| Objective:In order to study the effect of Baihui-penetrating-Qubin treatment on proliferation of neural stem cells on intracerebral hemorrhage rats and the possible mechanism of this effect,BrdU+/Nestin+positive cells were tested by immunofluorescence assay.Then immunohistochemical analysis and western blot were used to observe the expression of positive cells and protein levels of Shh,Ptch1 and Gli1,in order to study the effect of Baihui-penetrating-Qubin treatment on the expression of main factors in Sonic hedgehog pathway on intracerebral hemorrhage rats.Methods:The study was divided into blank group,model group,agonist group,acupuncture group.Except blank group,the other 3 groups were divided into 4 subgroups according 4 time points of 3d(72h),5d(120h),7d(168h),10d(240h)respectively.20 normal rats were randomly divided into blank group.Other 240 rats were performed by intracerebral hemorrhage surgey at day 1.After they were fully awake,Ludmila Belayev tests were used to select successful intracerebral hemorrhage models.All the 240 rats were involved and were randomly divided into each subgroup with 20 rats in each.Rats in agonist group were injected by purmorphamine(an agonist of Shh pathway)and rats in acupuncture group received Baihui-penetrating-Qubin acupuncture treatment each day.Rats in blank group recieved Ludmila Belayev tests only at 1d,and rats in other 3 groups recieved Ludmila Belayev tests again before sampling at corresponding time points.In order to mark newborn neural stem cells,5 rats in blank group and in each subgroup were seleted to perform BrdU injection.BrdU was injected i.p 5 times with 2 hours’ intervals at 32 hours earlier before sampling.Brains were sampled at corresponding time points,and BrdU+/nestin+positive cells were tested by immunofluorescence assay.15 tissues in blank group and in each subgroup of other 3 groups were divided into 3 groups:5 of them were used to detect pathologic damages by HE staining,5 of them were used to detect positive cells of Shh,Ptch1 and Gli1 by immunohistochemical assay,the others were used to detect protein expression of Shh,Ptch1 and Glil by western blot assay.The results above were analysed by statistical analysis system.Results:1.Baihui-penetrating-Qubin acupuncture treatment can improve defects of nerve function on intracerebral hemorrhage rats,and Ludmila Belayev scores in acupuncture group were lower than in model group in corresponding time points,which showed statistical significance(P<0.01 at 3d,7d,10d.P<0.05 at 5d).Compared with agonist group in corresponding time points,Ludmila Belayev scores in acupuncture group had no statistical significance(P>0.05).2.Pathogenic manifestations in model group at 3d showed a large number of erythrocytes,edema,necrosis,inflammation and vacuoles between cells.Above pathologic damages were relief with time extending,and basically disappeared at 10d.Compared with model group,damages in agonist group and acupuncture group were more abirritant at corresponding time points.However,damages between these two groups at corresponding time points remained no difference.3.The amount of BrdU+/Nestin+positive cells in acupuncture group were higher than in model group at corresponding time points except 3d,which showed statistical significance(P<0.01 at 7d and 10d.P<0.05 at 5d).Compared with agonist group,BrdU+/Nestin+positive cells in acupuncture group had no statistical significance at 7d and 10d(P>0.05).4.Shh positive cells were largely expressed in acupuncture group and the results had statistical significance at 7d and 10d compared with model group(P<0.05).However,there was no statistical significance between acupuncture group and agonist group at all time points(P>0.05).5.Shh protein expression was remarkably high in acupuncture group compared with model group at corresponding time points and the expression increased with time extending.The results had statistical significance compared with model group except day 3(P<0.01 at 7d and 10d.P<0.05 at 5d).However,there was no statistical significance between acupuncture group and agonist group at all time points(P>0.05).6.Ptch1 positive cells were decreased in acupuncture group with time extending and were less than in model group at all time points.The results had statistical significance at 3d,5d and 7d compared with model group(P<0.01 at 3d.P<0.05 at 5d and 7d).However,there was no statistical significance between acupuncture group and agonist group at all time points(P>0.05).7.Ptch1 protein expression decreased with time extending in acupuncture group and was remarkably low compared with model group at 5d,7d and 10d,which had statistical significance(P<0.05).But there was no statistical significance between acupuncture group and agonist group at all time points(P>0.05).8.Gli1 positive cells were largely expressed in acupuncture group and increased with time extending.The results showed statistical significance compared with model group at 7d and 10d(P<0.05).However,there was no statistical significance between acupuncture group and agonist group at all time points(P>0.05).9.Gli1 protein expression was remarkably high with time extending in acupuncture group.The results had statistical significance compared with model group at all time points(P<0.05 at 3d,7d and 10d.P<0.01 at 5d).However,there was no statistical significance between acupuncture group and agonist group at all time points(P>0.05).Conclusions:1.Baihui-penetrating-Qubin acupuncture treatment can obviously improve defects of nerve functions and improve a series of pathological injuries on intracerebral hemorrhage rats.2.Baihui-penetrating-Qubin acupuncture treatment can obviously promote neural stem cells proliferation on intracerebral hemorrhage rats.3.Baihui-penetrating-Qubin acupuncture treatment can obviously activate Shh signal pathway by upregulating Shh,Gli1 positive cells and protein content and downregulating Ptch1 positive cells and protein content on intracerebral hemorrhage rats.4.The mechanism of Baihui-penetrating-Qubin acupuncture treatment to neural stem cells proliferation,improvement of pathological injuries and defects of nerve functions on intracerebral hemorrhage rats may attribute to activating Shh signal pathway. |
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