| Background:Atherosclerosis(AS)is the primary pathological basis of various cardiovascular diseases,and the cardiovascular and cerebrovascular events due to AS are still major challenges to global public health.Therefore,controlling and delaying disease progression positively has an important social significance.Traditional Chinese Medicine mostly attributes AS to the categories of "chest arthralgia","pulse arthralgia" or "stroke",and its formation is closely related to the theory of blood stasis.Xuefu Zhuyu Decoction is a famous prescription created by Wang Qingren,a physician in the Qing Dynasty,for the treatment of "various syndromes of blood stasis in the chest".The combination of various medicines in this Decoction can promote blood circulation and remove blood stasis,regulate Qi and relive pain,protect vascular endothelium,inhibit inflammatory response,and resist platelet aggregation.Therefore,it is widely used in the treatment of coronary atherosclerotic heart disease.Previous researches on the anti-atherosclerosis targets of Xuefu Zhuyu Decoction were based on hypothesis-driven medical research model,in other words,the hypotheses were proposed and then would be verified,which had some limitations.With the emergency of omics analysis techniques,we have a more systematic and comprehensive understanding of diseases and drug intervention mechanisms.Transcriptomics could comprehensively reveal the gene expression of an individual at a specific time or in a specific tissue,bringing significant changes to medical research.Objective:Transcriptomic analysis was conducted to explore genes and signaling pathways significantly related to AS,as well as possible intervention targets of Xuefu Zhuyu Decoction in the treatment of AS.Methods:(1)ApoE-/-mice were fed with high fat to establish animal models of atherosclerosis.At 8 weeks,the plaque formation was observed by HE staining and Oil Red O staining of aortic root to evaluate the success of the AS model.(2)AS mice models were divided into Model group,Xuefu Zhuyu group and Atorvastatin group,followed by high-fat diet and corresponding drugs respectively for 12 weeks.C57BL/6N mice were used as controls,followed by normal diet and corresponding volume of deionized water.The levels of blood lipid were detected by automatic biochemical analyzer to evaluate the effects of Xuefu Zhuyu Decoction on TC,TG,HDL and LDL.HE staining,Oil Red O staining,Movat staining,α-SMA immunohistochemical staining and CD68 immunofluorescence staining were performed to evaluate the effects of Xuefu Zhuyu Decoction on aortic plaque structure,lipid deposition and plaque components.(3)Mice aortic samples underwent RNA-sequencing on the HiSeq platform,then the libraries were constructed by Illumina TruseqTM RNA sample prep Kit.After obtaining the gene Read Counts,differential gene expression analysis was performed using edgeR and DEGseq according to the gene abundance,and then the functions of differential genes would be studied.Gene set enrichment analysis including functional and pathway analyses were performed based on Gene Ontology(GO)and KEGG pathway.Protein-protein interaction(PPI)network was visualized by STRING and Cytoscape3.7.1.The protein expressions of differential genes were detected by Western blotting(WB).(4)The drug freeze-dried powder of Xuefu Zhuyu Decoction was prepared and the Raw264.7 macrophages were activated with Lipopolysacharide(LPS)to build an in vitro model.The effect of Xuefu Zhuyu Decoction on mRNA expression of main differential genes would be verified and analyzed by quantitative real-time PCR.Results:(1)The serum TC,HDL and LDL levels of ApoE-/-mice fed with high-fat diet for 8 weeks were significantly higher than those of C57BL/6N mice(P<0.01).Part of the vascular wall of aortic root became thinning,meanwhile,lipid and foam cells were deposited in the vessel wall,which developed into atherosclerotic plaques.(2)Atorvastatin could significantly reduce the levels of TC and HDL(P<0.05),however,Xuefu Zhuyu Decoction had no significant effect on serum lipid levels(P>0.05).(3)The vessel wall of mice in Normal group was thick and intact,besides,the intima was smooth and undamaged.However,the vessel wall of mice in Model group,Xuefu Zhuyu group and Atorvastatin group were thin,and foam cells were aggregated and cholesterol crystals were formed in the plaques.Compared with the Model group,Xuefu Zhuyu Decoction and Atorvastatin could significantly reduce the lipid deposition and the number of macrophages in plaques(P<0.05),meanwhile,they could also significantly increase the collagen content in plaques(P<0.01).Xuefu Zhuyu Decoction and Atorvastatin had no obvious effects on the plaque area and the number of smooth muscle cells in plaques(P>0.05).(4)Compared with Normal group,the transcriptomics results showed that a total of 1094 genes were dysregulated in the aorta of Model mice,in which 953 genes were upregulated,and 141 genes were downregulated.Gene set enrichment analysis(GSEA)was performed against KEGG pathway,we found that the degree of enrichment of Cytokine-cytokine receptor interaction and Chemokine signaling pathway were high,and the genes were in the "up" state.When comparing with Model group,the expressions of differential genes in Xuefu Zhuyu group were also mainly enriched in the Cytokine-cytokine receptor interaction and Chemokine signaling pathway,including Ccl12,Ccl22,Cx3cr1,Ccr7,Ccr2,Tnfrsf25 and Gdf5.And the gene expression levels of these molecules tended to be normal after the intervention of Xuefu Zhuyu Decoction.When comparing with Model group,the expressions of differential genes in Atorvastatin group showed the strongest enrichment in Regulation of lipolysis in adipocytes.In addition,they were also significantly enriched in Cytokine-cytokine receptor interaction,including Cx3cr1,Tnfrsf25,Tnfrsf9,Gdf5.After the treatment of Atorvastatin,the gene expressions of these molecules gradually returned to normal levels.TRRUST combined with PPI suggested that Nfkb-1 was the upstream core transcription factor,which could significantly regulate the expression of chemokines.(5)Compared with Normal group,the expressions of CX3CL1,CX3CR1,CCR2 and NF-κB in the aorta of Model group were significantly increased(P<0.05 or P<0.01),while GDF5 expression obviously decreased(P<0.01).When comparing with Model group,Xuefu Zhuyu Decoction could measurably decrease the expressions of CX3CL1 and CX3CR1(P<0.05),but increase GDF5 expression(P<0.01).In addition,atorvastatin also could substantially decrease CX3CL1 expression(P<0.05),but increase GDF5 expression(P<0.05).(6)Compared with Control group,the mRNA expressions of CX3CL1,CCL2,CCR2,CCL12 and CCL22 were significantly up-regulated in LPS-stimulated RAW264.7 macrophages(P<0.05 or P<0.01).And the levels of CX3CL1,CX3CR1,CCL2,CCR2,CCR7,CCL12,CCL22 mRNA were significantly down-regulated(P<0.05 or P<0.01)after treating with Xuefu Zhuyu Decoction.Conclusions:Xuefu Zhuyu Decoction could regulate plaque components effectively,increase plaque stability and improve atherosclerotic lesion.Its drug efficacy might be significantly related to Cytokine-cytokine receptor interaction and Chemokine signaling pathway.Especially Cx3cr1/Cx3cl1,Ccr2/Ccl2,Ccr7,Ccl12,Ccl22 could be the targets for therapeutic interventions in atherosclerosis,which deserved further research. |
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