| ObjectiveVascular dementia is a cognitive dysfunction syndrome caused by a variety of cerebrovascular diseases.As a chronic progressive disease,the incidence of vascular dementia in China has increased year by year in recent years.At present,there is no prevention and long-term effective treatment for vascular dementia.Studies have shown that Siwu decoction has a certain degree of therapeutic effect.It can alleviate cognitive dysfunction of vascular dementia model rats,but the research on this protective mechanism is not sufficient.Vascular dementia can lead to the decline of learning and memory ability.It can lead to cognitive dysfunction and affect the expression of synapse-associated proteins.As a small extract of Siwu decoction,petroleum ether fraction of Siwu decoction can pass through the blood-brain barrier.In our study,we used petroleum ether part of Siwu decoction as the drug-administering component to explore the therapeutic effect of this component on vascular dementia.In this study,we established VD model rats and screened the specific expressed miRNAs to explore whether petroleum ether fraction of Siwu decoction treating vascular dementia by regulating miRNA.MicroRNA(miRNAs)as an endogenous non-coding small RNA are closely related to brain development and function.They also play an irreplaceable role in various cerebrovascular diseases.We further explored the mimic experiment of miR-134-5p for VD model rats by Morris water maze experiment.We used molecular biology experiments to further explore the downstream target genes of miR-134-5p and its related mechanism of synapse-associated protein reduction,and also clarify the direct targeting of miR-134-5p and Foxp2;Siwu decoction.petroleum ether site regulats miR-134-5p and the regulation relationship of miR-134-5p/Foxp2/Synl proved that the petroleum ether part of Siwu decoction and miRNAs may be involved in the treatment of vascular dementia.Methods1.We extracted the petroleum ether part of Siwu decoction and selected 220～250g male SD rats for the experiment.VD rats were established by permanent ligation of bilateral common carotid artery by 2V0 method.Rats were randomly divided into Control group,VD model group,Siwu decoction petroleum ether high-dose group,Siwu decoction petroleum ether low-dose group.Morris water maze test was performed after administration.After taking the cerebral cortex for miRNA gene chip analysis.Fresh cortex and hippocampus tissue were harvested and the expression of miR-134-5p in cortex and hippocampus of VD model rats were detected by RT-qPCR assay.2.We selected 220～250g male SD rats for the experiment.VD rats were established by permanent ligation of bilateral common carotid artery by 2V0 method.Rats were randomly divided into Control group,VD model group and miR-134-5p antagomir group.Morris water maze test was performed after the intracerebroventricular injection of miR-134-5p antagomir.The expression of synapse-associated proteins in cortex of rats which related to cognitive dysfunction in each group was detected by RT-qPCR assay and Western Blotting.3.The petroleum ether fraction of Siwu decoction was extracted,and different concentrations of drugs were added to PC12 cells which were added serum-free treatment.The expression of miR-134-5p in the petroleum ether part of Siwu decoction groups were detected by RT-qPCR assay.In vitro experiments were carried out to verify whether the drug affected the expression of miR-134-5p.4.miR-134-5p target genes were predicted and screened by multiple bioinformatics websites.Construction of rno-miR-134-5p mimic and inhibitor,as well as negative control of each group were transfected into PC12 cells.The expression of target gene(Foxp2)was detected by Western Blot assay.Dual luciferase reporter vector containing Foxp2-3’ UTR and rno-miR-134-5p binding site was constructed.Dual luciferase reporter vector combined with miR-134-5p mimic and inhibitor and its negative control were co-transfected into PC12 cells to further verify the targeting relationship between miR-134-5p and Foxp2.5.miR-134-5p mimic,inhibitor and their negative control were transfected into PC12 cells.We used Western Blot assay to detect the expression of synaptic-associated proteins.We constructed and transfected siRNA fragment of Foxp2 into PC12 cells.The expression of synaptic-associated protein were detected by Western Blot assay.Then ChIP experiment was carried out to verify the targeting relationship between Foxp2 and its downstream target gene Synl.Results1.VD model rats were established by permanent ligation of bilateral common carotid artery(2V0).Animal intraperitoneal injection of Siwu decoction petroleum ether site.The main chemical constituents of Siwu decoction petroleum ether were analyzed by GC-MS assay.The results of miRNA gene chip showed that there are 56 miRNAs in the cortex of the high dose group of Siwu decoction petroleum ether decreased significantly compared with the VD model group(P<0.05).It proved that the petroleum ether part of Siwu decoction had a targeted effect of miR-134-5p.Morris water maze test showed that the petroleum ether fraction of Siwu decoction could alleviate cognitive dysfunction of vascular dementia rats.Compared with Control group,the escape latency of VD model was significantly increased and the times of platform area crossing times were significantly decreased(P<0.001).It proved that VD model rats were established successfully.Compared with VD model group,the escape latency of high dose group and low dose group of Siwu decoction were decreased(P<0.001),and the times of platform area crossing were increased(P<0.05).2.VD model rats were established by permanent ligation of bilateral common carotid artery(2V0).The results of miRNA gene chip showed that there are 46 miRNAs in the cortex of VD model group increased significantly compared with the Control group(P<0.05).Morris water maze test showed that inhibition of miR-134-5p expression could alleviate cognitive dysfunction of vascular dementia rats.Compared with Control group,the escape latency of VD model was significantly increased and the times of platform area crossing times were significantly decreased(P<0.01).It proved that the establishment of vascular dementia rats were successful.Compared with the VD model group,the escape latency of miR-134-5p antagomir group were decreased(P<0.01)and the times of the platform area crossing were increased(P<0.05).3.Western Blot assay showed that the expression of Foxp2 and synaptic-associated protein in cortex tissue of VD model group were significantly increased than Control group(P<0.05).Compared with the VD model group,the expression of Foxp2 and synaptic-associated protein of miR-134-5p antagomir group were significantly increased(P<0.001).4.Dual luciferase reporter assay showed that miR-134-5p mimic decreased luciferase activity of pLUC-Foxp2 after co-transfection of miR-134-5p and pLUC-Foxp2(P<0,001),and miR-134-5p inhibitor increased the luciferase activity of pLUC-Foxp2(P<0.001).The targeting between miR-134-5p and Foxp2 was preliminarily verified.Western Blot assay showed that compared with its negative control group,the expression of Foxp2 in miR-134-5p mimic group was decreased(P<0.01),and the expression of Foxp2 in miR-134-5p inhibitor group transfected group was increased(P<0.05).5.miR-134-5p mimic,inhibitor and their negative control were transfected into PC12 cells.We used Western Blot assay to detect the expression of synaptic-associated proteins.Western Blot showed that the expression of synaptie associated protein was inhibited in miR-134-5p mimic group(P<0.05)and increased in miR-134-5p inhibitor group(P<0.05).We constructed and transfected siRNA fragment of Foxp2 into PC12 cells.After silencing Foxp2,the expression of synapse一associated protein was al5o inhibited(P<0.01).Chip assay confirmed that Foxp2 acts as a transcription factor binding to Synl promoter region-specific binding.It proved that Synl may be one of the downstream targets of Foxp2.Conclusion1.The petroleum ether fraction of Siwu decoction can improve the cognitive function of VD model rats and regulate the specific expression of miR-134-5p in the cortex of VD model rats.2.Low expression of miR-134-5p can improve the cognitive function of VD model rats by regulating the expression of synapse-associated proteins.3.Foxp2 is a potential target gene of miR-134-5p.4.miR-134-5p/Foxp2/Synl axis affects the cognitive impairment of vascular dementia. |
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