The Effects Of A1 Adenosine Receptor And The Underlying Neuroprotective Mechanism On Intracerebral Hemorrhage-induced Secondary Brain Injury In Rats

Objective: Adenosine,exactly the endogenous adenine nucleoside,mainly generated from AMP by 5'-nucleotidase,has been found widely distributed in the body and mediates many physiological functions,it affects the pathological and physiological process of different cells.Adenosine plays a variety of enzyme reaction and cell activities as a cofactor,and binds to its receptors to achieve its biological effect.Adenosine receptors(ARs)is one of the members of G protein coupled receptors,including four subtypes: A1,A2 a,A2b and A3.ARs are widely expressed in the brain tissue.Previous studies have shown that adenosine was a kind of the major important endogenous neuromodulators in the central nervous system,when intracerebral hemorrhage(ICH)happened,adenosine was released a lot.So this study was designed to observe the changes and determine the role of the adenosine receptors in ICH-induced secondary brain injury(SBI)and the underlying mechanisms.Methods: ICH model was established in Sprague-Dawley rats with the collagenase,and to mimic ICH in vitro,cultured primary rat cortical neurons were exposed to oxyhemoglobin at a concentration of 10 ?M.Firstly,the rats were killed at the different time point(6h,12 h,24h,48 h,72h and 1 week)after ICH and cerebral samples were collected for analysis.Western blot analysis in vivo and in vitro was applied to observe the changes of protein level for each subtype of adenosine receptors after ICH,immunofluorescence staining for confirmation.The subtype receptor changed significantly was selected for the the next experiment(the result showed in this tudy A1 adenosine receptors expression level changes significantly.The A1 adenosine receptor agonist N(6)-cyclohexyladenosine and antagonist 8-phenyl-1,3-dipropylxanthine were used to for further studies,after that western blot analysis was performed to detect the changes in the expression of albumin and active caspase-3,terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)and Fluoro-Jade B(FJB)were used to evaluate the neuronal death and degeneration,finally brain water content was also examined and these tests were all aimed to study the role of A1 adenosine receptor in the secondary brain injury following ICH.In order to study the possible underlying mechanisms by which A1 adenosine receptor attenuated SBI after ICH and whether the MAPK family participated in this process,the activation of p38,ERK and JNK was examined by western blot.Then we used antagonists of p38(SB203580)and Hsp27(KNK437)to further investigate the role of P38/MAPKAP2/Hsp27 in the action of A1 adenosine receptor.The phosphorylation levels of proteins in P38/MAPKAP2/Hsp27 pathway were tested by western blot analysis,and TUNEL and FJB studies were performed to investigate the condition of neuronal death and degeneration in the presence of these antagonists.Finally,in order to further confirm this underlying mechanism,Evans blue(EB)was also used to assess permeability of the blood-brain-barrier(BBB)after after blocking p38 or Hsp27.Results: The expression of A1 adenosine receptor was significantly promoted by ICH,while there weres no significant changes in the protein levels of the other 3 adenosine receptors.In addition,the protein level of A1 adenosine receptor could be increased by the agonist N(6)-cyclohexyladenosine and decreased by antagonist 8-phenyl-1,3-dipropylxanthine under ICH conditions.Activation of the A1 adenosine receptor showed a significant neuroprotective effect,attenuated neuronal degeneration and apoptosis in the subcortex,characterized by decreased TUNEL and FJB positive numbers and reduced protein level of active caspase 3 albumin,which was associated with increased phosphorylation level of P38,MAPK,MAPKAP2,and Hsp27.Inhibition of the A1 adenosine receptor resulted in the opposite effects,enhanced apoptosis of neurons and brain damage.Finally,in the study of the possible mechanisms,the experimental results showed that the neuroprotective effects of the A1 adenosine receptor agonist N(6)-cyclohexyladenosine was suppressed by antagonists of P38 and Hsp27,revealed the potential mechanism of neuroprotective effects of adenosine A1 receptor was related to activated P38-MAPKAP2-Hsp27 pathway.Conclusions: This study demonstrates that the expression of A1 adenosine receptor was increased by ICH.Activation of the A1 adenosine receptor by its agonist N(6)-cyclohexyladenosine could attenuate ICH-induced secondary brain injury via the activation of P38-MAPKAP2-Hsp27 pathway.