| Objective: HCN1 and HCN2 gene silencing rats was constructed by lentivirus vector to explore the effect of reducing the expression of HCN1 and HCN2 genes in rat on acute focal cerebral ischemia-reperfusion injury and related molecular mechanisms.Methods:(1)The experimental animals were allocated into four groups: Normol group,Scramble group,sh RNA-HCN1 group and sh RNA-HCN2 group.The lentiviral vectors were constructed by choosing suitable interference sequences for HCN1 and HCN2 gene sequences.The lentiviruses were injected into the right ventricle of 8-week-old SD rats by stereotaxic localization.The expression of GFP fluorescent protein in brain slices were observed by laser confocal microscopy after two weeks,and the expression of HCN1,HCN2 protein and m RNA were detected by immunoblotting and real-time fluorescent quantitative PCR to confirm the success of gene silencing rats model.(2)The rats model of middle cerebral artery occlusion(MCAO)were obstructed by nylon suture.The rats were allocated into five groups: Normol group,Sham+Scramble group,MCAO+Scramble group,MCAO+sh RNA-HCN1 group,MCAO+sh RNA-HCN2 group.(3)The neurobehavioral score and TTC staining were evaluated after 2 hours of ischemia and 22 hours of reperfusion.(4)The expression of apoptosis-related molecules included AIF,BCL-2,BAX,Caspase-3,P53 protein and m RNA were detected by real-time quantitative PCR and Western blotting after cerebral ischemia-reperfusion injury.RESULTS:(1)The results observed by laser confocal showed : In Normol group,few fluorescence was observed in brain tissue.Compared with Normol group,GFP fluorescent protein was high expressed in cortex and hippocampus of Scramble group,sh RNA-HCN1 group and sh RNA-HCN2 group.The results of western blot and real-time fluorescence quantitative PCR showed that the expression of HCN1 protein and m RNA in cortex and hippocampus of sh RNA-HCN1 group was lower than that of Scramble group(P<0.05).There was lower expression of HCN2 protein and HCN2 m RNA in cortex and hippocampus of sh RNA-HCN2 group compared with Scramble group(P<0.05).(2)The results of neurological score showed that: Compared with Sham+Scramble group,MCAO+Scramble group had higher neurological function score(P<0.01).Compared with MCAO+Scramble group,the neurological score of MCAO+sh RNA-HCN1 group was decreased(P<0.05),while the neurological score of MCAO+sh RNA-HCN2 group did not changed.The result of TTC staining showed that: Compared with Sham+Scramble group,the infarct volume of MCAO+Scramble group was increased(P<0.01).Compared with MCAO+Scramble group,the infarct volume of MCAO+sh RNA-HCN1 group and MCAO+sh RNA-HCN2 group was decreased after cerebral ischemia(P<0.05).(3)The results of Real-time fluorescence quantitative PCR showed that: a.In the hippocampus,Compared with Sham+Scramble group,the expression of NR1 m RNA in MCAO+Scramble group was no change,while the expression of NR2 A,NR2B,NR3 A and NR3 B m RNAs was decreased(P<0.05);Compared with the MCAO+Scramble group,the expression of NR1 m RNA in the MCAO+sh RNA-HCN1 group was decreased(P<0.05),the expressions of NR2 A and NR3 B m RNAs was increased(P<0.05),and the NR2 B and NR3 A m RNAs were no significant change.Compared with the MCAO+Scramble group,the expression of NR2 A m RNA in the MCAO+sh RNA-HCN2 group was increased(P<0.05),while the expression of NR1,NR2 B,NR3A,and NR3 B m RNAs were no significant change.b.In the cortex,compared with the Sham+Scramble group,the expression of NR1 m RNA in MCAO+Scramble group was increased(P<0.05),the expressions of NR2 A,NR2B and NR3 A m RNAs were decreased(P<0.05),and there was no significant change in the expression of NR3 B m RNA.Compared with MCAO+Scramble group,the expression of NR1 m RNA in MCAO+sh RNA-HCN1 group was decreased(P<0.05),and there was no significant change in the expressions of NR2 A,NR2B,NR3 A and NR3 B m RNAs.Compared with MCAO+Scramble group,the expression of NR1 m RNA was decreased in MCAO+sh RNA-HCN2 group(P<0.05),and the expressions of NR2 A,NR2B,NR3 A and NR3 B m RNAs was no significant changed.(4)The results of Real-time fluorescence quantitative PCR showed that: a.In the hippocampus,compared with the Sham+Scramble group,the expressions of AIF,BAX,Caspase-3 and P53 m RNAs in MCAO+Scramble group was increased(P<0.05),the expression of BCL-2 m RNA and the ratio of BCL-2/BAX was decreased(P<0.05).Compared with MCAO+Scramble group,the expression of AIF,BAX,Caspase-3 and P53 m RNAs in MCAO+sh RNA-HCN1 group was decreased(P<0.05),the expression of BCL-2 was increased(P<0.05),and the ratio of BCL-2/BAX was also increased(P < 0.05).Compared with MCAO+Scramble group,the expression of P53 m RNA was decreased in MCAO+sh RNA-HCN2 group(P<0.05),while the expression of AIF,BAX,BCL-2 and Caspase-3 m RNAs was no significant change(P<0.05).b.In the cortex,compared with Sham+Scramble group,the expression of AIF,BAX,BCL-2,Caspase-3 and P53 in MCAO+Scramble group was up-regulated(P<0.05),the expression of BCL-2 was down-regulated(P<0.05),and the ratio of BCL-2/BAX was decreased(P<0.01).Compared with MCAO+Scramble group,the expression of BAX,BCL-2,AIF,Caspase-3 and P53 m RNAs in MCAO+sh RNA-HCN1 group was no significant change.Compared with MCAO+Scramble group,the BAX m RNA in MCAO+sh RNA-HCN2 group was decreased(P<0.05),and the ratio of BCL-2/BAX was increased(P<0.05),while the expression of BCL-2,AIF,Caspase-3 and P53 m RNA did not changed.(5)The results of western blotting showed that: a.In the hippocampus,compared with the Sham+Scramble group,the expressions of BAX,AIF,Caspase-3 and P53 protein was increased(P<0.05).The expressions of BAX,AIF,Caspase-3 and P53 protein in the MCAO+sh RNA-HCN1 group was decreased compared with the MCAO+Scramble group(P<0.05).Similarly,compared with the MCAO+Scramble group,the expression of Caspase-3 protein in MCAO+sh RNA-HCN2 group was decreased(P<0.05),but there was no significant change in the expression of BAX,BCL-2,AIF,P53 protein.b.In the cortex,the expressions of BAX,AIF,Caspase-3,P53 protein was increased compared with the Sham+Scramble group(P<0.05).Compared with MCAO+Scramble group,there was no significant change in the expression of BAX,AIF,Caspase-3 and P53 protein of MCAO+sh RNA-HCN1 group.Compared with MCAO+Scramble group,the expression of BAX protein in MCAO+sh RNA-HCN2 group was decreased(P<0.05),but the expressions of AIF,Caspase-3 and P53 protein was no significant change.Conclusion:(1)Decreased expression of HCN1 gene in rat cortex and hippocampus could reduce the rat infarct volume and improve the rat neurological score after stroke by regulating the expressions of NDMA receptor subunits and apoptosis-related molecules.(2)Decreased expression of HCN2 gene could reduce the volume of cerebral infarction and increase the ratio of BCL/BAX protein after cerebral ischemia in rats,but the score of neurological function had no significant improvement. |
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