The Relationship Of Changes Of GR Activity And Hsp90 After Acute Lung Injury

Stress is an adaptation reaction of organism to resist trauma excitation.Glucocorticoid receptor(GR) is one of the most important molecular among the anti-stress signals.Hsp90, which is the essential molecular chapone of GR,acts as a molecular switch on GR function. In physiological conditions,Hsp90 can change the stereo-conformation of GR,and emerge its ligand-binding domain as a pocket to make GR at the stand-by condition,preparing to bind glucocorticoid(GC).In the molecular chapones,Hsp90 acts as a crucial regulator. GR need to bind it to gain the high GC affinity;without Hsp90,the LBD of GR will collapse and the binding efficacy is very low.Some researchers propose,it is possible that there are two kinds of GR in cells" one has high affinity(GR_H),and the other has low affinity(GR_L).The two receptors have different molecular weight and subcellular distribution.When the GR_H is decreased significantly by stress,GR_L act as the main receptor to create the protection effects.But this idear hasn't been confirmed for a long time,so we have the different presumption:it is possible that there is only one type of GR,which has one molecular weight and three-dimention structure,but it can construct different cytoplasm complex.One is the high affinity complex,which has certain composition,and the other is the low affinity receptor, which hasn't.It is confirmed in many models of injury that the binding affinity of GR is decreased sharply after trauma.But right now,there are many opinions on the reason of this phenominon.In literature,This is maybe the reason of why GR protein level sharp reducing. And the decreased affinity of GR,which generates stress intolerance,can form shock syndrome in the early period of severe injury.Some studies show,that a positive modulation will be the result of an optimal steroid receptor/Hsp90 ratio,whereas abnormally low or high ratios will negatively interfere with steroid-dependent responses.It is the possible key point of fine therapic affect that haw to regulate the ratio of these two molecular.If these hypotheses are real,we can use rational methods to accommodate the expression of Hsp90 in cytoplasm,and it's possible to correct the disproportion of Hsp90/GR ratio,increasing the "GR_H".For this reason,we designed this experiment to detect GR protein level using Western blotting and to detect the GR binding affinity using radioligand binding assay,trying to analysis the rule of GR regulation after stress.On the base of mouse model of oleic acid lung injtiry,we use hyperthermic stress and somatostatin to change the levels of Hsp90 and Hsp70,and initially demonstrate that GR must form complex with Hsp90 to get the high affinity of ligand binding,or it is the low affinity receptor.At the same time,we investigated the mechanism of Hsp90 on regulation of GR ligand binding affinity.The main results were as follows:1.We inject oleic acid(OA) through tail vein of mouse to produce acute lung injury models,and it showed typical appearance of lung edema by the general observation, pathology observation,arterial blood gas analysis,lung body index and lung water ratio.2.Western blotting assay shows that the protein levels of Hsp90 and Hsp70 increased significantly in lung cytoplasm of animal models.Although GR protein level increased transiently on 1h point,the level of GR decreased significantly on 3h,12h and 24h.The radioligand binding assay shows that binding capacity(B_max) of GR and ligand binding affinity decrease significantly.B_max on 3h and 12h after ALl decreases significantly compairing with NS control and B_maxOn 24h increases slightly which is still significantly higher than NS control.The Kd of GR is increased on 1h,but it has no significancy with NS control,then the Kd increases significantly on 3h,12h and 24h.3.Before injection of oleic acid,we used hyperthermic stress(44℃1h) for pretreatment.We notice that the geneal feature,pathology,arterial blood gas,LBI and RLW are improved comparing with OA vulnarate group in mice.The protein expression of GR, Hsp90 and Hsp70 is increased on all phases comparing with OA group.But the B_max and ligand binding affinity of GR have no significancy with OA group.4.In the third part of this paper,we used somatostatin for pretreatment.The indexes of above are improved comparing with OA group.Somatostatin could increase Hps90 level specifically,and had no effect on Hsp70.So we can conclude that Hsp90 is the essential component of regulating GR ligand binding affinity.The above experimental results show that in OA acute lung injury mouse models,GR ligand binding affinity and protein level decreasing are the main reason of intolerance of injury;hyperthermic stress can induce the synthesis of Hsps,and increase the protection affect of GR;and Hsp90 is the essmponent of this mechanism.