Abstract:Objective To observe the changes of inflammatory reaction in tail-suspension mice after infected by E.coli in spaceflight. Methods 40 C57BL/6 mice were randomly divided into four groups: control group(Con), control+E.coli T1-13 group(Con+T1-13), tail suspension group(TS), tail suspension+E.coli T1-13 group(TS+T1-13). The inflammatory cytokines TNF-α, IL-1β and IL-6 production in plasma and mRNA level in intestinal tissue were detected by ELISA and RT-qPCR, and HE staining was used to represent the morphology changes in small intestine tissue. Results Compared with the control group, the expression of inflammatory cytokines in plasma and intestinal tissue of all experimental groups were increased, and the TS+T1-13 group was most significant(P < 0.01 or P < 0.001). HE staining showed that the small intestine mucosa in the experimental groups were damaged in different degrees, and the damage of TS+T1-13 group was most serious. Conclusions The E.coli from spaceflight increased significantly the expression of inflammatory cytokines in plasma and intestinal tissue from infected tail-suspension mice, and brought more serious damages to the small intestinal mucosal barrier, which suggested that the inflammatory reaction would be increased in tail-suspension mice infected by E.coli from spaceflight.

Abstract:Objective To establish a tree shrew mode of breast tumor. Method Forty-five 3 to 4 month-old female tree shrews were orally gavaged with 20 mg 7, 12-dimethylbenz(a)anthracene (DMBA) or peanut oil per animal for three times. Following that, fifteen DMBA administrated tree shrews were implanted 90 day-release medroxyprogesterone acetate (MPA) pellets. The tree shrews were palpated once weekly to detect mammary tumors for 45 weeks after first DMBA administration. Results DMBA were able to induce breast tumors (12.5%) in tree shrews, and MPA increased the tumor incidence (50%) while no breast tumors were observed in the control group. Three induced breast tumors were intraductal papillary carcinomas and one was IDC by H&E stain. Conclusion All induced tumors are similar with spontaneous tumors in structure and molecular markers.

Abstract:Objective To understand the expression of OX62 and CD83 in the lungs of rats with COPD, and to investigate the effect of the CCL20 monoclonal antibody on it. Methods A total of 30 rats were randomly divided into three groups: normal, model, and CCL20 antibody treated. COPD was induced by cigarette smoke exposure 28 days and LPS solution injection twice. The rats were injected with CCL20 antibody in the last group on the first day. The rats were sacrificed on the 29th day. We investigated the pathomorphology of their lungs by HE staining and evaluated the DC distribution in their lungs by immunohistochemistry. Results The HE staining results of the COPD models are consistent with the typical pathological features of COPD patients, the lung pathology in CCL20 group was significantly attenuated than that in the COPD group. Compared with healthy control group, the OX62+DCs in the COPD model group was significantly increased (P < 0.05), and the CCL20 group was lower than that in the COPD group (P < 0.05). The CD83+ DCs of COPD group was lower than that in healthy control group (P < 0.05), and the difference was not statistically significant between COPD and CCL20 treated group (P > 0.05). Conclusion The pathogenesis of COPD may be related to the increase of OX62+DC and the decrease of CD83+DC, and this effect could be partly inhibited by CCL20 antibody.

Abstract:Objective To explore a gestational insulin resistance mice model establishing method, which is steady, reliable and similar to human being. Method Female KM mice at 5 weeks were randomly divided into high fat diet group(n=30) and normal diet group(n=30). High fat diet group was exposed to high diet for 4 weeks, and then male and female were mated(n:n=1:1) Then the pregnancy mice were intraperitoneal injected with 30 mg/kg/d STZ during the first 3 days, the control group was injected with equal dosage of citrate buffer solution(0.1 mol/L, pH=4.2) for 3 days. Their blood glucose, body weight, the amount of food intake and water intake were recorded at 3, 7, 14 and 19 d of the GDM model respectively. The serum INS, ADP, LEP, CRP were measured by ELISA. Results We successfully made the GDM mice model, and the pregnant mice showed significant signs of polyuria, polydipsia, hyperphagia and weight loss compared to the control model(P < 0.01). The blood glucose of GDM mice >11.1mmol/L, the serum cytokines (INS(1.50±0.25)Mu/L, ADP(0.65±0.13)μg/L, LEP(1.60±0.12)μg/L, CRP(37.54±4.70)μg/L) of GDM mice were significant difference compared to the control model(P < 0.01). Postpartum, the GDM mice blood glucose returned to the normal level. Conclusion Gestational diabetes mellitus mice model can be successfully developed by high fat diet with low dose STZ and three times induce, which preferable mimics the characteristic of gestational pathologic insulin resistance in human beings.

Abstract:Objective To investigate the possibility of utilizing the mixed bacteria liquid and the Escherichia coli (E.coli) liquid to establish the chronic salpingitis model of New Zealand rabbits, respectively. Methods Taken as the study object, the un-pregnant New Zealand rabbits (4~5 years old) were randomly divided into three groups: the normal group, the mixed bacteria experimental group and the E.coli experimental group. The trans-vaginal intrauterine intubation operation was performed for the injection of the mixed bacteria liquid and the E.coli liquid. Visual observation was to evaluate the gross pathological changes of the salpingitis and the pelvic cavity. HE staining and the light microscope were used to observe the micro-pathological changes of salpingitis. Results On the 15^th day after modeling, increased pelvic effusion, dense peritoneal adhesion, interstitial hyperplasia and infiltration of lymphocytes were observed in both experimental groups. Conclusion Through the trans-vaginal intrauterine intubation operation, the chronic salpingitis model of New Zealand rabbits could be successfully established either by using the mixed bacteria liquid or by using the E.coli liquid.

Abstract:Objective To study the effects of morroniside on the expression of the HGF and vWF of the ischemic ipsilateral cortex 7 days after ischemia reperfusion. Methods 25 male Sprague-Dawley rats were subjected to MCAO model with modified Zea Longa's method, then randomly divided into sham group (n=5), ischmia group (n=5), and morroniside groups (low, medium, and high dosage groups, n=5). Morroniside were then administered intragastrically once a day at dose of 30 mg/kg, 90 mg/kg and 270 mg/kg after operation. The expression of HGF and vWF of the ischemic ipsilateral cortex were detected by western blotting and Immunofluorescence 7 days after ischemia-reperfusion. Results Compared with the sham group, the expression of HGF in ischemia group increased significantly (P < 0.01) 7 days after MCAO. Compared with the ischemia group, the expression of HGF in the morroniside groups (30 mg/kg, 90mg/kg, 270 mg/kg) showed significant increased (P < 0.05, P < 0.01, P < 0.001). Compared with the sham group, the expression of vWF in ischemia group increased significantly (P < 0.001). Compared with the ischemia group, the expression of vWF in the morroniside groups (90 mg/kg, 270 mg/kg) showed significant increased (P < 0.01, P < 0.001). Conclusion Morroniside could increase the expression of HGF and vWF in the ischemic ipsilateral cortex, romoting the process of angiogenesis in focal cerebral ischemia rat.

Abstract:Objective To investigate the method to isolate and culture hepatic stellate cells (HSCs) for studying the cellular mechanisms of hepatic frbrosis. Methods HSCs were isolated by nycodenz density gradient centrifugation after the hepatocytes obtained from adult male gebils were digested with pronase, collagenase and DNase, infused via portal vein. The cell viability was determined by trypan blue exclusion test. The purity of HSCs was identified by detecting α-SMA, desmin immunohistochemical staining. Results The yield rate of HSCs was 0.5~1×10⁷ per gerbil liver, and the cell viability was more than 90%. The percentage of α-SMA-positive cells was more than 75% after 3 days primary culture and almost 100% cells were α-SMA and desmin positive in passage culture. Conclusion The successful protocol of primary culture of Mongolian gerbil HSC provide a technical support for research of relevant liver diseases and drug development in the future.

Abstract:Objective To measure the lethal dosage values (LD₅₀) of i.v. asarone injection for mice and to establish a standard for abnormal toxicity test of asarone injection to potentially reduce the occurrence of adverse drug reaction. Methods To obtain the LD₅₀ value, a weighted linear probit regression method (Bliss method) is employed. The limit of abnormal toxicity test is determined according to Appendix XI C in its 2010 edition of the Chinese pharmacopoeia. Results It is found that the LD₅₀ of intravenously asarone injection in mice ranges from 51.9 to 153.1 mg/kg. The abnormal toxicity test should be added as an additional item in the standard. Conclusions Based on analyses in this study, an appropriate limit of abnormal toxicity test is 15 mg/kg, which is also in line with current medical standard in China.

Abstract:Objective To explore inhibition of nicotine on apoptosis of chondrocytes induced by monosodium iodoacetate (MIA). Methods Rat primary chondrocytes were isolated by enzyme digestion, and the cells were treated with 10^-8, 10^-7, 10^-6, 10^-5 mol/L nicotine for 48 h. The cases were randomly divided into five groups, except for normal group, the other four groups were treated with 4 μmol/L MIA 24 h, and three groups were treated 10^-8, 10^-7, 10^-6 mol/L nicotine. The viability of chondrocytes was detected by MTT assay. The apoptosis of chondrocytes was examed by Annexin V-FITC/PI flow dual-staining method. The activity of cysteinyl aspartate specific proteinase 3 (Caspase 3) was measured by spectrophotography method. The activation of phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) and the expression of down-stream molecule Bax, Bcl-2 was assayed by western blot. Results 10^-7, 10^-6 mol/L nicotine increased chondrocytes' viability (P < 0.05), 10^-5 mol/L nicotine reduced chondrocytes' viability (P < 0.05), and 10^-8 mol/L nicotine didn't effect on chondrocytes' viability (P > 0.05). 10^-8, 10^-7, 10^-6 mol/L nicotine could increase MIA-induced chondrocytes' viability (P < 0.05), suppress MIA-induced chondrocytes' apoptosis and the activity of MIA-induced Caspase 3 (P < 0.05). Moreover, 10^-7, 10^-6 mol/L nicotine could increase the expression of PI3K and phosphorylation of AKT (P < 0.05), down-regulate the expression of Bax and up-regulate the expression of Bcl-2 in MIA-induced rat chondrocytes (P < 0.05). Conclusion These results suggested nicotine could exert anti-apoptosis in MIA-induced rat chondrocytes, which might be related to PI3K/AKT signal pathway.

Abstract:Objective To explore the source of endothelial cells in tumor vessels by A 549 tumor model with GFP nude mouse.Methods To establish the A 549 lung cancer models with GFP nude mice, expression of CD 31 was determined by immunofluorescence to label tumor vessels; to observe and take a picture of the tumor frozen section by confocal microscopy and invert microscope; expression of GFP in tumor vessels was determined by immunohistochemistry. Result The results of immunofluorescence showed: Tumor interstitial vascular endothelial cells or endothelial cells clusters and micro-vascular lumen size and shape are clearly visible by immunofluorescence, and part of vessels with no obvious lumen or irregular lumen. We can see green fluorescent in tumor cells of tumor tissue and endothelial cells which form of tumor vessels. The results of immunohistochemistry showed: expression of GFP was determined in cytoplasm of tumor stromal cells and endothelial cells in tumor vessels. Conclusion The endothelial cells which formed tumor neovessels that derived from GFP nude mice partly and the other part derived from tumor cells.

Abstract:Objective To determine the effect of Nuclear factor-erythroid 2-related factor 2(Nrf2) on acute hephrotoxicity induced by CCl4 in male rat. Methods 20 male Wistar rats were randomly divided into control group and CCl₄ group, 10 rats in each group, another 10 male Wistar rats were transgenic rats microinjection through the carrier, obtained the Nrf2-tk gene integration and specific transgenic rats, as the CCl₄+Nrf2 integration group. The groups was given 1% polysorbate 80 for 4 days, Then the CCl₄ and CCl₄+Nrf2 integration group were intraperitoneally injected with a single dose of CCl₄ 7.5 mg·kg^-1 and were killed 24 h after CCl₄ injection. The serum chemical parameters including asparate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were measured. Also malonaldehyde (MDA), glutathione (GSH), oxidized glutathione (GSSG) levels in the liver as well as glutathione (GSH)/oxidized glutathione (GSSG) ratios were detected. Histopathologic changes in the liver were examined. Results F1generation TK transgenic rats in liver and testis and other tissues and organs were not detected the transcription of Nrf2-tk, indicating that Nrf2-tk expression in tissues is specific good. Nrf2 significantly reduced serum AST, ALT and LDH levels in a dose-dependent manner. The results of MDA levels and GSH/GSSG ratios in liver and kidney showed that Nrf2 reduced CCl₄ -induced hepatic lipid peroxidation, and ameliorated glutathione depletion. The histopathologic results showed that Nrf2 restrained liver and kidney damage induced by CCl₄. Conclusion Nrf2 can effectively protect male rat from acute hepatotoxicity and nephrotoxicity induced by CCl₄.

Abstract:Objectives To explore effect of lipitor on apoptosis and phosphatidyl inositol-3-kinase (PI3K)/protein kinase B (AKT)/endothelial nitric oxide synthase (eNOS) signal pathway in high glucose-induced human umbilical vein endothelial cell (HUVEC). Methods The cases were randomly divided into normal control group, model control group (33.3 mol/L glucose), lipitor low, medium, high-dose group (0.1, 1, 10 μmol/L lipitor).The viability of HUVEC was detected by MTT assay. The morphology of HUVEC was photographed by inverted microscope. The apoptosis of HUVEC was examed by Annexin V-FITC/PI flow dual-staining method. The concertration of NO in HUVEC supernatant was exmaed by Gries method. The activation of PI3K/AKT and expression of eNOS was assayed by western blot. Results HUVEC was shrinkage, rounded and brighten, the viability of HUVEC decreased, early and late apoptosis rate of HUVEC increased significantly, the level of NO, eNOS, PI3K and AKT phosphorylation also reduced in model control group (P < 0.01). 1, 10 μmol/L lipitor improved HUVEC morphology, increased HUVEC' viability and expression of PI3K (P < 0.05). 0.1, 1, 10 μmol/L lipitor suppressed HUVEC' apoptosis, increased the concentration of NO, expression of eNOS and phosphorylation of AKT (P < 0.05). Conclusion These results suggested lipitor exert anti-apoptosis in high glucose -induced HUVEC, which might be related to PI3K/AKT/eNOS signal pathway.

Abstract:Objective To investigate the effect of ganoderic acid A(GA-A) on apoptosis, invasion and KDR expression of human U251 cells. Methods Ganoderic acid A(GA-A) was prepared, human U251 cells were treated with 0.1, and 0.5 mmol/L GA-A, and the experiment was divided into blank control, low concentration and high concentration group. The expressions of KDR mRNA and KDR protein was assayed by RT-PCR and Western blot.The effect of GA-A on the proliferation and invasion capability of U251 cells was determined by CCK-8 and transwell assay in vitro, respectively. Flow cytometry was used to detect the influence of GA-A on the cell cycle and apoptosis of U251 cells, and TUNEL staining was detected the cell apoptosis too. Results Compared with the control group, KDR mRNA and protein expression of high concentration and low concentration group were significantly decreased(P < 0.05), GA-A can significantly reduce the cell growth rate, reduce the proportion of cells in G1 phase and increase the proportion of S phase and G2/M phase, cells apoptosis was significantly increased in the high concentration and low concentration group (P < 0.01), and cells proliferation and invasion was significantly decreased (P < 0.05). Compared with low concentration group, the high concentration group induce cell apoptosis and inhibit the expression of KDR more significant (P < 0.05). Conclusions Ganoderma acid A can induce apoptosis in U251 cells, inhibit proliferation and invasion, and can inhibit the expression of KDR mRNA and protein, which may be one of the mechanisms of anti-tumor.

Abstract:Objective To study the subacute inhalation toxicity of Ivermectin TC, and obtain its non-observed adverse effect level(NOAEL). Methods It was performed on the doses of Ivermectin TC 190, 380, 750 mg/m³, the solvent control group (0.03% Tween-80 solution), the control group and additional group (there were 6 female and 6 male Sprague-Dawley rats for each group). The animals inhaled with nose-only exposure for 4 weeks (4 h/d, 5 d/week). The additional group should be observed another 14 days after exposing. At the end of experiment, the rats were killed, the routine and biochemical detection, the body weight and organ to body weight ratios were all measured. Results In the high exposure group, clinical signs of rats included hair fluffy, dull, salivation, tremors were recorded at the exposure period; in female rats, feed efficiency was decreased, ALT and liver to body weight ratio were increased; in male rats, BUN and ALT were increased, CHOL and body weight for the 4th week were decreased. Histopathological examinations revealed that swelling in the liver cell was seen in some female rats at high exposure group. Conclusion The results suggested that the NOAEL of Ivermectin TC in SD rats was 380 mg/m³ (4 h/d for 28 days).

Abstract:Objective To establish ELISA method for H-1 parvovirus, and to apply it in detection. Method Cultured the H-1 parvovirus in rat glioma cell line C6, prepared the viral antigen for coating. Used the purified viral antigen to establish the ELISA method, and compared the ELISA method with the ELISA kit from XpressBio company. Then applied the ELISA method in detection of 35 rat serums. Results The positive serum which be diluted to 1280 can be detected by the ELISA method, there have not cross reaction with positive serum of CPV, MVM and PPV, but there has cross reaction with KRV. 35 pieces of rat serums were detected by the ELISA method, they were all negative, the results were consistent with the kit from XpressBio company. Conclusions The sensitivity and species specificity of the ELISA method for H-1 parvovirus were suitable, the method can be used in detection of H-1 parvovirus in rat serum.

Abstract:Objective To provide ideal endometriosis animal models for research new treatment methods. Methods 64 Sexual mature without pregnant SD rats who has regular oestrous cycle were underwent operation that autologous endometrium were transplanted to peritoneum and subcutaneous with lancet gently cut tissue of where to transplant in the rut, and compared the model of peritoneum and subcutaneous after 4 weeks. Results The general success rate of autologous endometrium transplantation in rat estrus was 93.3%, and the peritoneum transplantation success rate was 51.7%, subcutaneous was 88.3%, the difference of the two place transplantation has statistical significance. Compare the two parts of volume of endometriosis, there was no statistically significant difference. Conclusion Using the lancet to establish the endometriosis model has a high success rate, and subcutaneous model is better than peritoneum.

Abstract:Objective To compare the conventional marrow collection with new marrow collection, in the number of cell, other tissues pollution and the background of the smear, providing the reference for future micronucleus test. Methods The mice was enthanasia and the sternums were taken. One group, using the conventional method of marrow collection, squeezing the marrow to the slide with fetal bovine serum; the other group, using 1-mL injector extracting fetal bovine serum 100μL, injecting into mice sternums and rushing out the bone marrow for circle smear. Results Two methods can meet the requirement of test, but the new marrow collection can acquire more number of cells, less the tissues pollution and more clear in the background of smear. Conclusions Comparing with the conventional marrow collection, the new method has more superiority to simplify the next cell counting.