The Study On Splenic Macrophage Polarity In Rats With Cerebral Ischemia

Dynamic changes of splenic macrophage polarity in MCAO rats (Part A)Background:Post-stroke infection is closely related to immune system imbalance,increased mortality and poor prognosis.Experimental studies show that the volume of spleen,the number of lymphocytes,the expression of HLA-DR,the level of TNF-a and IFN-? decreased,while the level of IL-10 increased at the early stage of cerebral ischemia.The macrophages showed different polarity in different micro-environment,M1 macrophages mainly promote inflammation response,while M2 macrophages exert anti-inflammatory effect,promote the repair of tissue injury.The peripheral M2 macrophages play the role of immunosuppression,M2 macrophages in cerebral ischemic area mainly play the role of tissue repair.The balance between peripheral immune system and inflammation in the cerebral ischemia is the key point of treatment of stroke and prevention of stroke associated infection.Therefore,to explore the splenic macrophage polarity changes in rats with middle cerebral artery occlusion(MCAO)is of great significance to further elucidate the mechanism of immunosuppression.Objective:To explore the dynamic changes of splenic macrophage polarization in rats with cerebral infarction.Methods:Male mature Sprague-Dawley rats were used for MCAO,and the animals were randomly assigned to the 6 experimental groups:sham operation group,MCAO 6h,12h,24h,48h,72h group.RT-qPCR was used to evaluate iNOS,Arg-1,YM1 and IL-10 mRNA levels of MCAO and sham-operated rats.Results:The high expression levels of iNOS and Arg-1 are used to distinguish between M1 and M2 macrophages.Compared with the sham operated group,iNOS mRNA levels in MCAO rats slightly decreased at 6h,at 12h began to increase,and reached to the peak at 24h,then drop to the levels of the sham operated group.The levels of Arg-1 mRNA in MCAO rats reached to the highest at 6h,then gradually declined,at 24h fell to the minimum,yet at 72h the levels still higher than the sham operated group.The expression of YM1 mRNA was no obvious change at both 6h and 12h,and then slowly rose,reached to a peak at 48h,but there was no significantly difference between MCAO groups at each point and sham operated group.The expression levels of IL-10 mRNA at MCAO 6h rose to the highest,and then steadily declined,at 24h fell to the bottom,increasing at 72h to higher than the sham operated group,whereas there was no obvious difference between MCAO 72h and sham operated group.Conclusions:In rats with MCAO,the induction of M2 regulatory macrophages was associated with stroke induced immune-depression in the early stage of cerebral infarctionThe mechanism of induction of M2 regulatory macrophages in rats with MCAO (Part B)Background:More and more evidences show that the activation of sympathetic nervous system after cerebral infarction,the increased catecholamine regulates immune response via ?2 adrenergic receptor on the surface of immune cell.There is evidence to show catecholamine could increase the expression of M2 macrophage,which increase the phagocytic activity and anti-inflammatory effect,mainly though beta 2 adrenergic receptor through non-traditional pathway(PI3K/Akt),rather than the traditional the signal pathway(cAMP/PKA).This article focus on whether sympathetic nerve system is associated with the induction of M2 regulatory macrophages at the early stage of cerebral infarction,and further explore the molecular mechanism of the induction of M2 regulatory macrophage.Objective:To explore the molecular mechanism of the polarization of macrophages in the early stage of cerebral infarction.Methods:Male mature Sprague-Dawley rats were used for MCAO,and the animals at MCAO 6h were randomly assigned to the 4 experimental groups:MCAO+saline group,MCAO+Pro group,MCAO+oil group,MCAO+RU486 group.Quantitative reverse transcription-polymerase chain reaction was used to evaluate the iNOS mRNA levels,Arg-land IL-10 mRNA levels of all rats.The expression of MHC-?,p-Akt and p-PKA protein in splenic macrophage was measured by western-blotting.Results:Treated with propranolol resulted in the increase of the expression of iNOS mRNA compared to saline treated,while treated with RU486 had no effect on the expression of iNOS mRNA compared to oil treated.Interesting,the expression of Arg-1 and IL-10 mRNA decreased in the propranolol treated group compared to saline treated group.In contrast,there was no obvious difference between RU-486 treated group and oil treated group.Pre-treatment with GCR-blocker RU486 did not influence the expression of MHC? protein of macrophage.In contract,?-AR-blocker propranolol increased the expression of MHC? protein of macrophage.GCR-blocker RU486 had no effect on the expression of p-PKA and p-Akt protein compared to oil treated group,yet ?-AR-blocker propranolol could decline the expression of p-Akt protein compared to saline treated group,and the expression of p-PKA protein had no significant difference between propranolol treated group and saline treated group.Conclusions:Propranolol could improve the level of iNOS mRNA,decline the Arg-land IL-10 mRNA levels in the MCAO model.These results indicate that blockade of sympathetic nerve system could promote M1 macrophages,exert anti-inflammatory effect.Propranolol could increase the expression of MHC-? protein on the surface of splenic macrophage,while decrease the p-Akt protein expression,the expression of p-PKA protein had no significant difference.In brief,the over activation of sympathetic nerve system after cerebral ischemia is related to the induction of M2 regulatory macrophages though ? AR/PI3K-Akt signaling pathway.