Regulatory Effect Of Panax Notoginseng Saponins On Lingo-1 And EGFR/PI3K/AKT Pathways In MCAO Rats And SH-SY5Y Cells After Hypoxia-glucose Injury

Objective1.To explore the neuroprotective effect of Panax notoginseng saponins in SD rats who sufferded MCAO surgery and regulatory effect of Panax notoginseng saponins on Lingo-1 and EGFR/PI3K/AKT signal transduction pathways.2.To explore the protective effect of panax notoginseng saponins on hypoxia-ischemia in SH-SY5Y cells and regulatory effect of Panax notoginseng saponins on Lingo-1 and EGFR/PI3K/AKT signal transduction pathways.Methods1.In vivo:Rats were randomly divided into sham and surgery groups.The modified Longa method was used to prepare rat middle cerebral artery occlusion(MCAO)model and TTC staining for model assessment.The consciousness of the rats in the surgery group wasrestored.Longa scores were performed approximately 3 hours after surgery.1-3 points were included,and 0 points and 5 points were excluded.The rats were randomly divided into model group,PNS high dose group,PNS low dose group,nimodipine group.In the sham-operated group,rats were anesthetized with 3.5%chloral hydrate,and the median neck skin was incised.The muscles were bluntly dissected and CCA,ECA,and ICA were exposed.The skin was then sutured,iodophors were sterilized,and the insulation blanket was warmed until regained consciousness.The neurological scores of the rats in surgery groups were approximately the same(the neurological impairment was roughly the same).According to the equivalent measurement of body surface area of rats and humans,combined with previous studies of the study group members,PNS high-dose group was injected intraperitoneally with 7.2 mg/100 g;PNS low-dose group was injected intraperitoneally with 3.6 mg/100 g daily;nimodipine was 1.44 mg/100g gavage;sham operation group and model group were intraperitoneally injected with saline 1mL/100g daily.On day 1-7 postoperatively,neurological function scores were performed on the mNSS scale.The body weights were measured and body mass index was calculated.The brain protection of PNS was explored by Western Blot method to measure the protein of SYN and PSD95 in the cerebral cortex.The effects of Panax notoginseng saponins on the myelin inhibitory factor Lingo-1 and EGFR/PI3K/AKT signaling pathways in the infarcted side of the cerebral cortex 7 days after MCAO were further investigated by real-time PCR,immunohistochemistry and Western Blot.2.in vitro:Different hypoxia times and concentrations of Panax notoginseng saponins were set.The cell survival rate was determined by CCK8.The hypoxia time was 16 h and Panax notoginseng saponin concentration was 640 mg/L.Set grouping:normal group,model group,PNS group,PNS+AG1478 group,and AG1478 group.In accordance with the group given drug treatment and hypoxia treatment,CCK8 method was used to detect the survival rate of each group of cells,Annexin V-FITC/PI double staining method was used to measure SH-SY5Y cell apoptosis,and LDH activity was measured.The effects of panax notoginseng saponins on the signaling pathways of Lingo-1 and EGFR/PI3K/AKT in ischemic and hypoxic SH-SY5Y cells were further verified by qRT-PCR and Western Blot.Results1.TTC staining:The brain sections of sham-operated rats were homogeneous and bright red,and no ischemic lesions were seen.In the model group,continuous white infarcts were seen on the right side of the brain slices,and the left side was homogeneous bright red.The results show successful modeling.BMI evaluation:On the 1st day after surgery,the body mass index of the sham-operated group increased slightly,and the body mass index of the rats in each surgery group decreased significantly.Compared with the sham group,the model group decreased significantly(P<0.01).On the second and third postoperative days,the body mass index of the rats in the sham group continued to increase,and the body mass index of the rats in each group continued to decrease.There was no significant difference between the treatment groups and the model group(P>0.05).On the fourth postoperative day,although the body mass index of each surgical group continued to decrease,the body mass index of the small-dose PNS group(P<0.05)was higher than that of the model group;the high-dose PNS group(P<0.01)and the nimodipine group(P<0.01)Body mass index was significantly higher than that of the model group,and the difference was statistically significant.On the 6th day after operation,the body mass index of the low-dose PNS group(P<0.01)was significantly different from that of the model group.On the 7th day after surgery,the body mass index of PNS high-dose group began to stop falling.Evaluation of mNSS neurological function scores:In the sham-operated group,neurological deficit symptoms did not appear within 1-7 days.On the 1st day after operation,all the rats in the surgery group had severe symptoms of neurological deficit.The neurological function scores were all around 11 points,indicating that the degree of modeling was uniform.At 1 to 3 days after surgery,the mNSS neurological function scores of the rats in each surgery group gradually decreased,and there was no difference between the drug groups and the model group(P>0.05).On the 4th day after operation,the neurological score of mNSS in the nimodipine group was lower than that in the model group(P<0.05).The neurological function score of the nNSS in the high dose PNS group was significantly lower than that in the model group(P<0.01).On the fifth day after operation,the mNSS neurological function score of the low-dose PNS group was lower than that of the model group(P<0.05).The neurological function score of the mNSS in the nimodipine group was significantly lower than that of the model group(P<0.01).2.After 7 days of cerebral infarction in SD rats,the expression of SYN and PSD95 protein in the infarcted side of the cerebral cortex decreased significantly.At 7 days after operation,the expression of SYN and PSD95 in the cerebral cortex of the PNS low-dose group,high-dose PNS group,and nimodipine group was observed.Although the amount was lower than the sham group,it was significantly higher than the model group,and the difference was statistically significant(P<0.01).3.The contents of Lingo-1,EGFR,PI3K,and AKT in the cerebral cortex of the MCAO rats were detected by qRT-PCR after 7 days:The mRNA expression of Lingo-1 in the model group was significantly higher than that in the sham group after 7 days of SD rats.The difference was statistically significant(P<0.01).The expression of Lingo-1 mRNA in PNS low dose group,PNS high dose group and nimodipine group was significantly lower than that in model group(P<0.01),and the mean value of Lingo-1 mRNA in high dose PNS group was lower.In the PNS low dose group and nimodipine group.At 7 days after operation,the expression of EGFR,PI3K,and AKT mRNA in the rats of each surgery group increased slightly,but there was no statistical significance(P>0.05).Western Blot assay was used to detect the effects of Lingo-1 and p-EGFR/EGFR,p-PI3K/PI3K and p-AKT/AKT protein expression in MCAO rat brain:7 days after MCAO in SD rats,model group Lingo-1 Compared with the sham group,the expression of protein was significantly increased,and the difference was statistically significant(P<0.01).The expression of Lingo-1 protein in PNS low dose group,PNS high dose group and nimodipine group was significantly lower than that in model group.The average expression level of Lingo-1 protein in the large-dose PNS group and the nimodipine group was lower than that in the PNS low-dose group.At 7 days after MCAO in SD rats,the expression of p-EGFR/EGFR protein in the model group was significantly higher than that in the sham group(P<0.01).The expression of p-EGFR/EGFR protein was significantly higher in PNS low dose group,PNS high dose group,and nimodipine group than in model group.The mean levels of p-EGFR/EGFR protein expression in large doses of PNS and nimodipine were higher than those in small doses of PNS.The expression of p-PI3K/PI3K protein in the model group was significantly higher than that in the sham group(P<0.01).The expression of p-PI3K/PI3K protein in PNS low dose group,PNS high dose group and nimodipine group was significantly higher than that in model group.The average expression of p-PI3K/PI3K protein in PNS high dose group and nimodipine group was higher than that in PNS low dose group.At 7 days after MCAO in SD rats,the expression of p-AKT/AKT protein in the model group was significantly higher than that in the sham group(P<0.01).The expression of p-AKT/AKT protein in PNS low dose group,PNS high dose group and nimodipine group was significantly higher than that in model group.The average expression of p-AKT/AKT protein in PNS high dose group and nimodipine group was higher than that in PNS low dose group.Immunohistochemistry was used to measure the expression of Lingo-1,p-EGFR,p-AKT,and p-PI3K in the brain of MCAO rats 7 days after operation:The iinmunopositive substance was stained with dark brown particles,which was consistent with the Western Blot trend.4.With the prolonged hypoxia and hypoglycemia time of SH-SY5Y cells,the cell survival rate gradually decreased.16h was selected as the optimal hypoxic time.At this time,The cell survival rate is about 60%,which could not only cause oxygen-glucose deprivation injury,but also ensure the survival rate of most cells.When the PNS concentration was 640 mg/L,the cell survival rate was the highest,so the concentration of PNS was 640 mg/L as the concentration of the model drug.Set experimental groups:normal group,model group,PNS group,PNS+AG1478 group,and AG 1478 group.The results of CCK8 showed that oxygen-glucose deprived injury caused severe damage to SH-SY5Y cells,resulting in a cell survival rate of about 63.3%.PNS can effectively protect SH-SY5Y cells and increase cell viability(80.6%),compared with the model group.There was a statistically significant difference(P<0.01).When PNS group cells were added with AG1478,the protective effect of PNS on SH-SY5Y cells was weakened,and the survival rate was reduced to 65.9%.With AG1478 alone,the survival rate of the cells was significantly lower than that of the model group,and the difference was statistically significant(P<0.01).Effects of PNS on Apoptosis in SH-SY5Y Cells after Oxygen Deprivation and Hypoglycemia Treatment:After SH-SY5Y cells were injured by oxygen-glucose deprivation,the apoptosis rate increased significantly.PNS significantly decreased the apoptosis rate of the cells,and the difference was statistically significant(P<0.01).When PNS group cell culture medium was added with AG1478,the protective effect of PNS was obviously inhibited.Lactate dehydrogenase(LDH)activity assay results:Oxygen-glucose deprived injury caused severe damage to SH-SY5Y cells,and the LDH activity in the culture medium was significantly increased(P<0.01).PNS can effectively protect SH-SY5Y cells and reduce the LDH activity in the culture medium.Compared with the model group,PNS has statistical significance(P<0.01).When PNS group cells were added with AG1478,the protective effect of PNS on SH-SY5Y cells was weakened.When AG1478 alone was used,the LDH activity in the culture medium was significantly higher than that in the model group(P<0.01).5.Effects of Panax notoginseng saponins on the Expression of Lingo-1 and EGFR/PI3K/AKT mRNA in SH-SY5Y Cells Induced by Hypoxia and Hypoglycemia:Expression of Lingo-1 mRNA in SH-SY5Y Cells After Hypoxia and Hypoglycemia Injury Compared with the increase,the difference was statistically significant(P<0.01).The expression of Lingo-1 mRNA in PNS and AG1478+PNS group SH-SY5Y cells was significantly lower than that in the model group,and the difference was statistically significant(P<0.01).The expression of Lingo-1 mRNA in AG1478 group was not different from that in the model group.After SH-SY5Y cells were injured by oxygen-glucose deprivation,there was no difference in EGFR,PI3K,AKT mRNA expressions between the sham-operation group,PNS group,PNS+AG1478 group and AG1478 group and the model group.Effect of Panax notoginseng saponins on the Expression of Lingo-1,p-EGFR/EGFR,p-PI3K/PI3K and p-AKT/AKT Proteins in SH-SY5Y Cells after Hypoxia and Hypoglycemia The expression of Lingo-1 protein in the model group was significantly higher than that in the sham group(P<0.01).The expression of Lingo-1 protein in PNS group SH-SY5Y cells was significantly lower than that in the model group,and the difference was statistically significant(P<0.01).The expression level of Lingo-1 protein in the SHISY5Y cells of AG1478+PNS group was lower than that of the model group,and the difference was statistically significant(P<0.05).There was no difference in expression of Lingo-1 protein between the AG1478 group and the model group.After SH-SY5Y cells were injured by oxygen-glucose deprivation,the ratios of p-EGFR protein to EGFR total protein in the model group were significantly higher than those in the sham group(P<0.01).The ratio of p-EGFR protein to EGFR total protein in PNS group was significantly higher than that in model group,and the difference was statistically significant(P<0.01).The ratio of p-EGFR protein to EGFR total protein in the PNS+AG1478 group was not different from that in the model group(P>0.05).The ratio of p-EGFR protein to EGFR total protein in AG1478 group was significantly lower than that in the model group,and the difference was statistically significant(P<0.01).After SH-SY5Y cells were injured by hypoxia and hypoglycemia,the ratio of p-PI3K protein to PI3K total protein in the model group was significantly higher than that in the sham group(P<0.01).The ratio of p-PI3K protein to PI3K total protein in the PNS group was significantly higher than that in the model group,and the difference was statistically significant(P<0.01).The ratios of p-PI3K protein and PI3K total protein in PNS+AG1478 group and AG1478 group were not statistically significant compared with the model group(P>0.05).After SH-SY5Y cells were injured by oxygen-glucose deprivation,the ratio of p-AKT protein to AKT total protein in the model group was significantly higher than that in the sham-operated group(P<0.01).The ratio of p-AKT protein to AKT total protein in PNS group was higher than that in model group,and the difference was statistically significant(P<0.05).The ratio of p-AKT protein to AKT total protein in PNS+AG1478 group was not statistically significant compared with the model group(P>0.05).The ratio of p-AKT protein to AKT total protein in the AG1478 group was significantly lower than that in the model group,and the difference was statistically significant(P<0.01).ConclusionPanax notoginseng saponins have neuroprotective effect on MCAO rats and hypoxic-ischemic SH-SY5Y cells:Panax notoginseng saponins can promote the increase of body mass index and recovery of neurological function in MCAO rats,and increase the cerebral cortex SYN.The expression of PSD95 can increase the survival rate of ischemic and hypoxic SH-SY5Y cells,inhibit apoptosis and decrease the activity of lactate dehydrogenase.Panax notoginseng saponins can exert brain protection by decreasing the expression of myelin inhibitor Lingo-1 and promoting the activation of EGFR/PI3K/AKT signaling pathway.