| Objective: To explore the relationgship between pulmonary fibrosis in DM and heme oxygenase-1(HO-1) through detecting the change of HO-1 in lung tissue with diabetic rats, simultaneously, provide experimental data for therapying damage to lung in DM through observing the effects of rosiglitazone on pulmonary fibrosis and its influence to expression of heme oxygenase-1 (HO-1) in the lung tissue of diabetic rats.Methods: The experimental type 2 diabetic rats were established by low dose streptozotocin(STZ) injection and high fat diet. The SD rats were randomly divided into three groups: normal control, diabetic group and experimental group (treated with rosiglitazone), blood biochemical parameters and hydroxyproline were compared at weeks 10 and 24. The morphological changes of diabetic rat lung histopathological changes were examined by light microscope and transmission electron microscope. The site and level of expression of HO-1 protein were examined by immunohistochemistry staining and western blot . The level of HO-1 mRNA was examined by RT-PCR.Results:1. Symptoms and SignsThe diabetic rats occurred polyuria, polydipsia after injecting STZ. As the extention of disease, symptoms aboved worsened, together with trichoxerosis, faint politure, blunt response and decreased activity. Compared with the control group, there was no significance in the two groups although the body weight of diabetic rats decreased slightly. Also there was no obvious improvement of symptoms above-mentionded in the rosiglitazone group.2. Glucose clamp experimentThe level of GIR in control group is 10.93±0.59mg/kg/min, meanwhile, the level of GIR in the DM group is 5.89±0.43 mg/kg/min. The level of GIR in diabetic rats was significantly lower than control group(P<0.01), indicating that the DM rats existed insulin resistance. The experimental type 2 diabetic rats were established successfully.3. The change of biochemistry markersThe level of fasting plasma glucose, insulin, cholesterol in the DM group at 10 week were higher than those in control group(P<0.05). The level of fasting plasma glucose, fast insulin, triglyceride, cholesterol and HbA1c were significantly increased in DM group at the end of 24 week, compared with control rats(P<0.01). Compared with the diabetic rats at the end of 10 week, the level of fasting plasma glucose, fast insulin, triglyceride, cholesterol were significantly increased in DM group at the end of 24 week(P<0.01). Compared with those in the DM group, fasting insulin, cholesterol and triglyceride in the rosiglitazone group decreased significantly(P<0.01), but plasma glucose and HbA1c decreased slightly.4. Determination of rat tail blood pressureThe tail blood pressure in the DM group at 10 and 24 week were higher than that of the control in the corresponding time period(P<0.01). Compared with that in the DM group at 10w, the tail blood pressure in the 24w DM group was increased slightly, but had no statistical difference.5. Determination of hydroxyproline in lung tissueConcentrations of hydroxyproline in the DM group at 10 and 24 week were higher than that of the control in the corresponding time period(P<0.01). Concentrations of hydroxyproline was significantly increased in DM group at the end of 24 week, compared with diabetic rats at the end of 10 week(P<0.05). Compared with that in the DM group at 24w, the level of hydroxyproline decreased markedly in the rosiglitazone group(P<0.01).6. Masson stainingThere was jot fibroglia fibrilses between interstitial substances, extra-cellular matrixes circum-vasa in the control. At 10w, fibroglia fibrilses between interstitial substances, circum-vasa increased and distributed deranged, facio-density of collagen of lung in the DM was 0.1417±0.0020, was much high than that of control in the corresponding time period 0.0044±0.0007. Changes above-mentioned aggravated, the organism structure of lung distributed deranged, alveoli of lung were atrophied at 24w, even replaced by fibroglia fibrilses. Facio-density of collagen of lung in the DM at 24w was 0.2086±0.0007. Facio-density of collagen of lung in the rosiglitazone was 0.1212±0.0023, accumulation of collagen of lung tissues abatemented significantly to normal.7. Hematoxylin and eosin stainingAlveoli of lung composed with monolayer alveolar epithelium and basilemma and distributed uniformity, there were jot connective tissue between consecutive alveoli of lung of the control in the light microscope. The organism structure of lung in the DM rats distributed deranged, bronchus wall and alveolar wall were thicken, alveolar epithelial cells were indistinct, alveoli of lung were atrophied and collapsed. There were more interstitial substances, extra-cellular matrixes circum-vasa and desmocytes companied with inflammatory cell infiltration. Pathological alterations above-mentioned in the DM rats were more significant at 24w. The pathological changes of lung in the rosiglitazone group lessened obviously than the DM group, the bronchus wall and alveolar wall were thinner, but the organism structure of lung had not recovered to nomal.8. ImmunohistochemicastryThere were faint positive expression of HO-1 in alveolar macrophages and interstitial lung infiltrates the cytoplasm of inflammatory cells in the control. The expression of HO-1 in the lung tissue in the DM group increased slightly and was stained shallow at 10w, the staining optical density value was 0.1048±0.0691, which had no statistical difference in the corresponding time period 0.1100±0.0154. The positive staining optical density value in the DM at 24w was 0.1210±0.0021, which was lower than that of the control in the corresponding time period 0.1235±0.0026, but had no statistical difference. The positive expression of HO-1 in tissue of lung in the rosiglitazone group was 0.2940±0.0041, stained shallow, which was more than that of the control and DM group.9. Changes of transmission electronmicroscopeThere were a lot of microvilli in the typeⅡpenumonocyte, osmiophilic multi1amellar body mitochondria, rough endoplasmic reticulum, free ribosome in cytoplasm of the control rats. At 10w, the number of osmiophilic multi1amellar body mitochondria and microvilli in the typeⅡpenumonocyte induced, fibroglia fibrilses increased slightly, connective tissue proliferated, together with the edema of cytoplasm. Pathological alterations above-mentioned in the DM rats were more significant at 24w. In rosiglitazone group, the alterations above-mentioned lessened obviously than the DM group.10. The expression of HO-1mRNAThe expression of rosiglitazone group(0.94±0.02) was significantly higher than that of control group(0.52±0.01) and DM group(0.51±0.01) (P<0.01). Results of comparison between the control group and DM group showed that there was no significant difference respectively.11. Western blot analysis for HO-1 expressionThe expression of HO-1 protein in rosiglitazone group (0.85±0.029) was significantly higher than that of control group(0.43±0.007) and DM group(0.43±0.040)( P<0.01). Results of comparison between the control group and DM group showed that there was no significant difference respectively.Conclusions:1. Hyperglycemia in rats could be caused by feeding high fat food and injecting low dose streptozotocin(STZ), and then the GIR in glucose clamp experiment was conspicuously declined, which indicated a successful model of type 2 diabetes rats. The morphology changes in lung tissue could be found by light microscope and transmission electron microscope. Collagen accumulation in lung tissue could be tested by Masson staining and the determination of hydroxyproline in lung tissue , which indicated that the lung was also a target organ of diabetes, while pulmonary fibrosis was one of the main morphologic changes.2. The pathological alteration of tissue in lung in rosiglitazone group mitigated obviously, accumulation of collagen abatement, the expression of HO-1 increased, which indicating the intervention of rosiglitazone can markly reduce the procession of pulmonary fibrosis in diabetic rats. The mechanism is, at least in part, through the induction of HO-1. |
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