| ObjectiveDiabetes mellitus (DM) is one of the endocrinous metabolic disorder syndrome. The major biochemical character of DM is continuously high blood glucose. Gradually, the diabetic patients will develop complications of microangium and major vessels diseases, which may be fatal. The pathogenesis of DM vessel complications are currently thought to be related to some growth factors and vasoactive factors. The pathogenesis of the chronic complication is complex. There are hypotheses including protein nonenzymatic induced by hyperglycemia, abnormal pathway of polymeric alcohol and PKC signal pathway. Studies before demonstrated that hyperglycemia-PKC-MAPK pathway might be involved in the pathogenesis. In this study, we tried to provide some evidence of diabetic complications in lung by observing the pathologic changes, examining PKC activity and investigating the alteration of P38 MAPKinase.Methods1.Laboratory animals: 36 male Sprague- Dawley (SD) rats (150-170g) provided by Laboratory Animal Center of China Medical University, were randomly assigned into control and DM groups.2.Diabetic model was induced by a single intraperitoneal injection of streptozotocin (60mg/kg). The rats of control group were injected with equal volume of saline. Diabetes was defined when 2 days later as tail bloodglucose concentrations>16.7 mmol/L using the Otoluidine method. The rats were free to water and standard rat chow for 4 weeks. After that, all rats were anesthetized with pentobarbital and four pieces of lung tissues were removed from left lung of each rat,and processed for following studies .One was snap-frozen in liquid nitrogen and stored at -70 for enzyme assay. The other three were fixed in 0.4% polyformaldehyde/0.1%DEPC solution for in situ hybridiz-ation and immunohistochemical analysis staining.3.Special staining and HE routine stainingReticular fibers were detecteb with Gomori silver staining method. Collagenous fibers were detecteb with Weigert's resorcin=fuchsin staining method.4.PKC activities in lungs were examined by isotope method.By Takay method, tissues of lung were melted in grind buffer and ultrasonicated. Then, some supernatant fluid were taken into ATP reactive fluid, kept at 30癈 for 8 minutes, mixed with 75mmol/L PBS, washed for three times. Finally numbers were measured with liquid scintillation counter.5.Immunohistochemical staining.MMP2, MMP9,NF-B TGF-1 Staining was performed with polyclonal rabbit anti-rat antibodies of MMP2 MMP9, NF-B TGF-1 .Biotinylated goat anti-rabbit IgG was used as secondary antibody. Cryostar section were fixed in cold acetone for 10 min. Sections were then blocked with0.1% normal rat IgG to prevent nonspecific antibody staining of the secondary antibody. Sections were then incubated with primary antibodies for 1h at25. Polyclonal rabbit anti-TGF-1 ( 1:100 ) was used, After washing with phosphate-buffered saline(PBS),the secondary antibody, biotinylated rat anti-rabbit IgG(1:50), was applied to sections for 30 min.6. In situ hybridization.MAPKinase P38 oligonucleotide probe was labelled by digoxin, the sequence of which is 5'-GTGTG CCGAG CCAAA ATCCA GAATC-31. The procedure was performed using the method of Milani et al,with minor modification. For each tissue section, observers assessed the hybridization signals.7.NF- B, TGF- 1 levels analysed by Western blotting Westtern blot analysis of NF-B ,TGF-1 lung tissue pieces were snap-frozen in liquid nitrogen and stored at-80 .Proteins were extracted from lung samples after homogenization with RIPA solution (Santa CruzCA )and assayed by the Lowry method. Finally, membranes were incubated with enhanced chemoluminescence blotting substrate and exposed to X-ray film. Band intensity was measured by Leica Q500IW image analysis system.Results1 .Pathologic changes of lung tissues1.1 HE routine staining: thickened alveolar epithelium and pulmonary capillary basal lamina and proliferation of fibre in pulmonary interstitium were found.1.2 Collagenous fibre: in DM group, ther... |
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