Study On The Regulatory Mechanism Of Homocysteine On Rat Post-stroke Depression Based On Synaptic Plasticity And Neural Stem Cell Autophagy

ObjectiveTo observe the effect of homocysteine(Hcy)on depressive behavior in SD rats by establishing rat model of middle cerebral artery occlusion(MCAO),and investigate the possible regulatory role of the NMDA receptor pathway and the autophagy related neurogenesis in cerebral ischemia caused by combined intervention of Hcy with NMDA blocker MK-801 and autophagy inhibitor 3MA,respectively.Primary neural stem cells were cultured in vitro to detect the effect of Hcy on NSCs activity by establishing oxygen-glucose deprivation/reoxygenation model.The specific regulatory mechanism of Hcy on OGD/R NSCs autophagy pathway was explored through the combined intervention of Hcy and PI3K-AKT(or ERK)-dependent mTOR signaling pathway activator.This study will provide the experimental basis and new ideas for the prevention and treatment of Hcy-related post-infarction depression.Methods120 adult male SD rats weighing 170-180 g were randomly divided into sham-operated group(SHAM),middle cerebral artery occlusion group(MCAO),MCAO+HCY group,MCAO+HCY+3MA group,MCAO+3MA group,MCAO+HCY+MK-801 group,and MCAO+MK-801 group.There were 12 rats in each group,while there were the 24 rats in SHAM,MCAO and MCAO+HCY group.The rats in SHAM and MCAO group were injected with saline at 4 mL/kg/d via tail veins.The rats in MCAO+HCY,MCAO+HCY+3MA,and MCAO+HCY+MK-801 group were injected with 0.8 mg/mL Hcy solution via tail veins.The rats in MCAO+MK-801 and MCAO+HCY+MK-801 group were intraperitoneally injected with 3 mg/kg MK-801 solution before 15 min of surgery.The sucrose preference test(SPT)and the open field test(OFT)were used to detect the depressive behavior after 14 d MCAO surgery.The level of 5-HT,DA and NE in rat plasma were measured by High performance liquid chromatography.Transmission electron microscopy was used to observe the number and structure of synapses in hippocampus.The expression of NR1,NR2 A,NR2B,PSD-95,SNAP-25,VGLUT1 and Complexin I/II were detected by Western blot.The rats in MCAO+3MA and MCAO+HCY+3MA group were injected with 5 mM of 3MA solution(4 mL/kg)via tail vein for 5 d before surgery.The expression of LC3 in NSCs in SVZ area was determined by immunofluorescence in 3 d and 7 d,respectively.LDH and MTT were used to measure the viability of NSCs in vitro.The expression of LC3,Beclin 1,p-mTOR,mTOR,p-PI3 K,PI3K,p-AKT,AKT,p-ERK and ERK of NSCs were detected by Western blot.ResultsCompared with the MCAO group,the result of OFT after Hcy treatment indicated that the rats horizontal movement and the number of standing were reduced and the sucrose preference index was significantly decreased,high performance liquid chromatography results showed that a significant decrease in 5-HT,DA and NE levels,western blot results showed that the expression of SNAP-25,PSD-95,VGLUT1 and Complexin I/II were significantly decreased and the expression of NR1,NR2 A and NR2 B were significantly increased(P<0.05).Compared with the MCAO+HCY group,the rats horizontal movement,the number of standing,the sucrose preference index,the 5-HT,DA and NE levels,the expression of SNAP-25,PSD-95,VGLUT1 and Complexin I/II were all significantly increased,while the NR1,NR2 A and NR2 B levels were significantly reduced(P<0.05).Immunofluorescent co-staining of LC3 and Nestin showed that the LC3 puncta in NSCs of SVZ region was significantly higher in the MCAO+HCY group,compared to the MCAO+HCY+3MA group(P<0.05).NSCs were cultured in vitro to establish cell OGD/R model.Western blot results showed that the expression of LC3 and Beclin 1 were significantly increased,and the expression of p-PI3 K,p-AKT,p-ERK and p-mTOR were significantly decreased in the OGD/R group and further decreased in OGD/R+HCY group.Compared with OGD/R+HCY group,the results of LDH and MTT indicated that proper inhibition of autophagy could increase the cell viability of NSCs(P<0.05).Co-treatment Hcy with MHY1485(an activator of mTOR),IGF-1(an activator of PI3K),TPA and curcumin(two activators of ERK),respectively,the western blot showed that compared with OGD/R+HCY group,the expression of p-mTOR protein in OGD/R+HCY+MHY1485 group increased,while the expression of LC3 and Beclin 1 significantly decreased,the expression of p-mTOR protein in OGD/R+HCY+TPA/CUR group significantly increased,the expression of p-AKT and p-mTOR protein in OGD/R+HCY+IGF-1 group significantly increased(P<0.05).Compared with OGD/R group,the expression of p-mTOR protein in OGD/R+MHY1485 group significantly increased,while the expression of LC3 and Beclin 1 significantly reduced;the level of p-mTOR protein in OGD/R+TPA/CUR group and the expression of p-AKT and p-mTOR protein in OGD/R+IGF-1 group were both significantly increased(P<0.05).ConclusionsThe model of cerebral ischemia-reperfusion in rats was successfully established.It was found that homocysteine could reduce the number of synapses,the levels of synaptic-related proteins,5-HT,DA and NE in MCAO rats,promote expression of the NMDA receptor subunits,such as NR1,NR2 A,NR2B,and induce depressive behavior.These effects above were all reversed by the administration of MK-801,a NMDA receptor inhibitor.It is suggested that Hcy may impact synaptic plasticity through the NMDA receptor subunits expression,and aggravate depressive symptoms.Hcy inhibited the cell viability by stimulating the overactivated autophagy of NSCs in the SVZ region of ischemic brain.After treatment of 3MA,the autophagy induced by Hcy was inhibited.It was possible that Hcy might aggravate depressive symptoms by damaging the neurogenesis via stimulating autophagy of NSCs.The OGD/R model of NSCs was successful y established in vitro.Results showed that homocysteine could raise the levels of LC3 and Beclin 1,reduce p-PI3 K,p-AKT,p-ERK and p-mTOR proteins expression,and decrease NSCs viability.The administration of 3MA increased the viability obviously.After the treatment of PI3 K and ERK activators,the downstream proteins expression of ERK-and PI3K-AKT-dependent mTOR pathway was restored.After administration of MHY1485,the expression of LC3 and Beclin 1 decreased,suggesting that Hcy may negatively regulate autophagy through ERK-and PI3K-AKT-dependent mTOR pathway.