【作者】 吴迪；

【导师】 任立群；

【作者基本信息】 吉林大学 ， 药理学， 2022， 硕士

【摘要】 动脉粥样硬化(Atherosclerosis,AS)是众多心脑血管病的主要发病原因,多发于中老年群体,严重危害人类健康。其发病过程复杂,与多种细胞密切相关。他汀类药物是治疗AS的常用药物,但因其普遍具有毒副作用且价格高昂,因此亟待寻找一种更加安全的治疗药物。微小RNA(micro RNA,miRNA)是非编码RNA(noncoding RNA,nc RNA)的一种。miRNAs参与转录后基因表达调控,调节着人类三分之一的基因。越来越多的研究证明,miRNAs参与AS等心血管疾病的发生发展,为我们寻找治疗AS的药物及靶标提供了新思路。淫羊藿苷(Icariin,ICA)是传统中药小檗科植物淫羊藿(Epimedium)的主要提取物,具有抗炎、抗氧化、保护心血管系统等功能,但其对AS的治疗作用尚不甚明确。研究表明趋化因子FKN(Fractalkine,CX3CL1)、脾酪氨酸激酶Syk及p38MAPK信号因子均与AS发生发展密切相关,而将FKN、Syk、p38MAPK级联,探讨其对ICA抗AS的介导作用尚未见报道。因此,本研究以FKN/Syk/p38MAPK信号通路为切入点,在体外细胞模型中研究ICA抗AS的作用及机制,为寻找ICA治疗AS的新靶点提供实验依据。本研究分为3个部分:(1)FKN/Syk/p38MAPK信号通路在ICA抑制ox-LDL诱导HAVSMCs增殖与迁移中的作用研究。以ox-LDL诱导HAVSMCs复制体外AS细胞模型,应用MTT、流式细胞术、划痕实验、Transwell实验、Western blot等技术探讨FKN/Syk/p38MAPK信号通路在ICA抑制ox-LDL诱导的HAVSMCs增殖和迁移中的作用。结果发现:(1)ICA及FKN受体CX3CR1的拮抗剂JMS-17-2均可抑制ox-LDL诱导的HAVSMCs增殖和迁移,且拮抗剂的抑制作用更强;(2)CX3CR1拮抗剂JMS-17-2可以显著抑制p-Syk/Syk、p-Src/Src、p-p38/p38蛋白表达,说明FKN是FKN/Syk/p38MAPK信号通路的上游分子;(3)ICA及CX3CR1拮抗剂JMS-17-2均可显著抑制FKN/Syk/p38MAPK信号通路相关蛋白表达,且拮抗剂有更强的抑制作用;(4)ICA通过调控FKN/Syk/p38MAPK信号通路抑制ox-LDL诱导HAVSMCs增殖和迁移。(2)ICA治疗ApoE-/-小鼠动脉粥样硬化的miRNA芯片生物信息学分析及miR-133a-5p/FKN/Syk/p38MAPK信号通路的构建。对课题组前期建立的ICA治疗ApoE-/-小鼠AS的miRNA表达谱进行分析,对靶向FKN的miRNAs进行预测,将miR-133a-5p作为研究对象,对其进行靶基因预测和KEGG富集分析发现,FKN/Syk/p38MAPK在其富集的通路中;结合位点预测发现,FKN m RNA的3’UTR处有miR-133a-5p的潜在结合位点,且靶向性较好,最终确定miR-133a-5p作为研究对象。构建出miR-133a-5p/FKN/Syk/p38MAPK信号通路。采用RT-qPCR法对预测得到的miR-133a-5p对FKN的靶向负调控作用进行初步验证,结果发现:miR-133a-5p可以靶向负调控FKN。(3)miR-133a-5p在ICA抑制ox-LDL诱导HAVSMCs增殖与迁移中的作用研究。采用转染技术构建miR-133a-5p瞬转HAVSMCs细胞株,采用MTT、流式细胞术、划痕实验、Transwell实验、RT-q PCR及Western blot等实验方法探讨miR-133a-5p在ICA抑制ox-LDL诱导HAVSMCs增殖和迁移的作用。结果发现:(1)过表达miR-133a-5p可下调FKN m RNA表达,沉默miR-133a-5p可上调FKN m RNA表达,进一步验证miR-133a-5p可靶向负调控FKN;(2)沉默miR-133a-5p可促进ox-LDL诱导的HAVSMCs增殖和迁移,并逆转ICA对ox-LDL诱导的HAVSMCs增殖和迁移的抑制作用;(3)沉默miR-133a-5p能显著下调miR-133a-5p水平,上调FKN m RNA表达及FKN、CX3CR1蛋白表达,使p-Syk/Syk、pSrc/Src、p-p38/p38蛋白表达显著增加,并逆转ICA对上述基因和蛋白表达的作用;(4)ICA对ox-LDL诱导的HAVSMCs增殖和迁移的抑制作用是通过上调miR-133a-5p抑制FKN/Syk/p38MAPK信号通路实现的。综上所述,得出结论:(1)FKN是FKN/Syk/p38MAPK信号通路的上游分子。FKN/Syk/p38MAPK信号通路在ICA抑制ox-LDL诱导HAVSMCs增殖与迁移中发挥重要作用。(2)通过对课题组前期研究得到的ICA治疗Apo E-/-小鼠AS的miRNA芯片进行生物信息学分析,选择miR-133a-5p作为研究对象,最终构建出miR-133a-5p/FKN/Syk/p38MAPK信号通路。(3)ICA抑制ox-LDL诱导的HAVSMCs增殖和迁移可能是通过miR-133a-5p/FKN/Syk/p38MAPK信号通路实现的。miR-133a-5p可作为ICA抑制ox-LDL诱导HAVSMCs增殖与迁移的分子靶点。更多还原

【Abstract】 Atherosclerosis(AS)is the main cause of many cardiovascular and cerebrovascular diseases,which frequently occurs in middle-aged and elderly groups and seriously endangers human health.Its pathogenesis is complex and closely related to a variety of cells.Statins are commonly used in the treatment of AS,but due to their general toxic side effects and high price,it is urgent to find a safer treatment drug.microRNA(miRNA)is a kind of noncodingRNA(ncRNA).miRNAs are involved in the regulation of post-transcriptional gene expression and regulate one-third of human genes.More and more studies have proved that miRNA is involved in the occurrence and development of cardiovascular diseases such as AS.It provides a new idea for us to search for drugs and targets to treat AS.Icariin(ICA)is the main extract of Epimedium from berberis,which has antiinflammatory,antioxidant and cardiovascular protection functions.However,its therapeutic effect on AS remains unclear.Studies show that chemokine FKN(Fractalkine,CX3CL1),splenic tyrosine kinase Syk and p38 MAPK signal factor are closely related to the occurrence and development of AS,while the cascade of FKN,Syk and p38 MAPK to explore their mediating effect on ICA against AS is not been reported.Therefore,this study takes the FKN/Syk/p38 MAPK signaling pathway as the entry point to study the effect and mechanism of ICA inhibits AS in vitro cell model,so as to provide experimental basis for finding new targets for ICA treatment of AS.This research is divided into 3 parts:(1)The role of FKN/Syk/p38 MAPK signaling pathway in ICA inhibiting the proliferation and migration of HAVSMCs induced by ox-LDL.Using ox-LDL to induce HAVSMCs to replicate HAVSMCs in vitro AS cell models.MTT,flow cytometry,wound healing,Transwell assay and Western blot assay were used to investigate the role of FKN/Syk/p38 MAPK signaling pathway in ICA inhibiting the proliferation and migration of HAVSMCs induced by ox-LDL.The results showed that:(1)ICA and FKN receptor CX3CR1 antagonist JMS-17-2 could inhibit the proliferation and migration of HAVSMCs induced by ox-LDL,and the antagonist effect was stronger;(2)CX3CR1 antagonist JMS-17-2 could significantly inhibit p-Syk/Syk,p-Src/Src,p-p38/p38 protein expression,indicating that FKN is the upstream molecule of FKN/Syk/p38 MAPK signaling pathway;(3)ICA and CX3CR1 antagonist JMS-17-2 can significantly inhibit FKN/Syk/p38 MAPK signaling pathwayrelated protein expression,and antagonists have stronger inhibitory effects;(4)ICA inhibits ox-LDL-induced HAVSMCs proliferation and migration by regulating FKN/Syk/p38 MAPK signaling pathway.(2)Bioinformatics analysis of miRNA chip for ICA treatment of Apo E-/-mouse AS and construction and verification of miR-133a-5p/FKN/Syk/p38 MAPK signaling pathwayAnalyze the miRNA expression profile of the pre-constructed ICA treatment of Apo E-/-mouse AS by the research group,and the miRNAs targeting FKN were predicted and analyzed,and finally determined miR-133a-5p as the research object.KEGG enrichment analysis found that FKN/Syk/p38 MAPK is in its enriched pathway;binding site prediction found that there is a potential binding site of miR-133a-5p at the 3’UTR of FKN m RNA,which has good targeting ability.Therefore,miR-133a-5p/FKN/Syk/p38 MAPK signaling pathway was constructed.The predicted negative regulation effect of miR-133a-5p and FKN was preliminarily verified by RT-q PCR.The results showed that miR-133a-5p could target and negatively regulate FKN.(3)The role of miR-133a-5p in ICA inhibiting the proliferation and migration of HAVSMCs induced by ox-LDL.Transfection technology was used to construct miR-133a-5p transiently transfected HAVSMCs cell lines,and MTT,flow cytometry,wound healing,Transwell assay,RT-q PCR and Western blot assays were used to investigate the effect of miR-133a-5p on ICA inhibitiing of ox-LDL-induced proliferation and migration of HAVSMCs.The results showed that:(1)miR-133a-5p mimics can down-regulate FKN m RNA expression,and miR-133a-5p inhibitor can up-regulate FKN m RNA expression,further verifying that miR-133a-5p can target and negatively regulate FKN;(2)miR-133a-5p inhibitor can promote ox-LDL induced HAVSMCs proliferation and migration,and reversed the inhibitory effect of ICA for ox-LDL-induced HAVSMCs proliferation and migration;(3)miR-133a-5p inhibitor can significantly down-regulate the level of miR-133a-5p,up-regulate FKN m RNA expression and FKN and CX3CR1 proteins,the expression of p-Syk/Syk,p-Src/Src,p-p38/p38 protein were significantly increased,and the effect of ICA on the expressions of the above genes and proteins was reversed;(4)ICA inhibits the proliferation and migration of HAVSMCs induced by ox-LDL by up-regulating miR-133a-5p and inhibiting the FKN/Syk/p38 MAPK signaling pathway.From the above,it is concluded that:(1)FKN is an upstream molecule of FKN/Syk/p38 MAPK signaling pathway.FKN/Syk/p38 MAPK signaling pathway plays an important role in ICA inhibiting the proliferation and migration of HAVSMCs induced by ox-LDL.(2)Through the bioinformatics analysis of the miRNA chips for ICA treatment Apo E-/-mice obtained in the previous study of the research group,miR-133a-5p was selected as the research object,and miR-133a-5p/FKN/Syk/p38 MAPK signaling pathway was finally constructed..(3)The inhibition of ox-LDL-induced proliferation and migration of HAVSMCs by ICA may be achieved through the miR-133a-5p/FKN/Syk/p38 MAPK signaling pathway.miR-133a-5p can be used as a molecular target of ICA to inhibit the proliferation and migration of HAVSMCs induced by ox-LDL.更多还原