| Objective:To study the effect of Panax notoginseng saponins(PNS) injection on neurologic functional recovery and on the protein and mRNA expression of Nogo-A/NgR and the downstream Rho/ROCK signaling pathway in rats with middle cerebral artery occlusion(MCAO) at 14d and 28d observation periods. To investigate the influence of PNS on the protein expression and location of Nogo-A, NgR1, RhoA, ROCKII, SYP and PSD-95 and the mechanism of promoting neuroremodeling and synaptogensis recovery.Methods:Healthy mature male Sprague-Dawley (SD) rats were randomly divided into the control group, the surgery group,the Y27632 group and PNS and Y27632 group. Each group had 7 rats. All of the rats were used to establish MCAO model by improving Longa method except for the control group. The successful modeled and un-grouped rats were randomly divided into model group, PNS group and nimodipine group. The weight and neurological function score of the rats were assessed daily. Rats were executed at 14 days and 28 days and the brain tissues were taken out to observe the protein and m RNA expression of Nogo-A, NgRl, NgR2, RhoA, ROCKII with Western Blot and RT-PCR. HE staining and IHC were used to detect the morphology changes and the expression and location of Nogo-A, NgR1, RhoA, ROCKII,SYP and PSD-95 in the infarction peripheral region.Results:1 The weight of the rats in the surgery groups decreased compared with the control group in the 14d and 28d observation periods.(P<0.01). On the 14th day after MCAO the weight of the rats in the Y27632 group and the PNS+Y27632 group increased significantly compared with the model group(P<0.05, P<0.01), the weight in the PNS+Y27632 group was higher than the Nimodipine group(P<0.05). On the 28th day after MCAO, the weight in the treatment groups increased compared to the model group(P<0.05, P<0.01), moreover, the Y27632 group and the PNS+Y27632 group were higher than the PNS group(P<0.05, P<0.01).2 No neurological function damage was observed in the control group in this experiment. However the neurological function scores in the surgery groups were higher than the control group(P<0.01). After 14 days of MCAO, the scores in the Y27632 group and the PNS+Y27632 group were decreased compared to the model group(P<0.05). After 28 days of MCAO the score in the PNS+Y27632 group was still lower than the model group (P<0.05).3 On the 14th day and 28th day after MCAO, the cortical protein and mRNA expression of Nogo-A in the control group were the fewest, the expression in the model group was increased compared with the control group(P<0.05,P<0.01), the expression in the PNS group was higher than the control group(P<0.05, P<0.01)and lower than the model group (P<0.05). On the 14 days of MCAO, the protein expression of Nogo-A in the Y27632 group, the PNS+Y27632 group and the Nimodipine group less than the model group(P<0.01),and the PNS+Y27632 group and the Nimodipine group decreased compared to the PNS group(P<0.05), the mRNA expression of Nogo-A in the treatments groups decreased compared to the model group as well(P<0.05, P<0.01), however, compared to the control group the mRNA expression in the PNS group and the Y27632 group still increased (P<0.05). On the 28 days of MCAO, the protein expression in the Nimodipine group increased compared to the control group(P<0.05)and decreased compared to the model group(P<0.05), the expression was lower in the Y27632 group and the PNS+Y27632 group than the model group(P<0.05, P<0.01), the mRNA expression in the treatment groups decreased as well(P<0.05).4 On the 14th day and 28th day after MCAO, both of the NgRl protein and mRNA expression in the model group were higher than the control group(P<0.01). On the 14th day after MCAO, compared with the control group the NgR1 protein expression in the PNS group and the Y27632 group increased, the NgRl mRNA expression in the Y27632 group and the Nimodipine group increased too(P<0.05). The NgR1 protein expression in the PNS+Y27632 group and the Nimodipine group was lower than the model(P<0.01), in addition, the protein expression in Nimodipine group less than the PNS group(P<0.01) and the mRNA expression in the PNS+Y2763 group and the PNS group less than the model group(P<0.05).On the 28th day after MCAO, the protein expression in the treatments group decreased compared to the model group(P<0.05, P<0.01), the mRNA expression in the PNS+Y2763 group, the PNS group and the Nimodipine group decreased as well(P<0.05, P<0.01), the protein of NgR1 decreased obviously compared to the PNS group(P<0.05).5 On the 14th day after MCAO, the NgR2 protein expression was the lowest in the PNS+Y27632 group, which compared to the control group, the model group, the PNS group and the Y27632 group had the statistical significance(P<0.05, P<0.01), the NgR2 protein expression less than the model group(P<0.01),the NgR2 mRNA expression in the model group higher than the control group(P<0.05), the mRNA expression in the Nimodipine group less than the model group(P<0.05). On the 28th day after MCAO, the NgR2 protein expression was the highest in the model group(P<0.01), the protein expression decreased in the PNS+Y27632 group compared to the PNS and the Nimodipine group(P<0.05), the NgR2 mRNA expression in the model group higher than the control group(P<0.05).6 On the 14th day and 28th day after MCAO, both of the RhoA protein and mRNA expression in the model group were higher than the control group(P<0.05,P<0.01). On the 14th day after MCAO, the RhoA protein expression in the treatment groups decreased compared to model group(P<0.05, P<0.01), the protein expression in the Nimodipine group was higher than the control group and the Y27632 group (P<0.05)and lower than the model group(P<0.05), the RhoA mRNA expression in the treatment groups except for the PNS group all decreased compared to the model group(P<0.05).On the 28th day after MCAO, the RhoA protein expression in the treatment groups except for the Nimodipine group all lower than the model group(P<0.05), and the mRNA expression in the treatment groups all decreased compared to the model group(P<0.01).7 On the 14th day and 28th day after MCAO, both of the ROCKII protein and mRNA expression in the model group were higher than the control group(P<0.05, P<0.01). On the 14th day after MCAO, the ROCKII protein expression in the treatment groups all decreased compared to the model group(P<0.01), the ROCKâ…¡mRNA expression in the treatment groups except for the Nimodipine group all decreased compared to the model group(P<0.05). On the 28th day after MCAO, both of the ROCKII protein and mRNA expression in the treatment group except for the Nimodipine group were less than the model group(P<0.05, P<0.01), the mRNA expression in the Nimodipine group was higher than the Y27632 group(P<0.05).8 On the 14th day and 28th day after MCAO, in the control group the integrity structure of neurons, the neuronal soma and the axon were observed clearly and the immunoreactive substance of Nogo-A, NgR1, RhoA, ROCKII, SYP and PSD-95 were specifically latched on the cell membranes, cytoplasm and synaptic cleft, the positive protein expression in the control group was significantly different from the model group(P<0.01). In the model group most parts of neurons normal structures were disappeared in the infarction peripheral region, the mesh-like performance was more obvious and the immunoreactive substances looked like a round particles near the infarction central region. The expressions of Nogo-A, NgRl, RhoA, ROCKII protein were higher than the control group(P<0.01).On the 14th day and 28th day after MCAO, the SYP protein expression in the model group less than the control group(P<0.01). On the 14th day after MCAO the SYP protein expression in the PNS group, the Y27632 group and the PNS+Y27632 group were higher than the model group(P<0.05), in the treatment groups the PSD-95 protein expression decreased compared to the model group(P<0.05, P<0.01). On the 28th day after MCAO, the SYP and PSD-95 protein expression increased compared to the model group(P<0.05, P<0.01).Conclusion:1 It is very difficult to neurological function recovery after cerebral infarction. Though PNS is failed to completely cure the neurological damage in the observation of 14 days and 28 days after MCAO, we find the PNS and Y27632 have the same effect in promoting the recovery of the neurological function. Moreover, we take the points that PNS and Y27632 may have the synergistic action in promoting the neurological recovery and the combined treatment maybe improve the neurological function ahead of time. In addition, in the 28 days observation PNS is proved to have a strong improvement in neurological recovery all the time, Y27632 seems to have a weaken recovery effect after 14 days observation.2 PNS has the effect on inhibiting the protein and mRNA expression of Nogo-A, NgR1, NgR2, RhoA and ROCKII. The inhibition resemble the Y27632,a specific inhibitor of ROCK, and different from the Nimodipine. That may be the mechanism which PNS promotes the neuroremodeling and synaptogensis recovery by inhibiting the key molecules in the Nogo-A/NgRl and the downstream Rho/ROCKII signaling pathway. During the longer 28 days convalescence, we indicates that the combination treatment of PNS and Y27632 can enhance the stability of the axons regeneration and the synapsis remodeling.3 PNS enhances the expression of SYP and PSD-95 and improves the structure and function of cortex neurons in the infarction peripheral region by inhibiting the Nogo-A/NgR1 and the downstream Rho/ROCKII signaling pathway, which maybe the mechanism of arousing the neuroremodeling and synaptogensis recovery.
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