The Regulatory Role Of BAG-1—GR Signal Pathway On Anti-inflammatory Effect Of Glucocorticoids

Acute lung injury/acute respiratory distress syndrome(ALI/ARDS)is a common respiratory failure clinically characterized as acute hyoxemia, the pathogenic basis is reflections of systemic inflammatory response syndrome(SIRS) in lung, which is caused by various severe injuries,especially trauma and infection etc, and finally results in diffuse injuries of terminal breathing unit and exudative pneumonedema. Until now there is still no effective therapeutic principles of ALI/ARDS and the mortality reaches as high as 50-70%. Therefore, the mechanism for occurrence of ALI/ARDS has not been clarified. Some researchers believe that it might be associated with the uncontrolled development of SIRS. If controlling systemic inflammatory response promptly and correctly, We could depress the mortality of ALI/ARDS effectually. Glucocorticoid is one of the most popular medicine used to cure ARDS,and the executant of the GC effect is glococorticoid receptor(GR). Though GC have protective effects on lung, therapeutic efficacy of long-term in remedying ARDS patients is not optimistic, GC therapy can not depress incidence and mortality of ARDS. Recently,it was reported that there exists GC resistance in ARDS patients, the mechanisms are not clear, probably concerned with down-regulation of GR expression and (or) depression of GR functions. So it is very valuable for us to deeply explore the change and regulation of GR in SIRS/ALI rats, reveal mechanism of GC resistance. Methods 1. Healthy wistar rat were randomly divided into three groups: LPS group, LPS+Dex group and LPS+Dex+Dex group, which was further distributed into control, 1h, 2h, 4h, 8h, 12h and 24h time ponits. RIA, reverse transcription polymerase chain reaction (RT-PCR), Western blot, electrophoretic mobility shift assays (EMSA) were employed to examine serum corticosteroid, lung GR mRNA and GR expression and GR DNA binding activity. 2. LPS+Dex treated rats was randomly distributed into control, 4h, 8h and 12h group, Western blot was employed to examine the changes of Bcl-2-associated athanogene 1(BAG-1) expression in total protein and nuclear protein of lung tissue; Immune coprecipitation experiment was used to detect the interactions between BAG-1 and GR in vitro. 3. Rat alveolar macrophages (AMs) were isolated and cultured. AMs were divided inio three groups: LPS group, Dex group and LPS+Dex group, immunocytochemical stain and Western blot were employed to examine BAG-1 expression; AMs were transfected with BAG-1 eukaryotic expression plasmid pcDNA3.1-BAG and RNA interference expression plamid psiBAG-Ad-2, then BAG-1 overexpression and knockdown models of alveolar macrophages were established.. GRE transcription report system and ELISA were employed to examine TNF-αcontents in supernatant and GR transcriptional activation activity. Results 1. The contents of plasma corticosteroid significantly increased in the early injury stage of LPS induced SIRS rats, and reach the peak at 4h after injury, then decreased gradually, but still higer than that of normal control at 24 h; The expression lever of GR mRNA decreased, and returned to normal lever at 24 h after LPS treatment. The expression lever of GR also decreased, reached the lowest at 8 h, but still lower than that of normal control at 24 h. GR DNA binding activity decreased, and reached the lowest lever at 1 h, but still lower than that of normal control at 24 h. 2. GR DNA binding activity slightly enhanced at 1h after LPS+Dex treatmeant, then decreased at 2h, reached the lowest at 24h; GR DNA binding activity of LPS+Dex+Dex treatmeant group had no obvious exogenous differences versus LPS+Dex treatmeant group. 3. The BAG-1L expression in total protein of lung tissue significantly increased at 4h after LPS+Dex treatment, and maintained this lever at later time points; BAG-1S expression had no changes. BAG-1L not BAG-1S was detected in nuclear protein, and BAG-1L expression increased gradually in 12 h after the treatment. The immune coprecipitation experimen confirmed that there existed interactions between BAG-1L and GR after LPS+Dex treatment, and negative dependablities existed between BAG-1L expression and GR DNA binding activity. 4. Immunocytochemical stain indicated that BAG-1 expressio grains increased in kytoplasm of AMs after Dex treatment, and there were no changes after LPS treatment; But BAG-1 expressio grains significantly increased in nucelus after LPS+Dex treatment.. Also the consequences of Western Blot revealed that BAG-1L expression increased andBAG-1L translocated into nucleolus after LPS+Dex treatment. 5. BAG-1 eukaryotic expression plasmid pcDNA3.1-BAG and RNA interference expression plamid psiBAG-Ad-2 were successully constructed, and BAG-1 overexpression and knockdown models of AMs were established. The GR transcriptional activation activity was significantly inhibited in AMs of BAG-1 overexpression, and TNF-αcontents in supernatant significantly increased after LPS+Dex treatment; On the contrary, GR transcriptional activation activity was significantly increased in AMs of BAG-1 gene knockdown, and TNF-αcontents in supernatant significantly inhibited after LPS+Dex treatment. Conclusion 1. SIRS rats are in stringent state which displays high GC, low lever of GR expression and low DNA binding activity of GR, simultaneously endogenous GC resistance exists, which maybe associated with the decreased expression of GRH. 2. Exogenous GC can reinforce GR DNA binding activity transiently, which maybe associated with the activition of GRl. activity via exogenous GC; Exogenous GC resistance exists in 4h after LPS+Dex treatmeant, and is independent of GC contents. The mechanisms of GC resistance should be elucidated from other points of view further. 3. BAG-1L expression increases after LPS+Dex treatment, and BAG-1L coupled with GR translocates into nucleolus, which can result in the depression of GR DNA binding activity. This mechanism is probably one of the significant reasons causing glucocorticoid resistance. 4. BAG-1L expression increases and BAG-1L translocates into nucleolus after LPS+Dex treatment in AMs, which is basic condition of BAG-1-GR signal pathway actived. 5. BAG-1-GR signal pathway can negatively regulate GC anti-inflammationary effects. Repressing BAG-1L expression can eliminate GC resistance, which maybe helpful for recoving GC antiinflammationary effects...