Effect of caffeine on hyperoxic lung injury of premature rats and its relationship with p38 motigen￣acti￣vated protein kinase signal pathway / 中国小儿急救医学

Objective To study the effect of caffeine on hyperoxic lung injury of premature rats and its relationship with p38 motigen-activated protein kinase( MAPK) signal pathway. Methods Sixty Wistar premature rats were divided into 4 groups(n＝15) according to the random number table:air + normal sa-line group(A+N group),air + caffeine group( A+C group),hyperoxia + normal saline group( H+N group),and hyperoxia + caffeine group(H+C group). Among them,H+N group and H+C group were continually exposed to hyperoxia ( oxygen concentration was 60% ~70%). For A + C group and H + C group,the premature rats were injected with caffeine of 29 mg/(kg·d) into their peritoneal cavities every day after birth. For A+N group and H+N group,the premature rats were injected with normal saline of the same volume into their peritoneal cavities. In each group,the lung tissues of 5 premature rats were randomly select-ed on the third, seventh and fourteenth day respectively. The pathological changes of lung tissue, radiated alveolar count(RAC) and collagen content in lung tissue were observed under a light microscope. The wet/dry ratio ( W/D) was measured. Two-step immunohistochemistry was used to detect the distribution of p38MAPK in lung tissue. The content of phosphorylated p38MAPK( p-p38MAPK) protein was detected by western blot. Results Compared with the air groups,the lung tissues of premature rats in high oxygen expo-sure groups showed different degree of inflammatory changes on the third,seventh,and fourteenth day. The changes were more obvious with the prolonged exposure to hyperoxia. Pulmonary fibrosis was visible on the fourteenth day,which was improved after caffeine intervention. The RAC value of premature rats in hyperoxia exposure groups was significantly lower than that in air-exposure groups(P<0. 05),and the W/D ratio and collagen content in lung tissue increased significantly (P<0. 05),which were improved after caffeine inter-vention(P<0. 05). The results of two-step immunohistochemistry showed that the number of p-p38MAPK positive cells in the lung tissue of premature rats in hyperoxia exposure groups increased and widely distribu-ted, but decreased after caffeine intervention. The results of western blot showed that the content of p-p38MAPK protein in lung tissue of premature rats in hyperoxia exposure groups was significantly higher than that of air groups(P<0. 05),but it decreased after caffeine intervention(P<0. 05). Conclusion Hy-peroxia can promote the formation of pulmonary fibrosis by activating p38MAPK signal pathway. Caffeine can interdict the expression of p38MAPK to alleviate the fibrosis of lung tissue exposed to hyperoxia and thus protects the lung tissue.

Study on the role of allergic toxin C3a in epithelial mesenchymal transition of normal human bronchial epitahelium cells / 重庆医学

Objective To investigate the role of anaphylatoxin C3a on epithelial mesenchymal transition(MTT) in normal human bronchial epithelium cells and its molecular mechanism.Methods Normal human bronchial epithelium cells BEAS-2B were cultured,and divided into control group,rhC3a stimulation group,rhC3a+ C3a receptor(C3aRA)antagonist group,the morphological changes of cells were observed by microscope;cell proliferation was detected by MTT;the expression of TGF-β1 protein level in cell supernatant was evaluated by ELISA;the expression of C3aR mRNA and EMT related indicators mRNA changes were detected by RT-PCR;the expression of C3aR and Smad2/3,p38 MAPK pathway proteins were detected by Western blot.Results Cell morphology in 30,50 nmol/L rhC3a stimulation group was changed from normal cobblestone like to spindle shape,cell morphology in C3aRA antagonist group had no significant change when compared with the control group.The cell proliferation was reduced in 50 nmol/L rhC3a stimulation group(P=0.047);the levels of TGF-β1,p-Smad2/3,p-p38-MAPK protein were increased(P＜0.05),C3aR mRNA and protein levels were also significantly increased (P＜0.05) in 30 nmol/L rhC3a stimulation group when compared with control group,but the addition of 1 μmol/L C3aRA could reduce their expressions(P＜0.05).30 nmol/L rhC3a could induce the up regulation of α-SMA and N-cadherin mRNA,and decrease the expression of E-cadherin mRNA,adding 1 μmol/L C3aRA can antagonize this process (P＜0.05).Conclusion Anaphylatoxin C3a can induce EMT in normal human bronchial epithelium cells by combining C3aR,its mechanism may be involved in activating Smad2/3 and p38-MAPK pathway.

Spd1672 gene knockout significantly attenuates the virulence of Streptococcus pneumoniae / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of spd1672 gene in the infection process of Streptococcus pneumoniae.</p><p><b>METHODS</b>BALB/c mice were intraperitoneally infected by a spd1672 knockout strain and a D39 wild-type strain of S. pneumoniae, and the survival time of mice and blood bacterial counts were recorded. The adhesion and invasion ability of S. pneumoniae strains were assessed in A549 cells. Bactericidal assays were carried out to determine the resistance of spd1672 knockout strains and D39 wild strains, and the serum levels of inflammatory cytokines were detected in the infected mice.</p><p><b>RESULTS</b>The mice infected with spd1672 knockout strains showed a significantly longer median survival time, a higher survival rate, and a lower blood bacterial load than the wild strain-infected mice (P<0.05). Having a similar cell adhesion ability to the wild-type strain (P>0.05), the spd1672 knockout strain showed significantly lower cell invasion ability than the wild-type strain (P<0.05). The spd1672 knockout strain also had a reduced resistance to whole blood cells, and thw mice infected with spd1672 knockout strain exhibit lower levels of serum inflammatory cytokines than those infected with the wild-type strain.</p><p><b>CONCLUSION</b>Spd1672 gene is importantly related to the virulence of S. pneumoniae and plays important roles in modulating bacterial invasion, resistance to whole blood cells and proinflammatory responses.</p>

IgG mediated protection elicited by △A146 Ply against Streptococcus pneumoniae infections in murine models / 中华微生物学和免疫学杂志

Objective To evaluate whether immunization with △A146 Ply could confer protections against pneumococcal infections in murine models and to reveal the possible role of △A146 Ply-specific IgG in the protection elicited.MethodsBALB/c mice were immunized intraperitoneally with △A146 Ply or PBS plus alum.Fourteen days after the third immunization,mice were intranasally challenged with serotype 14 and 19F Streptococcus pneumoniae.Three days after inoculation,lungs were removed from mice and homogenized in PBS,followed by plated on red cell plates.Viable bacteria were counted after overnight incubation.As to the sepsis models,vaccinated mice were challenged intraperitoneally with different dosage bacteria of D39 and serotype 3 strain.The numbers of CFU log10 were compared by Mann Whitney U test and survival rates were analyzed using log-rank test.Passive protection was used to evaluate the role of △A146 Ply-specific IgG in the protection against otherwise lethal infection resulted from D39.Results ELISA analysis demonstrated higher titer specific antibody responses to △A146 Ply was produced after 3 times immunization.Mice boosted twice with △A146 Ply survived significantly longer than that for mice boosted once with △A146 Ply in Alum adjuvant,which were significantly longer than that of control group.Immunization with △A146 Ply was effective in reducing the numbers of pneumococcal strain 31614(serotype 14) and 31693(serotype 19F),which resulted in 50-and 20-fold decreases in bacterial load in the lungs respectively when compared to control protein-immunized mice.60% of vaccinated mice survived the infection with pneumococcal D39 of 1200 CFU.60% protection was achieved when mice intraperitoneally infected with D39 and received △A146 Ply-specific IgG,whereas no mice survived the infection when they were passively administered with △A146 Ply-specific IgG depleted antisera.Conclusion Immunization with △A146 Ply could confer protection against pneumococcal infections,and protection elicited was mediated by △A146 Ply-specific IgG.

Protective effect of ClpP fusion protein against invasive infection of different serotype Streptococcus pneumoniae in routine models / 中华微生物学和免疫学杂志

Objective To evaluate the protective effects elicited by ClpP fusion protein in animal protection tests. Methods Pneumococcal antigens were purified from recombinant Escherichia coli express-ing CIpP cloned gene. 6- to 8-week-old female BALB/c mice were immunized intraperitoneally with either ClpP or PBS plus alum. Every mouse received three doses of 20 μg antigen or PBS in 100 mg of alum adju-vant at 14 d intervals. Sera were collected from mice and analyzed by ELISA 1 week after the third immuni-zation, Intraperitoneal-challenge experiments with 12 different serotypes of Streptococcus pneumoniae were carried out 2 weeks after the third immunization, and we compared their median survival times and survival rates respectively by Mann Whitney U test and Fisher exact test. Results ELISA analysis demonstrated high titer specific antibody responses to ClpP. The median survival times for mice immunized with ClpP pro-tein antigens in adjuvant were significantly longer than those for mice that received the adjuvants alone. Con-clusion A highly expressed recombinant ClpP protein has been successfully obtained and proved to exert the protection against invasive pneumococcal infection without relation of serotype, suggesting ClpP can be a promising candidate vaccine.