| Objective1.To observe the effects of Xuesaitong injection on the neurological function of rats with cerebral middle artery occlusion(MCAO)after 28 days treatment,and to explore the effects of Xuesaitong injection on LINGO-1,EGFR,PI3K/AKT pathway,PTEN and GSK-3? in the damaged cortex.2.To observe the neuroprotective effect of Xuesaitong injection on oxygen and glucose deprivation reperfusion(OGD/R)damaged SH-SY5Y cells and to verify the effects of Xuesaitong injection and bpV on PTEN,PI3K/AKT pathway,and GSK-3? in OGD/R cultured SH-SY5Y cells.Methods1.MCAO model of SD rats was prepared by the modified Longa method.Rats in the sham-operated group did not insert the suture,only incised the skin and bluntly separated blood vessels and nerves.The successful establishment of MCAO model was evaluated by the TTC staining.2.According to the Longa score after surgery,the rats in the operated group were randomly separated into model,Xuesaitong low-dose,Xuesaitong high-dose and nimodipine groups(ie positive drug group).Different groups were given corresponding drug therapy.The sham and model group rats were ip.injected with the same dose sodium chloride.From the first day to the 28th day after the operation,all rats were weighed every morning before the administration,and scored according to the mNSS neurological function score table.To evaluate the effect of Xuesaitong on neurological function and weight recovery of rats at different time points(7,14 and 28 days)after MCAO.On days 28 after surgery,the rats were sacrificed after anesthesia and the rat brains were collected.The pathological changes of cerebral cortex in rats were observed by HE staining,the protein expressions of SYN and PSD-95 in the infarcted side of the cortex was detected by Western blot to explore the neuroprotective effect of Xuesaitong injection on cortical infarction area.qRT-PCR,immunohistochemistry(IHC)and Western blotting were applied to detect the protein and mRNA levels of LINGO-1,EGFR,PI3K,and AKT in the cerebral cortex of rats on day 28 after MCAO,and to explore the neuroprotective mechanism of Xuesaitong.3.qRT-PCR and Western Blot were used to detect the mRNA and protein expressions of PTEN and GSK-3? in the cerebral cortex of rats on day 28 after MCAO,and to further explore the neuroprotective mechanism of Xuesaitong.4.SH-SY5Y cells were cultured in vitro,CCK-8 was used to determine the optimal hypoxia time.After establishment of OGD/R damaged SH-SY5Y cell model.The optimal concentrations of Xuesaitong and bpV were determined by CCK-8.The SH-SY5Y cells were randomly separated into normal,model,Xuesaitong,bpV and Xuesaitong+bpV groups.The appropriate drugs were given according to the different groups.The normal group was given serum-free EBSS medium,and the model group was given sugar-free Earle's medium.The effects of Xuesaitong on the growth status of OGD/R injured SHSY-5Y cells were observed under microscope.The mRNA and protein levels of PI3K,AKT,PTEN,and GSK-3? in SH-SY5Y cells damaged by OGD/R were detected by western blotting and qRT-PCR to further verify the vivo experiments,and observe that when the activity of PTEN is inhibited,the expressions of PI3K/AKT pathway and GSK-3?.Results1.TTC staining displayed that the right brain of the sham-operated rat was uniformly red,indicating that there was no damage to the brain tissue;while the right brain tissue of the operated-rat was widely white,indicating that MCAO could induce infarction.The results of TTC staining showed that the MCAO model was successfully established.2.Before modeling,the body mass of rats in each group has no evident difference(P>0.05).The body weight of rats in the operated groups were obviously lower than that of the sham group on the 7th and 14th day after MCAO(P<0.05 or P<0.01).Compared to the sham group,the body weight of rats in all treatment groups had no obvious difference on the 28th day after MCAO(P>0.05).The body mass of the same group at different time points displayed that the weight of rats in the sham group on day 14 was higher than that on day 7,and there was no evidently difference between the two time points in the other groups(P>0.05).The body weight of rats in all groups on the 28th day was evidently higher than that of the rats in the same group on the 14th day(P<0.01).There was no significant difference in the EZ-Longa score between the rats in each group after waking up in the operation group(P>0.05).Compared with the sham group,rats in the operated group had obvious neurological damage(P<0.01).On day 7,compared to the model group,rats in Xuesaitong high-dose group had lower mNSS scores(P<0.05).The mNSS scores in all treatment groups were evidently lower compared to that in the model group after 14 days treatment(P<0.05).After 28 days treatment,the mNSS scores of rats in Xuesaitong treatment groups were obviously lower compared to that in the model group(P<0.05 or P<0.01);and compared to the the other two treatment groups,the mNSS scores in the Xuesaitong high-dose group were evidently lower(P<0.05).These results indicated that Xuesaitong could improve the neurological function of MCAO rats in a dose-dependent manner.The mNSS scores in the same group on days 7,14,and 28 after MCAO displayed that there was no evident difference of the mNSS scores between the 7th day and the 14th day(P>0.05);Except for the nimodipine group,the mNSS score on day 28 was obviously higher than that on day 14 in the same group(P<0.05).On day 28 after MCAO,HE staining showed that the morphology of brain tissue and cell structure in the sham group were intact.The brain tissues of model group showed large area damage,shallow tissue staining,nerve cell necrosis,nuclear pyknosis and deep staining.The brain tissues in the treatment groups were also damaged to some extent,however,compared to the model group,the degree of brain tissue injury was mild and more nerve cells were found.On 28 days after MCAO,compared to the model group,the expressions of PSD-95 in the treated groups were higher levels(P<0.01).Comparing each treatment,it was found that the expressions of PSD-95 in Xuesaitong high-dose group was evidently higher than that in Xuesaitong low-dose group and Nimodipine group(P<0.05 or P<0.01).Compared to the model group,except for Xuesaitong low-dose group,the SYN protein levels in other groups were higher(P<0.05 or P<0.01).Xuesaitong high-dose group had obvious advantage in improving SYN protein level compared to that in the Xuesaitong low-dose group(P<0.05).At 28 days after MCAO,qRT-PCR results showed that the expressions of LINGO-1,EGFR,PI3K and AKT mRNA had no difference in all groups(P>0.05).The results of IHC showed that the positive color of LINGO-1 was dark brown,and the positive LINGO-1 expression was very lower in the sham group.Compared to the model group,the positive LINGO-1 expressions in the treatment groups was evidently reduced(P<0.01).The level of LINGO-1 protein among all treatment groups had no obvious difference(P>0.05).The positive expressions of P-EGFR,P-PI3K and P-AKT were dark brown.The expressions of P-EGFR,P-PI3K and P-AKT in cerebral cortex in sham group were low.Compared to the model group,the P-EGFR,P-PI3K and P-AKT protein levels in all treatment groups were obviously up-regulated(P<0.01),there was no obvious difference between the treatment groups(P>0.05).Western blotting displayed that the expressions of LINGO-1 protein in the treatment groups was evidently decreased compared to the model group(P<0.01),keeping levels close to normal.There was no obvious difference in the expression of LINGO-1 among the treatment groups(P>0.05).The levels of P-EGFR,P-PI3K and P-AKT in treatment groups were higher than those in model group(tended to be higher,or P<0.05,or P<0.01).There was no obvious difference between the treatment groups(P>0.05).3.At 28 days after MCAO,the results of qRT-PCR showed compared to the sham group,the PTEN mRNA level of Xuesaitong high-dose group was evidently higher(P<0.05);and the PTEN mRNA level in other groups had no evident difference(P>0.05).The GSK-3? mRNA level among all groups had no obvious difference(P>0.05).Western blotting displayed that the protein levels of the P-PTEN/PTEN and P-GSK-3?/GSK-3? in the treatment groups were obviously higher than those in the model group(P<0.05 or P<0.01),there was no evident difference in the protein levels of P-PTEN/PTEN and P-GSK-3?/GSK-3? among all the treatment groups(P>0.05).4.The optimal hypoxia time of SH-SY5Y cells measured by CCK-8 was 7 h.The optimal drug concentrations of Xuesaitong and BPV to SH-SY5Y cells were 20 ug/mL and 1 umol/L,respectively.The effect of Xuesaitong,bpV and Xuesaitong+bpV on the growth status of SH-SY5Y cells injured by OGD/R was observed under microscope.Compared to the model group,the number of adherent cells in Xuesaitong,bpV and Xuesaitong+bpV groups was more,and most of the synapses still existed.There was no evident difference in cell morphology between the treatment groups.After SH-SY5Y cells were cultured for OGD 7 h/R 24 h,the results of qRT-PCR showed that there was no obvious difference in mRNA expressions of PI3K,AKT,PTEN and GSK-3? in normal,model,Xuesaitong,bpV and Xuesaitong+bpV groups(P>0.05).Western blotting displayed that the P-PI3K/PI3K,P-AKT/AKT,P-PTEN/PTEN,and P-GSK-3?/GSK-3? protein levels in all treatment groups were obviously higher than those in the model group(P<0.05,or P<0.01),and P-PI3K/PI3K,P-AKT/AKT,P-PTEN/PTEN and P-GSK-3?/GSK-3? among all treatment groups had no evident differences(P>0.05)Conclusions1.Xuesaitong could alleviate brain tissue damage in MCAO rats and promote nerve remodeling and regeneration,thereby increasing the body weight and improving the nerve function of MCAO rats2.The neuroprotective mechanisms of Xuesaitong included inhibition of LINGO-1 level,up-regulation levels of EGFR and PI3K/AKT signaling pathway,and inhibition of PTEN expression,further activation of PI3K/AKT pathway to inhibit the GSK-3? level,and then promotion of synaptic remodeling and regeneration.3.Xuesaitong could alleviate the damage of SH-SY5Y induced by OGD/R.By inhibiting the activity of PTEN,it activated the downstream PI3K/AKT/GSK-3? signaling pathway,thereby exerting long-term neuroprotective effects. |
PDF Full Text Request