| Objective1.To investigate the neuroprotective effect of Xuesaitong in middle cerebral artery occlusionrats,as well as the regulatory effect of lingo-1 and its downstream EGFR/PI3K/Akt signaling pathway and BDNF.2.To explore the in vitro protective effects of Xuesaitong on oxygen and glucose deprivation/reoxygenation SH-SY5Y cells,as well as its regulatory effects on the PI3K/Akt signaling pathway and BDNF.Methods1.The right middle cerebral artery focal infarction(MCAO)model was established by modifiedLonga method,using the male Sprague-dawley rats.After the rats were awakened,EZ Longa scale was used for scoring,and 1-3 points were included in the experiment.Then the rats were randomly divided into model group,low-dose PNS group,large-dose PNS group and nimodipine group.In the sham operation group,the skin of rat was incised along the middle of the neck to expose the common carotid artery,internal carotid artery and external carotid artery,and the skin was sutured.TTC staining method was used to evaluate the effect of MCAO modeling.2.According to the previous study and the conversion of equivalent drug dose between rats andhuman body surface area,the drug administration methods of rats in each group were as follows:1 mL/100g of normal saline was injected into rats intraperitoneally daily in the sham operation group and rats in the model group.The rats in the low-dose PNS group received intraperitoneal injection of Xuesaitong 3.6 mg/100g per day.The rats in the high-dose PNS group were given intraperitoneal injection of Xuesaitong 7.2 mg/100g per day.The rats in nimodipine group were given 1.44 mg/100g nimodipine daily by gavage.(1)During 1-14 days after operation,the general state,neurofunctional score and weight of the rats were observed and recorded daily.(2)After 14 days of modeling,brain tissues of rats were taken for HE staining to observe the pathological morphology of brain tissues.The protein expression of PSD95 and SYN in rats was detected by Western Blot,and the expression of BDNF was observed by quantitative real-time PCR(qRT-PCR)and immunohistochemical staining.(3)qRT-PCR,immunohistochemical staining and Western Blot were used to explore the effect of Xuesaitong on Lingo-1 and its downstream EGFR/PI3K/Akt signaling pathway3.To cultivate and establish the SH SY5Y cells oxygen-glucose deprivation/reoxygenation(OGD/R)model.Cell viability was determined by CCK8 assay to determine the optimal OGD time and the optimal intervention concentration.(1)The cultured cells were divided into three groups:normal group,model group,PNS group.The OGD/R model was established in the model group and the PNS group in advance.The expression of BDNF was detected by qRT-PCR and Western Blot.(2)The cultured cells were divided into five groups:normal group,model group,PNS group,PNS+LY294002 group and LY294002 group.In addition to the normal group,each group was given appropriate OGD/R treatment and appropriate drug intervention.qRT-PCR and Western Blot were used to detect the regulatory effect of Xuesaitong on PI3K/Akt signaling pathway.Results1.TTC staining showed that continuous pale infarcts were shown on the infarcted side(right side)of the MCAO surgery group,and the uniform red color on the left,indicated successful modeling.Body mass index:There was no significant difference in the weight among each group before surgery(P>0.05).On the 1st,3rd,7th and 14th day after the operation,the body mass index of rats in the model group,low-dose PNS group,high-dose PNS group and nimodipine group decreased significantly compared with the sham operation group(P<0.01).The body mass index of rats in the sham operation group gradually increased,which,on the contrary,gradually decreased over time in the model group and the low-dose PNS group.The body mass index of rats in the high-dose PNS group and nimodipine group gradually decreased from days 1 to 7,and maintained stability after 7 days without decreasing.The body mass index of rats in the sham operation group and the model group on the 14th day after surgery was significantly different from that on the first day after surgery.The body mass index of rats in the sham operation group was significantly increased(P<0.01),which was significantly decreased(P<0.05)in the model group.No significant change was observed in other groups(P>0.05).mNSS score:There was no significant difference in EZ Longa score in operation groups(P>0.05).The mNSS scores of low-dose PNS group,high-dose PNS group and nimodipine group were all higher than those in the sham operation group on days 1,3,7 and 14(P<0.01),and the mNSS scores of MCAO rats in each group gradually decreased.Compared with the model group,mNSS score of the high-dose PNS group was significantly reduced(P<0.05)on day 3.mNSS scores of MCAO rats in the model group,low-dose PNS group,high-dose PNS group and nimodipine group were significantly lower(P<0.05,P<0.01)on day 14.The difference of mNSS score on day 1 and day 14 in the high-dose PNS group was significantly higher than that in the model group(P<0.05).2.14 days after MCAO,PSD95 protein expression of model group was significantly decreased compared with the sham operation group(P<0.05).By contrast,PSD95 protein expression significantly increased in high-dose PNS group and nimodipine group compared with model group(P<0.05).It is also of average higher in low-dose PNS group compared with model group,but with no statistical difference(P>0.05).SYN protein expression of model group was significantly decreased compared with the sham operation group(P<0.05),SYN protein expression of high-dose group significantly increased compared with model group(P<0.01).The mRNA expression levels of BDNF in model group,low-dose PNS group,high-dose PNS group and nimodipine group were significantly lower than those in sham operation group(P<0.01).Compared with the model group,the mRNA expression of BDNF in the high-dose PNS group was significantly increased(P<0.01).Immunohistochemical staining results showed that immunopositive substances in the brain tissues of rats in the PNS high-dose group and nimodipine group were more widely distributed than those in the model group.3.qRT-PCR was used to detect the mRNA expressions of Lingo-1,EGFR,PI3K and Akt in rats after MCAO surgery on day 14.The mRNA expressions of Lingo-1 in model group was significantly higher than those in sham operation group(P<0.05).The mRNA expression of Lingo-1 in the high-dose PNS group and nimodipine group was significantly lower than that in the model group(P<0.01).The mRNA expression of Lingo-1 in the low-dose PNS group was not significantly lower than that in the model group(P>0.05).mRNA expression levels of EGFR,PI3K and Akt in model group,low-dose PNS group,high-dose PNS group and nimodipine group were higher than those in sham operation group,but there was no statistical difference(P>0.05).Western Blot was used to detect the expression of Lingo-1,p-EGFR/EGFR,p-PI3K/PI3K and p-Akt/Akt proteins in the rats 14 days after MCAO:14 days after MCAO surgery,Lingo-1 protein expression level of rats in the model group was significantly higher than that in the sham operation group(P<0.01),the expression level of Lingo-1 protein in the high-dose PNS group and nimodipine group was significantly lower than that in the model group(P<0.05).The expression level of p-EGFR/EGFR protein in the model group was reduced compared with the sham operation group(P<0.05).The ratio of p-EGFRJEGFR in high-dose PNS group and nimodipine group increased significantly comparing with the model group(P<0.05).The ratio of p-PI3K/PI3K protein in the model group was significantly lower than that in the sham operation group(P<0.05),and the ratio of p-PI3K/PI3K protein in the low-dose PNS group was significantly higher than that in the model group(P<0.05).The ratio of p-Akt/Akt protein in the model group was significantly lower than that in the sham group(P<0.01).The expression of p-Akt/Akt in the high-dose PNS group and nimodipine group was significantly higher than that in the model group(P<0.05).The expression of Lingo-1,p-EGFR,p-PI3K and p-Akt in the brain tissues of rats were detected by immunohistochemical staining.The immunopositive substances were stained dark brown and distributed in granular or spotty patterns,and the expression trend was consistent with Western Blot.4.According to the determination results of CCK8,7h was selected as the optimal oxygen-glucose deprivation time,and the survival rate of SH-SY5Y cells was 62.36%.The cells were not only damaged by OGD/R,but also ensured the vitality of most cells.The survival rate of SH-SY5Y cells was the highest when the Xuesaitong concentration was 20?g/mL.Effect of Xuesaitong on BDNF expression in SH-SY5Y cells of OGD/R:After OGD/R injury in SH-SY5Y cells,the mRNA expression of BDNF in the model group was significantly lower than that in the normal group(P<0.05).The mRNA expression of BDNF in the PNS group was significantly higher than that in the model group(P<0.01).Compared with the normal group,the BDNF protein expression in the model group was significantly decreased(P<0.05).The BDNF protein expression in the PNS group was significantly increased compared with the model group(P<0.01).5.Effect of Xuesaitong on PI3K/Akt pathway of SH-SY5Y cells with OGD/R injury:The cells were divided into five groups:the normal group,the model group,the PNS group,the PNS+LY294002 group and the LY294002 group.After OGD/R injury of SH-SY5Y cells,there was no difference in the expression of Akt mRNA among the normal group,the PNS group,the PNS+LY294002 group,the LY294002 group and the model group(P>0.05).The expression of p-Akt/Akt protein in the model group was significantly increased compared with normal group(P<0.05).p-Akt/Akt protein expression of PNS group was significantly increased compared with model group(P<0.05).p-Akt/Akt protein expression in LY294002 group was significantly reduced compared with model group(P<0.01).Conclusions1.Xuesaitong plays a role in promoting the growth of body mass index,improving nerve function,increasing the expression of SYN,PSD95 and BDNF in MCAO rats,and promoting the formation of synapses and the recovery of nerve function in cerebral infarction rats.2.Xuesaitong inhibits the overexpression of Lingo-1 in rats with cerebral infarction and activates the EGFR/PI3K/Akt signaling pathway by improving its phosphorylation level.It plays a neuroprotective role in rats.3.Xuesaitong could improve the activity of OGD/R SH-SY5Y cells,enhance the expression of BDNF and activate the PI3K/Akt signaling pathway.It plays a neuroprotective role in promoting the survival,regeneration of damaged nerve cells. |
PDF Full Text Request