The Protective Effects Of Xiehuo Bushen Decoction On The Cerebral Microvascular Endothelial Cells Of NSCs-transplanted Experimental Intracerebral Hemorrhagic Rats

Background and ObjectiveThe study aimed to observe the effects of Xiehuo Bushen Decoction on the expression of vascular cell adhesion molecule(VCAM-1), transforming growth factor beta (TGF-β1), Fas, Fas Ligand and apoptosis in rats brain of neural stem cells (NSCs)-transplanted experimental intracerebral hemorrhage and cultured interferon(IFN-γ)-induced cerebral microvascular endothelial cells (RCMECs), to explore the mechanism of Xiehuo Bushen Decoction (XHBS) in protecting RCMECs in NSCs-transplanted intracerebral hemorrhagic rat brains.Methods1. RCMECs were obtained from 7-10 days old neonate SD rats. The cultured RCMECs were randomly divided into control group, IFN-γgroup, serum control group and XHBS serum group. We observed the expression of VCAM-1 and TGF-β1 by enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR), the expression of Fas and FasL by immunohistochemistry and RT-PCR, as well as the apoptosis by TdT-mediated dUTP-biotin nick end labeling (TUNEL).2. Ninety rats were randomly divided into five groups including normal group (n=10), sham operation group (n=20), intracerebral hemorrhage group (n=20), NSCs-transplanted group (n=20) and XHBS-treated group(n=20). The intracerebral hemorrhage model was induced by injecting 0.4UⅦcollagenase into right globus pallidus(1.4mm posterior, 3.2mm lateral to bregma, and 5.6mm depth from the cortical surface) with stereotaxic apparatus. BrdU-labled NSCs were transplanted to subventricle zone (SVZ) 2 days later. The expression of VCAM-1 and TGF-β1, Fas, FasL and apoptosis was assayed by the same technique as those in in vitro respectively.Results1. VCAM-land TGF-β1 mRNA expressed remarkably in cultured RCMECs after induced with IFN-γ(P＜0.01 vs control group). Their expression were decreased after treated with XHBS serum (P＜0.05 vs IFN-γgroup), The concentration of VCAM-1 and TGF-β1 in supernatant changed similar to that of the mRNA. The cell viability of XHBS serum group were higher than that of IFN-γgroup by MTT (P＜0.05).2. The number of TUNEL positive cells which was very small in control group, increased markedly in IFN-γgroup (P＜0.01 vs control group) and decreased in XHBS serum group (P＜0.05 vs serum control group).3. The expression of Fas mRNA and protein in RCMECs was increased remarkably after induced by IFN-γ(P＞0.05 vs control group) and decreased in XHBS serum group (P＜0.01 vs serum control group). The expression of FasL was gradually up-regulated in IFN-γgroup, serum control group and XHBS serum group, and there is significant difference between IFN-γgroup and XHBS serum group (P＜0.05).4. VCAM-1 expressed at low level in normal group and sham operation group, it was up-regulated remarkably in intracerebral hemorrhage group and NSCs transplanted group. The expression of VCAM-1 was down-regulated significantly in XHBS-treated group (P＜0.05 vs NSCs-transplanted group). TGF-β1 mRNA also can be seen mildly in normal group and sham operation group (P＜0.05). It was higher in intracerebral hemorrhage group than in sham operation group (P＜0.05). The expression was up-regulated markedly in XHBS-treated group (P＜0.05 vs NSCs-transplanted group).5. There was no TUNEL positive cells in the rat brains of normal group and some in sham operation group. The number of TUNEL positive cells increased significantly in model group and NSCs-transplanted group, and there was no difference between the two groups. The number of apoptosis cells decreased markedly in XHBS-treated group(P＜0.05 vs NSCs-transplanted group).6. Fas expressed at low level in the rat brains of normal group and sham operation group. The expression of Fas mRNA and protein in intracerebral hemorrhage group and NSCs-transplanted group increased at 4d (P＜0.01 vs normal group and sham operation group). It expressed mainly in neurons and endothelial cells around hematoma. it was down-regulated remarkably in XHBS-treated group(P＜0.05 vs NSCs-transplanted group). There was little FasL mRNA expressed in normal group and sham operation group. The trend of FasL expression increased gradually in intracerebral hemorrhage group, NSCs-transplanted group and XHBS-treated group. The expression of FasL from 4d to 7d is ascendant.Conclusions1. Expression of VCAM-1, TGF-β1, Fas and FasL was up-regulated in IFN-γ-treated rat cerebral microvascular endothelial cells to simulate host-rejection in vitro, accompanied by celluar morphological change and viability reduction.2. Expression of VCAM-1, TGF-β1 and Fas/FasL was up-regulated in brain of NSCs-transplanted intracerebral hemorrhagic rats, which indicated intensification of inflammatory and immunological reaction.3. Xiehuobushen decoction can ameliorate host rejection following NSCs-transplantation in intracerebral hemorrhagic rat brains, which may be attributed to its inhibition of inflammation by reducing VCAM-1 activation and promoting TGF-β1 expression, and protection of rat brain microvascular endothelial cells by modifying Fas/FasL pathway.