摘要
目的探讨温阳复元方对miRNA-137/线粒体铁死亡通路的调控作用,阐明该方对脑缺血再灌注损伤(CIRI)的神经保护作用机制。方法线栓法制备大鼠CIRI模型,将144只SD大鼠随机分为假手术(SO)组、模型(I/R)组、补阳还五汤对照(BYHW)组、温阳复元方(WYFY)组、温阳复元方+miRNA-137模拟物(WYFY+mimic)组、温阳复元方+miRNA-137抑制物(WYFY+Inhibitor)组,每组按再灌注时间点分为3个亚组。采用Zea-Longa评分法进行神经功能缺损评分(NFS);TTC染色测量脑梗死体积;qRT-PCR和Western blot法观察miRNA-137、膜铁转运蛋白(FPN)和二价金属离子转运体1(DMT1)的mRNA及其蛋白表达水平。结果(1)NFS结果:I/R组NFS较SO组显著增加(P<0.01),与I/R组相比,BYHW组NFS仅在12 h时评分降低(P<0.05),而WYFY组在24 h和3 d的NFS明显降低(P<0.05或P<0.01),WYFY+mimic组NFS显著下降(P<0.01),WYFY+Inhibitor组在3 d时NFS降低(P<0.05);与WYFY组相比,WYFY+mimic组NFS仅在12 h时明显降低(P<0.05)。(2)TTC染色结果:I/R组梗死体积较SO组显著增大(P<0.01),BYHW组和WYFY组较I/R组明显减小(P<0.05或P<0.01),WYFY组梗死体积较BYHW组进一步减小(P<0.01);相较于WYFY组,WYFY+mimic组能进一步减小其梗死体积(P<0.05),而WYFY+Inhibitor组梗死灶明显增大(P<0.05)。(3)miRNA-137 mRNA表达水平:I/R组miRNA-137mRNA的表达较SO组显著下降(P<0.01),WYFY治疗后其表达量显著增加(P<0.01),且WYFY组其表达水平较BYHW组进一步增高(P<0.01);与WYFY组同期比较,WYFY+mimic组miRNA-137 mRNA表达量均进一步升高(P<0.01),WYFY+Inhibitor组抑制该表达(P<0.01)。(4)FPN mRNA及蛋白表达水平:I/R组FPN mRNA和蛋白表达水平较SO组均降低(P<0.01),WYFY组能显著增加其表达(P<0.01),该组在12 h和3 d时的表达量较BYHW组进一步增加(P<0.05或P<0.01);与WYFY组同期对比,WYFY+mimic组其表达水平在24 h及3 d时增加(P<0.01),而WYFY+Inhibitor组其表达水平均降低(P<0.01)。(5)DMT1 mRNA及蛋白表达水平:I/R组DMT1mRNA和蛋白表达量较SO组均显著增加(P<0.01);与I/R组比较,WYFY能降低其表达水平(P<0.01),且WYFY组在3 d时其表达量较BYHW组进一步降低(P<0.05);与WYFY组同期相比,WYFY+mimic组其表达水平在24 h和3 d时均降低(P<0.01),而WYFY+Inhibitor组其表达水平均显著增加(P<0.01)。结论温阳复元方可能通过上调miRNA-137的表达,进而上调铁转运蛋白FPN的表达,下调DMT1的表达,调节铁代谢,最终抑制线粒体铁死亡,发挥其神经保护作用,其疗效优于补阳还五汤。
Objective To investigate the regulatory effect of Wenyang Fuyuan Decoction(温阳复元方)on miRNA-137/mitochondrial ferroptosis pathway,and to clarify the neuroprotective mechanism of this recipe on cerebral ischemia-reperfusion injury(CIRI).Methods The rat CIRI model was prepared by suture method,and 144 SD rats were randomly divided into sham operation group(SO),model group(I/R),Buyang Huanwu Decoction(补阳还五汤)control group(BYHW),Wenyang Fuyuan Decoction group(WYFY),Wenyang Fuyuan Decoction+miRNA-137 mimic group(WYFY+mimic),Wenyang Fuyuan Decoction+miRNA-137 inhibitor group(WYFY+inhibitor),each group was divided into 3 subgroups according to reperfusion time point.Zea-Longa score was used to score neurological deficit(NFS);TTC staining was used to measure the volume of cerebral infarction;qRT-PCR and Western blot were used to observe the mRNA and protein expression levels of miRNA-137,FPN and DMT1.Results①NFS results:Comparison with SO group,NFS in the I/R group was significantly increased(P<0.01),compared with the I/R group,the score of the BYHW group was only decreased at 12 h(P<0.05),while the NFS of the WYFY group was significantly decreased at 24 h and 3 d(P<0.05 or P<0.01),and the NFS of the WYFY+mimic group was significantly decreased(P<0.01),NFS decreased in WYFY+inhibitor group at 3 d(P<0.05);compared with the WYFY group,the NFS of the WYFY+mimic group was significantly decreased only at 12 h(P<0.05).②TTC staining results:Comparison with SO group,the infarct volume in the I/R group increased significantly(P<0.01);Compared with the I/R group,the infarct volume of the BYHW group and the WYFY group was significantly reduced(P<0.05 or P<0.01),and the infarct volume of the WYFY group was further reduced compared with the BYHW group(P<0.01);compared with the WYFY group,the WYFY+mimic group could further reduce the infarct volume(P<0.05),while the WYFY+Inhibitor group significantly increased the infarct volume(P<0.05).③The expression level of miRNA-137 mRNA:Comparison with SO group,the expression of miRNA-137 mRNA in I/R group was significantly decreased(P<0.01),and its expression was significantly increased after WYFY treatment(P<0.01),and its expression level in WYFY group was further increased than that in BYHW group(P<0.01);compared with the WYFY group during the same period,the mRNA expression levels of the WYFY+mimic group were further increased(P<0.01),while the WYFY+Inhibitor group inhibited the expression(P<0.01).④FPN mRNA and protein expression levels:Comparison with SO group,the expression levels of FPN mRNA and protein in the I/R group were decreased(P<0.01),and the WYFY group could significantly increase its expression(P<0.01),and the expression level of this group at 12 h and 3 d was further increased than that of BYHW group(P<0.05,P<0.01);compared with the WYFY group during the same period,the expression level of WYFY+mimic group increased at 24 h and 3 d(P<0.01),while the expression level of the WYFY+inhibitor group decreased(P<0.01).⑤The expression level of DMT1 mRNA and protein:Comparison with SO group,the expression levels of DMT1 mRNA and protein in the I/R group were significantly increased(P<0.01);compared with the I/R group,WYFY could reduce its expression level(P<0.01),and the expression level of this group was further decreased than that of the BYHW group at 3 d(P<0.05);compared with the WYFY group at the same period,the expression level of WYFY+mimic group decreased at 24 h and 3 d(P<0.01),while the expression level of WYFY+inhibitor group was significantly increased(P<0.01).Conclusion Wenyang Fuyuan Decoction may play its anti-oxidative stress role by up-regulating the expression of miRNA-137,and then up-regulating the expression of iron transporter FPN,down-regulating the expression of DMT1,regulating iron metabolism,finally inhibiting mitochondrial ferroptosis,and playing its neuroprotective role,its curative effect is better than Buyang Huanwu Decoction.
作者
邓秋媚
吴林
袁莉
陈炜
黄秋霞
胡跃强
DENG Qiumei;WU Lin;YUAN Li;CHEN Wei;HUANG Qiuxia;HU Yueqiang(Guangxi University of Chinese Medicine,Nanning 530001,Guangxi,China;The First Affiliated Hospital of Guangxi University of Chinese Medicine,Nanning 530023,Guangxi,China)
出处
《辽宁中医药大学学报》
CAS
2024年第2期31-36,共6页
Journal of Liaoning University of Traditional Chinese Medicine
基金
国家自然科学基金项目(81973768)
广西自然科学基金重点项目(2019GXNSFDA245006)
广西中医脑病临床研究中心项目(桂科AD20238028)。
