Based On The MiR-133a-5p/FKN/Syk/p38MAPK Signaling Pathway To Study The Effect Of Icariin In Inhibiting The Proliferation And Migration Of HAVSMCs Induced By Ox-LDL

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.