Effect Of Xiehuo Bushen Decoction On The Expressions Of TGF-β1 And VCAM-1 Of Neural Stem Cells-transplanted Intracerebral Hemorrhage In Rats

Background:Intracerebral Hemorrhage is a severe disease in clinical work.Its morbility,mutilation rate and mortality rate are quite high,following with enormous expenses in homo-sapiens healthy and society medical treatment resource.The numerus of Endogenous neural stem cells is too finite to repair nerve injury after Intracerebral Hemorrhage,and there is no specific medicine to treat Intracerebral Hemorrhage at present in medicine realm.The neural stem cells not only have the ability to differentiate to nerve cells and glial cells but also have the ability to migrate to the injury region.Especially the establishment of neural stem cells line,which could offer nerve cells and glial cells infinitely.It could supply sufficient material to carry out succedaneous therapy,so the neural stem cells therapy present the hope of the recovery of nervous system injury after Intracerebral Hemorrhage and it has the initiative strategic significance in the therapy of Intracerebral Hemorrhage.Orthodox standpoint generally presents that the cerebrum is immune privilege zone because of the existence of blood-brain barrier. But when we are transplanting neural stem cells,the blood-brain barrier could be damaged,which could create massive inflamed neurogen, antibody and immunological activity material to Inducer the expression of major histocompability complex.Besides,neural stem cells generated by fractional cultivation in vitro has the character of immunogenicity.So how to degrade the reject reaction in the transplantation of neural stem cells becomes a focal point in our research. Objective:TGF-β1has the powerful immunosuppressive effect, while VCAM-1 is a significant immune-associated adhesion molecule. The experiment was designed to investigate the effect of Xiehuo Bushen Decoction on the expressions of TGF-β1and VCAM-1 in rats brain of neural stem cells-transplanted Intracerebral Hemorrhage,and to research the effectiveness and mechanisms of Xiehuo Bushen Decoction on the induction of immune tolerance.It could supply experimental foundation for the depression of immune rejection when carrying out succedaneous therapy of neural stem cells.Methods:Two hundred and sixty Sprague-Dawley Rats marked positive of panel reaction antibody were choosed and were randomly divided into two groups.Then the rats of every group were randomly divided into six groups.The six groups were:A group(normal group)(n=5),B group(sham operation group)(n=25),C group(model group)(n=25),D group(treated model group)(n=25),E group(transplanted group)(n=25),F group(treated transplanted-group)(n=25).The rats of C,D,E and F groups were induced to make IntraIntracerebral Hemorrhage model byⅦcollagenase,while collagenase was replaced by physiological saline in the rats of B group and we not enforced any intervention on the rats of A group.Fractional cultivation of neural stem cells:we disconnected neural stem cells from hippocamp of newly-horned Sprague-Dawley Rats,than cultured and passaged them in vitro.The transplantation of neural stem cells:cells were transplanted to subrentride zone(SVZ)of the rats of E and F groups after they were identified as neural stem cells.Administration:the rats of D and F groups were treated by Xiehuo Bushen Decoction.The expressions of TGF-β1mRNA and VCAM-1mRNA were assayed by reverse transcription-polymerase chain reaction(RT-PCR)2d,4d,7d,14d and 28d after the operation and the expressions of TGF-β1 and VCAM-1 protein were assayed by immunochemistry 2d,4d,7d,14d,28d after the operation in the six groups respectively.Results:1.The expression feature of TGF-β1mRNA:The expression of TGF-β1mRNA in the rats brain of A group was slight.At the 2nd day after the operation,the TGF-β1mRNA expression of B,C,D,E and F groups occurred,especially the F group is the highest among other groups(P＜0.01).The expression of TGF-β1 mRNA arrived the peak at the 4th day(P＜0.05)in B,C,D,E groups,then decreased gradually at 7th day and 14th day(P＜0.05)and recover to normal level at 28th day.There is no evident change at 4th day comparing to 2nd day of TGF-β1mRNA expression in F group,and it decreased gradually at 7th day and 14th day(P＜0.05),but there is a rebound expression at 28th day comparing to 14th day(P＜0.05).At the same time except 28th day,expression of TGF-β1mRNA in F and D group were significantly higher than E and C groups respectively(P＜0.05).2.The expression feature of VCAM-1mRNA:There was only a very slight expression of VCAM-lmRNA in the rats brain of A group,the expression of VCAM-1mRNA in the other groups all arrived the peak (P＜0.05)at 2nd day,especially the expression level of F group is highest among other groups(P＜0.01).The expression level of B group degrade to normal at 7th day,and C,D,E,F groups all degrade gradually at 7th day and 14th day(P＜0.05),till to the 28th day.At the same time except 28th day,expression of VCAM-1mRNA in F and D groups were significantly lower than E and C groups respectively(P＜0.05). 3.The protein expression feature of TGF-β1:The protein expression of TGF-β1 in the normal rats brain was slight.At the 2nd day after the operation,the positive cells of TGF-β1 was increased and arrived the peak at the 4th day(P＜0.05)in B,C,D,E groups.Then they decreased gradually at 7th day and 14th day(P＜0.05)and recovered to normal level at 28th day.The positive cells of expressed TGF-β1 were maximum at the 2nd day and there is no evident change at 4th day comparing to 2nd day in F group.The positive cells numerus decreased gradually at 7th day and 14th day(P＜0.05),but there is a rebound at 28th day comparing to 14th day(P＜0.01).At the same time,the protein expression of TGF-β1 in F and D group were significantly higher than E and C groups respectively (P＜0.05).The positive cells distributed at the perimeter of hematoma and subrentride zone,and maybe they were neurone,gitter-cell,macrophage according to the morphous.4.The protein expression feature of VCAM-1:The protein expression of VCAM-1 in the normal rats brain was slight.The expression positive cells arrived the peak at 2nd after the operation(P＜0.05),then dreased gradually.The positive cells was the maximum in all the groups at all the time points(P＜0.05).At the same time,the protein expression of TGF-β1 in F and D group were significantly lower than E and C groups respectively(P＜0.05).The positive cells distributed at the perimeter of hematoma and subrentride zone,and most of them were irregularity cells.Conclusions:1:The expressions of VCAM-1 and TGF-β1 of the Intracerebral Hemorrhage Sprague-Dawley Rats were increased,which demonstrated inflammatory reaction was strengthened after Intracerebral Hemorrhage.2:The expressions of VCAM-1 and TGF-β1were upregulated in brain of NSC-transplanted Intracerebral Hemorrhage Sprague-Dawley Rats,which demonstrated immunological reaction was strengthened after transplantation.3:Xiehuo Bushen Decoction can depress rejection after NSC-transplanted Intracerebral Hemorrhage Sprague-Dawley Rats and induce immune tolerance,which is related with the upregulation of TGF-β1 and downregulation of VCAM-1.