The Effects Of Xiehuo Bushen Decoction On The Expression Of IFN-γ/IL-4 In The NSCs-transplanted Intracerebral Hemorrhagic Rat's Brain

Background and Objective:Intracerebral Hemorrhage(ICH)is a severe cerebrovascular disease,which possesses a high rate of mortality and disability.Despite intensive research efforts,there are currently no effective treatments or therapies which can fully restore neurological deficits after ICH.Neural stem cells(NSCs)are self-renewing primordial cells that give rise to the differentiated progeny that constitute the mature nervous system,including neurons,astrocytes,and oligodendrocytes.The use of NSCs for therapeutic transplantation is a promising strategy for the treatment of intracerebral hemorrhage because of the potential for replacement of lost or dysfunctional cells.However,under certain circumstance NSCs can also express MHC molecular.Furthermore,the blood brain barrier(BBB)will be inevitably impaired after ICH and the immune privilege was damaged.Therefore,the NSCs have the possibility of being rejected after transplantation.It is the first and the most important step to avoid rejection as far as therapeutic transplantation is concerned.T helper 1(Th1)and T helper 2(Th2)cytokine profiles have conventionally been associated with allograft rejection and acceptance, respectively.CD4~+T helper cells can differentiate into at least two distinct phenotypes on activation.Th1 cells-produce the cytokines IL-2,IFN-γ, and TNF-α,and play an important role in transplant rejection and autoimmunity.Th2 cells produce IL-4,IL-5,IL-6,and IL-10,and facilitate antibody production by B cells.According to the classic Th1/Th2 paradigm,a Th1 to Th2 immune deviation is beneficial and fosters long term allograft survival.It has been one of the strategies of inducing immune tolerance by regulating the Th cells deviation. Pregnancy is actually the physiological 'allo-transplantation'.If Th1 response takes place on the fetal membrane,the foetus will be easily rejected and abortion is induced.As far as abortion is concerned,there are affluent effective therapeutical principles in the traditional Chinese medicine.Xiehuo Bushen decoction(XHBSD)which derived from the famous formula "Fetal Longevity"(used for treating abortion for a long time)is one of these traditional Chinese herbal formulas.Therefore,we presume that Xiehuo Bushen decoction has the effect of up-regulating Th2 and/or blocking Th1,resulting in inducing immune tolerance and suppressing the stem cells transplantation rejection.In this study,we investigate the effect of traditional Chinese medicine Xiehuo Bushen decoction on the expression of IFN-γ/IL-4 in intracerebral hemorrhagic rat's brain in an effort to determine whether XHBSD induced transplantation tolerance and the likely mechanism.Methods:First we isolated neural stem cells from new born SD rats' brains and then cultured,followed by identifying and labeling them with BrdU.Then PRA value was determined and 180 panel reactive antibody (PRA)positive SD rats were selected.One hundred and eighty rats were randomly divided into the six groups as follows:normal control group, model control group,sham control group,XHBSD therapy group, transplanted group and transplanted therapy group.By injecting collagenase typeⅦstereotaxically into right globus,ICH model was made.Two days after ICH,BrdU-labelled NSCs were transplanted into the homonymy SVZ in transplanted group and transplanted therapy group. XHBSD was orally administrated at a dose of 17.1 g/kg(eaquals to six times quantity of 70kg adults)twice per day in XHBSD therapy group and transplanted therapy group.On the 2nd,7th,14th,21st and 28th day, rats were euthanized and decapitated.Three rats' brains in each group have been used for detection of cytokine mRNA expressions of IFN-γand IL-4 by RT-PCR and the other 3 brains in each group have been used for analysis of IFN-γand IL-4 distribution.Results:1,NSCs isolation and identification:Fetal rats' cells isolated from the hippocampus tissues grew into plenty of neurospheres on 7th days.Immunocytochemistry revealed that the cell spheres were both BrdU and Nestin positive,certifying their NSCs ID.2,PRA test:under phrase microscope,we discovered that in positive reaction lymphocytes died,(dead cells became larger and their refraction were weak,looked like coal-balls),however,in negative reaction cells were still alive(alive cells were as large as ever and their refraction were perfect,looked like stars).We counted the number of positive reactions and calculated the PRA value.The rat's%PRA＞10%will be selected as experiment animals.3,IFN-γ/IL-4 distribution:Immunohistochemistry demonstrated that there was no detectable expression of IFN-γand IL-4 in normal control group and sham control group.The IFN-γexpressed and reached the peak at 2d in the other four groups,but the expression became weaker from 7d.Its expression in transplanted therapy group was lower than model control group and transplanted group from 7d to 28d(P＜0.05). There was nearly no expression of IL-4 in model control group and transplanted group and transplanted therapy group was the same the case at 2d.In transplanted therapy group IL-4 expressed merely at 7d and reached the peak at 14d,lasting to 21d.Though IL-4 expression became weaker,it was still higher than transplanted group(P＜0.01)4,IFN-γ/IL-4mRNA expression:RT-PCR revealed that there was nearly no expression of IFN-γand IL-4mRNA in normal control group and sham control group.The level of IFN-γin model control group, XHBSD therapy group,transplanted group and transplanted therapy group reached the peak at 2 d and then declined.The expression in HBSD therapy group was always lower than model control group, transplanted group and transplanted therapy group at different time points(P＜0.05).There was no significant difference between model control group and transplanted therapy group at 2d(P＞0.05),but from 7d,the expression of IFN-γmRNA became lower than model control group and transplanted group(P＜0.05).The expression of IL-4mRNA in model control group and HBSD therapy group was scarcely and there was no difference between them(P＞0.05).The level of IL-4mRNA in transplanted therapy group increased gradually and reached the peak at 7d,the difference was significant compared with model control group and transplanted group(P＜0.05)Conclusions:1,The approach of selecting recipient rats took PRA value as its selective standard.This method was convenient,effective and accurate.It simplified the procedure and as a result improved the efficiency of selecting rats considerably.2,Xiehuo Bushen decoction has the ability of effecting the IFN-γ/IL-4 genetic and proteomic expression in rats' brain,that is,reducing IFN-γactivation and promoting IL-4 expression.Therefore,it did favor of inducing Th1 to Th2 immune deviation,the immune tolerance and the inhibilation of transplantation rejection.