasone group, inflammation control group and normal group. The model of inflammatory injury in mice was set up by tail vein injection with the bacillus calmette-guerin (BCG) and LPS respectively prior and posterior to administration of those tested drugs. The tested drugs (the preparations of Angelica sinensis, sodium ferulate and dexamethasone) and normal saline were given respectively to the corresponding group. The pathological observation of the liver tissues in mice was made for the intensity of inflammatory liver injuries. The immunohistochemical detection and comparison were performed for the expression of ICAM-1 and E-selectin proteins in the liver tissues in those mice. RESULTS: The intensities of liver inflammatory injuries in mice from drug-treated groups were obviously lighter than that from the inflammation control group (P<0.01). The expressions of ICAM-1 and E-selectin proteins in the liver tissues of mice from the inflammation control group were not only significantly higher than that in normal control, but also obviously higher than that from drug-treated groups (P<0.01). CONCLUSIONS: Angelica sinensis and sodium ferulate alleviate inflammatory liver injury induced by injection of LPS. Their suppressive effects on inflammatory liver injury may be associated with the decrease in the expression of ICAM-1 and E-selectin proteins.     

无菌动物技术管理—无菌小鼠繁殖管理与监测

从日本中央实验动物研究所购买的２对ＩＱＩ无菌小鼠饲养在无菌隔离器中,饲养温度为２２～２４℃,相对湿度为５８～６０％,并成功地繁殖了２１４只无菌小鼠。无菌检验每２月进行一次,共检验４次,结果均为无菌生长。对鼠肝炎病毒和仙台病毒进行监测,结果为阴性。无菌小鼠剖解肉眼观察其盲肠巨大。     

Skeletal muscles of mdx mice were damaged after overload exercise

AIM:To observe the effects of overload exercise on skeletal muscles in X-linked muscular dystrophy(mdx) mice.METHODS:Mdx mice and C57 mice were carried out swimming and hanging tail movement tests (mdx mice as control did not exercise). It lasted for 13 minutes each time per day, and lasted 3 days. Evans blue was injected into tail vain. The mice were killed the next day, and the hind limbs were taken photographs after skins were flayed. The gastrocnemius muscles and diaphragms cryostat sections were made. Under a fluorescence microscope, Evans blue staining was seen. Then the sections were tested by routine HE staining, the histological change of muscles was analyzed under a light microscope.RESULTS:Many blue colored longitudinal lines were observed in skeletal muscles of mdx mice, whereas they were hardly seen in control mdx and C57 mice. Under a fluorescence microscope, some muscle fibers of mdx mice were stained with Evans blue, few muscle fibers of control mdx mice were stained, and C57 mice were not. Under a light microscope, HE staining of muscles showed some degenerated muscle fibers became round in shape and the myonuclei became condensed, or necrotic fibers had amorphous structures, most of them in the degenerated and necrotic fibers of diaphragms C57 mice did not have these changes.CONCLUSION:Overload exercise did harm to skeletal muscles of mdx mice; Vital staining with Evans blue is useful not only for distinguishing degenerating muscle fibers, but also for studying the degeneration process in dystrophin-deficient muscle.     

Comparative study on intraocular transplatation of three B16 melanoma cell lines in mice

AIM:To establish an animal model for studying the development and metastasis of melanoma.METHODS:C57BL/6 mice were used as host to receive melanoma cell transplantation. Three kinds of melanoma cell lines, B16F0, B16F1 and B16F10, cultured to prepare the cell suspensions, were transplanted into the mouse anterior chamber (AC) of the eye. The time of eyeball diabross, time of survival and metastasis of lymph node and lung were observed.RESULTS:The time of eyeball diabross in F10 group was earlier than that in other groups. The time of eyeball diabross was no difference between F0 and F1 groups. Metastasis was developed 18 days after transplantation in F1 and F10 groups, where the tumor cells was found in ipsilateral cervical lymph nodes. The melanoma cells metastasized to lung in all three groups 28 days after transplantation. The survival time in F0 group was longer than F1 and F10 groups. There was no difference in survival times between F1 and F10 group.CONCLUSION:The differences of three kinds of melanoma cell lines in tumor development and metastasis provided the evidence that was useful for choosing suitable animal model further to study the eye melanima.