| ObjectiveLung as one of diabetes target damage organs has not been drawn attention until 70's of this century. The damages widely involve lung function, biochemistry metabolism, pathology and so on .Lung local defense function deficit leads to the respiratory infect is common. Past research indicated that diabetes is the correlated dangerous factor which increases incidence and mortality rate of pneumonia caused by bacterium, virus and other pathogens. The alveolar macrophages (AMs)widely distributes in the pulmonary alveolus and the bronchial tube surface, which is the earliest and most often contact with antigen immune cells ,compose the first defense line of the lung. AMs can eliminate bacterium, virus, fungus more effectively compared to other cells. The change mechanism of alveolar macrophage as the tissue specificity at present is not clear. This article duplicates diabetes animal model, AMs were acquired by the bronchial alveolar lavage, from different course of the illness and determine and the release quantity of cytokine IL-1β,IL-12,TNF-α; and NO ,further observes the insulin treatment and in vitro high glucose culture effect.Materials and Methods1. MaterialsStreptozotocin(STZ) IL, rat IL-1β, IL-12, TNF-αELISA kits, NO kits, Middle Recombinant human Insulin, Blood Sugar Sensor.2. Methods Sixty healthy male wistar rats were randomly divided into 5 groups (12 per group): control group, DM 4 week group, DM 8 group, insulin treatment group, and a high glucose in vitro incubation group. Diabetes rats were received 60mg/kg STZ dissolved in ice Citric acid-Sodium citrate buffer (ph4.2) after 16 hours starvation, control rats received intraperitioneal injection of equivalent volumes of sodium citrate buffer. The high glucose in vitro incubation group rats were performed bronchoaveolar lavage directly, and the acquired AMs were cultured in RPMI 1640 medium contain 30 mmol/1 glucose.Preparation and culture of AMs: The rats were anesthetived and bronchoaveolar lavage (5ml×6) was performed, and the BALF incubated for 2 hours and purified AMs were obtained. Culture supernants were measured by specific ELISA kits for cytokine IL-1β,IL-12,TNF-α, and quantify the NO release with Griess method Observe the cock RBC phagocytosis rate of in vitro AMs with HE method.Statistical analysis of data was conducted using SPSS11.5 software .Data were expressed as mean±standard deviation, Statistical analysis between groups was performed using one-way analysis of variance (ANOVA) test .For multiple compare to the same control group followed by the test of Dunnett-t. P value less than 0.05 were considered to be statistically significant. Phagocytosis rate compare was performed using chi-square test.ResultsThe cock RBC phagocytosis rate of DM 4 and 8 weeks group (7.25±2.49%,5.25±1.83%) decreased significantly compared with the control group (9.63±2.20%, P <0.01) ;In vitro cultured AMs of DM 4 weeks group secreted less IL-12 (59.48±2.08pg/ml) than the control group (75.28+2.88pg/ml, P<0.01) ;The DM 8 weeks group secreted less IL-12 , TNF-α(66.38±3.12pg/ml,350.29±70.10pg/ml,respectively)than the control group(75.28±2.88pg/ml,475.72±92.6pg/ml respectively, all P<0.05) ;The content of IL-1βin DM 8 weeks group increased significantly(96.46±8.86pg/ml)compared with the control group(76.60±5.00 pg/ml, P<0.01) ,The 4 weeks group and the 8 weeks group released more NO (32.46±3.16μM,28.57±4.73μM ,respectively) than the control group (5.50±0.63μM, all P<0.01) ;Insulin therapy has the trend of recovering the changes but the differences between insulin treatment group and control group were not significant in statistics.Conclusions1.The cock RBC phagocytosis rate of DM rats decreased significantly compared with the control group, the changes are more obvious with the time prolonged.2.AMs from diabetes mice showed reduced release of IL-12 , TNF-α.3.IL-1β,NO release of AMs from diabetes mice is higher than control mice.4.Insulin treatment to DM 4 weeks improved the damages but the recovery was partial.5.AMs from the high glucose in vitro incubation group have the same trend of changes, but are not so obvious as the diabetes groups. |
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