| Aim:With the improvement of sanitation, development of vaccines and discovery of antibiotics, the infectious diseases are gradually declining. But the allergic diseases, such as bronchial asthma and atopic dermatitis, are progressively expanding. Bronchial asthma is a disease that is characterized by hyperresponsiveness and chronic inflammation in the airway, which has been taken as the most common disease. One hypothesis of etiology is hygiene hypothesis. With the improvement of sanitation, the chance of infection and exposure to the microorganism is decreased in the childhood, especially in the neonate. So the immune system is immature and disordered. These cause increased incidence of allergic disease. One of the immunological pathogenesis of asthma is imbalance of Th1/Th2. The quantity and functions of Th1 cells are decreased, and those of Th2 cells are increased. The cytokines, such as IL-4, IL-5 and IL-13, which are produced by Th2 cells, can improve the production of IgE and proliferation of eosinophil, basophil and mastocyte. All of these result in airway hyperresponsiveness. One of the methods to treat bronchial asthma is balancing the quantity and functions of T-helper cells. Administration of probiotics is expected to its advantage.According to the hygiene hypothesis, the moderate stimulation of microorganism is necessary for the formation of normal immune system. We selected Lactobacillus johnsonii Ncc533 (La1), which is one of the probiotics. Probiotics are live microorganisms which when administered in adequate amounts confer a healthy benefit on the host. There are three sorts of probiotics:Bifidobacterium, Lactic acid bacterium and Facultative anaerobic bacterium. They have different effects on human being. More clinical trials have been done on Lactobacillus rhamnosus and Bifidobacteria longum. The mechanism of these probiotics are different. Some trials suggested that lactic acid bacterium might improve immune function by increasing the number of IgA-producing plasma cells, or improving phagocytosis or increasing the proportion of T-lymphocytes and Natural Killer cells. It has been shown that the levels of IL-4, IL-5, IL-6 and IL-10 decreased in the trial to the control.In our present study, with administration of Lactobacillus johnsonii Ncc533 (La1),we researched the cytokines of IL-4 and IFN-y, produced by splenocytes of mice from the newborn to the adult. Then we established an asthma model and researched the serum levels of OVA-specific-IgE in different groups. We wanted to know if the La1-treated group had a different change in the excitation. Then we investigated the changes of IL-4 and IFN-y. We hoped to find a new way in treating the bronchial asthma.MethodsMice Twenty female and five male matured BALB/c mice were obtained from trial animals center of Shandong University (Shandong, PRC). The weight of female mice was from 24g to 28 g and the mean weight was 26.04g. The weight of male mice was from 26g to 30g and the mean weight was 27.86g. The copulatory ratio was 4:1 (female to male). The date of pregnancy was calculated from the date of observing verginal plugs.Reagents Monoclonal antibodies of IL-4-PE and IFN-y-PE were purchased from Immunotich. FixandPerm was obtained from Caltag(Shanghai, PRC). Ovalbumin(OVA, Grade V), phorbolester(PMA), ionomycin and monensin were obtained from Sigma (Shanghai, PRC). OVA-specific-IgE was purchased from DoBio(Shanghai, PRC). The suspension of La1 was obtained from the Nestle center(1×1012CFU/L). The MiniMACS was purchased from Miltenyi Biotech GmbH(Germany).Grouping All of the pregnant mice were randomly divided into two groups. For the mice of group 1, we injected the mice 2ml La1 suspension fluid through stomach tube, bid, lasting 2 to 3 weeks. For those of group 2, we injected the mice 2ml sterile-distrilled-water through stomach tube, bid, lasting 2 to 3 weeks. There were 56 offspring mice in group 1 and 62 in group 2. We selected randomly 7 mice for group 1’and 2’ from offspring mice of group 1 and 2, respectively. And then we separated splenocytes. We selected randomly 20 mice for group a and b from the other offspring mice of group 1 and 2, respectively. We injected the mice of group a lml La1 suspension solution through stomach tube after born, bid. For the mice of group b, we injected sterile-distrilled-water lml in the same way. After 4 weeks, we randomly selected 7 mice for group A and group B from group a and group b, respectively. Then we separated splenocytes. We selected 7 mice for group A’and B’from the other 13 mice, respectively. The total number of seven 4- to 6-weeks -old mice of group C’was the healthy control.Asthma model All the mice of group A’and B’were sensitized at the 1st and 14th day with 0.2ml sensitized solution(20μg OVA and 2mg Al(OH)3 were solute in 0.2ml PBS) intraperitoneal injection. At the 21st day, all the mice were put in a transplant closed glass container(30cm×18cm×13cm) and inspired aerosol of OVA PBS,20min, qd, lasting 5 days. The mice of group C’ were treated with sterile-distrilled-water at the same dose intraperitoneal injection. And they were not excited.Isolation of splenocytes We collected all the spleens, which were obtained from the mice of group 1’,2’, A, B, A’, B’and C’, and grinded with 300 mesh sieve. Then the splenocytic suspensions were centrifuged at 1700xg lasting 7min. The supernate was cast off. Then RCLB,10ml, was added to the solution. Put it aside for 4min. Then the solution was centrifuged at 1700×g again, lasting 7min. The supernate was cast off. The splenocytie suspension was acquired. To adjust the cell count to 1×106/mL with RPMI-1640(including 10% FBS) fluid.Isolation of CD4+ T cells 90μl buffer(PBS, including 5g/L OVA and 2mM EDTA) was added to the splenocytic suspension for every 107 cells. Then 10μl CD4+ magnetic beads were added to the solution. And the solution was incubated in 4-8℃for 15min and resuspended with 500μl buffer. The MiniMACS was installed. The splenocytic suspension was pass the separation column. And then it was rinsed 3 times with 500μl buffer. Then it was put on the suitable collection tube. And 1 ml buffer was added to the separation column. With the matching inner core, we obtained CD4+T lymphocytes in the collection tube.Antibody marking PMA(50μg/L), ionomycin(1mg/L) and monensin(2mg/L) were added to the acquired lymphocytes. Then stimulated cells were cultured for 4 to 6 hours. Then all the cells were collected and centrifuged at 7000×g lasting 5min and they were cultured in dark at room temperature for 10min after processed with 10ml FixandPerm. Then the direct monoclonic antibody of IL-4-PE was added to a half of the cells and the direct monoclonic antibody of IFN-y-PE was added to the other half of the cells. Both of the cells were cultured in dark at room temperature again for 30min. Then the cells were washed 1 time and prepared for detection.Flow cytometry(FCM) We assayed the lymphocytes which could secreted IL-4 and IFN-y in the acquired 20,000 lymphocytes with FCM. The wave of H+ excitation light was 400nm. The negative control was the same cells stained by IgG for every fluorescent channel. We calculated the percentage.Serum levels of OVA-specific-IgE With the technology of ELISA, we calculated the serum level of OVA-specific-IgE in all the groups.Pathological changes of the lung Methanal(40g/l)-fixed paraffin embedded lung tissue of all groups were stained by HE and observed the pathological changes by light microscopy.Results1. To assess the functions of La1, we firstly compared the levels of IL-4 and IFN-y in the offspring of La1- treated BALB/c pregnant mice to those in water-treated pregnant mice. The percentages of IL-4 and IFN-y in the offspring of La1- treated pregnant mice by 2 to 3 weeks did not show significant difference compared with the water-treated mice(p>0.05;and p>0.05).2. After 4 weeks of administration of La1, the percentage of IL-4 did not show significant difference (p>0.05, compared with the water-treated mice). And the percentage of IFN-y did not show significant difference, either (p>0.05, compared with the water-treated mice).3. In the excitation of asthma, some of the mice died. So there were 6,5,7 mice in group A’, B’and C’, respectively. The percentages of IL-4 in the La1-treated mice and water-treated mice were significantly increased compared with that in the control, respectively (p<0.001 and p<0.001). On the other hand, the percentage of IL-4 in the La1-treated mice was significantly decreased compared with that in the water-treated mice (p<0.001). The percentages of IFN-yin the La1-treated mice and water-treated mice were significantly decreased compared with that in the control, respectively (p<0.001 and p<0.001). But the percentage of IFN-y in the La1-treated mice showed significant difference compared with that in the water-treated mice (p< 0.001). Compared with the control mice, the serum levels of OVA-specific-IgE in the La1-treated and water-treated mice were significantly increased(p<0.05, and p< 0.05). There was significant difference in the serum levels of OVA-specific-IgE between La1-treated mice and water-treated mice(p<0.05).4. By light microscopy, mastocytes, alveolar macrophages, eosinophils, lymphocytes and meutrophil were infiltrated under the airway epithelial cells in the lung tissue of group A’ and B’mice. And edematous submucous tissue, constricted lumen and increased airway secretion were seen by light microscopy. The pathological changes were more severe in lung tissue of group B’than that in group A’. The pathological presentation of lung tissue in group C’ was normal. The structure of the alveola was clear. And the mucous epithelium of the bronchus was intact.ConclusionStudies on the functions of the probiotics from the neonate to the adult were limited. In our present study, we observed that administration of La1 could not change the immune system in the neonate. And continuous administration of La1 after birth, the immune system showed no significant difference compared to the control. The ratio of IL-4 and IFN-γwas changed, just like in the water-treated group. We could draw a conclusion that administration of La1 could not change the immune system from the pregnancy. On the other word, administration of La1 was safe in the pregnancy, especially to the fetus. But in the asthma model, administration of La1 could remarkably decrease the serum level of OVA-specific-IgE. And it could decrease the percentage of IL-4 and increase the percentage of IFN-γin CD4+ T cells in spleen, indicating a new treatment to the bronchial asthma. As we have known, the function of T cells is immature in the neonate. In our study, the percentages of IL-4 and IFN-ywere lower after birth than those in the adult. In the neonate and in the adult, the percentages of IL-4 and IFN-γdid not show significant difference in La1-treated mice compared with water-treated mice. But in other studies, it was shown that the matured B lymphocytes and monocytes were increased.The bronchial asthma is a chronic inflammation of airway hyperresponsiveness. The imbalance of Th1/Th2 could play an important role in the pathogenesis. In our asthma model, the percentage of IL-4 and the serum level of OVA-specific-IgE were increased and the percentage of IFN-γwas decreased in water-treated mice. The result was the same to the pathogenesis of asthma that we had known. The percentage of IL-4 was increased in La1-treated mice compared with the control. But the percentage of increase was lower than that in water-treated mice. It showed significant difference. The same result happened in the serum levels of OVA-specific-IgE. Both of results could demonstrate that administration of La1 could decrease the functions of Th2 lymphocytes. On the other hand, the percentage of IFN-γwas decreased in La1-treated mice compared with the control. But the percentage of decrease was lower than that in water-treated mice. It showed significant difference. This could demonstrate that administration of La1 could enhance the functions of Th1 lymphocytes.In summary, the administration of lactobacillus johnsonii Ncc533 (La1) had no effects on the immune system from the neonate to the adult in the normal mice. But in the asthma model, administration of La1 could significantly changed the percentages of IL-4 and IFN-γin the CD4+ T lymphocytes. These data may have potential impacts on clinical application of La1 in the treatment of patients with allergic disease, such as bronchial asthma. |
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