The Mechanism Of Neuroprotective Effect Of GLP-1/GIP Double-receptor Agonist On Cerebral Ischemia Reperfusion Injury In Diabetic Rats And Its Comparison With Liraglutide

Objective:1.To observe the neuroprotective effect(infarct size,m NSS score)of the novel GLP-1/GIP double-receptor agonist DA3-CH on diabetic cerebral ischemia reperfusion injury(I/R)rat model,and compare it with GLP-1 single-receptor agonist Liraglutide.2.To observe and compare the effects of DA3-CH and Lilaglutide on endoplasmic reperfusion stress and autophagy in diabetic cerebral ischemia reperfusion injury rats,so as to clarify the neuroprotective mechanism of DA3-CH and Lilaglutide on cerebral ischemia reperfusion injury in diabetic rats.Methods:Seventy-two male SD rats were randomly divided into 4 groups: blank group(Sham group,n=18),diabetic I/R model group(Saline group,n=18),diabetic I/R model group + liraglutide group(Lir group,n=18),and diabetic I/R model group +DA3-CH group(DA3 group,n=18).All the 4 groups were fed in the same environment.No special treatment was given in the Sham group,and the other 3 groups were given intraperitoneal injection with STZ for diabetes modeling.After successful modeling,the rats in the 4 groups were fed in the same environment for 7 days,and then the cerebral ischemia reperfusion injury model was constructed in the last 3 groups.4 groups of rats were fed in the same environment after 2hours of ischemia and reperfusion.DA3 group and Lir group were intraperitoneally injected with DA3-CH(10nmol/kg/d)and Liraglutide(10nmol/kg/d),respectively for 7 consecutive days.Sham group and Saline group were given equal volume normal saline at corresponding time points.7 days later,the blood glucose and m NSS scores in the tail vein of rats in each group were measured.Then,the rats in the 4 groups were killed,and the brains of the rats were taken by decapitation.The cerebral infarction area was measured by TTC staining method,and the percentage was obtained.Endoplasmic reticulum stress-related proteins(GRP78,CHOP)and autophagy related proteins(LC3,p62,beclin-1)were determined by immunohistochemical and Western Blot methods.Results:1.Blood glucose levels: The blood glucose levels of DA3 and Lir groups were lower than those of Saline group(Saline: 25.00 ±2.03,Lir: 19.50±1.00,DA3: 20.20±1.12).Compared with Saline group,there were significant differences in DA3 group(P < 0.001)and Lir group(P < 0.001),there were no significant differences in blood glucose levels between DA3 and Lir groups(P > 0.05).2.Infarct size: Compared with Sham,the infarct size of the other three groups was significantly different(P < 0.001),and that of DA3 and Lir groups was smaller than that of Saline group(Saline: 35.22 ±2.19,Lir: 28.65±6.55,DA3: 22.21±3.98).Compared with Saline group,there were significant differences in DA3 group(P < 0.001)and Lir group(P< 0.05)),the infarct size in DA3 group was smaller than that in Lir group(P < 0.05).3.Modified nervous system severity score(m NSS): m NSS scores of DA3 and Lir groups were lower than those of Saline group(Saline: 14.50±0.75,Lir: 11.50±9.37,DA3:9.70±0.38).Compared with Saline group,there were significant differences in DA3 group(P < 0.001)and Lir group(P < 0.001),m NSS scores in DA3 group were lower than those in Lir group(P < 0.001).4.Expression levels of endoplasmic reticulum stress-related proteins:Immunohistochemistry showed that the optical density of GRP78 in DA3 and Lir groups was lower than that in Saline group(Saline: 0.40±0.16,Lir: 0.24±0.05,DA3:0.11±0.02).Compared with Saline group,there were significant differences in DA3 group(P < 0.001)and Lir group(P < 0.01),the optical density of GRP78 in DA3 group was lower than that in Lir group(P < 0.05).CHOP optical density in DA3 and Lir groups was lower than that in Saline group(Saline: 0.45±0.13,Lir: 0.30±0.01,DA3: 0.18±0.01).Compared with Saline group,there were significant differences in DA3 group(P < 0.001)and Lir group(P < 0.01),CHOP optical density in DA3 group was lower than that in Lir group(P < 0.01).Western blotting showed that the expression level of GRP78 in DA3 and Lir groups was lower than that in Saline group(Saline: 0.97±0.03,Lir: 0.59±0.07,DA3: 0.36±0.06).Compared with Saline group,there were significant differences in DA3 group(P < 0.001)and Lir group(P < 0.001),the expression of GRP78 in DA3 group was lower than that in Lir group(P < 0.001).CHOP expression in DA3 and Lir groups was lower than that in Saline group(Saline: 1.15±0.16,Lir: 0.52±0.14,DA3: 0.32±0.08).Compared with Saline group,there were significant differences in DA3 group(P < 0.001)and Lir group(P < 0.001),CHOP expression in DA3 group was lower than that in Lir group(P < 0.05).5.Expression level of autophagy related proteins:Immunohistochemistry showed that Beclin-1 optical density in DA3 and Lir groups was lower than that in Saline group(Saline: 0.47±0.10,Lir: 0.26±0.05,DA3: 0.13±0.03).Compared with Saline group,there were significant differences in DA3 group(P < 0.001)and Lir group(P < 0.001),Beclin-1 optical density in DA3 group was lower than that in Lir group(P < 0.01).The optical density of p62 in DA3 and Lir groups was higher than that in Saline group(Saline: 0.03±0.03,Lir: 0.11±0.04,DA3: 0.22±0.06).Compared with Saline group,there were significant differences in DA3 group(P < 0.001)and Lir group(P < 0.05),the optical density of p62 in DA3 group was higher than that in Lir group(P < 0.01).LC3 optical density of DA3 and Lir groups was lower than that of Saline group(Saline: 0.40±0.07,Lir: 0.26±0.06,DA3: 0.13±0.03).Compared with Saline group,there were significant differences in DA3 group(P < 0.001)and Lir group(P < 0.001),LC3 optical density in DA3 group was lower than that in Lir group(P < 0.01).Western blotting showed that Beclin-1 expression in DA3 and Lir groups was lower than that in Saline group(Saline: 0.78±0.09,Lir: 0.33±0.07,DA3: 0.14±0.03).Compared with Saline group,there were significant differences in DA3 group(P < 0.001)and Lir group(P < 0.001),Beclin-1 expression in DA3 group was lower than that in Lir group(P < 0.001).The expression level of p62 in DA3 and Lir groups was higher than that in Saline group(Saline: 0.34±0.07,Lir: 0.52±0.06,DA3: 0.80±0.06).Compared with Saline group,there were significant differences in DA3 group(P < 0.001)and Lir group(P < 0.001),the expression of p62 in DA3 group was higher than that in Lir group(P < 0.001).The LC3II/LC3 I ratio of DA3 and Lir groups was lower than that of Saline group(Saline:2.09±0.13,Lir: 1.23±0.05,DA3: 0.48±0.08).Compared with Saline group,there were significant differences in DA3 group(P < 0.001)and Lir group(P < 0.001),LC3II/LC3 I expression levels in DA3 group were lower than those in Lir group(P < 0.001).Conclusion:1.The novel GLP-1/GIP double-receptor agonist DA3-CH can reduce the infarct size and m NSS score of diabetic cerebral ischemia reperfusion injury rat model,and has a significant neuroprotective effect,which is stronger than GLP-1 single-receptor agonist Liraglutide.2.DA3-CH and Liraglutide inhibit endoplasmic reticulum stress and autophagy levels in diabetic cerebral ischemia reperfusion injury rat models,which may be one of the mechanisms of their neuroprotective effects.