| Objective:1. To study the effect of Panax notoginseng saponins (PNS) injection on the remoldability of cortex and hippocampus, and the regulation effect on the expression of Nogo-A, NgR and downstream RhoA signaling pathway in rats with middle cerebral artery occlusion (MCAO).2. To investigate the influence of Panax notoginseng saponins injection on serum IL-1β and TNF-a and the correlation of inflammatory factors with nerve regeneration inhibitionors in MCAO rats.3. To investigate the influence of Panax notoginseng saponins injection on A beta25-35induced injury on the SH SY5Y cells and the adjustment function on the NGF, p75and TrkA expressions.(Dalhousie University).Methods:1.8rats of the control group were taken out randomly from112SPF Sprague-Dawley rats. The rest of the rats were used to establish MCAO model by improving Longa method, and randomly divided into the model group, PNS group, Y27632group, PNS+Y27632group and nimodipine group. All of rats were executed on the7th day after surgery. The general state, weight and neurological function score of the rats were assessed daily. H-E staining was used to detect the pathological and morphological changes. Immunohistochemistry staining was used to detect the expression of PSD-95, SYP, Nogo-A, NgR and RhoA. Western blot was used to detect the protein expressions of Nogo-A, NgRl, NgR2and RhoA. RT-PCR method was used to detect the mRNA level of Nogo-A, NgRl, NgR2and RhoA. ELISA method was used to test the content of IL-1β and TNF-a.2. SH SY5Y cells were divided into six groups, the control group, Aβ model group, PNS300mg/L group, PNS600mg/L group, and PNS300mg/L+Aβ group, PNS600mg/L+Aβ group.15μM Aβ25-35was used to induce SH-SY5Y cells injury, and MTT method was used to observe PNS function; immunofluorescence staining and Western Blot methods were adopted to observe notoginseng total saponin adjustment function on the protein expression of NGF, p75and TrkA.Results:1. The status of food and drinking intake of the control group rats was normal, and the body mass index (BMI) of this group rats increased stably after surgry. The weight of the rats in the MCAO surgery groups decreased significantly compared with the control group owing to surgical stress on the2nd day after the surgery(P<0.01). From the3rd day to5th day after the surgery, the weight of the rats in the surgery groups decreased continuously, and the model group was more obviously, but there was no significant difference compared with the other surgery groups (P>0.05). From the6t day to7th day after surgery, the food and drinking intake were increased, and the treated group was more obviously. The weight of the rats in PNS+Y27632group was higher than the model group (P<0.05), and the PNS and nimodipine group were also higher than the model group but there was no statistical significance (P>0.05).2. The rats in the control group showed normal status and no neurological function deficits at each time point. The neurological score of the rats in the surgery groups was higher than the control group on the first day after the MCAO surgry (P<0.01). On the second day after the surgry, the neurological function score decreased significantly in the Y27632group compared with the model group (P<0.05), and the PNS, PNS+Y27632and nimodipine group all decreased compared with the model group but with no significant difference (P>0.05). On the third and fourth days after the surgry, the neurological function score recovered slowly. The PNS, PNS+Y27632and nimodipine group showed significant decrease of the score compared with the model group (P<0.05). On the fifth and sixth day after the surgry, the neurological function score of the rats in the model group decreased slightly, with no significant difference compared with the treatment groups. On the seventh day, the neurological function score of the rats in the treatment groups was lower than the model group but the differences were not significant (P>0.05).3. The expressions of Syp and PSD-95in the central and peripheral zone of infarct, hippocampus area of the model group were less than the control group. The expression of Syp and PSD-95in the central zone of infarcts, peripheral zone of infarct, and hippocampus area of the rats in the PNS, Y27632, PNS+Y27632and nimodipine group were more than the model group. The immunohistochemical results indicated that Nogo-A, NgRl and RhoA distribute dispersively in the cortex as well as hippocampus of the rats in the control group. The expression of the Nogo-A, NgR1and RhoA at the central and peripheral infarcts as well as hippocampus of the rats in the model group increased compared with the control group. The expression of the Nogo-A, NgR1and RhoA at the central and peripheral infarcts as well as hippocampus area of the rats in the treatment groups decreased compared with the model group.4. The protein expression of Nogo-A, NgR1, NgR2and RhoA at cortex of the rats in the model group increased significantly compared with the control group (P<0.01or P<0.05). The expression of Nogo-A, NgRl and RhoA at cortex of the rats in the PNS and PNS+Y27632group decreased significantly compare with the model group (P<0.01or P<0.05). The expression of NgRl, NgR2and RhoA at cortex of the rats in the Y27632group decreased significantly compare with the model group (P<0.01or P<0.05). The expression of Nogo-A, NgRl and RhoA at cortex of the rats in the nimodipine group decreased significantly compare with the model group (P<0.01). The neurological function score of the rats has significant relation with the protein expression of RhoA.5. The expression of Nogo-A, NgR1and RhoA mRNA at cortex of the rats in the model group increased significantly compared with the control group (P<0.01or p<0.05), while the difference of the expression of NgR2mRNA in the control and model was not significant (P>0.05). The expression of Nogo-A, NgR1, NgR2and RhoA mRNA decreased at cortex of the rats in the PNS group, but only the expression of Nogo-A showed the significant difference compared with the model group (P<0.01). The expression of NgR1decreased significantly in the Y27632group compared with the model group (P<0.05). The expression of NgR1and RhoA mRNA in the PNS+Y27632and nimodipine group decreased significantly compared with the model group (P<0.01or P<0.05). The neurological function score of the rats has significant relation with the expression of Nogo-A mRNA.6.7days after MCAO surgery, the expression of serum IL-1β and TNF-α in the model group increased significantly compared with the control group (P<0.01). The treatment groups expressed lower than the MCAO model group, the difference was statistically significant (P<0.01or P<0.05). The correlation research of Rats serum IL-1β and TNF-a with Nogo-A, NgRl, NgR2and RhoA showed that IL-1β and TNF-a has certain correlation with NgRl and RhoA (P<0.01or P<0.05).7. The dentrite in the A beta model group was obviously shortened, or broken; cell volume increased; the number of cells decreased; cell activity decreased obviously, the difference was significant compared with the control group (P<0.05or P<0.01). Each PNS dosage group has obvious promotion function compared with the control group. Compared with model group, PNS has obvious protective effect, especially PNS high dose group, and the cell activity increased significantly compared with the model group (P<0.05or P<0.01) and showed a dose dependent trend (P<0.05or P<0.01).8. The expression levels of NGF and TrkA were lower and p75higher in A beta model group than the control group, and the difference were significant (P<0.01or P<0.05). PNS low-dose+A beta group had a higher NGF and TrkA expressions compared with the model group, but there was no significant difference (P>0.05); p75expression was slightly lower than the model group, the difference was significant (P<0.05). PNS high-dose+A beta group model, the expression of NGF and TrkA were higher compared with model group, and p75expression reduced significantly compared with model group, the difference were significant (P<0.01or P<0.05).Conclusion:1. The PNS injection alleviates the neurological deficits and enhances recovery of neurological function of the MCAO rats.2. The PNS injection may increase the expression of Syp and PSD-95of the MCAO rats to enhance regeneration of the axon.3. The PNS injection may decrease the expression of Nogo-A, NgR and RhoA protein and mRNA of the MCAO rats to enhance regeneration of the neuron. The neurological function score of the rats has significant relation with the expression of Nogo-A and RhoA.4. The PNS injection can reduce the content of serum inflammatory cytokines IL-1β and TNF-a of MCAO rats; IL-1β and TNF-a related with neural inhibitory factor Nogo-A, NgRl, and RhoA.5. The PNS injection protected SH SY5Y cells with Aβ25-35induced injury and increase the expression of NGF and TrkA and lower p75protein expression.
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