[Effects of Naomaitong combined with mobilization of bone marrow mesenchymal stem cells on neuron apoptosis and expressions of Fas, FasL and caspase-3 proteins in rats with cerebral ischemia].

OBJECTIVE: To observe the effects of Naomaitong, a compound traditional Chinese herbal medicine, combined with mobilization of bone marrow mesenchymal stem cells (BMSCs) on neuron apoptosis in rats with cerebral ischemia, and to explore the possible mechanism by detecting the expressions of Fas, FasL and caspase-3 proteins. METHODS: Two hundred and two SD rats were divided into sham-operated group, untreated group, recombinant granulocyte colony-stimulating factor (rG-CSF) group, Naomaitong group and Naomaitong plus rG-CSF group (combination group). Focal cerebral ischemia was induced by intraluminal middle cerebral artery occlusion using a nylon thread with some modification. Rats in the rG-CSF group and the untreated group were administered with rG-CSF 10 microg/(kg x d) by subcutaneous injection 3 d before and 2 d after the operation respectively, once a day, and rats in the Naomaitong group and the combination group were intragastrically administered Naomaitong before and after the operation until sacrificed. Two, three, seven and fourteen days after operation, count of CD34-positive cells in peripheral blood and CD34 expression in brain tissue were determined. General neural function score (GNFS) was evaluated. Neuron apoptosis, expressions of Fas, FasL and caspase-3 in rat's brain were all measured. RESULTS: Count of CD34-positive cells in peripheral blood and CD34 expression in brain tissue were high in the untreated group, and reached the peak at 3 d and 7 d respectively. CD34 expression in brain tissue was increased in each treated group, especially in the combination group. GNFS was increased at 3 d and 7 d in the untreated group, 7 d and 14 d in the rG-CSF group and the combination group. Expressions of Fas, FasL and caspase-3 were increased 2, 3 and 7 d after operation, while expression of FasL at 2 d in the rG-CSF group, expressions of Fas, FasL and caspase-3 in the combination group were decreased. Expressions of Fas, FasL and caspase-3 at 7 d and 14 d in the combination group were lower than those in the rG-CSF group. Meanwhile, expressions of Fas, FasL and caspase-3 were decreased in each group at 14 d as compared with those at 3 d. CONCLUSION: There exists interaction between Naomaitong and BMSC mobilization in the effect of improving nerve function and inhibiting neuron apoptosis in rats after cerebral ischemia. It is implied that Naomaitong combined with BMSC mobilization down-regulates the expressions of Fas and FasL in early phase and then inhibits the apoptosis cascade reaction caused by caspase-3, which causes further inhibition of Fas and FasL expression after cerebral ischemia.

[Effects of rhubarb aglycone on matrix metalloproteinase in cerebral ischemic tissue in rats with bone marrow mesenchymal stem cell transplantation].

OBJECTIVE: To observe the effects of rhubarb aglycone on pathological changes and activity of matrix metalloproteinase in cerebral ischemic tissue in rats with bone marrow mesenchymal stem cell (BMSC) transplantation, and to explore the action mechanisms of rhubarb aglycone in protecting against brain micrangium injury in rats. METHODS: The BMSCs were purified and amplified by methods of adherence and selection in vitro. One hundred and ninety rats were randomly divided into sham-operated group, untreated group, rhubarb aglycone group, BMSC transplantation group (abbreviated as transplantation group) and BMSCs combined with rhubarb aglycone group (abbreviated as combination group). Middle cerebral artery occlusion (MCAO) model was duplicated with nylon thread. Rats of transplantation and combination group were transplanted with BMSCs via carotid artery after 24-hour reperfusion. Rhubarb aglycone was used by intragastric administration in the rhubarb aglycone group and the combination group. The brain samples were taken at 7, 14 and 28 days after transplantation. Brain micrangium pathological changes were observed by light microscope, and immunohistochemical method was used to determine the expressions of immunoglobulin G (IgG), type IV collagen (Col IV), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1). RESULTS: Comparison with the normal control group revealed that brain micrangium in rats in the untreated group was obviously mutilated and damaged, the expression of IgG and MMP-9 increased, and showed a progressively enhanced tendency following the prolongation of reperfusion, while the expressions of Col IV and TIMP-1 were decreased, and TIMP-1 showed a attenuated tendency following the prolongation of reperfusion. Comparing with the untreated group, the improvements of brain micrangium structure in the rhubarb aglycone group (7 days after transplantation), the transplantation group (14 and 28 days after transplantation) and the combination group were significant; expression of IgG and activity of MMP-9 were decreased, while expressions of Col IV and TIMP-1 were increased in the rhubarb aglycone group and the combination group at each time point. The brain micrangium was integral and the expression of Col IV was enhanced in combination group (7 days after transplantation) as compared with those in transplantation group. MMP-9 activity in combination group (14 days after transplantation) was lower than that in the rhubarb aglycone group (14 days after transplantation), while expression of TIMP-1 in combination group also increased significantly as compared with that in transplantation group (28 days after transplantation). CONCLUSION: Rhubarb aglycone can decrease the degradation of basal lamina Col IV and the permeability of brain micrangium in cerebral ischemic rats with BMSC transplantation, and its mechanisms may be related to regulating the balance of MMP-9, especially by increasing the expression of TIMP-1.

[Effects of Naomaitong and thrombolysis on injuries of lung and stomach in rats with cerebral ischemia].

OBJECTIVE: To study the protective effects of Naomaitong, a compound traditional Chinese herbal medicine, used together with thrombolysis therapy, on injuries of the lung and stomach in rats with cerebral ischemia. METHODS: Rats were randomly divided into sham-operated group, untreated group, urokinase group (thrombolysis group), Naomaitong group, thrombolysis plus Naomaitong group. Cerebral ischemia was induced in rats by autonomous blood blot and inserted nylon thread. Rats were administrated with thrombolysis therapy through artery 3, 6 and 9 h after cerebral ischemia respectively. Twenty-four hours after the administration, mortality of the rats, and the brain and stomach hemorrhage ratios, as well as the pathological changes of the brain, lung and stomach were observed, and then cerebral infarct size (CIS) and lung water ratio (LWR) were measured. RESULTS: Compared with the sham-operated group, the rat mortality, and the brain and stomach hemorrhage ratios increased, the CIS enlarged, pathological changes of the brain, lung and stomach appeared obvious, and the LWR increased. Naomaitong plus thrombolysis treatment improved the changes above significantly. In the untreated rats with cerebral ischemia, injuries of the brain, lung and stomach were aggravated, the CIS enlarged and the LWR increased in the 9 h group as compared with those in the 3 h group. In the thrombolysis plus Naomaitong group, the pathological changes were improved, the CIS diminished and the LWR reduced. CONCLUSIONS: Injuries of the lung and stomach can be caused by cerebral ischemia, and the impairment was exacerbated following the prolongation of the ischemia. Thrombolysis therapy can cause brain and stomach hemorrhage. Thrombolysis therapy used early can perform protection against the injuries. Naomaitong, used together with thrombolysis, can reduce the mortality, the brain and stomach hemorrhage ratios, and perform protective effects on the injuries of cerebral ischemia.

[Preventive effects of emodin on cerebral ischemia injury and expression of the inflammatory factors in rats with cerebral ischemia].

OBJECTIVE: To assess emodin antagonism to cerebral ischemia injury, and to discuss the mechanism of emodin inhibiting the inflammatory cascade reaction from the levels and expressions of cytokines. METHOD: Rats were divided into sham-operated group, model group, Ligustrazine group and emodin groups (low, middle, high dosage). After focal cerebral ischemic model of cerebral middle artery occlusion was duplicated with nylon thread, we took the speciments after ischemia 6 hours, observed the changes of the evaluating score of neural symptoms, brain water ratio and cerebral infarction area, determined the levels of TNF-alpha, IL-beta and TGF-beta in rats brain tissue by radioimmunoassay, detected the expressions of TNF-alpha and VCAM-1 by immunohistochemistry, and measured VCAM-1-mRNA expression by in-situ hybridization. RESULT: Compared with sham-operated group, the evaluating score of neural symptoms, brain water ratio and cerebral infarction area of rats in model group were higher (P < 0.01) , the levels of TNF-alpha and IL-1beta of rats brain tissue in model group increased, while the level of TGF-beta was lower, and the expressions of TNF-alpha and VCAM-1 increased (P < 0.01). The evaluating score of neural symptoms, brain water ratio and cerebral infarction area improved obviously in every emodin group, especially in emodin low dosage group. Levels of TNF-alpha, IL-1beta and the expressions of TNF-alpha and ICAM-1 in emodin low dosage group and Ligustrazine group were lower, while the level of TGF-beta was higher. Compared with Ligustrazine group, the changes aboved are more significant in emodin low dosage group (P < 0.01). CONCLUSION: The increase of inflammatory cascade reaction mediated by various cytokines such as TNF, IL-1beta, ICAM-1 and the decrease of TGF protection are the important mechanism of cerebral ischemia injury. The mechanism of emodin antagonism to cerebral ischemia injury may be implemented by inhibiting inflammatory cascade reaction and increasing the brain protective factors, such as TGF.