Mechanism Of Panax Notoginseng Regulating Mir-370-3p/IRAK2/NF-κB Pathway To Improve Vascular Endothelial Function Injury In Hyperlipidemia

Atherosclerosis is an important pathological basis of cardiovascular and cerebrovascular diseases.In the initial stage of AS,vascular endothelial disorders induced by the accumulation of oxidized low-density lipoprotein are biomarkers and/or effects of THE occurrence and progression of AS.Improving vascular endothelial function is of great significance for the prevention and treatment of atherosclerosis.Panax Notoginseng（Burk.）F.H.Chen is a traditional medicine that activates blood circulation and resolves blood stasis,stops bleeding and tonifies deficiency.However,the key genes and the molecular mechanisms involved in its regulation are still unclear.In view of this,the aim of this study was to explore the effects of Panax notoginseng on vascular endothelial injury in hyperlipidemia and its related mechanisms by using bioinformatics methods combined with animal experiments and clinical trials,and to provide an objective basis for the clinical application of Panax notoginseng in the prevention and treatment of AS by improving vascular endothelial injury.ObjectiveA bioinformatic approach was used to screen the core genes of vascular endothelial injury,their upstream miRNAs and downstream effector molecules,and to construct key gene regulation relationships of endothelial injury.The mechanism was investigated in an in vivo environment by using animal experiments at different levels,including "markers of vascular endothelial injury-histomorphology-protein expression-gene expression",and was re-validated in clinical trials to clarify the mechanism of Panax ginseng’s effect on improving vascular endothelial injury in hyperlipidemia.MethodsPartⅠ: Exploring the core genes of endothelial dysfunction based on a combination of bioinformatics approachesThe GSE137578（platform number GPL21185）microarray was identified to meet the requirements of the study by searching the study title,screening the experimental design and reviewing the sample information and references.The expression matrix of the microarray was extracted using the Limma package and the data were normalised and visualised to ensure that the changes in gene expression in subsequent experiments were biologically meaningful.The WGCNA bioinformatics algorithm was used to functionally classify the genes in the GSE137578 microarray data by constructing a weighted gene coexpression network,hierarchical clustering analysis and sample feature association analysis,and to analyse and identify core gene sets（modules）that are highly correlated with vascular endothelial injury.To further explore the biological mechanisms and potential functions of the core gene sets,the GSEA method was applied to analyze the expression profiles in the gene sets as a whole,and the genes with high |ES| scores in The Leading-Edge Subset were used as the core genes for subsequent analysis.The KEGG database was searched for the signalling pathways involved in the regulation of IRAK2,and the biological mechanisms involved in its regulation were predicted in the light of previous studies in the literature.Part Ⅱ: Predictive validation of miRNA molecules that regulate genes central to vascular endothelial function impairmentThe miRNA target miRNAs of the core gene IRAK2 were predicted by miRNA bioinformatics prediction databases such as Target Scan,star Base and miRDB,combined with literature to screen miRNAs.miRNAhybrid database was used to predict the binding sites of miRNAs to IRAK2,and Blast was used to compare hasIRAK2 and The sequence similarity between has-IRAK2 and mus-IRAK2 was compared to confirm whether they were homologous.The binding relationship between IRAK2 and the target potential miRNA was verified by a dual luciferase gene reporter assay to lay the foundation for subsequent animal experiments.The KEGG database was searched for signaling pathways involved in IRAK2,and the possible pathways of IRAK2 involvement in vascular endothelial function injury were analyzed to predict the downstream regulation mode of IRAK2.Part Ⅲ: Effects of Panax Notoginseng on ox-LDL-induced endothelial dysfunction via miR-370-3p/IRAK2/NF-κB pathwayAn animal model of hyperlipidemia in APOE^-/-mice was constructed.The hyperlipidemic mice were given Panax ginseng powder by gavage to observe the effect of Panax ginseng on the improvement of endothelial impairment and the effect of Panax ginseng on the expression of effector molecules in the miR-370-3p/IRAK2/NF-κB pathway;the tail vein of the hyperlipidemic mice was also injected with miR-370-3p adeno-associated virus to establish an animal model of miR-370-3p overexpression and inhibition.The miR-370-3p inhibitory animal model was administered with Panax ginseng powder for 3 weeks,and the expression trends of markers of vascular endothelial function and upstream and downstream effector molecules were observed in this group of mice and the overexpressing group,so as to explore the mechanism of Panax ginseng’s action on miR-370-3p/IRAK2/NF-κB pathway to regulate vascular endothelial function.The study was carried out in the context of the "vascular endothelial function" approach.The study aims to explore this mechanism at different levels,including "markers of vascular endothelial injury-histomorphology-proteins-genes" : the improvement of endothelial injury was observed by measuring changes in ox-LDL levels in peripheral blood and markers of vascular endothelial injury（NO,ET-1,VWF,s VCAM and s ICAM-1）.The improvement in endothelial ultrastructure was observed by endothelial transmission electron microscopy;the morphological changes of endothelial pathology and protein expression were observed by HE,Masson pathological analysis and immunohistochemistry;the expression of proteins and genes in the miR-370-3p/IRAK2/NF-κB pathway was observed by Western blot and RT-q PCR.The changes of protein and gene expression in miR-370-3p/IRAK2/NF-κB pathway were observed by Western blot and RT-q PCR.In this study,we investigated the effect of Panax ginseng on improving vascular endothelial injury through miR-370-3p/IRAK2/NF-κB pathway and the mechanism.Part Ⅳ: Clinical study on the mechanism of vascular endothelial function impairment in hyperlipidemic patients through miR-370-3p/IRAK2/NF-κB pathway intervention by Panax notoginsengA before-and-after controlled clinical trial was designed to include patients with hyperlipidaemia who met the diagnostic criteria for dyslipidaemia in Chinese adults（Revised 2020）.The subjects were treated with Panax ginseng powder for 3 weeks.The expression of miR-370-3p,IRAK2,NF-κB and other genes in peripheral blood mononuclear cells before and after the administration of Panax ginseng was measured by RT-q PCR to further validate the mechanism at the clinical level.Results:PartⅠ1.WGCNA combined with GSEA enrichment analysis yielded the gene set Reactome signaling by interleukins as a significantly enriched gene set for ox-LDL-induced vascular endothelial function impairment.2.Enrichment analysis of the gene set Leading-Edge Subset of Reactome signaling by interleukins revealed that the gene IRAK2,with |ES|=0.565,had the highest enrichment score and was the core gene in this gene set,i.e.the gene that plays a central role in the biology of vascular endothelial function.3.IRAK2 is involved in the biological process of vascular endothelial injury.3.IRAK2 is involved in the regulation of multiple pathways centred on the Lipid and atherosclerosis（map05417）signalling pathway and is a key protein in the NF-κB signalling pathway.PartⅡ1.Bioinformatics combined with literature studies predicted that hsa-miR-370-3p is an IRAK2 target miRNA.hsa-miR-370-3p was predicted to have potential binding sites to the 993 and 330 seed regions of IRAK through the RNAhybrid database.This theoretically suggests that hsa-miR-370-3p may be a potential target miRNA for IRAK.2.The results of the dual luciferase reporter system showed that mmu-miR-370-3p binds to the 3’ UTR of the IRAK2 gene and that miR-370-3p specifically acts at this site to repress the expression of the IRAK2 gene.Blast comparison of has-IRAK2 and mus-IRAK2 sequences showed that has-IRAK2 and The sequence comparison between has-IRAK2 and mus-IRAK2 showed that has-IRAK2 and mus-IRAK2 are homologous genes and both can perform the same biological function,therefore,we can continue the animal experiments.3.The scientific hypothesis is that the miR-370-3p/IRAK2/NF-κB pathway may be a key pathway in vascular endothelial function impairment.Part Ⅲ1.Panax notoginseng can improve vascular endothelial function damage,affect miR-370-3p/IRAK2/NF-κB regulation level.Compared with the model group,Panax notoginseng could improve the expressions of ox-LDL,s ICAM,NO,s VCAM and v WF,and TC and other lipid indexes of vascular endothelial injury markers.It shows that Panax notoginseng can improve endothelial injury and reduce the adhesion of inflammatory cytokines;at the level of histomorphology,HE and Masson staining and perspective electron microscope observation showed that Panax notoginseng had an improvement effect on vascular endothelium and its ultrastructure.At the protein expression level,immunohistochemistry showed that Panax notoginseng reduced the expression levels of IRAK2 and NF-BP65 in aortic vessels;WB results showed that Panax notoginseng could reduce the phosphorylation levels of TAK1,IB and NF-κB protein,thereby inhibiting the activation and transduction of NF-B signaling pathway.Panax notoginseng can up-regulate the expression of miR370,down-regulate the expression of IRAK2,NF-B,TAK1 and IB,and affect the expression of miR-370-3p/ IRAK2/NF-κB pathway at the molecular level.2.Panax notoginseng can improve vascular endothelial function damage by regulating miR-370-3p/IRAK2/NF-κB pathway.Compared with the mmu-miR-370-3p AVV inhibition vector model and the AVV overexpression vector model,both of them could improve endothelial function damage index and blood lipid index,and improve tissue morphology and endothelial ultrastructure at the histomorphology level.Upregulated miR370 expression and down-regulated IRAK2 and NF-B expression at gene level;at the protein level,the phosphorylation levels of TAK1,IB and NF-κB were down-regulated,and the activation and transduction of NF-B signaling pathway were inhibited,thereby reducing the expression of s ICAM and s VCAM cell adhesion molecules.Part Ⅳ1.Through the pre-and post-clinical controlled exploratory experiment,it was found that Panax notoginseng had good efficacy and clinical safety in improving vascular endothelial injury in clinical subjects.Panax notoginseng can improve vascular endothelial injury and reduce the adhesion of s ICAM to s VCAM cell inflammatory factors.It can also reduce blood lipid indexes such as TC and increase HDL level.There is no significant change in safety indexes before and after treatment.2.Panax notoginseng can regulate the gene expression of effector molecules in miR-370-3p/IRAK2/NF-κB pathway,which is consistent with the conclusion of animal experiments,indicating that the mechanism of Panax notoginseng improving endothelial function injury through miR-370-3p/IRAK2 /NF-κB pathway has also been verified at the clinical level.ConclusionAnalysis of IRAK2 as a core gene with a central role in the biology of vascular endothelial injury was performed by combined mining of multiple bioinformatics algorithms.IRAK2/NF-κB is a key pathway for vascular endothelial function impairment,and miR-370-3p may be a key target miRNA upstream of IRAK2.dual luciferase reporter assays validated that IRAK2 is a target gene for mmu-miR-370 The double luciferase reporter assay verified that IRAK2 is a target gene of mmumiR-370-3p,and that mmu-miR-370-3p can bind to the 3’ UTR of IRAK2 gene to inhibit IRAK2 gene expression.The scientific hypothesis that the miR-370-3p/IRAK2/NF-κB pathway may be a key pathway in vascular endothelial function impairment and that Panax ginseng may regulate this pathway to improve vascular endothelial function impairment was then explored and validated.By constructing a mouse model of APOE^-/-hyperlipidemia and tail vein injection of miR-370-3p adeno-associated viral overexpression and suppression vector,we established a mouse model of miR-370-3p overexpression and suppression of APOE^-/-hyperlipidemia,and investigated the scientific hypothesis from the levels of "histomorphology-markers of vascular endothelial injury-protein expression-gene expression "We found that Panax ginseng could regulate the miR-370-3p/IRAK2/NF-κB pathway to improve vascular endothelial function in hyperlipidemia,and that upregulation of miR-370-3p could target IRAK2 expression and reduce NF-κB phosphorylation to reduce s ICAM and s VCAM cell adhesion factor chemotaxis to ameliorate endothelial cell injury and exert a protective effect.This mechanism was further validated at the clinical level.By comparing the expression changes of miR-370-3p,IRAK2 and NF-κB,markers of vascular endothelial injury and target genes in peripheral blood mononuclear cells in clinical hyperlipidemic subjects before and after the administration of Chinese herbal medicine Panax ginseng,we found that the expression levels of miR-370-3p increased compared with those before treatment,and the expression levels of IRAK2,TRAF6,TIRAP,TLR2,TAK1,I B and NF-κB expression levels were decreased compared with those before treatment,which corroborated with the findings of animal tests,indicating that the mechanism of Panax ginseng to improve vascular endothelial function impairment in hyperlipidemia through miR-370-3p/IRAK2/NF-κB pathway was also validated at the clinical level.