Lung Qi Deficiency Model Rats Mediated Tnf Alpha/p38mapk Signal Transduction Pathways Impact On Renal Tissue Aqps Expression

Purpose:The experiment intended application of molecular biology to observe the lung qi deficiency syndrome in rat kidney organization AQP1and AQP2expression. The same time, through studying the two kinds of blockers that TNF-α and p38MAPK of the affection on AQPs. Further explore the process of water metabolism by lung and kidney in Traditional Chinese Medicine, and effect on the molecular mechanisms of action, aims to provide a experimental evidence for treatment the clinical nephrotic edema through the lung.Material and method:Sixty male Wistar rats in SPF were randomly divided into six groups:normal control group (Group A), lung qi deficiency model group (Group B), etanercept intervention control group (Group C), etanercept intervention model group (Group D) SB203580intervention control group (Group E) SB203580intervention model group (Group F)(n=10).1. Model replication method:The rats of lung qi deficiency (B、C、D、E、F) were simulated by injecting lipopolysaccharide into the trachea twice together with cigarette smoking. The same time, based ou C、D、E and F,further impose different processing factors: group C from the first day, given a subcutaneous injection of0.9%NaCl injection0.5mg/kg every other day; group D from the first day (1h after sensitization, or excitation), given subcutaneous injection of TNF-alpha blockers (etanercept)0.5mg/kg every other day; group E intraperitoneal injection of0.9%NaCl (3mg·kg-1·d-1); group F p38MAPK blocker (SB203580) dissolved in dimethyl sulfoxide,3mg·kg-1·d-1by intraperitoneal injection.2. Used lung function analysis system for small animals to test FVC、Ri and Re.3. Enzyme-linked immunosorbentassay was used to measure the level of TNF-a and IgG in serum.4. RT-PCR was used to measure the expression of AQP1、AQP2gene in renal tissue.5. Western blot and Immunohistochemical was used to measure the protein contents of p-p38MAPK、NF-κB、IκB-αand CK2in renal tissue.6. All the date analyzed with GraphPad Prism5software. The result were expressed as One-way ANOVA was employed in the group comparison, The statiscal signifieance is defined as P<0.05.Results:1. Evaluation of lung qi deficiency model1.1Lung tissue morphological observation:normal lung tissue alveolar clean and uniform, bronchial epithelial cells were complete, as shown in scattered, small amount of inflammatory cell infiltration, glandular no hyperplasia, alveolar wall was no apparent hyperplasia. Lung tissue in the lung qi deficiency state alveolar expand,was presentd as emphysema, Bronchial epithelium can be see a large number of goblet cells. Epithelial discontinuous, the basement membrane hypertrophy and lots of inflammatory cells infiltration in the lumen.1.2Weight:modeling to14days, each group weight were progressively increased. Compared with group A, the weight of B、C、E increased has slowed (P<0.01). Modeling to28days, each group weight were still progressively increased. Compared with group A, the weight of B、C、E increased were slow down(P<0.01); compared with group C, group D weight slightly higher(P<0.01); compared with group E, group F weight slightly increased (P<0.01).1.3Urine output and urine specific gravity:Modeling to28days, compared with group A, the urine output of B、C、E reducrd obviously, urine specific gravity increased obviously (P<0.01); compared with group C, group D urine output increased and urine specific gravity reduced(P<0.01); compared with group E, group F output increased and urine specific gravity increased(P<0.01).1.4Pulmonary respiratory function change:modeling to28days,①Compared with group A, FVC of B、C、E diminished obviously(P<0.01); compared with group C, group D FVC increased obviously(P<0.01); compared with group E, although group F was rebounded, but the compared results were not statistically significant(P>0.05).②Compared with group A, Ri of B、C、E increased obviously(P<0.01); compared with group C, group D Ri reduced obviously(P<0.05); compared with group E, group F Ri reduced also obviously(P<0.01);③Compared with group A, Re of B、C、E increased obviously(P<0.01); compared with group C, group D Re reduced obviously(P<0.05); compared with group E, although group F was rebounded, but the compared results were not statistically significant(P>0.05).1.5Inflammatory target:modeling to28days, compared with group A, TNF-α of B、C、 E increased obviously(P<0.01); compared with group C, group D TNF-α reduced obviously (P<0.01); compared with group E, group F TNF-α reduced obviously(P<0.01).1.6Immunization target:modeling to28days, compared with group A, IgG of B、C、E reduced obviously(P<0.01); compared with group C, group D IgG increased obviously (P<0.01); compared with group E, group F IgG increased obviously(P<0.01).2. kidney tissue AQPs expressions in lung qi deficiency model ratsThe gene and protein expressions of kidney tissue AQP1and AQP2:modeling to28days, compared with group A, the gene and protein expressions of AQP1and AQP2of B、C、 E increase obviously(P<0.01); compared with group C, The gene and protein expressions of AQP1and AQP2with group D reduced obviously(P<0.01); compared with group E, The gene and protein expressions of AQP1and AQP2with group F reduced obviously, too (P<0.01).3. Kidney tissue protein contents of p-p38MAPK、NF-κB、IκB-α and CK2in lung qi deficiency model rats3.1P-p38MAPK expression in renal tissue:modeling to28days, compared with group A, p-p38MAPK of B、C、E increase obviously(P<0.01); compared with group C, group D p-p38MAPK reduced obviously(P<0.01); compared with group E, group F p-p38MAPK reduced obviously,too(P<0.01).3.2The expressions of NF-κB and IκB-α in renal tissue:modeling to28days, compared with group A, NF-κB of B、C、E increase obviously, IκB-α reduced obviously (P<0.01); compared with group C, group D NF-κB reduced obviously, IκB-α increase obviously (P<0.01); compared with group E, group F NF-κB reduced obviously, IκB-α increase obviously.(P<0.01).3.3CK2expression in renal tissue:modeling to28days, compared with group A, CK2of B、C、E increase obviously(P<0.01); compared with group C, group D CK2reduced obviously(P<0.01); compared with group E, group F CK2reduced obviously,too(P<0.01).Conclusion: 1. The weight of lung qi deficiency rat model increased slowly; urine output decreased and urine concentration increased; lung respiratory function declined; in plasma TNF-a content increased and IgG content declined obviously.2. Protein and gene expressions of AQP1and AQP2in lung qi deficiency syndrome rat model renal tissce were increased obviously. After intervention by etanercept and SB203580, protein and gene expressions of AQP1and AQP2in lung qi deficiency syndrome rat model renal tissce were reduced obviously.3. Kidney tissue protein contents of p-p38MAPK、NF-κB、IκB-α and CK2in lung qi deficiency model rats were increased obviously, IκB-a reduced obviously. After intervention by etanercept and SB203580, protein contents of p-p38MAPK、NF-κB、CK2reduced obviously, IκB-α increased obviously.